lecemoer
www.epa.gov/or
United States
Environmental Protection
Agency
Bio-response Operational
Testing and Evaluation
(BOTE) Project
Phase 1: Decontamination Assessment
IMJffil
Office of Research and Development
National Homeland Security Research Center
-------
&EPA
EPA-600-R-13-168
BIO-RESPONSE OPERATIONAL TESTING AND EVALUATION
(BOTE) PROJECT
PHASE 1: DECONTAMINATION ASSESSMENT
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, DC 20460
-------
NOTICE
The Bio-Response Operation Testing and Evaluation (BOTE) Project was a joint effort among
the Department of Homeland Security Science and Technology Directorate (DHS S&T), the U.S.
Environmental Protection Agency (EPA), and the Centers for Disease Control and Prevention
(CDC). DHS and EPA managed and funded this effort through an interagency agreement (IA
DW-89923315) with the Idaho National Laboratory (INL) for providing and establishing the test
bed and on-site execution support. Additional planning and execution support was provided by:
• Defense Threat Reduction Agency (DTRA) - funded by DHS S&T through an
interagency agreement with the Department of Defense (DOD), HSHQDC-10-X-00429.
• Honeywell, under contract HDTRA2-06-D-0001 with DTRA.
• ARCADIS-US, Inc., funded by EPA through EP-C-09-027 WA 1-13.
• EPA Region 10 Superfund Technical Assessment and Response Team (START)
Contract # EP-S7-06-02 with Ecology & Environment, Inc.
• Pacific Northwest National Laboratory (PNNL), funded by DHS S&T through an
interagency agreement with the Department of Energy (DOE), HSHQDC-07-X-00421/8.
• U.S. Geological Survey (USGS), funded by EPA through DW-1495774801-1.
• Pegasus Technical Services, Inc., funded by EPA through Contract # EP-C-11-006.
• Battelle under an interagency agreement between EPA and DOD, Defense Technical
Information Center, Chemical, Biological, Radiological, and Nuclear Defense Information
and Analysis Center (DW-9792323001, Contract No. SP0700-00-D-3180, Delivery Order
0679, Technical Area Task 886).
• DYNAMAC Corporation via the EPA's Decontamination Analytical & Technical Service
contract (EP-W-06-089, TO-02-07-09-0015).
• Sandia National Laboratory (SNL), funded by DHS S&T through an interagency
agreement with the DOE, HSHQPM10X00095.
• Lawrence Livermore National Laboratory (LLNL), funded by EPA through an interagency
agreement with DOE, DE-AC52-07NA27344.
• Chet Burks Productions, Inc., funded by EPA through Contract* EP-11-C-000101.
-------
Questions concerning this document or its application should be addressed to:
Christopher E. Russell*, DHS
BOTE co-Program Manager
*formerly DHS, currently a
government contractor
chris.russell1@us.army.mil
Shawn P. Ryan, EPA
BOTE co-Program Manager
ryan.shawn@epa.gov
Shannon D. Serre, EPA
BOTE co-Program Manager
and Decontamination
Assessment Group Lead
serre.shannon@epa.gov
Leroy Mickelsen, EPA
pH Adjusted
BleachDecontamination
Process Development Lead
mickelsen.leroy@epa.gov
Dino Mattorano, EPA
Sampling Planning and
Execution Group Lead
mattorano.dino@epa.gov
Paul Lemieux, EPA
Waste Management and Cost
Analysis Group Lead
lemieux.paul@epa.gov
Erin Silvestri, EPA
Exposure Assessment
Group Lead
silvestri.erin@epa.gov
-------
DISCLAIMER
This report has been peer and administratively reviewed and has been approved for publication
as an EPA document. It does not necessarily reflect the views of the EPA. No official
endorsement should be inferred. The EPA does not endorse the purchase or sale of any
commercial products or services. This report includes photographs of commercially available
products. The photographs are included for purposes of illustration only and are not intended to
imply that the EPA approves or endorses the product or its manufacturer.
IV
-------
FOREWORD
Following the tragic events of September 11, 2001, new legislation, Presidential Directives and
national plans called upon the U.S. Environmental Protection Agency (EPA) to support
government-wide efforts to prevent, protect, mitigate, respond to, and recover from terrorist
attacks, natural disasters, catastrophic incidents and other hazards. Although these homeland
security responsibilities can be traced to EPA's traditional role in consequence management,
they took on renewed focus and urgency as the Agency confronted evolving threats and
hazards.
Biological agents such as Bacillus anthracis ("anthrax") became a major concern following
Amerithrax -several incidents that impacted both public health and safety when several
buildings became contaminated and people died from exposures to anthrax spores. Under
Homeland Security Presidential Directive-10, EPA was tasked with leading, in coordination with
the other agencies and departments, the development of specific standards, protocols, and
capabilities to address the risks of contamination following a biological attack. The Directive
required the development of strategies, guidelines, and plans for the decontamination that would
be needed in response to such an incident. Building preparedness required a concerted effort to
understand the current cleanup capabilities and the research that was needed to address
knowledge gaps.
Over the past decade, EPA's research has advanced understanding of the nature and risks of
biological agent contamination and examined the need to develop reliable technologies that can
effectively contribute to short- and long-term remediation solutions. Based on interagency
planning and assessments, EPA has focused primarily on anthrax due to its persistence and the
challenges associated with cleaning it up. We believe that building the capacity to respond to
and decontaminate indoor and outdoor areas following an anthrax incident is critical to
understanding remediation of other biological threats as well. And yet, depending on the extent
of contamination in a wide area incident, current and often untested capabilities may be
significantly challenged and prove to be impracticable. EPA, therefore, continues to explore and
develop technologies that will enhance remediation capabilities for responding to large-scale
biological incidents.
This report documents the results of one of these efforts to advance the practice of biological
agent cleanup. The Bio-response Operational Testing and Evaluation (BOTE) Project was a
multi-agency effort designed to operationally test and evaluate, at the scale of a moderately
sized building, a response to a B. anthracis spore release from initial public health and law
enforcement investigation through environmental remediation. The effort involved more than 300
participants from across the government, including representatives from the Department of
Homeland Security; EPA; Centers for Disease Control and Prevention and the Laboratory
Response Network; Department of Energy National Laboratories; Department of Defense -
Defense Threat Reduction Agency; Federal Bureau of Investigation; Coast Guard; and National
Guard.
-------
The BOTE Project was divided into two phases: a field-level decontamination assessment and
an operational exercise. This report documents the results from Phase 1, designed to bring the
latest clean up-related research to the field to operationally transfer, assess, and study current
remediation capabilities. The results of this phase were expected to contribute to response and
remediation activities utilized during the functional exercise that followed as Phase 2. An After
Action Report has been put together separate from the Phase 1 report, to document Phase 2.
The results of the BOTE Project build national resilience against biological attacks, both in terms
of evaluating technologies at the operational scale and as a collaborative interagency response
and recovery effort. EPA is pleased to make these findings available, which is an important step
towards fulfilling the Agency's Homeland Security responsibilities and achieving EPA's overall
mission to protect human health and the environment.
Juan Reyes
Acting Associate Administrator
EPA's Office of Homeland Security
VI
-------
ACKNOWLEDGMENTS
The BOTE Project's Phase 1 required the collaboration of numerous Federal and contractor
personnel for planning and successful execution. The project could not have been successfully
accomplished without the collective commitment and contributions of all involved.
The following are acknowledged for their role in the primary data analysis and authorship of this
report:
M. Worth Calfee, Ph.D., EPA, Office of Research and Development
Bruce A. Hinds, DTRA, DTRAJ9-CXT
Paul M. Lemieux, Ph.D., EPA, Office of Research and Development
Cmdr. Dino Mattorano, EPA, Office of Solid Waste and Emergency Response
Leroy Mickelsen, EPA, Office of Solid Waste and Emergency Response
Scott Minamyer, EPA, Office of Research and Development
Tonya Nichols, Ph.D., EPA, Office of Research and Development
Shawn P. Ryan, Ph.D., EPA, Office of Research and Development
Shannon D. Serre, Ph.D., EPA, Office of Research and Development
Sanjiv R. Shah, Ph.D., EPA, Office of Research and Development
Erin Silvestri, EPA, Office of Research and Development
Sarah Taft, Ph.D., EPA, Office of Research and Development
Brett Amidan, PNNL
Brent Pulsipher, PNNL
Battelle, contractor to EPA
Coleman Scientific Consulting, subcontractor to Battelle
Cubic Applications, Inc., contractor to DHS
Dynamac Corporation, contractor to EPA
The following are acknowledged for their project planning and execution leadership:
Lance Brooks, formerly with DHS S&T
M. Worth Calfee, Ph.D., EPA, Office of Research and Development
Erica Canzler, EPA, Office of Solid Waste and Emergency Response
Michael Carpenter, INL
Maj. James Clegern, DTRA
Bruce A. Hinds, DTRA, DTRAJ9-CXT
Raymond Ippolito, National Guard Civil Support Team
Laura Jevitt, formerly with CDC
CDR Paul Judice, DTRA, DTRAJ9-CXT
Sang Don Lee, Ph.D., EPA, Office of Research and Development
vii
-------
Paul M. Lemieux, Ph.D., EPA, Office of Research and Development
Cmdr. Dino Mattorano, EPA, Office of Solid Waste and Emergency Response
Leroy Mickelsen, EPA, Office of Solid Waste and Emergency Response
Scott Minamyer, EPA, Office of Research and Development
Stephen Morse, Ph.D., CDC
Tonya Nichols, Ph.D., EPA, Office of Research and Development
Lt. Col. Nik Putnam, DTRA
Frank Roberto, INL
Jacky Rosati Rowe, Ph.D., EPA, Office of Research and Development
Christopher E. Russell, formerly with DHS, S&T Directorate
Shawn P. Ryan, Ph.D., EPA, Office of Research and Development
Frank Schaefer, Ph.D., EPA, Office of Research and Development
Shannon D. Serre, Ph.D., EPA, Office of Research and Development
Sanjiv Shah, EPA, Office of Research and Development
Erin Silvestri, EPA, Office of Research and Development
Sarah Taft, Ph.D., EPA, Office of Research and Development
Angie Weber, CDC
The additional contributions of the following are acknowledged for scientific planning,
coordination, and execution:
EPA, Office of Research and Development
Brian Attwood, Ph.D. (formerly EPA)
Eletha Brady-Roberts
Hiba Ernst, Ph.D.
Vincente Gallardo, Ph.D.
Kevin Garrahan, Ph.D.
Jacky Rosati Rowe, Ph.D.
Joseph P. Wood
EPA, Office of Solid Waste and Emergency Response
William Albrecht (formerly EPA)
Michele Burgess
Mario lerardi
James Michael
Marissa Mullins
Curtis Snook, M.D. (formerly EPA)
EPA, Office of Chemical Safety and Pollution Prevention
Carlton "Jeff" Kempter (formerly EPA)
Stephen Tomasino, Ph.D.
VIM
-------
EPA, Regional Offices
Deborah McKean, Region 8
Mike Nalipinski, Region 1
Dave Rees, Region 10
Richard Rupert, Region 3
Terry Stilman, Region 4
Wendy O'Brien, Region 8
Non-EPA contributors
Martin Meltzer, CDC
Robert Knowlton, SNL
Sharna Rossberg, INL
Michael Carpenter, INL
Jesse Bennett, INL
Kelly Bonjour, INL
Stephen Reese, INL
Region 10 START
Booz Allen Hamilton, Inc.
Cubic Applications, Inc.
SAIC, Inc.
Honeywell Technologies Solutions, Inc. (HTSI)
In total, over 3,000 samples were collected during the BOTE Project, Phase 1. The participation
of the following in this critical project activity is acknowledged:
EPA
Stephen Ball, EPA Region 4 On-scene Coordinator
Myles Bartos, EPA Region 3 On-scene Coordinator
Jeff Bechtel, EPA Region 2 On-scene Coordinator
Romy (Lee) Campisano, EPA, Office of Research and Development
Margaret Chong, EPA Region 2 On-scene Coordinator
Joe Davis, EPA Region 7 On-scene Coordinator
Brent England, EPA Region 1 On-scene Coordinator
Rich Fetzer, EPA Region 3 On-scene Coordinator
Charlie Fitzsimmons, EPA Region 3 On-scene Coordinator
Jordan Garrard, EPA Region 4 On-scene Coordinator
Jayson Griffin, EPA, Office of Solid Waste and Emergency Response
Elsbeth Hearn, EPA Region 1 On-scene Coordinator
Matt Huyser, EPA Region 4 On-scene Coordinator
Steve Jarvela, EPA Region 3 On-scene Coordinator
Janice Kroone, EPA Region 7 On-scene Coordinator
H. Alan Lindquist, EPA, Office of Research and Development
Jeanelle Martinez, EPA, Office of Solid Waste and Emergency Response
Dennis Matlock, EPA Region 3 On-scene Coordinator
IX
-------
Don Mclaughlin, EPA Region 3 On-scene Coordinator
Steve Merritt, EPA Region 8 On-scene Coordinator
Craig Meyers, EPA Region 8 On-scene Coordinator
Jose Negron, EPA Region 4 On-scene Coordinator
Kathy Nickel, EPA, Office of Research and Development
Lukas Oudejans, Ph.D., EPA, Office of Research and Development
Randy Schademann, EPA Region 7 On-scene Coordinator
Manuel Schmaedick, EPA Region 7 On-scene Coordinator
Megan Schuette, EPA Region 7 On-scene Coordinator
Mia Pasquerella, EPA Region 1 On-scene Coordinator
Subash Patel, EPA Region 4 On-scene Coordinator
Dominic Ventura, EPA Region 3 On-scene Coordinator
Chris Wagner, EPA Region 3 On-scene Coordinator
Stephanie Wenning, EPA Region 3 On-scene Coordinator
Stuart Willison, EPA, Office of Research and Development
Charlena Yoder, EPA, Office of Research and Development
U. S. Marine Chemical Biological Incident Response Force (CBIRF)
CPL Zachary S. Anaya
LCPL Jonathan A. Cullbreath
LCPL Herbert L. Dowlearn
National Guard Civil Support Teams
James Kivlehan, National Guard 1st Civil Support Team
Jason Maguire, National Guard 1st Civil Support Team
Tyler Pobiedzinski, National Guard 1st Civil Support Team
David Whitaker, National Guard 1st Civil Support Team
SGT Christian Gonzales, National Guard 8th Civil Support Team
SGT Matthew Mitchell, National Guard 8th Civil Support Team
SSG Amadeus Weeks, National Guard 8th Civil Support Team
MAJ Mark Bianchi, National Guard 12th Civil Support Team
SFC Harvey, National Guard 12th Civil Support Team
SSG Jordan, National Guard 12th Civil Support Team
SSG Christopher Mason, National Guard 12th Civil Support Team
SSG Matthew Maguire, National Guard 12th Civil Support Team
SSG Robert Mingolla, National Guard 12th Civil Support Team
SFC Michael Owens, National Guard 12th Civil Support Team
SGT David Turner, National Guard 12th Civil Support Team
SSG Darren Odom, National Guard 24th Civil Support Team
SSgt Kim Teakwan, National Guard 24th Civil Support Team
SSG Joe Cavada, National Guard 33rd Civil Support Team
SFC Dericko Gaither, National Guard 33rd Civil Support Team
SSG Earl Johnson, National Guard 33rd Civil Support Team
SGT Tanisha Mercado, National Guard 33rd Civil Support Team
-------
SGT Tyrone Perry, National Guard 33rd Civil Support Team
SGT Joelray Campbell, National Guard 41st Civil Support Team
SGT Scott Terrill, National Guard 41st Civil Support Team
CRT Bliven, National Guard 42nd Civil Support Team
SGT Kenna, National Guard 42nd Civil Support Team
MSgt Kuberacki, National Guard 42nd Civil Support Team
SGT Kubas, National Guard 42nd Civil Support Team
SSG Robert Ricks, National Guard 42nd Civil Support Team
2LT Stern, National Guard 42nd Civil Support Team
SGT Joseph G Berenzden, National Guard 43rd Civil Support Team
SSG Edward J. Edgerton, National Guard 43rd Civil Support Team
SGT Robert L. Nelson, National Guard 43rd Civil Support Team
1LT John R. Ptak, National Guard 43rd Civil Support Team
SGT Jason Michael Barfield, National Guard 45th Civil Support Team
1LT Shawn Paul Chance, National Guard 45th Civil Support Team
SSG Tony Edward Dooley, National Guard 45th Civil Support Team
SGT Stephen Anthony Evans, National Guard 45th Civil Support Team
SGT Gregory Allen Manning, National Guard 45th Civil Support Team
SSG William Michael Mclntyre, National Guard 45th Civil Support Team
SGT David Joseph Owen, National Guard 45th Civil Support Team
SFC Wendell Dean Reed, National Guard 45th Civil Support Team
SSG Jeremy Murry, National Guard 46th Civil Support Team
SGT Dan Pose, National Guard 46th Civil Support Team
SSG Sarah Allder, National Guard 48th Civil Support Team
SGT Ketner Jackson, National Guard 48th Civil Support Team
SGT Michael Mitchell, National Guard 48th Civil Support Team
SSG Robert Kirk, National Guard 48th Civil Support Team
SSgt Erik Partridge, National Guard 48th Civil Support Team
TSgt Raymond Simpson, National Guard 48th Civil Support Team
Nicholas Kenton, National Guard 54th Civil Support Team
Terek Taillon, National Guard 54th Civil Support Team
1LT Dustin Nash, National Guard 73rd Civil Support Team
TSgt Jarod Smith, National Guard 73rd Civil Support Team
SPC Claude Williams, National Guard 73rd Civil Support Team
Alex Smith, National Guard 81st Civil Support Team
Shane Anderson, National Guard 83rd Civil Support Team
Casey Carnahan, National Guard 83rd Civil Support Team
Jason Green, National Guard 83rd Civil Support Team
Jed Reeves, National Guard 83rd Civil Support Team
Nate Velin, National Guard 83rd Civil Support Team
SSG Zackery Hauf, National Guard 84th Civil Support Team
SGT Chris Rosentreter, National Guard 84th Civil Support Team
SSG Robert McCree, National Guard 102nd Civil Support Team
SGT Dutch Inman, National Guard 103rd Civil Support Team
XI
-------
SSgt Nina Kolyvanova, National Guard 103 Civil Support Team
U.S. Coast Guard
Greg Livingston
Karl Siegmund
Jeremy Thomas
Region 10 START
The sample analyses were performed by the Laboratory Response Network, INL, LLNL, and the
EPA Office of Chemical Safety and Pollution Prevention (Ft. Meade). The efforts of the following
for coordination and execution are acknowledged:
ARCADIS-US, Inc., contractor to the EPA
Pegasus, contractor to the EPA
Dale Griffin, USGS
Jarful Hassan, EPA, Office of Chemical Safety and Pollution Prevention
Jason Duncan, EPA, Office of Chemical Safety and Pollution Prevention
Laura Jevitt, formerly with CDC
Stephen Morse, CDC
Frank Roberto, INL
Kara Cafferty, INL
Angie Weber, CDC
Staci Kane, Ph.D., LLNL
Sonia Letant, Ph.D., LLNL
Gloria Murphy, LLNL
Teneile Alfaro, LLNL
Eddie Salazar, LLNL
Julie Avila, LLNL
Thomas Bunt, LLNL
Lalena Wallace, U.S. Army's Edgewood Chemical Biological Center
Lisa Smith, U.S. Army's Edgewood Chemical Biological Center
Dallas County Health and Human Services
Idaho Bureau of Laboratories
Florida Department of Health Bureau Laboratories
Minnesota Department of Health
State Hygienic Lab at the University of Iowa
Virginia Division of Consolidated Laboratory Services
Unified State Laboratories: Utah Public Health
Wadsworth Center, New York State Department of Health
XII
-------
Acknowledgment is also greatly warranted for those who were responsible for the health and
safety of the hundreds of personnel on site to conduct this project, specifically:
LCDR David Blauser, DTRA, DTRAJ9-CXT
CAPT Marshall Gray, EPA, Office of Research and Development
Stephen Musson, EPA, Office of Research and Development
INL Emergency Medical Technicians
On-site video documentation of the BOTE Project was coordinated and conducted by:
Chet Burks Productions, Inc.
The following are acknowledged for their technical editing of this report:
Joan Bursey, Ph. D., grantee to EPA
Booz Allen Hamilton, Inc. (Research Triangle Park, NC)
XIII
-------
TABLE OF CONTENTS
Notice ii
Disclaimer iv
Foreword v
Acknowledgments vii
Table of Contents xiv
List of Appendices xxvii
List of Tables xxviii
List of Figures xxxiv
Acronyms and Abbreviations xli
Executive Summary xlv
1. I ntroduction 1
1.1. Background 2
1.2. Study Objectives 4
1.2.1. Objective 1: Decontamination Efficacy Assessment 5
1.2.1.1. Decontamination Technologies Efficacy Assessment 6
1.2.1.2. Material Impact 6
1.2.1.3. Waste Management 6
1.2.1.4. Decontamination Line Wash Water - Handling, Treatment, and
Disposal 8
1.2.2. Objective 2: Demonstration and Assessment of Biological Sampling
and Analysis Methods 8
1.2.2.1. Surface Sampling 9
1.2.2.2. Rapid Viability-Polymerase Chain Reaction 9
1.2.2.3. Aggressive Air Sampling 9
1.2.3. Objectives: Overall Cost Analysis 10
1.2.4. Objective 4: Assessment of Potential Exposure 11
xiv
-------
1.2.4.1. Assessment of Bacillus Spore Migration from Inside to Outside
a Contaminated Building 11
1.2.4.2. Assessment of Reaerosolization 12
1.2.4.3. Exposure Assessment Plan 14
2. Materials and Methods 15
2.1. Facility 15
2.2. Test Organism 26
2.3. Spore Dissemination 27
2.4. Reference Dissemination and Surface Loading Determination (Referee
Methods) 29
2.5. Sampling Methods and Equipment 31
2.5.1. General Sampling Schedule 33
2.5.2. Sample Collection Teams and PPE 34
2.5.3. Summary of Sample Collection Methods used during the BOTE
Project 34
2.5.4. Surface Sampling Methods 34
2.5.4.1. Cellulose Sponge-stick Wipes 35
2.5.4.2. Macrofoam Swabs 36
2.5.4.3. Vacuum Socks 36
2.5.4.4. Versalon® Wipes 36
2.5.5. Air Sampling Methods 36
2.5.5.1. SKC BioSamplers® 36
2.5.5.2. Model 3314 UV-APS™ Spectrometer 38
2.5.5.3. Dycor XMX/2L-MIL Aerosol Collection System 39
2.5.5.4. Mattson-Garvin Model 220 Slit-to-Agar Sampler 40
2.6. Test Design and Sampling Methodology 41
2.6.1. Decontamination Efficacy Assessment 43
2.6.2. Wash Water Collection and Treatment 43
2.6.3. Assessment of the RV-PCR Method 46
2.6.4. Aggressive Air Sampling Assessment 46
xv
-------
2.6.4.1. Aggressive Air Sampling Methodology 46
2.6.4.2. Aggressive Air Sampling Area Preparation 48
2.6.4.2.1. Pre-cleaning Surfaces 48
2.6.4.2.2. Establishing Isolation 48
2.6.4.2.3. Establishing a Negative Pressure Environment 49
2.6.4.3. Sampling Strategy 49
2.6.4.4. Sampling Procedures 50
2.6.4.5. Sampling Team Organization and Roles 50
2.6.4.6. Initial Sampling Team Training Session 51
2.6.4.7. Sampling Equipment and Supplies 52
2.6.4.8. Sample Collection 52
2.6.5. Assessment of Bacillus Spore Migration from Inside to Outside a
Contaminated Building 53
2.6.5.1. Selection of Sample Matrix 53
2.6.5.1.1. Laboratory Preparation of Sample Matrix and
Containers 54
2.6.5.1.2. Preparation of Sampling Kits 54
2.6.5.2. Sand Sampling Process 55
2.6.5.3. Placement and Retrieval of Sand Dishes 55
2.6.6. Reaerosolization Assessment and Measurement Protocols 59
2.6.6.1. Sampling Design and Layout 60
2.6.6.2. Testing and Measurement Protocols 60
2.7. Sample Tracking and Shipping 64
2.8. Cross-Contamination Reduction Methods 65
2.9. Sample Analysis Methods 67
2.9.1. Surface Sample Analysis Methods 67
2.9.1.1. Analysis of Environmental Surface Samples by the LRN 67
2.9.1.2. Analysis of Surface and Referee Samples by INL 69
2.9.1.2.1. Processing of Settling Plates 69
XVI
-------
2.9.1.2.2. Analysis of RMCs 69
2.9.1.2.3. Wipe Sample Processing 69
2.9.1.2.4. RMC and Wipe Sample Serial Dilutions and Plating 70
2.9.1.2.5. RMC and Wipe Sample Filter Plating 70
2.9.1.3. Plate Incubation and Counts 70
2.9.2. Rapid Viability-Polymerase Chain Reaction Analysis of Wipe Samples... 71
2.9.2.1. Sample Type and Sampling Plans 72
2.9.2.2. Sample Receiving 72
2.9.2.3. Sample Processing: Spore Recovery from Wipe 73
2.9.2.4. Rapid Viability-Polymerase Chain Reaction Sample Processing
and Analysis 76
2.9.2.5. Analysis of Concentrated Enrichment Culture 76
2.9.2.6. Bg DMA Standards for Real-time PCR 77
2.9.2.7. Bg Real-time PCR Analysis 77
2.9.2.8. Data Interpretation and Reporting 77
2.9.2.9. Culture Sample Processing and Analysis 78
2.9.3. Air Sample Analysis Methods 79
2.9.3.1. SKC BioSampler® 79
2.9.3.1.1. Analysis Method 79
2.9.3.1.2. Data Reduction 80
2.9.3.2. Dycor®XMX/2L-MIL Aerosol Collection Systems 80
2.9.3.2.1. Analysis Method 80
2.9.3.2.2. Data Reduction 81
2.9.3.3. Mattson-Garvin Model 220 slit-to-agar 81
2.9.3.3.1. Analysis Method 81
2.9.3.3.2. Data Reduction 81
2.9.3.4. Ultraviolet Aerodynamic Particle Sizer 82
2.9.4. Analysis of Sand Samples for the Study of Bacillus Spore Migration
from Inside the Building to Outside 82
XVII
-------
2.9.4.1. Division of Sand Samples between USGS and EPA 82
2.9.4.2. Spore Concentration and DMA Extraction 82
2.9.4.3. Quantitative PCR Detection of B. globigii 83
2.9.4.4. EPA Analytical Specifics 84
2.9.4.5. EPA PCR Inhibition Analysis 85
2.9.4.6. USGS Analytical Controls 85
2.10. Decontamination Methods 86
2.10.1. Fumigation by STERIS Corporation VHP® 86
2.10.2. Surface Decontamination Approach using pH-Adjusted Bleach 86
2.10.3. Fumigation by Sabre Technical Services, LLC, with Chlorine Dioxide
Gas 87
2.10.4. Treatment of Decontamination line Wash Water 87
3. Results 89
3.1. Reference Method Results 89
3.1.1. Settling Plates 89
3.1.1.1. Round 1 Settling Plate Results 89
3.1.1.2. Round 2 Settling Plate Results 89
3.1.1.3. Round 3 Settling Plate Results 89
3.1.2. Reference Material Coupons 90
3.1.2.1. Round 1 RMC Results 91
3.1.2.2. Round 2 RMC Results 92
3.1.2.3. Rounds RMC Results 94
3.1.2.4. Comparisons of RMC Results 96
3.2. Surface, Air, Sand, and Water Sampling Results 97
3.2.1. Surface Sampling Results 97
3.2.1.1. MFP (Baseline Assessment) Surface Sampling Results 98
3.2.1.2. Round 1 Surface Sampling Results 99
3.2.1.2.1. Round 1 Pre-decontamination Surface Sampling
Results 99
XVIII
-------
3.2.1.2.2. Round 1 Post-decontamination Surface Sampling
Results 100
3.2.1.3. Round 2 Surface Sampling Results 101
3.2.1.3.1. Round 2 Pre-decontamination Surface Sampling
Results 101
3.2.1.3.2. Round 2 Post-decontamination Surface Sampling
Results 102
3.2.1.4. Round 3 Surface Sampling Results 102
3.2.1.4.1. Round 3 Pre-decontamination Surface Sampling
Results 102
3.2.1.4.2. Round 3 Post-decontamination Surface Sampling
Results 103
3.2.2. Results from Rapid Viability-Polymerase Chain Reaction Analysis 146
3.2.2.1. LLNL RV-PCR Results 146
3.2.2.1.1. Samples from the MFP Event 148
3.2.2.1.2. Samples from Round 1 148
3.2.2.1.3. Samplesfrom Round 2 148
3.2.2.1.4. Samplesfrom Round 3 149
3.2.2.2. EPA-OPP-MLB Results 149
3.2.3. Air Sampling 170
3.2.3.1. Aggressive Air Sampling Results 170
3.2.3.2. Reaerosolization Assessment Results 174
3.2.3.2.1. IndoorAirSKC BioSampler® Data 174
3.2.3.2.1.1. Descriptive Statistics 174
3.2.3.2.1.2. Statistical Analysis 179
3.2.3.2.2. Indoor Surface Sampling in Reaerosolization Study
Rooms 181
3.2.3.2.2.1. Indoor Surface Sampling Data for
Reaerosolization Study Rooms - Stage 4 182
3.2.3.2.2.2. Indoor Surface Sampling Data for
Reaerosolization Study Rooms - Stage 5 185
3.2.3.2.3. Particle Measurements 187
XIX
-------
3.2.4. Bacillus Spore Migration from Inside the Building to Outside Results.... 195
3.2.4.1. Limits of Detection 195
3.2.4.1.1. EPA Instrument Limit of Detection 196
3.2.4.1.2. EPA Environmental Limit of Detection 197
3.2.4.1.3. USGS Instrument Limit of Detection 198
3.2.4.1.4. USGS Environmental Limit of Detection 199
3.2.4.2. Sand Analysis Results for Spore Migration Study 200
3.2.4.2.1. EPA Data Transformation 200
3.2.4.2.2. USGS Data 203
3.2.4.2.3. Collected Blank Samples 204
3.2.4.2.4. Results for Inside the Secondary Enclosure, External
to the Building 204
3.2.4.2.5. Results for Samples from Inside the Building and
Associated Inhibition Testing 205
3.2.4.2.6. USGS Inhibition Testing 207
3.2.4.2.7. EPA Sample Deviation 207
3.2.4.2.8. USGS Sample Deviation 208
3.2.4.3. Statistical Analysis of Sand Sample Results 208
3.2.4.3.1. Statistical Comparison of EPA vs. USGS Methods 208
3.2.4.3.2. Statistical Analysis Comparing Decontamination
Treatment Rounds 209
3.2.4.3.3. Statistical Analysis of Stages 210
3.2.4.3.4. Statistical Analysis of Sample Placement 211
3.2.4.3.5. Statistical Analysis of Possible Carryover
Contamination between Rounds 213
3.2.5. Decontamination Line Wash Water Treatment Assessment Results 214
4. Data Assessment and Discussion 218
4.1. Decontamination Methods Assessment 218
4.1.1. STERIS VHP® Fumigation 218
4.1.1.1. Process Description 218
xx
-------
4.1.1.2. Facility Contamination 220
4.1.1.3. Setup and Preparation 220
4.1.1.4. Fumigation Conditions 224
4.1.1.5. Scrubbing and Aeration 225
4.1.1.6. Demobilization 226
4.1.1.7. Temperature and Relative Humidity 226
4.1.1.8. H2O2 Measurements 229
4.1.1.9. VHP® Decontamination Results 231
4.1.1.10. Bl Results 231
4.1.1.10.1. Chemical Indicators 233
4.1.1.10.2. Surface Sampling Results 233
4.1.1.10.2.1. Field Blanks 233
4.1.1.10.2.2. Pre-decontamination Sampling 233
4.1.1.10.2.3. Post- Decontamination Samples 236
4.1.1.10.3. Decontamination Efficacy 241
4.1.1.11. Materials Effects 241
4.1.1.12. Summary of Fumigation with VHP® 241
4.1.2. Surface Decontamination Approach using pH-Adjusted Bleach 242
4.1.2.1. Decontamination Process 242
4.1.2.1.1. Background and Purpose 242
4.1.2.1.2. Process Description 242
4.1.2.1.3. Facility Contamination 243
4.1.2.1.4. Decontamination Personnel 243
4.1.2.1.5. Safety, Health, and Facility Preparation 243
4.1.2.1.6. Special Items 253
4.1.2.1.7. pH-Adjusted Bleach Building Decontamination
Procedure 255
4.1.2.1.8. pH-Adjusted Bleach Building Decontamination
Procedure Logistics 264
XXI
-------
4.1.2.2. pH-Adjusted Bleach Decontamination Results 265
4.1.2.2.1. Collection and Analysis Procedures for Surface
Samples 265
4.1.2.2.2. Sampling Results 265
4.1.2.2.2.1. Field Blanks 266
4.1.2.2.2.2. Pre-Decontamination Sampling 266
4.1.2.2.3. Decontamination Efficacy 269
4.1.2.2.4. Surface Decontamination Results 269
4.1.2.2.5. HVAC Decontamination Results 273
4.1.2.2.6. Data Limitations 275
4.1.2.3. Summary of the pH-adjusted Bleach Decontamination Process.. 276
4.1.3. Fumigation by Sabre Technical Services, LLC, with CIO2 276
4.1.3.1. Process Description 276
4.1.3.2. Facility Contamination 277
4.1.3.3. Planning and Design 277
4.1.3.4. Fumigation Conditions 279
4.1.3.5. Scrubbing and Aeration 280
4.1.3.6. Demobilization 280
4.1.3.7. Temperature and Relative Humidity 283
4.1.3.8. CIO2 Measurements 284
4.1.3.9. CIO2 Decontamination Results 293
4.1.3.9.1. Biological Indicator Results 293
4.1.3.9.2. Surface Sampling Results 297
4.1.3.9.2.1. Field Blanks 297
4.1.3.9.2.2. Pre-decontamination Sampling 297
4.1.3.9.2.3. Post-decontamination Sampling 300
4.1.3.9.3. Material Effects 303
4.1.3.10. Summary of Fumigation with CIO2 303
4.2. Decontamination Line Wash Water Treatment 304
XXII
-------
4.3. Discussion of Rapid Viability-Polymerase Chain Reaction Results 304
4.4. Aggressive Air Sampling 309
4.5. Assessment of Reaerosolization 312
4.5.1. Reaerosolization Assessment Discussion 312
4.5.2. Reaerosolization Assessment Summary 314
4.6. Waste Management 315
4.6.1. General Waste Management Approach 315
4.6.2. On-Site Activities 316
4.6.3. Waste Categorization 317
4.6.4. Results 320
4.6.5. Discussion 322
4.6.6. Conclusions 326
4.7. Cost Analysis 327
4.7.1. Cost Analysis Approach 327
4.7.1.1. Costs that were included 327
4.7.1.2. Sampling 328
4.7.1.3. Laboratory Analysis 328
4.7.1.4. Decontamination 329
4.7.1.5. Waste Management 329
4.7.1.6. Building Refit 329
4.7.1.7. Incident Command 330
4.7.1.8. Costs not included 330
4.7.2. Conceptual Description of Cost Analysis 330
4.7.2.1. Sampling and Analysis Costs 331
4.7.2.2. Decontamination Costs 331
4.7.2.3. Restoration Costs 332
4.7.2.4. Total Cost Per Round 332
4.7.3. Sources of Data 332
XXIII
-------
4.7.3.1. Labor Cost Approach 334
4.7.3.2. Sampling/Analysis Cost Approach 337
4.7.3.3. Facility Decontamination Cost Approach 337
4.7.3.4. Refurbishment Cost Approach 338
4.7.3.5. Assumptions and Caveats 338
4.7.4. Results 340
4.7.4.1. Sampling and Analysis Costs 340
4.7.4.2. Facility Decontamination Costs 344
4.7.4.2.1. Cost of Labor for Decontamination and Removal 344
4.7.4.2.2. Fixed Cost and Material Costs of Decontamination
Contractors 344
4.7.4.3. Waste Management Costs 345
4.7.4.3.1. Overall Cost of Decontamination 353
4.7.4.3.2. Refurbishment Costs 354
4.7.4.3.3. Summary of Total Costs 357
4.7.5. Summary and Discussion 357
4.8. Potential Spore Migration outside a Contaminated Building 359
4.8.1. Conclusions 362
4.8.2. Future Considerations 364
4.8.2.1. Sampling Considerations 364
4.8.2.2. Analysis Considerations 364
4.9. Development of an Exposure Assessment Plan 365
5. Statistical Analysis of Pre- and Post-decontamination sampling Results,
Decontamination Efficacy, and cost assessment 370
5.1. Introduction and scope 370
5.2. Summary of Key Findings 370
5.3. Spatial Distribution Analyses 371
5.3.1. Pre-Decontamination Analyses 373
5.4. Post-Decontamination Analyses 381
XXIV
-------
5.5. Conclusions from Statistical Analysis 387
6. Quality Assurance and Quality Control 388
6.1. Background 388
6.2. Quality Assurance Objectives 388
6.3. Key QA and QC Activities and Reporting 389
6.3.1. Test Organism 389
6.3.2. Spore Dissemination and Reference Testing 389
6.3.3. Sample Collection, Tracking and Handling 390
6.3.4. Cross-Contamination Reduction Methods 391
6.3.5. Sample Analysis Methods 391
6.3.5.1. Analysis of Samples by the LRN 391
6.3.5.2. Analysis of Samples by INL 392
6.3.5.3. Analysis of Samples for the Rapid Viability-Polymerase Chain
Reaction Study 392
6.3.5.4. Analysis of Samples for the Sand Study 392
6.3.6. Collected Blank Samples 392
6.4. Technical Systems Audit 394
6.4.1. Summary of Observation and Findings 394
6.4.1.1. Sample Handling 394
6.4.1.2. Sample Storage 394
6.4.1.3. Water Concentrator 394
6.4.1.4. Waste Removal Process 394
6.5. Performance Evaluation Audit 394
6.6. Data Quality Assessment 400
7. Conclusions 402
7.1. Assessment of Decontamination Methods 402
7.2. Demonstration and assessment of biological sampling methods 405
7.3. Overall Cost Analysis 407
7.4. Assessment of Potential Exposure 407
xxv
-------
7.4.1. Assessment of Reaerosolization 408
7.4.2. Assessment of Bacillus Spore Migration from Inside to Outside a
Contaminated Building 408
7.4.3. Exposure Assessment Plan 408
7.4.4. Summary 408
8. References 410
XXVI
-------
LIST OF APPENDICES
Appendix A: INL Facility PBF-632 Test Room Photos and Inventory of Contents
Appendix B: Bg Spore Characterization Study Results
Appendix C: Surface Sampling Protocols
Appendix D: Aggressive Air Sampling (AAS) Protocol
Appendix E: LRN Bg Analysis Protocols for the BOTE Project
Appendix F: Sand Sample Preparation, Collection, and Extraction/Analysis Protocols
Appendix G: Report of Sample Results - LRN Samples
Appendix H: Waste Management and Cost Analysis Spreadsheet
Appendix I: I BAG Sensor Data
Appendix J: Sampling Plan Analysis
Appendix K: Standard Operating Procedure: Rapid-Viability Polymerase Chain Reaction
(RV-PCR) Method for the Bio-Response
Appendix L: Spatial Analysis Methodology
Appendix M: The iPad as an Electronic Laboratory Notebook
XXVII
-------
LIST OF TABLES
Table 2-1. Definitions for anticipated contamination levels and decontamination
technologies 15
Table 2-2. List of closed-circuit camera locations within PBF-632 24
Table 2-3. Location of I BAG Sensors during Dissemination 28
Table 2-4. Referee Samples for the BOTE Project 31
Table 2-5. Summary of sampling methods and the uses related to the BOTE Project
objectives 32
Table 2-6. Sampling event dates 33
Table 2-7. Type and number of samples collected during each BOTE Project round 42
Table 2-8. AAS sample locations, types, and numbers 50
Table 2-9. Sand sampling site locations 56
Table 2-10. Description of sand sample placement and purpose 57
Table 2-11. BOTE Project sand sample schedule 59
Table 2-12. SKC BioSampler® stages 61
Table 2-13. Staggered timing design for SKC BioSamplers® during dissemination (Stage
2) and pre-decontamination surface sampling (Stage 4) for all three rounds 62
Table 2-14. Staggered timing design for SKC BioSamplers® during post-decontamination
surface (Stage 5) sampling for all three rounds 63
Table 2-15. Bg primer and probe sequences 83
Table 2-16. Differences between EPA and USGSqPCR parameters 84
Table 2-17. EPA PCR analytical controls used for each of the sixteen 96-well plate assays. .. 84
Table 2-18. Samples selected for BOTE Project PCR inhibition due to decontamination
agent test 85
Table 2-19. USGS PCR analytical controls 86
Table 3-1. Summary of settling plate results from Round 2 90
Table 3-2. Summary of RMC results from Round 1 91
Table 3-3. Summary of RMC Results from Round 2 93
Table 3-4. Summary of RMC results from Round 3 95
XXVIII
-------
Table 3-5. LRN samples (swabs, sponge-stick wipes, vacuum socks) collected in the
MFP event 104
Table 3-6. LRN swab samples collected in the MFP event 105
Table 3-7. LRN sponge-stick wipe samples collected in the MFP event 106
Table 3-8. LRN vacuum sock samples collected in the MFP event 107
Table 3-9. LRN pre-decontamination surface sampling results for Round 1 113
Table 3-10. LRN pre-decontamination blank results for Round 1 115
Table 3-11. Round 1 pre-decontamination blank samples with Bg detected 116
Table 3-12. LRN post-decontamination surface sampling results for Round 1 119
Table 3-13. LRN post-decontamination blank results for Round 1 121
Table 3-14. Round 1 post-decontamination blank samples with Bg detected 122
Table 3-15. LRN pre-decontamination surface sampling results for Round 2 125
Table 3-16. LRN pre-decontamination blank results for Round 2 127
Table 3-17. Round 2 pre-decontamination blank samples with Bg detected 128
Table 3-18. LRN post-decontamination surface sampling results for Round 2 131
Table 3-19. LRN post-decontamination blank results for Round 2 133
Table 3-20. Round 2 post-decontamination blank samples with Bg detected 134
Table 3-21. LRN pre-decontamination surface sampling results for Round 3 137
Table 3-22. LRN pre-decontamination blank results for Round 3 139
Table 3-23. Round 3 pre-decontamination blank samples with Bg detected 140
Table 3-24. LRN post-decontamination surface sampling results for Round 3 143
Table 3-25. LRN post-decontamination blank results for Round 3 145
Table 3-26. Summary of LLNL samples received and processed by event type 147
Table 3-27. LLNL RV-PCR results for MFP samples 150
Table 3-28. LLNL RV-PCR results for Round 1 pre-decontamination samples 151
Table 3-29. LLNL RV-PCR results for Round 1 post-decontamination samples 152
Table 3-30. LLNL RV-PCR results for Round 2 pre-decontamination samples 155
Table 3-31. LLNL RV-PCR results for Round 2 post-decontamination samples 157
Table 3-32. LLNL RV-PCR results for Round 3 pre-decontamination samples 160
XXIX
-------
Table 3-33. LLNL RV-PCR results for Round 3 post-decontamination samples 161
Table 3-34. EPA-OPP-MLB RV-PCR results for MFP samples 165
Table 3-35. EPA-OPP-MLB RV-PCR results for Round 1 pre-decontamination samples 166
Table 3-36. EPA-OPP-MLB RV-PCR results for Round 1 post-decontamination samples 167
Table 3-37. EPA-OPP-MLB RV-PCR results for Round 2 pre-decontamination samples 169
Table 3-38. Aggressive air sampling results for Round 1 171
Table 3-39. Aggressive air sample results for Round 2 172
Table 3-40. Aggressive air sample results for Round 3 173
Table 3-41. Descriptive statistics for air concentration of Bg spores (CFU/ft3), calculated by
Round, Stage, and Room. 176
Table 3-42. Arithmetic means of surface and air concentrations of Bg spores collected and
analyzed by dilution plate method in Stage 4 (pre-decontamination), by round,
room, surface, surface type, and sample type 183
Table 3-43. Arithmetic means of surface and air concentrations of Bg spores collected and
analyzed by filter plate method in Stage 5 (post-decontamination), by round,
room, surface, surface type, and sample type* 186
Table 3-44. Summary of UV-APS event information 195
Table 3-45. Average results from EPA instrument LOD 196
Table 3-46. Average results from EPA spiked sand samples 198
Table 3-47. Average results from USGS instrument LOD 199
Table 3-48. Results from USGS spiked sand samples expressed as average genomic
equivalents in the 100 uLof eluentfrom the MO BIO PowerSoil® Kit 200
Table 3-49. EPA laboratory analysis code descriptions for the BOTE sand samples based
on mean cycle threshold value 201
Table 3-50. All EPA coded data: Round, Stage, Location, and Replicates 202
Table 3-51. USGS cycle threshold values obtained from the instrument 203
Table 3-52. Summary of the BOTE Project EPA qPCR QA results 204
Table 3-53. Codified EPA results within the secondary enclosure (and outside the building.. 205
Table 3-54. Number of sand samples collected within the building (first and second floors)
according to their codified EPA results 206
Table 3-55. Summary of qPCR decontamination agent inhibition tests of selected indoor
sand samples 207
xxx
-------
Table 3-56. EPA mean, maxima, and minima for all collected sand samples 208
Table 3-57. Summary of the number of samples within each of the positive and non-
detected classifications according to EPA analysis, by treatment round
(considering all samples) 210
Table 3-58. Summary of the number of samples within positive and ND classifications
according to EPA analysis, by collection stage (considering all samples) 211
Table 3-59. Number of samples with detected Bg by location, round, stage and detection
status based on averaged EPA analysis of individual samples 212
Table 3-60. Results for wash water collected in Round 1 after dissemination and pre-
decontamination 215
Table 3-61. Results for wash water collected in Round 2 after dissemination and pre-
decontamination 215
Table 3-62. Results for wash water collected in Round 2 during pH-adjusted bleach
decontamination 216
Table 3-63. Results for wash water collected in Round 2 after pH-adjusted bleach
decontam ination 216
Table 3-64. Results for wash water collected in Round 3 after dissemination and pre-
decontam ination 217
Table 4-1. Chronology of fumigation events with VHP® on April 19, 2011 225
Table 4-2. Results from first floor following decontamination with VHP® 236
Table 4-3. Results from second floor post-decontamination sampling 238
Table 4-4. NAM ports and direction of flow during decontamination and drying phases 248
Table 4-5. Negative surface samples collected after spore dissemination, yet prior to
decontamination with pH-adjusted bleach 266
Table 4-6. Surface samples with detectable Bg collected following decontamination with
pH-adjusted bleach 270
Table 4-7. HVAC surface samples collected after dissemination, yet prior to
decontamination with pH-adjusted bleach 274
Table 4-8. HVAC surface samples collected following decontamination with pH-adjusted
bleach 275
Table 4-9. Average facility temperature measurements from Sabre 283
Table 4-10. Average facility temperature measurements from EPA 284
Table 4-11. CIO2 sampling locations 285
Table 4-12. Results from first and second floor following decontamination with CIO2 300
XXXI
-------
Table 4-13. Summary of LLNL BOTE Project data by event type including blanks and
controls 307
Table 4-14. Summary of EPA-OPP-MLB BOTE Project data by event type, including
blanks and controls 308
Table 4-15. Summary of AAS and surface sampling results 311
Table 4-16. Waste categories 318
Table 4-17. BOTE Project waste management measurements and frequency 319
Table 4-18. Waste data from BOTE Project Phase 1 321
Table 4-19. Sources of data 333
Table 4-20. Worksheets from the cost analysis workbook 333
Table 4-21. Labor categories and loaded hourly labor rates 335
Table 4-22. Labor mix of teams of personnel 336
Table 4-23. Caveats and assumptions in cost analysis 339
Table 4-24. Mean and standard deviation of sampling activities 341
Table 4-25. Summary of sampling and analytical cost analysis 342
Table 4-26. Estimated cost per entry for facility decontamination 344
Table 4-27. Decontamination contractor fixed costs and material costs 345
Table 4-28. Quantities and categories of waste collected 346
Table 4-29. Summary of waste management scenarios 347
Table 4-30. Estimated transportation costs 348
Table 4-31. Estimated costs of waste handling, packaging, labeling, and characterization.... 349
Table 4-32. Estimated waste disposal costs 349
Table 4-33. Materials pre-populated into building prior to spore dissemination 356
Table 4-34. Summary of restoration costs 356
Table 4-35. Data availability by medium and exposure unit per decontamination round 369
Table 5-1. ANOVA results for the pre-decontamination data. Due to non-normality of the
data, analyses were performed on ranks and log (CFU/cm2). (Only factors
that were significant or in a significant interaction were included in the final
analysis.) 375
Table 5-2. The decontamination effectiveness for each decontamination method 382
XXXII
-------
Table 5-3. The sample characteristics of the pH-adjusted bleach and CIO2 samples that
showed positive growth 382
Table 5-4. ANOVA and Fisher's Exact Test results of the VHP® post-decontamination
data. (Only factors that were significant or close to significant were included in
the final model.) 385
Table 5-5. VHP® post-decontamination results for each floor 385
Table 5-6. VHP® post-decontamination results for each room type 385
Table 5-7. VHP® post-decontamination results for each sample method 386
Table 6-1. Aliquots of the stock spore solution used to spike performance evaluation audit
samples were subjected to tenfold dilution plating to determine the actual
number of spores in the inoculum 396
Table 6-2. Recovery results from LRN performance evaluation audit swab samples sent to
seven of the eight participating LRN laboratories 397
Table 6-3. Recovery results from LRN performance evaluation audit vacuum sock samples
sent to seven of the eight participating LRN laboratories 398
Table 6-4. Recovery results from LRN performance evaluation audit sponge-stick wipe
samples sent to seven of the eight participating LRN laboratories 399
Table 6-5. Mean and percent recovery values (all data) by sample type pooled across
laboratories 400
Table 6-6. Mean and percent recovery values (outliers excluded) by sample type pooled
across laboratories 400
XXXIII
-------
LIST OF FIGURES
Figure 2-1. Site layout of PBF-632 16
Figure 2-2. INL Building PBF-632, Floor 1 17
Figure 2-3. INL Building PBF-632, Floor 2 18
Figure 2-4. Schematic diagram of PBF-632 first floor 20
Figure 2-5. Schematic diagram of PBF-632 second floor 21
Figure 2-6. Location of air sampling equipment in Rooms 101A and 102 22
Figure 2-7. PBF-632 before and after setup of outer secondary enclosure 23
Figure 2-8. BOTE Project site configuration 26
Figure 2-9. Nebulizers on the first floor releasing into the air intake on the HVAC system 29
Figure 2-10. Timeline of sampling events within each round 33
Figure 2-11. Instrumentation setup and remote design using the BioSeq-12 38
Figure 2-12. Dycor XMX/2L-MIL Aerosol Collection System 40
Figure 2-13. Mattson-Garvin Model 220 Slit-to-Agar Air Sampler 41
Figure 2-14. Photograph of AAS being performed in Room 105 during Round 1 48
Figure 2-15. Schematic of sample placement during the BOTE Project 58
Figure 2-16. Summary of manual RV-PCR protocol steps and pictures of equipment used
to process samples 74
Figure 2-17. Combined BOTE Project protocol for RV-PCR and culture analyses including
details of culture analysis 75
Figure 3-1. Summary of samples taken for LRN analysis during the MFP event 108
Figure 3-2. Sample map for Floor 1 during MFP 109
Figure 3-3. Sample map for Floor 2 during MFP 110
Figure 3-4. Sample map for Floor 1 during Round 1 pre-decontamination sampling 111
Figure 3-5. Sample map for Floor 2 during Round 1 pre-decontamination sampling 112
Figure 3-6. Summary of samples taken for LRN analysis during pre-decontamination
sampling in Round 1 114
Figure 3-7. Sample map for Floor 1 during Round 1 post-decontamination sampling 117
Figure 3-8. Sample map for Floor 2 during Round 1 post-decontamination sampling 118
xxxiv
-------
Figure 3-9. Summary of samples taken for LRN analysis during post-decontamination
sampling in Round 1 120
Figure 3-10. Sample map for Floor 1 during Round 2 pre-decontamination sampling 123
Figure 3-11. Sample map for Floor 2 during Round 2 pre-decontamination sampling 124
Figure 3-12. Summary of samples taken for LRN analysis during pre-decontamination
sampling in Round 2 126
Figure 3-13. Sample map for Floor 1 during Round 2 post-decontamination sampling 129
Figure 3-14. Sample map for Floor 2 during Round 2 post-decontamination sampling 130
Figure 3-15. Summary of samples taken for LRN analysis during post-decontamination
sam pling in Round 2 132
Figure 3-16. Sample map for Floor 1 during Round 3 pre-decontamination sampling 135
Figure 3-17. Sample map for Floor 2 during Round 3 pre-decontamination sampling 136
Figure 3-18. Summary of samples taken for LRN analysis during pre-decontamination
sampling in Round 3 138
Figure 3-19. Sample map for Floor 1 during Round 3 post-decontamination sampling 141
Figure 3-20. Sample map for Floor 2 during Round 3 post-decontamination sampling 142
Figure 3-21. Summary of samples taken for LRN analysis during post-decontamination
sampling in Round 3 144
Figure 3-22. Box plots of Round 1 (VHP®) air concentration data for Bg spores (CFU/ft3),
by stage (S1 through S5) and room (101Aand 102) 177
Figure 3-23. Box plots of Round 2 (pH-Adjusted Bleach) air concentration data for Bg
spores (CFU/ft3), by stage (S1 through S5) and room (101A and 102) 178
Figure 3-24. Box plots of Round 3 (CIO2) air concentration data for Bg spores (CFU/ft3), by
stage (S1 through S5) and room (101A and 102) 179
Figure 3-25. Plot of particle concentrations collected real time from the UV-APS and
IBACs for Round 1, Room 101A 189
Figure 3-26. Plot of particle concentrations collected real time from the UV-APS and
IBACs for Round 1, Room 102 190
Figure 3-27. Plot of particle concentrations collected real time from the UV-APS and
IBACs for Round 2, Room 101A 191
Figure 3-28. Plot of particle concentrations collected real time from the UV-APS and
IBACs for Round 2, Room 102 192
Figure 3-29. Plot of particle concentrations collected real time from the UV-APS and
IBACs for Round 3, Room 101A 193
xxxv
-------
Figure 3-30. Plot of particle concentrations collected real-time from the UV-APS and
IBACsfor Round 3, Room 102 194
Figure 3-31. UV-APS release distribution 195
Figure 3-32. EPA instrument LOD including standard curve and exponential fit trend line 197
Figure 3-33. EPA LOD in sterile sand including standard curve and exponential fit trend
line 198
Figure 3-34. USGS instrument LOD standard curve 199
Figure 3-35. Schematic of the building and secondary enclosure layout for the BOTE
Project Phase 1 213
Figure 4-1. Schematic of first floor showing location of fans, sensors, and aerators 222
Figure 4-2. Schematic of second floor showing location of fans, sensors, and aerators 223
Figure 4-3. Photo of T4 generator with gas delivery lines going into PBF-632 224
Figure 4-4. Inlet NAMs on leftside of figure and outlet NAMs on right side of figure 226
Figure 4-5. Temperature profile for the first floor 227
Figure 4-6. Temperature profile for the second floor 228
Figure 4-7. RH on the first floor 228
Figure 4-8. RH on the second floor 229
Figure 4-9. Hydrogen peroxide concentration on the first floor 230
Figure 4-10. Hydrogen peroxide concentration on the second floor 230
Figure 4-11. Results for Bis on the first floor 232
Figure 4-12. Results for Bis on the second floor 232
Figure 4-13. Spatial distribution of first floor pre-decontamination characterization sample
results 234
Figure 4-14. Spatial distribution of second floor pre-decontamination characterization
sample results 235
Figure 4-15. Spatial distribution of first floor post-decontamination in Round 1 239
Figure 4-16. Spatial distribution of second floor post-decontamination in Round 1 240
Figure 4-17. Photograph of decontamination personnel suited in Level C PPE 244
Figure 4-18. Photograph of dumpster located near the outside stairs, used for second floor
waste 245
xxxvi
-------
Figure 4-19. Photographs of the NAMs during decontamination. Left photo shows the entry
side of the facility; right photo shows the far end of the facility (adjacent to the
Decontamination Line) 247
Figure 4-20. Photographs of the NAMs during drying. Left photo shows the entry side of
the facility; right photo shows the northeast side of the facility.) 247
Figure 4-21. Schematic of NAM connections to the facility. Left drawing shows the entry
side of the facility; right drawing shows the exit side of the facility. See Table
4-4 for legend 248
Figure 4-22. Mixing of the pH-adjusted bleach solution in 55-gal drums 249
Figure 4-23. Photograph of the Pro-Chem sprayer 250
Figure 4-24. Photographs of the backpack sprayer 251
Figure 4-25. Photograph of decontamination personnel cutting items into smaller sizes to
fit into waste bags for removal prior to spraying the facility 252
Figure 4-26. Photograph of decontamination personnel placing porous items into waste
bags prior to spraying the facility 253
Figure 4-27. Photograph of decontamination personnel ready to enter building with self-
contained breathing apparatus and supply air line respirators 256
Figure 4-28. Photograph of pH-adjusted bleach spraying using the chemical sprayer 257
Figure 4-29. Photograph of decontamination personnel operating the sump pump to
transfer pH-adjusted bleach runoff collected inside the facility to the exterior of
the structure 258
Figure 4-30. Photograph of the pH-adjusted bleach runoff collected by wet vacuuming one
day after the spray procedure 259
Figure 4-31. Photograph of the four heaters used to heat the facility during the pH-
adjusted bleach post-decontamination drying phase 260
Figure 4-32. Average first floor temperatures recorded by 17 HOBO® data loggers
dispersed throughout the rooms during the post-decontamination drying
phase of the pH-adjusted bleach round. Facility drying began on May 1, 2011,
with the activation of internal heaters and circulation fans. Fans and heaters
were deactivated on May 4, 2011. Post-decontamination surface sampling
commenced on May 5, 2011 261
Figure 4-33. Average second floor temperatures recorded by 18 HOBO® data loggers
dispersed throughout the rooms during the post-decontamination drying
phase of the pH-adjusted bleach round. Facility drying began on May 1, 2011,
with the activation of internal heaters and circulation fans. Fans and heaters
were deactivated on May 4, 2011. Post-decontamination surface sampling
commenced on May 5, 2011 262
XXXVII
-------
Figure 4-34. Average first floor RH recorded by 17 HOBO® data loggers dispersed
throughout the rooms during the post-decontamination drying phase of the
pH-adjusted bleach round. Facility drying began on May 1, 2011, with the
activation of internal heaters and circulation fans. Fans and heaters were
deactivated on May 4, 2011. Post-decontamination surface sampling
commenced on May 5, 2011 263
Figure 4-35. Average second floor RH recorded by 18 HOBO® data loggers dispersed
throughout the rooms during the post-decontamination drying phase of the
pH-adjusted bleach round. Facility drying began on May 1, 2011, with the
activation of internal heaters and circulation fans. Fans and heaters were
deactivated on May 4, 2011. Post-decontamination surface sampling
commenced on May 5, 2011 264
Figure 4-36. Spatial distribution of first floor pre-decontamination characterization sample
results 267
Figure 4-37. Spatial distribution of second floor pre-decontamination characterization
sample results 268
Figure 4-38. Spatial distribution of first floor post-decontamination in Round 2 271
Figure 4-39. Spatial distribution of second floor post-decontamination in Round 2 272
Figure 4-40 Photo showing the inner tent membrane (primary tent) on the right side of
the manlift and the secondary tent over the metal lattice structure 278
Figure 4-41. Photo showing the location of Sabre's equipment 279
Figure 4-42. Schematic of first floor of PBF-632 showing location of fumigant sampling
lines, fans, and sensors 281
Figure 4-43. Schematic of second floor of PBF-632 showing location of fumigant sampling
lines, fans, and sensors 282
Figure 4-44. Temperature profile during CIO2 fumigation on May 14, 2011 287
Figure 4-45. RH profile during CIO2 fumigation on May 14, 2011 288
Figure 4-46. CIO2 concentration (red) and CT (blue) profiles at sample Location 1: Sensor
Web (solid line) and titration by Sabre (markers) 289
Figure 4-47. CIO2 concentration (red) and CT (blue) profiles at sample Location 2: Sensor
Web (solid line) and titration by Sabre (markers) 290
Figure 4-48. CIO2 concentration (red) and CT (blue) profiles at sample Location 5: Sensor
Web (solid line) and titration by Sabre (markers) 291
Figure 4-49. CIO2 concentration (red) and CT (blue) profiles at sample Location 6: Sensor
Web (solid line) and titration by Sabre (markers) 292
Figure 4-50. CIO2 CT profile at seven different locations measured by the Sensor Web
sensors 293
XXXVIII
-------
Figure 4-51. Results for Bis on the first floor (black circles indicate Bis that were negative
for growth; red circles indicate Bis that were positive for growth) 295
Figure 4-52. Results for Bis on the second floor (black circles indicate Bis that were
negative for growth; red circles indicate Bis that were positive for growth) 296
Figure 4-53. Spatial distribution of first floor pre-decontamination characterization sample
results 298
Figure 4-54. Spatial distribution of second floor pre-decontamination characterization
sample results 299
Figure 4-55. Spatial distribution of first floor post-decontamination in Round 3 301
Figure 4-56. Spatial distribution of second floor post-decontamination in Round 3 302
Figure 4-57. Photos of ring stand clamps and quick connects showing some signs of
oxidation 303
Figure 4-58. Schematic diagram showing one possible distribution of spores during
sample processing of a wipe sample with a low spore level. The archive
sample tube represents spores remaining with the wipe sample after the two
spore extraction steps are completed. Spores associated with the wipe and in
the remaining extraction buffer are shown 306
Figure 4-59. Arithmetic means of air versus surface sample measurements from Stage 4
(different points correspond to different surfaces, surface types, and sample
types) 313
Figure 4-60. Waste management concept 316
Figure 4-61. Daily solid waste generation 322
Figure 4-62. Distribution of waste by activity (normalized to 100%). The top plot is liquid
and the bottom plot is solid waste 324
Figure 4-63. Solid waste by category 325
Figure 4-64. Liquid Waste by Category 326
Figure 4-65. Breakdown of sampling and analytical costs 343
Figure 4-66. Breakdown of waste management costs ("low" disposal difficulty scenario).... 351
Figure 4-67. Breakdown of waste management costs ("medium" disposal difficulty
scenario) 352
Figure 4-68. Breakdown of waste management costs ("high" disposal difficulty scenario)... 353
Figure 4-69. Breakdown of decontamination costs (using "medium" disposal difficulty
scenario) 354
Figure 4-70. Breakdown of overall cost contributions 357
xxxix
-------
Figure 4-71. Conceptual site model. * Inhalation exposures during Bg spore dissemination
(aerosolization) will be assessed only in Rooms 101A and 102 367
Figure 5-1. Kriged estimates (scale explained in Appendix L) of the contamination on the
first floor prior to the pH-adjusted bleach decontamination (square = vacuum
sock, diamond = swab, circle = sponge-stick) 372
Figure 5-2. Variogram for the first floor contamination prior to the pH-adjusted bleach
decontamination 373
Figure 5-3. Box plots of extracted contamination (CFU/cm2 in log scale) for each pre-
decontamination round (middle box represents middle 50% of data, middle
line in box represents median, red square represents mean) 376
Figure 5-4. Box plots of extracted contamination (CFU/cm2 in log scale) for each floor
across all three pre-decontamination rounds (boxes represent middle 50% of
data, middle line in box represents median, red square represents mean) 377
Figure 5-5. Box plots of extracted contamination (CFU/cm2 in log scale) for each sampling
method across all three pre-decontamination rounds (boxes represent middle
50% of data, middle line in box represents median, red square represents
mean) 378
Figure 5-6. Box plots of extracted contamination (CFU/cm2 in log scale) for each room
type across all three pre-decontamination rounds (boxes represent middle
50% of data, middle line in box represents median, red square represents
mean) 379
Figure 5-7. Box plots of extracted contamination (CFU/cm2 in log scale) for each sampled
object across all three pre-decontamination rounds (boxes represent middle
50% of data, middle line in box represents median, red square represents
mean) 380
Figure 5-8. Interaction Plot of Sampling Method and Pre-Decontamination Rounds
Measured in CFU/cm2 381
Figure 5-9. Interaction plot of sampling method and floor for the VHP® events (y-axis is the
mean DE for each combination of the factors) 386
Figure 5-10. Overall decontamination method cost and performance 387
xl
-------
ACRONYMS AND ABBREVIATIONS
AAS
ANOVA
ATCC
AWBERC
Ba
Bg
Bl
BOTE
BROOM
BSC
CBRN
CDC
CPU
Cl
CIO2
CMAT
COA
COC
COTS
CST
CT
Ct
DD
DE
DHS
DMA
DOD
DOE
DTRA
EDTA
EPA
EPC
ET
FAS
FBI
GEq
H2O2
HCI
HAZWOPER
HEPA
HVAC
I BAG
1C
ICx
INL
I PC
LLNL
aggressive air sampling
analysis of variance
American Type Culture Collection
Andrew W. Breidenback Environmental Research Center
Bacillus anthracis
Bacillus atrophaeus subspecies globigii
biological indicator
Bio-Response Operational Testing and Evaluation
Building Restoration Operations Optimization Model
biological safety cabinet
Chemical, Biological, Radiological, and Nuclear
Centers for Disease Control and Prevention
colony forming unit(s)
chemical indicator
chlorine dioxide gas
(EPA's CBRN) Consequence Management Advisory Team
Constant Output Atomizer
chain of custody
commercial off-the-shelf
Civil Support Team, National Guard
concentration x time
cycle threshold (time)
diffusion dryer
decontamination efficiency
Department of Homeland Security
deoxyribonucleic acid
Department of Defense
Department of Energy
Defense Threat Reduction Agency
ethylenediaminetetraacetic acid
U.S. Environmental Protection Agency
exposure point concentration
exposure time
filtered air supply
Federal Bureau of Investigation
genomic equivalent(s)
hydrogen peroxide
hydrochloric acid
Hazardous Waste Operations and Emergency Response
High Efficiency Particulate Air
heating, ventilation, and air conditioning
real-time aerosol particle monitor
Incident Command
ICx Technologies, Inc.
Idaho National Laboratory
internal positive control
Lawrence Livermore National Laboratory
xli
-------
LOD
LR
LRN
MFP
MLB
MSW
N/A
NA
NAM
NC
ND
NHSRC
NRC
NRT
NIC
NTU
OPP
OSHA
OSWER
PBST
PC
PCR
PDA
PEL
PNNL
POTW
PPE
QA
QAPP
QC
qPCR
RCRA
RFID
RH
RMC
RV-PCR
SD
SNL
SOP
STA
START
swab
TB
TNTC
TSA
TSB
UNG
USCG
USGS
UV
limit of detection
log reduction
Laboratory Response Network
mandatory full participation
Microbiology Laboratory Branch
municipal solid waste
not applicable
not analyzed
negative air machine
negative control
non-detect
National Homeland Security Research Center
National Research Council
National Response Team
no template control
nephelometric turbidity unit
Office of Pesticide Programs
Occupational Safety and Health Administration
Office of Solid Waste and Emergency Response
phosphate buffered saline plus Tween® 20
positive control
polymerase chain reaction
personal digital assistant
permissible exposure limit
Pacific Northwest National Laboratory
publicly owned treatment works
personal protective equipment
quality assurance
Quality Assurance Project Plan
quality control
quantitative polymerase chain reaction
Resource Conservation and Recovery Act
radio frequency identification
relative humidity
reference material coupon
rapid-viability polymerase chain reaction
standard deviation
Sandia National Laboratory
standard operating procedure
Mattson-Garvin Model 220 slit-to-agar air sampler
Superfund Technical Assessment and Response Team
macrofoam swab
true blank
too numerous to count
tryptic (trypticase) soy agar
tryptic soy broth
uracil-N-glycosilase
U.S. Coast Guard
U.S. Geological Survey
ultraviolet
xlii
-------
UV-APS ultraviolet aerodynamic particle sizer
VHP® vaporized hydrogen peroxide
XMX Dycor® XMX/2L-MIL Aerosol Collection System
xliii
-------
Units
|jg microgram(s)
|j|_ microliter(s)
|jm micrometer(s)
|jM micromolar
Btu British thermal unit(s)
°C degree(s) Celsius
CFM cubic foot/feet per minute
cm centimeter(s)
cm2 square centimeter(s)
cm3 cubic centimeter(s)
°F degree(s) Fahrenheit
fg femtogram(s)
ft foot/feet
ft2 square foot/feet
ft3 cubic foot/feet
g grams
gal gallon(s)
gpm gallons per minute
hp horsepower
hr hour(s)
in inch(es)
kg kilogram(s)
km kilometer(s)
L liter(s)
Lpm liter(s) per minute
Ib pound(s)
m meter(s)
m"1 per meter
m2 square meter(s)
m3 cubic meter(s)
mg milligram(s)
mi mile(s)
min minute(s)
ml_ milliliter(s)
mm millimeter(s)
mM millimolar
N number of particles
ng nanogram(s)
pg picogram(s)
ppm part(s) per million
ppmv part(s) per million by volume
psi pounds per square inch
psig pound(s)-force per square inch gauge
rpm revolutions per minute
SCFM standard cubic foot/feet per minute
sec second
xliv
-------
EXECUTIVE SUMMARY
The Bio-response Operational Testing and Evaluation (BOTE) Project was a multi-agency effort
designed to operationally test and evaluate a response to a biological incident (release of
Bacillus anthracis [Ba] spores, the causative agent for anthrax) from initial public health and law
enforcement response through environmental remediation. The BOTE Project consisted of two
distinct phases, both using the same non-pathogenic simulant (or surrogate) for Ba. Phase 1
was a field-level decontamination assessment, and Phase 2 was an operational exercise
involving key federal agencies responsible for the forensic investigation, public health
assessment, and remediation following a biological incident. The effort included the coordinated
project planning, support, and/or involvement from:
• Department of Homeland Security, Science and Technology Directorate (DHS S&T);
• U.S. Environmental Protection Agency (EPA);
• Centers for Disease Control and Prevention (CDC);
• Department of Energy (DOE) National Laboratories;
• Department of Defense (DOD) Defense Threat Reduction Agency (DTRA); and
• Federal Bureau of Investigation (FBI).
This report addresses Phase 1 of the BOTE Project. Phase 1 was designed to address site
remediation after the release of a Ba simulant, Bacillus atrophaeus spp. globigii (Bg), within a
facility in an operational setting, drawing upon the recent advances in both the biological
sampling and decontamination areas. The four principal objectives of the BOTE Project Phase 1
included:
• Objective 1: Conduct and evaluate field-level application of three decontamination
technologies/protocols, bringing the results of laboratory-scale studies to the field;
• Objective 2: Demonstrate that biological sampling and analysis methods evaluated in
previous studies provide accurate characterization of Ba simulant concentration
challenges for detection/identification purposes;
• Objective 3: Collect and analyze the results from the decontamination study and
perform a cost analysis of all aspects of the remediation approaches; and
• Objective 4: Determine the exposure associated with reentry into a building that has
been contaminated with surrogate Ba spores and subsequently decontaminated.
These objectives were addressed in three rounds of testing during Phase 1, with each round
employing a different decontamination method as a primary study parameter. Three
decontamination methods were chosen based on results that had been obtained in laboratory
studies.
The BOTE Project test facility was a two story unoccupied office building with approximately
4,025 square feet (ft2) per floor (90,000 cubic feet [ft3] total volume for the facility). Rooms on
each floor were set up identically and configured to contain a mixture of porous and nonporous
xlv
-------
materials, to simulate both commercial office and residential environments. Each floor had an
independent heating, ventilation, and air conditioning (HVAC) system that functioned only as an
air handling unit for each floor of the facility (i.e., no heating or cooling). The facility was
surrounded by a secondary containment enclosure to assist in preventing cross-contamination
during the project activities.
Each of the three rounds in Phase 1 included dissemination of the Bg spores throughout the
facility, characterization sampling, decontamination, waste management, and post-
decontamination sampling. After each round, the facility was set back up (re-set) to its initial
configuration for the start of the next round. For each round, Bg surface loadings (as determined
by pre-decontamination surface sampling), room configurations, and sampling procedures were
maintained consistent. Dissemination for each round was designed to result in a relatively higher
level Bg surface loading (1E4to 1E6 colony forming units [CFU]/ft2) on the first floor and a lower
level (1E2 to 2E2 CFU/ft2) on the second floor. The three decontamination methods that were
used were:
• Round 1: fumigation with hydrogen peroxide (H2O2) vapor by STERIS Corporation,
using their vaporized hydrogen peroxide (VHP®) technology;
• Round 2: surface decontamination using pH-adjusted bleach; and
• Round 3: fumigation with chlorine dioxide gas (CIO2) by Sabre Technical Services,
LLC.
To achieve the objectives of the BOTE Project Phase 1, a total of 3,259 samples were collected
using different environmental sampling methods for spores. These sampling methods included
surface, air, sand, and wash water media. Surface samples (cellulose sponge-stick wipes,
swabs, vacuum socks, and Versalon® wipes) were used to assess the concentration of spores
pre- and post-decontamination; these data (with the exception of the Versalon® wipes) were
utilized primarily to assess the effectiveness of each decontamination method. The Versalon®
wipes were used to evaluate the EPA's rapid-viability polymerase chain reaction (RV-PCR)
analytical method. Air sampling data were used to provide feedback on the dissemination
method, to assess the potential for indoor reaerosolization during remediation activities (e.g.,
sampling), to assess the use of aggressive air sampling (AAS) as a post-decontamination
sampling method, and to assist in the development of the exposure assessment framework.
Sand samples were placed outside the building but within the secondary enclosure, to assess
whether viable Bg spores might have migrated outside the facility during the project activities.
Wash water samples were utilized to assess the efficacy of onsite treatment of the wash water
to inactivate Bg spores. The Building Restoration Operations Optimization Model (BROOM) tool
was used to track sample collection, sample location, sample types, sample matrices, date,
time, samplers used, and other pertinent data.
The project sampling strategy was designed to maximize the use of the data generated to
achieve the four objectives stated above. A brief summary of the findings from the BOTE Project
Phase 1, correlated according to the objectives, is provided below.
xlvi
-------
Assessment of Decontamination Methods
Within each round, the specific decontamination method was required to be completed within
three days, from setup through return of the facility for post-decontamination sampling. In Round
2, an additional three days were allotted for the facility to dry after spraying with pH-adjusted
bleach and prior to surface sampling. For each method, the decontamination contractor or
performer decided upon their decontamination strategy, e.g., which materials inside the building
to remove prior to decontamination and treat ex situ as waste and how to perform distribution or
application of the decontaminant. This strategic decision included determination of conditions
considered appropriate for effective decontamination within the BOTE Project scenario, e.g.,
concentration of fumigant or solution, contact or dwell time, relative humidity, temperature. For
each decontamination method, the efficacy, waste generation, and effects on the facility were
determined. The approximate total cost of the decontamination method employed was also
assessed.
The decontamination efficacy in each round was determined qualitatively via comparison of the
number of surface samples in which Bg was detected pre- and post-decontamination.
Additionally, quantitative surface sample results provided an indication of the relative surface
loading pre- and post-decontamination. In general, fumigation with CIO2 or using the
decontamination procedure incorporating spraying with pH-adjusted bleach resulted in similar
overall effectiveness in reducing detection of viable Bg throughout the facility. Both methods
were considerably more effective than fumigation with VHP® as implemented in the BOTE
Project Phase 1. Each decontamination method was performed a single time in the BOTE
Project; the results and conclusions should be considered based upon the implementation as
described. Potential variance in effectiveness due to differences in implementation of each
method on subsequent uses was not determined in this project. Highlights of the
decontamination processes and results are presented in the following sections.
Fumigation by STERIS VHP®
STERIS Corporation's VHP® technology was used for the decontamination process in Round 1.
The fumigation contractor decided to leave all materials in place, i.e., in situ decontamination of
all facility contents. Fumigation of the facility, including the HVAC system on each floor, was
accomplished by initially plumbing one VHP® generation system into the air handling unit on
each floor. Fans were also placed throughout the facility to facilitate distribution of H2O2. The
target decontamination parameters selected by the fumigation contractor, having had the ability
to consider EPA research products and past use of VHP®, were set as 250 parts per million by
volume (ppmv) of H2O2 throughout the facility for 90 minutes or a cumulative exposure of 400
ppmv-hours (hr) at 65 °F or higher. Additionally, Spor-Klenz® Ready to Use was sprayed on any
surface on which equipment was placed during the decontamination process setup.
The fumigation concentration, temperature, and relative humidity (RH) were monitored at
numerous locations throughout both floors of the facility. Throughout the fumigation process
(when H2O2 was injected into the facility), the temperature remained above 65 °F and RH above
30% (no RH requirement was set). The target H2O2 concentration was not achieved at all
monitored locations (three per floor), despite a mid-process change to have both VHP®
generators plumbed into the first floor HVAC system.
xlvii
-------
Following aeration of the facility to a safe concentration of H2O2 after decontamination, a total of
276 surface samples were collected and shipped for analysis via the Laboratory Response
Network (LRN) for the assessment of the decontamination efficacy. Of these samples, roughly
one third (94) contained detectable viable Bg. Average surface loadings of detectable viable Bg
were reduced by approximately 1 log on each floor as a result of the decontamination process.
These results indicate that the H2O2 exposure was not sufficient to inactivate all the spores at
the high or low challenge/test levels (surface loading). A significantly greater percentage of
samples with detected Bg were obtained from the residential rooms compared to the other types
of rooms. A total of 90 chemical indicators were placed in the facility, and all indicated that they
had been exposed to H2O2 at the end of fumigation. Ninety biological indicators (Bis) were also
placed throughout the facility. (Bis do not accurately predict decontamination efficacy of
environmental contamination; rather, Bis are useful for identifying when sporicidal conditions
have not been achieved.) Of the 90 Bis, 56 showed no growth at the 6 log level.
No damage to the building contents was observed from exposure to the VHP® process. A total
of 1,350 pounds (Ibs) of solid waste was generated from the sampling activities. Liquid waste
was also generated during this round (e.g., during sampling) from rinsate recovered from the
personnel decontamination area ("Decontamination Line"). The Decontamination Line consisted
of three chambers including an entry area connected to the building, a personnel wash down
area, and an exit area leading to the outside.
Surface Decontamination Approach using pH-Adjusted Bleach
For the second round of Phase I, a surface decontamination process using readily available
supplies from local hardware stores was utilized. A sporicidal liquid (pH-adjusted bleach) was
used to treat waste items removed from the facility and surfaces within the facility. Elements of
the decontamination approach were recently used to remediate residential structures following
natural B. anthracis contamination events (i.e., Danbury, CT; and Durham, NH)m and were used
in addition to volumetric decontamination strategies within federal facilities following the anthrax
letter incident of 2001[2].
After Bg spore dissemination and characterization sampling, the decontamination procedure
began with the removal of all porous materials (ceiling tiles, furniture, carpet, etc.) from the
facility for subsequent treatment with pH-adjusted bleach (1 part bleach, 1 part white vinegar,
and 8 parts water; used within three hours after preparation), bagging, and disposal. Waste
items were sprayed during bagging, and bags were moved to a waste staging area before
exiting the facility through the personnel Decontamination Line or through a roll-off dumpster.
During this source reduction step, approximately 68 cubic yards (7,100 Ib) of waste was
removed from the facility. Within this round, most of the waste was generated during the
decontamination phase, more specifically during source reduction. In contrast, during Rounds 1
and 3, the bulk of the waste was generated during sampling operations (i.e., personal protective
equipment [PPE] waste).
The source reduction step was followed by spray application of pH-adjusted bleach to all
remaining surfaces in the facility using a gas-powered sprayer. All surfaces were completely
wetted with pH-adjusted bleach for a target 10-minute (min) contact time. The HVAC return air
xlviii
-------
ducts were decontaminated using a modified spray nozzle attached to the pH-adjusted bleach
chemical sprayer line. The spray nozzle was pulled through the duct while dispensing a mist of
pH-adjusted bleach. The treatment was then repeated five minutes later as the spray nozzle
was pulled in the opposite direction during its retrieval from the duct. Decontamination of the
supply HVAC ducts was notionalized (not conducted in reality, but considered with respect to
implication on the overall cost and time of the process), as this flexible ductwork would be
removed for decontamination in an actual remediation. The HVAC was not operated during the
decontamination operation.
Chlorine gas concentrations within the facility necessitated the use of level-B PPE (self-
contained breathing apparatus) during decontamination spraying operations; chlorine gas is
known to be generated in the pH-adjusted bleach solution and readily off-gases. Typically, the
decontamination crew consisted of six entry personnel and two support personnel.
Decontamination personnel entered through one end of the building and exited through a
Decontamination Line at the opposite end. Negative air machines (NAMs) were used to control
airflow into and within the facility, to reduce the chlorine gas concentrations within the facility, to
aid in drying after decontamination, and to prevent cross-contamination. Electric heaters and
box fans were also deployed within the facility after decontamination to expedite the drying
process.
Overall, the surface sampling results indicated that the pH-adjusted bleach decontamination
procedure was effective for reducing contamination. Only 8 out of 244 (3.2%) post-
decontamination surface samples were positive for viable Bg. Interestingly, seven of these eight
positive samples were from the less-contaminated second floor. All seven of these samples
required filter-plate methods for detection, suggesting a low level of contamination. HVAC return
duct decontamination procedures were also effective at removing contamination, as all five
HVAC samples collected post-decontamination were non-detect.
Fumigation with CIO2
After Round 2, the facility was again re-set to its planned configuration, and Bg spore
dissemination and characterization sampling was conducted for Round 3. Sabre Technical
Services, LLC ("Sabre") was selected to fumigate the facility with CIO2 as the Round 3
decontamination process. This decontamination method was chosen because of its high degree
of effectiveness in laboratory testing and previous use in Ba remediation actions. Due to having
only one day of aeration following fumigation, Sabre decided to remove (treat ex situ) a few
porous items (mattresses and foam cushions) due to their anticipated residual off-gassing. All
other materials were left in place and the facility was fumigated at a target concentration of
3,000 ppmv CIO2 for three hours (target concentration-time product [CT] of 9,000 ppmv-hr at a
minimum temperature of 65 °F and RH of 65%). These conditions were set by Sabre in
consideration of their past experience and published EPA research findings. Sabre generated
the CIO2 on site in a closed loop process while maintaining negative pressure on the facility via
tenting and the use of a NAM (scrubbing via carbon adsorption). The gas entered via the emitter
on the first floor and returned via the duct on the second floor. Gas distribution was done using
the HVAC system on each floor, with additional box fans placed throughout the facility to assist
in gas distribution.
xlix
-------
Measurements of CIO2 concentration, temperature and RH were made at several locations on
each floor. The data indicated that the target minimum CIO2 concentration and CT were
achieved at all monitoring locations throughout the first and second floors. The average
temperatures on both floors were maintained above the target, as well as the RH on the first
floor. However, the RH on the second floor was significantly lower than the first floor (15-20 %
lower RH) and at or slightly below the target value of 65%.
In addition to the process data measurements, Bis were placed throughout the facility to provide
an indication that sporicidal conditions had been achieved. Only one Bl was positive for the
surrogate organism from the first floor after fumigation; this Bl was located inside a filing cabinet.
However, 31 of 45 Bis on the second floor were positive. These results suggest that the
conditions (i.e., RH) on the second floor were not sufficient to result in a 6-log reduction in
spores on the Bis.
However, the surface loading of Bg spores on the second floor was targeted at 1E2 to 2E2
CFU/ft2; the actual loading was on average 1.4E4 CFU/ft2 based upon pre-decontamination
surface sampling. Of the 265 post-decontamination surface samples that were collected for
analysis by the LRN, only six had detectable Bg (three samples from each floor). Only one of
these samples had detectable Bg via spread plate analysis; the other five required more
sensitive filter plate analysis for the detection of Bg in the samples. Spore loadings on these few
post-decontamination samples were extremely low, all less than 9 CFU/ft2.
Overall, this decontamination process was effective, with nearly all facility contents left in place.
Further, because this building had been used in previous studies it was difficult to assess
whether any oxidation of the structural components had occurred as a specific result of these
tests. Materials that were placed in the building for these tests were inspected for damage. The
only objects that showed any damage were ring stand clamps and quick connects on gas
sample lines. All other surfaces and materials retained their original condition and color.
Additionally, a total of 877 Ib of waste was generated from the sampling and decontamination
activities. As in all rounds, liquid waste was also generated from rinsate recovered from the
personnel Decontamination Line.
Decontamination Line Wash Water Treatment
While chlorine bleach has been shown to be effective for Ba inactivation in buffered water, wash
water such as Decontamination Line wash water may present a different challenge. The BOTE
Project provided an opportunity to collect realistic wash water to assess chlorine bleach
treatment methods. Water from the Decontamination Line was collected for each sampling event
and chlorinated to test the effectiveness of such treatment. The water was sampled and tested
by Idaho National Laboratory (INL) technical personnel before and after treatment for pH, total
suspended solids, free chlorine, turbidity, chemical oxygen demand and the presence of Bg
spores.
Use of an ultrafiltration concentrator allowed collection of concentrated water samples. However,
the high turbidity of the wash water under the conditions experienced made the operation of the
concentrator difficult due to filter clogging. Additionally, due to the small number of viable spores
present in the wash water, evaluation of the efficacy of thechlorine bleach treatment method for
I
-------
the collected water was not initially possible. Subsequently, a greater than three log inactivation
(i.e., log reduction) was achieved using the proposed protocol when the wash water was spiked
with Bg spores (the level of spores added was only high enough to demonstrate a three log
inactivation; a six log inactivation was not observed).
Demonstration and Assessment of Biological Sampling and Analysis Methods
One objective of the BOTE Project was to demonstrate that surface sampling methods
previously evaluated for effectiveness of spore detection could be used to provide accurate
characterization of Bg (i.e., Ba simulant) concentration for detection/identification purposes.
Sub-objectives included the demonstrated use of current surface sampling methods, the
evaluation of the RV-PCR analytical method, and the use of AAS for post-decontamination
sampling.
The following sampling and analysis methods were used and are described below:
• Surface sampling methods used pre- and post-decontamination;
• RV-PCR analysis for selected samples pre- and post-decontamination; and
• AAS post-decontamination.
Surface Sampling
Surface sampling was the primary method used to collect samples to determine surface
concentrations or loadings (CFU per ft2) of Bg spores. The purpose of surface sampling was to
characterize the extent of contamination both pre- and post-decontamination within each round
to determine the effectiveness of the decontamination for the three technologies. Sampling
methods including cellulose sponge-stick wipes, swabs, and vacuum socks were the primary
collection methods used to evaluate the three decontamination technologies consistent with
current validated or recommended sampling for Ba spores. These samples were analyzed via
the LRN.
Versalon® wipes (gauze wipes, rather than cellulose sponge-stick wipes) were also collected
throughout the facility both pre- and post- decontamination to evaluate EPA's RV-PCR analytical
method (see Appendix K for details). The Versalon® wipes were used for consistency with the
previously-developed RV-PCR analysis protocol. Analysis of these samples occurred at
Lawrence Livermore National Laboratory (LLNL) and EPA's Microbiology Laboratory Branch
(MLB) of the Office of Pesticide Programs (OPP) at Ft. Meade, MD. Additional Versalon® wipes
were also collected in two rooms (Rooms 101A and 102) to determine surface contamination
concentrations pre- and post-decontamination for the reaerosolization study, with samples
analyzed onsite by the INL Microbiology Laboratory.
The measured surface loading for all rounds on Floor 1 was within the target range of 1E4 to
1E6 CFU/ft2. The measured surface loading for all rounds on Floor 2 was an order of magnitude
higher than the target range of 1E2 to 2E2 CFU/ft2. Although the surface loading on Floor 2 was
above the target range, there was still a significant difference between the measured surface
loadings on the two floors. Establishing this difference was the primary objective for the
decontamination efficacy assessment. Additional statistical analysis confirmed that the Bg
-------
spores were disseminated on each floor in such a manner that the first floor was significantly
more contaminated than the second floor for each of the three events.
RV-PCR
RV-PCR is a research method developed under an interagency agreement between EPA and
the LLNL to detect and identify live Ba spores rapidly during a bioterrorism event. The RV-PCR
method is a combination of both culture and real-time PCR and supports additional
preparedness to meet the challenge of analysis of a large number of samples. The current
version of the RV-PCR method had not previously been tested with large numbers of samples in
a field setting and, especially, had not been tested using post-decontamination field samples.
The opportunity afforded by the BOTE Project was to demonstrate the performance of the RV-
PCR method with field samples, both before and after decontamination treatments.
A total of 264 Versalon® wipe samples were spliot and analyzed using the RV-PCR and the
traditional microbiological culture methods (see Appendix K for details) to detect the presence of
viable Bg spores. The RV-PCR method was shown to work well for Bg spores exposed to
decontaminants at real-world application levels and for Versalon® wipe samples containing
background debris and indigenous microbial populations. Greater than 97% agreement was
observed between RV-PCR and culture results (detect/non-detect for Bg) for the field test that
included samples with low spore levels (at or below the detection limit of the traditional culture
method) after treatment with fumigants and surface disinfectants. The nine-hour endpoint
appeared to be sufficient to detect any spores that might have been delayed in germination due
to decontaminant exposure.
Aggressive Air Sampling
The main objective of AAS in the BOTE Project was to determine if, after application of
decontamination technology, disturbing indoor surfaces resulted in the detection of
reaerosolized residual spores via air sampling. AAS offers the potential to reduce the post-
decontamination sampling burden by collecting bulk air samples that could be used to determine
if contamination exists following decontamination. An AAS protocol was utilized that
incorporated two air sampling collection methods, including Dycor® XMX/2L- MIL Aerosol
Collection Systems and Mattson-Garvin Model 220 slit-to-agar air samplers to provide a
secondary evaluation of decontamination effectiveness.
AAS was conducted in two first-floor rooms after post-decontamination surface sampling in each
round. The procedure incorporated the use of a leaf blower to disturb surfaces within the room,
oscillating fans to keep reaerosolized Bg spores airborne and the two different air samplers
mentioned above. All air samples were analyzed on-site by the INL Microbiology Laboratory.
The operation was conducted successfully after all three decontamination rounds, and AAS
sample results were comparable to surface sample results. AAS results after Round 1
(fumigation with VHP®) showed the highest concentrations of spores detected in the air; the
lowest spore concentrations were detected for Round 3 (fumigation with
Overall Cost Analysis
One of the main objectives of the BOTE Project was to develop a methodology to estimate the
-------
overall cost of the application of various decontamination technologies as a function of
materials, time (including labor hours), and other resources.
The cost analysis approach attempted to capture incident- and site-specific information, so that
the results could be extrapolated to other incidents, using appropriate scaling factors based on
labor hours, numbers of samples, size of affected areas, and quantities of waste that were
generated.
A detailed cost analysis was performed for the use of three different decontamination
technologies for the inactivation of Bg spores (although cost estimates were based on assuming
the occurrence of an actual Ba incident). The parameters examined in this cost analysis include
sampling activities, application of decontamination technologies for the building and personnel
entering and leaving the building, equipment rentals and consumables, and waste management.
Based on subsequent analysis of the cost data, the following major cost-related observations
were noted:
• Sampling and analysis costs were the largest contributors to the overall cost. This
statement must add the caveat that this was a research operational testing and
evaluation project, and that estimates of laboratory labor hours and materials were
based only on a single laboratory's submission of level of effort data. In a real incident for
a building this size, fewer samples would most likely be taken. However, sampling and
analysis costs are still anticipated to be a major cost factor to consider.
• The costs of the decontamination processes alone (the actual fumigation or surface
decontamination) were roughly equivalent for all three rounds. Overall costs for the
fumigation methods (VHP® and CIO2) were very similar (between $800,000 and
$900,000), while the cost for the pH-adjusted bleach was nearly $1,200,000. The pH-
adjusted bleach decontamination process employed in this effort was more expensive
than either of the fumigation technologies, due largely to waste management costs.
These costs are specific to the processes as they were employed in the BOTE Project.
• Waste management costs were a significant component of all three technologies,
particularly for the pH-adjusted bleach decontamination process; waste characterization
sampling was the largest single component of waste management costs. Almost all of
the waste generated during the fumigations was a result of personnel decontamination
operations. In addition, waste management costs could be reduced significantly if the
State allows disposal of treated and/or decontaminated items in a Resource
Conservation and Recovery Act (RCRA) Subtitle D landfill or allows the wastewater to be
sent to a publicly owned treatment works (POTW); and
• The cost of personnel decontamination was also a significant contribution to cost due to
the standby procedures that required Decontamination Line personnel to be on-site at all
times when entries into the building were considered.
Assessment of Potential Exposure
Exposure to Ba spores during a release may be due to the primary release or secondary
exposure due to contact with reaerosolized spores or a contaminated surface. Spread of
-------
contamination (e.g., via reaerosolization and dispersion, inside to outside facilities [or vice
versa]) may further increase the potential for exposure. The BOTE Project provided an
opportunity to investigate some aspects of exposure in a field setting:
• Assess Bacillus spore migration from inside to outside a contaminated building.
• Investigate potential spore reaerosolization inside a contaminated building.
• Develop the concept of an exposure assessment plan.
Assessment of Reaerosolization
Inhalation is the route of exposure to Ba spores that has the highest likelihood of causing
adverse health effects. The challenges of estimating airborne spore concentrations from the
reaerosolization of deposited spores are influenced by many factors including residual
aerosolized spores, surface loading, surface material type, activity levels, and other site-specific
characteristics. The purpose of the reaerosolization study within the BOTE Project was to
provide a preliminary assessment of the potential reaerosolization of Bg during remediation
activities, and not to quantify actual potential exposure.
For this effort, surface and air concentrations of the Bg spores were assessed by standing
sampling stations in two rooms before (following Bg spore dissemination) and after the
application of a decontamination technology in each decontamination round. The activities
associated with the disturbance of the settled Bg spores were limited to the activities of the
sampling personnel and were not controlled or scripted to simulate typical residential or office
activities.
Reaerosolized spores were measured at both high (pre-decontamination) and low (post-
decontamination) levels of Bg spore surface contamination in some cases (i.e., not in all
rounds). There were no significant differences in Bg spore concentrations associated with
sampling heights or locations within the rooms. The findings do suggest that reaerosolization
and its impacts should be considered during response and remediation activities.
Assessment of Bacillus Spore Migration from Inside to Outside a Contaminated Building
The BOTE Project spore migration study was designed to examine the potential transportation
of disseminated spores from the initial area of contamination inside the building to outside the
facility (however, inside the tent enclosure). Sand samples (Petri dishes containing sterilized
sand to provide outdoor "soil" reservoirs) were placed directly outside the test facility, within the
secondary containment enclosure and around the building near entrances, exits and high traffic
areas.
The detection of Bg deoxyribonucleic acid (DMA) in previously uncontaminated sand samples
outside the building suggests that spores have the potential to migrate out of a contaminated
building and settle into the surrounding environment. This migration was outside the facility but
within the secondary enclosure. No samples were taken outside the secondary enclosure. The
study did not differentiate when exfiltration occurred from the facility (i.e., during dissemination
or subsequent remediation activities).
liv
-------
Exposure Assessment Plan
Based upon post-hoc interagency review of the exposure assessment methodology, the
objective of the Exposure Objective Plan was revised to the development of an exposure
assessment plan that can be used to determine the exposure associated with reentry into a
building that has been contaminated with surrogate Ba spores and subsequently
decontaminated. A methodology for qualitative characterization of inhalational exposure
associated with an adult's re-entry into the BOTE Project facility contaminated with Bg spores,
before and after decontamination, was developed. The current methodology takes into account
the specific site and the utilization of both indoor air and surface sample analytical data. During
development of the methodology, several areas of uncertainty and variability were
acknowledged and included: lack of knowledge of recovery efficiencies for sampling methods;
how to approach analysis of non-detect data; choice of statistical software and data distributions
used to calculate exposure point concentrations; use of existing reaerosolization data from the
literature; and choice of surrogate used. These areas of uncertainty and variability will need to
be examined prior to implementation of the exposure assessment methodology to limit the
potential bias in the final exposure calculation. While the exposures calculated using this initial
methodology will be limited by uncertainties and should be considered qualitative. This is the
first effort of its kind and the lessons learned from development of the methodology are critical to
moving the science forward to determine the gaps and research needed to develop a
quantitative exposure assessment methodology for B. anthracis.
Summary
The intent of Phase 1 of the BOTE Project was to develop an improved understanding of
response strategies for a single building, ultimately to extrapolate for use in wide area
remediation. This project was the first field level evaluation of decontamination technologies
under similar conditions outside a laboratory-controlled environment. The decontamination
efficacy, cost, labor, and waste analyses provide invaluable information to decision-makers
regarding time and resources required for each decontamination approach. Furthermore, the
BOTE Project provided an opportunity for improving the readiness for mitigating the effects of a
release of a bioagent over a wide area by allowing for:
EPA cross-regional training and biosampling experience;
Collaboration across regions and government agencies; and
Real-world experience with biological agent decontamination.
The information and experience obtained during Phase 1 was used in Phase 2, the interagency
response and remediation exercise, to aid in the development of sampling, risk mitigation,
decontamination, and waste management plans. (Phase 2 involved the interagency response to
a covert release of Ba (simulant); the exercise initiated with public health and federal law
enforcement notification and completed through facility remediation.) Overall, the BOTE Project
provided the opportunity to assess the interagency's current response and remediation
capabilities and areas of need for future capability enhancements.
Iv
-------
-------
1. INTRODUCTION
The Bio-response Operational Testing and Evaluation (BOTE) Project was a multi-agency effort
designed to operationally test and evaluate biological incident (anthrax release) response from
initial public health and law enforcement response through environmental remediation. The
effort included the coordinated project planning, support, and/or involvement from:
• Department of Homeland Security Science and Technology Directorate (DHS S&T);
• Environmental Protection Agency (EPA);
• Centers for Disease Control and Prevention (CDC);
• Department of Energy (DOE) National Laboratories;
• Department of Defense (DOD) Defense Threat Reduction Agency (DTRA); and
• Federal Bureau of Investigation (FBI).
The project was established through initial interactions between DHS S&T and EPA/Office of
Research and Development's National Homeland Security Research Center (NHSRC) in
partnership to further develop research products to support interagency Homeland Security
responsibilities.
Homeland Security-related research efforts by the EPA, DHS S&T, and others have culminated
in products that have contributed to significant advances in understanding of biological agent
remediation. The impacts of these products and developed expertise have been realized in field
responses (such as the natural anthrax responses in Region 1m), table top exercises, and
EPA/Office of Solid Waste and Emergency Response (OSWER) guidance and policy
documents. This research has helped improve the nation's preparedness and capability to
respond to a biological incident, setting the foundation for improving the readiness to mitigate
the effects of the release of a bioagent over a wide area.
Further significant advances in preparedness achieved through research and development were
recognized as requiring a scaled-up systems-oriented approach to research efforts in an
operational environment. Building on past DHS S&T collaborative efforts with EPA, the BOTE
Project was conceived within NHSRC and supported by Homeland Security-relevant Program
Offices throughout EPA and by EPA Regional offices.
The BOTE Project was divided into two distinct phases: (1) a field-level decontamination
assessment and (2) a functional operational evaluation. In Phase 1, three decontamination
methods that had shown effectiveness against Ba spores in laboratory and/or field use were
tested under field-relevant conditions. The three decontamination methods utilized
independently in three separate testing events (referred to as rounds) were: (Round 1)
fumigation with hydrogen peroxide (H2O2); (Round 2) a decontamination process incorporating
the spraying of surfaces with pH-adjusted (or amended) bleach; and (Round 3) fumigation with
chlorine dioxide gas (CIO2). Test parameters for each round included the decontamination
method, level of contamination, and contaminated environment (e.g., office setting, residential
area, heating, ventilation and air conditioning (HVAC)). An assessment of effectiveness and a
cost effectiveness analysis of application of each decontamination method (or process) were
1
-------
completed. The intent was to develop an improved understanding of response strategies on a
single building for use in wide area remediation.
In Phase 2, an interagency exercise simulating a response to a covert Ba release in a facility
was conducted. This interagency response included law enforcement, public health,
decontamination, and facility clearance. The exercise included coordination with the
local/state/regional response community and the establishment of both a Technical Working
Group and an Environmental Clearance Committee. The exercise was planned in accordance
with the guidance provided in the Homeland Security Exercise and Evaluation Program.
This report discusses the results of Phase 1 of the BOTE Project, the field-level
decontamination assessment. Information on Phase 2 can be found in the Exercise After Action
Report131.
1.1. Background
The release of Ba spores from envelopes mailed through the U.S. Postal Service system in
2001 (henceforth Amerithrax, after the FBI case code-name'41) resulted in the first bioterrorism-
related anthrax cases in the U.S.'51 Twenty-three facilities were confirmed to be contaminated to
at least some degree.'61 In total, remediation occurred over several years'61, and the total
recovery costs have been reported as nearing a billion dollars'71. In this context, remediation
refers to the entire cleanup process of which sampling, decontamination, and waste
management are a part. Decontamination costs alone (not overall remediation costs) have been
estimated to have totaled at least $290 million'81.
Response to the incidents and the resulting cleanup activities required unprecedented cross-
government efforts and led to the realization that development of capabilities to lessen the
impact of future bioterrorism incidents was vital. Although considerable experience was gained
from the many Amerithrax cleanup efforts, review of these efforts concluded that improved
methods were needed for remediation following contamination with Ba spores.'91 Significant
uncertainty remains regarding the number of spores that constitutes an infectious dose'61, the
efficiency of sampling and analysis methods, the relationship between inhalation infectious dose
and surface sampling, the most effective and appropriate decontamination methods, and
accepted waste handling, treatment, and disposal methods.
Comments from government reports and congressional inquiries pointed out that sampling and
decontamination methods were not standardized or validated and that deficiencies were
observed when attempts were made to locate and characterize Ba contamination.
Recommendations were made to standardize and validate procedures that could be used to
characterize biological agent contamination and follow on with efficient decontamination
measures that would effectively clear buildings and associated areas.'10"121
To address some of the sampling-related concerns, two interagency efforts were completed to
test and verify some of the progress made in the sampling and analysis area. The
methodologies applied and the results from these two studies are presented in the DHS S&T
and Joint Programs Executive Office-Chemical and Biological Division reports'13'14]. The first
-------
effort has been termed "INL-1" and the second "INL-2". Both efforts were conducted in a two-
story office building (PBF-632) at the INL facilities located west of Idaho Falls, ID.
INL-1 was conducted in 2007 with the primary objectives of understanding the differences
among sampling strategies (e.g., judgmental, probabilistic, and hybrid) and assessing the
effectiveness of sampling methods (e.g., wipes, swabs, and vacuum socks) in an operational
setting. For each test during INL-1, the facility was contaminated with one gram (1 g) of B.
atrophaeus subspecies globigii (formerly Bacillus globigii [Bg]) spores as a surrogate for Ba
spores, sampled (characterization sampling), decontaminated via fumigation with CIO2, and
sampled again (clearance sampling). From the summation of all tests, the three sample
collection methods tested (wipes, swabs, and vacuum socks) were shown to be capable of
collecting positive samples with a range of concentrations of spores. The statistical analysis in
the study showed that wipes have greater overall organism recovery rates than vacuum socks
or swabs. These results were independent of the sampling strategy used.
INL-2 was conducted in 2008 as a follow-up to INL-1. The primary objectives of INL-2 were to
operationally evaluate judgmental and probabilistic sampling strategies for characterization, as
well as evaluate and compare probabilistic and hybrid (judgmental and probabilistic) sampling
strategies for clearance in the building. In summary, results from INL-2 indicated that there was
no significant fundamental difference between detection rates using judgmental versus
probabilistic sampling strategies when examining the overall contamination of all rooms.
In addition to these operational sampling studies, significant efforts have also been ongoing to
address the limitations in decontamination methods (overall site remediation) experienced in the
2001 incidents. This work has included contributions made as a result of research to better
understand the effectiveness and improve implementation of decontamination methods'151.
These studies have focused on several different fumigants and liquid chemistries, evaluating
effectiveness as a function of material types and decontaminant application conditions.'16"181
Such studies have been primarily on the bench-top scale using standardized test methods
adapted for the scale of the study. Some efficacy studies on a larger scale, investigating
application procedures in addition to the sporicidal properties of a decontaminant, have more
recently been undertaken'191. Studies have also been conducted to understand the demand of
building materials for fumigants to assess the generation capacity requirements necessary to
achieve target gas/vapor concentrations in the enclosed volumes being treated'20'211. The impact
of decontaminants on materials and equipment has also been assessed for many of the most
effective decontamination chemicals and processes'22'231. The combination of effectiveness as a
function of the materials, materials impacting the ability to achieve effective conditions, and the
impact of decontamination on materials/equipment ties directly to the waste management
requirements for a specific site. Understanding of the interconnections and trade-offs between
the decontamination and waste management options contributes significantly to the site-specific
decisions to be made for an effective, yet efficient, remediation effort. While information is
necessary to understand the interconnection and trade-offs, true cost and time impacts can be
understood only from larger scale testing, functional exercises and real incidents.
-------
In addition to the remediation activities discussed above, several remediation efforts pertaining
to residences contaminated with natural Bacillus anthracis (Ba) spores have also contributed to
advances in the understanding of successful decontamination approaches. While these efforts
pertained generally to contamination from working with contaminated imported animal hides,
several impacts have been realized from these experiences. For example, the successful
implementation of a treatment process for the contaminated wooden shed in Danbury, CT, in a
2007 response'11 led to the development of a joint research project between EPA responders
and researchers. The objective of this research project was to assess the effectiveness of the
treatment steps (e.g., vacuuming, spraying with a pH-adjusted bleach solution, washing,
scrubbing, and rinsing) individually and in combinations. The goal of these assessments was to
understand the most effective combination of steps and situation-specific benefits of utilizing
such an approach.
When considering the decontamination plan to be used at a contaminated site, applicable
sampling data from the field may be used to perform a risk assessment to inform risk
management decisions. Microbial exposure assessments are conducted as part of the risk
assessment process to identify exposure pathways and determine the extent to which the
applicable population is exposed to a biological agent of concern. Although guidelines for risk
and exposure assessment such as the guidelines provided by EPA's risk assessment guidance
for Superfund sites'241, EPA's framework for ecological risk assessment'251, the National
Research Council's (NRC's) paradigm for human health risk assessments'261 and the NRC's
framework for risk-based decision making'271, and the NRC's Exposure Science in the 21st
Century guidance'281 do exist, guidelines specific to microbial exposure assessment are lacking.
While the International Life Sciences Institute has developed a framework for microbial risk
assessment[291, the framework is limited to waterborne pathogen exposure and does not cover
exposure in indoor settings. Because of the lack of available standardized protocols or
methodologies and data usability criteria, the field of microbial exposure assessment remains
limited to qualitative rather than quantitative assessments.
The effectiveness of the decontamination strategy for facilities is tied directly to the efficiency of
the sampling strategy. Decontamination effectiveness is determined by the ability to assess the
amount of contamination pre- and post-application of the decontamination strategy. In the
aftermath of the aforementioned activities and findings, the BOTE Project was developed to
address site remediation after the release of Bacillus spores within a facility in an operational
setting, drawing upon the advances in both the general sampling and decontamination areas
over the past several years.
This section of the report provides an outline of the specific test objectives and methodologies
for the BOTE Project.
1.2. Study Objectives
The BOTE Project Phase 1 was a field-level decontamination assessment managed by the EPA
and DHS with the DOD/DTRA serving as the interagency coordinating study directorate. Phase
1 included an assessment of three decontamination methods and a cost analysis of the test and
subsequent sampling results. The three decontamination methods were chosen by a group of
4
-------
subject matter experts from EPA and DHS S&T based upon laboratory study results, field
experience, gaps in operational understanding, and the project objectives. The three methods
ultimately decided upon were fumigation with vaporized hydrogen peroxide (VHP®), a treatment
process including the use of pH-adjusted bleach, and fumigation with CIO2. Results from the
BOTE Project Phase 1 contributed, in part, to the Incident Command (IC)/Unified Command
decision making in the BOTE Project Phase 2. (Phase 2 involved the interagency response to a
covert release of Ba (simulant); the exercise iniatied with public health and federal law
enforcement notification and completed through facility remediation. Information on Phase 2 can
be found in the Exercise After Action Report'31.)
The four principal objectives of the BOTE Project Phase 1 decontamination assessment
included:
• Objective 1: Conduct and evaluate field-level studies of three decontamination
technologies/protocols;
• Objective 2: Demonstrate that biological sampling and analysis methods evaluated in
previous studies provide accurate characterization of Ba simulant concentration
challenges for detection/identification purposes.
• Objective 3: Collect and analyze the results from the decontamination study and
perform a cost analysis of all aspects of the remediation approaches.
• Objective 4: Determine the exposure associated with reentry into a building that has
been contaminated with surrogate Ba spores and subsequently decontaminated.
Some of the main objectives listed above had sub-objectives. The specific study objectives and
sub-objectives are described below.
1.2.1. Objective 1: Decontamination Efficacy Assessment
The main objective of the decontamination efficacy assessment was to conduct and evaluate
field-level facility remediation studies using various decontamination technologies. A
considerable amount of research has been done in the area of indoor decontamination. This
research has been completed mostly in a laboratory environment looking at the efficacy of
several approaches to decontaminate building materials. Several methods with high efficacy
results in the laboratory were identified for testing at the field level where not all of the variables
can be controlled. In addition to the facility decontamination assessment, the effectiveness of
chlorine for inactivating spores in personnel Decontamination Line wash water generated during
the remediation of the building was also assessed. The objective of this assessment was to
develop a standard operating procedure that can be used in the field.
Key sub-objectives were to:
• Assess the effectiveness of the decontamination technologies as a function of room type
(materials common to a commercial, residential, or industrial setting).
• Identify any damage to the building or materials/objects that are located inside the building
arising from the application of the decontamination technologies.
-------
• Determine waste generation and waste management approaches.
• Evaluate the effectiveness of wastewater/wash water collection, treatment, and disposal
procedures.
The results from the decontamination efficacy assessment, combined with the other objectives
in this project, will provide decision makers with information that will allow them to make
informed decisions when selecting a decontamination strategy in the event of a biological
incident.
Some background on the key sub-objectives of Objective 1 is presented below.
1.2.1.1. Decontamination Technologies Efficacy Assessment
Remediation strategies for the cleanup of a biological agent will vary depending upon site-
specific considerations such as types of materials contaminated, extent of contamination, and
whether the release occurred indoors or outdoors. Testing to date has revealed that efficacy of
a particular technology can depend to a large degree on the materials with which the spores are
in contact. Generally, liquid technologies are more effective on hard nonporous surfaces such
as glass, metal, and laminate. Porous surfaces such as carpet, concrete, and wood prove to be
the most challenging for such technologies'171. Discussions of efficacy test results have focused
on porous versus nonporous materials because several studies have indicated this gross
parameter as explaining observed differences in decontamination efficacy with respect to
material types'30'181. In addition, decontaminants may react with certain material types and
reduce the effectiveness of the decontamination process. For example, H2O2 has been shown
to break down rapidly on contact with galvanized metal'311. The ability to test the effectiveness of
the decontamination approach on relevant or representative material types is essential. For this
reason, the BOTE Project facility was furnished with a variety of porous and nonporous
materials that would be found in both commercial and residential settings.
1.2.1.2. Material Impact
Building decontamination following a biological agent release can be performed using different
decontamination techniques such as fumigation of the building with CIO2 or VHP® or with a
liquid decontaminant. Although a decontaminant may be very effective at inactivating or
removing the intended contamination, the decontaminant may have negative effects on the
material or equipment being decontaminated. The impact of decontamination methods on
materials and building items must, therefore, be a consideration when determining which
method to employ in a particular decontamination scenario. The impact of decontamination
methods on both material and equipment using CIO2 and H2O2 fumigation on building materials
has been studied'32'331. Assessments for the same fumigants have also recently been completed
for sensitive electronics and other high value materials'22'231. In the BOTE project, visual
observations were used to identify any damage to building materials/objects arising from the
use of the three decontamination approaches.
1.2.1.3. Waste Management
The proper management of waste from the cleanup after a biological event is a key element of
the remediation process. Different decontamination strategies result in different waste quantities
6
-------
and characteristics, and the management of those wastes can significantly affect the overall
remediation timeline, resource requirements, and costs.
The likely waste streams that will be generated from a Ba response would predominantly
include: 1) personal protective equipment (PPE) items such as suits, gloves, and footwear
covers ("booties"); 2) sampling waste; 3) porous materials removed either before or after
decontamination operations; and 4) aqueous waste streams. Decontamination strategies will
have a profound effect on the quantities of potential residual contaminating agent in the waste
streams. The amount of residual contaminating agent will in turn impact the available disposal
pathways and waste management costs.
One of the waste management challenges that the BOTE Project presented was the need to
address waste issues (costs, quantities, logistics, etc.) as if the waste had been contaminated
with Ba, even though a nonpathogenic surrogate was used ("notional" waste). In addition, as a
constraint of the tests, all waste that was generated needed to be handled in accordance with
INL waste management practices ("real" waste). This effort manifested itself mainly as a need to
keep any biohazard-labeled bags (supplied to the sampling teams in the sampling kits) out of
the trash. Discarding the biohazard-labeled bags in the trash would not be consistent with waste
handling procedures associated with a real Ba contamination incident. The waste management
practices from a real anthrax contamination incident would be determined by the waste
acceptance criteria of the State where the incident occurred and how the responders
characterize the waste, as well as the owner-operators of the waste management facilities who
have to ultimately accept the waste. For example, in the recent naturally-occurring Ba response
in Durham, NHm, the State indicated that if the pH-adjusted bleach decontamination process
was followed properly, the waste could be disposed of as solid waste in a Resource
Conservation and Recovery Act (RCRA) Subtitle D facility without any additional waste
characterization sampling. It is advantageous, from a waste management cost standpoint, to
make sure that the decontamination process (and sampling, if required) results in the least
restrictive (i.e., Subtitle D) waste designation, thus reducing remediation costs. However, there
is no guarantee that waste disposal capacity would be available in the State where the incident
occurred, which may necessitate decisions by State regulatory personnel from States outside
the State where the incident occurred. For this and other reasons, there is a need for pre-
incident waste management planning so that these issues can be identified and resolved prior
to an incident. The results from this study may be expected to pertain to a single building
contamination incident. There may be significant additional complexities in the event of a wide-
area incident involving many buildings.
It is necessary to properly estimate the cost of and issues related to management of the
different "notional" waste streams generated during the BOTE Project while still properly
addressing IN Us requirements for the "real" waste streams. To perform this estimate, a waste
management approach was developed that identified, quantified, and characterized the waste
after it left the building, but prior to placing the waste in the dumpster for disposal through INL's
waste management program. (This approach is described in detail in Section 4.5.) In this
approach, the waste was treated as Ba-contaminated waste up to the point at which it entered
-------
its temporary waste management staging area in preparation for final disposition in the INL
waste management process.
1.2.1.4. Decontamination Line Wash Water- Handling, Treatment, and Disposal
During past Ba-related cleanups, sampling and decontamination personnel were washed down
upon exiting the buildings with disinfection/cleaning solutions prior to removing PPE. In typical
operations of this type, the Decontamination Line wash water generated from these processes
is collected in containers and treated prior to discharge to a publicly owned treatment works
(POTW), when possible. In some of these Ba remediation efforts, local POTWs would not
accept the Decontamination Line wastewater because of the nature of the organism and public
perceptions of potential risks. For example, in 2001, approximately 14,000 gal of
Decontamination Line wash water were collected during the cleanup of seven Ba spore-
contaminated Capitol Hill buildings in Washington, DC[341. The water was stored in 55-gal drums
while on site, then transferred to tanker trucks and transported to Fort Detrick, MD, after the
local wastewater utility declined to accept the water. The wash water was treated at Fort
Detrick's on-site wastewater treatment facility. In a smaller scale incident, state POTWs did
agree to accept Decontamination Line wash water, highlighting the differing scenarios.
Regardless, the cost and effort potentially associated with limitations on discharge of the wash
water accentuate the need for on-site treatment of the water. Therefore, Ba spore inactivation
studies need to be conducted with water that is typical of what would be generated in such a
remediation scenario. Additional scientific data verifying the effectiveness of treatment will
increase confidence of POTWs to allow them to accept the water. The BOTE Project provided
an opportunity to collect realistic wash water to assess chlorine bleach treatment methods.
1.2.2. Objective 2: Demonstration and Assessment of Biological Sampling and
Analysis Methods
Critical measurements for the BOTE Project included the determination of viable spores at
different times within a test round, specifically before and after decontamination. Testing for
viable spores was done for the surface, air, water, and sand samples collected in the BOTE
project. Surface and air sampling were primary methods used in conjunction with other
objectives, i.e., determination of decontamination efficacy and the exposure assessment. The
primary purpose of the surface sampling was to generate data to determine decontamination
efficacy. In addition, key sub-objectives were to:
• Demonstrate use of current surface sampling methods.
• Evaluate the performance of the rapid-viability polymerase chain reaction (RV-PCR)
analytical method.
• Assess the performance of aggressive air sampling (AAS) for post-decontamination
sampling.
Some background on the key sub-objectives of Objective 2 is presented below.
-------
1.2.2.1. Surface Sampling
Surface sampling was the primary measurement method to assess viable surrogate Ba
presence per unit area prior to and after application of a decontamination method. Comparison
of surface sampling results pre-decontamination and post-decontamination allowed a
determination of the effectiveness of the decontamination method that had been applied in a
specific testing round. Pre- and post- decontamination sampling was done using collocated
sampling locations to allow for resolution of material types or locations that were potentially
problematic for the decontamination method. The sampling strategy was developed to provide
an opportunity to resolve decontamination effectiveness based upon the facility setting, i.e., a
residential setting with typical porous materials such as furniture, appliances, and bedding; a
commercial space with typical office materials; an industrial setting representing a workshop;
and an office mail room. In addition, the surface sampling was an essential measurement as a
reference for all other BOTE Project studies discussed below.
Surface sampling strategies for biological agents (i.e., Ba spores) incorporate different
techniques depending upon the surface type and intended use of the results. The BOTE Project
surface sampling methods utilized typical surface sampling methods, with the strategy biased to
best achieve the primary project objectives (see Section 2.6.1). This sampling strategy also
offered the opportunity to assess the detection of viable surrogate Ba spores as a function of
sampling method, particularly during pre-decontamination sampling at both a high and a low
surface loading of surrogate Ba spores.
1.2.2.2. Rapid Viability-Polymerase Chain Reaction
The strategy also offered the opportunity to assess new analysis methods (i.e., RV-PCR) for
both pre- and post-decontamination samples. The RV-PCR is a research method developed
under an interagency agreement between EPA and the Lawrence Livermore National
Laboratory (LLNL) of the DOE to detect and identify the presence of live Ba spores rapidly
during a bioterrorism event. Briefly, the RV-PCR is a combination of a reliable broth culture
method (for viability determination) and the commonly-used real-time PCR (for highly sensitive,
specific, and rapid detection and identification)'35"371. The RV-PCR method presents an
additional factor of preparedness to meet the challenge of analyses of large numbers of
samples. The current version of the RV-PCR methodology had not previously been tested with
a large number of actual field samples and especially with post-decontamination field samples.
The opportunity afforded by the BOTE Project was to demonstrate the performance of the RV-
PCR method with field samples.
1.2.2.3. Aggressive Air Sampling
The main objective of AAS in the BOTE Project was to determine if, after application of
decontamination technology, disturbing indoor surfaces resulted in the detection of
reaerosolized residual spores via air sampling. This procedure was being tested as a
supplemental measurement for the determination of effectiveness of the decontamination
process.
The sampling methodology used documented AAS techniques'381 that physically disturbed
surfaces inside an enclosure and sampled large volumes of air using different sampling
-------
equipment to corroborate the success or failure demonstrated by the results from the previous
surface sampling. In a real Ba contamination event, the target clearance level would most likely
require that all final air samples be free of viable spores to achieve the ultimate goal of
successful remediation of the facility and restoring the facility to productive use. Any finding of a
viable spore of Ba would be considered a potential indication of ineffective decontamination that
may necessitate further decontamination and retesting of the area where the sample was
collected. In some past Ba remediation actions, after a facility was opened and reoccupied, a
transitional monitoring program, as recommended by the Occupational Safety and Health
Administration (OSHA), was instituted.'391
The determination of whether disturbing indoor surfaces resulted in reaerosolization of residual
spores was accomplished by achieving the following goals:
• Explore the feasibility of using a risk-based strategy consisting of multiple air sampling
techniques to verify the effectiveness of three decontamination technologies (post-
decontamination) and post-remedial surface sampling activities for future comparative
studies, and
• Evaluate possible protocols to establish facility clearance after decontamination using
the three technologies in each study area.
The objectives of the AAS methodology conducted at the BOTE Project testing facility are
summarized below and addressed in detail in Section 2.6:
• Ensure that sampling technicians are adequately protected during the sampling process;
• Establish the necessary parameters to conduct a successful evaluation of AAS by
establishing critical barriers and creating adequate negative pressure;
• Use different sampling techniques and media to ensure a more robust strategy and to
explore the strengths of each method;
• Use AAS techniques and high sampling flow rates as a sampling approach supplemental
to surface sampling for supporting clearance decisions; and
• Collect sufficient sample volumes of air through sampling media that will be analyzed
subsequently for the target organism (Bg).
1.2.3. Objectives: Overall Cost Analysis
The main purpose of the cost analysis in the BOTE Project was to estimate the overall cost of
the application of various decontamination technologies as a function of materials, time
(including labor hours), and other resources.
The cost analysis made the general assumption that, although certain pieces of information
derived from the BOTE Project are incident- and site-specific, the information can still be
extrapolated to other incidents, using appropriate scaling factors based on labor hours, numbers
of samples, size of affected area, and quantities of waste that are generated. The parameters
examined in this cost analysis include sampling activities, application of decontamination
technologies for the building and personnel entering and leaving the building, and equipment
rentals and consumables. Some costs that are critical to the analysis (i.e., waste management)
10
-------
could not be assessed based purely on the BOTE Project. Some BOTE-derived costs may be
unrealistic because the BOTE Project used a Ba spore surrogate Bg (not actual Ba spores).
Costs such as these were estimated using the BOTE Project data where appropriate, with
adjustments to include the impact of actual Ba contamination added using information from past
Ba responses and best engineering judgment. Although waste management costs could not be
estimated based purely on the BOTE Project, the BOTE Project data provided the information to
allow the EPA to better estimate what the waste management costs would likely be.
1.2.4. Objective 4: Assessment of Potential Exposure
Exposure to Ba spores during a release may be due to the primary release, or exposure may be
secondary due to contact with reaerosolized spores or a contaminated surface. Spread of
contamination (e.g., via reaerosolization and dispersion, inside to outside facilities [or vice
versa]) may further increase the potential for exposure. The BOTE Project provided an
opportunity to investigate some aspects of exposure in a field setting. Key sub-objectives were
to:
• Assess Bacillus spore migration from inside to outside a contaminated building.
• Investigate potential spore reaerosolization inside a contaminated building.
• Develop the concept of an exposure assessment plan.
Some background on the key sub-objectives is presented below.
1.2.4.1. Assessment of Bacillus Spore Migration from Inside to Outside a
Contaminated Building
The fate and transport of Ba spores in indoor and outdoor environments is not well understood.
Even less is known about spore migration into and out of buildings. The BOTE Project provided
a test bed to evaluate the potential for bacterial spores of a microbial agent dispersed inside a
building to migrate to the outside.
A review on the persistence of select agents by Sinclair et al.'401 found references that
suggested that Bacillus species spores (Including Ba) have the potential to remain viable in soil
for many years. For instance, viable Ba spores were repeatedly recovered in samples taken on
Gruinard Island for as long as 40 years post-inoculation'411. One study also showed that soil
samples containing Ba spores that had been sealed and stored for up to 68 years still contained
viable spores'421.
Following the intentional release of many Ba spores, Turnbull'431 reports the possibility of lasting
environmental contamination and ensuing reaerosolization of spores. Ibraham'441 found that
reaerosolized spores may be transported for up to 20 hours (hr) in the air, with the duration of
the transport contingent on the meteorological conditions at the time of the release.
While there have been numerous studies on spore transport within ventilation systems and in
buildings, a review of the literature resulted in few studies of the potential for outdoor transport
of an interior release of spores. One study by Sextro et al.[45] hypothetically modeled the spread
of anthrax in office settings to examine the fate and transport of Ba spores. The modeling
11
-------
predicted that during the first 48 hr, more than 90% of the disseminated spores may stay inside
a contaminated facility, while approximately 6% of the spores would be transported outside the
facility. The study also acknowledged that the number of spores migrating outside the facility
could increase if the model were run to include increased activity within the facility or were
extended to take into account longer time durations'451.
Spores deposited and persisting outdoors have the potential to reaerosolize and pose a threat
to populations living downwind. Doolan et al/46; examined the epidemiological characteristics of
the U.S. Capitol bioterrorism anthrax exposures and found immune responses in individuals
who had been outside an epidemiologically defined exposure zone. This observation potentially
suggests that the migration of spores outside the exposure zone might have been caused either
by environmental influences or by cross-contamination from individuals not properly
decontaminated. Taking this background information into consideration, spores are postulated
to migrate from a building through air circulation/ventilation systems, loose seals around doors
and windows, and human movement. The BOTE Project spore migration study was designed to
shed light on the potential transportation of disseminated spores from the initial area of
contamination inside the building to the outside (however, within the facility enclosure). The
study sought to determine the extent to which sterile sand samples were contaminated when
placed exterior to a contaminated building.
Both the methods and results from two separate laboratories, each using a different analytical
method to obtain qualitative data indicative of the presence/absence of Bg deoxyribonucleic
acid (DMA) deposited into the laboratory-prepared sand samples, are discussed in this report. In
addition to providing information on the methods used to collect and analyze the sand samples
from a field site, the data resulting from analysis of the samples were used to assess the
transport of aerosolized spores from the initial area of contamination. The results of this
preliminary study will inform additional research to better characterize spore migration from
indoor environments to outdoor areas.
1.2.4.2. Assessment of Reaerosolization
Following a bioterrorism event, assessment of human health risks from exposure to
microorganisms requires reliable data on exposure pathways including transport mechanisms
and potential routes of exposure such as inhalation. One mechanism being studied is the
reaerosolization of deposited spores or particles back into the air due to human or mechanical
activity following an initial release. To date, the risk of infection from exposure to Ba spores via
reaerosolization is unclear'471. Meselson et al.'481 reported on the Sverdlovsk anthrax outbreak of
1979 and concluded that attribution of the inhalational anthrax cases to reaerosolization of 6.
anthracis spores was unlikely. However, following the anthrax attacks of 2001, Weis et al.'491
observed reaerosolization through collected air and surface samples in the Hart Senate Office
Building under low activity levels typical of an office environment. The authors concluded that
the Ba spores used in the attack reaerosolized under active office conditions, but also
acknowledged that quantifying the associated risk of developing inhalational anthrax is
uncertain. From an exposure perspective, quantifying reaerosolization of spores is likely
complicated by dynamic spatial and temporal aspects as well as by the influence of numerous
variables. For example, Price et al.'501 noted that reaerosolization is affected by the specific
12
-------
surface onto which spores are deposited and the cause of the reaerosolization. The level of
spores deposited on the surface and properties of the spore or powder used in the initial release
also likely influence reaerosolization.1511 Additional examples of how variables such as activity
level, surface type, temporal considerations, and surface loadings affect reaerosolization of
particles and spores (fungal and bacterial) are briefly provided in the following paragraphs.
Reaerosolization is dependent on activity level and, accordingly, extent of reaerosolization
is likely to vary considerably'521. Ferro et al.[531 found that the reaerosolization of particulate
matter was influenced by the type and intensity of the activity as well as the number of
active persons. Gomes et al.[541 reported that the air swirls associated with walking
affected particle reaerosolization one to three orders of magnitude more than the floor
vibrations associated with walking. Oberoi et al.[551 observed that the net mass of
reaerosolized particles associated with human foot stomping in place while rotating around
the person's center axis was two times greater than foot stomping in place without
rotating.
Surface type may influence reaerosolization. For example, fungal (Penicillium
chrysogenum) spore reaerosolization was higher after walking on cut pile carpet (typical
residential carpet) than after walking on loop pile carpet (typical commercial carpet) or
vinyl tile'561. At contamination levels of 1E6 colony forming units (CFU) per cubic meter
(m3), differences in P. chrysogenum spore reaerosolization were not observed between
the vinyl tile and loop pile carpet'561. Interestingly, quartz particles and laboratory-produced
cockroach allergen dust had greater reaerosolization rates from linoleum than from
carpet'541. Krauter and Biermann'571 reported reaerosolization of B. atrophaeus spores from
steel and plastic at similar levels under constant airflow.
Contaminant loading on surfaces could also affect reaerosolization. After walking on
contaminated surfaces, more P. chrysogenum spores were observed in air when surface
loadings were higher'561. Gomes et al.'541 noted that the proportion of dust particles
reaerosolized from a surface decreases with higher loadings of dust, although a higher
total number of particles may be reaerosolized as loading is increased.
Some of the reaerosolization studies indicate a decrease in reaerosolization over time. For
example, Buttner et al.'561 noted that the reaerosolization of P. chrysogenum spores from
floors due to walking was reduced with repeated disturbances. Gomes et al.'541 also
reported that for a ten-minute vibration and air swirl disturbance, reaerosolization of
particles occurred primarily during the initial two minutes even though dust particles
remained on the surface the entire ten minutes. The initial reaerosolization rate of 6.
atrophaeus spores exposed to airflow in a ventilation system was rather reduced within 30
minutes'571. Oberoi et al.'551 reported that particles were readily reaerosolized from carpet
during the initial ten seconds of human activity (e.g., walking and foot stomping). Over
time, larger particles (mass mean diameter = 7.7 micrometers [urn]) settle while smaller
particles (mass mean diameter = 2.7 urn) continue to be airborne and to move on air
currents'551.
13
-------
The BOTE Project presented an opportunity to gain a better understanding of reaerosolization
of spores from surfaces during remediation activities. For this effort, surface and air
concentrations of the Bg spores were measured before (following Bg spore dissemination) and
after the application of the decontamination technology for each decontamination round in
Rooms 101A and 102, both configured to represent typical office settings. Surface samples for
Bg spores were collected using various sampling methods, indoor air concentrations of Bg
spores were measured, and continuous particle measurements were taken in these two rooms.
For each decontamination technology, samples were collected at various stages before and
after decontamination so that the reaerosolization of settled spores into the ambient air could be
characterized. The activities associated with the disturbance of the settled Bg spores were
limited to the activities of the sampling personnel and were not controlled or scripted to simulate
typical residential or office activities. The data were not collected under isolated (i.e., activity-
specific or surface material-specific) conditions. The contribution of Bg spore loadings on
specific objects/materials to the overall Bg spore concentrations in air could not be established.
Nevertheless, the factors affecting the reaerosolization of Bg spores during the BOTE Project
were collectively considered so the distribution of Bg spores on surfaces and in the air might be
considered as representing rather real-world conditions.
1.2.4.3. Exposure Assessment Plan
The potential for exposure to Ba spores via inhalation, dermal, or gastrointestinal pathways'581 is
a concern when considering re-entry into a building that has been intentionally or unintentionally
contaminated. Through microbial exposure assessment, the relationship between the biological
agent of concern, the environmental setting, and the affected population is determined by
developing an exposure profile'291. The exposure assessment helps provide quantitative or
qualitative input into the risk characterization'291. But, to date, no standardized protocols or
methodologies exist for conducting quantitative microbial exposure assessments.
The BOTE Project provided a unique opportunity to develop a site-specific methodology that
could be used to prepare a qualitative characterization of potential inhalation exposure
associated with reentry into the building pre- and post-decontamination using the semi-
quantitative/qualitative data generated in a field setting. Based upon post-hoc interagency
review of exposure assessment methodology, the objective was revised to the development of
an exposure assessment plan that can be used to determine the exposure associated with
reentry into a building that has been contaminated with surrogate Ba spores and subsequently
decontaminated. Key assumptions for handling the analytical data and exposure calculations
are being derived from chemical risk assessment guidelines and standard microbiological
practices. Analysis of the data set using this methodology will provide a qualitative inhalation
exposure assessment and is critical to moving the science forward and determining the
gaps/needs for quantitative exposure assessments. While the samplers did not wear personal
monitors to measure true exposure, the collected data from the air samplers indicate spore
reaerosolization and possible exposure hazard. The preliminary exposure calculations and
subsequent follow-on evaluations of the data can help guide data usability considerations and
statistical treatment of data for exposure analysis. Results and lessons learned can be used to
help inform development of an exposure assessment framework for microbial agents.
14
-------
2. MATERIALS AND METHODS
Details of the test facility, organism, and dissemination method are discussed in this section.
Three rounds of Phase 1 testing were conducted from April 11 to May 19, 2011. The project
was carried out in accordance with a Test Plan and Quality Assurance Project Plan (QAPP)
endorsed by EPA, DHS, and DTRA[59].
Each of the three rounds in Phase 1 included dissemination, characterization sampling,
decontamination, waste management, and post-decontamination sampling. For each round,
simulant levels, room configurations, and sampling procedures were virtually the same inside
the test facility. Bg dissemination for each round resulted in a high level of contamination on the
ground floor and a low level of contamination on the top floor, and each round employed
different decontamination technologies (see Table 2-1).
Table 2-1. Definitions for anticipated contamination levels and decontamination
technologies.
Contamination Levels
Low (top floor)
High (first floor)
100to200CFU/ft2
1 .OE5 to 1 .OE7 CFU/ft2
Decontamination Technologies
Round 1 (Apr 16-23, 2011)
Round 2 (Apr 25 - May 6, 201 1)
Round 3 (May 10-17, 2011)
Fumigation with VHP® (STERIS Corporation, Inc.) (H2O2)
Treatment process incorporating pH-adjusted (amended)
bleach
Fumigation with chlorine dioxide gas (CIO2) (Sabre Technical
Services, LLC)
Prior to Round 1, a mandatory full participation (MFP) round was conducted as a dry run for
dissemination and sampling. The MFP provided an opportunity to test operational systems
developed for the project (e.g., related to communication and coordination) and to train
sampling teams inside the facility while collecting background (pre-test) samples. The MFP was
conducted on April 14-15, 2011.
2.1. Facility
The test facility for the BOTE Project was PBF-632, a two-story unoccupied office building
owned by INL and located on INL property approximately 45 miles (mi) west of Idaho Falls, ID.
A site view of the INL test facility is shown in Figure 2-1. For future reference in this report, the
north end of the facility is located on the left side in the figure; the south end is on the right side
(i.e., facing the blue tank). Hence, the west side of the building is visible in the figure.
15
-------
Figure 2-1. Site layout of PBF-632.
The interior of the facility was prepared in a manner consistent with the objectives of the project,
i.e., having specific areas containing different materials in common on each floor. The layout of
both floors is illustrated in Figure 2-2 and Figure 2-3. Each floor was approximately 4,025
square feet (ft2). The first floor was made up of 11 rooms consisting of a reception area, men's
and women's restrooms, a mechanical room, and a hallway. The second floor was made up of
15 rooms consisting of two storage rooms, men's and women's restrooms, a mechanical room,
and a hallway. In this facility, each floor had an independent HVAC system. The ceiling,
approximately 8 feet (ft) in height, incorporated a "dropped ceiling" design that utilized ceiling
tiles to separate the occupied space from the utilities that run above the ceiling. The area above
the ceiling on the first floor is approximately 25 inches (in), and the area above the ceiling on the
second floor is approximately 53 in. The total building volume is approximately 90,000 cubic feet
(ft3).
16
-------
Figure 2-2. INL Building PBF-632, Floor 1.
17
-------
u
Figure 2-3. INL Building PBF-632, Floor 2.
18
-------
The first and second floors each had three rooms (Rooms 106, 108, 110, 208, 210, 212) that
were configured to represent a commercial office setting (see Figure 2-4 and Figure 2-5). Each
of the three "office" rooms on each floor contained furnishings representative of that type of
setting, such as:
• Flooring: Wood laminate.
• Furnishings: Desk, chair, filing cabinet (with one drawer full of papers), book case (one
shelf occupied by books), dividing partition, and computer with monitor.
• Walls: Latex-painted wallboard.
• Ceiling: Ceiling tile.
• Materials: Paper, files, books, etc.
The first and second floors each had three rooms configured to represent residential settings
(kitchen setting [Rooms 105 and 209], bedroom setting [Rooms 109 and 213], and living room
setting [Rooms 107 and 211]) (see Figure 2-4 and Figure 2-5). The types of furnishings in these
settings included materials such as:
• Flooring: Carpet.
• Furnishings: Couch, bed, tables, chairs, TV, kitchen oven/range, refrigerator, sink, and
countertop.
• Walls: Latex-painted wallboard.
• Ceiling: Ceiling tile.
• Materials: Paper, magazines, etc.
One room on each floor (Rooms 104 and 204) was configured to represent an industrial setting
(see Figure 2-4 and Figure 2-5). These "industrial" rooms contained a workbench and tools set
up to resemble an industrial workshop setting, with an epoxy-covered floor (epoxy over wood in
Room 204 and over concrete in Room 104). Approximately 1.8 pounds (Ib) of pine shavings
(animal bedding) was distributed in the industrial room to represent an organic load common to
such settings. One room on each floor (Rooms 103 and 207) was configured to represent a
mailroom and included a holding unit with mail slots containing paper and envelopes (see
Figure 2-4 and Figure 2-5). Photos of each of the rooms can be found in Appendix A.
Two additional rooms (Rooms 101A and 102) on the bottom floor were configured as
commercial offices for the reaerosolization study, with the following furnishings:
• Flooring: Carpet.
• Furnishings: Desks, chairs, metal filing cabinets, plastic Ultraviolet-Aerodynamic
Particle Sizer® (UV-APS) case.
• Walls: Painted wallboard.
• Ceiling: Ceiling tile.
Room 101A had dimensions of 20ft x 10 ft x 8 ft high and Room 102 had dimension of 11 ftx
13 ft x 8 ft high. There was one door to Room 101 A, and access was through Room 101. Room
102 had direct access to the main hallway through a single door. In these rooms, stands for the
19
-------
SKC BioSamplers® were bolted to the floor in three locations per room (See Figure 2-6). One
UV-APS was placed on top of a desk in each room (Figure 2-6).
The remainder of the rooms on each floor did not contain any furnishings. The walls in the
rooms that did not have painted wallboard installed were comprised of a plastic/polymeric
material. The plastic-walled rooms represent the remainder of the building other than the ten
rooms on the bottom floor and the eight rooms on the second floor that were set up in the
configurations described above. The floor material in all other areas (i.e., not the commercial,
residential, industrial or mailroom settings) was epoxy-covered concrete (first floor) or epoxy-
covered wood (second floor), except for the furnace rooms on each floor (concrete). A complete
inventory of the contents of each room can be found in Appendix A.
1
- i i
!
f commercial
Figure 2-4. Schematic diagram of PBF-632 first floor.
20
-------
Figure 2-5. Schematic diagram of PBF-632 second floor.
21
-------
4" MM
/
/
f^~ SSf * * """"^ *^^^HI
»— ^ «~~~IA-^. I T —r^— «"~~"~^*-^ - , «^^ ^^^Vi
|[^
to 1
«KC «n« •*
•SB
l^rl
,
•
il JIT- ifX
r» 1 ' ' '
•
ŁL^& 2*
*
R I 1
^ ^
^
Orr« flrra
m Biosamp 4
» 4
;;
> 4
...
\
X-!
-
m
•3? >i r— r
, T >
i
•
s
1— J
4 - ^B
^F i
D • D
'~V S vT"'*
01
| V »«*!•»
Ni ^DD
131 L a1 •.
0
» 4
-1.*
• *SN
.
2%=
I^sa- /
:
™X
^//2-
t * *
i
\ !!.._
ft
i
•! '
4^^^
•
•••'*'
i.
L
j
Figure 2-6. Location of air sampling equipment in Rooms 101A and 102.
22
-------
The facility had dual HVAC systems, one system for each floor. Neither HVAC system had a
heating or air conditioning component installed in the system but the HVAC system was
otherwise functional with a supply and return plenum on each floor. Both the HVAC supply duct
and return line were constructed of galvanized metal. The supply duct contained an insulating
material on the inside of the duct, leading from the boiler. The supply side had been modified
several times since the building was constructed, and insulation was not present throughout the
entire supply line. The return duct was added prior to the BOTE Project, for the INL-1 and INL-2
studies, to provide more control of return air flow within the building. The HVAC contained
standard air filters on the intake side.
The PBF-632 facility had a polymeric secondary containment structure (secondary enclosure)
outside the building, as shown in the right photo in Figure 2-7. This containment served as a
barrier to assist in preventing contamination of the surrounding area during testing, i.e., one
method for cross-contamination control. Additionally, as discussed in Section 2.10.3, the facility
was tented (under the secondary enclosure, directly on the building) during the fumigation with
CIO2.
Figure 2-7. PBF-632 before and after setup of outer secondary enclosure.
The facility was equipped with a closed-circuit camera system that recorded activities in the
facility throughout the project. For this system and other equipment, a wireless network
(802.11a and an 802.11b) was set up inside PBF-632 with coverage to the five surrounding
support trailers provided by INL (described below). This network was connected to the wired
gigabit network switch inside the building and transported data to the trailers. The wireless
system incorporated standard encryption protocols to limit network access. The internal radios
were powered by the Ethernet switch through Power Over Ethernet. The external radios had
line-of-sight to the trailers and transmitted signals directly to the trailer network terminal through
the 802.11 a backhaul radios. The Engenius EOA3630 access point radio was used for the
internal network. Three radios were placed on each floor in the hallway at evenly spaced
intervals across the PBF-632 building. Two Engenius EOA7535 radios were placed at a
minimum of 100 ft from each end of the PBF-632 building, and a third radio was located at the
23
-------
command-control trailer. Fourteen Vivotek FD7141 Pan, Tilt, Zoom network cameras were
located within the PBF-632 building, with six each on the first and second floors. One was also
located at the entrance of the facility and at the exit containing the personnel Decontamination
Line tent. All of the cameras were enclosed in a weather resistant dome and wired to a Power
over Ethernet switch. Video from the cameras could be viewed, recorded and controlled at the
command-control trailer and viewed in the decontamination support trailer. Recorded video was
archived to a computer hard drive. Video was recorded during any period when event personnel
were active within the structure. A list of the locations of the cameras is reported in Table 2-2.
Table 2-2. List of closed-circuit camera locations within PBF-632.
First Floor
Entry (ingress) door (south end)
Personnel Decontamination line (egress door)
Room 105
Room 106
Room 107
Room 108
Room 109
Room 110
Second Floor
Room 208
Room 209
Room 210
Room 211
Room 21 2
Room 21 3
An overhead view of the test site and proximity of the various assets is shown in Figure 2-8.
This aerial view shows the location of the facility in relation to the support trailers for the project.
The ingress to the facility was located on the south end. Ingress to the first floor was permitted
through an entrance located under the stairwell to the second floor or via interior stairwell from
the second floor. Ingress to the second floor was permitted only from the exterior stairwell under
the secondary enclosure. The interior stairwell was located toward the north end/west side of
the facility; an airlock was placed on the second floor landing to aid in the prevention of cross-
contamination between floors. This air lock at the top of the stairs isolated one room, the
second-floor men's restroom, which was right across the hall from the stair entry. Personnel had
to enter the air lock to access this second-floor men's room. For contamination control, no entry
to the second floor was permitted from the first floor. The egress from the facility was from the
first floor located on the north end/west side of the facility. Egress occurred through the
personnel Decontamination Line, represented by Location 5 in Figure 2-8.
The command and control trailer (Location 1 in Figure 2-8) served as the main briefing location
for all on-site activity during the project. The sampling and decontamination support trailer
(Location 7 in Figure 2-8) was divided into two distinct areas separated by a soft wall. Samples
were brought in through the east side of the trailer and passed into the west side of the trailer
24
-------
through the window in the soft wall. Personnel on the west side received the samples for
tracking, shipping or processing. A control room for monitoring decontamination and waste
management activities was also located on the east side of this trailer. This area was used to
track waste generated from each decontamination method and monitor project activities via the
closed- circuit TV feed.
Personnel entering the facility used the sampling prep trailer (Location 2 in Figure 2-8) to don
their PPE. This trailer contained all PPE needed during the project. The trailer was divided into
two distinct areas: one area for readying sampling personnel and one for preparing sampling
materials. Additionally, the office space in the trailer was used for sampling team coordination,
e.g., preparation of sampling maps and on-site support for the sample tracking system (i.e., the
Building Restoration Operations Optimization Mode (BROOM)'601, described in Section 2.7).
Location 8 (in Figure 2-8) served as a break and recovery trailer. This area was provided for
sampling and decontamination crews to rest and recover before or after their entry into the
facility. Sampling team training, pre-briefing, and debriefing were also held in this trailer.
The red outline in the figure shows the exclusion zone around the facility utilized during
decontamination activities (fumigation or spraying of pH-adjusted bleach). This zone was set to
avoid exposure of personnel to gaseous toxic hazards per the BOTE Project Health and Safety
Plan1591.
25
-------
1. Command Trailer
2. Sampling Prep Trailer
3. PBF-632 Ingress
4. PBF-632 Egress
5. Decon Line
6. Decon Support Trailer
7. Sampling/Decon Support
Trailer
8. Recovery Trailer
9. Emergency Rally Area
10. General Parking Area
11. PBF-638 (not used in the
BOTE Project)
12. Water Tower
13. Pump House
Red outline is exclusion zone.
Orange lines show personnel
operational flow.
Figure 2-8. BOTE Project site configuration.
2.2. Test Organism
To meet the objectives of the project, the use of a nonpathogenic surrogate organism
representative of Ba with respect to decontamination resistance and physical properties was
required. A number of building dispersal and fumigation studies using Bg spores as a surrogate
for Ba spores have been conducted at the INL PBF-632 facility since the Amerithrax incident of
2001H3,14] prom eacfo Of these studies, information on Bacillus spore dispersion, site
characterization, fumigation, on-site laboratory capacity, and site clearance has been learned.
Bg is a Biosafety Level 1 organism and is not a Select Agent (www.selectagents.com).
However, Bg serves as a nonpathogenic surrogate for Ba. The Bg spore preparation was
obtained from DOD's Critical Reagents Program Antigen Repository and was prepared by
growing Bg (American Type Culture Collection (ATCC) 9372; also known as B. atrophaeus and
B. subtilis var. niger) in tryptic soy broth (TSB) supplemented with magnesium sulfate until 80 to
90% sporulation occurred. After purification and drying, the spores were dry-blended with
Aerosil R812S fumed silica particles. The spore-silica mixture, with a ratio of 80% dry spore
material to 20% silica, was jet-milled to a uniform particle size. The final powdered spore
preparation contained approximately 1011 spores/gram (g)[61].
26
-------
Prior to its use, the Bg spore preparation was evaluated by the EPA's Aerosol Testing Facility
and BSL-2 Microbiology Laboratory in Research Triangle Park, NC, and the BSL-2
Biocontaminant Suite at EPA Andrew W. Breidenbach Environmental Research Center
(AWBERC) in Cincinnati, OH, using the following metrics:
• Viability (CFU/g on tryptic soy agar [TSA]).
• Appearance of spore preparation using phase contrast microscopy.
• Aerosol particle size distribution of the spore preparation.
• CFU/g of spore preparation after heat shock compared to a positive control.
• CFU/g of spore preparation and positive control after exposure to hydrochloric acid
(HCI).
• Colony morphology on TSA of spore preparation compared to positive control.
• Confirmatory quantitative polymerase chain reaction (qPCR) comparison of spore
preparation and positive control.
• DMA sequencing of the qPCR products of the spore preparation and positive control.
The results of these analyses can be found in Appendix B.
2.3. Spore Dissemination
In accordance with the BOTE Project objectives, the surface loading of viable Bg spores prior to
the testing of each decontamination method was intended to be approximately 1.0E6 CFU of Bg
per ft2 in the rooms on the first floor and 1 .OE2 CFU of Bg spores per ft2 in the rooms on the
second floor. The surface loadings were determined by surface sampling (see Section 2.5.4)
after each release of Bg spores into the facility, prior to the application of a decontamination
method.
Dissemination of the Bg spores was performed by FLIR Systems, Inc. (27700 SW Parkway
Ave., Wilsonville, OR 97070; formerly ICx Technologies, Inc., 4343 Pan American Freeway NE,
Albuquerque, NM 87107) prior to each test event. A wet aerosol dispersion of Bg spores was
used for this dissemination. The aerosol generator used for the dissemination was a
microcontroller driven medical nebulizer (Aeroneb Go 7070), manufactured by ICx (now FLIR
Systems, Inc.). The nebulizer was a battery-powered aerosol generator that produces aerosol
by the application of ultrasonic energy to a microporous disc. The generator was controlled by
an on-board microcontroller controlling the output from micrograms (ug) to g/minute (min). The
release of the desired amount of the stock preparation of Bg spores occurred within several
minutes of activation of the aerosol generator.
During the release, ICx (now FLIR Systems, Inc.) Instantaneous Biological Analyzers and
Collectors (IBACs) were used to provide a measure of the particle concentrations in the air.
These measurements provided on-site real-time feedback related to the release that could be
compared to expectations based upon pre-test data. A total of 21 IBACs were utilized
throughout both floors to measure the concentration gradient for each trial. The locations of the
IBACs are shown in Table 2-3, and results are shown in Appendix I. On each floor, two I BAG
sensors were placed in the hallway near the HVAC returns, and the remaining eight sensors
were placed individually within rooms. The I BAG sensors were positioned near the center of the
27
-------
room, placed either on the floor or on a desk/chair. The location of the IBACs was kept
consistent to monitor the particle concentrations during each run. Because there were more
offices than available I BAG sensors, several rooms were not monitored. More information on the
IBACs is provided in Section 2.4.
Table 2-3. Location of IBAC Sensors during Dissemination.
First Floor
Reception Area
Furnace Room
Room 101A
Room 103
Room 104
Room 105
Room 106
Room 107
Room 108
Room 109
Room 110
Second Floor
Hallway Outside Janitor's Closet
Room 201
Room 206
Room 207
Room 208
Room 209
Room 210
Room 211
Room 21 2
Room 21 3
The dissemination procedure was as follows:
1. IBAC sensors were deployed throughout both floors of the building. All sensors were
remotely monitored in real-time by a laptop located in the command-control trailer
utilizing the pre-wired Ethernet connections.
2. A final walk-through of PBF-632 was performed to ensure that no personnel were in the
building. All windows and building entrances were checked to ensure that they were
sealed.
3. Bg spore powder was weighed out in advance and stored in a sample vial. Distilled
water was then added to the Bg powder to provide a liquid suspension for the
nebulizers. This solution was thoroughly mixed using a vortexer. The amount released
on the top floor was 0.5 milligrams (mg) and 200 mg on the bottom floor to achieve the
desired surface loading targets on each floor.
4. The solution was transferred into the nebulizer well using a measuring pipette. The
nebulizer well was capped and then placed into the furnace filter (see Figure 2-9).
5. The IBAC sensors were set up and allowed to collect data for at least 30 minutes prior to
the release to characterize the particulate matter background inside the facility.
28
-------
6.
7.
8.
Nebulizers (ten on the first floor and one on the second floor) were carried into the
building by an FLIR Systems, Inc., employee and set up at the release point on each
floor. The release point was at the return filters of the HVAC system for each floor. The
FLIR Systems, Inc., employee left the building via the PBF-632 egress location and
closed the door. The dissemination was triggered by the FLIR Systems, Inc. employee
by applying power to the nebulizers using a cord located outside the facility. The outer
facility secondary enclosure was sealed and a sign was put up saying "Testing in
Progress - Do Not Enter". No test personnel were allowed to enter the facility at this
time. The HVAC fan on both floors was on and operational during the duration of the
release event. The HVAC fans were turned off two hours following dissemination; this
duration had been determined during pre-testing to achieve the target loading on each
floor. The HVAC fan for both floors was controlled externally using a breadboard that
was located in the command trailer.
I BAG sensor(s) were monitored during dissemination to ensure that the release was
successful.
Particles were given time to settle overnight (12-14 hr) before sampling teams entered
the building the next morning.
Figure 2-9. Nebulizers on the first floor releasing into the air intake on the HVAC system.
2.4. Reference Dissemination and Surface Loading
Determination (Referee Methods)
Reference (or referee) samples were utilized to provide an indication that the dissemination
process was successful at meeting the target surface loading criteria for the first and second
29
-------
floors (see Table 2-1). Real-time particle measurements were taken at 20 locations throughout
the facility during dissemination using the FLIR Systems, Inc., IBACs to provide an indication of
the success of the spore release (as mentioned in Section 2.3).
The I BAG provided near real-time and fully automatic detection of concentrated biological
aerosols. Air and aerosolized particles were pulled into the I BAG at a rate of 3 liters (L)/min by
means of a diaphragm pump. The particles passed through an optical illumination region where
they were excited by a continuous-wave blue laser diode. Elastically scattered light and auto-
fluorescence-produced light were observed simultaneously on independent optical channels. All
acquired data were transmitted through a network cable as well as stored on an internal flash
memory card.
Additionally, surface loadings (CFU/ft2) were indicated using reference material coupons
(RMCs) and TSA settling plates (Remel, Catalog Number R01917, 12 x 85 mm
"monoplates"). The inside diameter of the plate half with the TSA medium was 85 millimeters
(mm) (3.35 in). The RMCs were polished stainless steel rectangles measuring 1 in by 2 in (2.5
centimeters [cm] by 5 cm). The RMCs were sterilized via an autoclave with a 1-hr gravity
autoclave cycle at 121 degrees Celsius (°C), 15 pounds per square inch (psi) and then
packaged in groups of 30 per small plastic box (pipette tip box). The sterilized RMCs and TSA
settling plates were placed in the facility just prior to dissemination.
All three referee methods were used during the three decontamination events. The use of the
IBACs is discussed in the previous section; however, the description of the equipment is
included in this section. The RMCs and TSA settling plates were collected by the Surface
Sampling Teams (see Section 2.5.2) prior to surface sampling at each nearest location within a
room during the pre-decontamination (characterization) sampling in each round.
Each of the reference methods is shown in Table 2-4. The RMCs were used on both the first
and second floors; the TSA settling plates were used only on the second floor. Because the
maximum countable number of CPU on a settling plate was 300, use of settling plates on the
first floor was not considered useful as all plates were projected to be overgrown (too numerous
to count [TNTC]). Multiple RMCs were placed in each of the 18 study rooms prior to
dissemination. TSA settling plates were collocated with RMCs in the study rooms on the second
floor.
After collection, the RMCs and settling plates were sent to the Idaho National Laboratory (INL)
Microbiology Laboratory for analysis. The RMCs were extracted and dilution-plated in
accordance with the procedures described in Section 2.9. Both TSA settling plates and RMC
dilution plates were incubated at 37 °C for 18-24 hr. Following incubation, plates were
enumerated and correct morphology was confirmed via visual inspection. Surface loadings were
indicated by dividing the CFU/plate by the surface area of the collection media (i.e., 0.014 ft2 for
the RMCs and 0.061 ft2 for the TSA settling plates).
30
-------
Table 2-4. Referee Samples for the BOTE Project.
Referee Device
IBAC Sensor
Settling Plates
Stainless Steel
RMC
Description
Particle counter
that counts
particles in the air
Culture plate with
ISA (85 mm [3.35
in] diameter)
Polished stainless
steel squares
measuring 1 in by
2 in (2.5 by 5 cm)
Application
Used to provide real-time feedback
during dissemination in Rounds 1 -
3; the information was used to
corroborate previous air
concentrations during pre-test
dissemination trials.
Indicated target pre-decontamination
surface loadings (CFU/ft2) for each
round
Indicated target pre-decontamination
surface loadings (CFU/ft2)for each
round
Analysis
Real-time Particle
Count
Quantitative Analysis
(Incubation and
enumeration)
Quantitative Analysis
(Extraction, culture
plating, incubation,
and enumeration)
2.5. Sampling Methods and Equipment
The sampling of surfaces, air, sand and water for viable Bg spores was a critical component of
the measurement methods encompassing all objectives. To achieve all objectives, a number of
different sampling and subsequent analytical methods (see Section 2.9) were required. Table
2-5 lists the different sampling methods that were used, the locations where they were used, the
analysis location, and the relationship to the BOTE Project objectives. Several different
laboratories or Agencies were involved in analyzing the samples or raw data. For analysis of
surface samples, the Laboratory Response Network (LRN), INL, and LLNL were used. More
details on the laboratory analysis can be found in Section 2.9.1.
Sampling was conducted at multiple times within each round of testing listed in Table 2-1. For
Rounds 1-3, surface sampling was conducted after dissemination (pre-decontamination or
characterization sampling event) and after decontamination (post-decontamination or clearance
sampling event). Air sampling was conducted during dissemination for real-time feedback on the
release/contamination event for each round, during the aggressive air sampling event for each
round (after post-decontamination surface sampling) and at specific times during each round
specifically in the reaerosolization study rooms (Rooms 101A and 102). Wastewater sampling
from the water collected from the Decontamination Line was also done within each round. A
timeline of sampling events within each round is shown in Figure 2-10. The subsections below
describe all sampling methods in detail. All sampling was conducted in accordance with the
BOTE Project Test Plan/QAPP[59].
31
-------
Table 2-5. Summary of sampling methods and the uses related to the BOTE Project
objectives.
Media
Sampled
Surface
s_
<
•a
CO
W
s_
Ł
1
Sampling Method
Cellulose Sponge-stick
Wipes
Macrofoam Swabs
Vacuum Socks
Versalon Wipes®
Versalon Wipes®
SKC BioSamplers®
UV-APS
DycorXMX/2L-MIL
Aerosol Collection System
Mattson-Garvin Model 220
slit-to-agar
Sand-filled Petri plates
EPA Water Pathogen
Concentrator
Locations
All Rooms on
Floor 1 and 2
All Rooms on
Floor 1
Rooms 101 A
and 102
Rooms 101 A
and 102
Rooms 105 and
106;
Hallway outside
Rooms 105 and
106
Around the
outside of the
facility, within the
outer enclosure;
1st floor
reception; 2nd
floor hallway
Wash water
collected at the
Decontamination
Line
Analysis
Location
LRN
LLNL
INL
INL
INL
INL
INL
EPA and
uses
INL
Data Use
Pre-decontamination and post-decontamination
sampling to determine surface loading (CFU/ft2)
on each floor; used for assessment of
effectiveness of the decontamination method in
each test round and for residual contamination
amount after decontamination in the exposure
assessment
Pre-decontamination and post-decontamination
sampling for the assessment of RV-PCR
Pre-decontamination and post-decontamination
sampling as surface loading measurements for
the assessment of reaerosolization
Pre-decontamination and post-decontamination
time-integrated air measurements (CFU/ft3) for the
assessment of reaerosolization
Pre-decontamination and post-decontamination
real-time air measurements (particles/ft3) for the
assessment of reaerosolization
Post-decontamination, after surface sampling,
time-integrated air measurements (CFU/ft3) for the
assessment of AAS
Post-decontamination, after surface sampling,
time-integrated air measurements (CFU/ft3) for the
assessment of AAS
Assessment of the potential migration of viable Bg
outside the facility and assessment of Bg
detection methods in sand (soil)
Assessment of the effectiveness of chlorine to
inactivate spores in wash water from the
decontamination process (i.e., Decontamination
Line)
32
-------
Facility set-
up or reset
Reaerosolization airsampling
P re-
decontamination
surface sampling
Reaerosolization airsampling
Post-
decontamination
surface sampling
Dissemination
_Sand sample
collection
Decontamination
_Sand sample
collection
AAS
_Sand sample
collection
Figure 2-10. Timeline of sampling events within each round.
2.5.1. General Sampling Schedule
As discussed above, sampling (surface, air, sand, and water) was conducted at pre-specified
times within each round. The actual dates of sampling events are shown in Table 2-6.
Table 2-6. Sampling event dates.
Sampling
Event
Background
Pre-
decontamination
Post-
decontamination
Airsampling for
reaerosolization
assessment
AAS
Sand
Water
Test
Round
MFP
1
1
1
1
1
1
Dates
April 14-1 5, 2011
April 17-1 8, 2011
April 22-23, 201 1
April 16-1 8, 2011
and April 22-23,
2011
April 23, 2011
April 16,18 and
23,2011
April 16-1 8, 2011
and April 22-23,
2011
Test
Round
N/A
2
2
2
2
2
2
Dates
N/A
April 27-28, 2011
May 4-5, 201 1
April 26-28, 201 1
and
May 4-5, 201 1
May 6, 2011
April 25 and 26,
2011 and
May 6, 2011
April 26-28, 201 1
and
May 4-5, 2011
Test
Round
N/A
3
3
3
3
3
3
Dates
N/A
May 11-12, 2011
May 16-17, 2011
May 10-1 2, 2011
and
May 16-17, 2011
May 17, 2011
May 10, 12, and
17,2011
May 11-12, 2011
and
May 16-17, 2011
N/A = not applicable
33
-------
2.5.2. Sample Collection Teams and PPE
The sample collection effort was conducted by members of the National Guard Bureau Civil
Support Teams (CSTs), U.S. Coast Guard (USCG) Pacific Strike Team, U.S. Marine Corps
Chemical Biological Incident Response Force, INL, DTRA and EPA personnel. All sampling
personnel received classroom training regarding sampling protocols and also received hands-on
training on using the BROOM system.
Samplers wore level C PPE for sample collection which included Tyvek® suits, full-face
respirators (air purifying or powered air purifying), booties, and nitrile gloves. The seams,
including the Tyvek® hoods to the respirators, were taped to prevent exposure or cross-
contamination. The only exception was that the sand sampling team was not required to wear a
respirator for sample collection outside the facility (within the secondary enclosure) following the
facility decontamination in each round. Each sampler wore a base pair of gloves. Samplers then
donned several pairs of nitrile gloves over a base pair of gloves. One pair of gloves was
removed before each sample was taken. Samplers donned additional gloves when they were
down to the base pair. All personnel entering the test structure donned two pair of booties; one
was removed prior to entering the building, and the other was removed before exiting the facility
at the entrance to the Decontamination Line.
Additional information regarding sample team make-up and procedures is included with each
sampling method section. The use of the above mentioned PPE was ito simulate sampling
during an actual biological incident, although a benign surrogate was used in the BOTE Project.
2.5.3. Summary of Sample Collection Methods used during the BOTE Project
As discussed in Section 2.4 and summarized in Table 2-5, a number of different sample
collection methods were used to achieve the BOTE Project objectives. Surface samples were
used primarily to assess the contamination pre- and post-decontamination; the data were
utilized primarily to assess the effectiveness of each decontamination method. Air monitoring
data were utilized to provide feedback on the dissemination method (i.e., was it expected to
achieve the target surface loading levels?), to assess the potential for reaerosolization, and
during AAS. The sand samples were used to assess whether viable Bg spores might be
detected as escaping the facility (but within the secondary enclosure) during the project
activities. The water samples were utilized to assess the measurement methods for the potential
detection of viable Bg spores in wash water from the decontamination process (i.e.,
decontamination line and Round 2 decontamination process). In addition, referee sampling
methods were used to provide an indication of surface and air Bg concentration at specific times
during each round of testing (i.e., during and immediately after dissemination).
Detailed descriptions of each sampling method are included in the following sections.
2.5.4. Surface Sampling Methods
Surface sampling was the primary method used to collect samples to determine surface
concentrations or loading (CFU/ft2) of Bg spores. The purpose of surface sampling was to
characterize the extent of contamination (post-dissemination, also referred to as pre-
decontamination) following the dissemination of a surrogate and after the application of a
decontamination process (post-decontamination) to determine the effectiveness of the
34
-------
decontamination for the three technologies used in each study area. Hence, surface sampling
was done both pre- and post-decontamination within each round (see Figure 2-10) as well as
during the MFP. As summarized in Table 2-5, sponge-stick wipes, swabs and vacuum socks
were the primary collection methods used to evaluate the three decontamination technologies.
These types of samples were utilized because they have typically been used to characterize Ba
(or simulant) presence within facilities such as PBF-632'13'14]. All study rooms and accessory
rooms (i.e., those not having furnishings) were sampled before and after application of each
decontamination approach. In addition, the HVAC system was sampled.
Versalon® wipes were also collected throughout the facility both pre- and post- decontamination
to evaluate EPA's RV-PCR analytical method. Additional Versalon® wipes were also collected in
Rooms 101A and 102 to determine surface contamination concentrations pre- and post-
decontamination for the reaerosolization study.
Surface samplers were grouped into three person teams: the support/documentation person
(Person A), the Supplier (Person B), and the Collector (Person C) (see Appendix C for more
information on Sampler roles). Sampling carts were provided to each team and included: a
sample kit box (including swabs, sponge-sticks, wipes, vacuum socks, and templates, as
appropriate) for each room, a sample map detailing locations of each sample to be taken, bags
of gloves for each member of the sampling team, markers, flashlight, vacuum, garbage bags,
and separate bags into which to place collected samples. In addition to pushing/maneuvering
the cart, Person A carried a hand-held radio and the BROOM personal digital assistant (PDA)
that was used to scan samples, track the location of the sample, and record any observations of
samples taken.
The sample collection procedures for each surface sample method were based upon validated
CDC sampling methods (i.e., sponge-stick wipes and swabs)'62'631 or recommended procedures
(i.e., vacuum socks and wipes)'64'651 for Ba. The sampling method protocols are found in
Appendix C. Samplers utilized a paper template to standardize the collection area (size) for
each sample and sample type. Each template was used for one sample only and then
discarded. During pre-decontamination sampling, sampling teams used markers to outline the
perimeter of the sampling area after a sample had been collected. Templates for post-
decontamination samples were laid adjacent to the pre-decontamination sample (trying not to
overlap, if possible). Samplers used sampling maps to identify which sampling method should
be used on which surfaces and the order in which the samples were to be taken. Samplers took
care not to step over or disturb areas that had not yet been sampled.
Detailed descriptions of each sampling method are provided in the following subsections. All
sampling teams underwent on-site training, including proficiency testing, to promote use of the
prescribed techniques.
2.5.4.1. Cellulose Sponge-stick Wipes
Cellulose sponge-stick wipes were used to sample hard nonporous surfaces such as desk tops,
hard floors, tables, and nonupholstered chairs. An area of 10 in x 10 in was sampled with the
sponge stick, using a disposable cardboard template. Samplers collected in the following
35
-------
pattern: (1) using the flat side of the sponge stick, the surface was sampled using horizontal S-
strokes, covering the entire template area; (2) the sponge stick was then flipped over to the
opposite side to sample the surface in a vertical pattern, covering the entire template area; (3)
using a narrow edge of the sponge stick, the surface was sampled using the same S-strokes but
applied diagonally across the template; and (4) the tip of the sponge stick was then used to
sample the perimeter of the sampling area. The sponge-stick wipes come pre-moistened.
2.5.4.2. Macrofoam Swabs
Pre-moistened macrofoam swab kits were used on nonporous hard-to-reach surfaces including
air vents, computers, and pictures. Swabs were taken using a 2 in x 2 in disposable cardboard
template. Samplers utilized the same S-stroke horizontal, vertical, and diagonal sampling
procedure rotating the surface of the swab 90° with each step.
2.5.4.3. Vacuum Socks
Vacuum socks were used to collect samples on porous surfaces including carpet, unfinished
wood, concrete, cinderblock, cloth furniture, file cabinet drawers, book-laden bookshelves, and
ventilation filters. Vacuum sampling used a 2 ft x 2 ft disposable cardboard template and a fresh
disposable sock/nozzle attachment to collect the sample. Holding the nozzle at a 45° angle and
placed onto the sample area, samples were taken using the same S-strokes in the horizontal
and vertical direction.
2.5.4.4. Versalon® Wipes
As summarized in Table 2-5, Versalon® wipe samples were utilized for both the reaerosolization
study and assessment of RV-PCR. The pre-moistened wipes were used to sample hard
nonporous surfaces including, but not limited to, desktops, filing cabinets, hard floors, and the
UV-APS units (plastic). Wipes also used the 10 in x 10 in disposable cardboard template. The
wipe was folded into quarters, and, using two fingers, the sampler used the same four-step
sampling pattern of horizontal S-strokes, vertical S-strokes, and diagonal S-strokes of the
sample area. After each step, the wipe was folded inward to expose a clean sample collection
surface.
2.5.5. Air Sampling Methods
During the BOTE Project Phase 1, sampling teams conducted air sampling during the different
events within each round. Air samples utilizing SKC BioSamplers® and UV-APS measurements
were taken specifically in Rooms 101A and 102 related to the reaerosolization study. Air
samples using the Dycor XMX/2L-MIL Aerosol Collection System and Mattson-Garvin Model
220 slit-to-agar sampler were taken during the AAS assessment after post-decontamination
surface sampling within each round.
A description of these air sampling instruments and methods is found in the following
subsections.
2.5.5.1. SKC BioSamplers®
The SKC BioSampler® (SKC Inc., Eighty Four, PA) is a bioaerosol and airborne particle
collection device that traps airborne microorganisms in swirling liquid for subsequent analysis.
The SKC BioSampler® is made of glass and consists of three parts: inlet, nozzle section (with
36
-------
three tangential sonic nozzles), and collection vessel. The three nozzles of the SKC
BioSamplers® are designed to work as sonic orifices. Each orifice allows approximately 4.2
L/min of air to pass through when the pump establishes a downstream pressure of 15-30
pound-force per square inch gauge (psig). SKC BioSamplers® achieve a flow rate of
approximately 12-14 L/min during active sampling. During the study, a DryCal flowmeter
(DEFENDER 510, SKC Inc., Eighty Four, PA) was utilized to confirm flow, and values were
recorded.
The SKC BioSamplers® were remotely controlled and powered via the Bio Sampler Test System
(BioSeq-12) (developed by Honeywell Technology Solutions Inc., Morris Township, NJ) as
diagrammed in Figure 2-11. The BioSeq-12 is a protective housing case and vacuum manifold
system that contains multiple redundant vacuum pumps with inlet check valves. If one pump
failed, a check valve closed on the failed pump allowing the second pump to continue to pull a
vacuum on the system through metered flow restrictors and remotely controlled flow meters to
record and report air flow (used for data reduction) for the active samplers.
The vacuum pumps attached to each BioSeq-12 system were operated within sealed
ruggedized cases (Pelican Cases, Pelican Products, Tempe, AZ) to prevent biological
contamination and to guard against any potential corrosion caused by the decontamination
methods. These pumps were located exterior to Rooms 101A and 102 and were connected to
the instruments with inert nonconductive polyethylene tubing run through the walls. In addition,
the sealed cases were ventilated to allow for air intake as well as cooling of the pumps inside
the cases. The air drawn in from the ventilation system was passed through a High Efficiency
Particulate Air (HEPA) filter to prevent contamination in the system from outside the building.
All parts of the SKC BioSamplers® were sterilized via autoclave prior to use. All parts of the SKC
BioSamplers® were maintained as a set because each inlet, nozzle, and collection vessel is not
interchangeable with other SKC BioSamplers®. Each SKC BioSampler® part had an etched part
number to assist in maintaining organized SKC BioSamplers®.
Prior to placement for sampling, 15 milliliters (ml_) of sterilized Phosphate Buffer Solution plus
0.05% Tween® 20 (PBST), pH 7.4, was aseptically pipetted into the SKC BioSampler® collection
vessel. The SKC BioSampler® collection vessel was then assembled with a clean sterilized inlet
and nozzle in an aseptic area (in the off-site microbiology lab). The complete SKC BioSampler®
was then placed in a bag and wrapped in foam for transport. These SKC BioSamplers® were
then placed in the Pelican Cases and taken to the testing facility. Care was used to ensure that
the SKC BioSamplers® maintained an upright position at all times to avoid leakage or loss of the
sampling media during transport.
37
-------
Sampler;
BioSeq-12 Unit
External Pump
Figure 2-11. Instrumentation setup and remote design using the BioSeq-12.
All SKC BioSamplers® were labeled and tracked using bar codes attached to the actual
samplers and each sample location. This information was recorded and scanned by field
personnel using the BROOM PDA and iPad, both during SKC BioSampler® setup and during
sampler removal. The information recorded on the BROOM PDA and iPad was then uploaded
or synced to a database to maintain records of each SKC BioSampler® (sampling time and
location). More information on the use of the iPad can be found in Appendix M.
2.5.5.2. Model 3314 UV-APS™ Spectrometer
The Model 3314 UV-APS™ spectrometer (TSI Inc., Shoreview, MN) measures the aerodynamic
diameter, scattered light intensity, and fluorescence intensity of individual airborne particles in
real time. Samples are collected by drawing sheath (clean) and particle-laden (sample) air
through the UV-APS with respective flow rates of 4.0 L/min and 1.0 L/min. The aerodynamic
diameter based on time-of-flight principles and particle counts based on light scattering can be
found from the UV-APS laser measurements. The UV-APS also measures the fluorescence
properties of individual particles, thus allowing for the distinction of biological particles from
nonbiological particles. Particle fluorescence is excited by a pulsed ultraviolet laser and is
collected real-time using a photomultiplier tube. Because these instruments measure particle
number (particle number (N)/cm3), knowledge of particle density is required to convert to mass
concentration (mg/m3).
The UV-APS was initially factory-calibrated to identify sizing characteristics for each size bin
processed by the UV-APS. Additionally, a calibration unit was used to perform field-level day-of-
38
-------
test calibrations. The mobile calibration system consisted of a Model 3014B Filtered Air Supply
(FAS) (TSI Inc., Shoreview, MN), which filters, dries and regulates the air pressure provided to
the rest of the system setup. The FAS was connected to a Model 3076 Constant Output
Atomizer (COA) (TSI Inc., Shoreview, MN) that generates aerosols with particles of known size
from a liquid suspension. The output of the COA is evacuated through a Model 3062 Diffusion
Dryer (DD) (TSI Inc., Shoreview, MN), which dries the output using silica gel with minimal
aerosol loss. Pressurized air is forced through the FAS at 60 psig and stepped down to 32 psig
for routing to the COA. A suspension of nonfluorescent polystyrene latex beads at 1.034 urn
was used for primary calibration in the COA (at a concentration of 0.5 mL/L). The output from
the COA was then dried in the DD and sampled using the UV-APS system.
While no adjustments to the factory calibration were made during the calibration process on site,
the factory calibration was verified at the 1.0 urn range and at the 0.5 urn range using a
separate 0.5 urn bead suspension.
2.5.5.3. Dycor XMX/2L-MIL Aerosol Collection System
The Dycor XMX/2L-MIL Aerosol Collection System (Dycor Technologies Ltd., Edmonton,
Alberta, Canada) sampler is an aerosol separator and high mass flow concentrator system
designed for sampling spores under harsh field conditions (see Figure 2-12). The Dycor
XMX/2L-MIL Aerosol Collection System collects high volumes of air, strips away the large dust
particles and very small micro-debris and concentrates the particles of interest (respirable range
of 1 to 10 urn diameter) through a two-stage virtual impactor. The Aerosol Collection System
samples the air at a rate of 530 L/min, and the particles are collected onto a dry 37 mm three-
piece cassette filter with a 0.8 |o,m pore mixed cellulose ester filter, which is then removed and
soaked in sterile PBST solution for further analysis. According to the manufacturer, the Aerosol
Collection System has demonstrated collection efficiencies between 55 and 88% for 1.9 to 5 urn
particles in wind speeds of 2 kilometers (km)/hr and 54 to 84% in wind speeds of 8 km /hr.
Sample collection can be triggered remotely by a switch or by a biodetector such as the Dycor
Technologies Ltd. C-FLAPS (Dycor Technologies Ltd., Edmonton, Alberta, Canada). The
collection system is a portable device that weighs 17 kilograms (kg) (37.5 Ib) and measures 58
cm x 46 cm x 33 cm (22.8 in x 18.1 in x 12.9 in).
Calibration of the flow rate was performed before and after sampling using two types of
calibrated mass flow meters with capacities to measure from 0 to 1,000 liters per minute (Lpm).
After collection, each filter was left in the sample cassette filter holder and sent to the
appropriate laboratory for analysis.
39
-------
Figure 2-12. Dycor XMX/2L-MIL Aerosol Collection System.
2.5.5.4. Mattson-Garvin Model 220 Slit-to-Agar Sampler
The Mattson-Garvin Model 220 slit-to-agar (Barramundi Corp., Homosassa Springs, FL)
sampler is a device that utilizes a rotating stage that holds a Petri plate. The air impacts the
surface of the agar with whatever bioaerosols are present and the organisms impinge directly
onto the nutrient agar. The plate is then incubated, and the organisms are allowed to grow. This
process eliminates the need for extraction of spores from a sampling medium. The slit-to-agar
sampler draws air through a 0.152 mm slit at 28.3 L/min (1ft3/min) and impinges the particles
upon an agar surface 2 to 3 mm below the slit. The distance from the calibrated slit to the agar
surface is critical for proper impingement. The cut point particle size was 0.53 urn. The agar is
contained in a standard commercially available 150 mm disposable Petri plate that is rotated by
a synchronous drive motor. The rate of rotation can be varied by the interchangeable drive
motors. For the BOTE Project (and in situations where low or no target organisms are expected,
as is the case with post-decontamination sampling), the 60-min drive motor was utilized,
resulting in one revolution of the plate in 60 min. After sampling, the plate was incubated and
the colonies counted. This count reflects the number of target organisms collected from the
sampled air. No dilution or plating steps were required. Results were expressed as viable
particles or CFU per unit of air, and a time-concentration relationship was determined.
40
-------
Figure 2-13. Mattson-Garvin Model 220 Slit-to-Agar Air Sampler.
2.6. Test Design and Sampling Methodology
To accomplish the objectives defined in the BOTE Project, utilizing the sampling methods
described in Section 2.5 required different yet nonexclusive sampling and test designs related to
each objective. For each objective, sampling strategies were developed to determine the types,
numbers, and locations of the samples. The total numbers of samples of each type for each
round are presented in Table 2-7. The numbers of quality control (QC) samples (i.e., blanks) are
reported in parentheses. These numbers are in addition to the sample numbers listed for each
sampling event (e.g., MFP, Round 1 Pre- or Post-decontamination). When N/A is reported, the
sampling method was not used in the specified sampling event. The laboratory in parentheses
next to the sampling method indicates the location where the samples were analyzed (e.g., INL,
LRN). The sampling and testing designs or strategies related to each major objective/sub-
objective guiding the development of the matrix shown in Table 2-7 are described in the
following subsections.
41
-------
Table 2-7. Type and number of samples collected during each BOTE Project round.
Sampling Method
(Laboratory)
RMCs (INL)
Settling Plates (INL)
Sponge-stick wipes
(LRN)
Vacuum socks (LRN)
Swabs (LRN)
Versalon® wipes (INL)
Versalon® wipes (LLNL
and EPA [Ft. Meade])
SKC BioSampler® (INL)
XMX (INL)
STA (INL)
Sand (EPA and USGS*)
Wash water grab sample
(INL)
Wash water ultrafiltration
sample (INL)
Floor
1
2
2
1
2
1
2
1
2
1
1
2
1
1
1
Secondary
Containment
1
2
NA
NA
MFP
NA
NA
NA
14
(3)
12
(2)
14
(3)
13
(2)
5
(1)
5
(2)
5
(1)
8
(2)
7
(2)
NA
NA
NA
NA
NA
NA
NA
NA
Round 1
Pre
30
24
24
95
(11)
77
(8)
49
(10)
47
(7)
13
(8)
10
(8)
20
(2)
10
(8)
NA
72
(24)
NA
NA
40
(2)
2
2
4
4
Post
NA
NA
NA
93
(10)
79
(7)
48
(10)
46
(8)
11
(5)
9
(5)
20
(2)
59
(8)
NA
18
(6)
9
(3)
9
(3)
20
2
2
NA
NA
Round 2
Pre
30
23
24
109
(10)
95
(8)
45
(7)
18
(5)
10
(5)
9
(6)
20
(2)
21
(8)
NA
72
(24)
NA
NA
40
(2)
2
2
8
8
Post
NA
NA
NA
130
(10)
105
(8)
8
(3)
4
(3)
1
(1)
0
20
(2)
38
(8)
NA
18
(6)
9
(3)
9
(3)
20
(1)
2
2
4
4
Round 3
Pre
30
24
(3)
24
89
(16)
77
(8)
44
(10)
42
(8)
11
(5)
10
(5)
20
(2)
10
(6)
NA
72
(24)
NA
NA
40
(3)
2
2
4
4
Post
NA
NA
NA
85
(11)
77
(8)
42
(10)
40
(8)
11
(5)
10
(5)
20
(2)
60
(8)
NA
18
(6)
9
(3)
9
(3)
20
(1)
2
2
NA
NA
Total
90
71
(3)
72
615
(71)
522
(49)
250
(53)
210
(41)
62
(30)
53
(31)
125
(13)
206
(48)
7
(2)
270
(90)
27
(9)
27
(9)
180
(9)
12
12
20
20
*U.S. Geological Survey
42
-------
2.6.1. Decontamination Efficacy Assessment
A primary objective of the BOTE Project was to assess the effectiveness of three different
decontamination methods for a facility as a function of controlled parameters (e.g., bacterial
spore surface loading [i.e., degree of contamination], facility type [e.g., residential or
commercial]). Surface sampling results from pre- and post-decontamination were compared to
assess the effectiveness of the decontamination method that was applied within a test round. A
statistically-based sampling plan was designed for this assessment. This design focused on a
determination of the number of samples required to achieve the desired confidence in the ability
to discern differences in efficacy. This statistical analysis is included as Appendix J.
Three decontamination methods were chosen based on results that had been obtained in
laboratory studies. An interagency group examined the best available science for
decontaminating a facility that has been contaminated with a Bacillus species and selected
three separate approaches:
• fumigation with H2O2 using STERIS vaporized hydrogen peroxide (VHP®);
• surface decontamination using pH-adjusted bleach (amended bleach ); and
• fumigation with CIO2.
The BOTE Project provided an opportunity for operational testing of the performance of these
three decontamination approaches. A general overview of each decontamination method is
included in Section 2.10.The decontamination methods and the results are discussed in detail in
Sections 4.1.1, 4.1.2, and 4.1.3 for Rounds 1 through 3, respectively.
2.6.2. Wash Water Collection and Treatment
For the BOTE Project, water from the washdown of personnel exiting the contaminated building
and going through the decontamination line was collected for each sampling event in a 55-
gallon (gal) drum and chlorinated to test the effectiveness of chlorine treatment of wash water
under field conditions. The wash water was analyzed for pH, Total Suspended Solids, Free
Chlorine, Turbidity, and Chemical Oxygen Demand and the presence of Bg spores before and
after chlorination. Sampling and analysis were conducted by INL on site or in the INL
Laboratory. Because relatively few spores were expected to be present in the collected PPE
washdown water, a field-portable ultrafiltration water concentrator device developed by EPA and
INL[661 was used to concentrate Bg spores into 450 ml_ wash water samples for analysis. Grab
samples of wash water from the barrels were also collected and analyzed.
The BOTE Project Decontamination Line consisted of three chambers including an entry area
connected to the building, a personnel washdown area, and an exit area leading to the outside.
As they exited the building and before entering the Decontamination Line, sampling personnel
doffed outer gloves and booties and deposited them in a waste container. Sampling personnel
then turned over contaminated equipment and waste materials to Decontamination Line
personnel who disinfected them with bleach wipes (Dispatch® Hospital Cleaner Disinfectant
Towels with Bleach, The Clorox® Company, Oakland, CA). Sampling personnel then entered
the PPE washdown area, stepping into a pool where they were sprayed down with tap water
through a hose connected to a building faucet. Decontamination Line personnel scrubbed the
43
-------
feet of the individuals using a scrub brush dipped in a bucket of tap water containing Spartan
Blue-Glo detergent (Spartan Chemical Company , Inc., Maumee, OH) before they exited the
pool and entered the exit area. Periodically, collected water was transferred to a 55-gal drum
using a submersible pump. Sampling personnel doffed PPE and disinfected masks in the exit
area prior to exiting. Water was generally collected over two days of sampling activity to obtain
the amount needed for the subsequent inactivation studies. Collected wash water remaining
after each sampling event was turned over to INL for disposal.
Decontamination Line PPE wash water was collected, sampled, and treated at five different
points during the BOTE study as follows:
• Following dissemination in Round 1;
• Following dissemination in Round 2;
• During pH-adjusted bleach decontamination;
• During post pH-adjusted bleach decontamination sampling; and
• Following dissemination in Round 3.
For each sampling event, PPE decontamination wash water was pumped from the collection
pool into a 208 L (55 gal) metal drum with a plastic disposable liner insert. Each drum liner was
marked on the inside using an indelible ink marker to indicate water level when 146 L (38.5 gal)
had been added (calculated based on dimensions of the actual drum used). Each drum was
filled to the 146 L mark. If less than 146 L was collected during the sampling event,
nonchlorinated tap water was added to fill the drum to the 146 L mark and the amount of water
added was noted. One drum containing 146 L of water was collected for each sampling event,
and sampling and analysis were conducted when this volume was achieved.
To ensure adequate mixing of chlorine and additives, an electric barrel mixer was attached to
the side of the drum and run periodically while the sampling and inactivation procedures were
being conducted. Because the wash water contained surfactant from the foot washing
procedure, 5-7 ml_ of Antifoam A (Y-30, Sigma A5278, Sigma-Aldrich Corp., St. Louis, MO) was
added and the wash water in the drum was mixed for a period of two minutes at the beginning
of the sampling procedure to minimize foaming that could impair operation of the ultrafiltration
concentrator device. Immediately after mixing, the temperature of the water in the drum was
measured using a digital thermometer (Traceable® 15-077-9E, Control Company, Friendswood,
TX) and noted.
Three 600 mL and one 1 L grab samples were collected from the drum. For each 600 mL
sample collected, aliquots were distributed into appropriate vials for the individual analyses. The
1 L sample was used for total suspended solids analysis. The following water quality
parameters were measured following the instructions in the manufacturers' operation manuals
compiled by INL and described in the BOTE QAPP[59]:
• pH level (YSI Professional Plus pH Meter, Serial number #11B 101328, YSI Inc.,
Yellow Springs, OH).
Total Suspended Solids (Orbeco-Hellige MC500 Colorimeter, Orbeco-Hellige
Inc., Sarasota, FL).
44
-------
Free Chlorine level, analyze immediately (DPD Method using Orbeco-Hellige
MC500 Colorimeter).
Turbidity level (Orbeco-Hellige TB200 Turbidimeter, Orbeco-Hellige Inc.,
Sarasota, FL).
Chemical Oxygen Demand (Acid-dichromate Method using Orbeco-Hellige
MC500 Colorimeter).
Bg enumeration following the LRN "B. anthracis Spore Environmental Wipe
Processing Procedure" included in the BOTE Project QAPP[591. (Note: sample
volumes for the membrane filter were 10 mL done in triplicate for wash water
samples). Samples were not heat-treated for any of the five sampling events.
Upon completion of grab sample collection, the barrel mixer was switched on and the
ultrafiltration concentrator device operated following procedures described in the BOTE Project
QAPP[59] to process 72 L (19 gal) of the wash water contained in the collection drum. The
concentrator device uses the principles of tangential flow filtration to concentrate microbes and
other particles present in a large volume of water to a smaller volume sample. The
concentration step was added because the Bg spore concentration of the grab samples was
expected to be below the detection limit of the microbiological analytical method for sampling
events that did not include the spiking of spores to the collected wash water prior to sampling.
Concentrating samples was expected to increase the likelihood of capturing an adequate
number of spores needed to measure the effectiveness of chlorine inactivation. In this study,
concentrated samples had a volume of 450 mL. Bg enumeration of the concentrated sample
was performed by INL.
Following this initial pre-chlorination concentration step, the remaining water in the drum
(approximately 72 L) was treated by chlorinating using bleach. Before adding the bleach
solution, the mixer was switched on. The volume of bottled bleach (Clorox® bleach, 6% sodium
hypochlorite) needed to achieve a 1/20 dilution was calculated to be 3.8 L (1 gal) of bleach to 72
L of water. Bleach was added to the drum and a laboratory timer set to 15 min was started. After
one minute of mixing following the addition of chlorine, the water temperature was measured,
and a grab sample was collected and analyzed for pH and free chlorine concentration. The
volume of sodium thiosulfate needed to quench the chlorine residual was calculated to be 977 g
anhydrous or 1,533 g hydrated form added to 72 L (the amounts were based on the assumption
that 2 moles of sodium thiosulfate quenches 1 mole of sodium hypochlorite). At 15 min, sodium
thiosulfate was added to the drum, and the free chlorine level was measured. If the free chlorine
level was still above the detection limit, more sodium thiosulfate was added, and the free
chlorine level was measured again. Samples were collected from the drum and water quality
parameters were measured following the procedures described above. Samples were analyzed
for Bg.
The remaining quenched wash water in the drum was concentrated using the ultrafiltration
concentrator device to obtain a 450 mL sample and analyzed for Bg.
Log reduction (LR) was calculated as follows:
45
-------
LR = Iog10 (No/NT)
Equation 2-1
where N0 is the starting number of cells, and NT is the number of cells at time T.
2.6.3. Assessment of the RV-PCR Method
Versalon® wipe samples were collected and analyzed for the presence of Bg spores before and
after decontamination with fumigants and surface disinfectants. A selected number of Versalon®
wipe samples from both pre- and post-decontamination sampling were analyzed by the RV-PCR
method at LLNL. The accuracy of the RV-PCR method was evaluated by comparison with
traditional culture-based methods. Accordingly, the sample extract following spore removal was
split into two equal parts, with one part used for RV-PCR analysis and the other part used for
culture analysis after appropriate concentration. Additionally, as a practice run, the scientists
from Microbiology Laboratory Branch (MLB) of the EPA Office of Pesticide Programs (OPP) at
Ft. Meade, MD, also processed and analyzed a subset of the samples following the same
protocol.
2.6.4. Aggressive Air Sampling Assessment
The objective of the use of AAS in the BOTE Project was to assess the results post-
decontamination compared to surface sampling as a function of air sampler type. In general, the
AAS process consists of isolating a location, applying air pressure to a surface with a leaf
blower to attempt to reaerosolize any spores on the surface and collect airborne spores via high
volume air sampling. This sampling methodology has been used for biothreat agent air sampling
by drawing a known volume of air through a dry filter and/or impacting particles directly upon
agar. The filters and/or agar plates are then sent to a laboratory for analysis. These sampling
and analysis procedures, which may be varied or changed as required depending on site
conditions, equipment limitations or limitations imposed by the procedure are standard (i.e.,
typically applicable) methods. The AAS process employed in the BOTE Project is described in
detail in the following subsections.
2.6.4.1. Aggressive Air Sampling Methodology
An AAS protocol was utilized that incorporated two air sampling collection methods, including
Dycor® XMX/2L-MIL Aerosol Collection Systems (XMX) and Mattson-Garvin Model 220 slit-to-
agar (STA) air samplers, to provide a secondary evaluation of decontamination effectiveness.
The sample collection procedures for each of these methods were based upon accepted
protocols from the USEPA's Asbestos Hazard Emergency Response Act requirements, good
industrial hygiene practices, and past methods recommended by Technical Working Groups
during the 2001 Anthrax attacks. AAS was conducted by applying the applicable methods
related to USEPA's Asbestos Hazard Emergency Response Act requirements.'381
Aggressive air sampling was conducted in two first-floor rooms during Rounds 1, 2, and 3, after
the post-decontamination surface sampling was completed. Prior to the beginning of air
sampling, forced air equipment (i.e., one horsepower mechanical leaf blowers) was used to
direct a jet of air toward all surfaces in a room for a period of 20 min to dislodge and re-
aerosolize any remaining contamination. In addition, two 16 in oscillating floor fans were
46
-------
positioned in the room to increase air circulation and maintain mixing of air in the room.
Subsequently, three high volume air samplers (two XMXs and one STA) collected air samples
to measure airborne Bg in the room. In addition, one XMX and one STA sampler collected air
samples in adjoining areas outside the rooms to determine if there was any migration of
contamination outside the rooms. INL analyzed all AAS air samples. A photograph of an
example room is shown in Figure 2-14; visible are the two oscillating fans, STA (on the stove),
one of the two XMX units (middle of floor) and sampling personnel using the leaf blower.
Previous large-scale facility fumigations required that AAS be done throughout the entire facility,
even in areas where no Ba contamination was found during the facility contamination
characterization process. The rationale for this approach is that spores, once made airborne by
activities within a facility, have the potential to move anywhere within the facility via existing air
currents. It is therefore important to ensure that post-contamination AAS is performed
everywhere in the facility, even in areas where contamination has not previously been identified.
However, for the BOTE Project, the AAS was not conducted as a mandatory clearance method
or primary measurement for decontamination efficacy assessment. The AAS was therefore
conducted only in two designated rooms. In general, to ensure that AAS data are characteristic
of all usage airspace throughout a facility, all rooms and hallways would be evaluated.
Detailed description of sampling equipment and sample collection procedures for each method
can be found in Appendix D, and detailed sample analysis materials and procedures can be
found in Section 2.9. The total number of samples collected by event, room, and method can be
found in Table 2-8.
47
-------
Figure 2-14. Photograph of AAS being performed in Room 105 during Round 1.
2.6.4.2.
Aggressive Air Sampling Area Preparation
2.6.4.2.1. Pre-cleaning Surfaces
No pre-cleaning of surfaces was done as part of the AAS procedure in the BOTE Project. This
decision was based upon the following circumstances: (1) the study rooms were newly
refurbished, and levels of dust and debris were therefore very low; and (2) AAS was used after
decontamination procedures were completed. However, in past Ba remediation actions, prior to
AAS, rooms or areas that had abundant surface residue (dust and debris) that may have
interfered with sampling media analysis due to increased particle loading were further cleaned
by washing, wiping and/or HEPA-vacuuming.
2.6.4.2.2. Establishing Isolation
Integral to the successful completion of past AAS efforts during Ba remediation actions was the
assurance that airflow within the specific area being sampled remained isolated from adjoining
areas. In the BOTE Project test facility, isolation was maintained only between the two floors
and not between rooms or areas on each floor. Some isolation between rooms may have been
provided by the drop ceiling within each room. In addition, the room door was kept closed when
the rooms were sampled to minimize the migration of air outside the rooms, and the facility
HVAC unit was not operated during the AAS operation. The drop ceiling was removed as part of
the Round 2 decontamination procedure; therefore, the drop ceiling was not in place for the
Round 2 AAS in the sampled rooms.
48
-------
Any air infiltration through window frames, other penetrations in the walls, and floors must be
minimized. Minimizing air leaks will lessen the amount of air to be exhausted to maintain
adequate negative pressure in the enclosure. The BOTE Project facility secondary enclosure
maintained negative pressure environments between the interior of the facility and the
surrounding areas outside the secondary enclosure.
2.6.4.2.3. Establishing a Negative Pressure Environment
Due to the small size of the rooms (under 2,500 ft3 in volume), the large flow rate of the negative
air machines (NAMs) on-site (2,000 cubic feet per minute [CFM]), the logistical constraints of
reconfiguring the NAMs, and the constraints on time, the rooms that were sampled were not
maintained under negative pressure to the neighboring rooms or the interior of the facility during
the AAS operation.
For reference, the premise of AAS is to collect samples that have the highest probability of
detecting any potential reaerosolized spores. To accomplish this objective, NAMs and isolation
practices may be used to establish a pressure differential of at least 0.02 in (0.5 mm) of water,
and room air exchanges should be kept at a minimum to ensure that as many respirable
particles in the disturbed air (that potentially contains spores) pass through or onto the aerosol
sampling media. To determine the number of NAMs needed for each enclosure to be sampled,
calculations that determine volume of the enclosure, required airflow, and NAM capacity are
used. Critical barriers established with the facility can help establish a negative pressure
enclosure that maintains the spatial integrity of the area being sampled. HEPA-filtered portable
ventilation units (NAMs) should be placed at one end of the enclosure and used to achieve the
negative pressure environment by exhausting air outside the enclosure. "Make-up air" should
come mainly from an airlock at the opposite end of the enclosure. This air should be HEPA-
filtered to prevent any contaminants from entering the facility. This setup places the air inside
the enclosure at a negative pressure relative to the outside air.
While AAS was conducted inside each of the two rooms, additional air samples were collected
with XMX and STA samplers placed outside the rooms in an adjacent zone (hallway) to
document if any potential migration of contamination had occurred. AAS was performed
simultaneously in each of the two rooms on the first floor. If AAS was conducted in more
rooms/areas in the facility, the operation would have started in areas previously known to be
least contaminated and moved to those areas known to be more contaminated.
2.6.4.3. Sampling Strategy
While the facility (not specific rooms) was maintained under negative pressure, all surfaces
were aggressively agitated, and the room air was continuously disturbed while air samples were
being collected. The goal was to use air sampling methods that maximize the likelihood of
detecting any residual contamination.
To evaluate the AAS procedure, two rooms were sampled (Rooms 105 and 106). Each of the
two rooms (105 and 106) to be sampled is 18.6 m2 or 226 ft2 and 42.7 m3 or 1,700 ft3. The dro
ceiling in each of the rooms resulted in a lower actual room volume than utilizing facility
49
-------
specifications of 3 m or 10 ft ceiling heights. However, the drop ceiling tiles were removed
during the pH-adjusted bleach decontamination and were not present during that AAS event.
A total of 90 samples (72 air samples and 18 field blanks) were collected during the AAS
procedure and analyzed for Bg by culture and enumeration. Thirty samples were collected
following each decontamination event. The 90 samples were collected as detailed in Table 2-8.
Table 2-8. AAS sample locations, types, and numbers.
Facility Area
Room 105
Room 106
Hallway outside Rooms
105 and 106
Total per
decontamination event
Total
Number of XMX Samples
6 one-hr + Field Blank
6 one-hr + Field Blank
3 one-hr + Field Blank
15 + 3 Field Blanks
45 + 9 Field Blanks
Number of STA Samples
3 one-hr + Field Blank
3 one-hr + Field Blank
3 one-hr + Field Blank
9 + 3 Field Blanks
27 + 9 Field Blanks
Two rooms were sampled during each of the three decontamination events, and each of the
eight collectors (four XMXs and two STAs in the two rooms and one XMX and one STA in the
hallway outside the two rooms) collected three samples, resulting in 24 air samples per
decontamination event. In addition, six field blanks were collected for each decontamination
event, resulting in a total of 30 samples collected (18 XMX samples [12 inside rooms, 3 hallway,
3 blanks] and 12 STA samples [6 inside rooms, 3 hallway, 3 blanks]) for each decontamination
event. The total number of samples collected for the three decontamination events resulted in
90 samples. Lastly, two media blank samples were submitted for analysis.
2.6.4.4. Sampling Procedures
The AAS sampling procedures described in the following subsections were prepared to provide
standardized methods for industrial hygienists or other trained samplers under the direction of
sampling experts to use when sampling for Ba spores. These procedures are meant to be used
for collection of samples in indoor environments. AAS guidance should be updated as new
information becomes available. Product manufacturer recommendations, LRN guidance and
best professional judgment were followed. Detailed sampling equipment and sample collection
procedures for each method can be found in Appendix D.
2.6.4.5. Sampling Team Organization and Roles
Personnel were grouped into three, three-person AAS teams, with one person assigned as the
team supervisor to coordinate activities. Each of the two rooms to be sampled had an
independent sampling team; an additional team was assigned to conduct the sampling in the
hallway. The sampling teams were formed during an initial training session for AAS. Sampling
teams were organized to perform each aspect of the air sampling process effectively, including
identification of sampling points, placement of sampling equipment, collection of samples,
50
-------
decontamination of sample containers and equipment, and packaging and shipment of air
samples.
The roles of AAS collection team members were carefully defined to ensure conformance with
good aseptic techniques. Each team had two sample collection people and one
support/documentation person. Sample collection team members were responsible for donning,
removing and disposing of nitrile gloves donned specifically for sampling purposes over
standard PPE. Each person put on and removed a new pair of gloves at each sampling location.
Once each sample collection team member donned a pair of gloves for sampling, that person
replaced these gloves immediately if they touched any surface or object other than their
equipment/supplies. All discarded gloves were placed in a large plastic bag that was taken out
of the building and decontaminated. Once the sample collection person donned a new pair of
nitrile gloves at a given sampling location, he did not touch anything other than the aerosol
sampler filter assembly piece and the sample filter cassette being installed or removed or, in the
case of STAs, the agar plate.
The support/documentation person was responsible for handling and setting up all sampling
equipment and supplies, and for the proper documentation of all sampling activities, including
completion of checklists and initiating the chain of custody using the BROOM PDA. The
support/documentation person handled sample containers and sample bags for the sample
collection people, but at no time did the support person come into direct contact with the sample
filter or agar plate. The support/documentation person made sure nothing passed over the
sample container or sample bag and kept the sample containers and sample bags closed at all
times when not specifically required being open for the purpose of sampling.
2.6.4.6. Initial Sampling Team Training Session
Prior to beginning AAS, an initial training session was conducted for all personnel involved. This
initial training session included a detailed explanation and thorough demonstration of the
Standard Operating Procedures (SOPs) developed for AAS and surface sampling in the BOTE
Project (see Appendices C and D). The SOPs for surface sampling that also applied to AAS
tasks included sample location marking, equipment placement, sample preparation and
collection, sample decontamination, and shipment.
As part of initial training, each sampling team member was required to demonstrate proficiency
in performing each applicable SOP. Proficiency was shown through satisfactory completion of a
hands-on demonstration of each applicable SOP. The initial training session included an in-
depth discussion of the importance of following proper aseptic technique when collecting and
handling samples to prevent possible cross-contamination. The most common sources of cross-
contamination were from dust, air movement and people. Sampling personnel were properly
trained to prepare and handle sampling equipment and materials to minimize cross
contamination potential from these sources. A written record was made of all personnel who
had successfully completed initial training for AAS procedures.
51
-------
2.6.4.7. Sampling Equipment and Supplies
All XMX air sample filters were loaded into the sample filter cassette assemblies under aseptic
laboratory conditions by the manufacturer prior to being brought to the site for labeling.
Preparation of STA samples was not necessary because commercially manufactured agar
plates were used. A staging area where materials needed to be stored and assembled to
complete sample collection was established in the Sampling and Decontamination Support
Trailer, outside the facility (see Figure 2-8). All sampling supplies were individually packed and
pre-labeled prior to entering the facility to begin sampling activities both to reduce the potential
for cross-contamination and to reduce the complexity of performing sample collection activities
while wearing the prescribed PPE. To limit the potential for cross-contamination further, only
equipment and supplies to be used during a given day's sampling activities were brought into
the facility.
2.6.4.8. Sample Collection
During the BOTE Project, AAS was conducted on the day of surface sampling or the day after,
surface sampling was complete, prior to the receipt of any surface sampling results. In real
contamination scenarios, AAS would likely begin after the results of clearance surface sampling
had been received (based upon past use), and a determination had been made that no viable
Ba spores (culture technique) had been found. If viable Ba spores were detected through
surface sampling, AAS would not begin until further remedial activities were performed in that
area, and subsequent surface sampling had demonstrated that no residual contamination
remained.
Prior to the start of each post-decontamination sampling event, the volumetric flow rate of each
air sampler (XMX and STA) was measured with a high volume dry cell airflow calibrator (0-
1,000 Lpm, Aalborg, Orangeburg, NY), adjusted as needed, and recorded. Next, the calibration
sampling media in each air sampler were replaced with sampling media for collecting the first
AAS sample within the round. Approximately fifteen minutes prior to the start of the sampling
event, all team members made building entries and were responsible for bringing equipment
and supplies into the facility. Once sampling personnel arrived at their designated sampling
locations in the facility, all team members set up and plugged in equipment. Once all aerosol
samplers at the location were ready to sample, one team member remained in each room,
closed the office door, turned on the oscillating fans and simultaneously activated all aerosol
samplers. The team member remaining in each room agitated all surfaces with a 1-horsepower
leaf blower (i.e., Toro Power Sweep Electric Blower, Model # 51585, The Home Depot, Atlanta,
GA) for a period of 20 min. When the agitation phase was completed, all aerosol samplers
continued to sample for three total hr, collecting three, one-hour samples each. The filters were
aseptically removed from the filter assembly and placed in pre-labeled sample containers.
Sample start and stop times were recorded for determination of the total air sampling time.
After the first iteration of sample collection (one hr), the sample collection team members
donned a new pair of gloves and aseptically removed the sampling media from each air sampler
and placed the exposed sampling media in individual pre-labeled sample containers or bags. A
new collection medium was put in each air sampler for collection of the second one-hr sample.
During the three total hr of sample collection, sample collection team members were
52
-------
responsible for installing and removing the XMX filter assembly into and from the XMX sampling
manifold and the STA agar plate into and from the aerosol samplers and nothing else, until
sampling was completed. Aerosol sampler airflow rates were monitored periodically during the
sampling period. At the conclusion of the three-hr sampling period, all aerosol samplers were
stopped and the final sample filters aseptically removed from the filter assembly pieces and
placed in pre-labeled sample containers or bags.
The support/documentation person assisted the sample collection team members in opening
and closing the sample container or agar plate sample bag. Once the sample container or agar
plate bag had been sealed, the support/documentation person placed a tamperproof custody
seal on the bag. The custody seal listed the date and time of sample collection and the initials of
the support/documentation person. The sample container or agar plate sample bag was then
placed into an outer Ziploc® bag to prevent decontamination solutions from entering or
smudging the sample ID number during decontamination of the outermost sample container. All
air samples were sent to the INL Microbiology Laboratory for analysis.
2.6.5. Assessment of Bacillus Spore Migration from Inside to Outside a
Contaminated Building
Laboratory-prepared sand samples (Petri dishes containing sterilized sand) were placed directly
outside the test facility, within the secondary enclosure, around the building near entrances,
exits and high traffic areas. Duplicate samples were collected from each location at specified
times during each round within Phase 1 of the BOTE Project. Samples were collected before
dissemination to assess background levels of the test organism, after dissemination to assess
spore transport, and after decontamination of the interior of the building to assess if spores
remained detectable in dishes outside the facility over the course of the round (because only the
interior of the building was decontaminated, no significant decrease in detectable spores outside
the building was expected to occur). Additionally, two sets of laboratory-prepared sand samples
were placed within the building to acquire field positive samples, one set on each floor.
The details of the methods are described in the sections below.
2.6.5.1. Selection of Sample Matrix
Six in situ soil samples had been collected exterior to the BOTE Project building in the fall of
2010. Surface grab samples were analyzed by USGS using the same techniques as described
in Appendix F. Four of the six samples collected were positive for Bg at estimated
concentrations ranging from 30-900 spores/g of soil. Due to the presence of elevated
background levels of Bg in the test area, a clean soil matrix was required. Pro-Corn® silica sand
(Cat. # 4315024) purchased from a local hardware store (Cincinnati, OH) was used as the
capture medium. The decision to utilize fresh sterile sand was based on several factors.
Because the soil from the BOTE Project test site had been characterized by the USGS prior to
conducting the BOTE Project and was found to be contaminated with Bg from previous
exercises, sterile samples were needed to reduce the potential for confounding background
contamination. A matrix needed to be selected to limit inhibition of the analytical technique
(qPCR). qPCR is a rapid molecular biology technique that identifies the presence of a specific
DMA sequence in a sample; the sequence selected in this study targeted the recF gene of Bg
53
-------
DNA[351. This method does not indicate viability of the sampled cell or spore, and known
interferences and inhibitors of the qPCR process such as calcium, excessive levels of
magnesium, and humic acid are likely to be found in native soil. A balance must be achieved
between the primers, reaction temperatures, DMA, and magnesium for a successful PCR assay
to take place'671. The amount of PCR product may be reduced if magnesium levels are
inadequate'671 or magnesium is unable to bind with the DMA polymerase'681. However, excessive
magnesium concentrations can actually prevent complete denaturing of the DMA and reduce
the available product as well'671. Calcium can also compete with magnesium and reduce the
reaction efficiency and total amount of PCR product'691. Humic acids are a product of
biodegradation of organic matter in soil. For environmental samples, these compounds are the
most frequently mentioned inhibitor of PCR and can cause false negative results'70'71]. Studies
suggest that humic acids may also hinder PCR efficiency by limiting the amount of DMA
template available for the reaction'691. Previous studies confirm these geochemical properties of
sand vs. soil'721.
Geochemical analysis of the sterilized sand was performed by Midwest Laboratories, Inc.
(Omaha, NE). The average total concentrations as determined through dissolution and
inductively Coupled Argon Plasma detection were 8,037 (±1,372) parts per million (ppm), 183 (±
56) ppm, and 1.38 (± 0.24) ppm for calcium, magnesium, and zinc, respectively.
Microbiological analysis was also performed on the sterilized sand to confirm the absence of Bg
by the EPA laboratory (Cincinnati, OH) prior to the start of the BOTE Project. Aliquots of
sterilized sand were cultured in TSB overnight. After incubation, the enriched broth was plated
onto agar plates and incubated overnight again before the final determination. All sand aliquots
were deemed sterile after double enrichment. USGS separately assessed sterile sand samples
for Bg presence prior to the start of the BOTE Project through two separate analyses. DMA was
directly extracted from aliquots of sand, while additional aliquots of sand were enriched in TSB
overnight at room temperature before DMA extraction. No Bg DMA was detected within the sand
samples. Therefore, the reference sand was free of Bg prior to the experiment and was a
suitable matrix for the remainder of the project.
2.6.5.1.1. Laboratory Preparation of Sample Matrix and Containers
Fifty gram aliquots of sand were placed into aluminum weigh boats and then heat-sterilized (250
°C for 10 hr). After sterilization, 50 g of the cooled sand was aseptically transferred to sterile
polystyrene (150 mm) Petri dishes at EPA (Cincinnati, OH). The top and bottom of each Petri
plate wase then sealed with Parafilm® (Pechiney Plastic Packaging Company, Chicago, IL) and
secured with cellophane tape. The sealed samples were bagged in lots of ten, boxed, and
shipped to INL prior to the project.
2.6.5.1.2. Preparation of Sampling Kits
Once on site, each laboratory-prepared Petri dish containing sand was labeled with a barcode
(placed on the bottom of the Petri dish). Each sand sample was then placed in a Ziploc® bag by
itself, and a corresponding barcode was placed on the outside of the bag. Dishes were then
organized into sample placement boxes according to sampling round and event. Each round
had samples stacked in one box for pre-dissemination (background) sample placement and
54
-------
then a second box that contained placement kits for placing both the post-dissemination and the
post-facility-decontamination samples at the same time. The Round 1 (VHP®) background
samples were not individually bagged but instead were stacked in the sample collection box
without any bag. This design flaw, corrected for Round 2 and Round 3, might have introduced
contamination to the samples (i.e., blanks for Round 1 were positive). The Round 1 samples
were subsequently removed from statistical analysis. Collection kits (individually bagged empty
Petri dish and 14 in x 1 in [35.6 cm x 2.5 cm] piece of Parafilm®) were also assembled into
boxes before each sample collection event and round and were labeled with barcodes. All
supplies were stored under ambient conditions prior to placement.
2.6.5.2. Sand Sampling Process
Laboratory-prepared sand samples were placed within the secondary enclosure, around the
building near entrances, exits, and high traffic areas. Duplicate samples (Samples A and B)
were collected from each location at the specified times during the event. Samples were
collected before dissemination, after dissemination, and after decontamination. The samples
collected within the secondary enclosure following decontamination were not directly
decontaminated. Two sets of laboratory-prepared sand samples were placed within the building,
one on each floor, to acquire field positive samples within the two concentration environments.
These laboratory-prepared sand samples were placed prior to spore dissemination and were
collected after dissemination and after decontamination. The purpose of collection of the indoor
samples after decontamination was to assess PCR inhibition due to the presence of
decontamination agents. See Table 2-10 for a full description of sample collection/placement
procedures, sample purpose, and blanks.
2.6.5.3. Placement and Retrieval of Sand Dishes
EPA sampling personnel placed the laboratory-prepared sand samples in the designated
sampling locations. As each of the sand samples was placed, the lid was removed and
discarded. The sand samples were placed within orange-painted trays to aid visibility and
tracking of samples and to minimize location inconsistencies among the three testing rounds.
Within the building, the sand samples were placed in locations that minimized the risk of
disturbance due to foot traffic and activity. On the first floor, the sampling tray was located in the
reception area (see Figure 2-2) under the counter. The tray on the second floor was placed on
the floor in a corner of the hallway (see Figure 2-3). Trays were taped down within the building
to minimize movement. Samples were placed in an order that avoided walking by the sample
tray multiple times to prevent contamination during the placement process. Two personnel were
needed for the placement process: one to carry and provide supplies and one to place the sand
samples. Placement with two people averaged approximately 45 minutes per event.
Sampling personnel collected samples from the trays at the designated time: pre-dissemination,
post-dissemination, or post-decontamination. Samples were retrieved in the same order as
placement for each decontamination technology event. A list of the sampling site locations is
reported in Table 2-9. Retrieval started with Tray 1 and personnel moved counterclockwise
around the building (Figure 2-15). Personnel then entered the second floor of the building and
collected at location B2, passed through the airlock and down the stairwell to the first floor to
collect B1, and then exited from the tent to the decontamination area.
55
-------
Site blanks and trip blanks were retrieved midway through the sampling process, at Tray 5,
away from tent entrances throughout the exercise. Site blanks were opened on site,
immediately closed, and retrieved. Trip blanks were carried to the sample site but remained
unopened. Additional site blanks were collected during post-decontamination sampling during
Round 2 (pH-adjusted bleach decontamination process) and Round 3 (fumigation with CIO2)
and post-dissemination during Round 3 (see Table 2-10). Tracking labels with barcodes were
affixed to the individual sand samples and each tray. After exposure, the sand samples were
capped with new lids, sealed with Parafilm® and office tape, and individually bagged. During
retrieval, labels were scanned into the BROOM system for tracking the timestamp and tray
location.
The BOTE Project protocol used for the retrieval process can be found in Appendix F. Three
personnel were required for sample retrieval, one supplier, one collector, and a separate
individual to operate the BROOM tool. A third person dedicated to the BROOM allowed for the
collection process to move efficiently. Retrieval of all samples averaged approximately 1.25 hr
per event. See Table 2-11 for actual dates and times of collection.
Table 2-9. Sand sampling site locations.
Label
1
2
3
4
5
6
7
8
9
10
B1
B2
Sample Site Description
Secondary Enclosure sample located in the northwest corner of the Secondary
Enclosure, to the left side of personnel doorway.
Secondary Enclosure sample located just north of Floor 1 entrance, under the
staircase.
Secondary Enclosure sample located just south of Floor 1 entrance near the walkway.
Secondary Enclosure sample located in the southwest corner of the Secondary
Enclosure, near the negative air machine (NAM) tubing.
Secondary Enclosure sample located along the south wall of the Secondary
Enclosure, near a UV-APS pump. Trip blanks and site blanks were also collected at
this location.
Secondary Enclosure sample located at the southeast corner of the Secondary
Enclosure, near NAM tubing.
Secondary Enclosure sample located along the eastern wall of the Secondary
Enclosure, between the two building doorways.
Secondary Enclosure sample located just to the east of the Secondary Enclosure exit.
Secondary Enclosure sample located just to the west of the Secondary Enclosure
exit.
Secondary Enclosure sample located along the north wall of the Secondary
Enclosure, next to a UV-APS pump.
Control positive sample located within the building on the first floor.
Control positive sample located within the building on the second floor.
56
-------
Table 2-10. Description of sand sample placement and purpose.
Round
1
2
3
Sample Set
Pre-Dissemination
Post-
Dissemination
Post-
Decontamination
Pre-Dissemination
Post-
Dissemination
Post-
Decontamination
Pre-Dissemination
Post-
Dissemination
Post-
Decontamination
Schedule
Place during R1 setup/retrieve before
R1 dissemination
Place before R1 dissemination/retrieve
after R1 dissemination reference
sampling
Place before R1 dissemination/retrieve
after R1 decontamination reference
sampling
Place during R2 setup/retrieve before
R2 dissemination
Place before R2 dissemination/retrieve
after R2 dissemination reference
sampling
Place before R2 dissemination/retrieve
after R2 decontamination reference
sampling
Place during R3 setup/retrieve before
R3 dissemination
Place before R3 dissemination/retrieve
after R3 dissemination reference
sampling
Place before R3 dissemination/retrieve
after R1 decontamination reference
sampling
Total # of Samples
(Not including blanks)
Secondary Enclosure: 20
Building: 0
Secondary Enclosure: 20
Building: 2
Secondary Enclosure: 20
Building: 2
Secondary Enclosure: 20
Building: 0
Secondary Enclosure: 20
Building: 2
Secondary Enclosure: 20
Building: 2
Secondary Enclosure: 20
Building: 0
Secondary Enclosure: 20
Building: 2
Secondary Enclosure: 20
Building: 2
Purpose
Assess spore pre-dissemination levels
N/A
Assess spore migration from building and
spore presence following dissemination
Assess concentration of deposited spores into
test containers; provide positive control
Assess spore presence amassed throughout
entire round via spore migration from building
Determine if analytical interference occurs
from decontamination process
Assess spore pre-dissemination levels
N/A
Assess spore migration from building and
spore presence following dissemination
Assess concentration of deposited spores into
test containers; provide positive control
Assess spore presence amassed throughout
entire round via spore migration from building
Determine if analytical interference occurs
from decontamination process
Assess spore pre-dissemination levels
N/A
Assess spore migration from building and
spore presence following dissemination
Assess concentration of deposited spores into
test containers; provide positive control
Assess spore presence amassed throughout
entire round via spore migration from building
Determine if analytical interference occurs
from decontamination process
Blanks
Site blank
and trip
blank
N/A
N/A
Site blank
and trip
blank
N/A
Site blank
Site blank
and trip
blank
Site blank
Site blank
57
-------
West to East Wind
20-25 MPH
Tent
Exit&
Decontamination
Area
Tent
Tent Equipment
Entrance
LJ
Concrete Barriers
| | UV-APS and associated tubing
f 1 Negative Air Machine and associated tubing
1-10 TentSamples
Bl- B2 Building Control Positive Samples Floor 1 and Floor 2
* Control Blanks
Access
Road
Figure 2-15. Schematic of sample placement during the BOTE Project.
58
-------
Table 2-11. BOTE Project sand sample schedule.
Date / Time
April 14, 1700*
April 16, 0817
April 16, 0945
April 16, 1317
April 18, 1123
April 19-21
April 23, 0838
April 23, 1200*
April 25, 0805
April 25, 0901
April 25, 1415
April 26, 1551
April 28 - May 4
May 6, 0822
May6, 1413
May 10, 0834
May 10, 1100*
May 10, 1515
May 12, 0804
May 13-15
May 17, 1300
Action
Place R1 Pre-Dissemination Samples
Retrieve R1 Pre-Dissemination Samples
Place R1 Post-Dissemination and Post-
Decontamination Samples
R1 Dissemination
Retrieve R1 Post-Dissemination Samples
R1 VHP® Decontamination
Retrieve R1 Post-Decontamination Samples
Place R2 Pre-Dissemination Samples
Retrieve R2 Pre-Dissemination Samples
Place R2 Post-Dissemination and Post-
Decontamination Samples
R2 Dissemination
Retrieve R2 Post-Dissemination Samples
R2 Amended Bleach Decontamination
Retrieve R2 Post-Decontamination Samples
Place R3 Pre-Dissemination Samples
Retrieve R3 Pre-Dissemination Samples
Place R3 Post-Dissemination and Post-
Decontamination Samples
R3 Dissemination
Retrieve R3 Post-Dissemination Samples
R3 CIO2 Decontamination
Retrieve R3 Post-Decontamination Samples
Time In
Place
39.25 hr
49.75 hr
167 hr
45 hr
30 hr
262.5 hr
90.25 hr
45 hr
170hr
Number of
Sampling
Personnel
2
3
2
2
3
2
3
2
3
3
3
3
2
3
3
*Denotes estimated time, data not available.
2.6.6. Reaerosolization Assessment and Measurement Protocols
Air sampling was conducted to capture and potentially characterize reaerosolization. Airborne
Bg spore concentrations were measured in indoor air in two rooms (Room 101A and 102) of the
BOTE Project test facility using SKC BioSamplers®. The SKC BioSamplers® collected airborne
spore samples from three locations per room and three heights per sampling location at five
59
-------
different time stages. Additionally, the UVAPS units took continuous measurements in these
reaerosolization study rooms. The sampling design for the reaerosolization study is detailed in
the following subsections.
2.6.6.1. Sampling Design and Layout
The SKC BioSamplers® were installed in both rooms (Room 101A and 102) prior to
dissemination. Forty-eight SKC BioSamplers® were initially placed in each room, with the final
twelve samplers placed in each room after decontamination occurred. Four samplers were
placed at each of the four specified heights (12 in (low), 24 in (blank), 36 in (medium) and 48 in
(high) from the floor) at each of the three locations in each room. There were three SKC
BioSamplers® run at a time at each sampling location in the room as specified below. Due to the
configuration of the SKC BioSamplers®, lack of vacuum pressure, and the minimal air
movement in the room, passive sampling was not expected to occur with these instruments.
The blank SKC BioSampler® inlets were left uncovered during test events with no vacuum
hoses connected.
2.6.6.2. Testing and Measurement Protocols
Fifteen-min long SKC BioSampler® samples were collected at five different time stages
(background, dissemination, prior to surface sampling, during characterization [pre-
decontamination] surface sampling, and after decontamination [post-decontamination] surface
sampling) for each decontamination round (See Table 2-12). Sampling was limited to 15 min per
SKC BioSampler® due to sampler fluid limitations; further sampling would have resulted in
evaporation of the sampling media and would have skewed the results. During each of the three
decontamination rounds, the first samples (Background/Stage 1) were drawn 30 min prior to the
initial dispersion of spores into the building. The second stage of sampling (Dissemination/Stage
2) was taken 10 min after dispersion of the spores in the building. The second stage had three
individual sampler sets; each set was sampled for 15 min totaling 45 min total sampling time for
the second stage. The third set of samples was taken 30 min prior to the entrance of personnel
into the facility for characterization (pre-decontamination) surface sampling (before Pre-
Decontamination Surface Sampling/Stage 3). The fourth set of samples was taken during
characterization surface sampling (Pre-Decontamination Surface Sampling/Stage 4), again
each set was staggered at intervals of approximately 15 min, totaling 45 min total sampling time.
This stage was coordinated with the surface sampling team's movement through the room to try
to capture reaerosolization of deposited spores (see Table 2-12). The fifth set of samples was
taken post-decontamination during clearance (post-decontamination) surface sampling (Post-
Decontamination Surface Sampling/Stage 5). Three field blank samples were collected per
room for each of the five sampling stages; field blank samplers were not turned on. The
staggered timing design attempted to be as representative as possible across the times,
sampling locations, and sampling heights (see Table 2-13 and Table 2-14).
After the sampling was complete, SKC BioSamplers® were collected in whole units and not
disassembled. The SKC BioSamplers® were collected, sealed within a bag, wrapped with foam
and transported using a Pelican Case. All individual sample containers were sealed to prevent
contamination during transport and properly labeled with the sample identification. Samples
were transported immediately after collection/removal from rooms. Collection/removal of the first
60
-------
four stages of samples occurred immediately following pre-decontamination surface sampling.
The fifth stage of samples was placed prior to post-decontamination surface sampling and
collected/removed and transported immediately following post-decontamination surface
sampling. All SKC Bio-samplers® were labeled and tracked using scanable barcodes on each
individual sampler.
Table 2-12. SKC BioSampler® stages.
Timing
Description
Stage 1: Background
T-30 min to Dissemination
T-0 Dissemination
Samples Taken Prior to
Dissemination
Initiate Dissemination
Stage 2: Dissemination
T+10 min After Dissemination
Initiation
Samples Taken During
Dissemination
Stage 3: Before Surface Sampling
T-30 min Prior to Entry
Samples Taken Prior to Personnel
Entry
Stage 4: Pre-Decontamination Surface Sampling
T+15 min Post Entry
Samples Taken During Wipe
Sampling
Stage 5: Post-Decontamination Surface Sampling
T+15 min Post Entry
Samples Taken After
Decontamination During Wipe
Sampling
61
-------
Table 2-13. Staggered timing design for SKC BioSamplers® during dissemination (Stage
2) and pre-decontamination surface sampling (Stage 4) for all three rounds.
Round
1
1
1
1
1
1
2
2
2
2
2
2
3
3
3
3
3
3
Room
1
1
1
2
2
2
1
1
1
2
2
2
1
1
1
2
2
2
Time
min
0-15
15-30
30-45
0-15
15-30
30-45
0-15
15-30
30-45
0-15
15-30
30-45
0-15
15-30
30-45
0-15
15-30
30-45
Location 1
Height
L
M
H
M
L
H
H
L
M
L
M
H
M
L
H
H
L
M
Location 2
Height
M
H
L
H
M
L
M
H
L
H
L
M
L
H
M
L
M
H
Location 3
Height
H
L
M
L
H
M
L
M
H
M
H
L
H
M
L
M
H
L
62
-------
Table 2-14. Staggered timing design for SKC BioSamplers® during post-decontamination
surface (Stage 5) sampling for all three rounds.
Round
1
1
1
1
1
1
2
2
2
2
2
2
3
3
3
3
3
3
Room
1
1
1
2
2
2
1
1
1
2
2
2
1
1
1
2
2
2
Time
min
0-15
15-30
30-45
0-15
15-30
30-45
0-15
15-30
30-45
0-15
15-30
30-45
0-15
15-30
30-45
0-15
15-30
30-45
Location 1
Height
L
H
M
M
H
L
H
M
L
L
H
M
M
H
L
H
M
L
Location 2
Height
M
L
H
L
M
H
M
L
H
H
M
L
H
L
M
L
H
M
Location 3
Height
H
M
L
H
L
M
L
H
M
M
L
H
L
M
H
M
L
H
In addition, two UV-APS units were placed in PBF-632 building on the first floor. One UV-APS
was placed inside Room 101 A, and the second UVAPS was placed in Room 102. To prevent
contamination, the UV-APS units were enclosed within a ventilated Pelican Case and
maintained operating temperatures within the range of 50-104 degrees Fahrenheit (°F). Both
UV-APS units were remotely operated through an RS-232 to Ethernet media converter and
wired into the Ethernet switch located outside the building. Computer control of the system was
performed through manufacturer-supplied software (Aerosol Instrument Manager® Software for
Aerodynamic Particle Sizer).
The UV-APS collected samples continuously from 60 min prior to dissemination until building
clearance. The Pelican Case was left in the facility for VHP® decontamination (Round 1) but
was removed from the facility for the pH-adjusted bleach decontamination process (Round 2)
and fumigation with CIO2 (Round 3).
63
-------
2.7. Sample Tracking and Shipping
The BROOM1601 PDA was used by sample collection teams to track sample location, sample
types, sample matrices, date, time, samplers, and other pertinent data. BROOM comprises both
a hardware and software solution set. The hardware is commercial off-the-shelf (COTS)
equipment that was used in the data collection phase (i.e., during sampling). The software was
used both on the COTS hardware and on a laptop to communicate with the COTS hardware
and provide the data management and data analysis capabilities. As part of the hardware, a
PDA was used by the sampling teams to manage data acquisition. A laser range finder
connected to the PDA was used to determine indoor x, y, z locations. The software on the PDA
provided the following capabilities during the BOTE Project sample collection:
Predefined sampling locations shown on the facility floor plan on the PDA, with
pan and zoom capabilities;
Laser rangefinder positioning and touch screen capability to record or locate
(respectively) the position of a sample;
Documentation of the sampling method (e.g., swab, sponge-stick wipe, wipe, or
vacuum sock), the area sampled, the orientation of the sampled surface (e.g.,
vertical or horizontal upward), the texture of the surface (e.g., smooth or porous),
the type of surface (e.g., wallboard or carpet), and notes about any observations;
Scan unique barcode label on each sample collected;
Capture and storage of pictures for each sample entry; and
Signature capture of the user on completion of sampling for record of chain of
custody.
The PDA data were uploaded to the BROOM system server located in the sampling prep trailer
(Location 2 in Figure 2-8). The server software provided the following capabilities that were
used during the BOTE Project:
• Ability to query the data by a number of means (e.g., pop-up information
balloons);
Filter or cull the data to be displayed (e.g., look at swab samples only);
Display any photos that were taken during sampling;
Design of sampling plans (e.g., statistical, random, or gridded);
Sampling design optimization using geostatistical models and optimization
routines;
Geostatistical analyses to produce maps of the nature and extent of
contamination, uncertainty maps, and the probability of exceedance of a user-
specified concentration;
• A unique geostatistical algorithm that accounts for the influence of walls and
open doorways;
Mapping using an inverse-square distance method; and
• Ability to import/export in various formats (e.g., .csv, Excel, XML, KML).
Relevant sample identification data such as barcode ID, location information, and surface and
sample type were captured on the PDA by the sampling teams. This information was then
64
-------
uploaded and stored in the BROOM database. The database also stored any photos taken
using the PDA during the sampling process.
Environmental surface samples (sponge-stick wipes, Versalon® wipes, swabs, vacuum socks)
were collected by EPA-led sampling teams during the BOTE Project as described in the
previous section. The samples were collected using pre-labeled and barcoded
sample/specimen kits provided by EPA. The sampling kits included all supplies needed to
collect, label and package each sample. As each sample was collected, sampling personnel
entered the data into the BROOM PDA and scanned the bar code on the sample bag. Next, the
samples were placed into a specimen transport bag, decontaminated and packaged
appropriately for shipping. The BROOM system was used to generate chain of custody (COC)
forms, which were completed on site and shipped with the samples. The samples were
transported overnight in a cooler at 35 °F to the specified laboratories listed in Table 2-5, to
conduct the analysis (EPA, LRN, LLNL, and USGS). Samples were packaged and shipped in
high quality Styrofoam boxes with cardboard overpack (i.e., Polyfoam Packers orThermosafe).
Each cooler contained a temperature and relative humidity (RH) data logger (Onset Computer
Corp., HOBO U10 #1110-003). Samples were gathered and shipped by INL Laboratories
through an EPA Interagency Agreement (DW89923315).
All sand samples were collected by EPA-led sampling teams during the BOTE Project as
described in the previous section. Collected sand samples were transferred to the INL support
staff for packaging and shipping after scanning the bar- code and decontaminating the outer
transport bag. No ice or cold packs were used for shipment. Samples were shipped using
FedEx® priority overnight to the EPA AWBERC. A total of 214 sand samples were collected and
transported to EPA. COC forms generated by the BROOM system were included with each
shipment.
2.8. Cross-Contamination Reduction Methods
Several methods were employed during the BOTE Project to prevent cross-contamination and
included secondary enclosure of the facility, personnel entry methods, and decontamination of
samples and personnel leaving the building. Each method will be discussed below.
The first effort to prevent cross-contamination included the use of a polymeric secondary
enclosure structure which was fitted over PBF-632 by INL as shown in Figure 2-7. This structure
served as a barrier to assist in preventing contamination of the surrounding area during the
testing. An additional secondary containment tent was also placed directly over the building by
Sabre during fumigation with CIO2.
The second method utilized to prevent the spread of contamination was establishing a single
entry point to the facility and a single exit point at a location different than the entry point. It is
recognized that this idea may not be possible in all situations due to various factors such as
building structure (limited entry points), space available to set up decontamination line, location
and magnitude of contamination, and wind direction.
65
-------
The third method of cross-contamination prevention included precautions taken by sampling
personnel prior to, during, and following sampling. Samplers donned all PPE except respirators
in the sample preparation trailer (Location 2, Figure 2-8). (Note that the the trailer was divided
into two distinct areas: PPE storage/donning and sample preparation.) PPE consisted of full
face-piece air purifying respirator (or powered air purifying respirator) with P100 cartridges,
hooded Tyvek® suits with attached booties, and a base pair of nitrile gloves. The base pair of
gloves and respirator were typed to seal openings. Respirators and used cartridges remained
outside the sample preparation trailer (and break and recovery trailer) at all times to prevent
contamination from entering the trailer via used respirators.
After entering the secondary enclosure structure and before entering the PBF-632 facility,
samplers doffed one pair of booties to aid in preventing secondary contamination from outside
the test bed. This is especially important for clearance sampling so that any potential outdoor
contamination is not brought into the facility. Once in the building, samplers used a fresh pair of
gloves prior to handling each new sample or any time personnel may have touched a
contaminated surface. Use of a three-person sampling team, which included Person A (BROOM
operator), Person B (supplier), and Person C (collector), allowed aseptic techniques to be used
during sampling. Person A handled the BROOM PDA and all communication on the two-way
radio. Person B provided prepared sampling materials to Person C and was the only person to
handle sample bags before and after sampling. Person C handled only the sampling device
(swab, sponge-stick, wipe, vacuum sock). After collection, the samples were placed into large
bags, one for each sample type, which were attached to each cart used by each sampling team.
Sampling teams entered the building in stages, with the first team collecting samples in the
hallway or other areas that might be disturbed by foot traffic. Samplers followed the sampling
maps provided, taking care to sample first near doorways or in areas that might be disturbed by
foot traffic. Sampling teams on the first floor did not enter the second floor to prevent bringing
spores from the higher contamination level to the lower contamination level. Personnel on the
second floor moved through an airlock to enter the stairwell and then proceeded to the first floor
exit.
The last effort to prevent cross-contamination included a Decontamination Line. After all
samples were collected, and the BROOM PDA operator signed the COC form and uploaded the
BROOM data, samplers proceeded to the first floor exit. Samplers doffed the last pair of booties
before exiting the building and entering the Decontamination Line. Samplers kept one pair of
nitrile gloves over their base pair while in the Decontamination Line. Roles were assigned for
each member of the sampling team to streamline the decontamination process. Person A broke
down cardboard sample boxes and wiped unused sample kits with Dispatch® bleach wipes
(Medline Industries, Mundelein, IL). Person A then entered the Decontamination Line. Person B
wiped each sample bag with Dispatch® bleach wipes and then packed the sample bags into
large Ziploc® bags. (Note: Dispatch® bleach wipes are not proven sporicidal for use against Ba
spores. The bleach wipes were used here for contamination control related to the use of the
surrogate spores, Bg.) The exterior of the large Ziploc® bags was also wiped with Dispatch®
wipes and then transferred outside the Decontamination Line for shipment to the appropriate
analytical laboratory. Person B then proceeded to the Decontamination Line. Person C was in
66
-------
charge of wiping down the cart, vacuum, flashlight, and markers with Dispatch® wipes and
disposing of trash. If Person C completed tasks before Person B, Person C assisted with the
task of wiping sample bags to allow Person B to go through the Decontamination Line to move
personnel quickly through decontamination.
The Decontamination Line was set up in a tent immediately outside the secondary enclosure
structure and consisted of washing, rinsing, and doffing stations. Personnel exiting the facility
first removed the top layer of gloves and booties and placed them into trash bags located at the
facility exit and entry way to the Decontamination Line. The personnel then stepped into the first
pool; samplers were rinsed with water and their feet were washed and scrubbed with soapy
water by the decontamination team. Care was taken to step into the second pool and not back
into the first pool after each foot was scrubbed. Walkers were placed between pools to aid in
balance. The second pool was used to rinse personnel with clean water. After stepping into the
third pool, samplers removed the last layer of gloves, were assisted with removing the Tyvek®
suit, and the decontamination team wiped down the exterior of the sampler's respirator with
Dispatch® bleach wipes. After stepping out of the pools to the final Decontamination Line area,
samplers were assisted with removing and cleaning (the interior) their respirators. Respirator
cartridges were either discarded or tape was placed over the opening to prevent contamination
outside the facility.
2.9. Sample Analysis Methods
The LRN laboratories, INL, LLNL, EPA, and the USGS conducted sample processing for the
Phase 1 project samples (See Table 2-5 and Table 2-7). The analysis methods for each sample
type are described in the sections below.
2.9.1. Surface Sample Analysis Methods
Surface samples were analyzed for viable Bg by either the LRN or INL, in accordance with
standardized procedures (see Appendix E). All surface samples intended for use in the
assessment of decontamination effectiveness were analyzed via the LRN. Surface samples
used for assessment of RV-PCR and reaerosolization were analyzed by LLNL and INL,
respectively. All samples were analyzed for quantifiable viable Bg using dilution plating
methods. Surface samples with less than 30 CPU present at the lowest dilution were to be filter-
plated to attain lower detection limits. The analysis procedures are described in detail in the
following subsections. The difference in the selection point for triggering filter plating was
dependent upon the intended use of the data. LRN data only were used for the assessment of
decontamination effectiveness. This difference is not significant with respect to reported data by
any analysis method used in the BOTE Project.
2.9.1.1. Analysis of Environmental Surface Samples by the LRN
The LRN is a network of Federal, State, and Local laboratories that was formed to increase the
Nation's capacity to respond to bioterrorism incidents as well as to new and emerging diseases
that threaten public health. The LRN's mission is to develop, maintain and strengthen an
integrated national and international network of laboratories that can respond quickly to needs
for rapid testing, timely notification and secure messaging of results associated with acts of
biological or chemical terrorism and other high priority public health emergencies.
67
-------
The following LRN laboratories participated in the BOTE Project Phase 1, analyzing 1,937
samples:
• Dallas County Health and Human Services;
• Idaho Bureau of Laboratories;
• Florida Department of Health Bureau Laboratories;
• Minnesota Department of Health;
• State Hygienic Laboratory at the University of Iowa;
• Virginia Division of Consolidated Laboratory Services;
• Unified State Laboratories: Utah Public Health; and
• Wadsworth Center, New York State Department of Health.
To replicate to the greatest extent practicable the procedures likely to be used following an
actual bioterrorism incident, the LRN was tasked with receiving and analyzing surface samples
from the BOTE Project. Procedures for sample processing and culture analysis were identical to
the methods outlined for Ba but amended for Bg where necessary.
At a minimum, LRN laboratories conducted all procedures with Bg according to BSL-2
guidelines established in the Biosafety in Microbiological and Biomedical Laboratories, 5th
edition'731. Samples were processed using existing LRN procedures for sponge-stick wipes,
swabs, and vacuum socks (draft, interim procedure) for identification of Ba. Methods were
modified, where necessary, to reflect analysis of Bg. LRN analyzed samples by culture method
only (no PCR), because previous contamination events conducted at the INL facility (PBF-632)
used in the BOTE Project resulted in residual Bg DNA in the environment. A detailed description
of the LRN sample analysis procedures is included in Appendix E.
For the BOTE Project Phase I, samples from the pre-decontamination (characterization)
sampling events in each round were processed using dilution plating. Post-decontamination
samples were processed by dilution plating, as well as filter plating to enhance detection of Bg
which may be at low concentrations. Because viable spore concentrations were expected to be
low on the second floor, pre-decontamination samples from the second floor were also filter-
plated.
Eight different LRN laboratories (listed above) were used in Phase 1 of the BOTE Project as
listed above. Following arrival at the destination laboratory, custody was transferred according
to COC procedures and samples were unpackaged, inventoried, and processed. The initial plan
was for samples collected during characterization events to be processed using dilution plating
only for first floor samples and dilution plating plus filter plating for second floor samples. In
reality, not all second floor samples received the prescribed filter plate analysis, presumably due
to labeling errors, illegible labels, and/or laboratory worker misinterpretation of sample labels.
The intial plan for post-decontamination samples was dilution plating as well as filter plating
(when spread plates returned results below 30 CFU) as prescribed for all samples to ensure
detection of Bg which may be at low concentrations. Again, not all samples that required filter
68
-------
plating were filter plated. There was little impact on the BOTE project due to the few samples
that were not filter plated and should have been according to the original project plan. The
impact is discussed in more detail in Section 6.
2.9.1.2. Analysis of Surface and Referee Samples by INL
INL provided the analysis for the TSA settling plates and RMCs collected during pre-
decontamination surface sampling. INL also analyzed Versalon® wipes from the reaerosolization
study, taken in Rooms 101A and 102. Details regarding these analyses are in the sections
below.
2.9.1.2.1. Processing of Settling Plates
Upon receipt at the laboratories, settling plates were immediately incubated at 35 ± 2 °C for a
maximum of three days. Plates were counted as described in Section 3.6 after incubation.
2.9.1.2.2. Analysis of RMCs
To prepare the laboratory work areas, all laboratory benches and associated equipment were
pre-sterilized, autoclaved, or wiped with a pH-adjusted bleach solution followed by 70%
isopropyl alcohol. For RMC samples, three replicate TSA plates were labeled with the specific
sample identification number and dilution (10~1, 10"2, 10"3). Also, two 15 mL conical test tubes
were labeled with the sample identification number and dilution (10"1 and 10"2) for each RMC.
To process the RMCs, 20 mL of PBST was pipetted into each of the conical tubes containing
the collected RMC. The tubes were then capped and shaken for 30 min at 300 revolutions per
minute (rpm) in a platform shaker (such as New Brunswick Stackable Incubator Shaker I2500,
New Brunswick Scientific, Enfield, CT). The RMCs were removed from the tubes while allowing
excess liquid to drain back into the tubes. The samples were then dilution and filter plated, as
described in Sections 2.9.1.2.4 and 2.9.1.2.5, respectively.
2.9.1.2.3. Wipe Sample Processing
Upon receipt of the Versalon® wipes from the reaerosolization study at the INL laboratories,
wipe samples were refrigerated at approximately 4 °C until analyzed within 72 hr of receipt. The
viable count method was used to obtain Bg counts of samples by diluting the samples and
plating to enumerate the number of CPU present.
To prepare the laboratory work areas, all laboratory benches and associated equipment were
pre-sterilized, autoclaved, or wiped with a pH-adjusted bleach solution followed by 70%
isopropyl alcohol. For each wipe sample, three replicate TSA plates were labeled with the
specific sample identification number and dilution (10~1, 10"2, 10"3).
Also, for each wipe sample, one sterile 50 mL conical test tube was labeled with the specific
sample identification number and 10° dilution. Two additional 15 mL conical test tubes were
labeled with the sample identification number and serial dilution series (i.e., one with 10"1 and
one with 10"2).
To process the wipe samples for culture, the wipe samples were transferred in a biological
safety cabinet (BSC) to sterile 50 mL conical tubes labeled with the appropriate sample
69
-------
identification number and 10° dilution, and 20 ml_ of PBST was added. The tubes were capped
and shaken for 30 min at 300 rpm in a New Brunswick shaker (Eppendorf AG, Hamburg,
Germany). The wipes were then removed from the tubes, wringing excess liquid back into the
tubes. The samples were then dilution and filter plated, as described in Sections 2.9.1.2.4 and
2.9.1.2.5, respectively.
2.9.1.2.4. RMC and Wipe Sample Serial Dilutions and Plating
After vortexing on high for 30 seconds, 1 ml_ of the 10° sample was removed and placed in the
tube labeled 10"1 with 9 ml_ of PBST. After vortexing on high for 30 seconds, 1 ml_ of the 10"1
sample was removed and placed in the tube labeled 10"2 with 9 ml_ of PBST. All wipe and RMC
sample dilutions were retained at 4 °C until additional dilutions were found to be unnecessary.
After vortexing each dilution tube well, 100 uL of all dilutions were removed with a pipette and
placed on the corresponding labeled TSA plates and spread with sterile, disposable Lazy-L cell
spreaders (Catalog # 89042-018, VWR International, LLC, Radnor, PA). With the triplicate
sample dilution plates, a negative control of PBST was plated to check sterility.
2.9.1.2.5. RMC and Wipe Sample Filter Plating
To increase the chance of detection, all wipe and RMC samples were also processed for
capture on microfunnel filter membranes and cultured. Pall Microfunnel filters (Catalog # 55095-
060, VWR International, LLC, Radnor, PA) with 0.45 urn pore size were placed on the vacuum
manifolds and moistened with 5 mL PBST. After opening and closing the vacuum valve, 10 mL
of PBST was placed in each filter cup with 10 mL of the 10° dilution sample. The vacuum valves
were opened and the suspension was vacuumed through the filter at a pressure <20 cm
mercury. Following vacuuming, the filters were removed and placed on the surfaces of TSA
plates making sure that there was good contact with the agar and no visible air pockets.
2.9.1.3. Plate Incubation and Counts
All settling plates and dilution and filter plates were incubated at 35 ± 2 °C for a maximum of
three days. All plates were examined within 18-24 hr after the start of the incubation and again
at three days. After the incubation period, all colonies were enumerated for countable plates
(30-300 CFU) and recorded with two significant figures on the viable count worksheet for the
dilutions plated. CFU on each plate were counted and recorded as follows:
• If the CFU count was <300/plate, recorded actual number.
• If the CFU count was >300/plate, recorded as TNTC.
• If the CFU count was <30/plate, then the filter plating data were used. If filter plating was
not completed, then the spread plate data was used and noted as such.
• If no growth of suspect colonies, recorded as non-detect (ND).
The middle range countable plates were used to determine the CFU/mL by using the following
formula (Equation 2-2):
CFU
(average , . j (dilution factor)
CFU/mL = P
volume plated
70
-------
. CPU on Plate 1 + CPU on Plate 2 + CPU on Plate 3
Average CPU/plate =
Equation 2-2
All calculations and counts were recorded in the appropriate laboratory notebook and the viable
count worksheet and archived. All quantitative results were recorded in Excel spreadsheets
according to sample identification number and sent to the EPA project officer after analysis and
quality control checks.
For some samples, filter plating either was not used or resulted in ND (when dilution plating
yielded data for Bg). In these cases, as noted in the results (Section 3) or discussion (Section
4), the actual dilution plate counts were used below the lower quantitation limit. The use of data
below the lower quantitation limit as an estimated count is supported by the ASTM Method
D5465-93'741.
2.9.2. Rapid Viability-Polymerase Chain Reaction Analysis of Wipe Samples
The RV-PCR method is a research method developed under an interagency agreement
between EPA and the LLNL of the DOE to rapidly detect and identify the presence of viable Ba
spores from samples collected during a bioterrorism event. Briefly, the RV-PCR is a
combination of a reliable broth culture method (for viability determination) and the popularly
used real-time polymerase chain reaction (PCR) method (for highly sensitive, specific, and rapid
detection and identification)'35"371. The RV-PCR method uses the difference in real-time PCR
cycle threshold (Ct) value between DMA extracts processed from the sample culture at the
starting time point (TO) and the ending time point, after nine hours incubation (T9) as well as the
Ct at T9 to detect viable target spores in the sample (in this case, Bg spores). The method lyses
only vegetative cells and not spores, so that only DMA from cells is detected and the DMA from
spores (live or dead) does not interfere with the determination of viability.
A protocol for combined RV-PCR and culture analyses from the same wipe sample was used in
the BOTE Project Phase-l evaluation of the RV-PCR method; protocol steps are outlined in
Figure 2-15 and Figure 2-16. A step-by-step detailed protocol is also provided in Appendix K (as
an attached SOP). Briefly, samples were shipped from the field test site and processed on the
sample receipt date unless changes to the shipment schedule impacted staff availability. The
RV-PCR protocol with a nine hour incubation and DMA extraction and purification (modified
Promega MagneSil® method'751) was used. In addition, culture analysis was performed on the
same sample after removal of spores from wipe samples as described in the SOP developed for
the BOTE Project (see Appendix K). The spore extraction step was repeated once with separate
extracts pooled to provide sufficient volume to conduct both RV-PCR and culture analyses on
the same sample. To accommodate parallel culture analysis, the sample extract following spore
removal was split in half with half used for culture and half used for RV-PCR. The portion for
RV-PCR analysis was processed by the following steps: 1) collection of spores by filtration; 2)
washes to remove soluble contaminants/inhibitors; 3) addition of growth medium and aliquoting
of the culture for a baseline PCR measurement; 4) nine hour incubation of the remainder for
germination and outgrowth of viable spores; 5) aliquoting of the culture for an endpoint PCR
measurement. The portion for culture analysis was concentrated by centrifugation prior to
71
-------
performing the following steps: 1) serial dilution and plating; 2) collection of spores using
microfunnel filtration and plating of the filter membrane; and 3) enrichment of the remaining
suspension. If colonies were not observed from serial dilution or filter membrane plates but the
enrichment culture appeared turbid (i.e., due to bacterial growth), the culture was re-streaked
onto agar plates in an attempt to obtain Bg colonies.
Bg colony counts (CPU) were based on morphology and presence of orange pigmentation.
Colony counts were corrected for dilution factor to determine CFU/mL and CPU/sample. Real-
time PCR was used to confirm selected Bg colonies (two per sample if CPU were observed) or
the enrichment culture (if CPU were not observed but the culture was turbid). The protocol for
combined culture and RV-PCR analysis from the same sample was standardized prior to BOTE
Project sample analysis because it was a new protocol. The results of culture and RV-PCR
methods were compared using detection/non-detection of viable Bg spores (i.e., qualitative
analysis). The details on the materials and methods are presented below.
2.9.2.1. Sample Type and Sampling Plans
Versalon® Wipe samples used in the study consisted of 2 in x 2 in wipes purchased from
Kendall Inc. (Cat. No. 8042, 50% rayon and 50% polyester gauze). Pre-wetted sterile wipes
were prepared at LLNL and provided to field sampling personnel for the BOTE Project sample
collection. Briefly, a 2 in x 2 in sterile gauze wipe was placed into a 30 ml_ tube with screw cap,
and 1.5 ml_ of sterile PBST buffer was added to the wipe. Each tube was barcoded (barcode
label parallel to tube graduations), placed into a 4 in x 6 in Ziploc® bag, and a replicate barcode
was placed on the bag (a third replicate barcode was kept at LLNL). The barcoded tubes in
bags were grouped into batches of 25 and placed into larger Ziploc® bags and stored at 4 °C
until shipment to INL. In addition, 20 wipe samples remained at LLNL for use as true blanks
(TBs), and 10 wipe samples were shipped to the Ft. Meade Laboratory for use as TBs. Prior to
shipment to field personnel, random wipe samples were tested for sterility by washing in buffer
and plating onto TSA. No growth was observed after incubation for two days at 35 °C.
2.9.2.2. Sample Receiving
Samples received from the field included QC samples that were opened during sampling, but
were not used to wipe surfaces. No indication was made on the COC forms to distinguish
surface sampling versus QC wipes, to conduct a blind study. During receiving, outer surfaces of
bags containing sample tubes were decontaminated by wiping the outsides with a Disptach®
bleach wipe. The outside of the tubes containing the samples were decontaminating in the
same manner as it was removed from the bag and placed into a tube rack. The barcode was
scanned according to the position in the rack, and a written record was also maintained
including a physical description of the sample (i.e., amount of debris present). One sample was
processed at a time, and gloves were changed between samples to prevent cross-
contamination. Prior to conducting spore recovery, a sterile forceps was used to place a mesh
support over the wipe sample to keep the wipe clear of pipetting activities. Previous work
showed that extraction efficiency could also be enhanced when mesh was used to hold the wipe
to the side of the tube while buffer was washed through the wipe sample during vortexing.
72
-------
2.9.2.3. Sample Processing: Spore Recovery from Wipe
For each sample tube rack accommodating up to 24 tubes, up to 21 samples were processed
along with one negative control (NC) and one positive control (PC), and at least one TB. PCs
consisted of wipes spiked with Bg spore suspensions (Apex Laboratories, Apex, NC). While a
level of approximately 5E2 to -1E3 CPU/sample was targeted, actual CPU determined by plating
were approximately 1E5 CPU per sample initially (MFP and Round 1 test samples), later
adjusted to 50-100 CPU per sample (for Round 2 and Round 3 test samples). The NCs were
wipes spiked with PBST buffer only. In addition, one to three IBs were included for each set of
samples. To each wipe sample in a 30 ml_ tube, 20 ml_ of cold Extraction Buffer with Tween®
(70% of 0.25 mM KH2PO4/0.1% Tween® 80 [pH 7.4] and 30% ethanol; final pH -9.5) was
added, and the tubes were vortexed for 20 min on a platform vortexer (Multi-tube Vortexer,
VWR Part# 58816-115, Radnor, PA) to remove spores from the sample matrix. Cold buffers
were used to minimize the potential for spore germination prior to incubation that could
contribute to a PCR response at TO. Fourteen ml_ were then transferred to a 50 mL tube and a
second spore recovery step was conducted by addition of 14 mL cold Extraction Buffer without
Tween® (70% of 0.25 mM KH2PO4 [pH 7.4] and 30% ethanol). After brief vortexing, the second
extraction volume was combined with the first to give 28 mL total volume to allow sufficient
volume for equally splitting the sample between RV-PCR and culture analysis methods.
More detailed information on RV-PCR sampling and analysis can be found in Appendix K.
73
-------
Collect and wash spores in filter cup
Add 3.5 mLTSB medium to filter cup
Vortex filter cups
Take TO aliquot for
DMA concentration and purification
Incubate filter cups
9hr,35°C,230rpm
Vortex filter cups
Take T9 aliquot for
DMA concentration and purification
Real-time PCR analysis of
TO and T9 aliquots:
Data reporting
Figure 2-16. Summary of manual RV-PCR protocol steps and pictures of equipment used
to process samples.
74
-------
Wipe with support in 30-mL sample tube
Add 20-mL Extraction Buffer with Tween
Vortex 20m in
Mix briefly; Transfer 14-mLto 50-mLtube
Add 14-mL Extraction Buffer w/o Tween
Vortex 10min
Mix briefly; Transfer 14-mLto same 50-mLtube
Mix by pipetting; Transfer 13-mLto filter cup
for RV-PCR analysis
Transfer 13-mL from remaining extract solution
to another50-mL tube for culture analysis
Manual RV-PCR Protocol
Centrifuge 30 min, 4000rpm, 4°C
Remove9.5mLto waste (or archive)
Suspend pel let in remaining 3.5 mL
(10° suspension)
UseO.SmL of 10° suspensionfordilutionseries
Plate3repsof100|jLonTSAplates(10-1final)
~2.7mL for further processing (10° suspension)
I '
Perfo rm d i I ution series
(0.5mLin4.5mL)
Butterfield Buffer (1 a1 and 10-2)
Incubate at 35°C, 18-24hr
CountSgcolonies
Fi Iter 0.5 mL thro ugh
Microfunnel filter
Place fi Iter onTSA plate
(10° final)
Plate3repsof100|jLonTSA
plates (10-2and 1Q-3final)
Bg = Bacillus atrophaeus subsp. globigii
±.
Transfer remaining 10°suspension
to 15-mLtube;
Centrifuge 30 min, 4000rpm, 4°C;
Transfer supernatant to waste
Incubate at 35°C, 18-24hr
Co untSg colonies
±.
Add 5-mLTSBmedium; Suspend
pellet; Incubate at 35°C, 18-24hr
Incubate at 35°C, 18-24hr
CountSgcolonies
Two p resumptive BG coloniesfor each sample were confirmed by real-time PCR.
If no colonies were available and the enrichment culture was turbid, the
enrichment culture was tested by real-time PCR.
If turbid, streak onto TSAfor isolation
Check for Sgcolonies
Figure 2-17. Combined BOTE Project protocol for RV-PCR and culture analyses including details of culture analysis.
75
-------
2.9.2.4. Rapid Viability-Polymerase Chain Reaction Sample Processing and
Analysis
After mixing, 13 mL of the suspension was transferred to a filter cup, and spores were collected
on a 0.45 urn filter using a vacuum manifold and a vacuum pump. The remaining spore extract
was processed by traditional culture analysis described in the following section. After filtration,
filter cups were washed with 20 mL of cold filter-sterilized 210 mM KH2PO4 buffer (pH 6.0)
followed by washing with 7 mL of cold 25 mM KH2PO4 buffer (pH 7.4). Filter cups were sealed
on the bottom, 3.5 mL cold TSB medium was added, and top caps were added. The filter cup
manifold was vortex-mixed for 10 min, after which 1 mL (TO) aliquots were withdrawn from filter
cups for processing by magnetic bead-based DMA extraction and analysis by real-time PCR
(see section below)'751. The filter cups were sealed on the top and incubated for nine hr (T9) at
35 °C and 230 rpm. At T9, another 1 mL aliquot was withdrawn and processed as described for
the TO aliquot. In addition, 100 uL aliquots at TO and T9 were processed using a heat lysis
protocol (including incubation at 95 °C for 10 min). Heat lysis DMA extraction was performed in
parallel for all samples as another check on performance of the magnetic bead-based DMA
extraction protocol. Results from heat lysis DMA extraction are reported only where there was a
discrepancy between RV-PCR analysis using magnetic bead-based DMA extraction and culture
analysis.
2.9.2.5. Analysis of Concentrated Enrichment Culture
Additional culture analysis was conducted in cases where positive RV-PCR results [cycle
threshold > 6 and cycle threshold (T9) > 39] and/or cycle threshold (T9) values less than 45
were obtained, but initial culture results were negative (no Bg colonies were evident and/or
results were negative from initial real-time PCR analysis of the enrichment culture). The
additional analysis described below was performed to provide more accurate data for
comparison of the two methods, including a more accurate assessment of the false positive and
false negative percentages. The LRN protocol includes steps to analyze the enrichment culture
using a rapid boil DMA extraction method in the event that colonies are not evident (and
therefore cannot be confirmed by real-time PCR). However, the protocol does not include
concentration of the enrichment culture prior to analysis, so only about 1/50th of the total
enrichment culture volume is processed (100 uL of the total 5 mL culture), and only about 1/20th
of the crude DMA extract volume is analyzed by PCR (5 mL of the total 100 mL). In this study, 1
mL of culture was concentrated by centrifugation and resuspension of the pellet in 0.1 mL
(representing a concentration factor of 10 over the LRN protocol). However, in some cases,
negative Bg PCR results were still obtained. Therefore, for samples showing discrepancies
between methods, the remaining enrichment culture was harvested by repeated centrifugation
and the resulting pellet was suspended in 1 mL of Tris buffer (10 mM, pH 8) (Tris-
ethylenediaminetetraacetic acid (EDTA) buffer solution, Teknova, Hollister, CA) and processed
using the 1 mL magnetic bead-based DMA extraction protocol. The protocol provided an
additional concentration factor of 1/5th and also produced a cleaner DMA extract than that
produced by the LRN rapid boil protocol.
76
-------
2.9.2.6. Bg DNA Standards for Real-time PCR
The DNA standards were generated for the Bg strain. DNA was extracted from cultured cells
using a MasterPure™ Complete DNA and RNA Purification Kit (Epicentre® Biotechnologies Inc.,
Madison, Wl), followed by RNase treatment. The DNA concentration was measured with a
Qubit™ fluorometer (Life Technologies Corporation, Grand Island, NY) using the PicoGreen™
assay (Invitrogen™, Quant-iT™ dsDNA HS assay kit for Qubit fluorometer, Cat. No. Q32854,
Carlsbad, CA). Standard concentrations were prepared in PCR-grade water. On each sample
PCR plate, three replicates of 1 nanogram (ng) per 25 ml_ PCR, three replicates of 100
femtogram (fg) per 25 ml_ PCR, and one no-template control were included.
2.9.2.7. Bg Real-time PCR Analysis
Five microliter (uL) sample aliquots were transferred to a 96-well PCR plate with 20 ml_ of PCR
mix. PCR mix was prepared for the Bg primer-probe set according to conditions detailed in
Annex B of Appendix F, using TaqMan™ 2X Universal Master Mix (Applied Biosystems, Cat.
No. 4305719, Carlsbad, CA). After mixing and centrifugation, PCR was run using the ABI 7500
Fast platform (Applied Biosystems® 7500 Fast Real-Time PCR System, Carlsbad, CA). The
PCR thermal cycling parameters were as follows: 2 min at 50 °C for Uracil-N-Glycosilase (UNG)
incubation, 10 min at 95 °C for AmpliTaq Gold™ activation, followed by 45 amplification cycles
(5 seconds (sec) at 95 °C for denaturation and 20 sec at 60 °C for annealing/extension). For
RV-PCR, each sample was analyzed in triplicate with the Bg primer/probe set. The ROX
reference dye contained in the ABI Universal Master Mix was used to normalize the fluorescent
reporter signal. Automated analysis settings (baseline and threshold) were used throughout. If
triplicate PCR results were not consistent (e.g., 1 of 3 or 2 of 3 positive), PCR was repeated
until consistent results (i.e., three out of three PCR reactions were positive) were obtained.
2.9.2.8. Data Interpretation and Reporting
For RV-PCR, initial and final PCR cycle thresholds (Ct (TO) and Ct (T9), respectively), were
used in the algorithm to determine whether viable (live) spores were present in the sample.
Average values from triplicate analyses were used. TO and T9 results that were ND
("Undetermined" with the PCR system software) were set to 45 to calculate a ACt value. For the
BOTE Project, a Ct [T9] < 39 with a ACt (Ct[TO] - Ct[T9]) > 6 was set as a cutoff value for
positive detection of viable Bg spores. The ACt > 6 criterion represents an increase in DNA
concentration at T9 relative to detectable DNA at TO, if any, as a result of the presence of viable
spores in the sample that germinated and propagated during the nine hours of incubation in
growth medium.
The presumptive Bg CFU were determined based on colony morphology, and counts between
25 and 250 were recorded. For plates or filters containing more than 250 colonies, the number
was recorded as TNTC. Presence of Bg colonies on any of the sample culture plates (from
serial dilution and filter membrane plates) including those that were TNTC represented a
positive result (Bg detected) for that sample. CFU values were corrected for the dilution factor
and expressed as CFU/sample. Real-time PCR data obtained from selected Bg colonies (two
per sample) and/or the enrichment culture were also reported. Analysis of enrichment cultures
was conducted only if no Bg colonies were detected for serial dilution or filter membrane plates,
and cultures were turbid. The sample was also considered positive by culture analysis if the
77
-------
real-time PCR Ct value of the enrichment culture was < 35. The false positive percentage was
determined by dividing the number of samples with positive RV-PCR results but negative culture
results by the total number of samples and multiplying by one hundred. The false negative
percentage was calculated by dividing the number of samples with negative RV-PCR results but
positive culture results by the total number of samples and multiplying by one hundred.
Excel spreadsheets were generated to streamline sample analysis and reporting including
results from positive and negative PCR controls. Sample results by sample barcode were
reported via Excel spreadsheet for each sample batch. Data reports consisted of both RV-PCR
cycle threshold values (for TO and T9) and plate counts corrected for sample dilution as well as
qualitative data (positive/negative) for both RV-PCR and traditional culture analysis. Control
results from field and laboratory blanks/controls were also included in the reports. As per the
SOP (see Appendix K), the results (data) were submitted using the Data Report Form during the
BOTE Project. The data forms were generated and pasted into this report and are therefore
presented as figures.
Because the RV-PCR method is qualitative, the comparison between RV-PCR and the culture
method was performed in terms of positive/negative determination (detection/non-detection) of
viable Bg spores. However, average cycle threshold values (with standard deviations) and
average plate count data (CPU/sample) were also reported.
2.9.2.9. Culture Sample Processing and Analysis
The remaining suspension was used for culture analysis. Briefly, 13 mL was transferred to a 50
ml_ conical tube, followed by centrifugation to pellet the spores. After centrifugation, 9.5 mL of
the supernatant were removed to give the same concentration factor as that obtained for RV-
PCR analysis (i.e., 13 mL filtered and resuspended in 3.5 mL in the filter cup). The pellet was
then resuspended and used to generate a serial dilution for plating. Two 10-fold dilutions (1E-1
and 1E-2) as well as the undiluted spore suspension (1EO) were cultured on TSA plates in
triplicate and incubated 18-24 hr at 35 °C. In addition, 0.5 mL of undiluted spore suspension
was collected onto a filter membrane using a filter funnel apparatus, with the resulting filter
placed onto a TSA plate for incubation (18-24 hr, 35 °C). Finally, the remaining undiluted spore
suspension was centrifuged, the supernatant was removed, and the pellet was suspended in 5
mL TSB medium to create an enrichment culture; the culture was incubated for 18-24 hr at 35
°C and 200 rpm. Colony counts were obtained the next day and corrected for dilution to
determine the number of CPU per sample. If CPU were not evident and enrichment cultures
were turbid, 10 uL aliquots were sampled by sterile inoculation loop and streaked for isolated Bg
colonies onto TSA plates (18-24 hr, 35 °C). For each sample with presumptive Bg colonies
and/or a turbid enrichment culture, Bg real-time PCR analysis of colony DMA (from two colonies
for each sample if available) or the enrichment culture DMA extract was used to confirm the
culture results. The CDC LRN protocol based on the rapid boil DMA extraction method was
used and Bg real-time PCR cycle threshold values less than 35 were used to confirm that the
sample contained viable Bg spores.
78
-------
2.9.3. Air Sample Analysis Methods
Air samples were collected from the SKC BioSamplers®, XMX, STA and the UV-APS. The first
three methods required laboratory analysis of the collected samples to report air concentrations.
Laboratory analyses of these collected samples were performed by INL. The UV-APS data
required post-analysis to report air concentrations as a function of particle size. Details of the
sample and data analysis methods are reported in the following subsections.
2.9.3.1. SKC BioSampler®
2.9.3.1.1. Analysis Method
Upon receipt at the INL Microbiology Laboratory, samples were refrigerated at approximately 4
°C until analyzed within 72 hr of receipt. The viable count method was used to obtain Bg counts
of samples by diluting the samples and plating to enumerate the number of CPU present.
To prepare the laboratory work areas, all laboratory benches and associated equipment were
pre-sterilized, autoclaved, or wiped with a pH-adjusted bleach solution followed by 70%
isopropyl alcohol. For each sample, three replicate TSA plates were labeled with the specific
sample identification number and dilution (1E-1, 1E-2, 1E-3). Also, for each sample, three sterile
15 mL conical test tubes were labeled with the specific sample identification number and dilution
(1EO, 1E-1, 1E-2).
To process the SKC BioSamplers® samples for culture, the samples were first removed from
their foam sleeve from the transport case. The samples were then placed in a rack inside a BSC
and disassembled. Each liquid sample was measured, removed from the SKC BioSampler®
bottom collection vessel with a graduated 10 mL disposable pipette and transferred to the sterile
15 mL conical tube labeled with the appropriate sample identification number and 1EO dilution.
After vortexing on high for 30 seconds, 1 mL of the 1EO sample was removed and placed in the
tube labeled 1E-1 with 9 mL of PBST. After vortexing on high for 30 seconds, 1 mL of the 1E-1
sample was removed and placed in the tube labeled 1E-2 with 9 mL of PBST. All SKC
BioSampler® sample dilutions were retained at 4 °C until additional dilutions were determined to
be unnecessary. After vortexing each dilution tube, 100 uL of all dilutions were removed with a
pipette and placed on the corresponding labeled TSA plates (e.g., 1E-1 tube dilution plated on
the 1E-2 TSA plate), and spread with sterile, disposable Lazy-L cell spreaders. Along with the
triplicate sample dilution plates, a negative control of PBST was plated to check sterility.
To increase the chance of detection, all SKC BioSampler® samples were also processed for
capture on Microfunnel filter membranes and cultured. Pall Microfunnel filters (Pall Corp., Port
Washington, NY) with 0.45 urn pore size were placed on the vacuum manifolds and moistened
with 5 mL PBST. After opening and closing the vacuum valve, 10 mL of PBST was placed in
each filter cup along with 10 mL of the 1EO SKC BioSampler® samples. The vacuum valves
were opened and the suspension was vacuumed through the filter at a pressure <20 cm Hg.
Following vacuuming, the filters were removed and placed on the surfaces of TSA plates
making sure that there was good contact with the agar and no visible air pockets.
79
-------
All dilution and filter plates were incubated at 35 ± 2 °C for a maximum of three days. All plates
were examined within 18-24 hr after the start of the incubation and within 72 hr of sample
collection. CPU on each plate were counted and recorded as follows:
• If the CPU was <300/plate, recorded actual number.
• If the CPU was >300/plate, recorded as TNTC.
• If no growth of suspect colonies, recorded as ND.
After the incubation period, all colonies were enumerated for countable plates (30-300 CPU)
and recorded with two significant figures on the viable count worksheet for the dilutions plated.
The middle range countable plates were used to determine the CFU/mL by using Equation 2-2.
All calculations and counts were recorded in the appropriate laboratory notebook and the viable
count worksheet and archived. All quantitative results were recorded in Excel spreadsheets
according to sample identification number and sent to the EPA project officer after analysis and
quality control checks.
2.9.3.1.2. Data Reduction
Viable Bg spore concentrations were determined for the SKC BioSampler® samples using
dilution plating and filter plating. For each airborne particulate sample, the dilution plate result
was used except when below the typical quantifiable range (i.e., the mean dilution plate count
for each serial dilution was <30 CPU). More CPU are generally detected using the dilution plate
method than the filter plate method when 30 or more colonies are detected using the dilution
plate method. Because each CPU represents one or more culturable spores, the higher number
would be expected to be more accurate. If the mean dilution plate counts were <30 CPU, then
nonzero filter plate results were used because dilution plates with mean counts below 30 have
high variability and, historically, dilutions with counts of 30 to 300 were considered to be in a
quantifiable range. The SKC BioSampler® CPU results were first transformed to CFU/m3 by
dividing the CPU sample measurement (rounded to the nearest integer) by the product of the
measured flow rate of the sampler (L/min) and the sampling duration (15 min), then dividing by
1,000 to convert L to m3 and rounded to the nearest integer. This result was then converted to
CFU/ft3 by dividing by 35.3 (i.e., the number of cubic feet in one cubic meter) and rounding to
the nearest integer. For each SKC BioSampler® sample, the dilution plate results were used in
the subsequent analysis except when TNTC or equal to 0-30 CPU. In the latter case, the filter
result was used.
2.9.3.2. Dycor® XMX/2L-MIL Aerosol Collection Systems
2.9.3.2.1. Analysis Method
Upon receipt at the INL Microbiology Laboratory, samples were refrigerated at approximately 4
°C until analyzed within 72 hr of receipt. XMX samples were processed under aseptic
conditions, typically inside a BSC. Briefly, the filter cartridge was reopened to expose the filter. A
blunt instrument such as a slot screwdriver was used to pry apart the lower joint between the
lower and middle portion of the cartridge, which holds the filter in place. Sterile forceps with
flattened ends (filter forceps) were used to lift the filter carefully from the cartridge. The filter was
then placed into a 50 ml_ conical tube containing 20 ml_ of extraction buffer (i.e., PBS). The lid
80
-------
was returned to the 50 ml_ conical tube, and the filter was allowed to soak in the buffer for 10
min. The filter was vortexed for 2 min and then placed on a wrist action shaker for 15 min.
Following extraction, the fluid was decanted into a new, sterile 50 mL conical tube. The volume
collected was recorded in the laboratory notebook.
The viable count method was used to obtain Bg counts of samples by diluting the samples and
plating to enumerate the number of CPU present. To prepare the laboratory work areas, all
laboratory benches and associated equipment were pre-sterilized, autoclaved, or wiped with a
pH-adjusted bleach solution followed by 70% isopropanol. For each sample, three replicate TSA
plates were labeled with the specific sample identification number and dilution (1E-1, 1E-2, 1E-
3). Also, for each sample, three sterile 15 mL conical test tubes were labeled with the specific
sample identification number and dilution (1EO, 1E-1, 1E-2).
2.9.3.2.2. Data Reduction
Viable Bg spore concentrations were determined for the XMX samples using dilution plating and
filter plating. For each airborne particulate sample, the dilution plate result was used except
when below the typical quantifiable range (i.e., the mean dilution plate count for each serial
dilution was <30 CFU). More CFU are generally detected using the dilution plate method than
the filter plate method when 30 or more colonies are detected using the dilution plate method.
Because each CFU represents one or more culturable spores, the higher number would be
expected to be more accurate. If the mean dilution plate counts were <30 CFU (i.e., considered
"non-detect" or 0 CFU), then nonzero filter plate results were used because dilution plates with
mean counts below 30 have higher relative uncertainty and, historically, dilutions with counts of
30 to 300 were considered to be in a quantifiable range. The results were first transformed to
CFU/m3 by dividing the CFU sample measurement (rounded to the nearest integer) by the
product of the measured flow rate of the sampler (L/min) and the sampling duration (min), then
dividing by 1,000 to convert Lrs to m3 and rounded to the nearest integer. This result was then
converted to CFU/ft3 by dividing by 35.3 (i.e., the number of ft3 in one m3) and rounding to the
nearest integer. For each sample, the dilution plate results were used in the subsequent
analysis except when TNTC or equal to 0-30 CFU. In the latter case, the filter result was used.
2.9.3.3. Mattson-Garvin Model 220 slit-to-agar
2.9.3.3.1. Analysis Method
STA samples were comprised of 150 mm x 15 mm TSA agar plates. Following the collection
procedure, the lid for each Petri dish sample was carefully placed onto the agar-containing
portion. The plates were then transported to the INL laboratory where they were incubated at
35°C for 20 ± 4 hr. Following incubation, CFU were enumerated visually and recorded in the
notebook.
2.9.3.3.2. Data Reduction
The data were transformed by dividing the recorded CFU abundance by the volume of air
sampled. Thus, data are reported as CFU per volume of air sampled.
81
-------
2.9.3.4. Ultraviolet Aerodynamic Particle Sizer
Software (Aerosol Instrument Manager®) provided by the UV-APS manufacturer (TSI, Inc.,
Shoreview, MN) was used to review and evaluate the UV-APS measurements performed in
Rooms 101A and 102. The raw data files were exported to a text-based format in units of
particles per cm3. These data contained 52 size bins ranging from 0.5 micrometer (|o,m) to 20
lorn. The UV-APS data were imported into spreadsheets and further smoothed by calculating a
running average over ten-sample intervals.
For comparison with the UV-APS data, the data from the I BAG monitors located in the first floor
mechanical equipment room (HVAC-1) and Room 101A were also evaluated. I BAG monitors
count particles in sizes ranging nominally from 1.0 |o,m to 10 |o,m in diameter, and the data are
collected in the form of total counts, i.e., not distinguished by size bins. The raw data files
contained particle concentrations in units of particles per L at one second intervals. To reduce
the number of data points to be used for plot generation, the values that occurred on the minute
were extracted, converted to units of #/cm3 and smoothed via 10-min running averages for
comparison with the UV-APS data within the plots below. In some cases, noisy data were
further modified by ignoring apparent outliers in determining the running averages.
2.9.4. Analysis of Sand Samples for the Study of Bacillus Spore Migration from
Inside the Building to Outside
The exposed sand samples were analyzed for the presence of Bg DNA by EPA (Cincinnati, OH)
and USGS, each utilizing different processing techniques. The primers used during PCR dictate
the specificity and sensitivity of the final limit of detection (LOD). However, the DMA extraction
method influences the quantity and quality of template DMA. EPA indirectly extracted DMA from
spores within the sample, while USGS directly extracted DMA from an aliquot of the sand
sample. Direct and indirect DMA extraction techniques each have associated advantages and
disadvantages. Direct extraction yields DMA from any organism within the sample and may
therefore dilute the DMA target of interest among excess DMA. Indirect extraction uses a
washing step to separate the spores from a sample prior to DMA extraction, increasing the
proportion of the target DMA within the final extract. However, the washing step increases the
chance of spore loss prior to detection.
2.9.4.1. Division of Sand Samples between USGS and EPA
Sand samples were aseptically divided between USGS and EPA. The BOTE Project protocol for
division of the sand samples can be found in Appendix F. Aliquots (5 g) of each sample were
sent to the USGS laboratory. No ice or cold packs were used for shipment. The remaining
portion of each sample (-45 g) was held at EPA for analysis.
2.9.4.2. Spore Concentration and DNA Extraction
Each sand sample aliquot kept at EPA was weighed, and the data were recorded before indirect
DNA extraction. Spores from the EPA aliquots were first separated from the sand and
concentrated (See Appendix F). In short, the entire sand sample (mean sample weight 43.4 ±
2.3 g) was washed with PBST. After vigorous mixing and a settling period, the spore-laden
supernatant was transferred to a centrifuge tube. The sample was then centrifuged to pellet the
spores. EPA then used the entire spore pellet for DNA extraction. In comparison, the USGS
82
-------
method directly extracted DMA from 0.25 g of the original 5 g ± 0.1 g sand sample that was
received.
Both EPA and USGS extracted Bg DMA using the MO BIO vacuum-based protocol from the
PowerSoil™ DMA Isolation Kit (MO BIO Laboratories, Inc., Carlsbad, CA). This kit was selected
to process the samples due to its ability to limit humic acid and other PCR-inhibitor carryover
per previous laboratory evaluation. Research has shown that bead-beating-based protocols
such as the Powersoil® DMA Isolation Kit are effective assays for extraction of DMA from
environmental samples of spores and vegetative cells'761. Two uL of kit eluent (100 uL total)
were utilized per qPCR reaction. The extraction protocol is found in Appendix F.
2.9.4.3. Quantitative PCR Detection of B. globigii
EPA and USGS evaluated the extracted sample DMA for the presence of Bg DMA by qPCR
analysis using the specifications outlined by Kane et al. (2009)[3Sl. This analysis utilized Bg42F
and Bg104R, forward and reverse primers with probe Bg60FT to detect a 63-base pair amplicon
of the recF gene of Bg (Table 2-15). LLNL developed and tested the sensitivity of these primer
sequences'351. The probe was labeled with fluorescein amidite on the 5 ft end and the quencher
tetramethylrhodamine on the 3 ft end. All real-time digital data files were cataloged and backed
up on an external drive.
Both laboratories utilized the same primer sequences and cycle numbers. However, there were
slight differences between the primer concentration and cycle times (Table 2-16). While an in-
depth study was not conducted to determine the actual differences between the two methods
utilized, a comparison of the instrument LODs suggests that there was not a significant
difference between the two methods.
Table 2-15. Bg primer and probe sequences.
Label
Bg42F
Bg104R
Bg60FT
Sequence
5'-CGCGCCCGAGGACTTAA -3'
5'-ATGTCAAGGAAACCGCCGTC -3'
5'-FAM-TCTCGTAAAGGGCAGCCCGCAAG-TAMRA-3'
83
-------
Table 2-16. Differences between EPA and USGS qPCR parameters.
Parameter
Thermocycler
Reaction Volume, ul_
Probe Concentration , |jM
Primer Concentration, |jM
95 °C Denature Time, min
60 °C Annealing Time/ Cycle, s
EPA
ABI Prism® 7900HT
20
0.4
0.5
1
60
USGS
ABI StepOne™
25
1
1
10
15
2.9.4.4. EPA Analytical Specifics
EPA used an ABI Prism® 7900HT (Life Technologies Corporation, Carlsbad, CA) for qPCR
analysis. Each reaction consisted of 2 uL template DMA within a 20 uL PCR reaction. For each
sample, three replicates per dilution were assayed for 45 cycles. Each PCR run utilized a 96-
well plate that contained 14 samples, 3 no template controls (NTCs), and amplification positive
and negative controls (three of each; see (Table 2-17). Specificity of qPCR detection was
determined by comparison of results to nontarget DMA (Escherichia coli) and positive control
DMA from Bg spore preparations that had formerly been characterized. A total of 16 qPCR 96
well plates were required to assay all the samples. Results for samples and controls are listed in
Section 3.2.4.
Table 2-17. EPA PCR analytical controls used for each of the sixteen 96-well plate
assays.
Control ID
Bg BOTE DMA (1 0,1 00 GEq)
Bg BOTE DMA (1 ,01 0 GEq)
E. coli DMA (E-2 dilution)
NTC
Purpose
Amplification positive PCR Control
Amplification positive PCR Control
Amplification negative PCR Control
Negative PCR Control
Frequency
3 PCR reactions per run
3 PCR reactions per run
3 PCR reactions per run
3 NTC samples per run
Criteria for acceptance of negative controls required that all replicates be reported as ND. The
acceptance criterion for positive controls was for the cycle threshold time to be within 5% of the
prior determined cycle threshold time. Samples with higher target cell numbers will have a lower
cycle threshold time, while those with lower target cell numbers will have a higher cycle
threshold time. The Bg BOTE DMA control positive stock solution was obtained by culturing Bg
84
-------
spores used in the BOTE Project and extracting the DMA within a controlled laboratory setting.
The concentrations were measured as genomic equivalents (GEq).
2.9.4.5. EPA PCR Inhibition Analysis
PCR inhibition due to the decontamination chemicals used during the BOTE Project was tested
by spiking selected samples with an internal positive control (IPC). One post-decontamination
sample from each floor and round that showed a loss of sensitivity when compared to its pre-
decontamination counterpart and one post-dissemination sample from the first floor of each
decontamination technology round were selected as controls (see Table 2-18). The I PC
consisted of -10 GEq of Bg control DMA. The goal was to have a low concentration of target
DMA in each spiked reaction tube that was reliably within the detectable limit.
Table 2-18. Samples selected for BOTE Project PCR inhibition due to decontamination
agent test.
Sample ID
48
49
91
117
119
164
190
212
Description
Round
Round
Round
Round
Round
Round
Round
Round
1/VHP®/decontamination/B1 b
1/VHP®/decontamination/B2a
2/pH-adjusted bleach/dissemination/B1 a
2/pH-adjusted bleach /decontamination/61 a
2/pH-adjusted bleach /decontamination/B2a
3/CIO2/dissemination/B1 a
3/CIO2/decontamination/B2a
3/CIO2/decontamination/B1 b
2.9.4.6. USGS Analytical Controls
The USGS used the ABI StepOne™ (Life Technologies Corporation, Carlsbad, CA) with a 48
well format for all qPCR analysis. Within each 25 uL qPCR reaction, USGS utilized 2 uL of
template DMA. Duplicate reactions followed the specifications outlined by Kane et al.[351. DMA
concentrations of Bg standards were measured using an Invitrogen Qubit® Fluorometer
(Invitrogen Life Technologies, Grand Island, NY). Various standard concentrations were
prepared in PCR-grade water, and three dilutions of the standards were run with each set of
qPCR plates in addition to a negative control (Table 2-19).
85
-------
Table 2-19. USGS PCR analytical controls.
Control ID
Bg DMA (32.5 GEq)
Bg DMA (325 GEq)
Bg DMA (3250 GEq)
NIC
Purpose
Amplification positive PCR Control
Amplification positive PCR Control
Amplification positive PCR Control
Negative PCR control
Frequency
2 PCR reactions per run
2 PCR reactions per run
2 PCR reactions per run
2 NIC samples per run
2.10. Decontamination Methods
Three decontamination methods were chosen based on results that had been obtained in
laboratory studies. An interagency group examined the best available science for
decontaminating a facility that had been contaminated with a Bacillus species and selected
three separate approaches:
• fumigation with H2O2 using STERIS VHP®;
• surface decontamination using pH-adjusted bleach (amended bleach); and
• fumigation with CIO2.
The BOTE Project provided an opportunity to test the performance of these three
decontamination approaches operationally. The decontamination methods are discussed in
Sections 4.1.1, 4.1.2, and 4.1.3.
2.10.1. Fumigation by STERIS Corporation VHP®
The first decontamination method utilized in this project was the full-facility fumigation with
hydrogen peroxide vapor. Both the BIOQUELL HPV and STERIS Corporation VHP® processes
were considered for use in this project. Based upon the results of laboratory testing and past
use, STERIS Corporation ("STERIS") was selected to fumigate the facility using their VHP®
technology. The facility configuration, as described in Section 2.1, contained a mixture of porous
and nonporous surfaces throughout the building. The details of the decontamination process are
introduced briefly below and discussed in detail in Section 4.1.1.
During the VHP® process, an aqueous solution of H2O2 is flash-vaporized into a stream of
dehumidified heated air. This stream of H2O2 vapor is then injected into the area to be
decontaminated. The fumigation proceeds until a sufficient exposure of the VHP®, in terms of
time and concentration, to the contaminated space has been achieved. After sufficient exposure
has been achieved, the concentration of VHP® remaining in the space is reduced by the use of
a decomposition catalyst, natural decomposition and the introduction of fresh air.
2.10.2. Surface Decontamination Approach using pH-Adjusted Bleach
The second round of the BOTE Project utilized a decontamination process that employed pH-
adjusted bleach as the decontaminant. The details of the decontamination process are
introduced briefly below and discussed in detail in Section 4.1.2. The process started with the
generation of air flow in and out of the facility using a filtered NAM to reduce any airborne Bg in
86
-------
the facility. This filtered flow was maintained throughout the process to attempt to remove any
airborne Bg that might be generated via reaerosolization due to decontamination activities in the
facility. Decontamination personnel in Level C PPE then entered the facility to remove all porous
materials that were deemed to present a significant challenge for successful decontamination
using pH-adjusted bleach. These items were bagged, sprayed thoroughly with pH-adjusted
bleach, and removed from the facility for treatment as waste. The interior of the facility was then
sprayed with pH-adjusted bleach by decontamination personnel in Level B PPE. The spraying
was completed with a chemical sprayer and thoroughly wetted all surfaces in the facility. The
day after the majority of the spraying was completed, standing water was vacuumed up, and
heaters and fans were used to promote drying.
2.10.3. Fumigation by Sabre Technical Services, LLC, with Chlorine Dioxide Gas
The third and final round of the BOTE Project utilized fumigation with CIO2. Sabre Technical
Services LLC ("Sabre") was selected to fumigate the facility with CIO2 based upon technical
considerations and cost. The same facility configuration as in the previous two rounds was
used, as described in Section 2.1, and the facility contained a mixture of porous and nonporous
surfaces throughout the building. The details of the decontamination process using CIO2 are
discussed in Section 4.1.3.
Sabre utilizes a wet generation system by mixing water, sodium hypochlorite, hydrochloric acid,
and sodium chlorite to generate CIO2 in the liquid phase. A liquid-air stripper is then used to
transfer the CIO2 from the liquid phase to the vapor phase. The vapor phase CIO2 is then blown
into a facility using a 7,000 CFM fan. Following fumigation, the CIO2 must be removed from the
air. Sodium hydroxide is added to the generation system to neutralize the CIO2, and then the air
in the facility is polished using an activated carbon scrubber.
2.10.4. Treatment of Decontamination line Wash Water
Chlorine bleach has been shown to be effective for Ba inactivation in buffered water at certain
contact times'77"791. However, wastewater represents a different matrix for which the
effectiveness of chlorine is not well known. Wastewater generated during washdown may
contain components with a chlorine demand that could lessen the effectiveness of chlorine in
the inactivation of Ba spores.
EPA is currently conducting bench-scale studies to determine the effectiveness of chlorine to
inactivate a Ba spore surrogate, Bg, in wash water generated during a decontamination event.
Bg is more resistant to chlorine than Ba and serves as a conservative indicator of inactivation.
The research involves testing a Ba decontamination technique recommended by the NRT[801.
The NRT method calls for 1 part (by volume) household bleach and 1 part white vinegar for
every 10 parts wash water. The chlorine concentration resulting from this method is
approximately 0.5 % sodium hypochlorite, assuming a starting concentration of 6% sodium
hypochlorite in household bleach. The pH will be 7. At pH 7, the bleach solution contains a
greater amount of hypochlorous acid, a more effective disinfecting agent for Ba spores than the
hypochlorite ion. The recommended contact time is 1-2 hr.
In preparation for the BOTE Project wash water inactivation study, EPA conducted bench-scale
inactivation research at the EPA Test and Evaluation facility in Cincinnati, OH, using water that
87
-------
was representative of incident cleanup activities (report in development). This study included
bench-scale tests using a 5 L glass reactor and subsequently a scaleup to 55-gal drums.
Simulated decontamination wash water was obtained by washing down laboratory walls, floors
and other external surfaces and then spiking with Bg spores resulting in a concentration of
1.0E6 spores/1 ml_. This research indicated that a > six log inactivation (i.e., log reduction) can
be achieved under laboratory conditions using a 5% concentration of bleach (0.25 % sodium
hypochlorite) without the addition of vinegar.
For the BOTE Project, water from the washdown of personnel exiting the contaminated building
and going through the Decontamination Line was collected for each sampling event in a 55-gal
drum and chlorinated to test the effectiveness of chlorine treatment of wash water under field
conditions. The calculation of Bg spore log reduction was possible only for the last sampling
event on May 12, 2011, for which 0.15 mL of an estimated 1.5E8 CFU/mL suspension was
spiked into the wash water. All other sampling events resulted in non-detection of spores. For
the spiked sampling event, log reduction was calculated in accordance with Equation 2-1.
88
-------
3. RESULTS
This section of the BOTE Project report outlines the sampling results. Use of these results
relative to the project objectives is discussed in Section 4. All sample results are reported in
Appendix G.
For analysis of results in this chapter, these data were analyzed by one-way analysis of
variance (ANOVA) for each round. Bonferroni post-hoc tests were subsequently conducted to
evaluate each contrast. Significance was assessed using a p-value equal to 0.05. SigmaPlot 11
(Systat Software Inc., San Jose, CA) was utilized for the above mentioned statistical analyses.
Average results are provided, along with one standard deviation (SD) in parentheses (±SD).
3.1. Reference Method Results
3.1.1. Settling Plates
If the settling plate count was > 300 CPU, the plate result was recorded as TNTC. For all of the
countable plates (up to 300 CPU), the results were transformed to CFU/ft2 by dividing the CPU
final laboratory counts by the surface area of the plate. Settling plates were collected only on the
second floor to provide an indication of dissemination concentrations and potential gradients in
the facility. No plates were used on the first floor because surface loadings were expected to
yield TNTC on all plates and would not be of particular use. Further, the settling plate results
were used to provide an indication of the surface loadings expected in the rooms prior to
surface sampling. Surface sampling results were ultimately used to determine if the target
loading requirements were achieved and for assessment of decontamination efficacy.
3.1.1.1. Round 1 Settling Plate Results
All settling plate results on the second floor were identified as TNTC, indicating that surface
loadings were expected to be > 4.9E3 CFU/ft2 (i.e., 3.0E2 CFU divided by 0.061 ft2 [surface
area of 85 mm TSA settling plate]).
3.1.1.2. Round 2 Settling Plate Results
Settling plates ranged from 2.1E3 to TNTC (> 4.9E3) CFU/ft2. The average loading based upon
the settling plate data was 3.6E3 (± 9.6E2) CFU/ft2; TNTC values were included as 4.9E3
CFU/ft2. The settling plate results per second floor room are listed in Table 3-1.
3.1.1.3. Round 3 Settling Plate Results
All settling plate results on the second floor were identified as TNTC, indicating that surface
loadings were expected to be > 4.9E3 CFU/ft2 (i.e., 3.0E2 CFU divided by 0.061 in2 [surface
area of 85 mm TSA settling plate]).
89
-------
Table 3-1. Summary of settling plate results from Round 2
Room
Room 206
Room 206
Room 206
Room 207
Room 207
Room 207
Room 208
Room 208
Room 208
Room 209
Room 209
Room 209
Room 210
Room 210
Room 210
Room 211
Room 211
Room 211
Room 21 2
Room 21 2
Room 21 2
Room 21 3
Room 21 3
Room 21 3
Barcode
4565
4567
4574
4544
4573
4577
4547
4548
4575
4550
4578
4589
4546
4569
4584
4583
4586
4588
4570
4572
4576
4549
4571
4579
Settling Plate
Area (ft2)
0.061
0.061
0.061
0.061
0.061
0.061
0.061
0.061
0.061
0.061
0.061
0.061
0.061
0.061
0.061
0.061
0.061
0.061
0.061
0.061
0.061
0.061
0.061
0.061
Result (CPU)
TNTC
TNTC
TNTC
TNTC
2.1E2
2.1E2
2.6E2
1.7E2
2.9E2
2.0E2
1.7E2
2.2E2
2.4E2
2.9E2
1.9E2
1.3E2
1.3E2
1.5E2
2.6E2
2.4E2
1.7E2
1.6E2
1.5E2
2.1E2
Result
(CPU/ ft2)
TNTC
TNTC
TNTC
TNTC
3.5E3
3.4E3
4.2E3
2.8E3
4.8E3
3.2E3
2.8E3
3.6E3
4.0E3
4.7E3
3.1E3
2.1E3
2.2E3
2.5E3
4.3E3
3.9E3
2.8E3
2.6E3
2.5E3
3.4E3
TNTC = Too Numerous to Count (> 4,900 CFU/ft2)
3.1.2. Reference Material Coupons
RMCs were placed on both Floor 1 and Floor 2 (collocated with TSA settling plates). The RMCs
were collected during the pre-decontamination (characterization) surface sampling phase in
each round and prior to surface sampling at nearest locations within a specific room (as with the
TSA settling plates). The RMCs were sent to the INL Microbiology Laboratory to be extracted
and dilution plated. The ability to dilution plate the RMCs provided for a higher dynamic range
with respect to the indication of surface loading (hence their use on both floors). For all of the
countable plates (30-300 CFU), the results were transformed to CFU/ft2 by dividing the CFU
final laboratory counts by the surface area of the RMC (0.012 ft2).
90
-------
3.1.2.1. Round 1 RMC Results
The results for the Round 1 RMCs placed prior to dissemination and picked up before pre-
decontamination surface sampling at nearest locations in a particular room are reported in Table
3-2. The mean RMC loading for first floor rooms was 3.7E5 (±1.6E5) CFU/ft2. This average was
within the target range specified for surface loading on the first floor (1E4 to 1E6 CFU/ft2). The
mean RMC loading for second floor rooms was 1.1 E4 (±4.5E3) CFU/ft2. All quantified RMC
samples associated with the second floor rooms were higher than the target spore loading of
1E2 to 2E2 CFU/ft2. However, the results did indicate a significant difference (p <0.001)
between first and second floor surface loadings, which was the critical criterion for the
decontamination efficacy study.
Table 3-2. Summary of RMC results from Round 1
Room
Room 101 A
Room 101 A
Room 101 A
Room 102
Room 102
Room 102
Room 103
Room 103
Room 103
Room 104
Room 104
Room 104
Room 105
Room 105
Room 105
Room 106
Room 106
Room 106
Room 107
Room 107
Room 107
Room 108
Room 108
Room 108
Room 109
Room 109
Room 109
Room 110
Barcode
1047
1062
1064
1056
1066
1069
1004
1022
1053
1070
1077
1087
1003
1076
1095
1058
1082
1083
1075
1081
1119
1059
1079
1084
1078
1080
1160
1033
RMC Area (ft2)
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
Result (CPU)*
3.2E3
3.5E3
3.4E3
7.8E3
3.0E3
3.2E3
4.4E3
3.1E3
6.0E3
3.7E3
5.6E3
3.9E3
4.8E3
6.4E3
4.6E3
3.6E3
6.0E3
4.2E3
2.8E3
2.5E3
2.3E3
1.1 E4
3.7E3
3.2E3
6.0E3
3.2E3
6.8E3
2.8E3
Result
(CFU/ft2)
2.7E5
3.0E5
2.8E5
6.5E5
2.5E5
2.7E5
3.7E5
2.6E5
5.0E5
3.1E5
4.7E5
3.3E5
4.0E5
5.3E5
3.8E5
3.0E5
5.0E5
3.5E5
2.3E5
2.1E5
1.9E5
9.2E5
3.1E5
2.7E5
5.0E5
2.6E5
5.7E5
2.3E5
91
-------
Room
Room 110
Room 110
Room 206
Room 206
Room 206
Room 207
Room 207
Room 207
Room 208
Room 208
Room 208
Room 209
Room 209
Room 209
Room 210
Room 210
Room 210
Room 211
Room 211
Room 211
Room 21 2
Room 21 2
Room 21 2
Room 21 3
Room 21 3
Room 21 3
Barcode
1072
1085
1074
1090
1091
1051
1092
1113
1055
1073
1154
1018
1019
1063
1024
1045
1046
1031
1097
1121
1025
1050
1138
1040
1133
1157
RMC Area (ft2)
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
Result (CPU)*
3.9E3
2.9E3
1.2E2
1.2E2
1.2E2
1.0E2
1.0E2
1.0E2
2.0E2
1.2E2
2.0E1
4.0E1
1.4E2
6.0E1
2.6E2
1.2E2
1.6E2
2.0E2
1.6E2
1.0E2
1.6E2
1.0E2
1.8E2
2.0E2
1.4E2
1.6E2
Result
(CFU/tt2)
3.3E5
2.5E5
1.0E4
1.0E4
1.0E4
8.3E3
8.3E3
8.3E3
1.7E4
1.0E4
1.7E3
3.3E3
1.2E4
5.0E3
2.2E4
1.0E4
1.3E4
1.7E4
1.3E4
8.3E3
1.3E4
8.3E3
1.5E4
1.7E4
1.2E4
1.3E4
*CFU results based on
counts were <30 CPU,
dilution plating with mean CPU plate counts of 30-300 CPU; if lowest dilution plate
filter plate results were used if available.
3.1.2.2. Round 2 RMC Results
The results for the Round 2 RMCs placed prior to dissemination and picked up before pre-
decontamination surface sampling at nearest locations in a particular room are reported in Table
3-3. The mean RMC loading for first floor rooms was 1.4E4 (±3.8E4) CFU/ft2, considering ND as
equal to 0 CPU. This average is within the target range specified for surface loading on the first
floor (1E4 to 1E6 CFU/ft2). However, all RMC samples associated with Rooms 102, 105, 106,
107, 108, 109, and 110 were ND for Bg spores. Excluding these NDs, the mean loading for
RMCs on the first floor was 1.1E5 (±3.6E4) CFU/ft2. This value was more consistent with the
Round 1, Floor 1 results and consistent with the target loading criteria. However, the majority of
RMC samples on the first floor in Round 2 were reported as ND for unknown reasons. The
RMCs were meant to provide an indication of the surface loading; ultimately, surface sampling
was used to determine if the criterion was met and for the decontamination efficacy
92
-------
assessment. Based upon surface sampling results (see below), the RMC results for this round
did not impact the project or data analysis.
The mean RMC loading for second floor rooms was 7.0E3 (±4.8E3) CFU/ft2. All quantified RMC
samples associated with the second floor rooms were higher than the target spore loading of
1E2 to 2E2 CFU/ft2. One RMC on the second floor was ND for Bg. Excluding the inclusion of
this value as 0 CPU, the mean loading for second floor RMCs was 7.3E3 (±4.7E3) CFU/ft2.
When the unexplained NDs are included, the results did not indicate a significant difference (p
=1.0) between first and second floor surface loadings. Excluding the NDs, the results did
indicate a significant difference (p<0.001) between first and second floor surface loadings.
These results were meant to provide an indication of surface loading; the surface sampling
results were the critical criteria for the decontamination assessment.
Table 3-3. Summary of RMC Results from Round 2
Room
Room 101 A
Room 101 A
Room 101 A
Room 102
Room 102
Room 102
Room 103
Room 103
Room 103
Room 104
Room 104
Room 104
Room 105
Room 105
Room 105
Room 106
Room 106
Room 106
Room 107
Room 107
Room 107
Room 108
Room 108
Room 108
Room 109
Room 109
Room 109
Barcode
1028
1110
1134
1140
1155
1158
1029
1153
1159
1114
1147
1152
1023
1112
1141
1116
1131
1156
1101
1123
1137
1100
1124
1151
1108
1132
1148
RMC Area (ft2)
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
Result (CPU)*
1.1 E3
7.6E2
ND
ND
ND
ND
1.5E3
ND
ND
ND
ND
1.7E3
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Result
(CFU/ft2)
9.2E4
6.3E4
ND
ND
ND
ND
1.3E5
ND
ND
ND
ND
1.4E5
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
93
-------
Room
Room 110
Room 110
Room 110
Room 206
Room 206
Room 206
Room 207
Room 207
Room 207
Room 208
Room 208
Room 208
Room 209
Room 209
Room 209
Room 210
Room 210
Room 210
Room 211
Room 211
Room 21 2
Room 21 2
Room 21 2
Room 21 3
Room 21 3
Room 21 3
Barcode
1043
1094
1109
1021
1088
1146
1012
1030
1144
1037
1120
1126
1042
1068
1118
1015
1032
1089
1049
1117
1007
1009
1057
1014
1052
1129
RMC Area (ft2)
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
Result (CPU)*
ND
ND
ND
1.0E2
2.4E2
2.0E1
1.0E2
1.4E2
6.0E1
4.0E1
2.0E1
4.0E1
1.4E2
1.0E2
6.0E1
2.0E1
1.2E2
8.0E1
1.8E2
6.0E1
1.0E2
1.0E2
ND
1.2E2
2.0E1
6.0E1
Result
(CFU/ft2)
ND
ND
ND
8.3E3
2.0E4
1.7E3
8.3E3
1.2E4
5.0E3
3.3E3
1.7E3
3.3E3
1.2E4
8.3E3
5.0E3
1.7E3
1.0E4
6.7E3
1.5E4
5.0E3
8.3E3
8.3E3
ND
1.0E4
1.7E3
5.0E3
*CFU results based on
counts were <30 CPU,
ND = non-detect.
dilution plating with mean CPU plate counts of 30-300 CPU; if lowest dilution plate
filter plate results were used if available.
3.1.2.3. Round 3 RMC Results
The results for the Round 3 RMCs placed prior to dissemination and picked up before pre-
decontamination surface sampling at the nearest locations in a particular room are reported in
Table 3-4.The mean RMC loading for first floor rooms was 2.1E5 (±7.3E4) CFU/ft2. This
average was within the target range specified for surface loading on the first floor (1E4 to 1E6
CFU/ft2). The mean RMC loading for second floor rooms was 1.2E4 (±6.1E3) CFU/ft2. All
quantified RMC samples associated with the second floor rooms were higher than the target
spore loading of 1E2 to 2E2 CFU/ft2 (see Table 3-4). However, the results did indicate a
significant difference (p <0.001) between first and second floor surface loadings, which was the
critical criterion for the decontamination efficacy study. Both first and second floor RMC results
94
-------
were highly consistent with the results from Round 1. This consistency further highlights an
unexplained anomaly with respect to the RMC results for Round 2.
Three blanks (negative controls) were included during Round 3, as shown in Table 3-4. These
blanks were included to assess potential contamination during handling and transport of the
RMCs. All three blank RMCs were ND for Bg.
Table 3-4. Summary of RMC results from Round 3
Room
Room 101 A
Room 101 A
Room 101 A
Room 102
Room 102
Room 102
Room 103
Room 103
Room 103
Room 104
Room 104
Room 104
Room 105
Room 105
Room 105
Room 106
Room 106
Room 106
Room 107
Room 107
Room 107
Room 108
Room 108
Room 108
Room 109
Room 109
Room 109
Room 110
Room 110
Room 110
Room 206
Room 206
Barcode
1026P
1145P
1162
1020
1048
1067
1001 p
1035P
1061P
1130
1135
1161
1060P
1115
1136
1005
1036
1149
1008
1039
1096
1006
1065
1093
1016
1054
1143
1103
1139
1027
4804
4805
RMC Area (ft2)
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
Result (CPU)*
4.4E3
4.6E3
2.9E3
2.3E3
2.5E3
3.2E3
2.6E3
4.4E3
3.2E3
2.2E3
2.9E3
4.2E2
2.2E3
2.1E3
2.0E3
2.2E3
2.0E3
1.9E3
1.5E3
1.6E3
2.3E3
2.6E3
2.1E3
2.0E3
1.7E3
2.2E3
2.8E3
2.3E3
3.1E3
2.9E3
6.0E1
1.0E2
Result
(CPU/ ft2)
3.7E5
3.8E5
2.5E5
1.9E5
2.1 E5
2.7E5
2.2E5
3.7E5
2.7E5
1.8E5
2.4E5
3.5E4
1.8E5
1.8E5
1.6E5
1.8E5
1.6E5
1.6E5
1.2E5
1.4E5
1.9E5
2.2E5
1.8E5
1.7E5
1.4E5
1.8E5
2.4E5
1.9E5
2.6E5
2.4E5
5.0E3
8.3E3
95
-------
Room
Room 206
Room 206
Room 207
Room 207
Room 207
Room 207
Room 208
Room 208
Room 208
Room 209
Room 209
Room 209
Room 210
Room 210
Room 210
Room 211
Room 211
Room 211
Room 21 2
Room 21 2
Room 21 2
Room 21 3
Room 21 3
Room 21 3
Room 21 3
Barcode
4812
4806T
1086
1099
1125
481 0T
1002
1107
1128
1013
1038
1111
4807
4808
4811
1044
1098
1102
1010
1041
1150
1017
1034
1105
481 3T
RMC Area (ft2)
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
Result (CPU)*
2.0E1
ND
1.6E2
1.4E2
4.0E1
ND
8.0E1
1.0E2
1.4E2
1.2E2
1.4E2
2.2E2
2.8E2
2.6E2
2.2E2
1.8E2
2.0E2
1.2E2
1.8E2
2.6E2
1.6E2
2.2E2
4.0E1
1.2E2
ND
Result
(CPU/ ft2)
1.7E3
ND
1.3E4
1.2E4
3.3E3
ND
6.7E3
8.3E3
1.2E4
1.0E4
1.2E4
1.8E4
2.3E4
2.2E4
1.8E4
1.5E4
1.7E4
1.0E4
1.5E4
2.2E4
1.3E4
1.8E4
3.3E3
1.0E4
ND
*CFU results based on dilution plating with mean CPU plate counts of 30-300 CPU; if lowest dilution plate
counts were <30 CPU, filter plate results were used if available.
f = Quality control blank samples.
(3 = Lowest serial dilution values were below the quantitation limit of 30 CPU; however, filter plate results were
TNTC. Therefore, the serial dilution values were reported.
3.1.2.4. Comparisons of RMC Results
For Floor 1, the difference between the average RMC values were considered statistically
significant for each round (p<0.001 for all post-hoc comparisons). Round 3 had the highest
average loading, and Round 2 had the lowest. For Floor 2, the average RMC loading for Round
2 was lower than that of Round 1 and 3. The differences were not suggested to be statistically
significant at the 95% confidence value. Likewise, the settling plates for Round 2 also had the
lowest average CFU; Round 2 was the only round in which all plates were not TNTC.
96
-------
3.2. Surface, Air, Sand, and Water Sampling Results
During the BOTE Project Phase 1, sampling teams collected a total of 3,259 samples across all
media (surface, air, sand and water), including quality assurance (QA) samples (blanks). A total
of 2,801 test and 458 blank samples were collected. Sampling teams collected 83 test and 18
blank samples during the MFP event (background samples). The teams collected a total of
1,130 (975 test, 155 blank), 1,039 (911 test, 128 blank), and 989 (832 test, 157 blank) samples
during Rounds 1-3, respectively. A summary and breakdown of air and surface samples
collected is shown in Table 2-7.
Surface samples including sponge-stick wipes, Versalon® wipes, macrofoam swabs (swabs),
and vacuum socks were taken during two events within each round: pre-decontamination and
post-decontamination. The pre-decontamination sampling is generally referred to as
characterization sampling. The post-decontamination surface sampling is often synonymous
with clearance sampling. These samples were used for the determination of decontamination
efficacy within each round.
Air sampling was conducted for several purposes during the BOTE project. First, XMX and
STAs were used to conduct an assessment of AAS. Second, the UV-APS and SKC
BioSampler® were used in support of a reaerosolization study. The results from the SKC
BioSamplers® were also used during an exposure assessment.
The sand dish results were used to assess the potential for viable spores to be transported from
inside the facility to outside the facility during the BOTE Project.
Lastly, wash water from the Decontamination Line and waste water from the pH-adjusted bleach
decontamination process were analyzed for viable Bg. The intent of these results was to test the
effectiveness of chlorine to inactivate spores in the Decontamination Line wash water and
assess on-site wash/wastewater treatment.
The results from the surface, air, sand, and water sampling and analysis are presented in the
subsections below. The analysis of these data with respect to the study objectives is detailed in
Section 4.
3.2.1. Surface Sampling Results
Surface sampling was conducted for three primary purposes: (1) assessment of
decontamination efficacy, (2) reaerosolization study, and (3) RV-PCR method evaluation. For
the assessment of decontamination efficacy, swab, sponge-stick wipes, and vacuum sock
samples were collected and analyzed by the LRN. Versalon® wipe samples were collected and
analyzed by INL for the reaerosolization study and by LLNL for the RV-PCR method evaluation.
The results from the surface samples analyzed by the LRN or INL are reported below for each
round. Results from the samples for the RV-PCR evaluation are reported in Section 3.2.2.
Details for all samples and sample results, compiled from the BROOM database, can be found
in Appendix G.
All quantitative results should be considered with appropriate variance (i.e., confidence
intervals). While contributions to variance for microbial sampling and analysis are
97
-------
acknowledged, the quantitative variance has not been established to date for any sampling or
analysis methods. Comparison of quantitative values presented in this report is done using an
internal determination of the variance in the data due to the number, types, and replication of
sampling performed. This analysis is suitable for the internal comparisons used with the BOTE
Project; however, the reported confidence levels should not be assumed as standardized for
use elsewhere.
3.2.1.1. MFP (Baseline Assessment) Surface Sampling Results
During the MFP event, sample teams collected 63 surface samples comprised of swabs,
sponge-stick wipes, and vacuum socks. An additional 13 blank (QC) samples of these types
were also collected. The total number of samples (surface and blank) for each floor along with
the number of samples in which Bg was detected are reported in Table 3-5. The facility was
widely contaminated with detectable Bg prior to the start of the BOTE Project rounds. Nearly
48% of all surface samples sent to the LRN laboratories resulted in detectable Bg. A higher
percentage of baseline assessment samples from the second floor contained detectable Bg.
Data similar to the data described above are presented for swab samples in Table 3-6, sponge-
stick wipes in Table 3-7, and vacuum socks in Table 3-8. These data are also shown graphically
in Figure 3-1. The locations of all samples are shown in Figure 3-2 (Floor 1) and Figure 3-3
(Floor 2). Overall, the highest percent detection was from vacuum sock samples (Floor 1 and
Floor 2) and sponge-stick wipe samples (Floor 2). These data should not be taken to indicate
the effectiveness or efficiency of a particular sampling type, as comparable samples were not
taken across each type. Swabs were used for small areas or hard to reach locations such as
HVAC supply diffusers or light fixtures, sponge-stick wipes were used over larger areas on
nonporous surfaces, and vacuum socks were used over the largest areas for porous materials.
Table 3-5 through Table 3-8 also show the number of samples that showed detection of Bg on
spread plates and/or filter plates. Samples with less than 30 CFU on the lowest dilution spread
plate were filter plated. Therefore, some samples may have had Bg detected on both spread
and filter plates. Additionally, not all samples that had less than 30 CFU on the lowest dilution
spread plate were filter plated (as discussed in Section 2.9.1.1). In the MFP event, two samples
that had less than 30 CFU were not filter plated. Both samples had an average of approximately
9 CFU on the lowest dilution spread plates (triplicate plates at each dilution for each sample).
These data were used as estimated CFU counts for subsequent surface concentration analysis,
consistent with ASTM D5465-931741. As a point of reference, both samples with missing filter
plate data were from Floor 1 vacuum sock samples. The average surface concentration using
these two data points as estimated values is 10 (±47) CFU/ft2. Considering these two values at
the quantification limit (30 CFU), the average loading is 1.4E2 (±3.7E2) CFU/ft2. These
averages consider ND values as equal to zero. Using the ttest[81], the two-tailed p-value is
0.0556 and the difference between the two averages is not quite statistically significant at a 95%
confidence level.
Detectable Bg was found in 11 of 33 samples on Floor 1 and 20 of 33 samples on Floor 2. One
of the samples with detectable Bg was one of the six blank samples from Floor 2. This sample
was ND by spread plating and had 2 CFU detected by filter plating. Of the other 19 samples
98
-------
with detectable Bg from Floor 2, 10 used filter plate data and resulted in an average of 62 (±66)
CPU. Three samples were at or no more than twice the CPU determined in the blank sample.
The other seven samples were at least ten times greater than the blank sample. While any
detectable Bg in a blank raises concerns with respect to laboratory and field contamination
control measures, the very low CPU detected in the single sample provided a strong indication
that MFP results were not impacted by any source of confounding contamination.
The conclusion was that the average surface concentration determined during the MFP did not
impact the BOTE Project, since the values were not greater than the target surface loading
desired for Rounds 1-3. The background contamination in the facility was determined not to add
significantly to the contamination after Round 1 dissemination (compare MFP and Round 1 pre-
decontamination results). Surface concentrations in the MFP were an insignificant fraction of the
concentrations measured after dissemination in Round 1, 2, or 3.
The results from the wipe samples analyzed by INL for the reaerosolization study, targeted in
Rooms 101A and 102, are reported in Section 3.2.3.2.2.
3.2.1.2. Round 1 Surface Sampling Results
There were two surface sampling events in Round 1: pre-decontamination (characterization)
and post-decontamination (clearance).
3.2.1.2.1. Round 1 Pre-decontamination Surface Sampling Results
During Round 1 pre-decontamination sampling, sample teams collected 291 surface samples
comprised of swabs, sponge-stick wipes, and vacuum socks. An additional 52 blank (QC)
samples of these types were also collected. The locations and types of samples collected are
shown in Figure 3-4 (Floor 1) and Figure 3-5 (Floor 2).
The total number of samples collected on each floor with the number of samples in which Bg
was detected are reported in Table 3-9. In total, Bg was detected in approximately 97% of the
surface samples. The percent detection was equivalent for both floors; in total, Bg was detected
in 283 samples. In 270 samples, Bg was detected and quantified in the spread plate analysis.
Filter plating was used for the quantitation of 13 samples (i.e., the spread plate data were below
the quantitation limit). In six samples, Bg was detected only by filter plating (i.e., spread plating
was ND). Nineteen samples that should have been filter plated according to the BOTE Project
sample analysis plan were not filter plated. However, in all cases, these samples had detectable
Bg via spread plating; the resulting spread plate CFU were used below the quantitation limit in
these cases, as discussed previously.
A summary of Bg detection by sample type and floor is shown in Figure 3-6. Bg was detected in
more than approximately 98% of all sponge-stick wipe and vacuum sock samples collected in
PBF-632. Considerably fewer swab samples were taken; swab sampling was focused on
sampling supply vents, computer monitors, and small areas of the floor or ceiling (e.g., along the
lights or drop ceiling supports).
The average CFU/ft2 was 3.1E5 (±5.0E5) on Floor 1 and 1.3E4 (± 5.0E4) on Floor 2. The
measured surface loading on Floor 1 was within the target criteria of 1E4 to 1E6 CFU/ft2. This
99
-------
measured loading was consistent with the loading estimated from the RMCs; there was no
statistically significant difference between the surface sampling and RMC average values (t-test,
two-tailed p-value = 0.5479).'811 (Note that the t-test, as per the cited reference, was used for this
analysis due to both normality and equal variance failing for ANOVA. A confidence level of 99%
was used here, in consideration that t-tests inflate the Type I error.) The measured surface
loading on Floor 2 was two orders of magnitude higher than the target range of 1 .OE2 to 2.0E2
CFU/ft2. The RMCs, again, provided a good estimation of the surface loading; there was no
statistically significant difference between the surface sampling and RMC average values (t-test,
two-tailed p-value = 0.8282).'811 Supporting these results, three-way ANOVA considering both
floors together did not indicate a statistically significant difference between the RMCs and
surface sampling (p-value = 1.0). Although the surface loading on Floor 2 was above the target
range, there was still a significant difference between the measured surface loadings on the two
floors (p-value < 0.001), as determined via three-way ANOVA.
A total of 10 blank samples out of 52 from the pre-decontamination sampling resulted in
detectable Bg (Table 3-10). For six of the samples, Bg was detected only by filter plating. Seven
of the blank samples with detected Bg were from Floor 1 sampling teams. The results from each
of the blank samples in which Bg was detected are shown in Table 3-11. The blank results show
only very low levels of contamination compared to the bulk of the surface samples taken on
each floor.
3.2.1.2.2. Round 1 Post-decontamination Surface Sampling Results
During Round 1 post-decontamination sampling, sampling teams collected 276 surface samples
comprised of swabs, sponge-stick wipes, and vacuum socks. An additional 45 blank (QC)
samples of these types were also collected. The locations and types of samples collected are
shown in Figure 3-7 (Floor 1) and Figure 3-8 (Floor 2).
The total number of samples for each floor along with the number of samples in which Bg was
detected are reported in Table 3-12. In total, Bg was detected in approximately 34% of the
surface samples. Percent detection was significantly greater on Floor 1 (51%) than on Floor 2
(13%). In 47 samples, Bg was detected and quantified in the spread plate analysis. Filter plating
was used for the quantitation of 47 samples (i.e., the spread plate data were below the
quantitation limit). In 43 samples, Bg was detected only by filter plating (i.e., spread plating was
ND). Nine samples that should have been filter plated per the BOTE Project sample analysis
plan were not filter plated. However, in all cases, these samples had detectable Bg via spread
plating; the resulting spread plate CFU were used below the quantitation limit in these cases as
discussed previously. The Floor 1 average loading after decontamination was 8.5E3 CFU/ft2
with a standard deviation of 1.2E5. The average surface loading determined by the vacuum
sock samples was two orders of magnitude lower than the average surface loading determined
by swabs and sponge-stick wipes; this difference led to the high standard deviation.
A summary of Bg detection by sample type and floor is shown in Figure 3-9. The highest
percentage of samples in which Bg was detected were from sponge-stick wipes and vacuum
socks. Considerably fewer swab samples were taken; swab sampling was focused on sampling
100
-------
supply vents, computer monitors, and small areas of the floor or ceiling (e.g., along the lights or
drop ceiling supports).
A total of four blank samples out of 45 from the post-decontamination sampling resulted in
detectable Bg (Table 3-13). For three of the samples, Bg was detected only by filter plating. All
blank samples with detected Bg were from Floor 1 sampling teams. The results from each of the
blank samples in which Bg was detected are shown in Table 3-14. The blank results show only
very low levels of contamination compared to the bulk of the surface samples taken on each
floor. For example, even the two blank samples that had over 1.0E3 CFU on each sample are
two orders of magnitude lower than the average CFU per sample determined from Floor 1 pre-
decontamination samples.
3.2.1.3. Round 2 Surface Sampling Results
There were two surface sampling events in Round 2: pre-decontamination (characterization)
and post-decontamination (clearance).
3.2.1.3.1. Round 2 Pre-decontamination Surface Sampling Results
During Round 2 pre-decontamination sampling, sample teams collected 268 surface samples
comprised of swabs, sponge-stick wipes, and vacuum socks. An additional 41 blank (QC)
samples of these types were also collected. The locations and types of samples collected are
shown in Figure 3-10 (Floor 1) and Figure 3-11 (Floor 2).
The total number of samples for each floor with the number of samples in which Bg was
detected are reported in Table 3-15. A summary of Bg detection by sampling type and floor is
shown in Figure 3-12. In total, Bg was detected in approximately 97% of the surface samples.
This percentage was identical to the value determined in Round 1 pre-decontamination
sampling. There was a slightly higher percentage of detection for Floor 1 sampling than for Floor
2 sampling. In total, Bg was detected in 259 samples. In 242 samples, Bg was detected and
quantified in the spread plate analysis. Filter plating was used for the quantitation of 17 samples
(i.e., the spread plate data were below the quantitation limit). In seven samples, Bg was
detected only by filter plating (i.e., spread plating was ND). Ten samples that should have been
filter plated per the BOTE Project sample analysis plan were not filter plated. However, in all
cases, these samples had detectable Bg via spread plating; the resulting spread plate CFU
were used below the quantitation limit in these cases as discussed previously (see Section
3.2.1.1. regarding ASTM D5465-93[74]).
The average CFU/ft2 was 2.1E5 (±2.9E5) on Floor 1 and 4.8E4 (±2.1E5) on Floor 2. The
measured surface loading on Floor 1 was within the target criteria of 1E4 to 1E6 CFU/ft2. This
measured loading was consistent with that estimated from the four Floor 1 RMCs with Bg
detected (see Section 3.2.1.1); there was no statistically significant difference between the
surface sampling and RMC average values (t-test, two-tailed p-value = 0.4838).'811 As in Round
1, the measured surface loading on Floor 2 was two orders of magnitude higher than the target
range of 1E2 to 2E2 CFU/ft2. The RMCs, again, provided a good estimation of the surface
loading; there was no statistically significant difference between the surface sampling and RMC
average values (t-test, two-tailed p-value = 0.2918).'811 Supporting these results, three-way
101
-------
ANOVA considering both floors together did not indicate a statistically significant difference
between the RMCs and surface sampling (p-value = 1.0). Although the surface loading on Floor
2 was above the target range, there was still a significant difference between the measured
surface loadings on the two floors (p-value = 0.003).
The Floor 1 pre-decontamination surface loadings in Round 1 and Round 2 were within the
acceptable target loading range, and both were within the same order of magnitude; the
difference was not suggested to be statistically significant (p-value = 0.296). Likewise, there was
no statistically significant difference suggested between the Floor 2 pre-decontamination results
in Round 1 compared to Round 2 (p-value > 0.05).
A total of two blank samples out of 41 from the pre-decontamination sampling resulted in
detectable Bg (Table 3-16). For one of the samples, Bg was detected only by filter plating. Both
blank samples with detected Bg were from Floor 1 sampling teams. The results from each of the
blank samples in which Bg was detected are shown in Table 3-17. The blank results show only
very low levels of contamination compared to the bulk of the surface samples taken on each
floor.
3.2.1.3.2. Round 2 Post-decontamination Surface Sampling Results
During Round 2 post-decontamination sampling, sampling teams collected 244 surface samples
comprised of swabs, sponge-stick wipes, and vacuum socks. An additional 25 blank (QC)
samples of these types were also collected. The locations and types of samples collected are
shown in Figure 3-13 (Floor 1) and Figure 3-14 (Floor 2).
The total number of samples for each floor along with the number of samples in which Bg was
detected are reported in Table 3-18. A summary of Bg detection by sample type and floor is
show in Figure 3-15. In total, Bg was detected in approximately 3% of the surface samples.
Percent detection was significantly greater on Floor 2 (6%) than on Floor 1 (1%). In one sample,
Bg was detected and quantified in the spread plate analysis. Filter plating was used for the
quantitation of seven samples (i.e., the spread plate data were below the quantitation limit). In
seven samples, Bg was detected only by filter plating (i.e., spread plating was ND). All samples
that required filter plating per the sample analysis plan were filter plated.
One blank sample out of 25 from the post-decontamination sampling resulted in detectable Bg
(Table 3-19). Bg was detected only in the filter plating of the sample, i.e., spread plating results
were ND (Table 3-20).
3.2.1.4. Round 3 Surface Sampling Results
Two surface sampling events occurred in Round 3: pre-decontamination (characterization) and
post-decontamination (clearance).
3.2.1.4.1. Round 3 Pre-decontamination Surface Sampling Results
During Round 3 pre-decontamination sampling, sampling teams collected 273 surface samples
comprised of swabs, sponge-stick wipes, and vacuum socks. An additional 52 blank (QC)
samples of these types were also collected. The locations and types of samples collected are
shown in Figure 3-16 (Floor 1) and Figure 3-17 (Floor 2).
102
-------
The total number of samples for each floor along with the number of samples in which Bg was
detected are reported in Table 3-21. A summary of Bg detection by sampling type and floor is
shown in Figure 3-18. In total, Bg was detected in approximately 96% of the surface samples.
This percentage was nearly identical to the value determined in Round 1 and Round 2 pre-
decontamination sampling. There was a slightly higher percentage of detection for Floor 1
sampling than for Floor 2 sampling. In total, Bg was detected in 262 samples. In 247 samples,
Bg was detected and quantified in the spread plate analysis. Filter plating was used for the
quantitation of 15 samples (i.e., the spread plate data were below the quantitation limit). In eight
samples, Bg was detected only by filter plating (i.e., spread plating was ND). Nine samples that
should have been filter plated per the BOTE Project sample analysis plan were not filter plated.
However, in all cases, these samples had detectable Bg via spread plating; the resulting spread
plate CFU were used below the quantitation limit in these cases as discussed previously.
The average CFU/ft2 was 2.4E5 (±5.4E5) on Floor 1 and 1.4E4 (±3.9E4) on Floor 2. The
measured surface loading on Floor 1 was within the target criteria of 1E4 to 1E6 CFU/ft2. This
measured loading was consistent with the loading estimated from the Floor 1 RMCs; no
statistically significant difference was observed between the surface sampling and RMC
average values (t-test, two-tailed p-value = 0.6249).'811 As in Round 1, the measured surface
loading on Floor 2 was an order of magnitude higher than the target range of 1E2 to 2E2
CFU/ft2. The RMCs, again, provided a good estimation of the surface loading; no statistically
significant difference was observed between the surface sampling and RMC average values (t-
test, two-tailed p-value = 0.6353).'811 Supporting these results, three-way ANOVA considering
both floors together did not indicate a statistically significant difference between the RMCs and
surface sampling (p-value = 0.260) Although the surface loading on Floor 2 was above the
target range, there was still an insignificant difference between the measured surface loadings
on the two floors (p-value < 0.001).
No statistically significant difference was indicated between the Floor 1 surface loadings for
Round 3 compared to Round 1 (p-value > 0.05) or Round 2 (p-value > 0.05). Likewise, no
statistically significant difference was indicated between the Floor 2 surface loadings for Round
3 compared to Round 1 (p-value > 0.05) or Round 2 (p-value > 0.05).
A total of four blank samples out of 52 from the pre-decontamination sampling resulted in
detectable Bg (Table 3-22). For two of the samples, Bg was detected only by filter plating. All
blank samples with detected Bg were from Floor 1 sampling teams. The results from each of the
blank samples in which Bg was detected are shown in Table 3-23. Three of the blank results
show only very low levels of contamination compared to the bulk of the surface samples taken
on each floor. One sponge-stick wipe blank sample (from Room 108) was of the same order of
magnitude as the surface samples.
3.2.1.4.2. Round 3 Post-decontamination Surface Sampling Results
During Round 3 post-decontamination sampling, sampling teams collected 265 surface samples
comprised of swabs, sponge-stick wipes, and vacuum socks. An additional 47 blank (QC)
samples of these types were also collected. The locations and types of samples collected are
shown in Figure 3-19 (Floor 1) and Figure 3-20 (Floor 2).
103
-------
The total number of samples for each floor along with the number of samples in which Bg was
detected are reported in Table 3-24. A summary of Bg detection by sampling type and floor is
shown in Figure 3-21. In total, Bg was detected in approximately 2% of the surface samples.
Percent detection was identical on both floors, i.e., irrespective of the difference in pre-
decontamination surface loading. In one sample, Bg was detected and quantified in the spread
plate analysis. Filter plating was used for the quantitation of five samples (i.e., the spread plate
data were below the quantitation limit). In those five samples, Bg was detected only by filter
plating (i.e., spread plating was ND). All samples that required filter plating per the sample
analysis plan were filter plated.
None of the blanks out of 47 from the post-decontamination sampling resulted in detectable Bg
(Table 3-25).
Table 3-5. LRN samples (swabs, sponge-stick wipes, vacuum socks)
collected in the MFP event.
MFP
Blanks
Floor 1 Blanks
Floor 2 Blanks
Surface Samples
Floor 1 Surface
Samples
Floor 2 Surface
Samples
Pre-Decontamination
Total
Number of
Samples
Collected
76
13
7
6
63
33
30
Total
Number
with
Detected
CPU
31
1
0
1
30
11
19
Total
Number
with
Detected
CPU on
Spread
Plate
20
0
0
0
20
10
10
Total
Number
with
Detected
CPU on
Filter
Plate
19
1
0
1
18
2
16
Percentage of
Detection
40.8%
7.7%
0.0%
16.7%
47.6%
33.3%
63.3%
104
-------
Table 3-6. LRN swab samples collected in the MFP event.
Swabs
Swab Blanks
Floor 1 Swab
Blanks
Floor 2 Swab
Blanks
Swab Surface
Samples
Floor 1 Swab
Surface
Samples
Floor 2 Swab
Surface
Samples
Pre-Decontamination
Total
Number of
Samples
Collected
13
3
1
2
10
5
5
Total
Number
with
Detected
CPU
0
0
0
0
0
0
0
Total
Number
with
Detected
CPU on
Spread
Plate
0
0
0
0
0
0
0
Total
Number
with
Detected
CPU on
Filter
Plate
0
0
0
0
0
0
0
Percentage of
Detection
0.0%
0.0%
0.0%
0.0%
0.0%
0.0%
0.0%
105
-------
Table 3-7. LRN sponge-stick wipe samples collected in the MFP event.
Sponge-stick Wipes
Sponge-stick Wipes
Blanks
Floor 1
Sponge-stick
Wipe Blanks
Floor 2
Sponge-stick
Wipe Blanks
Sponge-stick Wipes
Surface Samples
Floor 1
Sponge-stick
Wipes Surface
Samples
Floor 2
Sponge-stick
Wipes Surface
Samples
Pre-Decontamination
Total
Number of
Samples
Collected
31
5
3
2
26
14
12
Total
Number
with
Detected
CPU
10
1
0
1
9
1
8
Total
Number
with
Detected
CPU on
Spread
Plate
6
0
0
0
6
1
5
Total
Number
with
Detected
CPU on
Filter
Plate
8
1
0
1
7
0
7
Percentage of
Detection
32.3%
20.0%
0.0%
50.0%
34.6%
7.1%
66.7%
106
-------
Table 3-8. LRN vacuum sock samples collected in the MFP event.
Vacuum Socks
Vacuum Socks
Blanks
Floor 1
Vacuum Socks
Blanks
Floor 2
Vacuum Socks
Blanks
Vacuum Socks
Surface Samples
Floor 1
Vacuum Socks
Surface
Samples
Floor 2
Vacuum Socks
Surface
Samples
Pre-Decontamination
Total
Number of
Samples
Collected
32
5
3
2
27
14
13
Total
Number
with
Detected
CPU
21
0
0
0
21
10
11
Total
Number
with
Detected
CPU on
Spread
Plate
14
0
0
0
14
9
5
Total
Number
with
Detected
CPU on
Filter
Plate
11
0
0
0
11
2
9
Percentage of
Detection
65.6%
0.0%
0.0%
0.0%
77.8%
71 .4%
84.6%
107
-------
14
14
14
• Total Number of Samples Collected
• Total Number with Detected CPU
13
Floor 1 Swab Floor2Swab Floor 1 Sponge- Floor2 Sponge- FlooM Vacuum Floor2Vacuum
Surface Samples Surface Samples stickWipes stickWipes Socks Surface Socks Surface
Surface Samples Surface Samples Samples Samples
Figure 3-1. Summary of samples taken for LRN analysis during the MFP event.
108
-------
rvi
Concentration (CFU,'ft2) Sample Types
Qnull ® swab
Qzero O1°3 OlO6 ® Sponge-stick wipe
• 101 O1°2 •lO7 © Vacuum sock
Figure 3-2. Sample map for Floor 1 during MFP.
109
-------
Concentration (CFU/ft2) Sample Types
Qnull Q102 Q105 (D Swab
Qzero • 103 Q106 © Sponge-stick wipe
• 101 O102 ^107 © Vacuum sock
Figure 3-3. Sample map for Floor 2 during MFP.
110
-------
TYI ryi
sfl ""^a" "W'lr "F®"!^" ^i &v -®~'i
Concentration (CFU/ft2) Sample Types
Qnull Q102 Q105 ®swab
Qzem ©103 Q106 © Sponge-stick wipe
• 101 Q104 •lO7 © Vacuum sock
Figure 3-4. Sample map for Floor 1 during Round 1 pre-decontamination sampling.
111
-------
Concentration (CFU/ft2
Sample Types
Q
®swab
®103 Q106 0 Sponge-stickwipe
O1°4 •lO7 © Vacuum sock
Figure 3-5. Sample map for Floor 2 during Round 1 pre-decontamination sampling.
112
-------
Table 3-9. LRN pre-decontamination surface sampling results for Round 1.
Total Surface Samples
Swab
Sponge-stick
Wipe
Vacuum Sock
Floor 1 Surface Samples
Swab
Sponge-stick
Wipe
Vacuum Sock
Floor 2 Surface Samples
Swab
Sponge-stick
Wipe
Vacuum Sock
# of Samples
Collected
291
23
172
96
157
13
95
49
134
10
77
47
# with Bg
Detected
283
20
169
94
153
11
93
49
130
9
76
45
# with Bg
Detected and
Quantified by
Spread
plating
270
16
161
93
151
11
91
49
119
5
70
44
Filter
plating
13
4
8
1
2
0
2
0
11
4
6
1
#of
Samples
with Bg
Detected
Only by
Filter
Plating
6
2
4
0
0
0
0
0
6
2
4
0
Percent
Detection
97%
87%
98%
98%
97%
85%
98%
100%
97%
90%
99%
96%
Average Surface
Loading
Average
CFU/ft2
1.7E5
3.4E5
2.3E5
2.4E4
3.1E5
5.8E5
4.1E5
4.7E4
1.3E4
2.6E4
1.9E4
4.7E2
SD
4.0E5
7.1E5
4.2E5
1.5E5
5.0E5
8.8E5
5.0E5
2.1E5
5.0E4
4.3E4
6.3E4
5.0E2
Average Loading
per Sam pie
Average
CFU
1.3E5
9.4E3
1.6E5
9.7E4
2.3E5
1.6E4
2.8E5
1.9E5
8.5E3
7.3E2
1.3E4
1.9E3
SD
4.1E5
2.0E4
2.9E5
5.9E5
5.3E5
2.4E4
3.5E5
8.2E5
3.4E4
1.2E3
4.4E4
2.0E3
113
-------
100 i
Total Number of Samples Collected
Total Number with Detected CPU
Floor 1 Swab
Surface Samples
Floor 2 Swab Floor 1 Sponge- Floor 2 Sponge- Floor 1 Vacuum Floor 2 Vacuum
Surface Samples stick Wipes Surface stick Wipes Surface Socks Surface Socks Surface
Samples Samples Samples Samples
Figure 3-6. Summary of samples taken for LRN analysis during pre-decontamination sampling in Round 1.
114
-------
Table 3-10. LRN pre-decontamination blank results for Round 1.
Total Surface Samples
Swab
Sponge-stick Wipe
Vacuum Sock
Floor 1 Surface Samples
Swab
Sponge-stick Wipe
Vacuum Sock
Floor 2 Surface Samples
Swab
Sponge-stick Wipe
Vacuum Sock
#of
samples
collected
52
16
19
17
29
8
11
10
23
8
8
7
#with
Bg
detected
10
1
4
5
7
1
3
3
3
0
1
2
# with Bg detected
and quantified by
Spread
plating
4
0
3
1
3
0
3
0
1
0
0
1
Filter
plating
6
1
1
4
4
1
0
3
2
0
1
1
#of
samples
with Bg
detected
only by
filter
plating
6
1
1
4
4
1
0
3
2
0
1
1
Percent
detection
19%
6%
21%
29%
24%
13%
27%
30%
13%
0%
13%
29%
Average surface
loading
Average
CFU/ft2
1.6E2
5.2EO
4.2E2
2.4E-1
2.8E2
1.0E1
7.3E2
1.2E-1
9.6E-1
O.OEO
2.4EO
4.3E-1
SD
8.6E2
2.1E1
1.4E3
5.1E-1
1.1E3
2.9E1
1.8E3
2.2E-1
4.0EO
O.OEO
6.7EO
7.5E-1
Average loading
per sample
Average
CFU
1.1E2
1.4E-1
2.9E2
9.8E-1
1.9E2
2.9E-1
5.1E2
4.6E-1
1.1EO
O.OEO
1.7EO
1.7EO
SD
6.0E2
5.8E-1
9.8E2
2.1EO
8.0E2
8.1E-1
1.3E3
8.6E-1
3.2EO
O.OEO
4.7EO
3.0EO
115
-------
Table 3-11. Round 1 pre-decontamination blank samples with Bg detected.
Floor
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 2
Floor 2
Floor 2
Room
Corridor+Lobby
Corridor+Lobby
Room 102
Room 104
Room 104
Room 107
Room 108
Room 206
Room 210
Room 21 2
Sampling Method
Sponge-stick wipe
Sponge-stick wipe
Sponge-stick wipe
Vacuum Sock
Swab
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge-stick wipe
Vacuum Sock
CPU/sample
Spread
Plating
1.4E3
3.4E1
4.1E3
ND
ND
ND
ND
7.2EO
ND
ND
Filter
Plating
NA
NA
NA
2.4EO
2.3EO
4.0E-1
2.0EO
ND
1.3E1
4.8EO
ND = Not detected. NA = Not analyzed.
116
-------
rv rvi
"^ L-
Concentration (CFU/ft2)
Sample Types
Qnull Q102 Q105 Q swab
Qzero 0103 OlO6 © Sponge-stickwipe
^101 O^O4 ^107 © Vacuum sock
Figure 3-7. Sample map for Floor 1 during Round 1 post-decontamination sampling.
117
-------
Concentration (CFU/ft2) Sample Types
Qnull Q102 Q10S ®swab
Q103 O106 0 Sponge-stick wipe
O1°4 •lO7 © Vacuum sock
Figure 3-8. Sample map for Floor 2 during Round 1 post-decontamination sampling.
118
-------
Table 3-12. LRN post-decontamination surface sampling results for Round 1.
Total Surface Samples
Swab
Sponge-stick Wipe
Vacuum Sock
Floor 1 Surface Samples
Swab
Sponge-stick Wipe
Vacuum Sock
Floor 2 Surface Samples
Swab
Sponge-stick Wipe
Vacuum Sock
#of
samples
collected
276
20
162
94
152
11
93
48
124
9
69
46
#with
Bg
detected
94
5
48
41
78
3
42
33
16
2
6
8
# with Bg detected
and quantified by
Spread
plating
47
3
20
24
40
2
18
20
7
1
2
4
Filter
plating
47
2
28
17
38
1
24
13
9
1
4
4
#of
samples
with Bg
detected
only by
filter
plating
43
2
24
17
34
1
20
13
9
1
4
4
Percent
detection
34%
25%
30%
44%
51%
27%
45%
69%
13%
22%
9%
17%
Average surface
loading
Average
CFU/ft2
8.5E3
1.8E4
1.2E4
1.6E1
1.3E4
8.9E2
2.1E4
2.8E1
2.8E3
3.9E4
1.1E1
3.4EO
SD
1.2E5
7.8E4
1.5E5
6.0E1
1.6E5
2.0E3
2.0E5
8.1E1
3.1E4
1.2E5
6.4E1
1.5E1
Average loading
per sample
Average
CFU
5.0E3
5.0E2
8.5E3
6.3E1
9.1E3
2.5E1
1.5E4
1.1E2
8.8E1
1.1E3
7.9EO
1.4E1
SD
8.2E4
2.2E3
1.1E5
2.4E2
1.1E5
5.6E1
1.4E5
3.2E2
8.7E2
3.2E3
4.4E1
5.9E1
119
-------
45.2%
93
Total Number of Samples Collected
Total Number with Detected CPU
Floor 1 Swab
Surface Samples
Floor 2 Swab
Surface Samples
Floor 1 Sponge- Floor 2 Sponge- Floor 1 Vacuum Floor 2 Vacuum
stick Wipes Surface stick Wipes Surface Socks Surface Socks Surface
Samples Samples Samples Samples
Figure 3-9. Summary of samples taken for LRN analysis during post-decontamination sampling in Round 1.
120
-------
Table 3-13. LRN post-decontamination blank results for Round 1.
Total Surface Samples
Swab
Sponge-stick Wipe
Vacuum Sock
Floor 1 Surface Samples
Swab
Sponge-stick Wipe
Vacuum Sock
Floor 2 Surface Samples
Swab
Sponge-stick Wipe
Vacuum Sock
#of
samples
collected
45
10
17
18
25
5
10
10
20
5
7
8
#with
Bg
detected
4
1
1
2
4
1
1
2
0
0
0
0
# with Bg detected
and quantified by
Spread
plating
1
1
0
0
1
1
0
0
0
0
0
0
Filter
plating
3
0
1
2
3
0
1
2
0
0
0
0
#of
samples
with Bg
detected
only by
filter
plating
3
0
1
2
3
0
1
2
0
0
0
0
Percent
detection
9%
10%
6%
11%
16%
20%
10%
20%
0%
0%
0%
0%
Average surface
loading
Average
CFU/ft2
1.3E1
6.0E1
1.9E-1
1.0E-1
2.4E1
1.2E2
3.3E-1
1.7E-1
O.OEO
O.OEO
O.OEO
O.OEO
SD
8.9E1
1.9E2
8.0E-1
3.0E-1
1.2E2
2.7E2
1.1EO
3.9E-1
O.OEO
O.OEO
O.OEO
O.OEO
Average loading
per sample
Average
CFU
5.7E-1
1.7EO
1.4E-1
3.8E-1
1.0EO
3.3EO
2.3E-1
6.9E-1
O.OEO
O.OEO
O.OEO
O.OEO
SD
2.6EO
5.3EO
5.6E-1
1.2EO
3.4EO
7.5EO
7.3E-1
1.6EO
O.OEO
O.OEO
O.OEO
O.OEO
121
-------
Table 3-14. Round 1 post-decontamination blank samples with Bg detected.
Floor
Floor 1
Floor 1
Floor 1
Floor 1
Room
Room 103
Room 107
Room 107
Room 110
Sampling Method
Sponge-stick Wipe
Vacuum Sock
Swab
Vacuum Sock
CFU/Sample
Spread
Plating
ND
ND
17
ND
Filter
Plating
2
5
3
2
122
-------
=JYL,.
••
Concentration (CFU/ft2) SampleTypes
Qnull Q102 O105 ®swab
^103 Q106 0 Sponge-stickwipe
O1°4 •107 © Vacuum sock
Figure 3-10. Sample map for Floor 1 during Round 2 pre-decontamination sampling.
123
-------
Concentration (CFLl/ft2)
Sample Types
Qnull Q102 Q105 ®swab
©103 Q106 © Sponge-stick wipe
Q104 •lO7 © Vacuum sock
Figure 3-11. Sample map for Floor 2 during Round 2 pre-decontamination sampling.
124
-------
Table 3-15. LRN pre-decontamination surface sampling results for Round 2.
Total Surface Samples
Swab
Sponge-stick Wipe
Vacuum Sock
Floor 1 Surface Samples
Swab
Sponge-stick Wipe
Vacuum Sock
Floor 2 Surface Samples
Swab
Sponge-stick Wipe
Vacuum Sock
#of
samples
collected
268
19
204
45
146
10
109
27
122
9
95
18
#with
Bg
detected
259
17
197
45
145
10
108
27
114
7
89
18
# with Bg detected
and quantified by
Spread
plating
242
15
186
41
144
10
107
27
98
5
79
14
Filter
plating
17
2
11
4
1
0
1
0
16
2
10
4
#of
samples
with Bg
detected
only by
filter
plating
7
2
5
0
0
0
0
0
7
2
5
0
Percent
detection
97%
89%
97%
100%
99%
100%
99%
100%
93%
78%
94%
100%
Average surface
loading
Average
CFU/ft2
1.4E5
2.9E5
1.4E5
5.0E4
2.1E5
5.3E5
2.1E5
8.3E4
4.8E4
2.7E4
5.9E4
5.9E2
SD
2.7E5
5.1E5
2.3E5
2.9E5
2.9E5
6.1E5
2.0E5
3.7E5
2.1E5
3.6E4
2.4E5
1.9E3
Average loading
per sample
Average
CFU
1.1E5
8.1E3
9.8E4
2.0E5
1.7E5
1.5E4
1.5E5
3.3E5
3.2E4
7.5E2
4.1E4
2.4E3
SD
4.9E5
1.4E4
1.6E5
1.2E6
6.5E5
1.7E4
1.4E5
1.5E6
1.5E5
9.9E2
1.7E5
7.5E3
125
-------
C3
GO
120 i
100 -
80
60
^ 40
20 •
Floor 1 Swab
Surface Samples
Total Number of Samples Collected
Total Number with Detected CPU
Floor 2 Swab
Surface Samples
Floor 1 Sponge- Floor 2 Sponge- Floor 1 Vacuum Floor 2 Vacuum
stick Wipes Surface stick Wipes Surface Socks Surface Socks Surface
Samples Samples Samples Samples
Figure 3-12. Summary of samples taken for LRN analysis during pre-decontamination sampling in Round 2.
126
-------
Table 3-16. LRN pre-decontamination blank results for Round 2.
Total Surface Samples
Swab
Sponge-stick Wipe
Vacuum Sock
Floor 1 Surface Samples
Swab
Sponge-stick Wipe
Vacuum Sock
Floor 2 Surface Samples
Swab
Sponge-stick Wipe
Vacuum Sock
#of
samples
collected
41
11
18
12
22
5
10
7
19
6
8
5
#with
Bg
detected
2
0
1
1
2
0
1
1
0
0
0
0
# with Bg detected
and quantified by
Spread
plating
1
0
0
1
1
0
0
1
0
0
0
0
Filter
plating
1
0
1
0
1
0
1
0
0
0
0
0
#of
samples
with Bg
detected
only by
filter
plating
1
0
1
0
1
0
1
0
0
0
0
0
Percent
detection
5%
0%
6%
8%
9%
0%
10%
14%
0%
0%
0%
0%
Average surface
loading
Average
CFU/ft2
5.1E-1
O.OEO
2.4E-1
1.4EO
9.5E-1
O.OEO
4.3E-1
2.4EO
O.OEO
O.OEO
O.OEO
O.OEO
SD
2.7EO
O.OEO
1.0EO
4.8EO
3.6EO
O.OEO
1.4EO
6.3EO
O.OEO
O.OEO
O.OEO
O.OEO
Average Loading
per Sample
Average
CFU
1.7EO
O.OEO
1.7E-1
5.6EO
3.2EO
O.OEO
3.0E-1
9.5EO
O.OEO
O.OEO
O.OEO
O.OEO
SD
1.0E1
O.OEO
7.1E-1
1.9E1
1.4E1
O.OEO
9.5E-1
2.5E1
O.OEO
O.OEO
O.OEO
O.OEO
127
-------
Table 3-17. Round 2 pre-decontamination blank samples with Bg detected.
Floor
Floor 1
Floor 1
Room
Room 109
Room 110
Sampling Method
Sponge-stick Wipe
Vacuum Sock
CFU/Sample
Spread
Plating
ND
6.7E1
Filter
Plating
3.0EO
4.2E1
128
-------
rv|_
=d [=
^
Concentration (CFU/ft2) Sample Types
Qnull Q102 Q105 ®swab
Qzem ©103 Q106 © Sponge-stick wipe
• 101 Q104 •lO7 © Vacuum sock
Figure 3-13. Sample map for Floor 1 during Round 2 post-decontamination sampling.
129
-------
Concentration (CFU/ft2)
Sample Types
Qnull Q102 Q10S ®swab
Qzero Q103 O106 0 Sponge-stick wipe
^101 O1°4 •lO7 © Vacuum sock
Figure 3-14. Sample map for Floor 2 during Round 2 post-decontamination sampling.
130
-------
Table 3-18. LRN post-decontamination surface sampling results for Round 2.
Total Surface Samples
Swab
Sponge-stick Wipe
Vacuum Sock
Floor 1 Surface Samples
Swab
Sponge-stick Wipe
Vacuum Sock
Floor 2 Surface Samples
Swab
Sponge-stick Wipe
Vacuum Sock
#of
samples
collected
244
1
235
8
135
1
130
4
109
0
105
4
#with
Bg
detected
8
0
7
1
1
0
1
0
7
0
6
1
# with Bg detected
and quantified by
Spread
plating
1
0
1
0
1
0
1
0
0
0
0
0
Filter
plating
7
0
6
1
0
0
0
0
7
0
6
1
#of
samples
with Bg
detected
only by
filter
plating
7
0
6
1
0
0
0
0
7
0
6
1
Percent
detection
3%
0%
3%
13%
1%
0%
1%
0%
6%
-
6%
25%
Average surface
loading
Average
CFU/ft2
2.0E-1
O.OEO
2.1E-1
7.8E-2
1.7E-1
O.OEO
1.8E-1
O.OEO
2.4E-1
-
2.4E-1
1.6E-1
SD
1.7EO
-
1.7EO
2.2E-1
2.0EO
-
2.1EO
O.OEO
1.1EO
-
1.1EO
3.1E-1
Average loading
per sample
Average
CFU
1.5E-1
O.OEO
1.4E-1
3.1E-1
1.2E-1
-
1.3E-1
O.OEO
1.8E-1
-
1.7E-1
6.3E-1
SD
1.2EO
-
1.2EO
8.8E-1
1.4EO
-
1.4EO
O.OEO
7.8E-1
-
7.7E-1
1.3EO
131
-------
140
120
100 -
s 80
0.8%
-
I
60
40
20 •
0.0%
1 n
Floor 1 Swab
Surface Samples
NA
_n o
Total Number of Samples Collected
Total Number with Detected CPU
Floor 2 Swab Floor 1 Sponge- Floor 2 Sponge- Floor 1 Vacuum Floor 2 Vacuum
Surface Samples stick Wipes Surface stick Wipes Surface Socks Surface Socks Surface
Samples Samples Samples Samples
Figure 3-15. Summary of samples taken for LRN analysis during post-decontamination sampling in Round 2.
132
-------
Table 3-19. LRN post-decontamination blank results for Round 2.
Total Surface Samples
Swab
Sponge-stick Wipe
Vacuum Sock
Floor 1 Surface Samples
Swab
Sponge-stick Wipe
Vacuum Sock
Floor 2 Surface Samples
Swab
Sponge-stick Wipe
Vacuum Sock
#of
samples
collected
25
1
18
6
14
1
10
3
11
0
8
3
#with
Bg
detected
1
0
0
1
0
0
0
0
1
0
0
1
# with Bg detected
and quantified by
Spread
plating
0
0
0
0
0
0
0
0
0
0
0
0
Filter
plating
1
0
0
1
0
0
0
0
1
0
0
1
#of
samples
with Bg
detected
only by
filter
plating
1
0
0
1
0
0
0
0
1
0
0
1
Percent
detection
4%
0%
0%
17%
0%
0%
0%
0%
9%
-
0%
33%
Average surface
loading
Average
CFU/ft2
6.4E-2
O.OEO
O.OEO
2.7E-1
O.OEO
O.OEO
O.OEO
O.OEO
1.5E-1
-
O.OEO
5.3E-1
SD
3.2E-1
-
O.OEO
6.5E-1
O.OEO
-
O.OEO
O.OEO
4.8E-1
-
O.OEO
9.2E-1
Average loading
per sample
Average
CFU
2.6E-1
O.OEO
O.OEO
1.1EO
O.OEO
O.OEO
O.OEO
O.OEO
5.8E-1
-
O.OEO
2.1EO
SD
1.3EO
-
O.OEO
2.6EO
O.OEO
-
O.OEO
O.OEO
1.9EO
-
O.OEO
3.7EO
133
-------
Table 3-20. Round 2 post-decontamination blank samples with Bg detected.
Floor
Floor 2
Room
Room 21 2
Sampling Method
Vacuum Sock
CFU/Sample
Spread
Plating
ND
Filter
Plating
6
134
-------
Concentration (CFU/ft2) SampleTypes
Qnull Q102 Q105 ©swab
Qzero Q103 OlO6 © Sponge-stick wipe
• 101 Q104 •lO7 © Vacuum sock
Figure 3-16. Sample map for Floor 1 during Round 3 pre-decontamination sampling.
135
-------
Concentration (CFU/ft2) Sample Types
Qnull Q102 Q105 ®swab
Qzero Q1°3 O106 ® Sponge-stickwipe
• 101 O1°4 •107 © Vacuum sock
Figure 3-17. Sample map for Floor 2 during Round 3 pre-decontamination sampling.
136
-------
Table 3-21. LRN pre-decontamination surface sampling results for Round 3.
Total Surface Samples
Swab
Sponge-stick Wipe
Vacuum Sock
Floor 1 Surface Samples
Swab
Sponge-stick Wipe
Vacuum Sock
Floor 2 Surface Samples
Swab
Sponge-stick Wipe
Vacuum Sock
#of
samples
collected
273
21
166
86
144
11
89
44
129
10
77
42
#with
Bg
detected
262
21
155
86
141
11
86
44
121
10
69
42
# with Bg detected
and quantified by
Spread
plating
247
18
145
84
139
10
85
44
108
8
60
40
Filter
plating
15
3
10
2
2
1
1
0
13
2
9
2
#of
samples
with Bg
detected
only by
filter
plating
8
1
7
0
1
0
1
0
7
1
6
0
Percent
detection
96%
100%
93%
100%
98%
100%
97%
100%
94%
100%
90%
100%
Average surface
loading
Average
CFU/ft2
1.3E5
3.9E5
1.3E5
8.3E4
2.4E5
7.0E5
2.2E5
1.6E5
1.4E4
3.4E4
1.8E4
3.7E3
SD
4.1E5
6.8E5
2.8E5
5.2E5
5.4E5
8.3E5
3.5E5
7.2E5
3.9E4
3.3E4
4.7E4
1.8E4
Average loading
per sample
Average
CFU
1.6E5
1.1E4
8.8E4
3.3E5
2.9E5
2.0E4
1.5E5
6.3E5
1.2E4
9.5E2
1.2E4
1.5E4
SD
1.2E6
1.9E4
1.9E5
2.1E6
1.6E6
2.3E4
2.4E5
2.9E6
4.8E4
9.2E2
3.2E4
7.1E4
137
-------
90 i
80
70 -
60
CO
I 50
GO
O
fe 40
|
^ 30
20 •
10 •
Total Number of Samples Collected
Total Number with Detected CPU
Floor 1 Swab
Surface Samples
Floor 2 Swab
Surface Samples
Floor 1 Sponge- Floor 2 Sponge- Floor 1 Vacuum Floor 2 Vacuum
stick Wipes Surface stick Wipes Surface Socks Surface Socks Surface
Samples Samples Samples Samples
Figure 3-18. Summary of samples taken for LRN analysis during pre-decontamination sampling in Round 3.
138
-------
Table 3-22. LRN pre-decontamination blank results for Round 3.
Total Surface Samples
Swab
Sponge-stick Wipe
Vacuum Sock
Floor 1 Surface Samples
Swab
Sponge-stick Wipe
Vacuum Sock
Floor 2 Surface Samples
Swab
Sponge-stick Wipe
Vacuum Sock
#of
samples
collected
52
10
24
18
31
5
16
10
21
5
8
8
#with
Bg
detected
4
0
2
2
4
0
2
2
0
0
0
0
# with Bg detected
and quantified by
Spread
plating
2
0
1
1
2
0
1
1
0
0
0
0
Filter
plating
2
0
1
1
2
0
1
1
0
0
0
0
#of
samples
with Bg
detected
only by
filter
plating
2
0
1
1
2
0
1
1
0
0
0
0
Percent
detection
8%
0%
8%
11%
13%
0%
13%
20%
0%
0%
0%
0%
Average surface
loading
Average
CFU/ft2
4.7E2
O.OEO
1.0E3
1.5EO
7.8E2
O.OEO
1.5E3
2.7EO
O.OEO
O.OEO
O.OEO
O.OEO
SD
3.4E3
O.OEO
4.9E3
4.7EO
4.3E3
O.OEO
6.0E3
6.2EO
O.OEO
O.OEO
O.OEO
O.OEO
Average loading
per sample
Average
CFU
3.3E2
O.OEO
7.0E2
6.0EO
5.5E2
O.OEO
1.1E3
1.1E1
O.OEO
O.OEO
O.OEO
O.OEO
SD
2.3E3
O.OEO
3.4E3
1.9E1
3.0E3
O.OEO
4.2E3
2.5E1
O.OEO
O.OEO
O.OEO
O.OEO
139
-------
Table 3-23. Round 3 pre-decontamination blank samples with Bg detected.
Floor
Floor 1
Floor 1
Floor 1
Floor 1
Room
Corridor+Lobby
Room 103
Room 107
Room 108
Sampling Method
Vacuum Sock
Vacuum Sock
Sponge-stick Wipe
Sponge-stick Wipe
CFU/Sample
Spread
Plating
3.3E1
ND
ND
1.7E4
Filter
Plating
NA
7.4E1
3.0EO
NA
ND = Not detected. NA = Not analyzed.
140
-------
n^.
! " \Łf^
Concentration (CFU/ft2) SampleTypes
Qnull Q102 Q10S ®swab
Qzero Q103 Q106 0 Sponge-stick wipe
0101 O1°4 •lO7 © Vacuum sock
Figure 3-19. Sample map for Floor 1 during Round 3 post-decontamination sampling.
141
-------
ft ,14. n @ i | i P*=
tŁ4^Lj^LJjflit-C
-^^ * ^
n^p"^^
^T _
CD
'•:'•' "^
_.*_*"•..-
-^-..JL^JfcJ=
^dfcj^
Concentration (CFU/ft2) SampleTypes
Qnull Q102 O105 ®Swab
Qzero Q1Q3 Q106 © Sponge-stickwipe
^101 O1°4 •lO7 © Vacuum sock
Figure 3-20. Sample map for Floor 2 during Round 3 post-decontamination sampling.
142
-------
Table 3-24. LRN post-decontamination surface sampling results for Round 3.
Total Surface Samples
Swab
Sponge-stick Wipe
Vacuum Sock
Floor 1 Surface Samples
Swab
Sponge-stick Wipe
Vacuum Sock
Floor 2 Surface Samples
Swab
Sponge-stick Wipe
Vacuum Sock
#of
samples
collected
265
21
162
82
138
11
85
42
127
10
77
40
#with
Bg
detected
6
0
4
2
3
0
2
1
3
0
2
1
# with Bg detected
and quantified by
Spread
plating
1
0
0
1
0
0
0
0
1
0
0
1
Filter
plating
5
0
4
1
3
0
2
1
2
0
2
0
#of
samples
with Bg
detected
only by
filter
plating
5
0
4
1
3
0
2
1
2
0
2
0
Percent
detection
2%
0%
2%
2%
2%
0%
2%
2%
2%
0%
3%
3%
Average surface
loading
Average
CFU/ft2
1.0E-1
O.OEO
1.3E-1
6.0E-2
5.0E-2
O.OEO
8.0E-2
1.0E-2
1.4E-1
O.OEO
1.8E-1
1.0E-1
SD
7.4E-1
O.OEO
8.9E-1
4.6E-1
4.0E-1
O.OEO
5.0E-1
9.0E-2
9.9E-1
O.OEO
1.2EO
6.6E-1
Average loading
per sample
Average
CFU
1.3E-1
O.OEO
9.0E-2
2.3E-1
5.0E-2
O.OEO
5.0E-2
5.0E-2
2.1E-1
O.OEO
1.3E-1
4.2E-1
SD
1.1EO
O.OEO
6.2E-1
1.9EO
3.3E-1
O.OEO
3.5E-1
3.5E-1
1.6EO
O.OEO
8.2E-1
2.6EO
143
-------
90
80
70
60
g 50 -
53 40
|
^ 30
20 •
10 •
85
2.4%
Floor 1 Swab
Surface Samples
Total Number of Samples Collected
Total Number with Detected CPU
Floor 2 Swab
Surface Samples
Floor 1 Sponge- Floor 2 Sponge- Floor 1 Vacuum Floor 2 Vacuum
stick Wipes Surface stick Wipes Surface Socks Surface Socks Surface
Samples Samples Samples Samples
Figure 3-21. Summary of samples taken for LRN analysis during post-decontamination sampling in Round 3.
144
-------
Table 3-25. LRN post-decontamination blank results for Round 3.
Total Surface Samples
Swab
Sponge-stick Wipe
Vacuum Sock
Floor 1 Surface Samples
Swab
Sponge-stick Wipe
Vacuum Sock
Floor 2 Surface Samples
Swab
Sponge-stick Wipe
Vacuum Sock
#of
samples
collected
47
10
19
18
26
5
11
10
21
5
8
8
#with
Bg
detected
0
0
0
0
0
0
0
0
0
0
0
0
# with Bg detected
and quantified by
Spread
plating
0
0
0
0
0
0
0
0
0
0
0
0
Filter
plating
0
0
0
0
0
0
0
0
0
0
0
0
#of
samples
with Bg
detected
only by
filter
plating
0
0
0
0
0
0
0
0
0
0
0
0
Percent
detection
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
Average surface
loading
Average
CFU/ft2
O.OEO
O.OEO
O.OEO
O.OEO
O.OEO
O.OEO
O.OEO
O.OEO
O.OEO
O.OEO
O.OEO
O.OEO
SD
O.OEO
O.OEO
O.OEO
O.OEO
O.OEO
O.OEO
O.OEO
O.OEO
O.OEO
O.OEO
O.OEO
O.OEO
Average loading
per sample
Average
CFU
O.OEO
O.OEO
O.OEO
O.OEO
O.OEO
O.OEO
O.OEO
O.OEO
O.OEO
O.OEO
O.OEO
O.OEO
SD
O.OEO
O.OEO
O.OEO
O.OEO
O.OEO
O.OEO
O.OEO
O.OEO
O.OEO
O.OEO
O.OEO
O.OEO
145
-------
3.2.2. Results from Rapid Viability-Polymerase Chain Reaction Analysis
The RV-PCR research method developed and optimized for surface wipe samples was
evaluated during the BOTE Project Phase |[36'37]. Samples collected from the event site at the
INL were shipped to LLNL and EPA-OPP-MLB laboratories. To evaluate the performance of the
RV-PCR method against the traditional microbiological culture method, the original protocol had
to be modified. Specifically, instead of using the whole sample for RV-PCR-based analysis, the
spore suspension was split into two equal parts after spore recovery from each of the BOTE
Project wipe samples with one part analyzed by RV-PCR and the other part analyzed by the
traditional culture method upon concentrating to the same volume. While this procedure might
have compromised the results for the low spore level samples, it was necessary for the most
appropriate method performance comparison. Therefore, any conclusions on the performance
of the RV-PCR research method must be carefully derived. Also, the current version of the RV-
PCR method provides qualitative analyses of the samples (detection/non-detection of live
spores), and the method had not previously been tested for post-decontamination samples.
A total of 262 BOTE Project samples were analyzed using the RV-PCR and the traditional
microbiological culture methods to detect the presence of viable Bg spores. The samples were
collected during the MFP (background samples) and Rounds 1-3 pre- and post-decontamination
sampling events. The BOTE Project samples collected at INL for the RV-PCR method-based
analysis were split between LLNL (214 samples) and the EPA-OPP-MLB Laboratory (50
samples). Also, as stated earlier, the participation of the EPA-OPP-MLB Laboratory in this effort
was mainly for the EPA scientists to acquire more practice and experience with the RV-PCR
method. Accordingly, for the evaluation of the RV-PCR research method, a majority of the
BOTE Project samples were shipped to LLNL. The identity of QC and real surface samples was
not known to either of the laboratories at the time of analysis. The same RV-PCR protocol was
followed for both the pre- and post-decontamination events. The results from each laboratory
are presented separately in the following sections of this report.
3.2.2.1. LLNL RV-PCR Results
A total of 214 samples (including QC samples) were analyzed at LLNL during the BOTE Phase I
(Table 3-26). The QC samples (also called field blanks) were opened during sampling but not
used to sample surfaces. The distribution of QC and surface samples was not known to LLNL
before the sample analysis, although the total number per event was verified. Due to the
complexity and difficulties with the sample shipment at INL, the desired sample distribution plan
as recommended by the statisticians could not be followed for some events. In addition to the
samples from INL, 20 TBs and 14 each of NCs and PCs were also analyzed. The TBs were
sterile pre-wetted wipes prepared in the same manner and at the same time as the wipes sent
to the field team to use for sampling. One PC, one NC, and at least one TB were included with
the samples on each tray/manifold. For a data table containing results for more than one
tray/manifold, more than one PC, NC, and TB are included in the table. The sample receipt
dates included: April 19, 2011 for MFP; April 19, 26, and 28, 2011 for Round 1; May 3 and 10,
2011 for Round 2; and May 14, 17, and 19, 2011 for Round 3. Samples were processed on the
receipt date unless there was a change from the original schedule resulting in a conflict with
staff availability. There were nine sample-processing dates and up to 48 samples and controls
were processed on the same day through both RV-PCR and traditional culture analyses. For
146
-------
some events, sample analysis (i.e., Round 3 post-decontamination samples) was split into two
days to accommodate the multiple steps required for culture analysis; however, the same
sample was processed concurrently by RV-PCR and culture to allow direct comparison of
results.
Table 3-26. Summary of LLNL samples received and processed by event type.
BOTE Project
Event
MFP
Round 1
Round 2
Round 3
Sample Type
MFP
QC Pre-
decontamination
Pre-
decontamination
QC Post-
decontamination
Post-
decontamination
QC Pre-
decontamination
Pre-
decontamination
QC Post-
decontamination
Post-
decontamination
QC Pre-
decontamination
Pre-
decontamination
QC Post-
decontamination
Post-
decontamination
Number of
Samples
17
2
0
3
41
6
14
8
39
6
10
8
60
Samples/Event
17
46
67
84
Total
Samples
214
The following section summarizes results for culture and RV-PCR analyses for the different
events. Data tables including both types of analyses are shown below for each event; in some
cases, the table for an event occupies multiple pages dependent on the sample number. RV-
PCR results were based on magnetic bead-based DMA extraction (Promega Magnesil® kit)
unless otherwise noted.
147
-------
3.2.2.1.1. Samples from the MFP Event
Results from MFP (background) samples showed that 17 of 17 (100%) agreed between culture
and RV-PCR analyses, with 14 positive and three negative for Bg (Table 3-27). Since the MFP
samples represented background levels from past dissemination events (INL-1 and INL-2
studies), they appeared to have very low viable spore levels. For the culture analysis, many of
the samples did not yield CFU on the ISA plates, but were positive only by analyzing the
enrichment culture using real-time PCR analysis. For these samples, RV-PCR analysis also
gave positive results showing agreement between methods (real-time PCR of the enrichment
culture and RV-PCR). In some cases (where noted in the tables), additional culture analysis
(beyond the current LRN protocol) was used to analyze a greater portion of the enrichment
culture and compare culture results with RV-PCR results more accurately. In the standard LRN
protocol, only a small fraction of the enrichment culture is analyzed by real-time PCR, whereas
RV-PCR uses a larger portion of the original spore suspension, therefore resulting in increased
sensitivity.
3.2.2.1.2. Samples from Round 1
Only two Round 1 pre-decontamination samples were received and analyzed at LLNL due to a
mix-up in sample distribution between LLNL and EPA-OPP-MLB during the sample shipment.
Results are shown in Table 3-28 including results for controls (same controls as shown in Table
3-27, but also included here for comparison purposes). These samples represented QC (field
blank) samples rather than wipe samples from surface sampling. For these samples, only one of
two (50%) showed agreement between methods due to a technical issue with one of the RV-
PCR samples (the filter cup leaked during incubation so spore outgrowth and subsequent cell
growth were compromised in this sample). Based on this issue, a change was instituted in the
protocol to cap the filter cup bottom before adding growth medium, rather than after. Because
capping is performed in the BSC, there is little risk for the additional handling of the filter cups
containing dry spores. After the protocol change was made, no issues with filter cup leakage
were noted.
For Round 1 post-decontamination samples, 41 of 44 (93%) were consistent between culture
and RV-PCR analyses (Table 3-29), including three samples that met the criteria for positive
detection based on heat lysis results, whereas the ACt values were < 6 for the magnetic bead-
based DNA extraction (as noted). Several MFP samples did not show CFU on the TSA plates
for culture analysis but were positive by PCR analysis of the concentrated enrichment culture.
Many of these samples were positive by RV-PCR. While two samples showed a PCR response,
these samples did not meet the criteria for positive detection by RV-PCR, namely Ct (T9) < 39
and ACt [Ct (TO)-Ct (T9)] > 6 (e.g., samples had ACt values of 5.6 and 3.7). In addition, one of
the samples was positive by RV-PCR but could not be confirmed by culture analysis. Such
discrepancies are expected since samples contained very low spore levels (no CFU measured
on plates) and, as previously mentioned, there are likely to be heterogeneities in partitioning
spores when splitting the extract for parallel culture and RV-PCR analyses.
3.2.2.1.3. Samples from Round 2
For Round 2 pre-decontamination samples, 19 of 20 samples (95%) showed consistent results
between culture and RV-PCR analyses, with only one sample not meeting the criteria for
148
-------
positive detection by RV-PCR (ACt value of 2.7 instead of > 6) (Table 3-30). As for the Round 1
post-decontamination samples mentioned above, this sample did not generate CPU on the ISA
plates from culture analysis and had low spore levels present. For the other Round 2 pre-
decontamination samples, 13 were positive for both methods, and six were negative for both
methods.
For Round 2 post-decontamination samples, there was 100% consistency between culture and
RV-PCR results, with 47 of 47 samples in agreement. Results are shown in Table 3-31. For
these samples, three samples were positive and 44 samples were negative for both methods.
Results for one negative control sample showed positive results for culture analysis likely due to
extra handling steps in the culture processing protocol.
3.2.2.1.4. Samples from Round 3
For Round 3 pre- and post-decontamination samples, there was 100% consistency between
culture and RV-PCR results, with 16 of 16 and 68 of 68 samples in agreement, respectively.
Results are shown in Table 3-32 for pre-decontamination samples and Table 3-33 for post-
decontamination samples. For pre-decontamination samples, 11 samples were positive and five
samples were negative for both methods. For post-decontamination samples, all 68 samples
were negative for both methods. In some cases (Table 3-33), negative control samples were
positive by culture analysis possibly due to cross-contamination. Protocol modifications
including extra glove changes were incorporated to prevent this error in the culture sample
analysis that followed.
3.2.2.2. EPA-OPP-MLB Results
A total of 50 samples were analyzed by the EPA-OPP-MLB Laboratory. Overall, the RV-PCR
method provided rapid results that were 86% consistent (43/50 samples) with results from
culture analysis. Among the seven samples showing disagreement between the methods, six
were culture positive and RV-PCR negative (12% false negative) while one was culture negative
and RV-PCR positive (2% false positive). A likely contributing factor to RV-PCR performance
involved inexperience with the magnetic bead-based DMA extraction procedure. Additional
experience and ongoing research on this method will address the problems with the
reproducibility of performance of the DMA extraction and purification protocol. However,
considering that the main purpose for participation of the MLB Laboratory in the BOTE Project
was to gain practice and experience with the RV-PCR method, the laboratory performed well.
The results are presented in Table 3-34, Table 3-35, Table 3-36, and Table 3-37.
149
-------
Table 3-27. LLNL RV-PCR results for MFP samples.
Sample
ID
1192
1203
1204
1205
1206
1207
1209
1211
1212
1213
1214
1215
1216
1218
1220
1221
1222
1461 (TB)
1462(TB)
1463(TB)
NC
PC
Culture (24-48 hr)
Average CPU/
Sample
0
0
0
0
0
0
0
0
0
3.6E1
3.6E1
0
1.8E1
1.1E2
0
2.3E2
0
0
0
0
0
3.7E5
Source
of
Culture
Result*
EC
EC
EC
EC
N/A
EC
RS
N/A
EC
EC
FF
EC
EC, RS
Plates
N/A
Plates
EC, RS
N/A
N/A
N/A
N/A
Plates
PCRof
Culture (Ct)**
23.4
32.8
21.5
31.6
N/A
30.3
17.2
N/A
21.1
19.1
20.9
33.4
17.7
19.4
Undetermined
18.4
17.0
N/A
N/A
N/A
N/A
18.1
Culture Result
(Pos/Neg)
Pos
Pos
Pos
Pos
Neg
Pos
Pos
Neg
Pos
Pos
Pos
Pos
Pos
Pos
Neg
Pos
Pos
Neg
Neg
Neg
Neg
Pos
RV-PCR (9 hr)
Average
ACt
15.6
7.2
9.5
11.2
0.0
18.2
18.2
0.0
16.0
17.7
17.6
14.3
16.9
15.8
0.0
13.4
16.8
0.0
0.0
0.0
0.0
24.5
Std
Dev
ACt
0.2
1.0
0.5
0.1
0.0
0.1
0.3
0.0
0.1
0.1
0.1
0.3
0.1
0.1
0.0
0.2
0.1
0.0
0.0
0.0
0.0
0.3
RV-PCR
Results
(Pos/Neg)
Pos
Pos
Pos
Pos
Neg
Pos
Pos
Neg
Pos
Pos
Pos
Pos
Pos
Pos
Neg
Pos
Pos
Neg
Neg
Neg
Neg
Pos
Notes
See Footnote (1)
See Footnote (1)
See Footnote (1)
See Footnote (1)
See Footnote (1)
*Positive (pos) or negative (neg) culture determination based upon the serial dilution (plates), filter funnel (FF) plate, and/or enrichment culture (EC), re-streak (RS)
from enrichment culture, or Promega-extracted concentrated EC (EC-PE). Green shading indicates when results were positive for Bg.
Positive culture results obtained if > 0 Bg colonies are presented on serial dilution or filter membrane plates, and/or Bg-positive PCR results are obtained (Ct < 35)
from colonies and/or the EC or RS.
N/A = not applicable; No CFU for analysis and EC not turbid and/or no growth from RS.
**Cycle threshold (Ct) values obtained from real-time PCR analysis of selected colonies and/or enrichment culture (EC).
Positive RV-PCR result based upon average ACt > 6 and T9 Ct < 39.
Footnote (1): To confirm culture result, the EC was concentrated 10-20 fold prior to PCR analysis.
Abbreviations: PC, positive control; NC, negative control; TB, true blank
150
-------
Table 3-28. LLNL RV-PCR results for Round 1 pre-decontamination samples.
Sample
ID
1196
1234
1461 (TB)
1462(TB)
1463(TB)
NC
PC
Culture (24-48 hr)
Average
CPU/Sample
0
3.6E2
0
0
0
0
3.7E5
Source of
Culture
Result*
EC
Plates
N/A
N/A
N/A
N/A
Plates
PCRof
Culture (Ct)**
16.0
18.0
N/A
N/A
N/A
N/A
18.1
Culture
Result
(Pos/Neg)
Pos
Pos
Neg
Neg
Neg
Neg
Pos
RV-PCR (9 hr)
Average
ACt
0.0
6.5
0.0
0.0
0.0
0.0
24.5
Std Dev
ACt
0.0
0.3
0.0
0.0
0.0
0.0
0.3
RV-PCR
Results
(Pos/Neg)
Neg
Pos
Neg
Neg
Neg
Neg
Pos
Notes
See Footnote (1)
*Positive (pos) or negative (neg) culture determination based upon the serial dilution (plates), filter funnel (FF) plate, and/or enrichment culture (EC). Green
shading indicates when results were positive for Bg.
Positive culture results obtained if > 0 Bg colonies are presented on serial dilution or filter membrane plates, and/or Bg-positive PCR results are obtained (Ct < 35)
from colonies and/or the EC or RS.
N/A = not applicable; No CFU for analysis and EC not turbid and/or no growth from RS.
**Cycle threshold (Ct) values obtained from real-time PCR analysis of selected colonies and/or enrichment culture (EC).
Positive RV-PCR result based upon average ACt > 6 and T9 Ct < 39.
Footnote (1): Sample ID No. 1196 dried out due to a capping problem. At T9, filter cup contents were resuspended in 1.5 ml_ medium for analysis.
Abbreviations: PC, positive control; NC, negative control; TB, true blank.
151
-------
Table 3-29. LLNL RV-PCR results for Round 1 post-decontamination samples.
Sample
ID
1185
1198
1199
1243
1246
1255
1257
1259
1261
1290
1291
1292
1298
1300
1301
1302
1303
1304
1305
1306
1475(TB)
1476(TB)
NC
PC
Culture (24-48 hr)
Average
CPU/Sample
0
0
0
3.0E1
3.0E1
0
3.0E1
0
0
0
0
0
1.3E3
3.0E1
0
0
0
0
5.3E1
1.8E1
0
0
0
2.9E5
Source of
Culture
Result*
EC-PE
N/A
N/A
Plates
Plates
EC
Plates
EC
EC-PE
EC-PE
EC
EC
Plates, FF
Plates
EC
N/A
EC
EC
Plates, FF
FF, RS
N/A
N/A
N/A
Plates
PCRof
Culture (Ct)**
25.2
N/A
N/A
15.7
13.7
25.6
14.6
21.9
25.8
23.8
25.6
25.5
12.9
13.6
25.4
N/A
35.0
23.8
13.3
14.5
N/A
N/A
N/A
14.0
Culture
Result
(Pos/Neg)
Pos
Neg
Neg
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Neg
Pos
Pos
Pos
Pos
Neg
Neg
Neg
Pos
RV-PCR (9 hr)
Average
ACt
7.1
0.0
0.0
6.5
6.5
5.6
6.1
0.0
7.8
8.6
10.9
12.9
8.3
6.7
4.2
0.0
9.6
14.8
20.2
16.0
0.0
0.0
0.0
23.6
Std Dev
ACt
1.8
0.0
0.0
0.9
0.9
0.3
1.3
0.0
1.2
0.6
0.1
0.2
0.7
0.8
2.0
0.0
2.2
0.1
1.8
1.7
0.0
0.0
0.0
0.0
RV-PCR
Results
(Pos/Neg)
Pos
Neg
Neg
Pos
Pos
Neg
Pos
Neg
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Neg
Pos
Pos
Pos
Pos
Neg
Neg
Neg
Pos
Notes
See Footnote (1)
RV-PCR Ave. ACt from 1 :20
dilution
Heat Lysis Ave. ACt = 3.7
See Footnote (1)
See Footnote (1)
See Footnote (2)
See Footnote (2)
Ave. ACt from 1:20 dilution;
Heat Lvsis Ave. ACt = 8.4
See Footnote (2)
*Positive (pos) or negative (neg) culture determination based upon the serial dilution (plates), filter funnel (FF) plate, and/or enrichment culture (EC), re-streak (RS)
from enrichment culture or Promega-extracted concentrated EC (EC-PE). Green shading indicates when results were positive for Bg. Positive culture results
obtained if > 0 Bg colonies are presented on serial dilution or filter membrane plates, and/or Bg-positive PCR results are obtained (Ct < 35) from colonies and/or
the EC or RS. N/A = not applicable; No CFU for analysis and EC not turbid and/or no growth from RS.
**Cycle threshold (Ct) values obtained from real-time PCR analysis of selected colonies and/or enrichment culture (EC). Positive RV-PCR result based upon
average ACt > 6 and T9 Ct < 39.
Footnote (1): EC concentrated 10-20-fold and DNA prepared using Promega extraction prior to PCR analysis to confirm culture result (EC-PE).
Footnote (2): PCR repeated to confirm RVPCR result; 1:20 dilution result shown.
Abbreviations: PC, positive control; NC, negative control; TB, true blank.
152
-------
Table 3-29 (continued). LLNL RV-PCR results for Round 1 post-decontamination samples.
Sample
ID
1307
1308
1309
1310
1311
1312
1374
1375
1377
1379
1382
1383
NC
PC
1163
1164
1165
1168
1171
1177
1179
Culture (24-48 hr)
Average
CPU/Sample
0
8.9E1
0
0
0
0
0
0
0
0
0
0
0
4.7E5
0
0
5.9E1
0
0
0
0
Source of
Culture
Result*
EC
Plates, FF
N/A
EC
N/A
EC
N/A
N/A
N/A
EC-PE
EC-PE
N/A
N/A
Plates
EC
RS
Plates
RS
N/A
RS
RS
PCRof
Culture (Ct)**
35.0
13.6
N/A
16.2
Undetermined
26.0
N/A
N/A
N/A
24.8
27.0
N/A
N/A
14.0
Undetermined
15.0
14.7
14.9
N/A
15.2
14.7
Culture
Result
(Pos/Neg)
Pos
Pos
Neg
Pos
Neg
Pos
Neg
Neg
Neg
Pos
Pos
Neg
Neg
Pos
Neg
Pos
Pos
Pos
Neg
Pos
Pos
RV-PCR (9 hr)
Average
ACt
6.7
7.9
0.0
8.7
6.4
7.7
0.0
0.0
0.0
12.3
12.6
0.0
0.0
24.9
0.0
5.9
8.8
6.1
0.0
8.6
4.9
Std Dev
ACt
0.4
0.6
0.0
0.3
0.3
0.3
0.0
0.0
0.0
0.5
0.1
0.0
0.0
0.1
0.0
0.6
0.2
0.4
0.0
0.7
1.0
RV-PCR
Results
(Pos/Neg)
Pos
Pos
Neg
Pos
Pos
Pos
Neg
Neg
Neg
Pos
Pos
Neg
Neg
Pos
Neg
Pos
Pos
Pos
Neg
Pos
Pos
Notes
RV-PCR Ave. ACt from 2
replicates (1 :20 dilution)
See Footnote (3)
See Footnote (3)
See Footnote (4): Heat Lysis Ave.
ACt = 8.3
See Footnote (4): Heat Lysis Ave.
ACt 8.7
*Positive (pos) or negative (neg) culture determination based upon the serial dilution (plates), filter funnel (FF) plate, and/or enrichment culture (EC), re-streak (RS)
from enrichment culture or Promega-extracted concentrated EC (EC-PE). Green shading indicates when results were positive for Bg. Positive culture results
obtained if > 0 Bg colonies are presented on serial dilution or filter membrane plates and/or Bg-positive PCR results are obtained (Ct < 35) from colonies and/or
the EC or RS. N/A = not applicable; No CFU for analysis and EC not turbid and/or no growth from RS.
**Cycle threshold (Ct) values obtained from real-time PCR analysis of selected colonies and/or enrichment culture (EC). Positive RV-PCR result based upon
average ACt > 6 and T9 Ct < 39.
Footnote (3): RVPCR results based on repeated PCR analysis with 1:20 dilution (1:10 dilution showed PCR inhibition); EC concentrated 10-20-fold and DNA
prepared using Promega extraction prior to PCR to confirm culture result (EC-PE).
Footnote (4): Low spore level, post-decontamination sample; sample positive by heat lysis RV-PCR.
Abbreviations: PC, positive control; NC, negative control; TB, true blank.
153
-------
Table 3-29 (continued). LLNL RV-PCR results for Round 1 post-decontamination samples.
Sample
ID
1180
1181
1182
1186
1210
1464(TB)
1465(TB)
1466(TB)
NC
PC
Culture (24-48 hr)
Average
CPU/Sample
0
0
3.0E1
0
3.6E1
0
0
0
0
3.4E5
Source of
Culture
Result*
FF, RS
EC
Plates, FF
EC
FF, EC
N/A
N/A
N/A
N/A
Plates
PCRof
Culture (Ct)**
34.6
Undetermined
14.4
19.9
14.1
N/A
N/A
N/A
N/A
13.5
Culture
Result
(Pos/Neg)
Pos
Neg
Pos
Pos
Pos
Neg
Neg
Neg
Neg
Pos
RV-PCR (9 hr)
Average
ACt
9.3
0.0
7.0
7.5
6.9
0.0
0.0
0.0
0.0
20.6
Std Dev
ACt
0.3
0.0
0.8
0.8
2.0
0.0
0.0
0.0
0.0
0.0
RV-PCR
Results
(Pos/Neg)
Pos
Neg
Pos
Pos
Pos
Neg
Neg
Neg
Neg
Pos
Notes
*Positive (pos) or negative (neg) culture determination based upon the serial dilution (plates), filter funnel (FF) plate, and/or enrichment culture (EC), or re-streak
(RS) from enrichment culture. Green shading indicates when results were positive for Bg.
Positive culture results obtained if > 0 Bg colonies are presented on serial dilution or filter membrane plates, and/or Bg-positive PCR results are obtained (Ct < 35)
from colonies and/or the EC or RS.
N/A = not applicable; No CFU for analysis and EC not turbid and/or no growth from RS.
**Cycle threshold (Ct) values obtained from real-time PCR analysis of selected colonies and/or enrichment culture (EC).
Positive RV-PCR result based upon average ACt > 6 and T9 Ct < 39.
Abbreviations: PC, positive control; NC, negative control; TB, true blank.
154
-------
Table 3-30. LLNL RV-PCR results for Round 2 pre-decontamination samples.
Sample
ID
1187
1293
1294
1345
1347
1351
1365
1366
1367
1369
1371
1372
1373
1378
1385
1467
1468
NC
PC
Culture (24-48 hr)
Average
CPU/Sample
0
1.5E5
2.2E5
0
2.3E5
1.9E5
2.2E5
2.2E5
0
1.5E5
0
1.9E5
0
5.2E4
1.3E5
0
0
0
5.3E1
Source of
Culture
Result*
N/A
Plates
Plates
N/A
Plates
Plates
Plates
Plates
N/A
Plates
N/A
Plates
EC, RS
Plates
Plates
N/A
N/A
N/A
Plates, FF
PCRof
Culture (Ct)**
N/A
15.1
15.6
N/A
15.5
15.5
15.2
15.4
Undetermined
15.4
N/A
14.9
21.3
14.8
15.2
N/A
N/A
N/A
15.6
Culture
Result
(Pos/Neg)
Neg
Pos
Pos
Neg
Pos
Pos
Pos
Pos
Neg
Pos
Neg
Pos
Pos
Pos
Pos
Neg
Neg
Neg
Pos
RV-PCR (9 hr)
Average
ACt
0.0
6.4
6.2
0.0
21.7
20.4
12.8
13.9
0.0
9.7
0.0
18.2
0.0
17.2
19.8
0.0
0.0
0.0
16.6
Std Dev
ACt
0.0
1.0
0.6
0.0
0.2
0.3
0.3
0.0
0.0
0.4
0.0
0.1
0.0
0.2
0.2
0.0
0.0
0.0
0.1
RV-PCR
Results
(Pos/Neg)
Neg
Pos
Pos
Neg
Pos
Pos
Pos
Pos
Neg
Pos
Neg
Pos
Neg
Pos
Pos
Neg
Neg
Neg
Pos
Notes
See Footnote (1): RV-PCR
1/1 0 dilution Ave. ACt = 8.2
See Footnote (2): Heat Lysis
1/10 dilution Ave. ACt = 2.7
See Footnote (1): RV-PCR
1/10 dilution Ave. ACt = 7.4
See Footnote (1): RV-PCR
1/10 dilution Ave. ACt = 3.4
*Positive (pos) or negative (neg) culture determination based upon the serial dilution (plates), filter funnel (FF) plate, and/or enrichment culture (EC), or re-streak
(RS) from enrichment culture. Green shading indicates when results were positive for Bg.
Positive culture results obtained if > 0 Bg colonies are presented on serial dilution or filter membrane plates, and/or Bg-positive PCR results are obtained (Ct < 35)
from colonies and/or the EC or RS. N/A = not applicable; No CFU for analysis and EC not turbid and/or no growth from RS.
**Cycle threshold (Ct) values obtained from real-time PCR analysis of selected colonies and/or enrichment culture (EC).
Positive RV-PCR result based upon average ACt > 6 and T9 Ct < 39.
Footnote (1): RV-PCR Ave. Delta Ct data from 1:20 dilution; 1:10 dilution showed inhibition.
Footnote (2): Low spore level, variability when sample split; RV-PCR negative by 1:10 dilution, 1:20 dilution, and undiluted heat lysis RV-PCR).
Abbreviations: PC, positive control; NC, negative control; TB, true blank.
155
-------
Table 3-30 (continued). LLNL RV-PCR results for Round 2 pre-decontamination samples.
Sample
ID
1363
1364
1368
1370
1380
1474
NC
PC
Culture (24-48 hr)
Average
CPU/Sample
5.9E4
0
0
2.0E4
1.7E5
0
0
5.3E1
Source of
Culture
Result*
Plates
N/A
N/A
Plates
Plates
N/A
N/A
Plates, FF
PCRof
Culture (Ct)**
16.6
N/A
N/A
16.5
16.2
N/A
N/A
15.6
Culture
Result
(Pos/Neg)
Pos
Neg
Neg
Pos
Pos
Neg
Neg
Pos
RV-PCR (9 hr)
Average
ACt
20.8
0.0
0.0
18.8
19.0
0.0
0.0
7.1
Std Dev
ACt
0.1
0.0
0.0
0.2
0.2
0.0
0.0
0.1
RV-PCR
Results
(Pos/Neg)
Pos
Neg
Neg
Pos
Pos
Neg
Neg
Pos
Notes
*Positive (pos) or negative (neg) culture determination based upon the serial dilution (Plates), or filter funnel (FF) plate. Green shading indicates when results were
positive for Bg.
Positive culture results obtained if > 0 Bg colonies are presented on serial dilution or filter membrane plates, and/or Bg-positive PCR results are obtained (Ct < 35)
from colonies and/or the EC or RS.
N/A = not applicable; No CFU for analysis and EC not turbid and/or no growth from RS.
**Cycle threshold (Ct) values obtained from real-time PCR analysis of selected colonies and/or enrichment culture (EC).
Positive RV-PCR result based upon average ACt > 6 and T9 Ct < 39.
Abbreviations: PC, positive control; NC, negative control; TB, true blank.
156
-------
Table 3-31. LLNL RV-PCR results for Round 2 post-decontamination samples.
Sample
ID
1260
1314
1315
1316
1317
1318
1319
1320
1322
1324
1325
1328
1329
1332
1337
1479(TB)
NC
PC
1341
1342
1344
1346
Culture (24-48 hr)
Average
CPU/Sample
0
0
0
1.8E1
0
0
0
0
0
0
0
0
0
0
0
0
1.8E1
7.1E1
0
0
0
0
Source of
Culture
Result*
N/A
N/A
N/A
FF
N/A
N/A
N/A
EC-PE
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
Plates, FF
Plates, FF
N/A
N/A
N/A
N/A
PCRof
Culture (Ct)**
N/A
N/A
N/A
16.8
N/A
N/A
N/A
27.4
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
15.3
15.1
N/A
N/A
N/A
N/A
Culture
Result
(Pos/Neg)
Neg
Neg
Neg
Pos
Neg
Neg
Neg
Pos
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Pos
Pos
Neg
Neg
Neg
Neg
RV-PCR (9 hr)
Average
ACt
0.0
0.0
0.0
9.0
0.0
0.0
0.0
6.9
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
11.4
0.0
0.0
0.0
0.0
Std Dev
ACt
0.0
0.0
0.0
0.3
0.0
0.0
0.0
0.2
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.3
0.0
0.0
0.0
0.0
RV-PCR
Results
(Pos/Neg)
Neg
Neg
Neg
Pos
Neg
Neg
Neg
Pos
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Pos
Neg
Neg
Neg
Neg
Notes
See Footnote (1)
See Footnote (2)
Cross-contamination for
culture portion only
*Positive (pos) or negative (neg) culture determination based upon the serial dilution (plates), filter funnel (FF) plate, and/or enrichment culture (EC). Green
shading indicates when results were positive for Bg.
Positive culture results obtained if > 0 Bg colonies are presented on serial dilution or filter membrane plates, and/or Bg-positive PCR results are obtained (Ct < 35)
from colonies and/or the EC or RS.
N/A = not applicable; No CFU for analysis and EC not turbid and/or no growth from RS.
**Cycle threshold (Ct) values obtained from real-time PCR analysis of selected colonies and/or enrichment culture (EC).
Positive RV-PCR result based upon average ACt > 6 and T9 Ct < 39.
Footnote (1): Culture-PCR result based on repeated analysis and 1:10 dilution.
Footnote (2): EC concentrated 10-20-fold and DNA prepared using Promega extraction prior to PCR, to confirm culture results (EC-PE).
Abbreviations: PC, positive control; NC, negative control; TB, true blank.
157
-------
Table 3-31 (continued). LLNL RV-PCR results for Round 2 post-decontamination samples.
Sample
ID
1348
1349
1352
1354
1355
1356
1357
1358
1359
1360
1361
1480(TB)
NC
PC
Culture (24-48 hr)
Average
CPU/Sample
0
0
0
0
0
0
0
0
0
0
0
0
0
8.9E1
Source of
Culture
Result*
N/A
N/A
N/A
N/A
N/A
N/A
EC-PE
N/A
N/A
N/A
N/A
N/A
N/A
Plates, FF
PCRof
Culture (Ct)**
N/A
N/A
N/A
N/A
N/A
N/A
25.0
N/A
N/A
N/A
N/A
N/A
N/A
15.1
Culture
Result
(Pos/Neg)
Neg
Neg
Neg
Neg
Neg
Neg
Pos
Neg
Neg
Neg
Neg
Neg
Neg
Pos
RV-PCR (9 hr)
Average
ACt
0.0
0.0
0.0
0.0
0.0
0.0
7.5
0.0
0.0
0.0
0.0
0.0
0.0
15.9
Std Dev
ACt
0.0
0.0
0.0
0.0
0.0
0.0
0.3
0.0
0.0
0.0
0.0
0.0
0.0
0.5
RV-PCR
Results
(Pos/Neg)
Neg
Neg
Neg
Neg
Neg
Neg
Pos
Neg
Neg
Neg
Neg
Neg
Neg
Pos
Notes
See Footnote (1)
*Positive (pos) or negative (neg) culture determination based upon the serial dilution (plates), filter funnel (FF) plate, and/or enrichment culture (EC). Green
shading indicates when results were positive for Bg.
Positive culture results obtained if > 0 Bg colonies are presented on serial dilution or filter membrane plates, and/or Bg-positive PCR results are obtained (Ct < 35)
from colonies and/or the EC or RS.
N/A = not applicable; No CFU for analysis and EC not turbid and/or no growth from RS.
**Cycle threshold (Ct) values obtained from real-time PCR analysis of selected colonies and/or enrichment culture (EC).
Positive RV-PCR result based upon average ACt > 6 and T9 Ct < 39.
Footnote (1): Culture EC concentrated 10-20-fold and DNA prepared using Promega extraction prior to PCR, to confirm culture result (EC-PE).
Abbreviations: PC, positive control; NC, negative control; TB, true blank.
158
-------
Table 3-31 (continued). LLNL RV-PCR results for Round 2 post-decontamination samples.
Sample
ID
1238
1313
1321
1323
1326
1327
1331
1333
1334
1335
1338
1339
1340
1343
1350
1473(TB)
NC
PC
1353
1362
Culture (24-48 hr)
Average
CPU/Sample
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
8.9E1
0
0
Source of
Culture
Result*
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
Plates, FF
N/A
N/A
PCRof
Culture (Ct)**
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
15.6
N/A
N/A
Culture
Result
(Pos/Neg)
Neg
Neg
Neg
Neq
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Pos
Neg
Neg
RV-PCR (9 hr)
Average
ACt
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
10.1
0.0
0.0
Std Dev
ACt
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.1
0.0
0.0
RV-PCR
Results
(Pos/Neg)
Neg
Neg
Neg
Neq
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Pos
Neg
Neg
Notes
*Positive (pos) or negative (neg) culture determination based upon the serial dilution (plates), or filter funnel (FF) plate. Green shading indicates when results were
positive for Bg.
Positive culture results obtained if > 0 Bg colonies are presented on serial dilution or filter membrane plates, and/or Bg-positive PCR results are obtained (Ct < 35)
from colonies and/or the EC or RS. N/A = not applicable; No CFU for analysis and EC not turbid and/or no growth from RS.
**Cycle threshold (Ct) values obtained from real-time PCR analysis of selected colonies and/or enrichment culture (EC).
Positive RV-PCR result based upon average ACt > 6 and T9 Ct < 39.
Abbreviations: PC, positive control; NC, negative control; TB, true blank.
159
-------
Table 3-32. LLNL RV-PCR results for Round 3 pre-decontamination samples.
Sample
ID
1241
1247
1266
1271
1273
1277
1278
1279
1280
1283
1285
1287
1478(TB)
NC
PC
1240
1275
1276
1472(TB)
NC
PC
1264
Culture (24-48 hr)
Average
CPU/Sample
0
9.6E4
7.6E4
0
1.7E5
2.2E5
2.1E5
0
1.8E1
2.0E5
7.1E3
1.4E4
0
2.4E2
1.8E1
2.1E4
1.3E5
0
0
0
5.3E1
0
Source of
Culture
Result*
N/A
Plates
Plates
N/A
Plates
Plates
Plates
N/A
Plates, FF
Plates
Plates
Plates
N/A
Plates, FF
Plates, FF
Plates
Plates
N/A
N/A
N/A
Plates, FF
N/A
Culture- PCR
(Ct)**
N/A
19.2
19.4
N/A
18.8
17.8
18.0
N/A
21.4
18.8
19.8
18.9
N/A
19.8
18.4
15.1
15.1
N/A
N/A
N/A
17.8
N/A
Culture
Result
(Pos/Neg)
Neg
Pos
Pos
Neg
Pos
Pos
Pos
Neg
Pos
Pos
Pos
Pos
Neg
Pos
Pos
Pos
Pos
Neg
Neg
Neg
Pos
Neg
RV-PCR (9 hr)
Average
ACt
0.0
21.9
21.7
0.0
9.6
20.5
20.4
0.0
22.5
10.4
21.2
20.8
0.0
0.0
12.0
14.4
16.7
0.0
0.0
0.0
7.8
0.0
Std Dev
ACt
0.0
0.2
0.1
0.0
0.4
0.2
0.1
0.0
0.1
0.4
0.1
0.1
0.0
0.0
0.3
0.2
0.8
0.0
0.0
0.0
0.6
0.0
RV-PCR
Results
(Pos/Neg)
Neg
Pos
Pos
Neg
Pos
Pos
Pos
Neg
Pos
Pos
Pos
Pos
Neg
Neg
Pos
Pos
Pos
Neg
Neg
Neg
Pos
Neg
Notes
See Footnote (1)
See Footnote (1)
See Footnote (1)
See Footnote (1)
See Footnote (1)
Cross-contamination for
culture portion only
*Positive (pos) or negative (neg) culture determination based upon the serial dilution (plates), or filter funnel (FF) plate. Green shading indicates when results were
positive for Bg.
Positive culture results obtained if > 0 Bg colonies are presented on serial dilution or filter membrane plates, and/or Bg-positive PCR results are obtained (Ct < 35)
from colonies and/or the EC or RS.
N/A = not applicable; No CFU for analysis and EC not turbid and/or no growth from RS.
**Cycle threshold (Ct) values obtained from real-time PCR analysis of selected colonies and/or enrichment culture (EC).
Positive RV-PCR result based upon average ACt > 6 and T9 Ct < 39.
Footnote (1): RV-PCR results based on 1:20 dilution (1:10 dilution showed PCR inhibition).
Abbreviations: PC, positive control; NC, negative control; TB, true blank.
160
-------
Table 3-33. LLNL RV-PCR results for Round 3 post-decontamination samples.
Sample
ID
1242
1244
1245
1248
1250
1251
1252
1253
1256
1263
1267
1268
1269
1270
1272
1274
1469(TB)
NC
PC
1281
1282
1284
1286
1330
1336
1402
1403
1404
Culture (24-48 hr)
Average
CPU/Sample
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1.8E1
3.6E1
0
0
0
0
0
0
0
0
0
Source of
Culture
Result*
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
Plates, FF
Plates, FF
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
PCRof
Culture (Ct)**
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
Undetermined
29.7
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
Culture
Result
(Pos/Neg)
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Pos
Pos
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
RV-PCR (9 hr)
Average
ACt
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
12.3
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
Std Dev
ACt
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.1
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
RV-PCR
Results
(Pos/Neg)
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Pos
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Notes
Cross-contamination
observed for culture onlv
*Positive (pos) or negative (neg) culture determination based upon the serial dilution (plates) or filter funnel (FF) plate. Green shading indicates when results were
positive forBg. Positive culture results obtained if > 0 Bg colonies are presented on serial dilution or filter membrane plates, and/or Bg-positive PCR results are
obtained (Ct < 35) from colonies and/or the EC or RS. N/A = not applicable; No CFU for analysis and EC not turbid and/or no growth from RS.
**Cycle threshold (Ct) values obtained from real-time PCR analysis of selected colonies and/or enrichment culture (EC).
Positive RV-PCR result based upon average ACt > 6 and T9 Ct < 39.
Abbreviations: PC, positive control; NC, negative control; TB, true blank.
161
-------
Table 3-33 (continued). LLNL RV-PCR results for Round 3 post-decontamination samples.
Sample
ID
1242
1244
1245
1248
1250
1251
1252
1253
1256
1263
1267
1268
1269
1270
1272
1274
1469(TB)
NC
PC
1281
1282
1284
1286
1330
1336
1402
1403
1404
Culture (24-48 hr)
Average
CPU/Sample
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1.8E1
3.6E1
0
0
0
0
0
0
0
0
0
Source of
Culture
Result*
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
Plates, FF
Plates, FF
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
PCRof
Culture (Ct)**
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
Undetermined
29.7
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
Culture
Result
(Pos/Neg)
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Pos
Pos
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
RV-PCR (9 hr)
Average
ACt
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
12.3
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
Std Dev
ACt
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.1
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
RV-PCR
Results
(Pos/Neg)
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Pos
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Notes
Cross-contamination
observed for culture onlv
*Positive (pos) or negative (neg) culture determination based upon the serial dilution (plates) or filter funnel (FF) plate. Green shading indicates when results were
positive forBg. Positive culture results obtained if > 0 Bg colonies are presented on serial dilution or filter membrane plates, and/or Bg-positive PCR results are
obtained (Ct < 35) from colonies and/or the EC or RS. N/A= not applicable; No CFU for analysis and EC not turbid and/or no growth from RS.
**Cycle threshold (Ct) values obtained from real-time PCR analysis of selected colonies and/or enrichment culture (EC).
Positive RV-PCR result based upon average ACt > 6 and T9 Ct < 39.
Abbreviations: PC, positive control; NC, negative control; TB, true blank.
162
-------
Table 3-33 (continued). LLNL RV-PCR results for Round 3 post-decontamination samples.
Sample
ID
1406
1407
1409
1410
1411
1412
1415
1470(TB)
NC
PC
1249
1418
1422
1423
1426
1427
1428
1429
1434
1435
1436
1437
1440
1442
1449
1471 (TB)
NC
PC
Culture (24-48 hr)
Average
CPU/Sample
0
0
0
0
0
0
0
0
0
1.2E2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
3.6E1
Source of
Culture
Result*
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
Plates, FF
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
Plates, FF
PCRof
Culture (Ct)**
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
29.5
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
15.5
Culture
Result
(Pos/Neg)
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Pos
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Pos
RV-PCR (9 hr)
Average
ACt
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
12.1
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
10.2
Std Dev
ACt
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.1
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.3
RV-PCR
Results
(Pos/Neg)
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Pos
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Pos
Notes
See Footnote (1)
See Footnote (1)
*Positive (pos) or negative (neg) culture determination based upon the serial dilution (plates), or filter funnel (FF) plate. Green shading indicates when results were
positive for Bg. Positive culture results obtained if > 0 Bg CFU are presented on serial dilution or filter membrane plates, and/or Bg-positive PCR results are
obtained (Ct < 35) from colonies and/or the EC or RS.
N/A = not applicable; No CFU for analysis and EC not turbid and/or no growth from RS.
**Cycle threshold (Ct) values obtained from real-time PCR analysis of selected colonies and/or enrichment culture (EC). Positive RV-PCR result based upon
average ACt > 6 and T9 Ct < 39.
Footnote (1): The PC results are from PCs prepared in different batches (spiked at either the 101 or 102 spores per wipe level).
Abbreviations: PC, positive control; NC, negative control; TB, true blank.
163
-------
Table 3-33 (continued). LLNL RV-PCR results for Round 3 post-decontamination samples.
Sample
ID
1450
1390
1391
1392
1393
1398
1399
1400
1401
1405
1439
1441
1443
1444
1472(TB)
NC
PC
1430
1431
1433
1445
1446
1447
1448
1474(TB)
NC
PC
Culture (24-48 hr)
Average
CPU/Sample
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
5.3E1
0
0
0
0
0
0
0
0
0
5.3E1
Source of
Culture
Result*
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
Plates, FF
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
Plates, FF
PCRof
Culture (Ct)**
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
17.8
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
15.6
Culture
Result
(Pos/Neg)
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Pos
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Pos
RV-PCR (9 hr)
Average
ACt
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
7.8
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
7.1
Std Dev
ACt
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.6
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.1
RV-PCR
Results
(Pos/Neg)
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Pos
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Pos
Notes
*Positive (pos) or negative (neg) culture determination based upon the serial dilution (plates), or filter funnel (FF) plate. Green shading indicates when results were
positive for Bg.
Positive culture results obtained if > 0 Bg colonies are presented on serial dilution or filter membrane plates, and/or Bg-positive PCR results are obtained (Ct < 35)
from colonies and/or the EC or RS.
N/A = not applicable; No CFU for analysis and EC not turbid and/or no growth from RS.
**Cycle threshold (Ct) values obtained from real-time PCR analysis of selected colonies and/or enrichment culture (EC).
Positive RV-PCR result based upon average ACt > 6 and T9 Ct < 39.
Abbreviations: PC, positive control; NC, negative control; TB, true blank.
164
-------
Table 3-34. EPA-OPP-MLB RV-PCR results for MFP samples.
Sample
ID
1201
1208
TB(1451)
NC
PC
Culture (24-48 hr)
Average
CPU/Sample
0
0
0
0
1.0E3
Source of
Culture
Result*
N/A
N/A
N/A
N/A
Plates
PCRof
Culture (Ct)**
N/A
N/A
N/A
N/A
ND
Culture
Result
(Pos/Neg)
Neg
Neg
Neg
Neg
Pos
RV-PCR (9 hr)
Average
ACt
0.0
13.5
3.0
5.0
14.1
Std Dev
ACt
0.0
0.5
2.7
1.2
0.4
RV-PCR
Results
(Pos/Neg)
Neg
Pos
Neg
Neg
Pos
Notes
*Positive (pos) or negative (neg) culture determination based upon the serial dilution (plates). Green shading indicates when results were positive for Bg.
Positive culture results obtained if > 0 Bg colonies are presented on serial dilution or filter membrane plates, and/or Bg-positive PCR results are obtained (Ct < 35)
from colonies and/or the EC or RS.
N/A = not applicable; No CPU for analysis and EC not turbid and/or no growth from RS.
**Cycle threshold (Ct) values obtained from real-time PCR analysis of selected colonies and/or enrichment culture (EC).
Positive RV-PCR result based upon average ACt > 6 and T9 Ct < 39.
Abbreviations: PC, positive control; NC, negative control; TB, true blank.
165
-------
Table 3-35. EPA-OPP-MLB RV-PCR results for Round 1 pre-decontamination samples.
Sample
ID
1217
1219
1223
1224
1225
1226
1227
1228
1229
1230
1231
1232
1233
1235
1236
1237
TB(1451)
NC
PC
Culture (24-48 hr)
Average
CPU/Sample
1.8E5
0
3.6E5
0
4.4E5
4.3E5
5.4E4
4.4EO
1.8E1
0
3.4E5
5.8E5
2.0E5
3.4E5
2.9E5
0
0
0
1.0E3
Source of
Culture
Result*
Plates
N/A
Plates
N/A
Plates
Plates
Plates
Plates
FF
EC
Plates
Plates
Plates
Plates
Plates
N/A
N/A
N/A
Plates
Culture- PCR
(Ct)**
ND
N/A
ND
N/A
21.7
ND
ND
22.4
21.5
19.5
ND
ND
ND
ND
ND
N/A
N/A
N/A
ND
Culture
Result
(Pos/Neg)
Pos
Neg
Pos
Neg
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Neg
Neg
Neg
Pos
RV-PCR (9 hr)
Average
ACt
19.4
0.0
21.8
0.0
10.4
13.3
0.0
0.0
13.8
0.0
12.9
23.9
0.0
28.0
12.5
0.0
3.0
5.0
14.1
Std Dev
ACt
0.2
0.0
1.8
0.0
0.2
0.3
0.0
0.0
0.1
0.0
2.4
0.1
0.0
0.0
0.2
0.0
2.7
1.2
0.4
RV-PCR
Results
(Pos/Neg)
Pos
Neg
Pos
Neg
Pos
Pos
Pos
Neg
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Neg
Neg
Neg
Pos
Notes
Footnote (1): Heat Lysis
Ave. ACt= 17.9
Footnote (1): Heat Lysis
Ave. ACt = 0
Footnote (1): Heat Lysis
Ave. ACt= 17.0
Footnote (1): Heat Lysis
Ave. ACt= 17.0
*Positive (pos) or negative (neg) culture determination based upon the serial dilution (plates), filter funnel (FF) plate, and/or enrichment culture (EC). Green
shading indicates when results were positive for Bg.
Positive culture results obtained if > 0 Bg colonies are presented on serial dilution or filter membrane plates, and/or Bg-positive PCR results are obtained
(Ct <35) from colonies and/or the EC or RS.
N/A = not applicable; No CFU for analysis and EC not turbid and/or no growth from RS.
ND = Not Determined.
**Cycle threshold (Ct) values obtained from real-time PCR analysis of selected colonies and/or enrichment culture (EC).
Positive RV-PCR result based upon average ACt > 6 and T9 Ct < 39.
Footnote (1): Sample also tested by heat lysis RV-PCR with results shown.
Abbreviations: PC, positive control; NC, negative control; TB, true blank.
166
-------
Table 3-36. EPA-OPP-MLB RV-PCR results for Round 1 post-decontamination samples.
Sample
ID
1166
1167
1169
1170
1172
1173
1174
1175
1176
1178
1183
1184
1188
1189
1190
1193
1194
1197
1239
1288
1295
Culture (24-48 hr)
Average
CPU/Sample
0
0
0
0
0
0
0
0
0
0
0
0
3.6E4
0
0
0
4.4E1
0
4.4E1
0
0
Source of
Culture
Result*
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
EC
Plates
N/A
N/A
N/A
Plates
N/A
Plates
N/A
N/A
PCRof
Culture (Ct)**
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
18.2
23.3
N/A
N/A
N/A
18.9
N/A
17.8
N/A
N/A
Culture
Result
(Pos/Neg)
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Pos
Pos
Neg
Neg
Neg
Pos
Neg
Pos
Neg
Neg
RV-PCR (9 hr)
Average
ACt
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
Std Dev
ACt
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
RV-PCR
Results
(Pos/Neg)
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Pos
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Notes
Footnote (1): Heat Lysis
Ave. ACt = 0
Footnote (1): Heat Lysis
Ave. ACt = 13.1
Footnote (1): Heat Lysis
Ave ACt = 0
Footnote (1): Heat Lysis
Ave ACt = 0
*Positive (pos) or negative (neg) culture determination based upon the serial dilution (plates), filter funnel (FF) plate, and/or enrichment culture (EC). Green
shading indicates when results were positive for Bg.
Positive culture results obtained if > 0 Bg colonies are presented on serial dilution or filter membrane plates, and/or Bg-positive PCR results are obtained (Ct < 35)
from colonies and/or the EC or RS. N/A = not applicable; No CFU for analysis and EC not turbid and/or no growth from RS.
**Cycle threshold (Ct) values obtained from real-time PCR analysis of selected colonies and/or enrichment culture (EC). Positive RV-PCR result based upon
average ACt > 6 and T9 Ct < 39.
Footnote (1): Sample also tested by heat lysis RV-PCR with results shown.
Abbreviations: PC, positive control; NC, negative control; TB, true blank.
167
-------
Table 3-36 (continued). EPA-OPP-MLB RV-PCR results for Round 1 post-decontamination samples.
Sample
ID
1296
1299
TB(1452)
NC
PC
Culture (24-48 hr)
Average
CPU/Sample
0
0
0
0
6.4E2
Source of
Culture
Result*
N/A
N/A
N/A
N/A
Plates
PCRof
Culture (Ct)**
N/A
N/A
N/A
N/A
ND
Culture
Result
(Pos/Neg)
Neg
Neg
Neg
Neg
Pos
RV-PCR (9 hr)
Average
ACt
0.0
0.0
0.0
0.0
12.5
Std Dev
ACt
0.0
0.0
0.0
0.0
3.4
RV-PCR
Results
(Pos/Neg)
Neg
Neg
Neg
Neg
Pos
Notes
*Positive (pos) or negative (neg) culture determination based upon the serial dilution (plates). Green shading indicates when results were positive for Bg.
Positive culture results obtained if > 0 Bg colonies are presented on serial dilution or filter membrane plates, and/or Bg-positive PCR results are obtained (Ct < 35)
from colonies and/or the EC or RS.
N/A = not applicable; No CPU for analysis and EC not turbid and/or no growth from RS.
ND = Not Determined.
**Cycle threshold (Ct) values obtained from real-time PCR analysis of selected colonies and/or enrichment culture (EC).
Positive RV-PCR result based upon average ACt > 6 and T9 Ct < 39.
Abbreviations: PC, positive control; NC, negative control; TB, true blank.
168
-------
Table 3-37. EPA-OPP-MLB RV-PCR results for Round 2 pre-decontamination samples.
Sample
ID
1384
1289
1262
1387
1258
1381
1254
1376
1386
TB(1455)
NC
PC
Culture (24-48 hr)
Average
CPU/Sample
6.7E5
0
5.6E5
6.3E5
0
2.3E5
3.0E5
3.6E5
3.6E5
0
0
5.5E2
Source of
Culture
Result*
Plates
N/A
Plates
Plates
N/A
Plates
Plates
Plates
Plates
N/A
N/A
Plates
PCRof
Culture (Ct)**
ND
N/A
ND
18.2
N/A
ND
ND
ND
ND
N/A
N/A
ND
Culture
Result
(Pos/Neg)
Pos
Neg
Pos
Pos
Neg
Pos
Pos
Pos
Pos
Neg
Neg
Pos
RV-PCR (9 hr)
Average
ACt
10.0
0.0
0.0
11.0
3.0
0.0
11.7
0.0
6.2
3.0
5.0
17.1
Std Dev
ACt
2.8
0.0
0.0
0.9
0.0
0.0
0.8
0.0
1.9
2.7
1.2
2.3
RV-PCR
Results
(Pos/Neg)
Pos
Neg
Pos
Pos
Neg
Neg
Pos
Neg
Pos
Neg
Neg
Pos
Notes
Footnote (1): Heat Lysis
Ave ACt = 25 7
Footnote (1): Heat Lysis
Ave ACt = 0
Footnote (1): Heat Lysis
Ave ACt = 0
*Positive (pos) or negative (neg) culture determination based upon the serial dilution (plates). Green shading indicates when results were positive for Bg.
Positive culture results obtained if > 0 Bg colonies are presented on serial dilution or filter membrane plates, and/or Bg-positive PCR results are obtained (Ct < 35)
from colonies and/or the EC or RS.
N/A = not applicable; No CFU for analysis and EC not turbid and/or no growth from RS.
ND = Not Determined.
**Cycle threshold (Ct) values obtained from real-time PCR analysis of selected colonies and/or enrichment culture (EC).
Positive RV-PCR result based upon average ACt > 6 and T9 Ct < 39.
Footnote (1): Sample also tested by heat lysis RV-PCR with results shown.
Abbreviations: PC, positive control; NC, negative control; TB, true blank.
169
-------
3.2.3. Air Sampling
This section reports the results from the air sampling performed during the BOTE Project for (1)
AAS, and (2) the reaerosolization study.
3.2.3.1. Aggressive Air Sampling Results
The INL Microbiology Laboratory reported all sample results as CPU. The quantification range
was 30-300 CPU per plate. Concentrations in CPU per cubic foot (ft3) (or m3) of air were derived
by dividing the reported CPU for each sample by the volume of air drawn through the samplers.
Each sample was collected for one hr; the volume of air drawn through the XMXs was therefore
1,123 ft3 (31.8 m3, 530 L/min x 60 min), and the volume of air drawn through the STAs was 60
ft3 (1.70 m3, 28.3 L/min x 60 min). At the minimum quantification limit (30 CPU), the
corresponding minimum quantifiable level for the STA was 0.50 CPU/ft3 (17.6 CFU/m3). For the
XMX, the laboratory analyzed 1/20th of the sample, so the minimum quantifiable level was 0.54
CPU/ft3 (18.9 CFU/m3). Technically, the LOD is 1 CPU. The estimated LCDs for the XMX and
STA are therefore 0.018 and 0.017 CFU/ft3 (0.63 and 0.59 CFU/m3), respectively. Values below
the lower quantification limit (for CFU) or minimum quantifiable level (for air concentrations)
were used as estimates, consistent with ASTM Method D5465-931741.
Background air samples in the facility were collected and analyzed prior to the Bg release,
duirng the MFP. All three XMX results were ND and one of the three STAs resulted in
detectable Bg (8 CFU). However, upon further investigation, six of the CFUs were discovered to
be located in the middle of the agar plate where particles would not impact. Sampling of the
STA samplers revealed that four of the nine were contaminated with Bg prior to sampling. All
STA samplers were decontaminated with pH-adjusted bleach and placed in the facility during
the VHP® fumigation in Round 1 to further sterilize the STA samplers.
Table 3-38. through Table 3-40. display the AAS results after each decontamination event.
Three one-hr samples were collected by each XMX and STA sampler during each sampling
event (except for the Hallway STA sampler during the hydrogen peroxide event, due to
equipment failure). This equipment failure reduced the statistical power of the data, however, it
did not nullify the test results. The three one-hr time intervals are noted in
Table 3-38., Table 3-39., and Table 3-40. as "H + 0" for the first hr of sampling, "H + 60" for the
second hr of sampling, and "H + 120" for the third hr of sampling. Rooms 105 and 106 had two
XMX samplers and one STA sampler, and the hallway had one XMX sampler and one STA
sampler during each sampling event. There are therefore two XMX sampler concentrations
reported during the three time intervals in each room. All results for blanks are reported in total
CFU. One XMX blank (Round 3, Room 105) had 1 Bg CFU. This result was reported as 20 CFU
because the laboratory analyzed only 1/20th of the total sample.
170
-------
Table 3-38. Aggressive air sampling results for Round 1
Location
Room 105
Room 105
Room 105
Room 105
Room 105
Room 105
Room 105
Room 105
Room 106
Room 106
Room 106
Room 106
Room 106
Room 106
Room 106
Room 106
Hallway
Hallway
Hallway
Hallway
Hallway
Hallway
Hallway
Hallway
Sample Type
XMX
XMX
XMX
STA
STA
STA
XMX-Blank
STA-Blank
XMX
XMX
XMX
STA
STA
STA
XMX-Blank
STA-Blank
XMX
XMX
XMX
STA
STA
STA
XMX-Blank
STA-Blank
Time Interval
(1st, 2nd, or 3rd hour
of sampling)
H + 0
H + 60
H + 120
H + 0
H + 60
H + 120
N/A
N/A
H + 0
H + 60
H + 120
H + 0
H + 60
H + 120
N/A
N/A
H + 0
H + 60
H + 120
H + 0
H + 60
H + 120
N/A
N/A
Concentrations
(CFU/ft3 or CPU/Blank)
1.4, 1.4
0.24,0.18
ND, 0.18
ND
ND
ND
ND
0.057
0.12,0.54
0.18,0.18
ND, ND
0.082
ND
ND
ND
ND
0.65
0.24
ND
ND
Not collected
Not collected
ND
ND
ND = Not detected. N/A = Not applicable.
171
-------
Table 3-39. Aggressive air sample results for Round 2
Location
Room 105
Room 105
Room 105
Room 105
Room 105
Room 105
Room 105
Room 105
Room 106
Room 106
Room 106
Room 106
Room 106
Room 106
Room 106
Room 106
Hallway
Hallway
Hallway
Hallway
Hallway
Hallway
Hallway
Hallway
Sample Type
XMX
XMX
XMX
STA
STA
STA
XMX-Blank
STA-Blank
XMX
XMX
XMX
STA
STA
STA
XMX-Blank
STA-Blank
XMX
XMX
XMX
STA
STA
STA
XMX-Blank
STA-Blank
Time Interval
(1st, 2nd, or 3rd hour
of sampling)
H + 0
H + 60
H + 120
H + 0
H + 60
H + 120
N/A
N/A
H + 0
H + 60
H + 120
H + 0
H + 60
H + 120
N/A
N/A
H + 0
H + 60
H + 120
H + 0
H + 60
H + 120
N/A
N/A
Concentrations
(CFU/ft3 or CPU/Blank)
0.054, 0.054
ND, ND
ND, ND
0.034
ND
ND
ND
ND
ND, 0.037
ND, 0.018
0.018, ND
ND
ND
ND
ND
ND
0.054
ND
ND
ND
ND
ND
ND
0.028
ND = Not detected. N/A = Not applicable.
172
-------
Table 3-40. Aggressive air sample results for Round 3
Location
Room 105
Room 105
Room 105
Room 105
Room 105
Room 105
Room 105
Room 105
Room 106
Room 106
Room 106
Room 106
Room 106
Room 106
Room 106
Room 106
Hallway
Hallway
Hallway
Hallway
Hallway
Hallway
Hallway
Hallway
Sample Type
XMX
XMX
XMX
STA
STA
STA
XMX-Blank
STA-Blank
XMX
XMX
XMX
STA
STA
STA
XMX-Blank
STA-Blank
XMX
XMX
XMX
STA
STA
STA
XMX-Blank
STA-Blank
Time Interval
(1st, 2nd, or 3rd hour
of sampling)
H + 0
H + 60
H + 120
H + 0
H + 60
H + 120
N/A
N/A
H + 0
H + 60
H + 120
H + 0
H + 60
H + 120
N/A
N/A
H + 0
H + 60
H + 120
H + 0
H + 60
H + 120
N/A
N/A
Concentrations
(CFU/ft3 or CPU/Blank)
0.018, ND
ND, ND
ND, ND
ND
ND
ND
0.57
ND
ND, ND
ND, ND
ND, ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND = Not Detected. N/A= Not applicable.
173
-------
3.2.3.2. Reaerosolization Assessment Results
3.2.3.2.1. Indoor Air SKC BioSampler® Data
3.2.3.2.1.1. Descriptive Statistics
Descriptive statistics for the SKC BioSampler® data (CFU/ft3) are provided by round, stage, and
room in Table 3-41. These statistics were empirically-based (i.e., not based on an assumed
statistical distribution fit to the data) and include the number of measurements, arithmetic and
geometric means, standard deviation, minimum and maximum observed measurement, and the
50th percentile.
Of the 54 samples collected in Stage 1 (background), only three samples yielded detectable
CPU measurements:
• A measurement of 10 CFU/ft3 for a sample collected in Round 2 (pH-adjusted bleach) at
Location #3 within Room 101 A, at a medium height.
• A measurement of 2 CFU/ft3 for each of two samples collected in Round 3 (CIO2) at
Location #1 within Room 101 A, at heights of medium and high.
These three detectable outcomes were at very low concentrations compared to concentrations
measured during spore dissemination (Stage 2). Hence, the three detectable outcomes did not
impact Stage 2 results. However, the three detectable outcomes do need to be considered
when drawing conclusions from Stage 5 data.
All measurements for samples taken in Stages 2, 3, and 4 were nonzero.
Of the 54 samples collected in Stage 5 (after decontamination), all Round 2 (pH-adjusted
bleach) and Round 3 (CIO2) samples yielded non-detectable results. In contrast, eight of the
nine samples in each room during Stage 5 of Round 1 (VHP®) yielded detectable
measurements (ranging from 2 to 8 CFU/ft3 in Room 101A and from <1 to 3 CFU/ft3 in Room
102). The Round 1, Stage 5 arithmetic mean Bg spore concentrations in air, based on the nine
samples in each room, were 4 CFU/ft3 in Room 101A and 2 CFU/ft3 in Room 102.
While the descriptive statistics in Table 3-41 present a tabular summary of the observed
airborne sample measurements, the box plots in Figure 3-22 through Figure 3-24 provide a
graphical portrayal of the overall distribution of these measurements across the range of
observed values and for different decontamination rounds, stages, and rooms with quartiles
being displayed relative to each other. Each figure represents a specific decontamination round
and contains box plots for each combination of stage and room (i.e., 5 stages x 2 rooms =10
box plots per figure). Within a box plot, the bottom and top of the boxes represent the 25th and
75th percentiles of the observed sample measurements, respectively, with the line within the box
representing the 50th percentile. The "whiskers" on either side of the box extend to the highest
(or lowest) measurement that falls within 1.5 times the interquartile range of the data (i.e., within
1.5 times the difference between the 75th and 25th percentiles). Points falling outside these
whiskers are noted as points on the plot; points falling far from the boxes may signify outliers.
These box plots are presented on a log-axis, with the labels on the axis denoting powers of 10
(i.e., axis labels of E1 through E6 denoting values of 10 through 1,000,000). Note that sample
174
-------
measurements of non-detect were represented by 1 CFU/ft3 in constructing these plots, and
most results for Rounds 1 and 5 are, therefore, represented by a single line at 1 CFU/ft3,
indicating all sample results that were ND.
The box plots show that after approximately a day and before re-entry for surface sampling,
substantial settling has occurred. Levels of spores in the air have declined from MOO,000
CFU/ft3 to about 10 to 100 CFU/ft3. Whether the residual spore density is due to continued
suspension of small particles or reaerosolization of spores cannot be determined from the data.
Whether sampling activities result in reaerosolization is unclear from the data. The mean spore
densities in the air after sampling activities were <100 CFU/ft3 and, in some cases, were <10
CFU/ft3.
The descriptive statistics and box plots demonstrate that the patterns of air measurements were
generally similar between the two rooms and decontamination rounds, with the expected large
difference occurring between Stage 2 (spore dissemination) and the other four stages. The
range of measurements in Stage 3 (before surface sampling) appears to be higher within the
CIO2 round (where the geometric means equaled 43 and 60 CFU/ft3 in the two rooms)
compared to the other two rounds (where the geometric means ranged from 3 to 13 CFU/ft3). In
addition, while the measurements tended to increase from Stage 3 (before surface sampling) to
Stage 4 (pre-decontamination) during the VHP® round and to increase slightly from Stage 3 to
Stage 4 during the pH-adjusted bleach rounds (Rounds 1 and 2), a general decreasing pattern
from Stage 3 to Stage 4 was observed with CIO2 (Round 3).
175
-------
Table 3-41. Descriptive statistics for air concentration of Bg spores (CFU/ft3), calculated by Round. Stage, and Room.
Round*
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
Stage1
1
1
2
2
3
3
4
4
5
5
1
1
2
2
3
3
4
4
5
5
1
1
2
2
3
3
4
4
5
5
Room
101A
102
101A
102
101A
102
101A
102
101A
102
101A
102
101A
102
101A
102
101A
102
101A
102
101A
102
101A
102
101A
102
101A
102
101A
102
N*
9(0)
9(0)
9
9
9
9
9
9
9(8)
9(8)
9(1)
9(0)
9
9
9
9
9
9
9(0)
9(0)
9(2)
9(0)
9
9
9
9
9
9
9(0)
9(0)
Arithmetic
Mean
O.OEO
O.OEO
3.2E5
2.1E5
4.0EO
8.0EO
2.0E1
3.2E1
4.0EO
2.0EO
1.0EO
O.OEO
2.7E5
2.5E5
1.5E1
2.1E1
1.6E1
1.4E1
O.OEO
O.OEO
<1.0EO
O.OEO
2.3E5
2.0E5
4.4E1
7.2E1
2.4E1
9.0EO
O.OEO
O.OEO
Standard
Deviation
O.OEO
O.OEO
3.0E5
1.1E5
2.0EO
4.0EO
7.0EO
8.0EO
2.0EO
1.0EO
3.0EO
O.OEO
2.3E5
7.9E4
2.1E1
3.0E1
6.0EO
3.0EO
O.OEO
O.OEO
1.0EO
O.OEO
1.7E5
8.2E4
1.2E1
6.2E1
8.0EO
2.0EO
O.OEO
O.OEO
Minimum
O.OEO
O.OEO
1.2E5
1.1E5
2.0EO
2.0EO
5.0EO
2.0E1
O.OEO
O.OEO
O.OEO
O.OEO
8.0E4
1.5E5
4.0EO
6.0EO
8.0EO
8.0EO
O.OEO
O.OEO
O.OEO
O.OEO
7.5E4
9.3E4
2.9E1
4.3E1
1.1E1
5.0EO
O.OEO
O.OEO
Maximum
O.OEO
O.OEO
8.4E5
4.7E5
7.0EO
1.5E1
3.0E1
4.6E1
8.0EO
3.0EO
1.0E1
O.OEO
7.4E5
3.4E5
7.2E1
1.0E2
2.4E1
1.9E1
O.OEO
O.OEO
2.0EO
O.OEO
5.2E5
3.2E5
6.3E1
2.4E2
3.8E1
1.2E1
O.OEO
O.OEO
50th Percentile
O.OEO
O.OEO
1.8E5
2.0E5
2.0EO
8.0EO
2.1E1
3.0E1
4.0EO
1.0EO
O.OEO
O.OEO
1.8E5
2.4E5
9.0EO
1.1E1
1.5E1
1.4E1
O.OEO
O.OEO
O.OEO
O.OEO
1.5E5
2.1E5
4.0E1
5.3E1
2.4E1
9.0EO
O.OEO
O.OEO
Geometric
Mean
—
—
2.4E5
2.0E5
3.0EO
7.0EO
1.8E1
3.1E1
4.0EO
2.0EO
1.0E1
—
2.0E5
2.3E5
1.0E1
1.3E1
1.5E1
1.3E1
—
-
2.0EO
—
1.8E5
1.9E5
4.3E1
6.0E1
2.3E1
8.0EO
—
-
Rounds represent decontamination technology used: 1 = VHP ; 2 = pH-Adjusted bleach; 3 = CIO2.
f Stages: 1=background; 2=spore dissemination; 3=before surface sampling; 4=pre-decontamination surface sampling; 5=post-decontamination surface sampling.
* Total number of samples entering into the calculations (collected from three locations per room, at three heights per location). For Stages 1 and 5, the number of
samples yielding nonzero measurements is given in parentheses.
176
-------
CD
o
IO
o
LU
111
LU
o
LU
O
LU
Round 1
S1 S2 S3 S4 S5 S1 32 S3 S4 S5
101A 102
Time Stage and Room
Figure 3-22. Box plots of Round 1 (VHP®) air concentration data for Bg spores (CFU/ft3),
by stage (S1 through S5) and room (101A and 102).
177
-------
CO
o
01
in
o
111
s
LU
3
LLJ
5
LJJ
Round 2
S1 S2 S3 S4 S5 S1 S2 S3 S4 S5
101A 102
Time Stage and Room
Figure 3-23. Box plots of Round 2 (pH-Adjusted Bleach) air concentration data for Bg
spores (CFU/ft3), by stage (S1 through S5) and room (101A and 102).
178
-------
Round 3
LJJ
m
o
a
+
UJ
o
Ul
o
UJ
S1 S2 S3 S4 S5 S1 S2 S3 S4 S5
101A 102
Time Stage and Room
Figure 3-24. Box plots of Round 3 (CIO2) air concentration data for Bg spores (CFU/ft3), by
stage (S1 through S5) and room (101A and 102).
3.2.3.2.1.2. Statistical Analysis
The statistical analysis was conducted by performing parametric ANOVA on log-transformed
CFU/ft3 measurements, hence the reference to testing for significant differences among
geometric means versus arithmetic means. (The measurements were log-transformed because
the range of observed measurements covered several orders of magnitude, and the inherent
assumptions necessary for the ANOVA were better satisfied after making a log transformation.)
The ANOVA model included fixed effects of round, stage, room, sampling height, and all two-
way interactions of these factors. The model also included a fixed effect of sampling location
within a room, but this factor was nested within the room effect because the three locations
differed from one room to the next. Significance of a two-way interaction implies that the
presence of significant differences between levels of one factor is dependent on the other factor.
When factors were statistically significant and had more than three levels, linear contrasts were
established and tested to identify those pairs of levels that were significantly different. All tests
were performed at the 0.05 significance level (95% confidence), while the significance levels of
the tests of linear contrasts were adjusted to control the false discovery rate to within 0.05. Only
air data associated with Stages 2, 3, and 4 (i.e., from dissemination to pre-decontamination)
were considered in the statistical analyses, as the air data associated with Stages 1 and 5 (i.e.,
background and post-dissemination) were dominated by ND results.
The ANOVA was performed twice: once including data for Stages 2 through 4, and again after
excluding Stage 2 data. The 54 Stage 2 measurements (samples collected during
179
-------
dissemination) were several orders of magnitude higher than the measurements from Stages 3
and 4 (samples collected before surface sampling and pre-decontamination) - the smallest
Stage 2 measurement was 7.5E4 CFU/ft3, while the largest of the 108 measurements from
Stages 3 and 4 was 2.4E2 CFU/ft3 (the next largest was 1.0E2 CFU/ft3). Because the effect of
stage on the measurements was so great, it was necessary to assess the effects of the other
factors both including and excluding the Stage 2 measurements.
The results of the statistical analyses follow (all references to statistical significance are at the
95% confidence level):
Effect of Stage. Differences in the geometric means among Stages 2 through 4 were highly
significant (p<0.0001), as data from Stage 2 were from three to four orders of magnitude higher
than any data from Stages 3 and 4 (Table 3-9). Regardless of whether Stage 2 measurements
were included or excluded in the analysis, the interaction of stage and round was highly
significant (p<0.0001), and the interaction of stage and room was significant at the 0.05 level,
indicating that the effect of stage needed to be assessed by room and round. In both analyses,
significant differences between Stages 3 and 4 were observed in the following situations:
• Within Room 101A (where the Stage 4 geometric mean was nearly 50% higher than in
Stage 3).
• Within Round 1 (VHP®, where the Stage 4 geometric mean was six times higher than in
Stage 3).
• Within Round 3 (CIO2, where the Stage 3 geometric mean was nearly double that of
Stage 4).
Differences in the geometric means between Stages 3 and 4 were not statistically significant for
either Room 102 or Round 2 (pH-adjusted bleach).
Effect of Round. As noted above, the BOTE Project test round effect interacted significantly with
the stage effect (p<0.0001). When measurement data for Stage 2 were included in the analysis,
significant differences among rounds were not observed within Stage 2, as the intent of
dissemination was to introduce an equivalent number of spores in each round. However,
regardless of whether Stage 2 data were included or excluded from the analysis, significant
differences were present between rounds within Stages 3 and 4.
• Within Stage 3 (before surface sampling), all three rounds differed significantly, with the
geometric mean for Round 3 (CIO2) being higher than for Round 2 (pH-adjusted bleach),
which in turn was higher than for Round 1 (VHP®).
• Within Stage 4 (pre-decontamination), Round 1 (VHP®) differed significantly from
Rounds 2 and 3, where the geometric mean for Round 1 (23 CFU/ft3) was nearly two-
thirds higher than the other two rounds (each having a geometric mean of 14 CFU/ft3).
While this outcome suggests that more spores may have remained in the air prior to
VHP® fumigation, the geometric mean prior to treatment with VHP® was observed to be
lower than the geometric mean prior to CIO2 treatment in Room 101 A. Thus, it appears
unlikely that the observed differences among decontamination rounds in aerosolized
180
-------
spores within Stage 4 are of practical importance. No significant difference occurred in
the geometric means between Rounds 2 and 3.
In both analyses, the interaction of room and round was significant at the 95% confidence level.
Similar to the bullets above, all three rounds differed significantly within Room 101A (with Round
3 having the highest geometric mean and Round 1 the smallest), while only Rounds 2 and 3
differed significantly within Room 102 (where Round 3 had a geometric mean of 22 CFU/ft3,
compared to 13 CFU/ft3for Round 2, when data for only Stages 3 and 4 were considered).
Effect of Room. In both analyses, the interaction of room effect and stage was significant
(p=0.011 in the analysis including Stage 2 data; p=0.002 in the analysis excluding Stage 2
data). When the analysis excluded Stage 2 data, the two rooms differed significantly only in
Stage 3, where the geometric means for Rooms 101A and 102 were 11 CFU/ft3 and 17 CFU/ft3,
respectively.
In addition, within the analysis excluding Stage 2 data, the interaction of room and round was
significant (p=0.0011). Here, the two rooms differed significantly only in Round 1, where the
geometric means for Rooms 101A and 102 were 7 CFU/ft3 and 14 CFU/ft3, respectively.
However, these rooms are similar in size and materials present, and the extent to which
differences in spore concentrations are present among rooms can vary under different
conditions. Different outcomes could possibly occur if greater disparity in the room setups were
present. Further research would be required to investigate this possibility.
Effect of Location Within Room. The effect of location within room was not significant at the 95%
confidence level.
Effect of Sampling Height. The statistical analysis found no significant differences among
different sampling heights, regardless of the levels of other factors. The geometric means were
very similar between the three heights: from 3.5E2 to 3.7E2 CFU/ft3 when Stage 2 data were
included and from 14 to 17 CFU/ft3 when Stage 2 data were excluded. These results suggest
that up to a height of 48 inches from the floor, aerosolized spores may be well mixed in rooms
similar in size to those considered in this study, and, therefore, the height at which the samplers
are placed within this space is not critical when characterizing aerosolized spores in such
rooms.
3.2.3.2.2. Indoor Surface Sampling in Reaerosolization Study Rooms
For complete details on the materials and methods used in the collection of indoor surface data,
please see Section 2.5.3.1 for Indoor Surface Sampling.
Surface samples for Bg spores within Rooms 101A and 102 of the BOTE Project facility were
collected via wipes, vacuum socks, and sponge-stick wipes. The wipe samples were analyzed
by INL, and the vacuum socks and sponge-stick wipe samples were analyzed by the LRN.
Surface samples were collected during the last two stages (Stages 4 and 5, corresponding to
pre- and post-decontamination) in each round. For the reaerosolization comparison, results for
blank samples were excluded, as were results for any samples collected using a vertical
sampling orientation. Although surface sample results (expressed in CFU) were generally
181
-------
reported for both spread plate and filter plate results, most data presented in this assessment
are based on the spread plate results. Surface loadings were expressed as CFU/ft2 sampled.
This study design did not allow for an appropriate estimation of the resuspension factor (as a
ratio of surface to air concentrations). There were multiple surfaces in the room and activity on
all surfaces was not equivalent. No effort was made in the test design to attempt to isolate air
concentrations coming from specific surfaces due to a specific activity (i.e., force). Hence, the
data do not allow for a simple estimation of a resuspension factor.
3.2.3.2.2.1. Indoor Surface Sampling Data for Reaerosolization Study Rooms - Stage 4
All surface sample results from Stage 4 were based on spread plate results. As might be
expected with sampling during this stage, where contamination levels remain high prior to
decontamination (post-fig spore dissemination), filter plating of samples was not necessary.
The analysis of Stage 4 data considered only those outcomes associated with 125 of the 128
surface samples that were collected using a horizontal upward sampling orientation. (The three
excluded samples had reported results of either TNTC or ND (meaning that the spread plate
count was below 30 CPU following any necessary dilutions, which, by protocol, were not
quantifiable.) The surfaces sampled were characterized by the object (cabinet, desk, UV-APS
[the surface of this instrument was sampled], floor, chair) and the texture of the surface (metal,
plastic, smooth, carpet, cloth).
• Among the wipe samples, only three combinations of object and texture were
represented, resulting in a total of 54 wipe sample measurements: ten observations per
each round/stage combination for Room 101A and eight observations per each
round/stage combination for Room 102.
• Of the 18 collected vacuum samples, the analysis included surface concentrations for 16
samples. These concentrations corresponded to 14 of the 15 floor carpet samples (two
to three carpet samples per round/room combination) and two of the three chair cloth
surface samples (one collected in Room 101A in each of the three rounds). One carpet
sample result labeled as TNTC was excluded and one non-quantifiable cloth sample
result from Round 2 was excluded.
• Of the 56 collected sponge samples, 55 samples reported nonzero surface
concentrations that could be used in the statistical analysis. (The result for one sample
taken from a desk in Room 102 in Round 3 was specified as TNTC and was therefore
excluded from analysis.) Of these 55 samples, nine samples were collected in Room
101A in each round, and from nine to ten samples were collected in Room 102 in each
round. These samples were collected from plastic, smooth, and metal surfaces from UV-
APS, cabinets, and desks (surfaces and drawers).
To assess how surface loadings differ among surface types, the 125 pre-decontamination
surface samples with results entered into the analysis were classified into six categories based
on the object and surface texture:
182
-------
• Cabinet, metal (41 samples, with three samples collected by each of wipe and sponge in
each round within Room 101A - with the exception of Round 3, where only two wipe
samples were collected - and four samples were collected each by wipe and sponge in
each round within Room 102).
• UV-APS, plastic case (18 samples collected via wipe and sponge, rather evenly divided
among rooms, rounds, and sample types).
• Desk, smooth laminate surface (47 samples collected via wipe and sponge - 31
samples in Room 101 A, and 16 samples collected in Room 102, nearly evenly divided
among rounds and sample types).
• Desk drawer, smooth metal surface (three samples collected via sponge in Room 102,
one sample per round).
• Floor, carpet (14 samples, with two or three samples collected using vacuum sampler in
each room in each round).
• Chair, cloth (two samples, collected in Rounds 1 and 3 from Room 101A).
Table 3-42 presents the arithmetic mean surface and airborne spore concentration data for the
pre-decontamination stage (Stage 4) for each of the 47 combinations of round, room, surface,
surface type, and sample type that had at least one quantifiable (nonzero) surface sample
measurement.
Within Table 3-42, the arithmetic means of the spore measurements in air are specific only to a
round and room. When considering reaerosolization, airborne spores collected in a given air
sample cannot be attributed directly to spores present on a particular surface within the room.
Table 3-42. Arithmetic means of surface and air concentrations of Bg spores collected
and analyzed by dilution plate method in Stage 4 (pre-decontamination). by round, room,
surface, surface type, and sample type
Round1
1
1
1
1
1
1
1
Room
101A
101A
101A
101A
101A
101A
101A
Surface
Cabinet
Cabinet
Desk
Desk
UVAPS
UVAPS
Chair
Surface
Type
Metal
Metal
Smooth
Smooth
Plastic
Plastic
Cloth
Sample
Type
Wipe
Sponge
Wipe
Sponge
Wipe
Sponge
Vacuum
N*
3
3
5
5
2
1
1
Arithmetic Mean
Surface
(CFU/ft2)
6.2E5
4.3E5
6.3E5
6.2E5
1.2E6
2.6E6
9.0E3
Air§
(CFU/ft3)
2.0E1
Standard Deviation
Surface
(CFU/ft2)
1.1E5
3.9E4
1.0E5
2.5E5
1.8E5
~
—
Air§
(CFU/ft3)
7.0EO
183
-------
Round1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
Room
101A
101A
102
102
102
102
102
102
102
102
102
101A
101A
101A
101A
101A
101A
101A
101A
102
102
102
102
102
102
102
102
102
101A
101A
101A
101A
101A
101A
101A
101A
101A
Surface
Floor
Surface
Type
Carpet
Sample
Type
Vacuum
All Surfaces and Sample
Types
Cabinet
Cabinet
Desk
Desk
Desk
Drawer
UVAPS
UVAPS
Floor
Metal
Metal
Smooth
Smooth
Smooth
Plastic
Plastic
Carpet
Wipe
Sponge
Wipe
Sponge
Sponge
Wipe
Sponge
Vacuum
All Surfaces and Sample
Types
Cabinet
Cabinet
Desk
Desk
UVAPS
UVAPS
Floor
Metal
Metal
Smooth
Smooth
Plastic
Plastic
Carpet
Wipe
Sponge
Wipe
Sponge
Wipe
Sponge
Vacuum
All Surfaces and Sample
Types
Cabinet
Cabinet
Desk
Desk
Desk
Drawer
UVAPS
UVAPS
Floor
Metal
Metal
Smooth
Smooth
Smooth
Plastic
Plastic
Carpet
Wipe
Sponge
Wipe
Sponge
Sponge
Wipe
Sponge
Vacuum
All Surfaces and Sample
Types
Cabinet
Cabinet
Desk
Desk
UVAPS
UVAPS
Chair
Floor
Metal
Metal
Smooth
Smooth
Plastic
Plastic
Cloth
Carpet
Wipe
Sponge
Wipe
Sponge
Wipe
Sponge
Vacuum
Vacuum
All Surfaces and Sample
N*
2
22
4
4
3
3
1
1
2
3
21
3
3
5
5
2
1
2
21
4
4
3
2
1
1
2
2
19
2
3
6
5
2
1
1
2
22
Arithmetic Mean
Surface
(CFU/ft2)
8.2E3
6.6E5
4.1E5
4.3E5
4.3E5
4.7E5
2.2E5
1.7E6
2.2E6
1.1E4
5.9E5
2.1E5
2.3E5
1.5E5
1.1E5
7.1E4
4.8E4
1.6E4
1.3E5
2.6E5
2.5E5
2.5E5
1.7E5
9.8E4
8.1E4
1.9E5
1.3E4
1.9E5
1.7E5
5.8E5
1.1E5
5.5E5
4.0E4
1.2E5
2.3E4
2.2E3
2.6E5
Air§
(CFU/ft3)
3.2E1
1.6E1
1.4E1
2.4E1
Standard Deviation
Surface
(CFU/ft2)
4.5E3
6.6E5
4.1E4
2.1E5
5.6E4
3.8E4
~
~
2.7E6
2.5E3
5.9E5
4.7E4
9.2E4
5.8E4
1.4E4
3.5E4
~
9.0E3
1.3E5
2.0E4
5.9E4
2.1E4
2.5E4
~
~
6.5E4
2.9E3
1.9E5
3.5E4
5.6E4
7.6E4
7.8E4
2.7E4
~
~
1.8E1
2.6E5
Air§
(CFU/ft3)
7.0EO
6.0EO
4.0EO
8.0EO
184
-------
Round1
3
3
3
3
3
3
3
3
3
Room
102
102
102
102
102
102
102
102
102
Surface
Surface
Type
Sample
Type
Types
Cabinet
Cabinet
Desk
Desk
Desk
Drawer
UVAPS
UVAPS
Floor
Metal
Metal
Smooth
Smooth
Smooth
Plastic
Plastic
Carpet
Wipe
Sponge
Wipe
Sponge
Sponge
Wipe
Sponge
Vacuum
All Surfaces and Sample
Types
N*
4
4
3
2
1
1
2
3
20
Arithmetic Mean
Surface
(CFU/ft2)
3.1E5
2.9E5
2.7E5
2.6E5
1.3E5
1.6E5
1.0E5
4.6E3
2.1E5
Air§
(CFU/ft3)
9.0EO
Standard Deviation
Surface
(CFU/ft2)
5.5E4
1.0E5
3.2E4
1.7E5
~
~
1.3E4
2.3E3
2.1E5
Air§
(CFU/ft3)
2.0EO
t Round 1=VHP , Round 2=pH-Adjusted bleach, Round 3=CIO2.
t N corresponds to the number of samples with results entered into the calculation of the surface sample
arithmetic mean.
§ 9 air sample results were used per each round and room to calculate the air sample arithmetic mean.
- not applicable given only one sample.
3.2.3.2.2.2. Indoor Surface Sampling Data for Reaerosolization Study Rooms - Stage 5
All post-decontamination surface samples (Stage 5) based on the spread plate analysis were
NDs (i.e., mean spread plate counts were <30 CPU and thus were considered nonquantifiable).
Detectable spores were identified using the filter plate analysis for a few Stage 5 samples,
although all of the samples for Round 2 (pH-adjusted bleach) were ND. One Round 3 (CIO2)
sample, which was taken from Room 101 A, was positive for Bg based on the filter plate (3
CFU/ft2). Sixteen Round 1 (VHP®) samples were positive for Bg in Stage 5 based on filter plate
results. Two of the samples resulting in detectable Bg were from Room 101A (a sponge sample
at 10 CFU/ft2 and a wipe sample at 29 CFU/ft2). Fourteen Round 1 samples from Room 102
were detectable for Bg including one vacuum sample (16 CFU/ft2), six sponge samples (ranging
from 3.0EO to 1.9E2 CFU/ft2), and seven wipe samples (ranging from 2.9E1 to 2.3E2 CFU/ft2).
When considering the filter plate results, the Stage 5 arithmetic mean concentration by room
and round is 0 CFU/ft2 for each room and round, with the exception of Round 3 (CIO2) Room
101A (<1 CFU/ft2) and Round 1 (VHP®) Room 101A (3 CFU/ft2) and Room 102 (57 CFU/ft2)
(see Table 3-43). While all three methods resulted in substantial reduction in the number of
viable and culturable spores recovered, in this experiment only the pH-adjusted bleach
treatment achieved the historic clearance requirement of "no detected spores." With <1 CFU/ft2
detected after CIO2 treatment, additional decontamination might be required. An important gap
is the extent to which 0 CFU/ft2 detected and <1 CFU/ft2 detected reflect a significant difference
in human health risk.
185
-------
Table 3-43. Arithmetic means of surface and air concentrations of Bg spores collected
and analyzed by filter plate method in Stage 5 (post-decontamination), by round, room,
surface, surface type, and sample type*.
Round1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
Room
101A
101A
101A
101A
101A
101A
102
102
102
102
102
102
102
102
102
101A
101A
101A
101A
101A
101A
101A
101A
102
102
102
102
102
102
102
101A
101A
101A
101A
101A
101A
101A
Surface
Cabinet
Desk
Desk
UVAPS
Workbench
Surface
Type
Metal
Plastic
Smooth
Plastic
Plastic
Sample
Type
Wipe
Wipe
Sponge
Sponge
Wipe
All Surfaces and Sample
Types
Cabinet
Cabinet
Desk
Desk
Desk
UVAPS
Floor
Workbench
Metal
Metal
Metal
Plastic
Smooth
Plastic
Carpet
Plastic
Sponge
Wipe
Sponge
Wipe
Sponge
Sponge
Vacuum
Wipe
All Surfaces and Sample
Types
Desk
Desk
Desk
UVAPS
Cabinet
Cabinet
Floor
Plastic
Smooth
Smooth
Plastic
Metal
Smooth
Smooth
Wipe
Sponge
Wipe
Sponge
Sponge
Wipe
Sponge
All Surfaces and Sample
Types
Desk
Desk
Desk
Cabinet
Cabinet
Floor
Plastic
Smooth
Smooth
Metal
Smooth
Smooth
Wipe
Sponge
Wipe
Sponge
Wipe
Sponge
All Surfaces and Sample
Types
Desk
Desk
Desk
UVAPS
Cabinet
Cabinet
Floor
Plastic
Smooth
Smooth
Plastic
Metal
Smooth
Carpet
Wipe
Sponge
Wipe
Sponge
Sponge
Wipe
Vacuum
N*
3
5
3
1
2
14
4
4
1
3
3
2
1
1
19
2
5
5
1
2
3
3
21
1
2
3
3
4
3
16
3
5
4
1
3
3
1
Arithmetic Mean
Surface
(CFU/ft2)
1.0E1
O.OEO
3.0EO
O.OEO
O.OEO
3.0EO
1.0E1
7.9E1
6.0EO
1.4E2
6.6E1
2.5E1
1.6E1
2.9E1
5.7E1
O.OEO
O.OEO
O.OEO
O.OEO
O.OEO
O.OEO
O.OEO
O.OEO
O.OEO
O.OEO
O.OEO
O.OEO
O.OEO
O.OEO
O.OEO
O.OEO
O.OEO
O.OEO
O.OEO
1.0EO
O.OEO
O.OEO
Air§
(CFU/ft3)
4.0EO
2.0EO
O.OEO
O.OEO
O.OEO
Standard Deviation
Surface
(CFU/ft2)
1.7E1
O.OEO
6.0EO
~
O.OEO
3.0EO
1.6E1
8.9E1
~
1.0E2
1.1E2
3.5E1
~
~
5.7E1
O.OEO
O.OEO
O.OEO
~
O.OEO
O.OEO
O.OEO
O.OEO
~
O.OEO
O.OEO
O.OEO
O.OEO
O.OEO
O.OEO
O.OEO
O.OEO
O.OEO
~
2.0EO
O.OEO
~
Air§
(CFU/ft3)
2.0EO
1.0EO
O.OEO
O.OEO
O.OEO
186
-------
Round1
3
3
3
3
3
3
3
3
Room
101A
102
102
102
102
102
102
102
Surface
Surface
Type
Sample
Type
All Surfaces and Sample
Types
Desk
Desk
Desk
UVAPS
Cabinet
Cabinet
Plastic
Smooth
Smooth
Plastic
Metal
Smooth
Wipe
Sponge
Wipe
Sponge
Sponge
Wipe
All Surfaces and Sample
Types
N*
20
1
4
3
2
4
4
18
Arithmetic Mean
Surface
(CFU/ft2)
<1.0EO
O.OEO
O.OEO
O.OEO
O.OEO
O.OEO
O.OEO
O.OEO
Air§
(CFU/ft3)
O.OEO
Standard Deviation
Surface
(CFU/ft2)
<1.0EO
Air§
(CFU/ft3)
O.OEO
* The samples presented in this table reflect only the samples with filter plate results; in some cases,
additional samples were collected and analyzed by spread plate only. All spread plate results post-
decontamination in Rooms 101A and 102 were considered non-quantifiable.
t Round 1=VHP®, Round 2=pH-Adjusted bleach, Round 3=CIO2.
t N corresponds to the number of samples with results entered into the calculation of the surface sample
arithmetic mean.
§ 9 air sample results were used per each round and room to calculate the air sample arithmetic mean.
- not applicable given only one sample.
3.2.3.2.3. Particle Measurements
Along with the SKC BioSampler® measurements, the UVAPS and I BAG took continuous real-
time airborne particle measurements in Rooms 101A and 102. Figure 3-25 through Figure 3-30
provides summary plots for each of the two rooms (101A and 102) for each test round (Round 1
through Round 3). UV-APS particle concentrations for individual bins between the particle sizes
of 0.5 urn and 5.0 urn are provided on the plots as well as the total counts. Periods during which
there are no UV-APS data are identifiable on the plots as straight lines connecting the last value
collected in a sampling interval to the first value of the next interval. The IBAC data are indicated
in the legend of each chart as "IBAC-" with the name of the room in which the device was
located. There was no IBAC monitor in Room 102 during the three rounds. On each plot,
important points in time are indicated by vertical dashed gray lines. These time periods are also
listed in Table 3-44. "Building Entered" refers to the time at which sampling personnel entered
the building, and "Sampling Period" refers to the times during which the sampling personnel
entered the two rooms used for detailed sampling (Rooms 101A and 102). These are the
periods when reaerosolization was likely to occur. These plots indicate that the total counts of
the UV-APS appear to match the IBAC results fairly well and that aerosol concentration appears
to be increasing over the period during which the sampling personnel were sampling in the
rooms. However, these data do not indicate whether or not the particles measured with these
two devices were the target spores (or other particles brought in with the samplers) and further,
whether the particles are viable or not.
187
-------
The release periods were also further analyzed to provide a better understanding of the particle
sizes of interest. An example of a plot showing the particle size distribution prior to the release
and a few time intervals leading up to the peak of the release on the April 16 test is provided in
Figure 3-31. Each line of data in this plot represents a period in time prior to release and leading
up to the peak concentration. This plot can help establish the difference between background
and release levels for the given particle sizes. The plot indicates that particle releases include
the particles in ranges from 0.723 urn up to approximately 5 urn. The dominant size was
approximately 1 urn.
188
-------
Round 1-10 la
iv is,j»Qii
ReteKe Apr 16 (ffl 13:18,MVACoH 9 15:18
Entry Apr 17 g« 9:18
(12:06-16:47)
-0.777
-1.596
-2.2S8
-3J78
-UVAPS-total
-WAC-lOla
4/lfe/llUAM i/16/1112W 4/17/11 12AM 4/17/11 IIPW 4/18/11 I/AM 4/18/11 ]J PM
Figure 3-25. Plot of particle concentrations collected real time from the UV-APS and IBACs for Round 1, Room 101 A.
189
-------
1000
HI
Dun
4/16/11 12 AM
•0.542
-0.777
-1J037
-2,2*8
- 3.278
-4. MS
- UVAPS total
(BA(-hvacl
4/W11 U PM
Figure 3-26. Plot of particle concentrations collected real time from the UV-APS and IBACs for Round 1, Room 102.
190
-------
1000
ICO
i
BoundZ-lOU
April 2S 26,2011
Reteaw Apr 25 I® 14:16,HVAC Olf 9 16,t6
001
4/2S/1112PW
4/26/11 12 AM
4/26/11 12 PM
0.542
0.777
= ljQ37
-1,596
—2,28*
— 3,278
— 4.698
WAPS10UI
IBAC HVAC 1 it
IBA<
Figure 3-27. Plot of particle concentrations collected real time from the UV-APS and IBACs for Round 2, Room 101 A.
191
-------
1000
ICO
I
Round J • ID?
Apr* 2S 26,2011
16,HVAC alf «p
001
4/M/illIPW
-0,542
-0.777
-1J037
-1.W6
-2.399
- J.27S
IBACHVACUt
4/26/11 12 AM
12 PM
Figure 3-28. Plot of particle concentrations collected real time from the UV-APS and IBACs for Round 2, Room 102.
192
-------
1000
leu
I/, .70II
fidrsw Muf 10 9 15:23, MVAC ofl
-------
1000
Round 3 - 102
111 ,
, IIVAC off ft 17:22
Entry Mjy 11 «s 8:42
[16:02-17:^]
S/10/1112AM 5/10/11
0,777
1.037
1.596
2.2SS
3,278
4,698
IBAC HVAC 1st
S/12/1112PM
Figure 3-30. Plot of particle concentrations collected real-time from the UV-APS and IBACs for Round 3, Room 102.
194
-------
Table 3-44. Summary of UV-APS event information.
Round
1
2
3
Date
April 16-1 7,
2011
April 25-26,
2011
May 10-12,
2011
Release Time
April 16, 13:18
April 25, 14:16
May 10, 15:22
Release Info
1st floor H VAC,
200 mg
2nd floor HVAC,
0.5 mg
Building
Entered
April 17,09:18
April 16,08:09
May 11, 08:42
Sampling Period
12:06-16:47
101a,09:28-11:31
102, 11:59-13:38
101a, 11:51 -14:34
102, 16:02-17:54
icon
-o 4/16/2011 13:15
4/16/2011 13:18
— 4/16/2011 13:21
—=—4/16/2011 13:26
(M
0,01
0001
456
Particle Diameter [|im)
U
Figure 3-31. UV-APS release distribution.
3.2.4. Bacillus Spore Migration from Inside the Building to Outside Results
Laboratory and statistical analysis results on the transport of nonpathogenic spores initially
disseminated inside the BOTE Project test facility to the outer perimeter of the building are
summarized in the sections below.
3.2.4.1. Limits of Detection
Instrument LOD and the LOD within the matrix were assessed by both the EPA and USGS
laboratories conducting the analyses. Instrument LODs were determined based upon analysis
of an aqueous solution of pure Bg genomic DMA, while the matrix LODs were determined by
195
-------
analyzing sterile sand samples spiked with known concentrations of Bg spores followed by
sample extraction, purification, and qPCR detection. The matrix LOD may also be considered
the matrix effect or environmental LOD[821.
3.2.4.1.1. EPA Instrument Limit of Detection
The EPA instrument standard curve for this analysis was determined after the sand sample
analyses. The standard curve was therefore used retroactively to determine the LOD based on
the acquired cycle threshold values. While the LOD curve was not established until after the
study, controls analyzed throughout the laboratory work were consistent, indicating that the
instrument was stable. The standard curve was obtained by analyzing a concentrated stock
solution of Bg DMA that was diluted eightfold. The DMA concentrations measured in GEq (the
mass of the Bg genome within a spore) ranged from 1.0E5 to 1.0E-2 GEq. Triplicate qPCR
analyses of each dilution ran for 45 cycles. Detectable results were attained down to the 1.02
GEq/reaction at an average cycle threshold time of 38.29 (standard deviation (SD) = 0.08; n=3).
Lower than ~1 GEq, the instrument registered as "undetected". The instrument lower limit of
detection was therefore determined to be a cycle threshold value of 38.3, or 1.02 GEq/reaction.
For the BOTE Project analyses, all averaged cycle threshold values greater than 38.3 were
considered NDs based on the instrument LOD. The standard curve for this analysis fit an
exponential trend line with good accuracy (see Table 3-45). The standard curve in Figure 3-32
demonstrates the effect on the EPA qPCR analytical procedure under ideal laboratory
conditions. A separate analysis was conducted to determine the matrix LOD for the entire
analysis process from extraction through qPCR detection (see Section 3.2.4.1.2).
Table 3-45. Average results from EPA instrument LOD.
GEq/ PCD Reaction
10,176
1,018
101.8
10.18
1.02
0.1
0.01
Cycle Threshold1
23.43 (0.07)
26.83(0.13)
30.44(0.16)
33.89 (0.43)
38.29 (0.08)
ND
ND
n=3 for these analyses with SDs given within the brackets.
196
-------
l.OOE-01
Cycle threshold
Figure 3-32. EPA instrument LOD including standard curve and exponential fit trend line.
3.2.4.1.2. EPA Environmental Limit of Detection
The overall recovery for the EPA sand extraction and analysis method was determined with
matrix spikes: 45 g aliquots of sterile sand spiked with liquid suspensions of known
concentration of Bg spores. Blind sand samples were spiked in triplicate with Bg spore
concentrations ranging from 1EO to 1E6 spores per gram of sand. DMA from the spores
collected from each of the 45 g aliquots of spiked sand was extracted by utilizing the same
procedure as used for the actual BOTE samples. The concentration of spiked spores present
within each of the sand samples was revealed to the analyst only after qPCR results had been
acquired. The study determined that a minimum of 1E4 spores/g sand is required for the
average cycle threshold value to be above the instrument LOD (Table 3-46). The standard
curve attained from the entire method fit an exponential fit regression (Figure 3-33).
197
-------
Table 3-46. Average results from EPA spiked sand samples.
[Final Spike]
Spores/ g Sand
1.7E6
1.8E5
1.9E4
1.8E3
1.9E2
1.8E1
1.7EO
O.OEO
Cycle threshold time1
30.1 (0.26)
33.8 (0.76
36.2 (0.86)
ND
ND
ND
ND
ND
n = 9 for these analyses with SDs given within the brackets.
Bg Spores / g sand
i n F+06
i n F+OS
i n F+09
i n F+ni
i n F+nn
^^
^\^
v = lE+15e-°-682x XXX
R2 = 0.9379
20 25 30 35 40
Cycle threshold
Figure 3-33. EPA LOD in sterile sand including standard curve and exponential fit trend
line.
3.2.4.1.3. USGS Instrument Limit of Detection
The instrument LOD was determined by USGS using a fivefold dilution of a solution of purified
Bg DMA prior to the start of the BOTE exercise. The DMA concentrations (measured in GEq, the
mass of the Bg genome within a spore) ranged from 1.5E1 to 1.5E5 GEq. All standards were
analyzed in duplicate with three negative controls consisting of PCR-grade water. Detectable
results were attained down to the 15 GEq dilutions at an average cycle threshold time of 32.88
(SD = 0.64). Lower than -15 GEq, the instrument registered "undetected". Therefore, the lower
limit of detection was determined to be a cycle threshold time value of 32.9 (see Table 3-49). All
198
-------
Ct values greater than 32.9 were considered NDs based on the instrument LOD. The standard
curve fit an exponential trend line with good accuracy (Figure 3-34).
The standard curve in Figure 3-34 demonstrates the LOD for the USGS qPCR analytical
procedures under ideal conditions. A separate analysis was conducted to determine the LOD for
the entire analysis process from extraction through qPCR detection (see Section 3.2.4.1.4).
Table 3-47. Average results from USGS instrument LOD.
GEq/ PCD Reaction
1.5x105
1.5x104
1.5x103
1.5x102
1.5x101
1.5x10°
0.0x10°
Cycle Threshold1
19.23(0.14)
22.20 (0.08)
25.61 (0.04)
29.99 (0.01)
32.89 (0.64)
Not Conducted
ND
n=2 for these analyses with SDs given within the brackets.
1^
S3 ••«
4» tl
(j w
«G W* i oF+09
1 0 l-OE^2
W
y = 3E+10e-°-652x
^\^^ R2 = 0.9938
^"\.
^^--^^
^^\
15 20 25 30 35
Cycle threshold
Figure 3-34. USGS instrument LOD standard curve.
3.2.4.1.4. USGS Environmental Limit of Detection
Similar to EPA, USGS analyzed sand samples (the samples were analyzed blind) spiked with
liquid suspensions of Bg spores ranging from 1EO to 1E6 spores/g sand. In total, 17
seeded/unseeded building sand samples (samples marked 1 through 16 and sterile sand) were
199
-------
sent to USGS, and these samples were analyzed for the presence of Bg using direct DMA
extraction and qPCR.
The MO BIO protocol (see Appendix F) using 2 uL of eluent as template was able to detect Bg
DMA only at the highest seed concentration (see Table 3-51). This analysis was run
concurrently with the BOTE Project samples. There was therefore no knowledge that the sand
matrix would have such a detrimental effect on the outcome of the study. Following these tests,
the USGS laboratory extracted a spiked sand sample under four other conditions. Even with
only a few replicates, it was apparent that the extraction method utilized impacts the final qPCR
outcome.
Table 3-48. Results from USGS spiked sand samples expressed as average genomic
equivalents in the 100 uL of eluent from the MO BIO PowerSoil® Kit.
Bg Spores/ g sand
1.8 E6
1.6 E5
1.7 E4
1.6 E3
2.0 E2
1.7 E1
1.7 EO
0.0 EO
GEq/ 100 [jl_ eluent
(n=4)
20
ND
ND
ND
ND
ND
ND
ND
3.2.4.2. Sand Analysis Results for Spore Migration Study
The EPA and USGS data are better suited as qualitative rather than quantitative data due to the
limited standard curve data obtained from each laboratory. The EPA standard curve was
determined after the BOTE Project sand sample analyses and retroactively fit to all acquired
data. USGS ran concurrent standards within each qPCR run; however, the narrow range of the
standard curve limited its utility. Because the standard curve data from both organizations were
somewhat uncertain for the stated reason, Bg results detected for the samples were treated
qualitatively and were assigned a relative degree of positive detection (a qualitative
assessment). Results for only those samples collected from the ten locations within the
secondary enclosure and outside the building were included in the data analysis. In addition,
results for the samples collected within the building are also presented, but were not included
within the overall statistical analysis.
3.2.4.2.1. EPA Data Transformation
The raw cycle threshold values for each triplicate reaction were exported to a file from the EPA
instrument. Samples that yielded two or more "undetected" values were considered ND by the
200
-------
instrument. The remaining averaged results for each sample were codified as degrees of
positive (0-5) using the designations listed in Table 3-49.
The analysis code was based upon the instrument LOD (cycle threshold of 38.3) as the lower
bound and the calculated GEq/reaction for the cutoff points; see Table 3-50 for all EPA coded
data results.
Table 3-49. EPA laboratory analysis code descriptions for the BOTE sand samples based
on mean cycle threshold value.
Mean Cycle Threshold Value
Undetected
>38.3to<45.0
>36 to <38.3
>34 to <36
>32 to <34
>30 to <32
<30
Description
Not detected by the instrument
Below the level of detection
Very weak positive
Weak positive
Positive
Strong positive
Very strong positive
Code
ND
0
1 +
2+
3+
4+
5+
GEq/PCR Reaction
ND
<1
1-3
3-10
10-40
40-150
>150
201
-------
Table 3-50. All EPA coded data: Round, Stage, Location, and Replicates.
Location
1
2
3
4
5
6
7
8
9
10
B1
B2
Site Blank
Trip Blank
Round 1
Pre-
Dissem*
A
4
2
2
ND
1
3
3
4
1
ND
2
2
B
2
1
3
5
2
ND
3
2
ND
ND
Post-
Dissem
A
4
2
2
ND
1
2
1
1
ND
ND
4
4
B
2
1
3
1
2
2
2
1
1
0
5
2
Post-
Decon**
A
2
3
2
ND
2
3
ND
1
1
4
3
1
B
1
2
3
ND
2
2
2
1
2
3
4
1
Round 2
Pre-
Dissem
A
4
ND
ND
1
2
ND
2
1
ND
2
B
ND
ND
ND
3
ND
ND
ND
ND
ND
1
Post-
Dissem
A
2
1
ND
ND
1
ND
1
1
2
1
3
1
ND
ND
B
1
2
ND
ND
1
1
1
1
2
ND
4
1
Post-
Decon
A
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
B
1
ND
ND
ND
1
ND
ND
ND
ND
ND
ND
ND
Round 3
Pre-
Dissem
A
ND
0
ND
ND
ND
ND
1
ND
ND
ND
ND
ND
B
ND
ND
ND
ND
ND
1
ND
ND
ND
ND
Post-
Dissem
A
1
1
1
ND
ND
ND
1
ND
ND
ND
3
1
ND
B
1
1
1
ND
1
ND
ND
1
1
ND
3
ND
Post-
Decon
A
1
ND
ND
ND
ND
1
ND
ND
1
ND
1
ND
ND
ND
B
2
ND
ND
ND
ND
ND
ND
1
1
1
ND
ND
*Dissemination.
*Decontamination.
202
-------
3.2.4.2.2. USGS Data
Standard curve analyses were conducted by USGS during each PCR run. The instrument LOD
was determined to be a cycle threshold value of 32.9 based upon the averaged results for a
DMA concentration of 32.48 GEq. However, a similar code was not developed for the USGS
data due to a lack of data points generated by the PCR instrument itself.
No detectable cycle threshold values were obtained by the USGS for any of the collected BOTE
Project sand samples. Even the samples collected from within the building from the first floor
with the highest dissemination concentration were below the LOD through the USGS analysis.
USGS extracted 216 sand samples collected during the BOTE Project exercise. A total of 432
qPCR reactions were processed from those extracts. Of those reactions, only 15 yielded results
from the thermocycler, yet none of the averaged cycle threshold values were found to exceed
the instrument LOD (cycle threshold 32.9). The cycle threshold values obtained from the USGS
results are listed in Table 3-51. Only the PCR positive controls (Table 2-19) gave consistent
results.
Table 3-51. USGS cycle threshold values obtained from the instrument.
Sample Description
Round/ Stage/ Sample Location - Replicate
Round 1 / Post-Dissemination / B1-B
Round 1 / Post-Dissemination / 5-A
Round 1 / Post-Dissemination / 6-A
Round 1 / Post-Decontamination / B1-B
Round 1 / Post-Decontamination / 5-B
Round 2 / Post-Dissemination / B1-A
Round 2 / Post-Dissemination / B1-B
Round 2 / Post-Dissemination / 4-A
Round 2 / Post-Dissemination / 5-B
Round 2 / Post-Dissemination / 6-A
Round 2 / Post-Dissemination / 7-B
Round 3 / Pre-Dissemination / 2-B
Round 3 / Post-Dissemination / B1-A
Round 3 / Post-Dissemination / B2-B
Round 3 / Post-Decontamination / B2-B
Reaction 1
ND
ND
41.35
41.84
ND
40.47
41.88
ND
38.89
ND
ND
ND
37.59
38.48
ND
Reaction 2
40.77
40.86
ND
ND
40.53
ND
40.36
38.45
ND
39.95
43.24
38.66
ND
ND
37.99
203
-------
3.2.4.2.3. Collected Blank Samples
Site blank and trip blank QA samples were collected during each round of the BOTE Project.
The purpose of the site blanks was to determine the potential for background contamination of
sampling media at the site. The site blank sample containers were opened on site and then
immediately closed and re-bagged for shipment to the laboratory for analysis. The purpose of
the trip blanks was to determine the potential for sample contamination over the course of an
entire sampling round. Trip blanks were shipped out to the site with the sampling media, held in
sample kit boxes during sample collection (but never opened) and then shipped with the
samples to the laboratory for analysis. All site and trip negative controls were reported as ND
with the exception of two collected during Round 1 for which corrective action was taken.
During the placement and collection process for Round 1, sampling personnel noted that the
sample dishes were not individually bagged. Additionally, notes were made regarding breakage
of sampling dishes following collection during shipment to the laboratory. These notes by the
sampling personnel and the detected signal from the blank samples were the basis for
considering all Round 1 samples as contaminated through sampler handling. Therefore, all
Round 1 sample results were eliminated from the data analysis conducted for this report.
Sample dishes for all other rounds were individually bagged to prevent contamination and all
other trip and site blanks collected during the project were ND.
In addition to the blank samples collected on site, positive (seeded) and negative (blank) control
samples were analyzed within each of the 16 EPA qPCR runs to ensure QC. Criteria for
acceptance of negative qPCR controls were that all replicate samples be ND. Two types of
negative controls were utilized in this study: NTC and Escherichia coli DNA. Likewise, two types
of positive controls were used: BOTE Bg DNA and an additional strain of Bg DNA previously
stored at the EPA laboratory. Acceptance of analytical results for positive controls required the
observed cycle threshold time to be within 5% of the prior determined cycle threshold value. As
shown in Table 3-52, all qPCR quality assurance (QA) results met the acceptance criteria.
Table 3-52. Summary of the BOTE Project EPA qPCR QA results
Control
NTC
E. coli DNA
Bg BOTE DNA
Bg EPA DNA
EPA
Mean Cycle
threshold time
(SD)
Undetected (0.00)
Undetected (0.00)
27.6 (0.37)
28.1 (0.18)
EPA
Code
ND
ND
5+
5+
No. Positive/
No. Analyzed*
0/51
0/51
57/57
51 /51
Number of Positive PCR Reactions per number analyzed, for sixteen individual 96 well PCR runs.
3.2.4.2.4. Results for Inside the Secondary Enclosure, External to the Building
The codified results for all 120 samples analyzed by the EPA (i.e., across samples "A" and "B"
taken at each of ten locations, three collection stages, and two decontamination treatment
rounds) are summarized in Table 3-53. In total, 63% (76/120) of the samples were classified as
204
-------
ND by the instrument. The lowest cycle threshold value found in the EPA analysis was a cycle
threshold value of 30.4, which indicates that no sample was classified as strong or very strong
detection. In addition, 70% of the collocated samples ("A" and "B") yielded consistent results.
Table 3-53. Codified EPA results within the secondary enclosure (and outside the
building.
Code
ND
0
1 +
2+
3+
4+
5+
Total
All Samples
n=3
76
1
33
8
1
1
0
120
"A" samples
n=3
38
1
15
5
0
1
0
60
"B" samples
n=3
38
0
18
3
1
0
0
60
"A" and "B" samples
n=6
29
1
23
6
0
1
0
60
3.2.4.2.5. Results for Samples from Inside the Building and Associated Inhibition
Testing
Sample containers were placed inside the building before spore dissemination and collected
after Bg dissemination and after building decontamination for each round of the BOTE Project.
These samples were analyzed to assess whether analytical interference occurred due to the
decontamination agents used. However, none of the sand samples were targeted for
decontamination. Any decontaminant they received was due to overspray or general presence,
not because the sample trays were specifically targeted for decontamination. One sample
location was on the first floor, and another location was on the second floor. The analytical
results for each sample collected inside the building on the two floors are detailed in
Table 3-54. Spores were detected by the EPA analysis in all but one of the samples collected
post-dissemination, and all but one of the post-decontamination samples were classified as ND.
205
-------
Table 3-54. Number of sand samples collected within the building (first and second
floors) according to their codified EPA results.
Code
ND
0
1 +
2+
3+
4+
Total
1st Floor
Post-
Dissemination
0
0
0
0
3
1
4
Post-
Decontamination
3
0
1
0
0
0
4
2nd Floor
Post-
Dissemination
1
0
3
0
0
0
4
Post-
Decontamination
4
0
0
0
0
0
4
Note: Results represent samples "A" and "B" collected during Rounds 2 and 3.
Inhibition tests were conducted with the samples collected within the building to determine if
residual decontamination chemical used during the BOTE Project interfered with the PCR
reactions, as discussed in Section 2.9.4.5. Eight "building during BOTE" sample extracts, one
sand sample from each floor (1 and 2) and each decontamination treatment round (VHP®, pH-
adjusted bleach, and CIO2 fumigation) collected post-decontamination were selected for
inhibition testing. In addition, one sand extract collected post-dissemination for Round 2 (pH-
adjusted bleach process) and Round 3 (CIO2 fumigation) was assessed. Triplicate reactions
using extracted template DMA from the original selected sand samples were run alongside
triplicate reactions of the sample extract spiked with 10 GEq of the standard Bg DMA (Table
3-55). The addition of Bg DMA allowed for a low but reliable concentration of target DMA to be
present within each spiked reaction tube; the target DMA averaged at a Ct of 33.9 (SD = 0.43)
or a 3+ coded response.
Based on these analyses, none of the three decontamination agents apparently caused qPCR
inhibition in the sand samples assessed during this study. In each qPCR reaction, the resulting
cycle threshold value decreased when compared to the sample extracts alone, or a previously
ND sample became detectable to the expected spiked concentration. While this simplistic
analysis shows that the qPCR analysis was not inhibited in these samples, this analysis cannot
be used to determine if the reagents may have affected the study results due to DMA
degradation or other effects.
206
-------
Table 3-55. Summary of qPCR decontamination agent inhibition tests of selected indoor
sand samples.
BOTE Sample
Round 1 /decontamination/Bib
Round 1/decontamination/B2a
Round 2/dissemination/B1a
Round 2/decontamination/B1a
Round 2/decontamination/B2a
Round 3/dissemination/B1a
Round 3/decontamination/B1b
Round 3/decontamination/B2a
Mean
Cycle
Threshold
Time of
Sample
(SD)
31.7(0.08)
38.5 (2.94)
33.0(0.16)
Undetected
(0.00)
Undetected
(0.00)
33.0 (0.22)
39.9 (4.60)
Undetected
(0.00)
Sample
Code
4+
0
3+
ND
ND
3+
0
ND
Mean Cycle
Threshold Time
with Inhibition
Spike (SD)
31.4(0.13)
33.3 (0.25)
32.3(0.18)
33.8(0.12)
34.0(0.18)
32.0 (0.49)
33.8 (0.51)
34.0(0.13)
Spike
Code
4+
3+
3+
3+
3+
4+
2+
3+
3.2.4.2.6. USGS Inhibition Testing
To date, no inhibition studies have been conducted by USGS.
3.2.4.2.7. EPA Sample Deviation
The EPA analytical results indicated the presence of low concentrations of Bg DMA in the
collected sand samples, many of which were near the instrument LOD. The SDs between
sample replicates ranged from a maximum of 2.18 to a minimum of 1.76 for the study as a
whole (Table 3-56).
207
-------
Table 3-56. EPA mean, maxima, and minima for all collected sand samples
Round 2
Mean
Maximum
Minimum
Round 3
Mean
Maximum
Minimum
Combined Averages
Mean
Maximum
Minimum
Cycle Threshold Time
36.2
41.3
30.4
37.0
43.3
32.8
36.5
43.3
30.4
SD
2.18
1.76
2.03
Code
1 +
0
4+
1 +
0
3+
1 +
0
4+
3.2.4.2.8. USGS Sample Deviation
Due to the limited data set, no intra-sample deviations for the USGS data could be tabulated.
3.2.4.3. Statistical Analysis of Sand Sample Results
For the codified data associated with samples collected within the secondary enclosure (and
outside the building), categorical data analyses were conducted to assess differences in the
distribution of sand sample classifications (detected/ND) that occurred between the analytical
laboratories (EPA, USGS), the study decontamination round, the stage of each round, and the
sample location. The strength of the data was limited due to a lack of standard curves
concurrently used within the EPA analyses and the overall lack of data from the USGS
analyses. Therefore, the resulting EPA and USGS data are best suited as qualitative rather than
quantitative data. The statistical analysis considered only data for the second and third
treatment rounds, due to concern about sample contamination that may have occurred in the
first treatment round.
3.2.4.3.1. Statistical Comparison of EPA vs. USGS Methods
Assessing the presence of statistically significant discordance between the EPA and USGS
approaches was affected by the lack of detectable outcomes by the USGS analysis, using
Fisher's Exact test'831, binomial tests were performed to assess the extent to which the
classifications made by EPA and USGS analysis for a given sample were independent of each
other (i.e., no statistical relationship is apparent in the outcome of a given sample). Because all
USGS results were ND, this analysis took the form of a test of whether the percentage of ND
outcomes within the EPA analysis (i.e., outcomes classified as "ND" or "0") differed significantly
from 50%, or, stated otherwise, was there a significant deviation from a 50:50 split between the
EPA samples being detected or non-detected? The following conclusions can be drawn from
208
-------
the binomial analysis performed on the classification outcomes for all 120 samples from Round
2 and Round 3:
• The percentage of ND outcomes from the EPA analysis was 60% in Round 2, not
significantly different from 50% at the 95% confidence level (p=0.155), and 68% in
Round 3 which was significantly different from 50% at the 95% confidence level
(p=0.006).
• From the EPA analysis, the percentage of non-detected outcomes was 75% during the
pre-dissemination collection period (Stage 1), 40% during post-dissemination (Stage 2),
and 78% during post-decontamination (Stage 3). These percentages were significantly
different from 50% at the 95% confidence level at pre-dissemination (p=0.002) and post-
decontamination (p=0.0007); the percentage at post-dissemination did not differ
significantly from 50% (p=0.268).
The results of this statistical analysis imply that the EPA and USGS analysis results cannot be
considered statistically independent in the third treatment round nor in Stages 1 and 3. In the
second treatment round and in the sampling that follows post-dissemination (Stage 2),
insufficient evidence existed to reject the null hypothesis that the two sets of outcomes were
independent of each other, given the lower non-detection rate under the EPA analysis in both
Round 2 and Stage 2 compared to the other treatment rounds and stages. Across all samples,
the EPA analysis determined that approximately 64% of the samples were ND, compared to
100% of the samples according to the USGS analysis. This difference may be due to the large
discrepancy between initial sample amount (45 g for EPA vs. 0.25 g for USGS) and the
difference in replicate numbers (three replicates for EPA, two replicates for USGS). Because the
EPA analysis yielded some detectable results, only the EPA results were used for further
analysis in this report.
3.2.4.3.2. Statistical Analysis Comparing Decontamination Treatment Rounds
Statistical analyses to test for significant differences in the proportion of ND results (i.e.,
outcomes classified as "ND" or "0") between Round 1 and Round 2 were performed using
Fisher's Exact test'831. Table 3-57 summarizes the classification data entering into this analysis.
According to this table, and as noted in the conclusions, 60 and 68 percent of samples in Round
2 and Round 3, respectively, were classified as ND. The association between the percentage of
ND and the testing round was not significant at the 95% confidence level (p=0.447).
A logistic regression analysis was fitted to the detected/ND outcome data from Round 2 and
Round 3 to assess the extent to which testing round and sampling stage were statistically
significant predictors for the proportion of NDs. This analysis allows the effect of one of these
factors to be assessed while taking account of the other effects in the logistic regression model.
The logistic regression model also included a random effect for the sample location; this effect
accounted for possible correlation in the outcomes among samples collected at the same
location over the course of the study (i.e., among treatment rounds and collection stages). Like
the outcome of Fisher's Exact Test, the logistic regression analysis found no significant effect of
testing round (i.e., no significant difference between the second and third decontamination
209
-------
technologies in the proportion of NDs in the sand samples at the 95% confidence level
(p=0.306)).
Table 3-57. Summary of the number of samples within each of the positive and non-
detected classifications according to EPA analysis, by treatment round (considering all
samples).
Code
ND
0
1 +
2+
3+
4+
5+
Total
Round 2
36
0
15
7
1
1
0
60
Round 3
40
1
18
1
0
0
0
60
Total
76
1
33
8
1
1
0
120
Note: Codes of ND and 0 represent "non-detected" outcomes
3.2.4.3.3. Statistical Analysis of Stages
Fisher's Exact test'831 was used to test for significant association between the proportion of ND
results and the sampling stages (Pre-Dissemination, Post-Dissemination of Bg in the building,
Post-Decontamination of the building; Table 3-58) for samples collected in Rounds 2 and 3.
When the test was performed separately for each decontamination technology (i.e., each
round), the difference in the proportion of ND samples among the testing stages was significant
for Round 2 (p<0.001) and Round 3 (p=0.019). The primary contributor to significance in both
decontamination rounds was the difference between post-dissemination (Stage 2) and the other
two stages. As noted in Table 3-58, the ND rate is lowest during post-dissemination, especially
in the second treatment, with the difference due primarily to a larger number of samples being
classified as weakly detected (1+) in post-dissemination.
When Fisher's Exact test was used to compare the percentages of NDs between the two rounds
independently for each testing stage, no significant differences were observed between the
rounds at the 95% confidence level for any testing stage. Hence, the proportion of ND results
was not significantly different between the Round 2 Pre-Dissemination and the Round 3 Pre-
Dissemination (p=0.065). Similar conclusions can be reached for the post-dissemination
(p=0.333) and post-decontamination (p=0.127) stages.
When the analysis was conducted across both (Round 2 and 3) testing rounds (i.e., the data
from both decontamination technologies were lumped together for each testing stage), the
association between the three stages was found to be highly significant (p=0.002). This
outcome was due primarily to the higher detection rates observed in post-dissemination
compared to the other two stages. The ND rate during post-dissemination is about half the rate
210
-------
observed during post-decontamination (Table 3-58), with the difference due primarily to a larger
number of samples being classified as weakly detected (1+) in post-dissemination.
The effect of sampling stage was further assessed using the same logistic regression analysis
model described in section 3.2.3.3.2, focusing this time on the significance of the sampling
stage effect in the model. The overall sampling stage effect was highly significant (p=0.0009).
As a result, pairwise comparisons among the three stages were performed within the model
fitting. The p-value of the logistic regression analysis comparing pre-dissemination to post-
dissemination was 0.020, while the p-value for comparing pre-dissemination to post-
decontamination was 0.79, and the p-value for comparing post-dissemination to post-
decontamination was 0.001. Because significance was determined here at the 0.05/3=0.0167
level (the three pair-wise comparisons were performed at this significance level to ensure that
the overall error rate among all three pairs was no higher than 0.05), only the post-
dissemination vs. post-decontamination comparison was determined to be significant at an
overall 95% confidence level. This difference was therefore the primary contributor to the overall
differences among stages.
Table 3-58. Summary of the number of samples within positive and ND classifications
according to EPA analysis, by collection stage (considering all samples).
Code
ND
0
1 +
2+
3+
4+
5+
Total
Round 2
Pre-Dissem*
12
0
3
3
1
1
0
20
Post-Dissem
6
0
10
4
0
0
0
20
Post-Decon**
18
0
2
0
0
0
0
20
Round 3
Pre-Dissem
17
1
2
0
0
0
0
20
Post-Dissem
10
0
10
0
0
0
0
20
Post-Decon
13
0
6
1
0
0
0
20
*Dissemination. """Decontamination.
3.2.4.3.4. Statistical Analysis of Sample Placement
Table 3-59 summarizes the average EPA results from each decontamination technology round,
collection stage, and sampling location, based upon whether or not the Bg concentration was
within the detectable limits (i.e., was classified as 1+ through 5+). As each round progressed,
the rate of detection increased from the samples collected during pre-dissemination to those
collected post-dissemination of spores. While a decrease in the number of samples with
detectable Bg was seen post-decontamination, care must be taken before attributing this
observed decrease to the decontamination technologies alone, as the sand samples within the
211
-------
secondary enclosure were not directly decontaminated during this study. As stated previously,
when an analysis was conducted looking at the detected results from both decontamination
technology rounds together, the association of detects to NDs between stages was significant
(p=0.001). However, no significant association was observed between the proportion of NDs
and location at the 95% confidence level, based on Fisher's Exact test (p=0.360).
Figure 3-35 shows where the sand sample locations were positioned along the outside of the
building within the secondary enclosure. Table 3-59 shows that the largest proportion of
detected results occurred at Location 1, near the secondary enclosure personnel entrance.
Here, five of the six sampling events led to detected outcomes.
Table 3-59. Number of samples with detected Bg by location, round, stage and detection
status based on averaged EPA analysis of individual samples.
Secondary
Enclosure Location
1
2
3
4
5
6
7
8
9
10
Total
Detected
ND
Round 2
Detected Samples
Pre-
Dissem*
N=2
1
0
0
2
1
0
1
1
0
2
8
12
Post-
Dissem
N=2
2
2
0
0
2
1
2
2
2
1
14
6
Post-
Decon**
N=2
1
0
0
0
1
0
0
0
0
0
2
18
Round 3
Detected Samples
Pre-
Dissem
N=2
0
0
0
0
0
1
1
0
0
0
2
18
Post-
Dissem
N=2
2
2
2
0
1
0
1
1
1
0
10
10
Post-
Decon
N=2
2
0
0
0
0
1
0
1
2
1
7
13
Total
8
4
2
2
5
3
5
5
5
4
43
77
*Dissemination. """Decontamination.
212
-------
Tent Egress
Tent
Personnel
Entrance
Under
Stairs
Tent
Equip in eiit
Entrance
\
Test Buildine
Nesntive Ail-
Ma chine nn (1
Tubing
UV-APS »nd
Tubing
Figure 3-35. Schematic of the building and secondary enclosure layout for the BOTE
Project Phase 1.
3.2.4.3.5. Statistical Analysis of Possible Carryover Contamination between Rounds
A few surface samples collected post-decontamination for Round 1 resulted in detectable Bg
spores at very significant levels. All surface samples collected post-decontamination for Rounds
2 and 3 were below the limit of detection. Because the Round 2 sand pre-dissemination
sampling occurred directly following the Round 1 decontamination treatment without any
additional decontamination, the number of detectable sand samples collected during the pre-
dissemination sampling of the bleach might be due to inefficient decontamination by VHP®
within the building. The prevalence of detected samples in Round 2 pre-dissemination was
therefore assessed to see how detected samples in Round 2 pre-dissemination compared to
detected samples present in Round 1 post-decontamination. Among the 20 different sampling
locations (two samples at each of the ten locations), five locations had samples showing
detected results in both sampling periods, 12 locations had samples showing detected results in
the Round 1 post-decontamination period but not detected in the Round 2 pre-dissemination
period, and three locations had samples showing not detected in the Round 1 post-
decontamination period and detected in the Round 2 pre-dissemination period. Results of
213
-------
applying a nonparametric sign test concluded that the median difference in average cycle
threshold response between the two periods was significantly negative (p=0.012), meaning that
the average cycle threshold response in Round 1 post-decontamination tended to be lower than
the response in Round 2 pre-dissemination. This result is consistent with saying that the
prevalence of detected results in Round 2 pre-dissemination was significantly higher than in
Round 1 post-decontamination at a given location. (A similar conclusion was not reached
between Round 2 post-decontamination and Round 3 pre-dissemination: the median difference
in average cycle threshold time was not significantly different from zero at the 95% confidence
level). There is statistical evidence that most sampling locations had an increase in detectable
spore counts between the end of Round 1 and the start of Round 2, and this increase of spores
may have been building spores redistributed during the building reset.
3.2.5. Decontamination Line Wash Water Treatment Assessment Results
Water quality results for all the sampling events are summarized in Table 3-60 through Table
3-64. Spore concentration results (before and after wash water chlorination) were NDs for the
first four sampling events; therefore, Bg spore log reduction (per Equation 2-1) was not possible
for these samples. This result is not surprising because personnel removed outer gloves and
booties, which would be expected to have the best likelihood of having attached spores, before
the PPE wash down procedure was performed. Given the preliminary results from the first four
decontamination runs, Bg spores were spiked into the collected wash water for the final
sampling event prior to sampling and analysis (0.15 mLof an estimated 1.5E8 CFU/mL
suspension). An initial level titer of 3 log CFU/mL resulted in a greater than 3-log reduction at
approximately 60 ° F (15 °C) and a pH of approximately 11 at the 15-minute contact time. The
estimated simple CT was 45,000 mg-min/L. Due to a cross- contamination problem, the results
from the concentrated sample analysis were not used to calculate log reduction. Only results
from grab sample analyses were used for these calculations.
The inactivation results are similar to EPA bench scale results, which show for similar conditions
a greater than 7 log reduction of Bg spores at a temperature of 23 °C and a 4.5 log to a greater
than 7 log reduction at a temperature of 4 °c[661. For the BOTE Project, wash water
temperatures ranged from 4 to 15 °C.
214
-------
Table 3-60. Results for wash water collected in Round 1 after dissemination and pre-
decontamination.
Measurement
Temperature (°C)
Free Chlorine (mg/L)
PH
Turbidity (NTU)**
Total Suspended Solids
(mg/L)
Chemical Oxygen
Demand (mg/L)
Pre-chlorine
NA
0.01, ND,0.05
111.4,116.2,108.3
117, 114, 114
NA
Time
Zero*
3.32
9.59
15-min
Chlori nation*
3.13
9.76
Quench
0.02
11.69
Post-
Chlorine
ND
97.7,
90.55,
117.7
95, 87,
87
*1 x10a dilution
*Nephelometric turbidity unit
Table 3-61. Results for wash water collected in Round 2 after dissemination and pre-
decontamination.
Measurement
Temperature (°C)
Free Chlorine (mg/L)
PH
Turbidity (NTU)**
Total Suspended Solids
(mg/L)
Chemical Oxygen
Demand (mg/L)
Pre-chlorine
3.8
0.12,0.09, 0.07
8.43
241.6,239.7
181, 180, 174
0.51,0.58,0.58
Time
Zero*
10.3
3.94,4.15
10.05
15-min
chlorination*
4.33, 4.44
Quench
0.08,
ND, ND
11.85
Post-
chlorine
10.3
0.24,
ND, ND
11.18
243.2,
225.3
166,
164,
167
5.00,
4.99,
5.01
*1 x 10° dilution
*Nephelometric turbidity unit.
215
-------
Table 3-62. Results for wash water collected in Round 2 during pH-adjusted bleach
decontamination.
Measurement
Temperature (°C)
Free Chlorine (mg/L)
PH
Turbidity (NTU)**
Total Suspended Solids
(mg/L)
Chemical Oxygen
Demand (mg/L)
Pre-chlorine
5.4
2.47
8.45
197.5, 196.4, 193.3
122, 123, 121
ND, ND
Time
Zero*
7.8
3.84
10.03
15-min
chlori nation*
8.3
3.24
9.5
Quench
11.3
0.1, ND
11.77
Post-
chlorine
ND, 0.03
11.7
172.1,
167.3,
169.8
103, 100,
99
5.17,5.18
*1 x10J dilution
*Nephelometric turbidity unit.
Table 3-63. Results for wash water collected in Round 2 after pH-adjusted bleach
decontamination.
Measurement
Temperature (°C)
Free Chlorine (mg/L)
PH
Turbidity (NTU)**
Total Suspended Solids
(mg/L)
Chemical Oxygen
Demand (mg/L)
Pre-chlorine
13.3
0.01, ND, ND
8.4, 8.4, 8.4
277.3, 279.7, 270.7
183, 181, 181
0.77,0.91
Time
Zero*
14.7
15-min
chlorination*
15.0
Quench
Post-
chlorine
0.02, ND,
ND
10.37,
10.46,
10.46
237.0,
252.8,
223.1
127, 126,
125
5.12,4.77
*1 x10J dilution
*Nephelometric turbidity unit.
216
-------
Table 3-64. Results for wash water collected in Round 3 after dissemination and pre-
decontamination.
Measurement
Temperature
(°C)
Free Chlorine
(mg/L)
PH
Turbidity
(NTU)**
Total
Suspended
Solids (mg/L)
Chemical
Oxygen
Demand
(mg/L)
Pre-chlorine
14.9
0.17, 0.09,0.08
8.4
294.9,309.4,301.9
207, 208, 208
0.05, 0.06, 0.05
Time
Zero*
16.7
2.94, 2.93
15-min
chlori nation*
3.01,3.00,2.98
Quench
ND/ND
Post-
chlorine
0.06, 0.02,
ND
11.2
325.7, 324.2,
322.6
184, 183,
183
5.71, 5.71,
5.71
*1 x10J dilution
*Nephelometric turbidity unit.
217
-------
4. DATA ASSESSMENT AND DISCUSSION
4.1. Decontamination Methods Assessment
One of the primary objectives of the BOTE Project was to operationally assess the effectiveness
of pre-selected decontamination processes. The methods were selected based upon subject
matter expert workgroup discussions, assessing the potential of the processes from existing
laboratory data and/or past field performance. The BOTE Project facility configuration and Bg
surface loadings were designed to assess the decontamination process effectiveness under
several scenarios, for future use in extrapolating results to incident and site-specific needs.
Based upon the subject matter expert workgroup discussions, three decontamination methods
were chosen for assessment in the BOTE Project. As discussed in Section 2.10, the following
decontamination processes were selected:
• fumigation with H2O2 using STERIS VHP®;
• surface decontamination using pH-adjusted bleach (amended bleach); and
• fumigation with CIO2.
Each decontamination process was assessed independently, used in three different test rounds.
As discussed in detail in Section 2, each round involved the setup (or reset) of the facility,
dissemination of Bg spores, pre-decontamination (characterization) sampling, application of the
decontamination process, post-decontamination (clearance) sampling, and facility assessment
(and re-set). In the first round, STERIS Corporation (Mentor, OH) was subcontracted by INL
under the EPA agreement to fumigate the facility with hydrogen peroxide using their VHP®
process. The EPA Region 10 START contractor was used to decontaminate the facility using
pH-adjusted bleach. This process was directed by EPA Region 1 and ten On-scene
Coordinators, supported by technical experts from EPA's Chemical, Biological, Radiological,
and Nuclear (CBRN) Consequence Management Advisory Team (CMAT, formerly the National
Decontamination Team) and EPA's National Homeland Security Research Center within the
Office of Research and Development. The third round decontamination process was a full
facility fumigation with CIO2, performed by Sabre Technical Services, LLC (Slingerland, NY), as
a subcontractor to INL under the EPA agreement.
Discussion of each process and the sampling results with respect to the assessment of
effectiveness is provided in the subsections that follow.
4.1.1. STERIS VHP® Fumigation
For the first round of Phase I, STERIS Corporation's VHP® technology was selected for the
decontamination process.
4.1.1.1. Process Description
During the VHP® process, an aqueous solution of hydrogen peroxide is flash-vaporized into a
stream of dehumidified heated air. This stream of hydrogen peroxide vapor is then injected into
the space to be decontaminated. In general, the process can be operated in a closed- or open-
218
-------
loop mode. In the closed-loop mode, process air is withdrawn from the enclosure, passed
through a catalyst to decompose any residual hydrogen peroxide and dehumidified before being
re-enriched with hydrogen peroxide vapor and returned to the enclosure. In the open-loop
mode, process air is drawn from outside the space to be decontaminated, dehumidified and
enriched with hydrogen peroxide vapor before being injected into the enclosure. Excess air in
the enclosure passes through a catalyst to decompose the hydrogen peroxide to safe levels
before being vented to the environment. In either mode, the injection process proceeds
continuously until a sufficient exposure of the VHP® to the contaminated space in terms of time
and concentration has been achieved. After the decontamination has been completed, the
concentration of VHP® remaining in the space is reduced by the use of a decomposition
catalyst, natural decomposition and the introduction of fresh air.
The target H2O2 concentration was 250 ppm for 90 minutes or a cumulative exposure of 400
ppm-hr at a temperature of 65 °F or higher. The selection of these fumigation criteria was
determined by STERIS, in accordance with their EPA registration label (EPA Reg. No. 58779-4)
for Vaprox® use as a sterilant. For fumigation of PBF-632, STERIS chose to use a separate
generator unit for each floor located externally to the building with the vapor injected into the
building air handler units. For each floor, the generation equipment consisted of one VHP®
M1000-T4 Biodecontamination System (STERIS, Mentor, OH) and one Munters HC-300
dehumidifier (Amesbury, MA). The T4 generator operates in an open-loop configuration, where
fresh air is dehumidified by the Munters HC-300 units before being injected with hydrogen
peroxide vapor and delivered into the building. The T4 generator operates in an injection rate
range of 4-96 g/min of hydrogen peroxide solution and airflow rates of 40-100 standard cubic
feet per minute (SCFM). Actual injection and air flow rates will be described in the following
section. The T4 is designed to inject either 35% or 59% solutions of hydrogen peroxide (Durox
LR®). The 59% solution was utilized by the vendor for this process. Use of the 59% solution
effectively increases the generation capacity of the unit and reduces the risk of condensation of
the injected vapor due to less concurrent water injection. To offset the air introduced into the
building by the fumigant generation process, an "exhaust skid" consisting of a HEPA filter,
catalyst, and fan was used.
The building HVAC fans (one for each floor) were operational and were used to facilitate
distribution of the fumigant throughout the building. To provide further air movement and to
ensure that the fumigant would penetrate into rooms not containing supply registers, 74 16-in
pedestal fans were used (see Figure 4-1 and Figure 4-2). Five high-volume catalyst units
(-1,200 CFM each) were activated during the aeration phase to increase the rate of hydrogen
peroxide decomposition and decrease the aeration time. Two Patron 30,000 British thermal unit
(Btu)/hr electric heaters (Cheektowaga, NY) were deployed in the building (first floor hallway) to
maintain the desired operational temperature of 65 °F.
To monitor environmental conditions within the building during fumigation, six sensor pods were
used. Each pod contained an electrochemical hydrogen peroxide sensor, a humidity sensor and
temperature sensor. Data were monitored and logged at a computer terminal outside the
building. EPA also had HOBO® U10 data loggers (Onset Computer Corp., Bourne, MA) installed
in each room to monitor the temperature and RH. To further measure the distribution of the
219
-------
fumigant and its efficacy, a number of biological indicators (Bis) and color-changing chemical
indicators (CIs) were placed in the building. The Bis were Tri-Scale Biological Indicators (part#
LOG-456) from Apex Laboratories, Inc. (Apex, NC). Each Bl consisted of three stainless steel
discs inoculated with G. stearothermophilus (#12980) at the 1E6, 1E5, and 1E4 levels,
respectively. The CIs used were STERIS, Inc. (Mentor, OH) model numbers NB305 and
PCC036.
A description of PBF-632 can be found in Section 2.1. This same configuration was used in all
three rounds.
4.1.1.2. Facility Contamination
Bg spores were disseminated on April 16, 2011, following the procedure described in Section
2.3. The target surface loading of 1E4 to 1E6 CFU/ft2 was desired on the first floor, and a
surface loading of 1E2 to 2E2 CFU/ft2 was desired on the second floor. The actual surface
loading was characterized by surface sampling as described in Section 2.5.4.
4.1.1.3. Setup and Preparation
On April 17, 2011, STERIS equipment was delivered to the test bed site via common carrier.
The T4 units were placed outside the outer membrane near the furnace room and the exhaust
skid was placed just outside the furnace room on Floor 1 as shown in Figure 4-1. The T4 units,
dehumidifier and the monitoring equipment were covered with a secondary enclosure to protect
them from the elements. The STERIS team consisted of four personnel who assisted with the
setup, fumigation and teardown of the equipment.
Before each round, each vendor was given the opportunity to walk through the facility to
determine if any items in the facility would absorb or consume the fumigant. The items that were
identified were then removed from the facility before fumigation to be treated in a different
manner. STERIS elected to leave all materials inside the facility during fumigation, so no
materials or furniture were removed from the building during this round. STERIS also elected
not to encapsulate (tent) the facility directly (i.e., on the building directly, underneath the
secondary enclosure).
On April 18, 2011, following characterization sampling, the placement of fans, sensors, and
aerators (also referred to as catalyst units) inside the facility was completed. Four personnel
staged equipment in the staging area at the west end of the building. All entries into PBF-632
following spore dissemination were in Level C PPE, which included a full-face air purifying
respirator with HEPA filters, Tyvek® suit, and nitrile gloves. The respirator, gloves and boots
were each taped to the suit using liquid chemical resistant tape to form a complete barrier.
Three STERIS personnel and two EPA personal entered the facility to stage equipment and
distribute Bis and CIs throughout the facility. One STERIS employee remained outside to pass
equipment into the facility. Equipment was then passed through the doorways on the first and
second levels to prevent contamination of the staging area. The second floor was set up first,
with a total of 36 pedestal fans placed throughout the floor to facilitate dispersion of the
vaporized H2O2. Two aerators were deployed in the hallway. The power cords for the aerators
were run outside the secondary enclosure through an access hole near the furnace room. Three
220
-------
sensor pods were deployed on this floor. The wiring for the sensor pods was also run to the
outside to allow for monitoring during fumigation. Before any equipment was placed on the floor,
the footprint in which it was to be placed was sprayed with Spor-Klenz® Ready to Use (STERIS,
Mentor, OH) to inactivate any spores that would be under the equipment and would hence not
be exposed to the hydrogen peroxide vapor. As the equipment was being set up, the Bis and
CIs were placed in the rooms and hallways of the second floor. Locations of the BIs/CIs were
recorded and are discussed in a later section. After all equipment and BIs/CIs were placed,
personnel moved down to the first floor. The airlock between the floors was left open during the
fumigation so that excess air from the second floor could flow to the exhaust skid connected to
the first floor. Equipment on the first floor was set up in a manner similar to the second floor,
deploying 38 fans, three aerators, and three sensor pods. In addition, two space heaters were
placed in the hallway of the first floor in order to provide heat to achieve the desired temperature
during fumigation. The position of all equipment on the first and second floors is shown in Figure
4-1 and Figure 4-2.
The distribution of hydrogen peroxide throughout the facility was accomplished by connecting
the flow from the T4 generators to the building HVAC systems (on each floor) using a 3-in
insulated plastic flexible duct. A separate line was installed for each floor. A hole was cut into
the side of the HVAC supply air plenum and a bulkhead fitting was installed to connect the hose
to the plenum. On the bottom floor, the hole was cut into one side of a dual plenum. On the top
floor, the hole was centered between the dual plenum supply sides.
A photo of one of the T4 generators is shown in Figure 4-3. The black lines coming off the top
and going to the center of the photograph are the insulated delivery lines that went into PBF-
632. The yellow flexible duct was connected to a dehumidifier and supplied dry air to the
generator.
221
-------
F
i
L
F
u~
omcE
—iioa
Aerator
Fan
FIRST FLOOR PLAN
(RH & T)
Figure 4-1. Schematic of first floor showing location of fans, sensors, and aerators.
222
-------
rr
F
ii
JL
F
ii
- -.H
'1
OFFICE
2C6
— l|
^ H il
*~ ~~ir~
/ •- ii
x- p ii
1
II
Fll
II
II
II
, II
F ||
•- V 1
F vj
F
F
'i
i
i
i!
2i
[
\
\
'12
\
*--
|
|
1
1
|
OR
K
;
\
\
o
—
-j-
HCE
!3A
1
V.
1
1
1
1
1
1
1
1
3
F
-1
F
II
""-
1
;L_
|~
i
1 <
iy
F
F
1 1
F
j
F
._ i
F '
EErrRDDhi
1 1 || || ^j^ (^
~i^i-iPif ~~n"
S '^QHEMS /^
RESTRQQhl U
L^""""=
a_oetr
ir __
/r R
-------
Figure 4-3. Photo of T4 generator with gas delivery lines going into PBF-632.
4.1.1.4. Fumigation Conditions
The facility was turned over to STERIS on the morning of Tuesday, April 19, to begin
fumigation. At 1130 hrs, safety personnel swept the building to ensure no one was inside prior
to commencement of fumigation. Immediately following the safety sweep, STERIS personnel
determined that one of the T4 units was experiencing a short circuit. Plans were made to
fumigate one floor at a time with the one remaining T4 unit. Fortunately, a technician was able
to determine the cause of the short circuit and was able to repair the malfunctioning unit.
Fumigation of both floors proceeded shortly thereafter at 1150 hr. A timeline of events that took
place during the fumigation is shown below in Table 4-1. The amount of hydrogen peroxide
injected was incrementally increased to prevent condensation in the lines. The concentration on
the first floor had not reached the target concentration of at least 250 ppm, so at 1700 hrs the
delivery lines to each floor were swapped to see if this exchange might help. The swap of
delivery lines did increase the concentration so a decision was made to plumb both delivery
lines into the first floor to increase the concentration of hydrogen peroxide on that floor. At 1950
hr, both supply lines were connected to the first floor for the next hour prior to shutting down the
generators. The natural degradation of hydrogen peroxide was observed for 30 minutes prior to
turning on the aerators to run overnight. Personnel departed the site at approximately 2200 hrs,
and a night watchman was left on site to ensure that no personnel entered the building.
224
-------
During the fumigation, monitoring was conducted around the perimeter of PBF-632
approximately 15 ft from the outer membrane. The frequency was initially 15-min intervals for
the first hour and then hourly after that to ensure that the concentration of hydrogen peroxide
remained below the permissible exposure limit (PEL) of 1 ppm. There was one instance where
the concentration exceeded the PEL, and this was near the exhaust ductwork for the UV-APS
systems located on the south side of the building. The ductwork was sealed with duct tape and
the hydrogen peroxide concentration around the perimeter returned to 0 ppm.
Table 4-1. Chronology of fumigation events with VHP® on April 19, 2011.
Time
1150
1218
1230
1235
1240
1424
1442
1705
1715
1952
2102
2131
2200
Action
VHP® generator started at 50 g/min on first floor
Second floor VHP® generator delayed by equipment problem
T4 generator repaired, and VHP® generator started at 50 g/min on second floor
Injection rate increased to 65 g/min on both floors
First floor injection stopped due to equipment problem
T4 generator repaired and VHP® generation resumed on first floor at 65 g/min
Injection rate increased to 75 g/min on both floors
Injection rate increased to 80 g/min on both floors
VHP® generation stopped to swap floors supplied by each generator
VHP® generators resumed at 80 g/min
Both T4s plumbed to supply first floor at combined 140 g/min
VHP® generation terminated
Aerators turned on
Personnel depart site
4.1.1.5. Scrubbing and Aeration
Once the fumigation was terminated on the evening of April 19, the aerators were turned on at
2130 hr. The aerators were allowed to operate overnight, and the concentration of H2O2 in the
building was monitored remotely on the following day. Once the hydrogen peroxide
concentration had dropped sufficiently, a number of entries with appropriate respirators were
made into the building to measure the H2O2 concentration using more sensitive handheld
monitors (Dra'ger PAC III, Draeger Safety, Pittsburgh, PA). On the morning of Thursday, April
20, hydrogen peroxide concentrations in some rooms still measured up to 6 ppm around some
materials measured using the handheld monitors. Four Novatek Novair 2000 NAMs (Novatek,
Exton, PA) were then connected to ducts that had been pre-installed to aid in aeration of the
building by bringing in fresh air (Figure 4-4). One NAM for each floor was connected on the west
side of the facility, set to supply 2,000 CFM. Likewise, one NAM for each floor was connected
on the east side of the facility and set to pull 1,000 CFM from the facility. The building was
pressurized slightly to prevent sucking in any spores that might be present outside the building.
H2O2 concentrations in the facility had dropped below the OSHA PELof 1 ppm, and two EPA
225
-------
and three STERIS personnel entered the building in Level C PPE at approximately 1600 hr to
remove the equipment and recover the BIs/Cls. The removal of the fans and aerating equipment
inside PBF-632 required approximately two hours. All internal lines were disconnected and
pushed outside the outer secondary enclosure.
Figure 4-4. Inlet NAMs on left side of figure and outlet NAMs on right side of
figure.
4.1.1.6. Demobilization
PBF-632 was turned back over to the BOTE Project team on Thursday, April 21. By the
afternoon of April 21, all equipment that had been placed inside the building had been removed
by three STERIS personnel in Level C PPE in the interior of the building and one STERIS
personnel on the exterior. STERIS personnel remained on site for Friday, April 22, and
disassembled and packaged the equipment that was located under a small protective
secondary enclosure. The equipment had been packaged and was shipped out at
approximately 1600 hr on the afternoon of April 22. Approximately half of one 15 gal container
of Durox LR (FMC Corporation, Philadelphia, PA) H2O2 solution remained, and approximately
5.5 gal remained of the other 15 gal container. The remaining H2O2 solution was returned with
the equipment to the STERIS facility.
4.1.1.7. Temperature and Relative Humidity
The heating system in PBF-632 was not functional, and the project required the use of
supplemental heating to raise the temperature in the facility to the desired temperature above
65 °F. Two Patron® 30,000 Btu/hr heaters were utilized to heat the facility in conjunction with
operation of the blower on the building HVAC system to distribute the heat. A diesel-powered
heater was used to heat the outer envelope (area between the building and the secondary
enclosure). The interior heaters were started on April 18, 2011, and allowed to operate
throughout the fumigation. The envelope heater was turned off at 1300 hr on April 19. The
226
-------
temperature throughout the fumigation was maintained above the 65 °F threshold; there was no
requirement on RH.
In addition to the three temperature and RH sensors that STERIS used, the EPA also collected
temperature and RH data in each room using HOBO® U10 data loggers. These data loggers
recorded temperature and RH every two minutes. Once the clearance sampling was completed,
the data loggers were removed and the data were recovered.
Temperature and RH measurements inside the facility were recorded both by STERIS and by
the EPA. The temperatures that STERIS recorded are shown in Figure 4-5 and Figure 4-6. The
RH values are shown in Figure 4-7and Figure 4-8. The start time of zero in the figures
corresponds to 1150 hr on April 19, 2011.
Figure 4-5. Temperature profile for the first floor.
227
-------
a so
I
H
75
300 400
Time (min)
Figure 4-6. Temperature profile for the second floor.
RHRoomlOS ^—RH Reception Are
200 300
Time (min)
Figure 4-7. RH on the first floor.
228
-------
I 35
i
RH Women's Rot
RH Room 201A RH Room 213
300 400
Time (min)
Figure 4-8. RH on the second floor.
4.1.1.8. H2O2 Measurements
The concentration of H2O2 was measured using three sensors on each floor as shown in Figure
4-9 and Figure 4-10. The sensors were hard-wired to a data acquisition system located near the
generator systems outside the building. The H2O2 concentration on the first floor never reached
the target (FIFRA registration) conditions (250 ppm for 90 minutes) for this product. The target
was 250 ppm for 90 minutes or a cumulative exposure of 400 ppm-hrs. It was not until after 500
minutes, when both T4 generators were plumbed into the first floor HVAC system, that the
concentration in Room 109 went over 250 ppm. Two of the sensors on the second floor
measured an H2O2 concentration of 250 ppm for several hours. The third sensor in the women's
room never measured higher than 200 ppm. The bathrooms did not have an HVAC register, so
getting H2O2 vapor distribution into these rooms was difficult.
229
-------
300 400
Time (min)
Figure 4-9. Hydrogen peroxide concentration on the first floor.
Figure 4-10. Hydrogen peroxide concentration on the second floor.
230
-------
4.1.1.9. VHP® Decontamination Results
A variety of methods were used to evaluate the decontamination efficacy. These methods
included the Bl and Cl results as well as the process monitoring (H2O2 concentration,
temperature, RH) data. Surface samples were collected prior to and following decontamination
for the ultimate determination of decontamination efficacy. This section presents the results of
the VHP® fumigation.
4.1.1.10. Bl Results
A total of 90 biological indicators from Apex Laboratories were set out, 44 on the first floor and
46 on the second floor. Each Bl consisted of three stainless steel discs inoculated with G.
stearothermophilus at the 1E6, 1E5, and 1E4 levels, respectively. The Bis were recovered by
two EPA personnel in Level C PPE and were analyzed by the INL Microbiology Laboratory.
The Bl locations are shown by the colored circles in Figure 4-11 and Figure 4-12. The 6 log Bis
were analyzed first to see if they had been inactivated ("no growth"). The Bis shown in green
were 6 log Bis that were inactivated by the hydrogen peroxide. If the 6 log Bl showed growth,
then the 5 log Bl was processed to see if it had been inactivated by the hydrogen peroxide. A
solid yellow circle denotes a 5 log Bl that had no growth after being exposed to hydrogen
peroxide. Likewise, if the 5 log Bl showed growth, then the 4 log Bl was processed. A solid red
circle denotes a 4-log Bl that had been inactivated. For the first floor, 27 of the 6 log Bis were
inactivated, followed by 16 of the 5 log and 1 of the 4 log Bis. For the second floor, 30 of the 6
log Bis were inactivated, followed by 15 of the 5 log and 1 of the 4 log Bis. Both of the 6 log Bis
in the women's restroom were inactivated even though the H2O2 concentration remained below
150 ppm throughout the test. For the rooms in which H2O2 was monitored, the women's
restroom and other rooms on the second floor (having < 150 ppm H2O2for the majority of the
fumigation) had effectiveness just as good as or better than indicated by the Bis compared to
rooms on the first floor that had higher H2O2 concentrations.
231
-------
_.. JYL--
~~
No Growth on Bis: «106 105 0104
Figure 4-11. Results for Bis on the first floor.
No Growth on Bis: SIO6 105 • Id4
Figure 4-12. Results for Bis on the second floor.
232
-------
4.1.1.10.1. Chemical Indicators
The CIs were collected at the same time as the Bis. All CIs on both floors had changed color
and matched the end point standard, indicating that they had been exposed to H2O2 vapor.
These indicators are more of a qualitative endpoint to indicate that the area was exposed to a
sufficient amount of H2O2. No conclusions can be drawn from the CIs except that H2O2 had
reached the location of each Cl.
4.1.1.10.2. Surface Sampling Results
Characterization sampling was conducted on April 17 and 18, 2011. Post- decontamination
sampling commenced on April 22-23, 2011. Swabs, sponge-sticks, and vacuum sock samples
were collected from various surfaces and structures not removed from the building during the
decontamination procedures. This section contains the results of the pre- and post-
decontamination sampling that occurred during Round 1, fumigation with VHP®.
4.1.1.10.2.1. Field Blanks
Field blank samples were collected during each sampling campaign to determine the potential
for background contamination of sampling media. Contamination could occur during sample
handling in the field or in the laboratory during sample processing. Field blanks (62) were
collected during the characterization sampling. Of the field blanks collected, four samples from
the second floor came back with detectable Bg counts of 4.1E3, 7EO, 3.4E1, and 1.4E3 CFU.
Field blank samples (43) were collected during post-decontamination sampling for Round 1; two
samples were found to have detectable Bg counts at 2 and 17 CFU.
4.1.1.10.2.2. Pre-decontamination Sampling
Bg spores were disseminated on April 16, 2011 following the procedure described in Section
2.3. A target surface loading of 1E4 to 1E6 CFU/ft2 was desired on the first floor, and a surface
loading of 1E2 to 2E2 CFU/ft2 was desired on the second floor. The actual surface loading was
characterized by surface sampling as described in Section 2.5.4. Surface sampling results are
shown in Figure 4-13 and Figure 4-14.
Pre-decontamination samples (399) were collected, a total of thirteen were ND (no viable
spores recovered). Ten of the 13 were from the less-contaminated second floor. A more
detailed description of the pre-decontamination sampling results is presented in Section 3.2.1.2.
233
-------
Concentration (Log Estimate)
0
Figure 4-13. Spatial distribution of first floor pre-decontamination characterization sample results.
234
-------
• I m.
Concentration (Log Estimate)
Figure 4-14. Spatial distribution of second floor pre-decontamination characterization
sample results.
235
-------
4.1.1.10.2.3. Post- Decontamination Samples
A total of 288 samples were collected following decontamination with VHP®. The results with
detectable Bg are shown in Table 4-2 and Table 4-3 and shown in Figure 3-7 and Figure 3-8 for
Floors 1 and 2, respectively. A total of 78 samples from the first floor and 16 from the second
floor came back with detectable Bg either from spread and/or filter plating. All sample results are
reported in Appendix G.
Table 4-2. Results from first floor following decontamination with VHP®.
Sample
ID#
3568
2233
2236
2916
3594
3629
3585
3644
2072
2722
2566
2653
3670
2679
2231
3621
4157
4064
2942
2900
3248
2734
2957
1973
3660
3608
3243
3238
3509
3506
3273
3242
3239
3676
3077
3013
Room
Bathroom-Men
Bathroom-Men
Bathroom-Men
Bathroom-Women
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Mechanical Room
Mechanical Room
Mechanical Room
Room 101
Room 101A
Room 101A
Room 101A
Room 101A
Room 101A
Room 101A
Room 101A
Room 101A
Room 102
Room 102
Room 102
Room 102
Room 102
Room 102
Room 102
Room 102
Room 102
Room 103
Room 103
Room 103
Sample
Method
Vacuum Sock
Sponge-Stick
Sponge-Stick
Sponge-Stick
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge-Stick
Sponge-Stick
Vacuum Sock
Sponge-Stick
Sponge-Stick
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge-Stick
Sponge-Stick
Sponge-Stick
Sponge-Stick
Sponge-Stick
Sponge-Stick
Vacuum Sock
Vacuum Sock
Sponge-Stick
Sponge-Stick
Sponge-Stick
Sponge-Stick
Sponge-Stick
Sponge-Stick
Sponge-Stick
Vacuum Sock
Sponge-Stick
Sponge-Stick
Sampled
Surface
Ceiling
Floor
Floor
Floor
Ceiling
Ceiling
Ceiling
Ceiling
Floor
Floor
Floor
Floor
Supply Vent
Floor
Floor
Ceiling
Floor-carpet
Floor-carpet
Desk
Workbench
Cabinet
Desk
Desk
Cabinet
Floor
Floor
Workbench
Cabinet
Cabinet
Desk
Desk
Desk
Cabinet
Ceiling
Table
Table
Spread
Plate
Result
(CFU/ft2)
1.0E2
9.2E2
ND
ND
8.0EO
1.7E1
1.3E1
4.2E1
2.9E1
5.7E1
2.2E1
8.2E1
4.0E2
3.5E2
5.0E2
3.7E2
2.8E1
1.6E2
ND
ND
ND
1.8E1
ND
ND
2.9E1
2.8E1
1.4E2
2.8E1
6.5E1
ND
1.1 E2
ND
2.6E1
4.0EO
ND
ND
Filter Plate
Result
(CFU/ft2)
5.8E1
2.0E2
1.9E1
4.0EO
ND
ND
1.0EO
ND
5.3E1
3.2E1
ND
3.8E1
TNTC
ND
ND
1.2E2
NA
NA
4.0EO
3.0EO
1.9E1
1.2E1
1.0E1
4.0EO
1.4E1
NA
4.6E1
4.0EO
ND
5.0EO
1.8E2
2.0EO
3.2E1
ND
1.5E1
3.6E1
236
-------
Sample
ID#
3513
3529
3528
3076
1597
3714
3495
3287
3283
3686
3718
3569
3981
3595
3066
1676
3631
3616
3725
2840
2839
3246
1682
3599
3067
3719
3978
3520
3522
3065
3627
3669
3563
3689
2658
2429
3655
3612
3648
3584
3868
3284
Room
Room 103
Room 103
Room 103
Room 103
Room 103
Room 104
Room 104
Room 104
Room 104
Room 105
Room 105
Room 105
Room 105
Room 105
Room 105
Room 105
Room 106
Room 106
Room 106
Room 106
Room 106
Room 106
Room 106
Room 107
Room 107
Room 108
Room 108
Room 108
Room 108
Room 108
Room 109
Room 109
Room 109
Room 109
Room 109
Room 109
Room 110
Room 110
Room 110
Room 110
Room 110
Room 110
Sample
Method
Sponge-Stick
Sponge-Stick
Sponge-Stick
Sponge-Stick
Swab
Vacuum Sock
Sponge-Stick
Sponge-Stick
Sponge-Stick
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge-Stick
Swab
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge-Stick
Sponge-Stick
Sponge-Stick
Swab
Vacuum Sock
Sponge-Stick
Vacuum Sock
Vacuum Sock
Sponge-Stick
Sponge-Stick
Sponge-Stick
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge-Stick
Sponge-Stick
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge-Stick
Sampled
Surface
Floor
Floor
Floor
Mail slot
Supply Vent
Ceiling
Wall
Table
Table
Floor-carpet
Floor-carpet
Ceiling
Floor
Floor
Table
Supply Vent
Shelves
Floor
Ceiling
File cabinet
Desk
Floor
Supply Vent
Ceiling
Table
Shelves
Chair
Shelves
Wall
Floor
Bed
Bed
Floor
Ceiling
Countertop
Countertop
Chair
Ceiling
Wall
File cabinet
File cabinet
File cabinet
Spread
Plate
Result
(CFU/ft2)
1.2E2
ND
ND
ND
6.0E3
ND
ND
ND
ND
ND
4.0EO
4.0EO
ND
ND
1.4E2
3.6E3
8.0EO
1.3E1
1.7E1
2.4E1
9.1E1
ND
ND
4.2E1
2.0E6
1.3E1
ND
2.8E1
1.9E1
ND
ND
ND
ND
ND
1.5E2
1.9E1
ND
ND
4.0EO
ND
ND
ND
Filter Plate
Result
(CFU/ft2)
1.3E1
3.8E1
2.0E1
8.0EO
2.7E2
2.0EO
5.0EO
7.0EO
3.0EO
2.0EO
2.0EO
2.0EO
1.0EO
1.0EO
1.4E2
3.0E3
1.0EO
3.0EO
9.0EO
2.6E1
5.1E1
1.8E1
1.8E2
2.0E1
NA
2.0EO
1.0EO
NA
NA
2.0EO
1.0EO
1.0EO
1.0EO
1.0EO
ND
ND
1.0EO
4.0EO
1.0EO
1.0EO
1.0EO
5.0EO
NA=Sample was not filter plated.
ND=Non-detect.
TNTC=Too numerous to count.
237
-------
Table 4-3. Results from second floor post-decontamination sampling.
Sample
ID#
3640
2049
3551
2047
2709
1628
3552
2600
3606
1696
3643
3702
3546
3547
2602
2347
Room
Mechanical Room
Mechanical Room
Room 201
Room 202
Room 203
Room 206
Room 208
Room 209
Room 210
Room 210
Room 211
Room 211
Room 213
Room 213
Room 213
Room 213
Sample Method
Vacuum Sock
Sponge-Stick
Vacuum Sock
Sponge-Stick
Sponge-Stick
Swab
Vacuum Sock
Sponge-Stick
Vacuum Sock
Swab
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge-Stick
Sponge-Stick
Sampled
Surface
Return Vent
Floor
Ceiling
Floor
Floor
Supply Vent
Floor
Table
Chair
Ceiling
Ceiling
Chair
Bed
Floor-carpet
Cabinet
Sink
Spread
Plate
Result
(CFU/ft2)
ND
ND
ND
ND
ND
ND
ND
ND
2.9E1
3.5E5
2.8E1
4.0EO
9.3E1
ND
3.3E2
4.2E2
Filter Plate
Result
(CFU/ft2)
1.0EO
2.2E1
1.0EO
3.0EO
3.0EO
9.0E1
1.0EO
6.0EO
1.1E1
ND
NA
ND
NA
1.0EO
NA
NA
NA=Sample was not filter plated.
ND=Non-detect.
238
-------
Concentration (Log Estimate)
Gray indicates areas with estimated zero CPU.
Figure 4-15. Spatial distribution of first floor post-decontamination in Round 1.
239
-------
Concentration (Log Estimate)
Gray indicates areas with estimated zero CPU.
Figure 4-16. Spatial distribution of second floor post-decontamination in Round 1.
240
-------
4.1.1.10.3. Decontamination Efficacy
Roughly a third of the samples on the first and second floor resulted in detectable Bg after
decontamination. The temperature was maintained above the desired set-point throughout the
testing. The main condition that was not achieved was the H2O2 concentration throughout the
facility for the specified amount of time, possibly a result of breakdown or adsorption of the H2O2
that was being injected into the facility, poor circulation of the H2O2, or simply not enough H2O2
was injected. These results indicate that the H2O2 exposure was not sufficient to inactivate all
the spores at either challenge level (surface loading). One improvement would have been to
increase the amount of H2O2 that was introduced on each floor by doubling the number of T4
generators or using a larger generator.
4.1.1.11. Materials Effects
A post-test inspection of the building was completed after clearance sampling. Materials that
had been placed in the building were inspected for damage. All surfaces and materials retained
their original condition and color. Based on this inspection, there did not appear to be any
damage to the building contents from exposure to the VHP® process.
4.1.1.12. Summary of Fumigation with VHP®
Fumigation of materials in the laboratory with H2O2 has shown the process to be efficacious for
inactivation of Bacillus spores. There are several advantages with this process because this
process does not require the removal of the porous materials from the facility prior to fumigation.
Experimental evidence has shown that porous materials can adsorb H2O2, thus creating a
demand and lowering the immediate concentration of H2O2 in a facility. Removal of porous
materials like mattresses and thick cushions may have reduced the adsorption of H2O2 and
resulted in a higher H2O2 concentration which could have resulted in a higher efficacy. The
aeration time could also have been reduced by the removal of heavy foam products, but leaving
the materials in the building would just require a longer aeration phase, as indicated by higher
H2O2 readings over the mattresses and cushions during the aeration phase. The building did not
need to be tarped or modified prior to decontamination. The crew from STERIS was able to
complete the project in three days. Another advantage is that after fumigation, the H2O2 is
broken down into water and oxygen, so there is no need for sorbents or neutralization following
fumigation.
The system that was used was not a field-deployable system, but a system designed to be
installed more permanently in a facility for routine fumigations. Specialized equipment and
experience is necessary to conduct the fumigation with H2O2.
A site visit by the STERIS crew would have allowed the crew to familiarize themselves with the
facility layout and HVAC design. The HVAC on each floor was used to distribute H2O2
throughout the facility (each floor independently). The connection on the bottom floor was not
optimized and resulted in a lower H2O2 concentration than expected. A higher H2O2
concentration may have resulted in a higher degree of inactivation of the Bg spores. Another
improvement would have been to increase the amount of H2O2 that was introduced on each
floor by doubling the number of T4 generators or using a higher capacity generator.
241
-------
Specialized equipment and training is required for the fumigation process using hydrogen
peroxide vapor. There are only a couple of companies that have the equipment necessary to
carry out the fumigation of a facility. The procurement process time may be an important factor
when considering response and recovery preparedness, as well as incident-specific options.
There may be large quantities of chemicals that would need to be transported and stored on
site; secondary containment measures must be taken to prevent any spills. Any materials in the
facility that will confound the decontamination may need to be removed prior to decontamination
for the process to be successful. Removal of materials from the facility may require additional
personnel and entries in PPE for material removal and treatment of removed material, adding to
the labor and waste management costs compared to what was done in this project.
4.1.2. Surface Decontamination Approach using pH-Adjusted Bleach
For the second round of Phase I, a surface decontamination process was utilized.
4.1.2.1. Decontamination Process
The EPA Region 10 START contractor was used to decontaminate the facility using pH-
adjusted bleach. This process was directed by EPA Region 1 and ten On-Scene Coordinators,
supported by technical experts from EPA's CMAT, formerly the National Decontamination Team
and EPA's National Homeland Security Research Center within the Office of Research and
Development. The process involved the removal of porous materials for subsequent treatment
with pH-adjusted bleach and disposal, followed by spraying all remaining surfaces in the facility
with a pH-adjusted bleach solution (amended bleach). During both the removal and spraying,
NAMs were used to assist in mitigating airborne Bg. This process and the subsequent results
are discussed in the following subsections.
4.1.2.1.1. Background and Purpose
In addition to the use of fumigation approaches for facilities contaminated with biological agents,
additional readily-available and approved methods are needed to improve the EPA's and the
Nation's preparedness for wide area remediation scenarios. Such available methods could
include a wide range of technologies (e.g., gases, liquids, foams, gels, ozone, etc.) and would
be effective against minimally contaminated to heavily contaminated surfaces.
Decontamination methods other than fumigation have been used previously and included
combinations of disposal of contaminated items, vacuuming, and the use of liquid sporicides
such as a pH-adjusted bleach solution. For example, a combined set of mechanical and
chemical procedures (vacuum, scrub/wash and pH-adjusted bleach) was used successfully in
the decontamination of a small wooden shed contaminated with "natural" B. anthracis spores
originating from animal hides during a drum-making process'11. Based upon these field results,
an effort was undertaken in the EPA laboratory to assess the effectiveness of specific process
steps'191. Understanding the effectiveness and assessing the capability on a field scale would
significantly increase EPA's readiness to respond to a wide area release.
4.1.2.1.2. Process Description
The procedures discussed in this chapter are considered the "low-tech" approach. The general
strategy of this approach was to bag and remove porous waste materials (i.e., ceiling tile,
242
-------
mattresses, couches, etc.) and then decontaminate the remaining items and surfaces by
spraying with liquid sporicidal chemicals (pH-adjusted bleach) using gas-powered chemical
sprayers. Following application of the sporicidal liquid, squeegees and wet-dry shop vacuums
were used to collect the excess liquids.
The US EPA Region 10 On-Scene Coordinator was the Incident Commander during the
decontamination procedures and directed the EPA START contractors to implement this pH-
adjusted bleach decontamination protocol in a manner consistent with what would be
undertaken during an actual "anthrax" removal action.
The facility, dissemination, sampling, and sample analysis will be discussed briefly here and are
discussed in more detail in Sections 2.1, 2.3, and 2.4, respectively.
4.1.2.1.3. Facility Contamination
On April 25th, 2011, Bg spores were disseminated as described in Section 2.3. The
dissemination team contaminated both floors of PBF-632 with Bg spores. A predetermined
amount of Bg was disseminated so that a high concentration (approximately 1E4 to 1E6
CFU/ft2) was dispersed and settled on the first floor, and a lower concentration (approximately
1E2 to 2E2 CFU/ft2) was dispersed and settled on the second floor. The amount of surface
loading was determined from pre-decontamination (characterization) samples collected April
26th and 27th before remediating the building.
4.1.2.1.4. Decontamination Personnel
The crew size was eight individuals consisting of six entry personnel and two support personnel.
The crew was part of the EPA Region 10 START. In addition, three support personnel (from
EPA's CMAT, the EPA Region 4 On-Scene Coordinator, the EPA Region 1 On-Scene
Coordinator) entered the facility and assisted in the decontamination. At a minimum, each crew
member had taken a site-specific training program ensuring that workers received the site-
specific hazard awareness training they needed to work safely at this site. Training was based
on the job hazard analysis in the Health and Safety Plan and other applicable standards. At a
minimum the crew members had initial 40-hr (OSHA) Hazardous Waste Operations and
Emergency Response (HAZWOPER) and current annual 8-hr refresher training. A minimum of
one team member had current first aid and cardiopulmonary resuscitation training. Each crew
member had a successful respiratory fit test in the model of mask worn.
4.1.2.1.5. Safety, Health, and Facility Preparation
On April 28 and 29, 2011, during facility preparation, workers wore Level C personal protection
which included a full-face air purifying respirator with HEPA filters, liquid chemical resistant
gloves, boots, and suit with hood (see Figure 4-17). The respirator, gloves and boots were each
taped to the suit using liquid chemical-resistant tape to form a complete barrier. Workers
entered from one end of the building and exited through a Decontamination Line, described in
Section 2.8, at the other end of the building.
243
-------
Figure 4-17. Photograph of decontamination personnel suited in Level C PPE.
A roll-off dumpster for the first floor wastes was positioned just outside the personnel
Decontamination Line from the building. All the solid waste exiting the first floor was passed
through the personnel Decontamination Line, identified as to its source and characteristics,
weighed, and then placed in a dumpster. For the second floor, a dumpster was located on the
ground just beyond the outdoor stairs at the building entrance so that waste could be ejected
from the second floor stair landing directly into the dumpster (see Figure 4-18). Second-floor
waste was not weighed and was assumed to be equal to the weighed first-floor waste. Waste
handling receptacles were positioned to minimize handling costs and to reduce ergonomic
stress.
244
-------
Figure 4-18. Photograph of dumpster located near the outside stairs, used for second
floor waste.
Electrical power to the facility outlets, HVAC and lights was turned off to reduce the risk of
electrical shock. The power was locked out according to IN Us safety procedure. Auxiliary low-
voltage lighting was placed in each room along with cameras and sampling equipment as
described in Section 2.1.
NAMs were used to control the flow of air in the facility to manage particulates (spores) that may
be reaerosolized during the process and to reduce chlorine gas concentration generated from
the pH-adjusted bleach spraying. Each NAM was equipped with HEPA filters (H1990
horsepower (hp) models, Novatek Novair 2000 and Abatement Technologies (Fort Erie, CA))
and each NAM had a low and high setting that corresponded to a nominal 1,000 and 2,000 CFM
air flow rate. The NAMs were turned on as soon as the characterization sampling was
completed and stayed on throughout the pH-adjusted bleach remediation process, including
drying. The NAMs were located outside the structure, including outside the secondary enclosure
structure, and a duct was attached from the NAMs to the secondary enclosure structure, then
from the secondary enclosure structure to the building using 12-in duct and duct fittings (see
Figure 4-19 and Figure 4-20). Flow was directed in one side of the building and out the opposite
side of the building for each floor. The layout of the NAM connections to the facility is shown in
Figure 4-21 and described in Table 4-4. For each floor and each side of the building, NAMs
were connected to the building through one window fitted with a plywood adaptor that replaced
the window pane. All air entering the building through the NAMs passed through HEPA filtration
to reduce transport of spores into the building, and all air exiting the building through the NAMs
passed through HEPA filtration to reduce transport of spores from the building.
Flow rate settings on the NAMs were adjusted to obtain relative negative pressure on the first
(high spore contamination) floor, as compared to the second (low spore concentration) floor,
245
-------
and both floors at negative pressure compared to the outside ambient pressure to reduce the
transport of spores from the high concentration areas. The first floor was set to 6,000 CFM (all
three NAMs on high) flowing out and 4,000 CFM (one NAM on high and two on low) flowing in.
The second floor was set to 6,000 CFM (all three NAMs on high) flowing out and 5,000 CFM
(two NAMs on high and one on low) flowing in (all flows are nominal). NAM air inlets on the first
floor were on the personnel entry side of the building. NAM air outlets on the first floor were on
the exit, Decontamination Line side of the building. Originally the second floor air flow was to be
in the same direction as the first floor air flow; however, for logistical reasons the dumpster was
located on the entry side of the building so the air flow on the second floor was oriented in the
same direction as the waste material flow, out the entry side of the building. NAM air inlets on
the second floor were on the personnel exit, Decontamination Line side of the building. NAM air
outlets on the second floor were on the entry side of the building. A total of 12 NAMs were used
during decontamination, and an additional two NAMs were available as backups. A total of four
NAMs were used during the drying phase, all pushing air flow into the facility. This orientation
during drying positively pressured the facility to minimize the potential for infiltration of any
environmental contamination into the building prior to post-decontamination (clearance)
sampling.
The NAM inlets on each floor all entered the building at one location. To distribute the inlet air
flow and accompanying pressure, one of the NAMs was allowed to flow into this room while the
other two NAM inlets were connected to 12-in ducts, one positioned approximately 1/3 of the
way to the other side of the building and the other approximately 2/3 of the way to the other side
of the building.
The final orientation of the NAMs during the drying phase is listed in Table 4-4, with
corresponding port locations shown in the schematic in Figure 4-21. Three of the NAMs used to
pull air during the decontamination phase were reversed to push air into the building for the
drying phase. Initially during the drying phase, three ports (LU1, LU2, RD3 and connecting
ducts) used for pulling air from the facility during the decontamination phase were then used to
push air into the facility during the drying phase. This change in airflow raised the concern that
viable spores might have been deposited in the duct during the decontamination phase and
subsequently re-contaminated the facility during drying. This orientation was maintained for
several minutes until the connections were revised to those shown in Table 4-4. After this
correction, only ducts that were previously used for air flow into the facility were used in the
drying phase to minimize the risk of recontamination of the facility. Wipe samples were taken
from the exhaust flow ducts (LU3 and RD3); both samples came back ND for viable spores.
These results and the low probability for deposited spores (if any) in the ducts to suddenly be
re-entrained lessened the concern about any potential cross-contamination that may have
occurred prior to going to the final NAM orientation used during the drying phase.
246
-------
Figure 4-19. Photographs of the NAMs during decontamination. Left photo shows the
entry side of the facility; right photo shows the far end of the facility (adjacent to the
Decontamination Line).
Figure 4-20. Photographs of the NAMs during drying. Left photo shows the entry side of
the facility; right photo shows the northeast side of the facility.)
247
-------
( LD1 H LD2 J( LD3 J
Entry Side
(Left Side)
Exit Side
(Right Side)
Figure 4-21. Schematic of NAM connections to the facility. Left drawing shows the entry
side of the facility; right drawing shows the exit side of the facility. See Table 4-4 for
legend.
Table 4-4. NAM ports and direction of flow during decontamination and drying phases.
Entry Side
Upstairs 1 (LU1)
Upstairs 2 (LU2)
Upstairs 3 (LU3)
Downstairs 1
(LD1)
Downstairs 2
(LD2)
Downstairs 3
(LD3)
During
Decontamination
Out
Out
Out
In
In
In
During
Drying
Not
Used
Not
Used
Not
Used
In
Not
Used
In
Exit Side
Upstairs 1 (RU1)
Upstairs 2 (RU2)
Upstairs 3 (RU3)
Downstairs 1
(RD1)
Downstairs 2
(RD2)
Downstairs 3
(RD3)
During
Decontamination
In
In
In
Out
Out
Out
During
Drying
In
Not Used
In
Not Used
Not Used
Not Used
248
-------
The pH-adjusted bleach solution consisted of 1 part bleach, 1 part white vinegar, and 8 parts
water. The solution was prepared in 50-gal batches and mixed, stored and used in polyethylene
55-gal drums (see Figure 4-22). The solution was monitored using pH paper (Whatman™, Type
CF - pH indicator paper, GE Healthcare, Waukesha, Wl) to maintain the pH between 6 and 7
(adding bleach or vinegar as needed to obtain desired pH). New batches were mixed as
needed. A record of when solutions were mixed was kept to ensure that batches were used
within three hours.
Bleach and vinegar were not combined directly together. Water was first added to the bleach
(Ultra Clorox® Germicidal, Item* 44600, purchased from The Home Depot, Idaho Falls, ID)
blend (2 parts water to 1 part bleach), then vinegar was added (Brand: Oasis Foods White
Vinegar, 4% acidity Bar Code: 7-3214611442-5, Item* FVW40HOA). Lastly, the remaining
water was added. The mixture was initially found to be too acidic so the formula was adjusted
slightly as follows: 5 gal bleach, 4.5 gal white vinegar, and 40.5 gal tap water to make a 50-gal
solution of pH-adjusted bleach.
Figure 4-22. Mixing of the pH-adjusted bleach solution in 55-gal drums.
Personal chlorine monitors (Dra'ger Pac-7000, Pittsburgh, PA) were used to evaluate worker
exposure. A monitor was used at the pH-adjusted bleach mixing area to spot test during mixing.
Monitors were also worn when workers used sprayers (backpack sprayers, 1 Lpm, Solo® 425
Piston Pump Sprayer, such as from Home Depot, Idaho Falls, ID; and gas powered sprayer,
Ultimate Washer, Inc., Jupiter, FL, Pro-Chem Sprayer, 300 psi, maximum 40 Lpm, 6.5-hp
Honda engine) (see Figure 4-23 and Figure 4-24) inside the building.
249
-------
Figure 4-23. Photograph of the Pro-Chem sprayer.
On initial building entry, outer booties were removed to reduce the potential for tracking
contamination into the building. A light spray of water with backpack sprayers was used around
entryways, doors and door frames. On initial entry into each room or hallway, a light spray of
water was also used. This light spray was used only to contain the spores in an attempt to
reduce their reaerosolization. The light spray of water was not used as a surface
decontamination process.
Upon initial building entry, a walkthrough of the rooms was conducted to ensure that the floor
plan was correct, furniture was located in the rooms roughly in the locations noted on the
diagrams and to make a video record of the inside of the building. Deviations from diagrams
were specifically noted as well as the initial condition of building materials or property. The
building and furniture were found as indicated in the diagrams and no adverse material or
building conditions were noted.
250
-------
Figure 4-24. Photographs of the backpack sprayer.
A waste staging area was set up on each floor to handle waste items removed from rooms on
that floor. To capture generated particles or chlorine that evolved from the pH-adjusted bleach
during the bagging and spraying of waste items, the waste staging area was located near the
NAMs outlet for that floor which was also adjacent to the dumpster exit. The waste staging area
was sprayed with pH-adjusted bleach using backpack sprayers in preparation for receiving and
processing waste items.
To help track time spent in each room and to help track waste handling, one team completed
the removal of items, books, papers, computers, printers, lamps, rugs, hand tools, etc., from one
room before moving to the next room. A separate team of two was in charge of removing ceiling
tiles. Radio frequency identification (RFID) chips (see Section 4.6.2 for more details) specific to
certain rooms on the first floor were inserted into each waste bag for waste weight tracking.
Items within cabinets and drawers were removed, bagged and handled just as the other items
within the room were handled. Upon entering a room, surfaces were lightly sprayed with water
using the backpack sprayer, including furniture and all items. After a light spray of all surfaces,
most items were bagged (some items needed to be cut into smaller sizes before bagging and
some large items were covered in plastic and taped closed) and moved to the waste staging
area (see Figure 4-25 as an example). Other nonporous items such as desks, book cases and
appliances were left in the room and decontaminated in place when the room was sprayed. At
the waste staging area, the bags were opened and items were sprayed using the chemical
sprayer, moved within the bags and sprayed to cover all exposed surfaces (see Figure 4-26 for
reference). However, the goal was to cover only the surfaces and not leave pools of liquid in the
bottoms of bags (the requirement to "not leave pools of liquid" in bags was instituted by INL so
that liquid waste limits were not exceeded and may or may not be required in an actual incident.
However, in an actual incident the volume of bleach sprayed into waste bags may be much
greater, as risks of reinfection will be weighted more heavily than liquid waste minimization).
The bags were closed, double-bagged, sprayed between bags before closing the outer bag, and
251
-------
finally the outside of the second bag was sprayed with pH-adjusted bleach before moving the
bag to the dumpster.
Figure 4-25. Photograph of decontamination personnel cutting items into smaller sizes to
fit into waste bags for removal prior to spraying the facility.
252
-------
Figure 4-26. Photograph of decontamination personnel placing porous items into waste
bags prior to spraying the facility.
4.1.2.1.6. Special Items
In addition to the other preparatory work, portable steps with wheels and hand rails were placed
on each floor to allow easy access for sealing HVAC supply registers and for ceiling tile
removal. Ceiling tiles from each room were removed, placed in bags, and moved to the waste
staging area. One exception: the men's restroom on the second floor was accessed through a
special door in the air lock at the top of the stairs. This room was originally overlooked and had
to be decontaminated, including the removal of ceiling tiles, at the very end of the
decontamination process. Similarly to the other items that were bagged, some space was left in
the ceiling tile bags so that tiles could be separated while they were being sprayed within the
bag during processing in the waste staging area. The bags of tiles were double-bagged,
sprayed between the inner and outer bag, outer bag sealed and the outside of the bag was
sprayed with pH-adjusted bleach before being moved out of the building to the dumpster.
All soft-surfaced items were removed, bagged and moved to the waste staging area. Waste
staging area personnel sprayed all surfaces of the items while in their bags, double-bagged,
sprayed between bags, sealed the outer bag and then sprayed the outside of the bag before
moving the bags to the roll-off dumpster.
A reciprocating saw was used to reduce the size of large soft-surfaced items for easier bagging
and handling. Fabrics on furniture were sprayed and bagged, moved to the waste staging area
and handled like other items. Sawdust and wood shavings were treated as soft-surfaced items.
Carpeting and other contaminated floor coverings (rugs) were sprayed to reduce
253
-------
reaerosolization, cut into three-ft sections, bagged, moved to the waste staging area where they
were sprayed again, double-bagged and disposed of in the roll-off dumpster. Bedding and other
fabrics not attached to furniture were treated as soft-surfaced items.
By the end of April 28, all ceiling tiles from both floors had been removed from the ceiling and
bagged (with the exception of the ceiling tiles on the second floor men's room), and
approximately 80% of the waste material from the second floor had been removed from the
building. Removal of waste material from the second floor was completed by mid-morning April
29, and then work began to remove waste materials from the bottom floor. Removal of waste
materials from the bottom floor was completed on April 30 at approximately 1015 hr. All waste
material removed from first floor was weighed, and the total amount of material removed from
the first floor was 3,550 Ib (see Section 4.5 for more information). Waste material from the
second floor was not weighed, with the assumption that the mass of waste from each floor
would be equivalent. Based on this assumption, the total weight of material removed prior to the
pH-adjusted bleach decontamination was ~ 7,100 Ib. The total volume of waste removed from
the building was estimated to be 68 cubic yards, based on having 3.25 dumpsters of material
(21 cubic yards per dumpster).
Power to the lights was turned off and lights were treated as hard-surfaced items. Attached light
fixtures were swung open as when bulbs are changed and sprayed with a10-min contact time
as part of the room decontamination (see Section 4.1.2.1.7). Hard surface furniture was
decontaminated in place similarly to the light fixtures. The exterior surfaces were sprayed with
pH-adjusted bleach and kept wet for a 10-min contact time. If there was too much furniture in a
room, some was removed to another area while decontaminating the room and the remaining
furniture. The moved furniture was decontaminated before it was moved back into that room.
Gross decontamination of dirt, grease and grime was not needed for this facility other than the
removal of wood shavings and saw dust.
On the morning of April 30th, the decontamination of the return air ducts was conducted using a
modified spray nozzle attached to the pH-adjusted bleach chemical sprayer line. This nozzle
was constructed of stainless steel and had a 0.048-in diameter orifice and a 90 degree spray
pattern in a hollow cone configuration (BETE® nozzle number L48, Greenfield, MA). To prevent
snagging and damage to the nozzle, it was suspended off the bottom of the duct by housing it
within a 6-in cage made of 1/4-in polyvinyl chloride pipe. The register duct openings were used
as sprayer entry ports for this process. A fifty-ft steel wire pulling tool was used to insert a nylon
cord the length of the duct through the duct register openings. On the second floor an additional
access point was cut into the duct using an 18-volt reciprocating saw (DeWalt DC385, DEWALT
Industrial Tool Co., Baltimore, MD) to aid in inserting the nylon cord in the duct (on the first floor
this procedure was not necessary). The nylon cord was tied to the nozzle cage and hand-pulled
through the duct while spraying pH-adjusted bleach inside the return duct. To insure a 10-min
wetted contact time, the duct was first sprayed while pulling in one direction and then sprayed a
second time after 5 min, pulling the nozzle in the opposite direction. A mist was observed
escaping from the ends of the duct and liquid was observed dripping from several seams in the
duct during the decontamination process.
254
-------
Supply registers were sealed because the supply side duct contained fiberboard insulation and
decontamination using pH-adjusted bleach was assumed to be problematic without first testing
in the laboratory. All supply side ducting to the rooms was notionally removed and treated as a
waste because the ducts were internally insulated. At the time of testing, a suitable method for
decontamination of such ducting had not yet been developed; removal of this ducting was
considered more cost-effective than attempting to decontaminate it in place with pH-adjusted
bleach. The supply registers remained sealed for the remainder of Round 2.
4.1.2.1.7. pH-Adjusted Bleach Building Decontamination Procedure
On the afternoon of April 30th, PPE was upgraded to Level B by replacing the Powered Air
Purifying Respirators with self-contained breathing apparatus and supplied airline respirators
(see Figure 4-27). Air was supplied to the air-line respirators by an EPA Region 10 Level A
support truck. The truck was outfitted with a cascade air system consisting of four 6,000 psi air
tanks connected in series. The system was designed to be able to support four personnel
working for eight hours, as well as refilling self-contained breathing apparatus tanks in
preparation for the expected high chlorine air concentrations during facility decontamination
(spraying with pH-adjusted bleach). All other PPE was kept the same.
On each floor, the pH-adjusted bleach decontamination process started on the side of the
building where the fresh air entered and moved from room to room until the last rooms were
sprayed with pH-adjusted bleach on the side of the building where the air exited the building.
The building HVAC system was off during the entire time of the pH-adjusted bleach spraying
process. The chemical sprayer was used for this task and all the spraying tasks that followed
from this point. The chemical sprayers produced 40 times more flow rate than the backpack
sprayers and increased productivity proportionally.
On this date, a very light spray of pH-adjusted bleach was used down the hall floor prior to
entering the rooms and then sprayed on surfaces on entering a room to keep reaerosolization to
a minimum. Upon entering a room, checks were made to ensure that the ceiling tiles had been
removed, the HVAC supply register had been sealed, all waste items had been removed, and
excess furniture had been removed before starting the decontamination of the room.
Many surfaces may have been sprayed during the entry into the room and when processing
items; however, this spray during room entry was not part of the building decontamination
process.
The goal of the decontamination process was to spray all surfaces (using the gas powered
chemical sprayer) with pH-adjusted bleach to achieve a 10-min contact time on all surfaces. The
flowrate from the Pro-Chem sprayer was approximately 3 gallons per minute (gpm). The
flowrate from the Solo backpack sprayer was approximately 1.3 Lpm. The initial plan was to
spray all surface areas completely (building structure ceiling, walls and floor, lights, and
remaining furniture including internal spaces such as desk drawers) in 5 min. Then those same
surfaces were to be re-sprayed, again taking approximately 5 min to complete the reapplication,
keeping the surfaces wetted for 10 continuous min. Each room was thoroughly wetted after just
one application and remained wetted for 10 min, so a second spraying was not administered in
255
-------
every room. (The time required to spray each room was typically 10-20 min). The spraying
process went room by room until every surface in the entire floor had received a 10-min wet pH-
adjusted bleach contact time. A sample photograph of the pH-adjusted bleach spraying using
the chemical sprayer can be seen in Figure 4-28.
Figure 4-27. Photograph of decontamination personnel ready to enter building with self-
contained breathing apparatus and supply air line respirators
256
-------
Figure 4-28. Photograph of pH-adjusted bleach spraying using the chemical sprayer.
For each floor, two personnel (equipped with supplied air line respirators) each operated a
separate hose from the chemical sprayer to apply the pH-adjusted bleach so that four hoses
were operated from the two chemical sprayers. There were instances where the pressure was
too low and adjustments had to be made to the pressure regulators of the sprayers. Each floor
also had a "leader" (equipped with self-contained breathing apparatus, which provided air for
about an hour) to assist with any issues.
When the spraying of the rooms was completed, the hallway and exit area used for removing
waste and moving hoses was sprayed with pH-adjusted bleach similarly to the rooms. The
building was left overnight in this wet condition. A total of 505 gal of pH-adjusted bleach was
sprayed that afternoon, and approximately 70 gal of prepared pH-adjusted bleach went unused.
Workers exited the building through the Decontamination Line as discussed in Section 2.8.
Several instances of skin irritation were reported by workers conducting the pH-adjusted bleach
spraying. The affected individual was immediately checked out by on-site paramedics, treated
and released from the aid station. There were no reports of inhalational exposures to chlorine,
but the skin irritation reports indicate that breaches in the PPE ensemble may have caused
exposure to liquid pH-adjusted bleach. Better quality taping of the ensemble, better selection of
suit size, the addition of a splash protection hood, or changes in the spraying process to reduce
the stress on ensemble seams may be indicated by these skin exposures.
At the end of April 30, one room (the men's bathroom) on the second floor, a room that could be
accessed only through the air lock system, had not been processed as the other rooms had
been processed. The ceiling tiles in this area had not been removed; there were no other "soft"
items in that room. Plans were adjusted to address decontaminating this room on the following
morning with several other final decontamination tasks. To save time and not delay the drying
257
-------
process, the decision was made to spray this room with ceiling tiles in place. In this room,
several ceiling tiles were removed so that the space above the drop ceiling could be sprayed
from below. In addition, this space was sprayed from the adjacent room.
The next morning, May 1st, the furnace room, men's bathroom, air lock, and space between the
building and secondary enclosure were sprayed with pH-adjusted bleach using the chemical
sprayer. A total of 65 gal of pH-adjusted bleach was sprayed that morning (with -35 gal left
unused). Following the spraying process, wet/dry shop vacuums fitted with HEPA filters were
used to pick up all standing water from floors and other horizontal surfaces. Areas inside
furniture, such as drawers and shelves, were emptied or wet-vacuumed as needed to remove
standing liquids. As needed, the wet vacuums were emptied into 55-gal lined drums. Waste
liquids inside the building were transferred to drums outside the building using a sump pump
(see Figure 4-29). A total of 105 gal of waste liquid was collected from the building (see Figure
4-30). In a response where clearance sampling is not required immediately following
decontamination, natural drying may be used and the need for and associated costs for wet
vacuuming, fans, heaters and additional ventilation may be modified.
Figure 4-29. Photograph of decontamination personnel operating the sump pump to transfer pH-
adjusted bleach runoff collected inside the facility to the exterior of the structure.
258
-------
f 1 I • /»
Figure 4-30. Photograph of the pH-adjusted bleach runoff collected by wet vacuuming
one day after the spray procedure.
The NAMs remained on during the decontamination and drying processes. During the drying
process, two NAMs on each floor were used to push air into the facility to maintain positive
pressure in the facility and promote air exchange (exfiltration) to enhance the drying process.
In addition to having the NAMs move air through the building, in the afternoon of May 1, 16
small household box fans and four Patron® 30,000 Btu/hr heaters (see Figure 4-31) were placed
in the building and turned on at 1730 hr. The box fans were distributed evenly, eight on each
floor to help circulate air in the rooms to help the drying process which was scheduled for three
days. All four heaters were placed on the first floor. Box fans and heaters were turned off May 4
at 1000 hr. Temperature and RH data were collected during the drying process and data are
plotted in Figure 4-32 though Figure 4-35. Ambient RH for May 1, 3, 4, and 5 was 48, 44, 26,
and 48%, respectively. From the RH data (Figure 4-34 and Figure 4-35) after the initial high
reading on the afternoon/evening of May 1, the RH data were evidently below the ambient RH
readings. These data showed the effectiveness of the drying process and higher temperatures
on indoor RH.
After drying and upon re-entry, the laminated and wood flooring demonstrated noticeable
swelling (sufficient to require replacement if it were in an actual residence). Bleach residue was
noticeable on horizontal surfaces but no appreciable damage was caused by the residue.
259
-------
Figure 4-31. Photograph of the four heaters used to heat the facility during the pH-
adjusted bleach post-decontamination drying phase.
260
-------
Figure 4-32. Average first floor temperatures recorded by 17 HOBO® data loggers
dispersed throughout the rooms during the post-decontamination drying phase of the
pH-adjusted bleach round. Facility drying began on May 1, 2011, with the activation of
internal heaters and circulation fans. Fans and heaters were deactivated on May 4, 2011.
Post-decontamination surface sampling commenced on May 5, 2011.
261
-------
Figure 4-33. Average second floor temperatures recorded by 18 HOBO® data loggers
dispersed throughout the rooms during the post-decontamination drying phase of the
pH-adjusted bleach round. Facility drying began on May 1, 2011, with the activation of
internal heaters and circulation fans. Fans and heaters were deactivated on May 4, 2011.
Post-decontamination surface sampling commenced on May 5, 2011.
262
-------
Figure 4-34. Average first floor RH recorded by 17 HOBO® data loggers dispersed
throughout the rooms during the post-decontamination drying phase of the pH-adjusted
bleach round. Facility drying began on May 1, 2011, with the activation of internal heaters
and circulation fans. Fans and heaters were deactivated on May 4, 2011. Post-
decontamination surface sampling commenced on May 5, 2011.
263
-------
Figure 4-35. Average second floor RH recorded by 18 HOBO® data loggers dispersed
throughout the rooms during the post-decontamination drying phase of the pH-adjusted
bleach round. Facility drying began on May 1, 2011, with the activation of internal heaters
and circulation fans. Fans and heaters were deactivated on May 4, 2011. Post-
decontamination surface sampling commenced on May 5, 2011.
4.1.2.1.8. pH-Adjusted Bleach Building Decontamination Procedure Logistics
After completion of the pH-adjusted bleach facility decontamination, several logistical
advantages and disadvantages of this procedure were apparent. This method requires no major
facility modifications prior to decontamination. No tenting or temperature/humidity control is
required during the procedure, although low temperature and high humidity may delay drying of
the facility and therefore its return to service. Bleach and vinegar are readily available from
multiple sources, so it is unlikely that decontaminant procurement would delay a response.
Sprayers, whether back-pack or electric/gas powered chemical sprayers, are also readily
available at typical local stores. In addition, highly specialized laborers are not required to
perform the procedure. However, HAZMAT-trained personnel with minimal on-site training may
be required due to the chlorine off-gassing. This procedure is labor-intensive and does require
numerous personnel. It is also difficult to ensure complete wetting of all surfaces in complex
264
-------
spaces, such as the space above the ceilings which contains a multitude of support beams,
electrical conduits, HVAC ductwork, and other structures. Person-to-person technique may vary,
resulting in differences in surface spray coverage. The spraying procedures required by the pH-
adjusted bleach method were physically taxing on personnel due to the strenuous activities
being performed in PPE. This strenuous activity places additional stress on PPE, particularly on
the seams where respirators, gloves, and boots were attached to the suit. Additional care in
taping PPE seams should be exercised and monitored. The need for Level B PPE to protect
from inhalation hazards associated with chlorine gas introduces additional logistical
requirements. In addition, some of the components of this procedure were difficult to complete.
For instance, some of the furniture was challenging to section with the reciprocating saw,
especially while in PPE. Springs within couches and chairs were difficult to cut as they moved
freely with the reciprocating blade, and the foam padding in these items frequently jammed the
teeth of the saw blades, rendering them ineffective. Bagging large items such as a mattress and
cubicle walls was also challenging. The procedure also required significant amounts of planning
and coordination prior to deployment. Numerous pieces of specialized equipment were required
for this procedure (i.e., compressed air for respirators, NAMs, gas-powered sprayers, backpack
sprayers, heaters, sump pump, large drums for mixing bleach, etc.). Overall, the procedure was
feasible and was administered in the allotted time.
4.1.2.2. pH-Adjusted Bleach Decontamination Results
Surface samples were collected prior to and following decontamination so that full-facility
surface inactivation efficacy could be evaluated. This section presents the results of the pH-
adjusted bleach decontamination evaluation.
4.1.2.2.1. Collection and Analysis Procedures for Surface Samples
Characterization sampling prior to decontamination included 312 surface samples (222 sponge-
sticks, 58 vacuum socks, 32 swabs) sent to eight LRN labs for processing and analysis. Surface
sampling following decontamination included 269 samples (253 sponge-sticks, 14 vacuum
socks, and two swabs). Of the pre-decontamination samples, 41 were field blanks and 271 were
test samples. Twenty-five of the post-decontamination samples were field blanks and 244 were
test samples. More information on the surface sampling and analysis methods can be found in
Section 2.5.3 (sampling methods), Section 2.9.1 (sample analysis), and Section 3.2.1 (surface
sampling results).
4.1.2.2.2. Sampling Results
Characterization (pre-decontamination) sampling was conducted April 26 and 27, 2011.
Decontamination of the facility was conducted on April 30 and May 1, 2011. Following a three-
day drying period, post-decontamination sampling commenced on May 5, 2011. Swabs,
sponge-sticks, and vacuum sock samples were collected from various surfaces and structures
not removed from the building during the decontamination procedures. Details of sample
locations are described in Section 3.2.1. This section describes the results of the pre- and post-
decontamination sampling that occurred during the pH-adjusted bleach decontamination.
265
-------
4.1.2.2.2.1. Field Blanks
Field blank samples were collected during each sampling campaign to determine the potential
for background contamination of sampling media. Contamination could occur during sample
handling in the field or in the laboratory during sample processing. Of the 66 field blank samples
collected during Round 2, two (one pre-decontamination and one post-decontamination)
samples resulted in detectable Bg. These results do demonstrate the ability of samples to
become contaminated with the test organism during sample handling or manipulation. Overall,
because a small portion (3%, this round) of the field blanks resulted in detectable Bg, erroneous
contamination is not expected to affect the interpretation of the test results negatively.
4.1.2.2.2.2. Pre-Decontamination Sampling
Of the 271 pre-decontamination test samples collected, nine were ND (no viable spores
recovered for either spread plate or filter plate analyses) (). One of these nine (Sample ID#
2830) was from the highly contaminated first floor (Room 107), while the remaining eight
samples were from the less contaminated second floor. The remaining 262 samples indicated
that the building contamination level varied widely as recoveries ranged from 1 CPU to "TNTC".
Overall, contamination levels were higher than the target levels of 1E2 CFU/ft2 on the second
floor, and consistent with the target of 1E6 CFU/ft2 on the first floor (Figure 4-36 and Figure
4-37). A more detailed description of the pre-decontamination sampling results is presented in
Section 3.2.1. The complete set of pre- and post-decontamination sampling data is presented in
Appendix G.
Table 4-5. Negative surface samples collected after spore dissemination, yet prior to
decontamination with pH-adjusted bleach.
Sample
ID#
1564
1644
2122
2224
2309
2588
2598
2830
2837
Floor
2
2
2
2
2
2
2
1
2
Room
208
212
Hallway
212
208
206
205
107
210
Sample Method
Swab
Swab
Sponge-Stick
Sponge-Stick
Sponge-Stick
Sponge-Stick
Sponge-Stick
Sponge-Stick
Sponge-Stick
Blank
No
No
No
No
No
No
No
No
No
Sampled
Surface
Monitor
Monitor
HVAC
Supply Vent
Wall
Wall
Table
Wall
Table
Wall
Spread
Plate
Result
(CPU)
ND
ND
ND
ND
ND
ND
ND
ND
ND
Filter
Plate
Result
(CPU)
ND
ND
ND
ND
ND
NA
NA
ND
NA
NA= sample was not analyzed via this analysis method.
ND = not detected.
266
-------
Concentration (Log Estimate)
Figure 4-36. Spatial distribution of first floor pre-decontamination characterization sample results.
267
-------
Concentration (Log Estimate)
0
Figure 4-37. Spatial distribution of second floor pre-decontamination characterization sample results.
268
-------
4.1.2.2.3. Decontamination Efficacy
Surface samples were collected prior to and following decontamination so that full-facility
surface inactivation efficacy could be evaluated. This section contains the results from the
decontamination of PBF-632 using a pH-adjusted bleach process.
4.1.2.2.4. Surface Decontamination Results
Of the 244 post-decontamination samples, only eight resulted in detectable Bg (viable spores
detected by either spread plate or filter plate analysis) (Table 4-6). All eight of these samples
were collected from floor surfaces; seven were collected by sponge-stick, one was collected by
vacuum sock. Seven of the eight samples with detectable Bg were collected from the second
floor, which received the lower initial spore contamination level (~1E2/ft2).
The results suggest that decontamination of surfaces with pH-adjusted bleach was highly
efficacious on the first floor and moderately efficacious on the second floor (despite the lower
spore load on the second floor, see below). Overall, only 3.2% of samples collected following
decontamination resulted in the recovery of viable spores. This percentage is comparable to the
field blank detection rate for Round 2 (two of 66 samples, 3.0%). Recovery of viable agent from
these eight samples was low, suggesting that, even in these areas with detectable Bg,
contamination was significantly reduced by the complete decontamination process.
Seven of the eight samples showing detectable Bg from non-blank samples were collected from
the second floor, which received the lower initial spore contamination level (1E2 to 2E2 per ft2).
Individual sampling results are shown in Figure 3-13 and Figure 3-14; spatial distribtions are
shown in Figure 4-38 and Figure 4-39. All eight post-decontamination samples showing
detectable Bg were collected from the floor, not surprising as horizontal surfaces were expected
to receive a higher contamination level than vertical surfaces, and floors constituted a majority
of the horizontal surface area sampled. The only sample from the first floor with detectable Bg
(#3085) collected after decontamination yielded 16 CFU from the spread plate method, yet zero
CFU from the filter plate method. This result is unexpected because the filter plate method
theoretically provides a lower limit of detection. We are unsure of the cause of this anomaly. The
locations of the eight post-decontamination second floor samples with detectable Bg appear to
be random. We were unable to attribute the cause of their location to proximity to in-room
supply ducts, to the amount of pH-adjusted bleach used on the second floor compared to the
amount used on the first floor, nor to proximity to exterior walls, electrical outlets, halls or
walkways, nor to furniture remaining in the rooms (Figure 4-38 and Figure 4-39). One
hypothesis for the second floor results is related to the men's restroom being decontaminated a
day after all the rest of the building was decontaminated. The original decontamination plan
called for starting the spraying at the fresh air end of the building (where the NAMs supplied
fresh air into the building) and decontaminating each room in sequence moving from the fresh
air inlet side of the building to the air outlet side so that any spores resuspended in the process
would travel to the exit side of the building, which had not yet been decontaminated. However,
the men's restroom on the second floor was on the air inlet side of the building. If any spores
were resuspended during the men's room decontamination, they would be pulled toward the air
exit and potentially re-contaminate the second floor in a random pattern.
269
-------
Table 4-6. Surface samples with detectable Bg collected following decontamination with
pH-adjusted bleach.
Sample
ID
3085
2751
2305
2529
3949
2733
2525
2611
Floor
1
2
2
2
2
2
2
2
Room
Men's
Bathroom
207
201A
206
212
203
209
210
Sample
Method
Sponge-
Stick
Sponge-
Stick
Sponge-
Stick
Sponge-
Stick
Vacuum
Sock
Sponge-
Stick
Sponge-
Stick
Sponge-
Stick
Blank
No
No
No
No
No
No
No
No
Sampled
Surface
Floor
Floor
Floor
Floor
Floor
Floor
Floor
Floor
Spread
Plate
Result
(CPU)
1.6E1
ND
ND
ND
ND
ND
ND
ND
Filter
Plate
Result
(CFU)
ND
6.0EO
3.0EO
3.0EO
3.0EO
3.0EO
2.0EO
2.0EO
ND = not detected.
270
-------
Concentration (Log Estimate)
Gray indicates areas with estimated zero CPU.
Figure 4-38. Spatial distribution of first floor post-decontamination in Round 2.
271
-------
Concentration (Log Estimate)
Gray indicates areas with estimated zero CPU.
Figure 4-39. Spatial distribution of second floor post-decontamination in Round 2.
272
-------
4.1.2.2.5. HVAC Decontamination Results
Fifteen of the 17 samples collected from the HVAC system surfaces prior to decontamination
resulted in detectable Bg (Table 4-7). Surface samples collected post-decontamination from
areas determined to be highly contaminated before decontamination (furnace filter and return
duct) suggested that no viable spores were recovered. These data suggest that the interior
decontamination procedure for the HVAC system was highly effective. Consistent with other
surface samples, no viable spores were recovered from any supply air vents at the termini of
each supply line. These surfaces were covered with plastic, and the exterior surfaces of this
plastic were decontaminated by the general spray procedure and not subjected to the targeted
HVAC system decontamination procedure.
Five post-decontamination samples were collected from HVAC components, three on the first
floor and two on the second floor. All five samples resulted in no viable spores recovered from
the HVAC surfaces (see Table 4-8). Post-decontamination sampling was not conducted inside
the HVAC supply duct. The supply duct was sealed prior to the pH-adjusted bleach
decontamination procedure. The removal and disposal of the supply duct was notionalized, so
the supply duct was not sampled following decontamination.
273
-------
Table 4-7. HVAC surface samples collected after dissemination, yet prior to
decontamination with pH-adjusted bleach.
Sample
ID#
1631
2378
2490
2668
3683
3946
1495
1526
1528
1553
1593
1599
1625
1645
1665
2270
2122
Floor
1
2
1
2
1
2
1
2
2
1
2
2
2
1
1
1
2
Room
Hallway near
Building Entry
Hallway near
Building Entry
Hallway near
Building Exit
Hallway near
Building Exit
Mechanical
Room
Mechanical
Room
Room 105
Room 210
Room 206
Room 110
Room 207
Room 21 2
Room 208
Room 106
Room 107
Corridor + Lobby
Hallway
Sample
Method
Swab
Sponge-Stick
Sponge-Stick
Sponge-Stick
Vacuum Sock
Vacuum Sock
Swab
Swab
Swab
Swab
Swab
Swab
Swab
Swab
Swab
Sponge-Stick
Sponge-Stick
Sampled
Surface
Inside Return
Duct
Inside Return
Duct
Inside Return
Duct
Inside Return
Duct
Furnace Filter
Furnace Filter
Supply Vent
Supply Vent
Supply Vent
Supply Vent
Supply Vent
Supply Vent
Supply Vent
Supply Vent
Supply Vent
Inside Return
Duct
Supply Vent
Spread Plate
Result (CFU)
4.0E4
4.0E3
3.2E5
1.3E4
7.8E6
3.2E4
4.4E4
1.9E3
NA
3.3E4
2.4E3
5.5E2
1.8E3
7.2E3
6.0E3
2.0E5
ND
Filter Plate
Result
(CFU)
TNTC
ND
NA
ND
TNTC
NA
TNTC
TNTC
NA
TNTC
TNTC
3.8E2
ND
TNTC
NA
NA
ND
NA= sample was not analyzed via this analysis method; Sample 1528 was not shipped for analysis (reason
unknown).
ND = not detected.
TNTC = too numerous to count.
274
-------
Table 4-8. HVAC surface samples collected following decontamination with pH-adjusted
bleach.
Sample ID
4032
3166
2732
2785
2666
Floor
1
1
1
2
2
Room
Mechanical
Room
Hallway near
Building Entry
Hallway near
Building Exit
Hallway near
Building Entry
Hallway near
Building Exit
Sample
Method
Vacuum
Sock
Sponge-
Stick
Sponge-
Stick
Sponge-
Stick
Sponge-
Stick
Sampled
Surface
Furnace
Filter
Inside
Return Duct
Inside
Return Duct
Inside
Return Duct
Inside
Return Duct
Spread
Plate Result
(CFU)
ND
ND
ND
ND
ND
Filter
Plate Result
(CFU)
ND
ND
ND
ND
ND
ND = not detected.
4.1.2.2.6. Data Limitations
Forty-eight samples resulting in less than 30 CFU during the spread plate procedures were not
subjected to the prescribed filter plating, a method with a lower limit of detection. While it is
unlikely that obtaining the filter plate data from these samples would greatly change the efficacy
results, more post-decontamination samples detectable for Bg may have been discovered if the
additional analysis had been conducted.
Consistent with the other decontamination rounds, sampling was not conducted in the space
above the drop ceiling before or after decontamination. Samples were collected from the top of
ceiling tiles prior to decontamination and on top of the light fixtures and drop ceiling support
frame after decontamination. None of the samples collected from the ceiling (support frame or
light fixture) resulted in detectable Bg following decontamination. The space above the drop
ceiling contains numerous wires, ducts, conduits, metal and wood framing, and other structures
that are difficult to wet completely by spraying, and therefore these structures may pose
challenges to liquid-based spray decontamination approaches.
In addition, four bags of the waste removed from the facility at the beginning of the
decontamination procedure were sampled by vacuum sock after being shipped to US EPA at
RTP, NC. Two replicate vacuum sock samples were collected from each bag. Samples
collected from two of the four bags (both replicates) indicated viable Bg remained within the
waste. In an actual incident, the volume of bleach sprayed into waste bags may have been
greater, as risks of re-infection would be weighted more heavily against liquid waste
minimization. Regardless, highly contaminated waste would have significant consequences with
regard to cost, logistics, and ease of waste disposal.
275
-------
4.1.2.3. Summary of the pH-adjusted Bleach Decontamination Process
Laboratory research has demonstrated the effectiveness of pH-adjusted bleach on a multitude
of materials. Commonly, however, pH-adjusted bleach demonstrates attenuated efficacy on
materials with a high organic content such as bare wood. During the current study, all porous
items were removed from the facility prior to the spray treatment. This approach reduced the
amount of difficult-to-decontaminate materials remaining in the facility during the spray
treatment procedures.
Overall, the results suggest that decontamination of surfaces with pH-adjusted bleach was
highly efficacious on the first floor and moderately efficacious on the second floor. Only eight of
244 post-decontamination samples resulted in detectable Bg and at very low surface loading
concentrations (requiring filter plating analysis for detection). HVAC return-side decontamination
procedures were also effective at removing contamination, as all HVAC samples post-
decontamination were ND.
With regards to logistics, the liquid-based decontamination procedure affords several
advantages over the fumigation methods, yet several disadvantages. Advantages of this
methods include: no major facility modifications (i.e., tenting) prior to decontamination, and thus
a remediation response can initiate rapidly following an incident; sprayers, bleach and vinegar
are readily available at retail stores; and highly specialized laborers are not required for this
procedure (although HAZMAT training is needed). Disadvantages of this method include the
fact that person-to-person technique may vary and result in differences in effectiveness; wetting
all surfaces thoroughly in a large facility is challenging; the procedure is physically demanding;
Level B PPE is required during spray procedures to protect workers from chlorine gas, removal
of disposable items is logistically challenging (especially for large items), time consuming, and
results in significant amounts of waste, and lastly, some specialized equipment (NAMs, heaters,
sump pumps, compressed air, supplied air respirators, etc.) was needed to complete the
procedure effectively. Overall, the procedure was feasible and was administered in the allotted
time.
4.1.3. Fumigation by Sabre Technical Services, LLC, with CIO2
The third and final round of Phase 1 utilized fumigation with CIO2 as the decontamination
method.
4.1.3.1. Process Description
Sabre Technical Services LLC ("Sabre") was selected to fumigate the facility with CIO2. The
same facility configuration as the previous two rounds was used (as described in Section 2.1)
and contained a mixture of porous and nonporous surfaces throughout the building. This
chapter describes the process that was used for Round 3.
As part of the process, Sabre worked with EPA and INL to determine the requirements of the
project, including a site visit by two Sabre engineers three weeks prior to the scheduled
fumigation. Sabre worked with BOTE Project management to define necessary project
resources and plan required fumigation resources. The goal was to fumigate the facility at a
CIO2 target concentration of 3,000 parts per million by volume (ppmv) for three hours resulting in
276
-------
a minimum CT of at least 9,000 ppmv-hr. After fumigation, the aeration phase would continue
until the concentration in the building was lower than the OSHA PE L of 0.1 ppmv. Once the
concentration in the facility was lower than this value, the facility would be turned back over to
the BOTE Project management team for post-decontamination (clearance) sampling.
Sabre utilizes a wet generation system by mixing water, sodium hypochlorite, hydrochloric acid,
and sodium chlorite to generate CIO2 in the liquid phase. A proprietary liquid-air stripper is then
used to transfer the CIO2 from the liquid phase to the vapor phase. The vapor phase CIO2 is
then blown into a facility using a 7,000 CFM fan. More information on the Sabre generation
process is described in US Patent* 7807101[84].
4.1.3.2. Facility Contamination
Bg spores were disseminated on May 10, 2011, following the procedure described in Section
2.3. The target surface loading of 1E4 to 1E6 CFU/ft2 was desired on the first floor and a surface
loading of 1E2 to 2E2 CFU/ft2 was desired on the second floor. The actual surface loading was
characterized by surface sampling as described in Section 2.5.4.
4.1.3.3. Planning and Design
The two-story fabricated steel building was covered with an external secondary enclosure, as in
the previous two rounds, to reduce wind shear on the building. The external secondary
enclosure allowed access to the building through a standard entryway as well as through a
rollup door. Both doors were located on the west side of the building. Egress was made through
the Decontamination Line door located on the north side of the building.
Sabre was provided three full days (May 13-15) to complete their process, which included
fumigating and aerating the facility prior to turning the facility back to the INL/EPA project team
by the end of the third day. This time did not include time to set up their equipment or to apply
an exterior tent to PBF-632. Sabre was allowed access to the exterior of the building for three
days prior to the scheduled fumigation to stage their equipment and to apply an exterior tent to
PBF-632. Sabre also elected to cover the entire building with a polyethylene tent system that
had direct contact with the building and was intended as primary containment for CIO2 (i.e.,
Sabre's tent was located between the building shell and the secondary enclosure). The primary
containment tent was installed on May 10-12, 2011, using five Sabre personnel and two
additional sub-contractors. This process required the use of a forklift and a scissor (manlift) lift to
position the materials on top of PBF-632. Personnel then draped the building and clipped the
seams using metal spring clips. A photo of the primary containment tent is shown in Figure
4-40. The secondary enclosure is shown draped over the lattice tubing.
Sabre's equipment and trucks were positioned on spill pads adjacent to the west end of the
PBF-632 (see Figure 4-41). The CIO2 generation system consisted of a 20 ft box truck with a
trailer that contained the air-liquid separation unit.
277
-------
Figure 4-40 Photo showing the inner tent membrane (primary tent) on the right side of
the manlift and the secondary tent over the metal lattice structure.
278
-------
Figure 4-41. Photo showing the location of Sabre's equipment.
Each decontamination vendor or lead in each round was given the opportunity to walk through
the facility to determine if any items in the facility would absorb or consume the fumigant or
hinder the decontamination process. Any items that were identified were then removed from the
facility before fumigation, to be treated by an alternative method. Sabre elected to have the
heavier foam items removed because the foam would absorb the CIO2 and extend the time
required for aeration. Had the total time allotted for fumigation and aeration been longer, these
items would have been left in the building. Because a total of three days was scheduled, the
following items were removed: two queen-size mattresses and the thicker foam cushions from
the couches and chairs. The total amount of material that was removed from the building
weighed 452 Ibs and took two personnel 30 min to remove. These items were notionally
decontaminated with liquid CIO2 and treated as waste.
Two 3/8-in gas sampling lines were installed on each floor at the locations shown in Figure 4-42
and Figure 4-43. The temperature and RH were monitored constantly using two HOBO® U12
data loggers (Onset Corp, Bourne, MA) that were positioned adjacent to the gas sampling lines.
The target temperature inside the facility was 65 °F or higher with a humidity of 65% or higher.
The EPA also collected temperature and RH data using HOBO® U10 data loggers positioned in
each room. The CIO2 concentration in the building was measured using a modified method SM-
4500-E[8Sl. EPA also measured the CIO2 concentration in the building using SensorWeb
(prototype) pods manufactured by SensorWare (Arcadia, CA).
4.1.3.4. Fumigation Conditions
As mentioned previously, the goal was to fumigate the facility at a CIO2 target concentration of
3,000 ppmv for three hours resulting in a minimum CT of at least 9,000 ppmv-hr at a minimum
temperature of 65 °F and RH of 65%.
279
-------
Sabre also began preparation of their generation system during the day on May 13 with the goal
of beginning the fumigation during the evening on that day. The facility was cleared by safety
personnel and locked out to prevent anyone from entering the building. Fumigation of the facility
began at 0000 hr on May 14. Once fumigation began, the number of personnel on site was
reduced to three Sabre personnel and three government employees to monitor the fumigation.
Fumigation continued until 0430 hr, at which time the process changed to the aeration phase.
During the fumigation monitoring was conducted around the perimeter of PBF-632
approximately 15 ft from the outer membrane. The frequency was initially 15-min intervals for
the first hour and then hourly thereafter to ensure that the concentration of chlorine dioxide
remained below the PEL of 0.1 ppm. There was one instance where the concentration
exceeded the PEL, and this exceedance was near the opening on the west side of the building
where the 36-in injection duct passed under the door. The height of the door was lowered and
the concentration returned to 0 ppm.
4.1.3.5. Scrubbing and Aeration
Once the desired concentration-time value of 9,000 ppmv-hr was achieved, the chlorine dioxide
generation process was terminated and the scrubbing phase began at 0430 hr. Caustic (sodium
hydroxide) was mixed into the sparger to neutralize the CIO2 in the liquid and to neutralize the
vapor-phase CIO2. At 0630 hr, the CIO2 concentration in the facility was below 12 ppmv on the
first floor and 30 ppmv on the second floor. At 1330 hr, HEPA-filtered air was pushed into the
facility and the gas from the building was pulled through a TIGG model N2500PDB activated
carbon scrubber (Oakdale, PA), which contained approximately 2,500 Ib of activated carbon
(TIGG, Oakdale, PA). The flow rate through the carbon was 3,000 CFM. This polishing step was
designed to reduce the CIO2 concentration in the building below the OSHA PEL of 0.1 ppmv.
The CIO2 concentration in the building had dropped below 0.1 ppmv at approximately 1330 hr
on May 14. However, the aeration continued until the following day when Sabre personnel and
the project safety officer verified that the concentration of CIO2 throughout the facility was below
the 0.1 ppmv level. The facility was cleared on the morning of May 15, and the facility was
turned over to the EPA at approximately 1000 hr.
4.1.3.6. Demobilization
Once the facility was turned over to the EPA on May 15, Sabre personnel remained on site on
the afternoon of May 15 and May 16 to break down their equipment, including removal of the
generation lines and moving their trucks and equipment away from the building. The inner
membrane tent was not removed from PBF-632 during this stage, but remained on the facility
throughout the remainder of this round.
280
-------
Sample
Line
Sensor FIRST FLOOR PLAN
(RH&T)
Figure 4-42. Schematic of first floor of PBF-632 showing location of fumigant sampling lines, fans, and sensors.
281
-------
J
Sample
Line
Fan
Sensor
(RH & T)
SECOND FLOOR PLAN
Figure 4-43. Schematic of second floor of PBF-632 showing location of fumigant sampling lines, fans, and sensors.
282
-------
4.1.3.7. Temperature and Relative Humidity
The heating system in PBF-632 was not functional, and the project required the use of
supplemental heating to raise the temperature in the facility to the desired temperature above
65 °F. Four (three bottom floor and one top floor) Patron® 30,000 Btu/hr heaters (Cheektowaga,
NY) were utilized to heat the facility in conjunction with operation of the blower on the building
HVAC systems (each floor) to distribute the heat. The heaters were activated on May 12 and
allowed to operate through 1600 hr on May 13. At this time, the heaters were turned off and
wrapped in plastic in preparation for the fumigation.
In addition to the two HOBO® data loggers that Sabre used, the EPA also collected temperature
and RH data in each room using HOBO® U10 data loggers. These data loggers recorded
temperature and humidity every two min. Once the post-decontamination (clearance) sampling
was completed, the data loggers were removed and the data were recovered.
The temperatures that Sabre recorded are shown in Table 4-9, and the average temperature for
each floor from EPA measurements is shown in Table 4-10. From these measurements, the
following time periods were used for the average measurements:
Preparation: May 13, 2300 - 2400;
Fumigation: May 14, 0000 - 0430;
Scrubbing: May 14, 0430 - 1330; and
Aeration: May 14, 1330 - 0945 (05/15).
Table 4-9. Average facility temperature measurements from Sabre.
Phase
Preparation
Fumigation
Scrubbing
Aeration
Room 101 A
Temp, °F
77.9
77.2
76.1
66.6
RH, %
73.5
75.3
81.0
67.3
Room 104
Temp, °F
78.6
77.3
76.0
67.1
RH, %
72.7
75.3
81.7
66.3
Room 201 A
Temp, °F
80.4
77.5
77.9
67.6
RH, %
64.4
70.3
75.8
68.8
Room 213
Temp, °F
85.2
82.5
80.4
72.4
RH, %
57.4
62.0
69.3
60.0
283
-------
Table 4-10. Average facility temperature measurements from EPA.
Phase
Preparation
Fumigation
Scrubbing
Aeration
First Floor
Temp, °F
77.1 ±1.1
76.2 ±1.2
75.0 ±2.0
66.8 ±1.1
RH, %
82.3 ± 3.7
79.6 ± 3.2
81 .8 ±3.4
59.9 ±4.2
Second Floor
Temp, °F
83.8 ±2.1
81.0 ±2.1
79.0 ±1.3
71 .4 ±1.4
RH, %
62.0 ±5.9
63.7 ±5.9
68.0 ±5.1
54.6 ± 3.6
4.1.3.8. CIO2 Measurements
Two types of measurements were performed to measure the concentration of CIO2 in the
building. Sensor Web pods and sensors were placed at eight different locations throughout the
test building. Four of eight pods and sensors were collocated with Sabre's gas sampling lines.
Each pod was paired with a specific sensor. This information is shown as pod number + sensor
number under the Sensor Web Measurements heading in
Table 4-11. The sampling locations are also described in
Table 4-11. The Sensor Web sensors and pods, hereafter referred to as pods, were set to
collect CIO2 concentration, RH, and temperature data every 30 sec. The pod from sampling
Location 1 malfunctioned and RH and temperature data were not collected. However, the CIO2
concentration was monitored and recorded. The pod in Location 4 malfunctioned and stopped
transmitting data; hence, no data were collected from Location 4.
284
-------
Table 4-11. CIO2 sampling locations.
Sampling
Location
1
2
3
4
5
6
7
8
Description
104 Office
101 A Office
First Floor Men's
Restroom
First Floor Hallway
21 3 Office
201 A Off ice
Second Floor Men's
Restroom
Second Floor
Hallway
SABRE
Sampling
Yes
Yes
No
No
Yes
Yes
No
No
Sensor Web
Measurements
P13 + S6
P12 + S9
P1 +S4
P11 +S8
P9 + S7
P4 + S3
P8 + S5
P3 + S1
Comments
Pod malfunctioned (No RH
and Temperature data)
Pod malfunctioned (No RH,
Temperature, and CIO2data)
Sensor Web pods and sensors were calibrated in the EPA's Research Triangle Park laboratory
after the field test. Eight of the pods and sensors were placed into an environmentally controlled
exposure chamber (55 in x 30 in x 48 in) to allow them to be calibrated with the modified SM
4500-E titration1851. The temperature and RH were controlled and monitored in the exposure
chamber throughout the calibration experiments using a HMP50 combination temperature/RH
probe (Vaisala, Woburn, MA), water-cooled fans, and a steam injection system. The chamber
temperature was maintained at 75 ± 4 °F and 75 ± 2 % RH throughout the calibration. After
placing the pods and sensors in the exposure chamber, various concentrations of CIO2 were
introduced. Target concentrations in the exposure chamber were achieved using a CIO2
generator from ClorDiSys Solutions, Inc. (Lebanon, NJ). The CIO2 generator maintained a
constant target CIO2 concentration in the exposure chamber and injected fumigant when the
concentration inside the chamber fell below a pre-set condition. Once the targeted concentration
was reached, the pods and sensors were allowed to equilibrate for 20 min. Three modified SM-
4500-E titrations were then pulled from the exposure chamber at each targeted concentration,
and the Sensor Web sensor data taken during the titration sampling period were averaged.
Sabre monitored the CIO2 level throughout the fumigation and scrubbing phases using modified
method SM-4500-E1851. This method is an amperometric titration to analyze chlorine, CIO2,
chlorite, and chlorate as a single value. A gas phase sample was collected in the 5% potassium
iodide buffered phosphate solution in an impinger at a flow rate of 1 L min"1. The phosphate
buffer solution (pH 7.2) was prepared with 25 g of potassium iodide and 500 mL of buffer
phosphate. After CIO2 gas was sampled, the buffer solution in the impinger was mixed with 150
mL of deionized water, and then 5 mL of a 6 N hydrochloric acid solution was added to the
solution. The solution was titrated with 0.1 N sodium thiosulfate. The titration volume was
285
-------
converted to calculate CIO2 concentration. The sampling duration varied from 1 min to 25 min,
depending on the previous CIO2 concentration level.
The CIO2 fumigation started at 2355 hr on May 13 and ended at 0430 hr on May 14. The CIO2
gas in the air was scrubbed from 0430 hr till 1330 hr on May 14th. The building was cleared after
1330 hr on May 14. The temperature and RH conditions during fumigation are shown in Figure
4-44 and Figure 4-45, respectively. Due to malfunctions of two pod systems, the figures show
the data from five sampling locations. The temperature was maintained between 75 to 85 °F
and an RH of 60 to 85% throughout the fumigation.
The CIO2 measurements were compared for two different methods (titration method and Sensor
Web sensors). The results are shown in Figure 4-46 through Figure 4-49 for Locations 1, 2, 5,
and 6, respectively, corresponding to the rooms listed in
Table 4-11. The figures contain the CIO2 concentration change as a function of time and also
CT (ppmv-hr) as a function of time. The results from both measurements showed that the
fumigation met an accumulated minimum 9,000 ppmv-hr CT clock value. The initial CIO2
concentration was well matched for both methods, but the sensor reading was always higher
than the titration method after one to two hours of CIO2 fumigation. The measurements from
sample Location 6 showed good agreement of both methods throughout the fumigation
compared to the other three sampling locations. In the field study setup, it is difficult to identify
why there is a measurement difference between the two methods; the difference between the
two methods needs further investigation.
Figure 4-50 shows the CT from all sampling locations monitored by Sensor Web sensors. The
results confirm that all seven locations met the accumulated minimum requirement of 9,000
ppm-hr CIO2 CT value.
286
-------
100
90
60
50
0
—104 office
—101A office
213 office
—2nd men WC
-201A office
—2nd floor CI02 inlet
4 6 8 10
Time of the Day (MDT)
12
14
Figure 4-44. Temperature profile during CIO2 fumigation on May 14, 2011.
287
-------
—104 office
-lOlAoffice
213 office
— 2nd men WC
201A office
—2nd floor CI02 inlet
0
6 8 10
Time of the Day (MDT)
12
14
Figure 4-45. RH profile during CIO2 fumigation on May 14, 2011.
288
-------
6000
5000
oUOOO
Ł 3000
01
u
O
~ 2000
1000
0
0
• Sabre CI02
—Sensor Web
« Sabre-ppmhr
—Sensor Web-ppmhr
4 6 8 10
Time of the Day (MOT)
12
14
25000
20000
15000
10000
5000
0.
a
Figure 4-46. CIO2 concentration (red) and CT (blue) profiles at sample Location 1: Sensor
Web (solid line) and titration by Sabre (markers).
289
-------
6000
5000
CL4QOO
-------
6000
• Sabre CI02
—Sensor Web
—Sensor Web-pprnhr
• Sabre-ppmhr
0
4 6 8 10
Time of the Day (MDT)
12
14
20000
18000
16000
14000
12000
10000 •=
u
8000 g
6000
4000
2000
Figure 4-48. CIO2 concentration (red) and CT (blue) profiles at sample Location 5: Sensor
Web (solid line) and titration by Sabre (markers).
291
-------
6000
5000
0
• Sabre CI02
—Sensor Web
—Sensor Web-ppmhr
• Sabre-ppmhr
4 6 8 10
Time of the Day (MDT)
12
14
16000
14000
12000
10000 -c
a.
8000 ~
b
6000 °
u
4000
2000
0
Figure 4-49. CIO2 concentration (red) and CT (blue) profiles at sample Location 6: Sensor
Web (solid line) and titration by Sabre (markers).
292
-------
30000
25000
~ 20000
—104 off ice
-101A off ice
-213 office
-2nd men WC
-201A office
-2nd floor CIO2 inlet
1st men WC
Q.
— 15000
-1
579
Time of the Day (MDT)
11
13
Figure 4-50. CIO2 CT profile at seven different locations measured by the Sensor Web
sensors.
4.1.3.9. CIO2 Decontamination Results
Surface samples were collected prior to and following decontamination so that full-facility
surface inactivation efficacy could be evaluated. Additional efficacy measurements included the
use of Bis. This section contains the results from the fumigation of PBF-632 with chlorine
dioxide.
4.1.3.9.1. Biological Indicator Results
Forty-five 1E6 B. atrophaeus on stainless steel Bis were set out by EPA on each floor prior to
fumigation. The Bis were obtained from Apex Laboratories (item number GRS-090; Lot #
G3190; Apex, NC). The Bis were recovered by EPA and were analyzed by the INL Microbiology
Laboratory.
The Bl locations are shown by the circles in Figure 4-51 and Figure 4-52. A solid black circle
shows a Bl that had no-growth after being exposed to CIO2, and the solid red circles show Bis
that showed growth following fumigation. For the Bis placed on the first floor, all of the Bis
except for one were inactivated by the fumigation. For the second floor, thirty-one of the forty-
five were not inactivated by the CIO2. The average temperature and RH during fumigation were
293
-------
81 °F and 61.7%, respectively. The only condition that differs between the first and second
floors is the slight drop in RH. Inactivation of Bacillus spores is significantly dependent upon RH.
As RH drops below 75%, the time or CT required for a six log inactivation (or log reduction)
(such as indicated by the Bis) increases drastically'861. This drop in RH is consistent with such
an impact, i.e., requiring greater than 9,000 ppmv-hr for complete inactivation of six-log Bis.
294
-------
X~T
A PI r^~~T
M«LV ;i
OFFICE
10S
IP
II
II
L
OFFICE
103
^
•B
i
1 FURNACE
1
1
1
OFFICE
1CZ
1
—U
Figure 4-51. Results for Bis on the first floor (black circles indicate Bis that were negative for growth; red circles indicate
Bis that were positive for growth).
295
-------
LJ
Figure 4-52. Results for Bis on the second floor (black circles indicate Bis that were negative for growth; red circles indicate
Bis that were positive for growth)
296
-------
4.1.3.9.2. Surface Sampling Results
Characterization sampling was conducted on May 11, 2011. Post-decontamination sampling
commenced on May 16-17, 2011. Swabs, sponge-stick wipes, and vacuum sock samples were
collected from various surfaces and structures not removed from the building during the
decontamination procedures. This section contains the results of the pre- and post-
decontamination sampling that occurred during Round 3, fumigation with CIO2.
4.1.3.9.2.1. Field Blanks
Field blank samples were collected during each sampling campaign to determine the potential
for background contamination of sampling media. Contamination could occur during sample
handling in the field or in the laboratory during sample processing. There were 85 field blanks
collected during the characterization sampling; three samples from the first floor resulted in
detectable Bg (with counts of 1.4E1, 4.1E1 and 1.7E4 CFU).
Of the 57 field blank samples collected during post-decontamination sampling for Round 3, all
57 samples came back ND for spores.
4.1.3.9.2.2. Pre-decontamination Sampling
Bg spores were disseminated on May 10, 2011, following the procedure described in Section
2.3. The target surface loading of 1E4 to 1E6 CFU/ft2 was desired on the first floor, and a
surface loading of 1E2 to 2E2 CFU/ft2 was desired on the second floor. The actual surface
loading was characterized by surface sampling as described in Section 2.5.4. Surface sampling
results are shown in Figure 4-53 and Figure 4-54.
Of the 399 pre-decontamination samples collected, a total of thirteen were ND (no viable spores
recovered). Ten of the 13 were from the less contaminated second floor. A more detailed
description of the pre-decontamination sampling results is presented in Section 3.2.1.4.1.
297
-------
Concentration (Log Estimate)
0
Figure 4-53. Spatial distribution of first floor pre-decontamination characterization sample results.
298
-------
Concentration (LogEstimate)
7
Figure 4-54. Spatial distribution of second floor pre-decontamination characterization sample results.
299
-------
4.1.3.9.2.3. Post-decontamination Sampling
A total of 344 surface samples were collected after fumigation with chlorine dioxide. Of the total
samples collected, only one sample, from Room 213, resulted in detectable Bg (17 CPU or 4
CFU/ft2) using spread plating. An additional five samples that had ND using spread plating had
detectable Bg with filter plating. The results for the post-decontamination sample in which Bg
was detected are shown in Table 4-12. The individual sample plots are shown in Figure 3-19
and Figure 3-20; the spatial distributions are shown in Figure 4-55 and Figure 4-56.
Table 4-12. Results from first and second floor following decontamination with CIO2.
Sample
ID#
3432
3619
3336
2758
4285
3357
Floor
1
1
1
2
2
2
Room
101A
105
110
207
213
Stairwell
Sample
Method
Sponge-Stick
Vacuum Sock
Sponge-Stick
Sponge-Stick
Vacuum Sock
Sponge-Stick
Sampled
Surface
File Cabinet
Floor-Carpet
File Cabinet
Wall
Bed
Floor-Smooth
Spread Plate
Result
(CFU/ft2)
ND
ND
ND
ND
4.0EO
ND
Filter Plate
Result
(CFU/ft2)
3.0EO
1.0EO
3.0EO
5.0EO
ND
9.0EO
ND=non-detect.
The discrepancy between the Bl inactivation and surface sampling results for the second floor is
noteworthy. Bis have been used as indicators of effectiveness in past fumigations'87'881.
However, laboratory-based fumigation studies suggest that Bis can severely underestimate the
effectiveness of decontamination for inactivation of spores in indoor environments'86'89'901. This
statement is true for similar challenge levels between the Bis and contaminated environmental
surfaces (e.g., each containing six-log of viable spores per swatch of material or Bl). This
difference (underestimation of the required CT for effective decontamination of most facility
surfaces) is likely because standard Bis (such as the Bis used in the BOTE Project) are typically
spores inoculated onto uniform materials with lower demand for the decontaminant (e.g.,
stainless steel). Spores deposited on typical environmental surfaces require a significantly
higher CT value to achieve the same degree of reduction in viable spore numbers'151.
The Bis used on both floors were at the six-log challenge level (containing ~1E6 spores per Bl).
The lower RH on the second floor presented conditions that were not suitable to achieve a six-
log reduction on the Bis at the CT value achieved. These results are consistent with laboratory
research'151. The Bg surface loadings on the second floor for Round 3 were, on average, 1.4E4
CFU/ft2. While the second floor fumigation conditions were not sufficient to achieve the six-log
reduction on the Bis required to achieve "no growth", the fumigation conditions were sufficient to
achieve the lesser log reduction required to reduce the Bg spores below detectable levels on
the facility surfaces.
300
-------
Concentration (Log Estimate)
Figure 4-55. Spatial distribution of first floor post-decontamination in Round 3.
301
-------
Concentration (Log Estimate)
Spatial distribution not able to be
calculated for gray areas.
Figure 4-56. Spatial distribution of second floor post-decontamination in Round 3.
302
-------
4.1.3.9.3. Material Effects
A post-test inspection of the building was completed after clearance sampling. Because this
building had been used in previous studies, it was difficult to assess whether any new oxidation
had occurred as a result of these tests. Materials that were placed in the building for these tests
were inspected for damage. The only objects that showed any damage were ring stand clamps
and quick connects on gas sample lines as shown in Figure 4-57. All other surfaces and
materials retained their original condition and color.
Figure 4-57. Photos of ring stand clamps and quick connects showing some signs of
oxidation.
4.1.3.10. Summary of Fumigation with CIO2
Fumigation of materials in the laboratory with CIO2 has shown the process to be efficacious for
inactivation of Bacillus spores. The Sabre process was shown to be able to effectively achieve
conditions necessary for spore inactivation throughout the facility. This process has the
advantage that it does not require the removal of any materials from the facility prior to
fumigation (although some materials were chosen to be removed to reduce the aeration time).
Overall, this process was successful in the decontamination of this facility. The efficacy results
from fumigation with CIO2 show that this process was effective for decontamination with only
one sample of 344 coming back positive with spread plating and an additional five with filter
plating. The Bis on the bottom floor were inactivated, with the exception of one that was located
inside a filing cabinet. Despite a 4 log reduction in detectable spores on the second floor, 31 of
303
-------
the 45 Bis came back positive. The RH in this case was insufficient to inactivate the 6 log Bis. In
future fumigations, the target RH should be raised to a minimum of 70% to ensure efficacy.
Specialized equipment and training is required to generate CIO2, and there are only a couple of
companies that have the equipment necessary to carry out the fumigation of a facility. Further,
only one company (Sabre) has shown the capability to generate conditions with a facility (CIO2
concentration, temperature, and RH) that are sufficient to inactivate Ba (and surrogate) spores
on relevant building materials (i.e., not just Bis). The procurement process time may be an
important factor when considering response and recovery preparedness, as well as incident-
specific options. This process used tarpaulins to contain the CIO2. The logistics of tenting larger
facilities would likely add additional time for facility preparation. There maybe large quantities of
chemicals that would need to be transported and stored on site; secondary containment
measures must be taken to prevent any spills. Any materials in the facility that are constructed
of mild (low carbon) steel would be expected to show signs of corrosion following exposure to
CIO2.
4.2. Decontamination Line Wash Water Treatment
Due to the small number of spores present in the personnel Decontamination Line wash water,
evaluation of the efficacy of the bleach treatment procedure for the collected waters was not
possible. The removal of outer gloves and booties before entering the washdown area may
have affected inactivation study results by reducing the number of spores contained in the wash
water. However, a greater than three log inactivation (i.e., log reduction) was achieved using the
proposed protocol when the wash water was spiked with the Bg spores.
Results from the spiked wash water were similar to the results obtained from laboratory
experiments using artificially generated wash water with similar water quality characteristics.
These findings suggest that the proposed inactivation procedure would be applicable for wash
water derived from similar PPE decontamination activities.
The physical and chemical characterization of the generated personnel Decontamination line
wash water provided valuable data regarding the water quality parameters representative of this
sample type. This information will be helpful in generating a typical wash water to be used in
laboratory experiments for evaluating various treatment procedures.
Use of the ultrafiltration concentrator allowed collection of concentrated samples. However, the
high turbidity of the wash water under the conditions experienced made operation of the
concentrator difficult due to filter clogging. For future wash water studies using the ultrafiltration
concentrator, improvements should be made so that turbid water is concentrated more
effectively.
4.3. Discussion of Rapid Viability-Polymerase Chain Reaction
Results
From the LLNL analysis, culture results for pre-decontamination samples typically showed 1E4-
1E5 CFU per sample; lower values (1E1 to 1E2 CFU) were occasionally observed, possibly
representing QC (field blanks) samples rather than actual surface samples, although the sample
304
-------
type was not made known. For MFP (or background) samples, five samples had 1E1 to 1E2
CPU per sample. For Round 1 post-decontamination samples, 11 samples showed 1E1 to 1E3
CPU per sample. Only one Round 2 post-decontamination sample showed CPU at 10 per
sample, whereas no Round 3 post-decontamination samples led to any CPU or culture-positives
by PCR analysis.
Overall, the RV-PCR method provided rapid results that were 97.6% consistent (209/214
samples) with results from culture analysis. The results are summarized in Table 4-13 including
calculation of false positive and false negative percentages for RV-PCR based on differences
with culture results. The overall false positive percentage for samples was 0.5% and overall
false negative percentage was 1.9%. As discussed above, each sample was split into two equal
parts (concentrated to the same extent), and most of the samples showing discrepancies in
results between methods represented samples with low spore levels that did not generate CPU
on culture plates. There were several samples that did not give any CPU on culture plates while
the RV-PCR gave positive results. Among those samples, there were 12 samples for which the
culture result was positive by real-time PCR analysis of the enrichment culture, and there were
13 samples for which the culture result was positive by real-time PCR analysis of the
concentrated enrichment culture. In a couple of cases, discrepancies were due to technical
issues while performing sample processing that later were addressed through protocol changes
to prevent issue reccurrence. These results indicated that the RV-PCR could be more sensitive
than the traditional plate culture methods, due mainly to the fact that RV-PCR allows the use of
the whole sample for the analysis.
In Table 4-13, data from Round 2 and 3 pre- and post-decontamination samples were
generated with some minor changes to the protocol including washing aliquots in buffer prior to
conducting DMA extraction (either by magnetic bead-based or heat lysis protocols), using cold
buffers and cold medium to prevent spore germination in TO aliquots and allowing coarser
particles to settle out prior to performing liquid transfers. While changes improved the data
quality, in some cases the additional handling steps added to the risk of cross-contamination
between samples that was manifested by some of the negative control samples showing
positive results for culture analysis (Table 4-13). To address the risk of additional handling
steps, a more frequent glove change procedure was instituted as well as changes in the final
protocol to reduce the number of steps involving mixing of sample contents and settling. The
mitigation measures appeared to be successful, given that contamination was not evident in the
subsequent sample processing efforts.
Overall, the high percent agreement is significant, given that the method had not previously
been tested with post-decontamination field samples containing relevant levels of debris. For
the post-decontamination samples, the percent agreement was slightly higher, 98% (156/159).
The BOTE Project samples contained a wide range of spore levels (< 1E1 to > 1E5
CPU/sample), with real-world debris loadings that were accurately detected by the RV-PCR
method. The agreement of the two methods is quite noteworthy given that two-thirds of the
samples contained low (<10 CPU/sample) to ND levels of viable spores from either background,
pre-release (MFP) sampling (8% of the total sample number) or from post-decontamination
sampling (74% of total sample number). Because the intended use of the method is for post-
305
-------
decontamination clearance analysis, the high accuracy observed with the gold-standard culture
method under relevant decontaminant scenarios provides a solid foundation for continued
optimization and application of the method for virulent Ba spores.
Furthermore, results from the EPA MLB Laboratory showed 86% agreement between culture
and RV-PCR analysis for 50 samples (see Table 4-14). The discrepancy in results was largely
attributed to limited practice with the RV-PCR protocol, and the BOTE Project was intended to
provide such an opportunity for the MLB Laboratory. Another likely contributing factor involved
inexperience and, thus, technical issues with the magnetic bead-based DMA extraction
procedure. Additional experience with the method and ongoing research will resolve the
problems with the interlaboratory reproducibility of performance of the DMA extraction and
purification protocol.
For low spore levels, more variability between culture and RV-PCR results was expected due to
factors such as spore clumping and pipetting variations. A hypothetical spore distribution in
different sample sub-sections is shown in Figure 4-58. In the BOTE Project protocol, the extract
was split so that -38% went to culture (13 mL), -38% went to RV-PCR (13 mL) and 24%
remained unprocessed or archived (8 mL). At TO, 1 mL was removed for processing, a volume
that represented 28% of the total volume in the filter cup.
Sample split
TO sample (1 ml)
Original wipe
sampletube
(archive)
B
Filter cup
for incubation
Combined spore
suspension
Plates
Filter Funnel
Enrichment culture
Culture
Centrifuge tube
Figure 4-58. Schematic diagram showing one possible distribution of spores during
sample processing of a wipe sample with a low spore level. The archive sample tube
represents spores remaining with the wipe sample after the two spore extraction steps
are completed. Spores associated with the wipe and in the remaining extraction buffer
are shown.
306
-------
Table 4-13. Summary of LLNL BOTE Project data by event type including blanks and controls.
BOTE
Project
Event
MFP
Round 1
pre-decon
Round 1
post-decon
Round 2
pre-decon
Round 2
post-decon
Round 3
pre-decon
Round 3
post-decon
Total
Total
Sample
No.1
17
2
44
20
47
16
68
214
Samples
RV-PCR
and
Culture
Pos.
14
1
30
13
3
11
0
72
RV-PCR
and
Culture
Neg.
3
0
11
6
44
5
68
137
RV-
PCR
Pos.
and
Culture
Neg.
0
0
1
0
0
0
0
1
RV-
PCR
Neg.
and
Culture
Pos.2
0
1
2
1
0
0
0
4
Total
Agreement3
(%)
17/17
(100%)
1/2
(50%)
41/44
(93.2%)
19/20
(95%)
47/47
(100%)
16/16
(100%)
68/68
(100%)
209/214
(97.6%)
False
Pos.3 (%)
0/17
(0%)
0/2
(0%)
1/44
(2.3%)
0/20
(0%)
0/47
(0%)
0/16
(0%)
0/68
(0%)
1/214
(0.5%)
False
Neg.3 (%)
0/17
(0%)
1/2
(50%)
2/44
(4.5%)
1/20
(5%)
0/47
(0%)
2/16
(0%)
0/68
(0%)
4/214
(1.9%)
True
Blanks
RV-
PCR
and
Culture
Neg.
3/3
0/0
5/5
2/2
3/3
2/2
4/4
20/20
Negative
Controls
RV-PCR
and
Culture
Neg.4
1/1
1/1
3/3
1/1
1/2
1/2
3/4
11/14
Positive
Controls
RV-PCR
and
Culture
Pos.
1/1
1/1
3/3
1/1
2/2
2/2
4/4
14/14
Total sample number includes surface and QC samples and does not include True Blanks or laboratory negative and positive controls.
2Summary of culture positive samples: One Pre-VHP RV-PCR sample leaked, therefore the result is not reliable—the protocol was modified to correct the issue for
additional sample analysis. Two Post-VHP samples, the culture portion required PCR analysis of the concentrated enrichment culture to obtain positive results;
One Pre-Bleach sample (the culture portion) showed no plate CPU and was positive by real-time PCR analysis of the enrichment culture only (RV-PCR showed T9
Ct values, but did not meet the criteria for a positive result).
3Note that each sample was divided into two equal parts for parallel RV-PCR and culture analyses. As a result, variability could have been observed for the
samples with low spore levels. Percentages were based on surface and QC samples and did not include True Blanks or laboratory negative and positive controls.
4For three of 14 negative controls, culture results were positive but RV-PCR results were negative, suggesting cross-contamination occurred for the culture portion.
Abbreviations: Pos, Positive; Neg, Negative; decon, decontamination.
307
-------
Table 4-14. Summary of EPA-OPP-MLB BOTE Project data by event type, including blanks and controls.
BOTE
Project
Event
MFP
Round 1
pre-decon
Round 1
post-decon
Round 2
pre-decon
Total
Total
Sample
No.1
2
16
23
9
50
Samples
RV-PCR
and
Culture
Pos.
0
12
1
5
18
RV-PCR
and
Culture
Neg.
1
3
19
2
25
RV-
PCR
Pos.
and
Culture
Neg.
1
0
0
0
1
RV-
PCR
Neg.
and
Culture
Pos.2
0
1
3
2
6
Total
Agreement3
(%)
1/2
(50%)
15/16
(93.8%)
20/23
(87%)
7/9
(77.8%)
43/50
(86%)
False
Pos.3 (%)
1/2
(50%)
0/16
(0%)
0/23
(0%)
0/9
(0%)
1/50
(2%)
False
Neg.3 (%)
0/2
(0%)
1/16
(6.2%)
3/23
(13%)
2/9
(22.2%)
6/50
(12%)
True
Blanks
RV-
PCR
and
Culture
Neg.
1/1
1/1
1/1
1/1
4/4
Negative
Controls
RV-PCR
and
Culture
Neg.4
1/1
1/1
1/1
1/1
4/4
Positive
Controls
RV-PCR
and
Culture
Pos.
1/1
1/1
1/1
1/1
4/4
Total sample number includes surface and QC samples and does not include True Blanks or laboratory negative and positive controls.
2Summary of culture positive samples: One Pre-VHP RV-PCR sample leaked, so the result is not reliable—the protocol was modified to correct the issue for
additional sample analysis. Two Post-VHP samples, the culture portion required PCR analysis of the concentrated enrichment culture to obtain positive results;
One Pre-Bleach sample, the culture portion showed no plate CPU and was positive by real-time PCR analysis of the enrichment culture only (RV-PCR showed T9
Ct values, but did not meet the criteria for a positive result).
3Note that each sample was divided into two equal parts for parallel RV-PCR and culture analyses. As a result, variability could have been observed for the
samples with low spore levels. Percentages were based on surface and QC samples and did not include True Blanks or laboratory negative and positive controls.
4For three of 14 negative controls, culture results were positive but RV-PCR results were negative, suggesting cross-contamination occurred for the culture portion.
Abbreviations: Pos, Positive; Neg, Negative; decon, decontamination.
308
-------
As mentioned, samples also often contained high debris levels that provided a challenge for
PCR-based analysis. However, RV-PCR protocols were shown to be robust for environmental
samples and, in most cases, the standard protocol gave consistent results with culture analysis.
Occasionally, additional sample dilution (1 to 20 rather than 1 to 10 dilution) was required to
obtain accurate RV-PCR results. In addition, the heat lysis DMA extraction protocol applied to a
few samples showed greater ACt values than the protocol based on magnetic bead-based DMA
extraction (see Table 3-29). Further optimization of the DMA extraction and purification portion
of the protocol is expected to address the observed PCR inhibition as well as the higher
apparent limit of detection for some post-decontamination samples. Protocol modifications
introduced after the Round 1 post-decontamination sample testing (used for subsequent Round
2 and 3 testing) also appeared to improve the magnetic bead-based method, but the protocol
modifications need to be evaluated systematically to ensure the accuracy of the RV-PCR
approach across all sample types and relevant decontamination scenarios.
The method was shown to work well for the surrogate B. atrophaeus (Bg) spores exposed to
decontaminants at real-world application levels and with wipe samples containing background
debris and indigenous microbial populations. The > 97% agreement between methods was
remarkable for a field test that included samples with low spore levels (at or below the detection
limit of the plating method) after treatment with fumigants and surface disinfectants. The T9
endpoint appeared to be sufficient to detect any spores that might have been delayed in
germination due to decontaminant exposure. Additional research has been planned for RV-PCR
analysis applied to B. anthracis spores exposed to decontaminants to confirm the robustness of
the method for post-decontamination scenarios.
4.4. Aggressive Air Sampling
In past decontamination activities for Ba, AAS augmented surface sampling for making
clearance decisions. In the BOTE Project, AAS was incorporated to assess the determination of
effectiveness using a decontamination method as compared to surface sampling. AAS used two
different air samplers for comparison, as no standard air sampling methodology currently exists
for use of AAS for Ba spores. The operation was conducted successfully for all three events and
sample results closely paralleled surface sample results (see Table 4-15). AAS after Round 1
(fumigation with VHP®) had the highest concentrations of spores detected in the air and surface
samples. AAS after Round 3 (fumigation with CIO2) had the lowest concentrations of spores
detected in the air and surface samples. Sampling after the CIO2 fumigation produced only one
sample (Room 105, XMX) resulting in detectable Bg, and the concentration was at the detection
limit. The XMX field blank for Room 105 also had one CPU. Notably, none of the surface
samples from Rooms 105 or 106 resulted in detectable Bg after fumigation with CIO2; likewise,
only one surface sample of a total of 273 surface samples resulted in detectable Bg anywhere in
facility.
During the first AAS sampling event in Round 1 (H+0), the concentrations achieved in Room
105 during the first two hours could have resulted in an exposure to over 90 Bg CPU to
unprotected individuals in the room during that time, based on breathing an average of 15 Lpm
(0.53 ft3/min) (90 CPU/ft3 x 0.53 ft3/min x 120 min).
309
-------
The first two AAS sampling events (after Round 1 [VHP®] and Round 2 [pH-adjusted bleach]
post-decontamination sampling) resulted in detectable Bg in the hallway where there were no
leaf blowing operations. Because the rooms were not under negative pressure, Bg spores could
have migrated outside the rooms into the hallway during the leaf blowing operations.
The XMX sample result concentrations were always higher than the corresponding STA
samples possibly due to the higher flow rates for the XMX samplers (530 Lpm) versus the STA
samplers (28.3 Lpm) causing higher XMX capture velocities coupled with the low concentrations
of Bg.
310
-------
Table 4-15. Summary of AAS and surface sampling results.
Round
1
1
1
2
2
2
3
3
3
Location
Room
105
Room
106
Hallway
Room
105
Room
106
Hallway
Room
105
Room
106
Hallway
Aggressive Air Sampling
Results (CFU/ft3)
XMX
H+0
1.4, 1.4
0.12,0.54
0.65
0.054, 0.054
ND, 0.037
0.054
0.018, ND
ND, ND
ND
H+60
0.24, 0.18
0.18,0.18
0.24
ND, ND
ND, 0.018
ND
ND, ND
ND, ND
ND
H+120
ND, 0.054
ND, ND
ND
ND, 0.018
0.018, ND
ND
ND, ND
ND, ND
ND
STA
H+0
ND
0.034
ND
ND
ND
ND
ND
ND
ND
H+60
ND
ND
-
ND
ND
ND
ND
ND
ND
H+120
ND
ND
-
ND
ND
ND
ND
ND
ND
Post-Decontamination
Surface Sampling Results
# Samples
with
Detectable
Sg/Total
Samples
7/10
7/13
0/14
0/9
0/8
0/10
1/10
0/12
0/13
Average
Concentration
(CFU/ft2) (SD)
3.8E2(1.1E3)
2.5E1 (5.5E1)
-
-
-
-
5.8E-2(1.8E-1)
-
-
311
-------
4.5. Assessment of Reaerosolization
4.5.1. Reaerosolization Assessment Discussion
The purpose of this analysis was to provide a preliminary view into the surface and airborne Bg
spore sample data and how reaerosolization may be characterized. Aerosolized Bg spores were
detected a day after dissemination, both before Stage 3 and after Stage 4 re-entry of personnel.
The mean Bg spore concentrations in air during Stages 3 and 4 (approximately 1.0E1 to 1.E2
CFU/ft3) are higher than background concentrations (Stage 1).
Baron et al.[911 reported on the development of a system for aerosol deposition of B. anthracis
and Bg spores (dried spores with a silica-based flow enhancer added) on surfaces and noted
that most spores settled within a few hours. More specifically, more than 99% of 1 to 2 urn
particles would settle within 10 hr[91]. With a dissemination of about 2.5E5 CFU/ft3, 2.5E4 CFU/ft3
would be expected to be left in the air after 10 hr. The residual aerosolized spores detected in
Stage 3 may reflect a continued settling of approximately 99% of the spores remaining at 10 hr
during the subsequent approximately 14 hr. However, the spores detected in Stage 3 could also
arise from settling and subsequent reaerosolization. Furthermore, the comparison between this
study and the Baron study might not be directly valid as the Baron et al. study used dried spores
whereas the Bg spores for the BOTE project were disseminated as a wet suspension. Which of
these alternative explanations (or a combination) is correct cannot be determined from the
collected data.
Stage 4 arithmetic mean Bg spore surface concentrations by room and round (see Table 3-42,
all surface and sample types) ranged from 1.3E5 CFU/ft2 (Round 2, Room 101A) to 6.6E5
CFU/ft2 (Round 1, Room 101A). As shown in Table 3-42, mean Bg spore surface concentrations
can vary considerably by surface and sample type. Certain materials (i.e., plastics) appear to
attract spores under certain conditions, hypothetically, when there is an electrostatic charge. A
relatively small portion of the disseminated Bg spores remained aerosolized or were
reaerosolized during Stage 4 (see Figure 4-59). The arithmetic mean Bg spore concentrations in
air ranged from 9 to 32 CFU/ft3 during Stage 4. Interestingly, during Stage 3 (an apparently
more quiescent period; collected approximately a day after Bg spore dissemination and before
surface sampling personnel entered), the arithmetic mean Bg spore concentrations in air ranged
from 4 to 72 CFU/ft3. Increasing air concentrations associated with increased activity (i.e.,
surface sampling) were apparent only during Round 1 as arithmetic mean spore concentration
in Stage 3 (4 to 8 CFU/ft3) increased in Stage 4 (20 to 32 CFU/ft3). However, no clear
relationship has been observed between spores recovered from surfaces and spores recovered
from air. The type/level of sampling activity during Round 1 may have been especially effective
at reaerosolization of the deposited spores. Variability among sampling teams may be a
contributing factor to the apparent differences observed with regard to reaerosolization.
Disseminated spores may remain airborne during the later stages, complicating the
interpretation of potential reaerosolization. In addition, the indications of reaerosolization are
rather limited temporally, and the extent and length of time that reaerosolization would continue
at this level is uncertain. Spore loadings and reaerosolization levels could change via natural
spore migration outside the building'501 or possible enhanced binding with surface materials'921.
Extrapolation of these settling/reaerosolization characteristics beyond the sampling time frame
312
-------
would be rather uncertain. Likewise, application of these settling/reaerosolization trends to
different sized buildings, with different materials and disturbance activities/levels, would be
uncertain as well.
dft
3R
~ on
4^ 3W
2
7T
F >•;
i •
I ?n
5 zu '
1
515
.
.n
$
E
_E
'= 30
<" <
. * ^ , *
""* "
—
0 500,000 1,000,000 1,500,000 2,000,000 2,500,000 3,000,000
Arithmetic Mean Wipe Measurement (CPU/ft*)
* Round 1, Room 101A * Round 1, Room 102 • Round 2, Room 101A
• Round 2, Room 102 • Round 3, Room 101A • Round 3, Room 102
Figure 4-59. Arithmetic means of air versus surface sample measurements from Stage 4
(different points correspond to different surfaces, surface types, and sample types).
Some spores may not have settled or may have reaerosolized during Stage 5 of Round 1 (post-
decontamination with VHP®). Arithmetic mean spore concentrations in air were 4 CFU/ft3 in
Room 101A and 2 CFU/ft3 in Room 102. These airborne concentrations were detected even
with very low concentrations of Bg spores detected on surfaces. All Stage 5 Round 1 surface
samples based on spread plate analyses were NDs (i.e., mean spread plate counts were <30
CFU), but there were detects based on the filter plate results (3 CFU/ft2 in Room 101A and 57
CFU/ft2 in Room 102).
313
-------
4.5.2. Reaerosolization Assessment Summary
Air and surface samples were collected from Rooms 101A and 102 during the BOTE Project to
better characterize the potential for reaerosolization of spores released in a real-world indoor
environment. Airborne Bg spores were detected the day after dissemination and, in some
cases, even after decontamination of the rooms.
Airborne Bg spores appeared to reaerosolize (or otherwise remain aerosolized for a period of
time) and mix readily throughout the rooms. There were no significant differences in Bg spore
concentrations associated with sampling heights or locations within the rooms.
Residual aerosolized or reaerosolized Bg spore concentrations were a fraction of the Bg spore
concentrations measured in air during active dissemination. Arithmetic mean Bg airborne spore
concentrations exceeded 2.0E5 CFU/ft3 during spore dissemination, but fell below 1.0E2
CFU/ft3 during the post-dissemination stages (after one day of settling).
Aerosolized spores were observed at both high (pre-decontamination) and low (post-
decontamination) levels of Bg spore surface contamination. The proportion of Bg spores that
apparently reaerosolize from surfaces appears to be lower at high levels of contamination than
at low levels of surface contamination. For example, during Stage 4 (all rounds), the arithmetic
mean Bg spore surface loadings (by room and round for all surfaces and sample types
combined) were >1.0E5 CFU/ft2, and the arithmetic mean air concentrations were <31 CFU/ft3.
During Stage 5 (Round 1), the arithmetic mean Bg spore concentration on surfaces was 57
CFU/ft2 in Room 102, while the arithmetic mean air concentration was 2 CFU/ft3. These results
can not rule out that residual aerosolized and viable spores also contribute to the air sampling
data.
As noted in the introduction, estimating airborne spore concentrations from the reaerosolization
of deposited spores is probably influenced by many factors including residual aerosolized
spores, surface loading, surface material type, activity levels, etc. Consideration of settling and
reaerosolization of spores should be included as part of future decontamination studies and
activities.
314
-------
4.6. Waste Management
The proper management of waste from the cleanup after a biological event is a key element of
the remediation process. Different decontamination strategies result in different waste quantities
and characteristics, and the management of those wastes can significantly affect the overall
remediation timeline, resource requirements, and costs.
One of the waste management challenges that the BOTE Project presented was that there was
a need to address waste issues (costs, quantities, logistics, etc.) for the project as if the waste
that was generated had been contaminated with Ba, even though the waste that was generated
was not contaminated with Ba ("notional" waste). In addition, as a constraint of the tests, the
waste that was generated needed to be handled in accordance with INL waste management
practices ("real" waste). This effort manifested itself mainly as a need to keep any biohazard-
labeled bags (supplied to the sampling teams in the sampling kits) out of the trash. Putting
biohazard bags into the trash would not be consistent with waste handling procedures in a real
Ba incident. The waste management practices from a real Ba contamination incident would be
determined by the State in which the incident occurred, and how that State characterizes the
waste. The disposal of the waste (and criteria for defining/classifying the waste) must also be
considered prior to selecting the decontamination technology. Ease of waste management for a
given technology must be balanced with factors of time, availability, and cost (e.g., if there is
only one suitable fumigation contractor and they are unavailable for six months but the facility
needs to be cleaned in one month, a different technology would need to be selected).
4.6.1. General Waste Management Approach
To estimate the cost of and issues related to management of the different "notional" waste
streams generated during the BOTE Project, while still appropriately addressing INL
requirements for the "real" waste streams, the waste management concept shown in Figure
4-60 was developed. "Initial Disposition" refers to what happened to the "notional" waste
immediately upon generation at the site. "Measurements" refers to the different measurements
(e.g., weight, point of origin) that were taken on the "notional" waste after its initial disposition.
"Temporary Management" refers to the near-term management of the "real" waste such as
temporary storage, mingling with or separation from other waste streams, etc. "Final
Disposition" refers to the waste management activities related to introducing the "real" waste
into INL's waste management processes. In other words, the waste was treated as Ba-
contaminated waste up until the point at which the waste entered its temporary waste
management stage in preparation for final disposition in the INL waste management process.
315
-------
Waste Stream
Waste is treated as "notional"
Initial Disposition
Measurements
Temporary
Management
Temporary
Management
Waste is treated as "real"
Figure 4-60. Waste management concept.
4.6.2. On-Site Activities
The operational implementation of the above waste management concept was put into practice
at the BOTE Project for all three rounds of Phase 1 testing.
Waste that originated in the Sampling Prep Trailer was periodically brought to the Sampling and
Decontamination Support Trailer in bags and weighed on a platform scale (My Weigh, Inc.,
Phoenix, AZ, PN VHD). Weights were recorded either directly into a laboratory notebook or into
an iPad device and transferred to the laboratory notebook as a backup. (More information on
the use of the iPad can be found in Appendix M.) Once weighed, the waste was transferred into
a nearby dumpster for eventual processing through the INL waste management process.
Solid waste that originated in the personnel Decontamination Line was bagged by the
Decontamination Line operations personnel and placed outside the personnel Decontamination
Line structure. The bags were weighed on the platform scale, the weights and point of origin
logged, and then the bags were put into the dumpster. Liquid waste that was collected from the
personnel Decontamination Line was accumulated in a 55 gal drum; when the drum was full, the
quantity was logged and the drum was replaced with an empty drum.
For the Round 2 (pH-adjusted bleach) decontamination, a significant amount of material was
bagged and removed from the building prior to the bleach spraying process. Tracking the point
of origin of the solid waste generated during the pH-adjusted bleach decontamination process to
the individual room that the materials came from was desirable. To implement the tracking
process, RFID chips, each with a unique serial number (Metalcraft, Inc., Mason City, IA, PN
40375-0001) were placed into the bags prior to closing. Bags were accumulated on a room-by-
316
-------
room basis so that their originating location could be tracked. The bags were then brought to a
staging area (one on each floor of the building), opened, and sprayed with pH-adjusted bleach,
then closed and double-bagged. Due to the contaminant control procedures put in place to
minimize cross-contamination of the less-contaminated parts of the building (Floor 2) and the
more contaminated parts of the building (Floor 1), the waste bags were directly thrown into a
dumpster from the second floor and not weighed. Waste from the second floor was assumed to
be equal to that from the first floor. All bags from the first floor were brought outside the
personnel Decontamination Line, the bags were weighed, waved in front of the RFID reader
device (Motorola, Inc., Libertyville, IL, PN RD5000); the room of origin was logged; then the
bags were placed in a dumpster separate from the waste generated at the personnel
Decontamination Line.
The soapy water generated in the personnel Decontamination Line was kept separate from the
wastewater that contained bleach from the pH-adjusted bleach decontamination process.
4.6.3. Waste Categorization
For the purposes of separating the waste streams into potential pathways for waste treatment
and disposal, the various "real" waste streams were categorized according to the categories
listed in Table 4-16, based on whether they had ever been contaminated by the Bg, whether
they were liquid or solid, and whether they had been subjected to the decontamination
processes from the various rounds. In addition, categories were created for waste generated as
a result of decontamination operations, such as PPE waste from the Decontamination Line, or
liquid bleach residues vacuumed up from the floor following the pH-adjusted bleach
decontamination process from Round 2. In a real biological contamination incident, once a
stream has been determined to be a waste, the next step is to characterize the waste in
accordance with the procedures established by the State in which the waste was generated.
These procedures are especially important for biological agents because biological agents are
not addressed as part of the Federal Hazardous Waste Framework within the RCRA[9Sl. As a
result, individual State waste management officials must be consulted regarding how these
agents are characterized/classified within their respective states. In addition, while the State has
a lot of say in the categorization of the waste, it is the owner/operators of waste management
facilities that ultimately have to accept the waste. A combination of the identification of state
waste management requirements of these waste streams and the waste acceptance criteria and
the willingness of owner/operators of waste management facilities to accept the waste
determines the final disposition of these waste streams. Pre-incident waste management
planning is therefore a real need, so that these issues can be identified and resolved prior to an
incident. In addition, the comparison of a single building approach does not address the
overwhelming complexities associated with a wide area anthrax release. Facilities outside the
State of the release may also be needed to support the response, therefore it becomes a more
accurate statement to discuss waste management in terms of the acceptance of the State and
owner/operators regardless of where the release occurs. The complexity of multi-state response
also further supports the need for pre-incident waste management planning.
317
-------
Table 4-16. Waste categories.
Number
Name
Description
1S Un contaminated
Solids
Solid materials that have never been contaminated. An example is the
PPE packaging waste from the Sampling Prep Trailer.
2S
Contaminated
Solids
Solid materials that are contaminated and have never been
decontaminated. None of this type of waste was generated during the
BOTE Project Phase 1.
3S Decontaminated
Solids
Solid materials that were once contaminated but have undergone
decontamination processes. There may be residual contamination, but
sampling and analysis would be required to determine whether residual
contamination still exists. A decision would still have to be rendered
because this material constitutes a waste. The waste would have to be
characterized to determine how it will be handled, labeled, transported,
treated and/or disposed of. An example is the material removed prior to
the Amended Bleach decontamination that was sprayed with bleach prior
to packaging.
4S Decontamination
Solid Waste
Solid materials that were generated through the decontamination process.
This waste would still require a waste characterization prior to disposal. An
example is the used PPE that came from the Decontamination Line, or
fans used in the VHP® fumigation process.
1L
Uncontaminated
Liquids
Liquids that have never been contaminated. None of this type of waste
was generated during the BOTE Project Phase 1 although some was
generated during the dissemination activities of Round 1 that involved
collecting water during the timeframe prior to the building being
contaminated.
2L
Contaminated
Liquids
Liquids that are contaminated and have never been decontaminated.
None of this type of waste was generated during the BOTE Project Phase
1.
3L
Decontaminated
(treated) Liquids
Liquids that were once contaminated but have undergone the treatment
processes. Residual contamination may exist, but sampling and analysis
would be required to determine whether residual contamination still exists.
A decision would still have to be rendered because this material
constitutes a waste. The waste would have to be characterized to
determine how it will be handled, labeled, transported, treated and/or
disposed of. The waste that fell into category 3L was the liquids generated
during the drying operation after the Amended Bleach decontamination.
4L
Decontamination
Liquid Waste
Liquids that were generated through the decontamination process. An
example is the recovered rinsate from the Decontamination Line or the
spent bleach solution vacuumed from the floor during the amended bleach
decontamination.
318
-------
Table 4-17 lists the various waste streams and describes the initial disposition, measurements,
frequency of measurements, temporary management/storage, and final disposition of the waste
materials. The final disposition of this waste is based on INL waste management procedures
with State input; however, if this were an actual incident involving a release of Ba, waste
management procedures would be developed in accordance with State requirements with
collaborative technical support from appropriate Federal agencies as requested.
Table 4-17. BOTE Project waste management measurements and frequency.
Waste Stream
PPE and Other
Solid
Decontamination
and Sampling
Residues
Personnel
Decontamination
Rinsate and
Collected Liquid
Residues from
Amended Bleach
Decontamination
Items Removed
Prior To/During
Amended Bleach
Decontamination
Sampling Waste
from Donning
Trailer
Waste
Category
(see Table
4-16)
2S, 3S, or
4S
2L, 3L, or4L
3S
1S
Initial Disposition
Collect in bag at end of
Decontamination Line
Collect in barrel at end of
DecontaminationLline;
soapy water from
Decontamination Line and
bleach from
Decontamination Line
were collected separately;
liquid residues from
amended bleach
decontamination was
collected separately
Package prior to removal
(Bag, Add RFID Tag, Move
to Staging Area Inside
Bldg., Add Bleach, Close
Bag, Double Bag); place
outside Decontamination
Line
Place in plastic bag
Measurements
Item Description
Weight
Volume
Item Description
Item Weight
Item Room
Description
Weight
Frequency
As bags were
filled,
Decontamination
Line Ops
personnel put bag
outside
Decontamination
Line
When barrel is full
As bags were
filled
As bags were
filled
ASSUMPTIONS:
No residues are either listed or characteristically hazardous per RCRA. However, if the residues are either listed or
characteristically hazardous, then disposal must be in accordance with RCRA Subtitle C requirements defined by the
State and the waste acceptance criteria of the waste management facility where the waste will be managed. If the
waste management facility is not in the same State where the incident occurred, then both State solid waste
regulatory offices must be consulted. Any pathogenic agent has been "rendered non-viable" through autoclaving or
similar process, and no free liquids are present. Liquid waste likewise is "rendered non-viable" and meets PLN-8104.
Most of the liquid (assuming non-RCRA), if not all, in the past went to the sanitary waste system via a pumper truck.
Any additional solid waste regulations of the State have been followed.
319
-------
4.6.4. Results
The waste generation data are subject to the caveat that, to the extent possible, generated
waste was assigned to the activities that resulted in the generation of that particular batch of
waste. However, due to the duration of some of the workdays during the BOTE Project Phase 1
testing, some activities were deferred until the following day, and this deferral may have resulted
in some waste being assigned to the activities associated with the following day. These minor
discrepancies may change the activity that the waste was assigned to but would have a
negligible effect on the overall cost analysis associated with the waste management.
Due to logistical issues with handling the removed materials that were collected from the second
floor of the building during Round 2 (pH-adjusted bleach decontamination process) while
maintaining the contaminant control procedures, the weights of the removed materials from the
second floor were not measured directly. Rather, the quantities of materials removed from the
first floor were doubled. In addition, although the laminate floors were not removed prior to the
pH-adjusted bleach decontamination process, the laminated floors were severely warped by the
pH-adjusted bleach spray and, in a normal decontamination situation, would have required
replacement. Also, the supply-side HVAC ductwork would have been removed as part of the
pH-adjusted bleach decontamination process and later replaced (see Section 4.1.2.1.6). The
laminate floors and HVAC duct were therefore notionally added to the waste stream for the pH-
adjusted bleach decontamination process, as well as to the refit cost in the cost analysis. These
issues all combined to result in the pH-adjusted bleach decontamination process generating
significant quantities of waste relative to the fumigation rounds. Table 4-18 lists the amount of
solid and liquid waste generated during each test activity, categorized as per Table 4-16. Note
that waste related to dissemination of the spores represents a total for all three rounds.
320
-------
Table 4-18. Waste data from BOTE Project Phase 1.
Ł
>
I
Category 1S
Uncontaminated Solid
(Ib)
Category 3S
Decontaminated Solid
(Ib)
Category 4S
Decontamination Waste
Solid (Ib)
3"
1
1
;g
"o
w
15
1
Category 1 L
Uncontaminated Liquid
(gai)
Category 3L
Decontaminated Liquid
(gai)
Category 4L
Decontamination Waste
Liquid (gal)
03
U)
1
1
;o
'D
o-
_i
15
1
Breakdown by Detailed Activity
Dissemination
VHP® - Characterization
Sampling
VHP®- Decontamination
VHP® - Clearance Sampling
AB- Characterization
Sampling
AB Decontamination -
Removal
AB Decontamination - Spray
AB Decontamination - Dry
AB - Clearance Sampling
CIO2 - Characterization
Sampling
CIC>2 - Decontamination
CIC>2 - Clearance Sampling
AB- Building Reset
70
17
7
20
21
6
4
0
16
11
0
0
0
0
0
0
0
0
10,142
2,157
0
704
0
0
0
0
0
210
447
188
247
154
178
0
325
101
0
315
0
70
227
455
208
268
10,302
2,339
0
1,045
112
0
315
0
66
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
105
0
0
0
0
0
0
0
211
55
74
177
26
528
0
101
137
0
38
0
66
211
55
74
177
26
633
0
101
137
0
38
0
Breakdown by Round
VHP®
AB
CIO2
Total
114
46
11
171
0
13,003
315
13,318
846
905
101
1,852
960
13,954
427
15,341
66
0
0
66
0
105
0
105
340
831
175
1,345
406
936
175
1,516
Breakdown by Aggregated Activity
VHP® Sampling
VHP® Decontamination
AB Sampling
AB Decontamination
CIO2 Sampling
CIO2 Decontamination
Other
37
7
37
10
11
0
70
0
0
704
12,299
315
0
0
398
447
572
332
101
0
0
436
455
1,313
12,641
427
0
70
0
0
0
0
0
0
66
0
0
0
105
0
0
0
285
55
278
554
175
0
0
285
55
278
659
175
0
66
AB = pH-adjusted bleach decontamination process; Other = collected during other activities (e.g., dissemination)
321
-------
Summarizing the daily solid waste data on the basis of total amount collected and plotting the
results yields Figure 4-61. The materials removed from the pH-adjusted bleach decontamination
process in Round 2 contributed significantly to the overall waste stream, as well as requiring
much more support for handling and packaging than the other days of the testing. Coordination
of waste management procedures with the State waste management officials is critical. For
example, in the recent naturally-occurring Ba response in Durham, NHm, the State indicated
that if the pH-adjusted bleach decontamination process were properly followed, the waste could
be disposed of as solid waste in a RCRA Subtitle D facility without any additional waste
characterization sampling. In a real Ba response, it is advantageous to ensure that the
decontamination process (and sampling, if required) results in the least restrictive (e.g., Subtitle
D) waste designation, thus reducing remediation costs.
12000
10000
pH-Adjusted Bleach Process
Date
Figure 4-61. Daily solid waste generation.
4.6.5. Discussion
Figure 4-62 shows the distribution of waste by activity (sampling and decontamination and
other) for liquid and solid waste. The vast majority of the waste from Round 2 with the pH-
adjusted bleach decontamination process was generated during the decontamination itself,
whereas for Round 1 and Round 3, a significant fraction of the waste was generated during
sampling operations. Note that any differences in quantities of waste generated during sampling
operations represent the variability of sampling waste generation (i.e., sampling activities for the
three rounds of the BOTE Project were virtually identical).
322
-------
CLO2 Decontamination
CLO2 Sampling
AB Decontamination
AB Sampling
VHP® Decontamination
VHP® Sampling
I Category IS - Uncontaminated -
Solid
I Category 2S - Contaminated - Solid
Category 3S - Decontaminated -
Solid
I Category 4S - Decon Waste - Solid
5000 10000
Quantity (Ib)
15000
Figure 4-63 shows the quantities of solid waste as a function of waste category. The largest
amount of solid waste generated occurred during Round 2 (pH-adjusted bleach
decontamination process) in which porous surfaces were removed, bagged, decontaminated ex
situ, and treated as waste (Category 3S). Waste generated during Round 1 (fumigation with
VHP®) included only category 4S waste such as PPE and sampling waste (e.g., packaging) for
both building decontamination and sampling. A small amount of decontaminated solid material
was also generated during the post-decontamination sampling in Round 2. No solid waste was
generated during building decontamination using CIO2, but a small amount of solid waste
(Category 4S) from PPE and sample kit packaging was generated during CIO2 sampling. Foam
materials (452 Ibs) were removed after the CIO2 fumigation (chairs, chair cushions, and
mattresses), but these materials were not included in the waste tally because they were not
damaged and would not have been replaced had there been an additional round following CIO2
fumigation.
323
-------
1400
1200
IDecon & Other
I Sampling
VHP
pH-Adjusted Bleach
Process
CL02
,§ 6000
2000
I Decon & Other
I Sampling
VHP®
pH-Adjusted
Bleach Process
CL02
Figure 4-62. Distribution of waste by activity (normalized to 100%). The top plot is
liquid and the bottom plot is solid waste.
324
-------
CLO2 Decontamination
CLO2 Sampling
AB Decontamination
AB Sampling
VHP® Decontamination
VHP® Sampling
I Category IS - Uncontaminated -
Solid
I Category 2S - Contaminated - Solid
Category 3S - Decontaminated -
Solid
I Category 4S - Decon Waste - Solid
0 5000 10000 15000
Quantity (Ib)
Figure 4-63. Solid waste by category.
Figure 4-64 shows the quantities of liquid waste as a function of waste category. The largest
amount of liquid waste was generated during Round 2 (pH-adjusted bleach decontamination
process) due to decontamination of the building using pH-adjusted bleach (Category 4L).
Decontamination liquid waste was also generated during all three rounds during sampling; this
waste consisted of the rinsate recovered from the Decontamination Line.
325
-------
CLO2 Decontamination
CLO2 Sampling
AB Decontamination
AB Sampling
VHP® Decontamination
VHP® Sampling
I Category 1L - Uncontaminated -
Liquid
I Category 2L - Contaminated - Liquid
Category 3L - Decontaminated -
Liquid
I Category 4L - Decon Waste - Liquid
0 200 400 600 800
Quantity (gal)
Figure 4-64. Liquid Waste by Category
4.6.6. Conclusions
The management of the waste from the BOTE Project presented several challenges, including:
• Difficulty weighing the waste from the second floor of the building during the pH-adjusted
bleach decontamination process while maintaining the contaminant control measures
necessary to prevent cross-contamination of samples;
• Difficulty extrapolating waste management practices from waste containing a
nonpathogenic organism to waste management of wastes containing Ba; and
• Difficulty specifying appropriate waste characterization strategies (i.e., sampling
procedures for bagged waste are not well-defined; bagging waste is logistically difficult,
and the effectiveness of using a surface sampling technique on bags of wetted building
materials is questionable).
The following observations and conclusions can be made:
• Waste management is an integral part of the decontamination process and must be
included as a specific function when response planning is done;
• Overall, the pH-adjusted bleach decontamination process generated the most waste for
both building decontamination and sampling efforts;
326
-------
• The use of RFID chips to track the source of the waste worked very well in spite of fears
that the presence of the pH-adjusted bleach in the bags might interfere with reading the
RFID chip information properly; and
• If the State agrees to accept liquid wastes in a POTW facility and the solid waste in a
RCRA Subtitle D facility, the waste management is greatly simplified.
4.7. Cost Analysis
A major objective of the BOTE Project was to estimate the cost of the application of various
decontamination technologies as a function of materials and labor effort. This estimation was
done by achieving the following objectives:
• Primary Objective: To conduct an analysis of the cost of the application of cleanup
technologies;
• Secondary Objective: To develop a tool or methodology that can be used to help guide
decision making for future events; and
• Secondary Objective: To provide input to a risk assessment to estimate residual
exposure potential as a function of remediation costs and decontamination technologies.
4.7.1. Cost Analysis Approach
The cost analysis approach makes the general assumption that although certain pieces of
information derived from the BOTE Project are incident- and site-specific, the information can
still be extrapolated to other incidents, using appropriate scaling factors based on labor hours,
numbers of samples, size of affected areas, and quantities of waste that are generated.
Although it would be desirable to be able to extrapolate the BOTE Project cost analysis to a
wide area incident, the goal of this analysis was to be able to extrapolate the analysis to other
single building incidents. The parameters examined in this cost analysis include costs related to
sampling activities, application of decontamination technologies for the building and personnel
entering and leaving the building, and costs related to equipment rentals and consumables.
Some costs that are critical to an analysis (e.g., waste management) were not able to be
assessed purely based on the BOTE Project; i.e., some BOTE Project-derived costs may be
unrealistic because the BOTE Project used a Ba surrogate and not actual Ba spores. Some cost
issues are highly dependent on the waste designation that is determined by the State, because
the initial presence of Ba in the materials may not preclude disposal of liquid waste in a POTW
facility or solid waste in a RCRA Subtitle D landfill provided that the State approves these
disposal pathways for waste that has undergone a decontamination or treatment process. In the
BOTE Project Phase 1, there was an underlying operational assessment that influenced many
decisions that impact costs. Costs that could not be assessed either by using data directly from
the BOTE study or from estimates based on best engineering judgment were not included in the
cost analysis.
4.7.1.1. Costs that were included
Costs estimated from the BOTE Project Phase 1 activities were assumed to be applicable to the
extrapolation of costs for another single building incident. Extrapolation of this cost analysis to a
wide area event was not within the scope of this effort, although future analyses may attempt to
make this extrapolation. The goal of this effort was to assess costs (labor + materials)
327
-------
associated with cleaning up a single building to a given level of residual number of viable spores
remaining on the surfaces and/or in the air based on a per application basis, if possible. If
possible, labor costs were based on an approximate loaded hourly labor rate and accounted for
both Federal and non-Federal workforce (i.e., contractor) efforts (labor costs associated with
Federal workers are frequently ignored because they are sometimes paid by funding sources
not normally associated with the response). This consideration is particularly important for cost
elements that are more easily scaled such as decontamination personnel, where the effect of
adding additional decontamination personnel could be examined. Costs for waste management
were based on type, quantity, and level of residual contamination, coupled with hypothetical
transportation, treatment, and disposal considerations. Some of the waste management cost
elements were notional rather than based on data from the BOTE Project because a surrogate
for Ba spores was used. Statistical analysis was performed to assign confidence intervals to the
cost estimates when compared to residual spores remaining after decontamination. Costs that
are considered in the analysis are discussed in the subsections below.
4.7.1.2. Sampling
Due to the research study nature of the BOTE Project, the sampling effort for the BOTE Project
was significantly greater than the sampling effort that would have been performed at a real Ba
incident. Cost analysis of the sampling effort broke the costs down so that future cost estimates
could be scaled as appropriate for an incident, but the overall sampling cost from BOTE should
not be construed to reflect the total sampling cost for a similar sized building. The BOTE
building had unique aspects (e.g., diversity of rooms) that would influence the sampling effort;
the cost analysis attempted to account for these unique aspects where possible.
• Travel costs for sampling teams (including lodging and per diem);
• Training time for sampling teams (it is assumed that even for a real Ba incident, there
would be site-specific training for sampling teams);
• Labor costs associated with planning sampling activities;
• Labor costs for sampling teams to don PPE and prepare for building entry;
• Labor costs for sampling teams entering the building and performing sampling activities;
• Labor costs for sampling teams exiting the building through the personnel
Decontamination Line and associated labor costs of Decontamination Line personnel;
• Labor costs associated with preparing the sampling kits that were used for sampling;
• Labor costs associated with supporting the sampling activities (e.g., BROOM, Incident
Command, Safety);
• Material costs associated with sampling, including the cost of PPE;
• Material costs associated with sampling teams entering the building; and
• Waste management costs associated with sampling.
4.7.1.3. Laboratory Analysis
• Costs associated with preparing and shipping samples to the laboratory;
• Labor costs for laboratory analysis;
• Material costs for laboratory analysis; and
• Labor costs associated with statistical analysis of the laboratory data.
328
-------
4.7.1.4. Decontamination
• Travel costs for decontamination and/or removal teams (including lodging and per diem);
• Labor costs for training of decontamination and/or removal teams (it was assumed that
even for a real Ba incident with experienced decontamination teams, there would be
some site-specific training required);
• Labor costs for decontamination and/or removal teams donning PPE and preparing for
building entry (PPE costs were separately tracked);
• Labor costs for decontamination and/or removal teams entering the building and
performing decontamination operations;
• Labor costs for decontamination and/or removal teams exiting the building through the
personnel Decontamination Line and associated labor costs of Decontamination Line
personnel;
• Labor costs associated with maintaining entry teams (e.g., Level B PPE support);
• Material costs associated with decontamination and/or removal teams entering the
building;
• Material costs for decontamination;
• Equipment rental costs;
• Fixed contractor costs for decontamination (e.g., contracts with fumigation vendors); and
• Waste management costs due to decontamination and removal teams entering the
building.
4.7.1.5. Waste Management
• Labor costs associated with development of plans for waste management,
transportation, health and safety, and communications (notional);
• Labor costs associated with coordination with regulatory authorities (notional);
• Waste transportation costs (notional but based on measurements of waste quantities);
• Waste disposal fees (notional but based on measurements of waste quantities);
• Waste handling, packaging, and labeling costs (notional, but based on measurements of
waste quantities); and
• Waste sampling and analysis costs (notional, but based on measurements of waste
quantities).
4.7.1.6. Building Refit
The costs of replacing the items in the building have a high degree of uncertainty. In this cost
analysis, no consideration was made as to who would actually be paying the costs for
replacement of items. The building owner would probably be responsible for refit costs, and their
insurance coverage would likely significantly impact what items get replaced. In addition, the
numbers of items and amount of material in the contaminated rooms will have a profound
impact on refit costs. For the BOTE Project, the rooms were relatively sparsely fitted with
representative items, but the numbers of items and amount of materials in the BOTE rooms was
likely to be significantly less than in a real residence or office. The cost estimate that was
performed assumed that all items in the rooms would be replaced. No assumptions were made
as to who would bear these costs.
329
-------
• Labor costs for replacing items removed from building either before or after
decontamination;
• Replacement costs for items in building that were removed either before or after
decontamination; and
• Replacement costs for items in building that were not removed but would have been
removed in a real situation (notional).
4.7.1.7. Incident Command
The costs of the 1C covered in this section include costs not directly accounted for in other cost
categories (e.g., sampling, decontamination).
• Travel costs of minimal 1C personnel (e.g., Safety, Incident Commander), including
lodging and per diem;
• Labor costs associated with maintaining minimal 1C through duration of each round; and
• Labor costs for health and safety monitoring of decontamination reagent levels inside the
building to determine when the building can safely be entered for clearance sampling.
4.7.1.8. Costs not included
A number of cost elements, although important, were not assessed in the cost analysis from the
BOTE Project. In general, these costs were either impossible to assess accurately due to their
site- and incident-specific nature, or were not likely to be a strong function of the
decontamination strategy. These costs included:
• Costs due to denial of access to facilities that have not been cleared;
• Costs associated with delays in reaching final decontamination or waste disposal
decisions;
• Costs associated with delay in cleanup due to limited availability of cleanup contractors;
• Costs associated with the closest waste management facilities being unwilling to accept
the incident-generated wastes for whatever reason (i.e., public, political, shareholder, or
other concerns);
• Most 1C costs;
• Costs associated with public panic (e.g., the types of costs reported in news media after
incidents);
• Costs associated with denial of access due to the public's refusal to reoccupy buildings
that have been cleared;
• Costs of items not directly related to the decontamination and building remediation; and
• Costs due to the extension of response/recovery timelines due to political
considerations.
4.7.2. Conceptual Description of Cost Analysis
The equations in this section represent a mathematical approach to collecting the various cost
elements and combining them into an overall cost. The cost estimate is broken down into
several main components: 1) sampling and analysis costs; 2) decontamination costs; and 3)
restoration costs.
330
-------
4.7.2.1. Sampling and Analysis Costs
Equation 4-1 describes the total costs for sampling:
Nes Ns
i=0
Cs=^ (CPS + CPDS + CPDWS) + Ł (Csu + CSM + CSAl + Csm ]
Equation 4-1
where Cs represents the sampling costs, CPS is the cost of sample team preparation (labor +
PPE) for each entry (i) into the contaminated building up to A/es that is the total number of
sample team entries, CPDs is the cost of post-entry personnel decontamination of the sampling
team (labor + materiel) for each entry, CPDws is the cost of waste management for the personnel
decontamination waste for each entry into the contaminated zone, and, for each type of sample
(/) up to the total number of samples (Ns), CSu is the cost of labor, CSMI is cost of materials, CSAI
is the cost of sample analysis (including packaging and shipping), and CSw is associated waste
management costs. Sampling costs were calculated for both the pre-decontamination
(characterization) and post-decontamination (clearance) sampling for each round of the BOTE
Project.
4.7.2.2. Decontamination Costs
Equation 4-2 describes the costs for decontamination:
Ned
~ / i \^PD ~~ -PDD ~~ PDWo ~~ DC ~~ -RLB ~~ RW ~~ DL ~~ DM ~~ DW
i=0
Equation 4-2
where CD represents the cost for decontamination, CPD is the cost of decontamination/removal
team preparation (labor + PPE) for each entry; into the contaminated building up to A/edthat is
the total number of decontamination/removal team entries into the building, CPDD is the cost of
post-entry personnel decontamination of the decontamination/removal team (labor + materiel),
CPDWD is the cost of waste management for the personnel decontamination waste for each entry
into the contaminated zone, CDC is the fixed cost of any third party decontamination contractors,
CR/.B is the cost of labor for pre-decontamination item removal, CRW is the cost of waste
management for the removed items, CDL is the cost of decontamination team labor, COM is the
cost of decontamination materials, including purchase and/or lease of equipment such as
backpack sprayers or NAMs, CDw is the cost of managing decontamination waste. Some of
these terms were zero for certain decontamination technology selections, and some of these
terms were notionalized (e.g., waste) because this effort used a simulant and not a real
331
-------
biological warfare agent. Where possible, a detailed assessment of cost was performed,
including such factors as electricity for NAMs, water, and shipment for supplies.
4.7.2.3. Restoration Costs
Equation 4-3 conceptually describes the costs for restoration of the facility and addresses costs
incurred after the decontamination until the building has been refitted with new or
decontaminated items. Some of these cost elements were notionalized, e.g., laminate flooring
was not replaced after the pH-adjusted bleach decontamination process but would have been
replaced in a real situation due to warping that occurred from the laminate becoming wet and
saturated from the decontamination process.
Equation 4-3
In Equation 4-3, CR represents the cost for restoring the facility to normal operation after the
decontamination, CRLA is the labor cost of removing materials post-decontamination, CRW is the
cost of managing those removed materials as waste, and Cy is the cost of refitting the facility
with new items (labor + materials).
4.7.2.4. Total Cost Per Round
Using these equations, the total cost per Round (C) was estimated using Equation 4-4:
C — Cs + CD + CR
Equation 4-4
There are several ways to normalize these data, including:
• Cost Per Room Type;
• Cost Per Unit Area; or
• Cost Per Unit Volume.
4.7.3. Sources of Data
The raw data were acquired from several sources. Table 4-19 lists the data acquired, the source
of the data, and how the data were QC-checked. The cost data were collected in an MS Excel
workbook (i.e., the Cost Analysis workbook) that contained multiple worksheets. These
worksheets can be found in Appendix H and are available if additional detail is desired on how
costs were estimated. Table 4-20 lists the worksheets and what data they contain.
332
-------
Table 4-19. Sources of data.
Measurement
Entry team personnel
Decontamination Line data
Entry team preparation time
Entry team times inside building
Material costs
Personnel Decontamination Line
operations data
Times to perform
decontamination operations
Times to take samples
Waste origination point
Waste quantity estimates
Primary Source
Notes from radio broadcasts
Radio broadcasts
Radio broadcasts
Copies of purchase orders,
emails
Test Group Director Notes
Radio broadcasts
Observer inside building
watching sampling teams
RFID chips inserted into bags of
waste prior to removing bags
from rooms; later tracked as
bags of waste left building using
RFIDreader
Platform scale near personnel
Decontamination Line; volumetric
estimate on full barrels
QC Method
Compared with Test Group
Director notes and Emergency
Medical Team notes
Occasional observations
Compare with Test Group
Director notes
Spot check random entries for
accuracy
Compared with notes
Compare with notes, Test Group
Director notes
Compare with notes, Test Group
Director notes
N/A
Scale periodically checked for
drift and zero with full bottle of
bleach
Table 4-20. Worksheets from the cost analysis workbook.
Worksheet Name
AB Building Refit
Activities
Analytical Costs
Cost Equations
Daily Activity List
Daily Waste Generation
Decontamination line
Ops
Entry Team
Decontamination Line
Time
Entry Team Prep Time
Knobs
Lumped Costs
Information in the Worksheet
Calculations for the Round 2 building refit [Equation 4-3]
The list of the activities that occurred throughout the BOTE Project Phase 1
Calculations for estimating analytical costs [Equation 4-1]
The main cost calculation sheet that estimated values for the terms in Equation
4-1, Equation 4-2, Equation 4-3, and Equation 4-4
A lookup table assigning the activities in the "Activities" worksheet to a given
date
Calculations of the amount of waste generated each day
Raw data - observations of the labor due to personnel Decontamination Line
operations
Raw data - observations of the amount of time each building entry team spent
in the personnel Decontamination Line
Raw data - observations of the amount of time each building entry team spent
donning their PPE and preparing for entry
The worksheet with the adjustable parameters for the cost analysis
Raw data - observations and calculations of various lumped costs that were
333
-------
Worksheet Name
Notes and Assumptions
Numbers of Samples
Purchase Orders
Resource Tracker
Sampling Data
Revisions
Room Sample Box
Prep Time
Room Sample Time
Salary Table
Summary
Team Entries
Team Makeup
Time Per Sample
Waste
Waste Cost
Waste Summary
Information in the Worksheet
not scalable based on labor hours or entries (e.g., travel, BROOM support)
A list of notes and assumptions
Calculations related to numbers of samples
Raw data - observations and calculations of various items purchased for the
BOTE Project
Raw data - observations taken by the observer inside the building during
sampling operations
A list of revisions to the Cost Analysis Workbook as the calculations were
developed
Raw data - observations of how long it took to prepare the sample boxes for
use in the different rooms in the building
Raw data - how long it took to sample each room
A table of salaries that were used to estimate labor rates of various efforts
Intermediate calculations of various terms used in the Cost Equations
worksheet
Calculations of numbers and duration of entries by various teams
This worksheet defined the makeup of each team and calculated the team's
loaded hourly labor rate based on the team makeup and the Salary Table
worksheet
Calculations of how long each type of sample took to acquire
Raw data - quantities of waste generated
Calculations of waste management costs
Calculations of amount of waste generated in each room and amount of waste
broken down by category
4.7.3.1. Labor Cost Approach
Labor costs were estimated using a loaded hourly labor rate approach that used designated
teams for various activities. Contractor hourly labor estimates'941 were based on values for the
labor categories shown in Table 4-21, and the Incident Commander or On-Scene Coordinator
was based on a GS-13 Step 5 rate found in the 2011 General Schedule Locality Pay Tables for
Raleigh-Durham-Cary, NC[9Sl. A multiplication factor of three was used to estimate total loaded
hourly rates from the base hourly salary values. This multiplier accounted for benefits and
management overhead associated with the employee. The personnel mix of teams that
comprised the efforts for various aspects of the response was based on mixtures of the
previously mentioned labor categories, deployed for varying numbers of hours. The teams that
were used are listed in Table 4-22.
334
-------
Table 4-21. Labor categories and loaded hourly labor rates.
Labor Category
PL1
PL2
PL3
PL4
TL1
TL2
TL3
EMT
On-Site
Coordinator/Commander
Job Classification
Engineer I
Engineer II
Engineer III
Engineer V
Engineering Aide I
Engineering Aide II
Engineering Aide III
Paramedic
GS-13Step5
Loaded Hourly
Rate ($)
86
102
124
170
66
79
88
58
147
335
-------
Table 4-22. Labor mix of teams of personnel.
Team Type
AAS Team
BROOM Team
Building UpfitTeam
Command Team
Data Analysis Team
Decontamination Line
Ops Team
Decontamination Line
Setup Team
Decontamination
Team (PPE Level B)
Decontamination
Team (PPE Level C)
Documentation/Plan
Writing Team
EPA Purchasing Team
Health and Safety
Team
INL Equipment
Purchase Team
Laboratory Analyst
Team
On-Scene
Coordinator
Regulatory
Coordination Team
Removal Team
Room Sample Box
Prep Team
Sample Kit Prep
Team
Sample Packaging
Team
Sample Planning
Team
Sampling Team
Statistical Analysis
Team
Waste Handling Team
Waste Sampling
Team
Water Sampling Team
osc/
Commander
1
1
1
0.33
0.33
1
0.25
1
1
0.33
0.33
1
EMT
1
PL1
1
1
0.5
1
3
3
PL2
3
0.25
1
PL3
2
3.33
2.33
0.25
3.33
3
PL4
1
2
0.67
0.67
1
0.25
2
0.67
1
1
1
TL1
3
1
1
TL2
3
3
3
TL3
3
2
0.25
1
3
1
#of
Teams
1
1
1
1
1
1
1
3
3
1
1
1
1
1
1
1
3
1
1
1
1
6
1
1
1
1
336
-------
4.7.3.2. Sampling/Analysis Cost Approach
The following costs were calculated based on the number of entries:
• Building entry costs;
• Team preparation costs; and
• Personnel decontamination costs.
The following costs were calculated based on the number of samples:
• Team labor for sampling;
• Materials for sampling, including PPE;
• Labor for analysis; and
• Materials for analysis.
The following costs were calculated based on totaling purchase order values or by estimation of
time coupled with loaded labor hours:
• Preparing sampling kits;
• Travel for sampling teams;
• Purchase of temperature/RH monitoring devices (e.g., HOBOs®);
• BROOM support; and
• Analysis and QA of data.
The waste management costs from sampling were estimated based on the amounts of different
types of waste generated and notional waste management decisions and costs that would arise
based on those decisions.
4.7.3.3. Facility Decontamination Cost Approach
The following costs were calculated based on the number of decontamination and/or removal
team entries (the majority of the items below pertain only to Round 2 [when the pH-adjusted
bleach decontamination process was used]):
• Team preparation; and
• Team labor during personnel decontamination.
The following costs are calculated based on the time that the entry teams spend inside the
building performing decontamination and removal activities:
• Labor for decontamination; and
• Labor for removal.
The following costs are calculated based on totaling the value of purchase orders and other
such expenses:
• Materials;
• Equipment purchase and rental;
• Travel for decontamination/removal teams;
337
-------
• Fixed (lumped sum) contractor costs (e.g., contracts with fumigation vendors in Rounds
1 and 3 included labor, materials, travel, etc., and were not broken down);
• 1C support (e.g., safety); and
• Purchase of temperature/RH sensors (e.g., HOBOs®) and other instrumentation.
The waste management costs from decontamination are estimated based on the amounts of
different types of waste generated and notional waste management decisions and costs that
would arise based on those decisions.
4.7.3.4. Refurbishment Cost Approach
Costs changed based on what was removed, decontaminated or treated, and disposed. In the
BOTE Project, some materials were removed prior to decontamination, and some were
removed after decontamination. Those activities generated waste that had characteristics based
on when the waste was removed and where in the facility or surrounding areas the waste
originated. Materials removed from the building were replaced prior to initiation of the
subsequent round. Costs associated with replacing those materials were estimated based on
average costs of materials in the different types of rooms and labor hours by INL personnel.
There were some materials (e.g., laminate flooring, electrical components) that were not
removed between rounds that most likely would have been removed in a real situation (e.g., the
pH-adjusted bleach warped the laminate floors; the CIO2 corroded many electrical components).
Costs associated with replacing those materials were calculated based on manufacturer
estimates for labor and materials. It was not possible to assess the corrosion damage that CIO2
did to electrical components and various other metal parts of the building completely because
the building had been fumigated with CIO2 repeatedly in earlier studies. This inability to assess
damage due to CIO2 fumigation completely may result in an underestimate of the replacement
cost of materials after CIO2 fumigation.
4.7.3.5. Assumptions and Caveats
Many assumptions and caveats must be noted in this cost analysis. Table 4-23 lists these
assumptions and caveats. The "Worksheet" column refers to the worksheet tabs in the MS
Excel Cost Analysis workbook that is found in Appendix H.
338
-------
Table 4-23. Caveats and assumptions in cost analysis.
Worksheet
All
AB Building Refit
AB Building Refit
AB Building Refit
Analytical Costs
Analytical Costs
Analytical Costs
Analytical Costs
Cost Equations
Cost Equations
Cost Equations
Cost Equations
Cost Equations
Lumped Costs
Lumped Costs
Lumped Costs
Lumped Costs
Lumped Costs
Caveat/ Assumption
Used a single average time per entry based on Test Group Director data for all
entries by all teams, with the exception of incidental entries. This average takes
into account the attempt to have teams inside the building for nominally three
hours, but, due to various issues, the teams could not remain for the full three
hours.
Damage to building structure and wiring could not be assessed partially due to lack
of access and partially due to inability to determine whether damage was incurred
during the BOTE Project or during previous CIO2 fumigations.
Laminate floor cost: $3.70/ft2 installed1961
Notional cost to install HVAC duct on both floors: from
http://www.homewyse.com/services/cost to install duct.html'971
There is a parameter on the "Knobs" worksheet that has the multiplier for Bio-
safety Level 3 (BSL-3) analysis versus BSL-2 Analysis
The laboratory analytical labor was based only on the responses from the Utah
laboratory - no additional laboratories responded with tracking data
Analytical Cost of A AS Samples = average of analytical costs of HEPA vacuum,
sponge-stick, swab, and EPA wipes
Analytical Cost of Waste Samples = average of analytical costs of HEPA vacuum,
sponge-stick, swab, and EPA wipes
Sampling cost does not include Bis, RMCs, TSA settling plates, sand, or SKC
BioSampler® samples
Average material cost per sample type = total materials for that sample type + total
lumped costs for that sample type + general sample costs distributed among
number of HEPA, wipe, swab, air, sponge samples
Decontamination contractor fixed costs = the sum of all lumped costs + purchase
order costs for each Round, plus one-third of the general decontamination costs
that are not attributed to any given Round
Purchase orders for sampling supplies and HOBOs® are equally distributed among
all samples of all main types (wipes, swabs, sponge-sticks, aggressive air)
Cost of Safety Team is included in 1C costs. Cost of decontamination from safety
team entering building is in decontamination cost.
505 gal of pH-adjusted bleach used; Remaining Stock - Vinegar 72 cases, 4-1 gal
bottles per case ($2.37 ea WalMart)
505 gal of pH-adjusted bleach used; Remaining Stock - Bleach 62 cases, 6 3-qt
bottles per case ($1 .98 ea WalMart)
Assume for travel cost estimates that sampling and BROOM teams fly in, rent one
car per team, stay duration of sampling (including lodging, meals, and incidental
expenses), fly out; one day travel each way
Assume for travel cost estimates that decontamination teams fly in, rent one car
per team, stay duration of decontamination (including lodging, meals, and
incidental expenses), fly out; one day travel each way
Assume that only travel being paid for samplers, decontamination, safety, On-
Scene Coordinator, command. Other travel (e.g., Decontamination line ops,
339
-------
Worksheet
Numbers of
Samples
QC
Room Sample Time
Summary
Time Per Sample
Waste
Waste
Waste
Waste Summary
Caveat/ Assumption
sample kit box prep, other helpers, from local labor)
Total number of samples for each Round was estimated by dividing the total
number of each type of sample by three to account for minor differences in sample
counts between nominally identical sampling in each round
Recuperation time after entries was estimated by adjusting to minimize the
difference between calculating the Decontamination Line time and the
decontamination team time using either total days or based on entries
Assumed 15 AAS samples per entry; three hours per team
To calculate waste distribution between sampling and decontamination, liquid and
solid were combined
Assumed 30 AAS samples per Round; three hours per team
Laminate Flooring = 21 ft2/carton; 35 Ib/carton = 1.67 Ib/ft2
Did not include porous materials removed after CIO2 clearance sampling because
it didn't have anything to do with the decontamination or clearance process, and
wouldn't have applied to reset
HVAC material = 24 gauge; 7.71 Ib/linear foot[98];
Laminate Flooring was notionally added to Removal Operations
4.7.4.
Results
4.7.4.1. Sampling and Analysis Costs
Due to the operational testing and evaluation nature of the BOTE Project Phase 1, the overall
costs of sampling and analysis were much higher than would be observed from a real incident.
One benefit from this large number of samples, however, is that statistical data for variability
could be extracted from the observations of the time and effort needed to acquire samples. To
account for the fact that the BOTE Project laboratory analytical efforts were done under BSL-2
conditions, whereas in an incident involving real anthrax, the laboratory analyses would be done
under BSL-3 conditions, a multiplier factor of 1.5 was applied to analytical cost estimates. Table
4-24 lists the mean and standard deviations from sampling and analysis activities. Table 4-25
summarizes the sampling and analytical results for Rounds 1 through 3. Figure 4-65 pictorially
shows a breakdown of the cost results for sampling and analysis. Standard deviations could not
be calculated for costs that did not have multiple values. The differences between the costs
associated with management of waste from the three Rounds probably reflects the variability of
the sampling operations overall and gives an indication of the precision of the cost estimation for
this activity.
340
-------
Table 4-24. Mean and standard deviation of sampling activities.
Sampling and Analysis Costs
Cost of Sample Team Preparation per Entry
Cost of Sample Team Personnel Decontamination per Entry
Cost of Sample Team per Entry
Cost of AAS Team Prep per Entry
Cost of AAS Team Decontamination per Entry
Cost of AAS Team per Entry
Labor Cost Per HEPA Sample Analysis
Material Cost Per HEPA Sample Analysis
Analysis Cost Per HEPA Sample Analysis
Labor Cost Per Sponge-Stick Sample Analysis
Material Cost Per Sponge-Stick Sample Analysis
Laboratory Analysis Cost Per Sponge-Stick Sample Analysis
Labor Cost Per Wipe (EPA) Sample Analysis
Material Cost Per Wipe (EPA) Sample Analysis
Laboratory Analysis Cost Per Wipe (EPA) Sample Analysis
Labor Cost Per Wipe (LLNL) Sample Analysis
Material Cost Per Wipe (LLNL) Sample Analysis
Laboratory Analysis Cost Per Wipe (LLNL) Sample Analysis
Labor Cost Per Swab Sample Analysis
Material Cost Per Swab Sample Analysis
Laboratory Analysis Cost Per Swab Sample Analysis
Labor Cost Per Aggressive Air Sample Analysis*
Material Cost Per Aggressive Air Sample Analysis*
Laboratory Analysis Cost Per Aggressive Air Sample Analysis*
Mean
($/sample)
252
697
720
273
725
779
53
29
288
34
20
239
30
19
231
30
19
640
29
21
219
57
18
245
Std Deviation
($/sample)
121
78
328
130
82
355
18
10
9
9
9
- average of HEPA, sponge-stick, wipe (EPA), wipe (LLNL), and swab analysis
341
-------
Table 4-25. Summary of sampling and analytical cost analysis.
Cost Description
Attributable Other
Sampling/Analytical Costs (e.g.,
materials purchased for a specific
type of sample, such as HEPA
socks)
Non-Attributable Other
Sampling/Analytical Related Costs
(e.g., materials purchased for
sampling and analysis but not able
to be attributed to a specific type of
sample, such as growth media,
data management and data
analysis costs)
Decontamination Line Labor
Sampling Labor
Material
Laboratory Analysis
Management of waste associated
with taking samples
Total Sampling Cost
Sampling Cost Std Dev
Round 1
($)
148,513
95,138
10,020
37,049
23,894
313,490
42,166
660,251
27,398
Round 2 ($)
148,513
95,138
10,736
39,564
23,894
313,490
25,725
646,324
28,412
Round 3 ($)
148,513
95,138
10,736
39,673
23,961
314,649
67,053
688,987
28,489
Average ($)
148,513
95,138
10,497
38,762
23,916
313,877
44,981
665,188
28,099
342
-------
$800,000
$700,000
$600,000
$500,000
VHP® pH-Adjusted
Bleach Process
CIO2
Waste Management
Analysis
Material
Sampling Labor
Decon Line Labor
Non-Attributable Other
Sampling/Analytical Related
Costs
Attributable Other
Sampling/Analytical Costs
Figure 4-65. Breakdown of sampling and analytical costs.
343
-------
4.7.4.2.
Facility Decontamination Costs
4.7.4.2.1. Cost of Labor for Decontamination and Removal
The labor costs for decontamination and removal for the fumigation technologies (Rounds 1 and
3) are largely rolled into the cost estimate for the fumigation contractors. The numbers of
sources of data for these portions of the activities were very limited (i.e., N=1 for each
decontamination Round). For example, the EPA START contractors were used for the pH-
Adjusted Bleach Process, but they are a rapid-response team which may be more expensive
than response contractors that might be used in a real incident. In addition, it is not known how
representative the fumigation contractor bids were, given the nature of the BOTE Project. The
fumigation contractors did enter the facility on occasion in preparation for the fumigations,
however, so there was effort expended by the on-site test group to facilitate their entries. Costs
for all three decontamination technologies including the fumigations therefore included
estimates for 1C, Safety, and Personnel Decontamination Line Operations. The pH-adjusted
bleach decontamination process (Round 2) required entries in Level B PPE which necessitated
additional support personnel outside the building. Table 4-26 lists the estimated costs per entry
for the various teams entering the facility.
Table 4-26. Estimated cost per entry for facility decontamination.
Labor Description
Decontamination Team Prep
Removal Team Prep
Decontamination Team
Personnel Decontamination
Removal Team Personnel
Decontamination
Decontamination Team Per
Entry
Removal Team Per Entry
Round 1
Mean
($/entry)
271
722
773
Standard
Deviation
($/entry)
129
81
352
Round 2
Mean
($/entry)
345
345
822
822
985
985
Standard
Deviation
($/entry)
165
165
92
92
449
449
Round 3
Mean
($/entry)
271
722
773
Standard
Deviation
($/entry)
129
81
352
4.7.4.2.2. Fixed Cost and Material Costs of Decontamination Contractors
The fixed costs and material costs for the decontamination contractors included the fixed price
contracts that were made with the fumigation contractors, setting up and disassembling the
personnel Decontamination Line, travel for the EPA Region 10 START contractors, labor
associated with the notional removal of the HVAC ductwork, and post-deployment
documentation performed by the START contractors. Table 4-27 summarizes these costs.
344
-------
Table 4-27. Decontamination contractor fixed costs and material costs.
Cost Description
Decontamination Contractor
Fixed Cost ($)
Material Cost for
Decontamination Team ($)
Round 1 ($)
105,493
1,587
Round 2 ($)
66,831
31,976
Round 3 ($)
169,093
1,587
4.7.4.3. Waste Management Costs
Waste management cost estimates were based on observed quantities of waste from various
parts of the BOTE Project facility. Liquid wastes and solid wastes were tracked separately, and
the source of the waste was identified. The waste was characterized as being from one of the
following sources:
• Building;
• PPE Donning Trailer; and
• Decontamination Line.
Due to logistical limitations, it was not possible to weigh the waste removed from the second
floor of the building during the pH-adjusted bleach decontamination process (Round 2). The
weighed quantities from the first floor were therefore doubled. This estimation was a reasonable
approach because the rooms on both floors were configured similarly. In addition to these three
sources, waste from the pH- amended bleach decontamination process (Round 2) that
originated in the building first floor was additionally attributed to a given room (if it originated
from one of the commercial, residential, mailroom, or shop spaces), or designated as "other".
See Section 4.6.2 for a description of how RFID tags were used during the pH-adjusted bleach
decontamination process to track the originating point of bags of waste.
Once the source and quantity of the waste were identified, the waste was assigned to different
categories (see Section 4.6.3) based on the activity that was occurring on a given day. The
categories that the waste was segregated into consisted of:
• Category 1S
• Category 2S
• Category 3S
• Category 4S
• Category 1L •
• Category 2L •
• Category 3L •
• Category 4L •
Uncontaminated - Solid;
Contaminated - Solid;
Decontaminated - Solid;
Decontamination Waste - Solid;
Uncontaminated - Liquid;
Contaminated - Liquid;
Decontaminated - Liquid; and
Decontamination Waste - Liquid.
Although Categories 2S and 2L were defined, after the sources of the waste were identified and
the quantity measured, there was no waste generated during any of the BOTE Rounds that fell
into those categories. Table 4-28 lists the quantities and categories for the wastes that were
collected.
345
-------
Table 4-28. Quantities and categories of waste collected.
Waste Category
Category 1S - Un contaminated - Solid (Ib)
Category 3S - Decontaminated - Solid (Ib)
Category 4S - Decontamination Waste - Solid (Ib)
Category 1 L - Uncontaminated - Liquid (gal)
Category 3L - Decontaminated - Liquid (gal)
Category 4L - Decontamination Waste - Liquid
(gal)
Total Solid (Ib)
Total Liquid (gal)
Round 1
114
846
66
340
960
406
Round 2
46
13,003
905
105
831
13,954
936
Round 3
11
315
101
175
427
175
Estimating the total costs of waste management from the various rounds required notionalizing
many of the waste management activities. None of the waste generated during the BOTE
Project was actually contaminated with Ba; therefore, as is described in Section 4.5, the waste
was characterized as if it were Ba-contaminated or had been treated/decontaminated Ba-
contaminated materials prior to placing the waste into the dumpster and entering the waste into
the INL waste management process.
Three hypothetical waste management scenarios were examined based on the difficulty of
managing the waste that affected estimated costs of transportation and disposal. This approach
was supported by process knowledge and experience from real responses to both intentional
and naturally-occurring anthrax incidents since 2001. All scenarios assumed that solid waste
that was never contaminated could be brought to a RCRA Subtitle D landfill ten miles away, and
that all liquid waste regardless of initial level of contamination could be brought to a local POTW
facility 10 miles away. Any solid waste that was initially contaminated but through either the
decontamination processes or later on-site treatment was decontaminated was assumed to be
brought to a RCRA Subtitle D landfill 200 miles away. The first scenario, "Low Difficulty",
assumed that all solid waste that was initially contaminated was disposed of as municipal solid
waste (MSW) in a RCRA Subtitle D landfill 200 miles away from the incident and assumed that
all liquid waste was brought to a local POTW facility 10 miles away for no increased charges
above and beyond what normal MSW or sanitary sewage would require. The second scenario,
"Medium Difficulty", assumed that decontaminated waste was indeed decontaminated but a 10x
multiplier surcharge was imposed on transportation and disposal. The third scenario, "High
Difficulty", assumed that decontaminated waste was assumed still to be contaminated, resulting
in a 100x multiplier surcharge on transportation and disposal. These waste management
scenarios are summarized in Table 4-29.
346
-------
Table 4-29. Summary of waste management scenarios.
Degree
of
Disposal
Difficulty
Low
Medium
High
Decontaminated
Waste
Classification
MSW
MSWwith
surcharge
Contaminated
Waste
Disposal Pathway
Uncontaminated
Solids Disposal
Local Landfill
(1 0 miles away)
Local Landfill
(1 0 miles away)
Local Landfill
(10 miles away)
Decontaminated
Solids Disposal
Remote Landfill
(200 miles
away)
Remote Landfill
(200 miles
away)
Remote Landfill
(200 miles
away)
Uncontaminated
and
Decontaminated
Liquids
Local POTW
facility (10 miles
away)
Local POTW
facility (10 miles
away)
Local POTW
facility (10
miles away)
Surcharge on
Transportation
/ Disposal
10x
100x
The notional waste management costs were defined as:
^waste-fixed "*" ^waste-transportation "*" ^waste-handling "*" ^waste-characterization "*" ^waste-disposal
Equation 4-5
where Cwaste-fixed is the fixed cost associated with waste management regardless of how much
waste is generated, Cwaste-transportation is the cost associated with transporting the waste to its
ultimate disposal site, Cwaste-handiing is the cost associated with handling, labeling, tracking, and
packaging the waste, Cwaste-characterization is the cost associated with sampling and analyzing the
waste, and Cwaste-disposai is the tipping fee at the disposal facility (in this case the disposal facility
was assumed to be either a landfill or a POTW wastewater treatment facility).
The components of Cwaste-fixed include such elements as:
• Development of Waste Management Plan (estimated 40 hr);
• Development of Transportation Plan (estimated 40 hr);
• Development of Tracking and Reporting Plan (estimated 40 hr);
• Health and Safety Plan and Oversight Costs (estimated 40 hr);
• Contract Oversight Costs (estimated 40 hours);
• Development of Communications and Community Outreach Plan (estimated 40 hr); and
• Coordination with Regulatory Agencies and Facilities (estimated 40 hr).
Based on those estimates of levels of effort to perform these tasks, Cwaste-fixed was approximately
$53,353. Based on experience during exercises as well as real response situations, the
development of the Waste Management Plan has a high probability to take much more time
347
-------
than this proposed number, particularly for a complicated incident involving a weaponized
biological contaminant or a wide-area incident.
The components of Cwaste-transportation included an estimate of $5/mile charges for waste disposal
trucking, based on a 2001 estimate of $3/mile[991 adjusted for inflation, with an additional
multiplier of 1 (for "Low" difficulty waste management), 10 (for "Medium" difficulty waste
management), or 100 (for "High" difficulty waste management). Table 4-30 lists the estimated
transportation costs for the three Rounds and the three disposal difficulty scenarios. Although
transportation costs for a wide-area incident would be expected to be proportional to the amount
of waste, for a smaller incident that would involve only a single large truck to carry all waste that
was generated, this proportionality is not going to be observed.
Table 4-30. Estimated transportation costs.
Waste Disposal
Difficulty
Low
Medium
High
Round 1 ($)
1,150
10,600
105,100
Round 2 ($)
1,100
10,550
105,050
Round 3($)
1,100
10,550
105,050
was estimated assuming a waste packaging rate of 100 Ib/hr or 100 gal/hr by the
waste packaging team. This number was scaled with the relative amount of waste. Cwaste-
characterizatbn was estimated based on an assumed 10-minute timeframe for the waste sampling
team to open the container, collect a waste sample, and close the container again. One sample
was assumed to be taken from every bag of waste (approximately 33.3 Ib), and one sample was
taken from every barrel of wastewater (55 gal). This approach was the waste sampling strategy
that was used in the BOTE Project Phase 2pl. Analytical costs were assumed to be the average
of HEPA vacuum, sponge-sticks, swabs, and wipe samples. Table 4-31 lists the estimates of
costs for handling, packaging, and characterizing the waste.
348
-------
Table 4-31. Estimated costs of waste handling, packaging, labeling, and characterization.
Cost Description
Handling, Packaging,
Labeling Costs
Sampling and Analytical
Costs
Round 1 ($)
5,260
10,192
Round 2 ($)
61,122
124,218
Round 3 ($)
2,316
5,096
i was estimated assuming a tipping fee of $100/ton of solid waste (based on a series
of Internet searches that yielded a wide range of estimated costs ranging from approximately
$50/ton to approximately $150/ton and $50/10,000 gal of liquid waste (based on assuming 50%
of the cost of Gary, NC, residential sewer rates), with an additional multiplier of 1 (for "Low"
difficulty waste management), 10 (for "Medium" difficulty waste management), or 100 (for "High"
difficulty waste management). Table 4-32 lists the estimated waste disposal costs for the three
rounds and the three disposal difficulty scenarios.
Table 4-32. Estimated waste disposal costs.
Waste Disposal
Difficulty
Low
Medium
High
Round 1 ($)
50
446
4,405
Round 2 ($)
702
7,003
70,006
Round 3 ($)
22
217
2,169
349
-------
Combining all of these waste management cost elements together and pictorially representing
them results in the charts shown in
$450,000
Transportation Costs
I Disposal Costs
Waste Sampling and
Analytical Costs
I Handling Costs
I Fixed Costs
pH-Adjusted
Bleach Process
CI02
Figure 4-66 through Figure 4-68 which represent the "Low", "Medium", and "High" disposal
difficulty scenarios. Some observations arise from these figures. First, for the "Low" and
"Medium" disposal levels of difficulty, the transportation and disposal fees do not contribute
significantly to the overall waste management costs, even with the 10x surcharge on
transportation and disposal in the "Medium" case. Even with the 100x surcharge on
transportation and disposal from the "High" disposal difficulty case, overall waste management
costs increase only approximately 30% from the "Low" case. Although transportation costs for a
wide-area incident would be expected to be proportional to the amount of waste, for a smaller
incident that would involve only a single large truck to carry all waste that was generated, this
proportionality will not be observed. Rather, the costs associated with handling the waste and
characterizing the waste are the most significant contributions across all waste management
scenarios. These added analytical requirements may overwhelm the capacity of the laboratory
available to support the response, especially for a wide area release or for a building with the
normal amount of contents. These costs escalate solely based on the amount of waste that is
generated, regardless of what disposal pathways are selected. These results also suggest that
developing a better strategy for characterizing the waste from events such as this with many
fewer samples could result in a significant cost savings, provided that the State regulatory
agencies concur with using some sort of reduced sampling scheme for the waste. Choosing to
dispose of the waste as contaminated material and foregoing waste characterization sampling
may not be significantly more expensive than doing extensive characterization to prove the
waste is not contaminated.
350
-------
Transportation Costs
I Disposal Costs
Waste Sampling and
Analytical Costs
I Handling Costs
I Fixed Costs
VHP
pH-Adjusted
Bleach Process
CIO2
Figure 4-66. Breakdown of waste management costs
("low" disposal difficulty scenario).
351
-------
$450,000
$400,000
$350,000
$300,000
$250,000
$200,000
$150,000
$100,000
$50,000
$-
VHP
pH-Adjusted
Bleach Process
CIO2
Transportation Costs
I Disposal Costs
Waste Sampling and
Analytical Costs
I Handling Costs
I Fixed Costs
Figure 4-67. Breakdown of waste management costs
("medium" disposal difficulty scenario).
352
-------
VHP
pH-Adjusted
Bleach Process
CIO2
Transportation Costs
I Disposal Costs
Waste Sampling and
Analytical Costs
I Handling Costs
I Fixed Costs
Figure 4-68. Breakdown of waste management costs
("high" disposal difficulty scenario).
4.7.4.3.1. Overall Cost of Decontamination
Combining all the costs associated with the application of the three different decontamination
technologies and using the "Medium" difficulty waste management scenario results in the chart
shown in Figure 4-69. The overall decontamination cost is largely driven by waste management
considerations.
353
-------
$400,000
Waste Management Costs
Cost of Decon Materials
I Decon Contractor Fixed
Costs
Cost of Removal Teams
Entering
I Cost of Decon Teams
Entering
I Cost of Decon Line
Operations
VHP
pH-Adjusted
Bleach Process
CIO2
Figure 4-69. Breakdown of decontamination costs
(using "medium" disposal difficulty scenario).
4.7.4.3.2. Refurbishment Costs
The materials pre-populated into the various rooms in the building, as well as the approximate
size of the rooms, are listed in Table 4-33. The amount of materials pre-populated into the
rooms was much less than would be found in a normal office or residential setting. No unique or
valuable items that would result in an underestimation of the refurbishment costs were
populated into the rooms. The costs of removal of materials and related costs such as waste
management and refurbishment may be artificially low. This underestimation is especially true
for Round 2 (pH-adjusted bleach decontamination process), which involved material removal
(and, consequently, waste management and refurbishment) as a significant part of the
decontamination process. The pH-adjusted bleach decontamination process costs would be
expected to be even higher had the building been furnished in a more realistic fashion. The
replacement of some items (e.g., laminate floor and HVAC ductwork) was handled notionally.
The CIO2 fumigation corroded electrical components and some of the building structural steel,
but the extent of this corrosion could not be attributed exclusively to the BOTE testing because
the building had been subjected to CIO2 fumigations prior to the BOTE Project. The replacement
of wiring and electrical components could potentially add a significant cost to the CIO2
fumigation. Several rooms on each floor were not pre-populated with materials and are not
listed here.
354
-------
Based on estimates for the cost of these various items and labor cost estimates from INL for the
level of effort to replace the items removed, coupled with manufacturer estimates of time and
materials to replace the laminate floor and HVAC ductwork, the refurbishment cost was
calculated. The results are shown in Table 4-34.
The fumigation decontamination technologies did not result in any refurbishment cost, which is
somewhat artificial. The VHP® fumigation contractor did not remove any materials prior to their
fumigation, in spite of the fact that there is evidence of material demand for H2O2 that may
impact the ability to achieve the required concentration for effective decontamination if the VHP®
generation system does not have sufficient capacity'1001. Possibly due to the building's previous
experiences with CIO2 fumigation in the INL-1 and INL-2 studies'13' 14], there was already a
significant amount of rust and oxidation on some of the building surfaces that made it impossible
to fully assess potential damage due to any or all of the decontamination methods.
355
-------
Table 4-33. Materials pre-populated into building prior to spore dissemination.
E
o
0
K
110
109
108
107
106
105
104
103
213
212
211
210
209
208
207
206
Floor
1
Floor
2
Configuration
Office
Residential
Office
Residential
Office
Residential
Shop
Mail room
Residential
Office
Residential
Office
Residential
Office
Mail room
Shop
HVAC Duct
HVAC Duct
Length (ft)
21
21
21
21
21
21
10
21
21
21
21
21
21
21
21
21
200
200
e
+-
•c
§
10
10
10
10
10
10
12
10
10
10
10
10
10
10
10
10
CM
e
re
Ł
<
210
210
210
210
210
210
120
210
210
210
210
210
210
210
210
210
Laminate Floor
1
1
1
1
1
1
I
re
O
1
1
1
1
1
1
"re"
-2J.
*
M
Q>
H
O)
'53
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
"re"
-2J.
M
j*:
0
o
m
20
20
20
20
20
20
20
20
20
Binders (ea)
10
10
10
10
10
10
Mail (pieces)
20
20
"re"
•32,
•c
Q
1
1
1
1
1
1
1
' - indicates that the entire ceiling of that room was populated with ceiling tiles
Table 4-34. Summary of restoration costs.
Restoration Costs
Labor Cost of Post-Decontamination
Material Removal
Cost of Replacing Removed Items
Restoration Cost
Round 1 ($)
0
0
0
Round 2 ($)
7,854
46,934
54,788
Round 3 ($)
0
0
0
356
-------
4.7.4.3.3. Summary of Total Costs
Combining all of the above cost elements with a component to account for the presence of a
minimal 1C structure (command, safety) during the duration of the decontamination processes
results in the chart shown in Figure 4-70, using the "Medium" waste disposal difficulty scenario.
For Figure 4-70, the sampling costs for all three rounds were averaged into a single number
because there was an apparent reduction in sampling cost as the BOTE Project progressed,
likely due to improved efficiency of the sampling personnel. This minor reduction was judged to
be a bias, because the sampling efforts for the three rounds were identical; the total sampling
costs for the three rounds were therefore averaged. Overall, the cost of the pH-adjusted bleach
decontamination process was significantly higher than the cost of CIO2 fumigation, which was
slightly higher than the cost of the VHP® fumigation. The sampling effort for the BOTE Project
was significantly higher than what would be done in a real incident, so the contribution to the
overall cost of BOTE due to sampling is much higher than in a real situation.
$1,200,000
11C Cost
Restoration Cost
I Decon Cost
I Average Sampling and
Analysis Cost
VHP® pH-Adjusted CIO2
Bleach Process
Figure 4-70. Breakdown of overall cost contributions.
4.7.5. Summary and Discussion
A detailed cost analysis was performed on the use of three different decontamination
technologies on an in-building release of Bg spores (although cost estimates are based on
assuming an actual Ba incident). The following activities were performed:
357
-------
• Cost data were acquired on time and materials required to perform a wide variety of
sampling activities including AAS and surface sampling;
• Cost data were acquired on time and materials required to perform sampling preparatory
activities, training of sampling personnel, sample chain of custody activities; sample
packaging and shipping activities; and laboratory analytical activities;
• Cost data were acquired on time and materials required to prepare building entry teams
and perform personnel decontamination operations after the teams left the building;
• Cost data were acquired on time and materials required to use three different
decontamination technologies on a contaminated building and restore the building to its
condition prior to contamination;
• Cost data were acquired on quantities and characteristics of waste that was generated
during sampling and decontamination operations; and
• Some elements of the cost analysis were notional in nature, including: extrapolation of
analytical costs to account for increased effort of performing analyses in a BSL-3
environment as opposed to the BSL-2 environment that the BOTE Project samples were
analyzed in; estimation of fixed costs associated with management of waste potentially
contaminated with Ba; estimation of waste characterization sampling and analytical
costs; estimation of replacement costs for building items such as the HVAC ductwork
and laminate floors; and estimation of waste transportation costs and waste disposal
tipping fees.
Based on subsequent analysis of the cost data, the following major cost-related observations
are noted:
• Sampling and analysis are huge contributors to the overall cost. This statement must
add the caveat that this was a research operational testing and evaluation project. In a
real incident for a building this size, fewer samples would most likely be taken. In
addition, analytical costs are somewhat uncertain because they were based only on
tracking of labor efforts from one LRN laboratory;
• Fumigation with VHP® was the least expensive decontamination technology, with CIO2
fumigation being only slightly more expensive than VHP®. The pH-adjusted bleach
decontamination process was significantly more expensive to apply than either of the
fumigation technologies, largely due to waste management costs, which could potentially
be reduced by being able to use RCRA Subtitle D landfills and POTWs for disposal;
• Building refitting cost estimates have a significant amount of uncertainty. The rooms
were not populated with a large number of items, so assessing the amount of damage
due to the three decontamination technologies was not always possible. It is very likely
that the decision of whether or not items are replaced after decontamination will be
based on who is paying for the replacement;
• Waste management costs were a significant component of all three technologies,
particularly for the pH-adjusted bleach decontamination process. Waste characterization
sampling was the largest single component of waste management costs. In addition,
waste management costs could be reduced significantly if the State allows disposal of
358
-------
treated and/or decontaminated items in a RCRA Subtitle D landfill or allows the
wastewater to be sent to a POTW facility;
• The cost of personnel decontamination was a significant contribution due to the need for
the Decontamination Line personnel being on site during any time when entries into the
building are considered, whether or not those Decontamination Line operations staff are
actually performing any personnel decontaminations; and
• Almost all of the waste generated during the fumigations was a result of personnel
decontamination operations.
Based on the cost analysis, the following recommendations are noted:
• Identifying ways to reduce the sampling labor burden could result in significant cost
savings;
• Identifying ways to minimize waste could result in significant cost savings;
• Identifying ways to accomplish personnel decontamination in such a way as to minimize
the amount of waste generated could result in significant cost savings;
• Pre-incident waste management planning will be a critical aspect of achieving cost
savings for the remediation;
• Identifying ways to perform the remediation while minimizing the number of entries into
the contaminated facility in PPE will enable the personnel Decontamination Line
operations personnel not to be on site, resulting in significant cost savings; and
• Identifying alternate strategies for minimizing the number of waste characterization
samples could result in significant cost savings.
The following important caveats must also be noted:
• The cost analysis was performed based on the assumption that only a single
decontamination method would be used on a given building, which may not be the case
(i.e., different parts of a building may be decontaminated in different ways, such as
combining a fumigation with a pH-adjusted bleach process);
• The materials that were populated into the rooms were meant to be representative of the
types of materials that would be found in a residential, commercial, or shop setting; the
quantities that were present were probably on the low end of the quantities that would be
found in a real setting;
• Receiving permission from the appropriate regulatory authorities to landfill some or all of
the waste directly to a local RCRA Subtitle D facility without additional waste
characterization sampling could have a profound impact on reducing the waste
management costs. A key provision of this permission will hinge upon whether the waste
is considered to be hazardous, infectious, biohazardous, or solid waste. The
classification of the waste will greatly impact disposal costs.
4.8. Potential Spore Migration outside a Contaminated Building
Although this analysis was preliminary, spores evidently have the potential to migrate out of a
contaminated building and settle into the surrounding soil. These results are for primary release
within the HVAC system, and the design of the study did not allow for direct determination of
359
-------
when and how the spores escaped the building (i.e., whether samples with detectable Bg were
due to escape of spores from the building during the initial release or disturbance of the spores
due to sampling activity within the building). Additional studies would be needed to determine
the point of release from the building. The detection of Bg genetic material in outdoor soil
indicates another exposure medium (outdoor soil) that could lead to other potential exposure
routes for human receptors (e.g., dermal contact with contaminated soil, incidental ingestion of
contaminated soil, and inhalation associated with the reaerosolization of contaminated soil).
However, with the limited dataset, estimation of the quantity of spores that migrate is difficult.
Key findings and limitations are of the spore migration study are discussed below.
Key Findings
• The LOD of the method limited overall spore recovery.
The matrix LOD analyses showed poor spore recovery and large variations in detection. EPA
demonstrated a matrix LOD of 1E4 spores/g of sand when all 45 g of sand were utilized, while
USGS, using the standard protocol for the MO BIO Soil extraction kit with 0.25 g of sand, had a
matrix LOD of 1E6 spores/g of sand. At these detection limits, EPA was able to detect the spore
concentration disseminated on the first floor (target concentration 1E6 spores/ft2) of the building,
but not the second floor (target concentration 1E2). The results show that the matrix LOD for the
USGS was higher than the highest disseminated concentration of spores during the study. The
two-order of magnitude difference in matrix LODs could be attributed to a number of factors.
Undoubtedly, the large variation in sample amount contributed to the observed differences. EPA
utilized the entire 45 g sand aliquot, 180x more sand than USGS utilized, increasing the total
amount of DMA and potential PCR inhibitors present within the sand samples.
The differences in sand quantities would also have affected the variability in samples. For over
30% of the sample locations, the classification of the "A" sample among the categories listed in
Table 3-53 disagreed with the classification of the "B" sample. There were only 13 sample sets
in both decontamination technology rounds where both "A" and "B" samples yielded detectable
quantities of Bg DMA. As a result, deposition variability is assumed to be a major component to
total variability in the collected data. This sample variability may have been introduced in the
field or in the laboratory during processing. In the field, samples were placed in very near
proximity. However, an exact duplicate of a sample was not possible. Air flow and sample
placement, in addition to laboratory analysis variability, may therefore have led to the observed
differences.
While both laboratories used the same extraction kit, qPCR primers, and thermocycler program,
differences in thermocycler instruments may also have had a small impact on the outcomes.
The literature demonstrates that extractions (e.g., for DMA) from soil can be difficult and can
often result in low yields and/or low concentrations of microbial DMA. EPA's method of
extracting the total aliquot of sand produced results comparable to the results reported by Ryu
et al.[101], who detected 1E4 CFU/g of Ba spores in soil samples. Similarly, in an assessment of
eight soil studies, Herzog et al.[82] found the mean matrix LOD for Ba to be 1.2E4 CFU/g of soil
with a wide variation in detection limits for spiked soils, ranging from 1E-1 to 1E8 CFU/g of soil.
360
-------
• Decontamination agents were not qPCR inhibitors.
Samples placed within the building were collected post-decontamination to determine if
analytical interference occurred in the qPCR analysis due to the decontamination agents.
Because these samples were placed in the second floor hallway and on the first floor reception
area where decontamination took place, they were exposed to the decontamination agent. The
qPCR reactions using extracted template DMA from the original samples collected within the
building following decontamination were spiked with a known concentration of Bg DMA. These
results did not show significant qPCR inhibition. However, to more fully demonstrate the lack of
inhibition and help to understand the true nature of the apparent decreases noted after
decontamination, a laboratory-based exposure and analysis study would need to be done.
• Duration of exposure was not an indicative factor for the overall detection rate.
Though the samples were in place for differing amounts of time (Table 2-11), for this analysis
the same types and amounts of activities were assumed to occur during the exposure periods.
While this assumption is not strictly factual, this assumption allowed for the data collected over
the course of the BOTE Project as a whole to be compiled. One known deviation from this
assumption occurred during the pH-adjusted bleach decontamination process during Round 2.
As part of the pH-adjusted bleach decontamination process, all upholstered items (office chairs,
cubicle partitions, couches, carpet, etc.), and ceiling tiles were removed from the building and
replaced. This removal and replacement accounts for the substantial amount of time required
for the pH-adjusted bleach decontamination process, including drying (-11 days). During this
process, the large equipment door was left open for ease of access for the decontamination
personnel, and a large dumpster was positioned inside the secondary enclosure in front of the
building entry door (near trays 2 and 3). Removing and replacing these items from within the
building also increased the amount and duration of human activity occurring around the pH-
adjusted bleach post-decontamination samples. More spores were expected to be within the
sand samples following the pH-adjusted bleach decontamination due to the increased human
movement during that activity; however, the results herein did not show a significant difference
in the proportion of the sand samples with detectable Bg collected post-pH-adjusted bleach or
post-CIO2 decontamination. Therefore, the Bg concentration within the collected sand samples
was either not significantly impacted by human movement into and out of the building, or the
added openings in the secondary enclosure prevented spore sedimentation.
• Limitations to VHP® decontamination may have impacted the pH-adjusted bleach
pre-dissemination sampling.
A few surface samples collected post-VHP® decontamination resulted in detectable Bg, and two
of these surface samples were greater than 1E6 CFU/m2. However, the samples collected from
the same surfaces post-pH-adjusted bleach and post-CIO2 decontamination were below the limit
of detection (See Section 4.9). Because the pH-adjusted bleach background sampling occurred
directly following the decontamination with VHP®, the number of detectable sand samples
collected during the pre-dissemination sampling of the pH-adjusted bleach may therefore be
due to insufficient decontamination by VHP® within the building. The spores seen within the pre-
dissemination sand samples may be building spores re-distributed during the building reset.
361
-------
• A clear spore migration pathway could not be identified from the collected data.
Sterile sand samples were placed outside the test building within the secondary enclosure.
Though these samples were placed in strategic locations near the building doorways, no
statistical conclusions could be made regarding the migration pathway of the Bg spores. In a
recent study conducted by Van Cuyk et al. (2012)[1021, B. thuringiensis spores released outside
the building as an organic pesticide migrated into nearby buildings with the highest
concentrations near the entrances and the HVAC filters. The lack of statistical conclusions in the
BOTE Project study may have been due to the facility setup. A roof vent was unintentionally left
open during most of the project. This vent was sealed shut just prior to Round 3
decontamination, but after spore dissemination in Round 3 and all stages of the Round 1 and 2
were concluded. During an actual event, any open door, window, or vent would be a point of exit
for airborne spores. In addition, in a real scenario, a secondary barrier would probably not be in
place during the initial release, and spores could thus be carried much greater distances than
were studied here. Finally, the test area had been used during previous events and was
contaminated with significant levels of Bg spores. Every effort was made to mitigate the
influence of contaminated in situ soil; sterile sand samples were placed within large sampling
trays for this study. The sterility of the sand was checked before the exercise, and trip controls
opened briefly on site ensured sampler handling did not contaminate the collected sand
samples within the previously contaminated environment. The large trays protected the exterior
of the Petri dishes from direct contact with the ground, and their bright orange color made
personnel aware of their presence and reduced activity in their proximity. Regardless of these
efforts, nothing could be done to prevent spores within the in situ soil from being reaerosolized
by personnel elsewhere within the secondary enclosure.
• The reason for the decrease in measured viable spores in the sand samples removed
after dissemination compared to those removed post-decontamination is currently
unknown.
Though an overall increase and decrease was noted between dissemination and post-
decontamination sampling, no specific efforts were conducted to decontaminate the sand
samples. The reason for this finding is unknown. Unless the DMA was damaged by the
decontamination chemicals, the DMA should have been detected via the qPCR. One possible
explanation is the presence of physical processes. Weis et al. (2002)[491 demonstrated the
potential for secondary aerosolization of B. anthracis spores from minimal movement, leading to
a hypothesis that spores were carried out of the building by physical processes including
people, air movement, or electrostatic forces leading to a decrease in spores. However, the
actual reason for the decrease in spore concentration seen in the sand samples is still unknown.
4.8.1. Conclusions
The contaminated building was inside a secondary enclosure with controlled areas of entrance
and egress, so care must be taken when interpreting the results. The secondary enclosure may
reduce infiltration and exfiltration effects, which in turn reduces migration from the building. The
secondary enclosure interfered with the natural dissemination of spores to the surrounding
areas, causing any escaped spores to be deposited between the exterior building walls and the
interior secondary enclosure walls. Sampling was limited to within the secondary enclosure. In
362
-------
addition, unlike in situ soils, laboratory-prepared sand samples consisting of Petri dishes filled
with sterile sand were set out of the way of direct foot traffic to avoid disturbance of the trays
during the exercise and, consequently, direct foot tracking might not be adequately captured
within the collected sand samples. However, the sample trays were within the vicinity of human
movement and were therefore exposed to activity-related resuspension. Regardless of these
design limitations, this study clearly showed that spores can be carried outdoors following an
indoor release.
• Spores potentially migrate out from a contaminated building into the surrounding
area.
Though the data included in this report are preliminary, there is evidence that spores have the
potential to migrate out of a contaminated building and settle into the surrounding area. The test
area had previously been contaminated with Bg spores, so sterile sand samples were utilized
during this analysis to reduce contamination by in situ soil. While EPA data cannot be used to
give a quantitative estimate of spores that migrate and it should be noted that disturbances of
natural soil surrounding the area could have also contributed to detections in the samples, the
data suggest that spores have the potential to migrate from a contaminated building. Van Cuyk
et al.[1021 came to a similar but opposite conclusion in their study of the capabilities of spores to
migrate into a building following an outdoor release. During future exercises, the potential for
soil contamination exterior to the building must be considered.
• The current data set can give only qualitative information.
The degree to which residual soil contamination may be a significant exposure pathway will
require further evaluation. One standard curve was assessed post-analysis and used for all EPA
data rather than standards within each qPCR analysis. The resulting data are therefore not
suitable for quantitative comparisons. The LOD gleaned from this analysis was used as the cut-
off value for all averaged Ct results, which were then categorized according to the degree of
positive value. Due to the lack of standard curve data within each PCR run, detected samples
can be assigned only a degree positive (a qualitative assessment) and cannot be quantified.
Furthermore, the PCR assay detects DMA regardless of spore viability. Spores may be present
in "NDs" at levels below the LOD, so an ND result does not indicate the absence of viable Bg
spores.
• This study did not address a method for soil decontamination.
While a decrease in the number of samples with detectable Bg post-decontamination was seen,
care must be taken before attributing this observed decrease to the decontamination
technologies alone, as none of the sand samples were directly decontaminated during this
experiment. Only the interior of the building was decontaminated, and, therefore, no significant
decrease in detectable Bg DMA outside the building within the places sampled was expected
prior to the study. The decrease in samples with detectable Bg post-decontamination could
have been caused by chemical DMA degradation due to decontamination overspray or vapors
flowing out into the secondary enclosure. Regardless of mechanism, the qPCR results show
that there was a decrease in DMA concentration coming from either viable or non-viable spores
363
-------
within the assessed sand samples. Follow-on work would be required to determine the efficacy
of the decontamination technologies in a soil matrix.
• Alternative processing methods could be studied to improve detection of Bg DNA.
USGS directly extracted a smaller quantity of sand to achieve a matrix LOD of 1E6 spores/g
sand, while EPA indirectly washed the spores from a large amount of sand prior to DNA
extraction to achieve a 1E4 spores/g sand matrix LOD. Using an extraction and analysis method
with a lower matrix LOD could greatly improve results.
4.8.2. Future Considerations
The following considerations should be taken into account when planning similar or follow-on
projects.
4.8.2.1. Sampling Considerations
• Ensure that all samples are individually bagged before placement to prevent cross-
contamination;
• Research sturdier Petri dishes to prevent breakage;
• Consider shipping methods with increased padding to help prevent breakage; and
• Ensure that each stage has at least one trip blank and site blank.
4.8.2.2. Analysis Considerations
• This study points out a need for extension of contamination testing to the exterior of a
building of concern. Following an actual release within a building, human activity and
airflow can cause the agent of concern to be released to a wide area and potentially
affect a significant number of bystanders. Future studies could help determine the
probability and extent of contamination.
• One of the significant limitations of qPCR analysis is its inability to determine the viability
of the organism from which the DNA is extracted. RV-PCR is a recently developed
analysis tool for determining spore viability in minimal time'351. RV-PCR combines culture
with PCR detection to determine both the viability and DNA specificity of a targeted
microorganism. To our knowledge, a method for analyzing DNA in soil samples using
RV-PCR has not been described in the literature. The feasibility of analyzing soils for
DNA content using RV-PCR should be explored because this technique may yield
pertinent information regarding the concentration and viability of assessed samples.
• Matrix LODs in sand and soil are a limiting factor to determining soil contamination. A
more efficacious method for extracting DNA from soil samples is needed. Future work
utilizing various extraction kits and eluent concentrations might help identify a more
appropriate methodology. While this study utilized sand as the soil medium, future work
using other soil media would be of significant interest as each medium could have
different DNA extraction efficiencies. As an example, carrier DNA during the extraction
process has been found to increase the total DNA yield during low-concentration
extractions (<10,000 genomes/mL).[1031
• Inhibition caused by results from the decontamination chemicals/process needs to be
more fully demonstrated. To help understand the true nature of the apparent decreases
noted after decontamination, a laboratory-based exposure and analysis study should be
done. This essential experiment could be accomplished by spiking sand with Bg DNA
and spores (separate experiments), then exposing each in a chamber (with limited air
movement) to various decontamination agents, then re-analyzing. The idea would be to
364
-------
prove that the decontamination chemicals had no effect and/or destroyed/impacted the
spore/DNA, yielding the lowered amounts seen after decontamination.
The possibility of physical processes within the secondary enclosure that decrease
spore presence needs to be addressed. In particular, if spores bind to surfaces such as
the secondary enclosure walls, the exterior of the secondary enclosure, or personnel
clothing, other areas of concern could be implicated during an actual event.
4.9. Development of an Exposure Assessment Plan
Following the Amerithrax events of 2001, there has been much interest in better addressing the
risk of inhalational anthrax from incidents involving the release of Ba spores into the
environment. However, no standard methodology exists for estimating the risk of exposure to 6
a spores or to estimate exposure concentrations based on site-specific sampling data. The
BOTE Project Phase 1 provided a rich dataset of sampling data to assess usability of collected
data at a contaminated site for developing a methodology for conducting an exposure
assessment. Because inhalational exposure to Ba spores is a significant health risk, this
exposure pathway will be the primary focus for the development of a site-specific inhalation
exposure assessment plan which could be used to perform a qualitative assessment of
exposure using semi-quantitative/qualitative data generated in a field setting. The intent is to
conduct an exposure assessment, only a part of the risk analysis process which generally
consists of hazard identification/problem formulation, dose assessment, exposure assessment,
and risk characterization. A preliminary methodology is being developed to characterize
potential exposure for re-entry into a building contaminated with Bg spores, before and after
decontamination. Key assumptions for handling the analytical data and exposure calculation are
being derived from chemical risk assessment guidelines and standard microbiological analytical
practices. Semi-quantitative/qualitative data on indoor surface and air samples that were
collected during the BOTE Project will be used for the inhalation exposure assessment.
One of the greatest challenges of risk assessment is addressing the uncertainties associated
with the process from sample collection to interpreting the analytical results combined with the
physical site characteristics and the variability of the exposed population(s). Analysis of the
BOTE Project dataset is the first attempt to identify the uncertainties associated with the
calculation of exposure (lack of knowledge of recovery efficiencies of sampling methods;
handling of ND data; use of reaerosolization factors from the literature; and choice of surrogate
agent used for the BOTE Project). Thus, the analysis of this dataset will provide a qualitative
assessment of inhalation exposure, but it is still critical to moving the science forward and to
determine gaps/needs for quantitative exposure assessments. While exposures calculated
using this derived exposure assessment methodology are limited by uncertainties, the use of
the data to address analytical method limitations and run sensitivity analyses is very beneficial.
This initial exposure assessment will also allow the objective evaluation of the assumptions and
decisions that were made during the planning process.
4.9.1 Exposure Assessment Methodology
The exposure assessment methodology is being developed to assess the exposures of adult
receptors to Bg spores via inhalation of Bg spores in indoor air including the potential
365
-------
reaerosolization of spores from indoor surfaces. The methodology will include considerations for
qualitative determination of intake doses of Bg spores via inhalation associated with re-entry
into the BOTE Project facility following Bg spore dissemination both before and after
decontamination for various exposure settings:
• Residential; and
• Office, mailroom, and industrial.
The exposure assessment will utilize analytical data (viable and culturable Bg spores as
measured by CPU) for air and surface samples collected before and after the application of
each decontamination technology (i.e., pH-adjusted bleach, fumigation with CIO2 gas, and
fumigation with VHP®).
The initial exposure assessment will also allow the objective evaluation of the assumptions and
decisions that were made during the planning process. Many of the exposure assessment
assumptions and decisions were based on typical chemical risk assessment approaches and
standard microbiological analytical practices. Key assumptions and decisions guiding the
exposure assessment methodology include:
• Using "0" for values below the historic "quantitation limit" and assessing sensitivity
around the "0";
• Using ProUCL software'1041 (that assumes continuous data) rather than models that
assume discrete data, e.g., exponential or beta-Poisson, to analyze discrete (CPU) data;
• Using the 95% upper confidence level of the mean as the exposure point concentration;
• Performing analyses with spread plate data alone, in the absence of available filter plate
data;
• Using Bg as a surrogate for Ba, rather than the more closely related B. thuringiensis;
• Using reaerosolization factors calculated for non-fig organisms or particles; and
• Using CPU results "as is"; adjustments were not made to reflect potential recovery
inefficiencies of the sampling methods
The inhalation exposure pathways identified for evaluation in the BOTE Project are illustrated in
the conceptual site model (Figure 4-71). Briefly, the two complete exposure pathways that will
be evaluated for the assessment include: 1) Bg spores are released in the BOTE Project facility
via aerosolization, contaminating the indoor air through which adult receptors are potentially
exposed via inhalation; and 2) Bg spores are released in the BOTE Project facility via
aerosolization and then become deposited onto indoor surfaces and subsequently reaerosolized
(via disturbance of the contaminated surface), re-contaminating the indoor air through which
adult receptors are potentially exposed via inhalation. Additional information on potential
exposure pathways is provided in the following paragraphs.
The source of contamination was Bg spores released as an aerosol within the BOTE Project
facility. Post-dissemination (pre-decontamination) sampling took place the day after Bg spore
dissemination (approximately 17 to 24 hr after dissemination). With the exception of rooms
101A and 102, this assessment will not focus on inhalational exposure that might occur during
the actual dissemination of Bg spores, but rather, inhalational intake doses will be estimated
366
-------
after the Bg spores had a chance to settle. Baron et al.[911 reported on the development of an
aerosol system for depositing Ba spore particles on surfaces, noting that most spores settled
within a few hours inside a chamber set up with a HEPA filter ventilation system . More
specifically, more than 99% of 1- to 2-um particles would settle within 10 hr. Inhalation
exposures will be determined on a limited basis (i.e., Rooms 101A and 102) during each
dissemination event.
Following the initial introduction of aerosolized Bg spores, the release mechanisms included
deposition and reaerosolization (e.g., disturbing the settled spores by walking across the
contaminated floor or wiping a contaminated desk surface). A portion of the Bg spores possibly
remained airborne following dissemination. Indoor air was the identified exposure medium and
was sampled directly for Bg spores in rooms 101A and 102. Indoor surfaces throughout the
building were also sampled for deposited Bg spores, and these data will be used to quantify
spores that might reaerosolize into indoor air.
The scope of this exposure assessment will be limited to the inhalation route of exposure in
adults and will not address children or pet receptors. Deposition of Bg spores on indoor surfaces
and the associated potential routes of exposure via dermal contact and incidental ingestion will
not be evaluated in this exposure assessment. Outdoor air concentrations of Bg spores were
not measured during this exercise. The assessment will focus only on Bg spores; post-
decontamination exposures to potential decontamination technology-related chemicals or
chemical by-products will not be evaluated.
Source
Airborne
Bg Spores
Released in
the BOTE
Facility
Release
Mechanism
Reaerosolization
Deposition onto
Indoor Surfaces
Aerosolization*
Exposure
Medium
Route of
Exposure
Receptor
Figure 4-71. Conceptual site model.
Inhalation exposures during Bg spore dissemination (aerosolization) will be assessed only in
Rooms 101Aand 102.
367
-------
A summary of available data differentiated by exposure unit that will be used to calculate
inhalational intake doses is shown in Table 4-35. Each exposure unit is a room or group of
rooms to which an adult receptor is exposed and for which an exposure point concentration
(EPC) is calculated and applied over the exposure time (ET). Table 4-35 also notes the
exposure setting (e.g., residential or office) associated with each exposure unit. Indoor air spore
concentration measurements were available only for two rooms representing an office exposure
setting. Spore concentration measurements from surfaces were available from many rooms
representing a variety of exposure settings and acquired using a variety of sampling methods.
4.9.2 Future Steps
The selected approaches represent an initial look at a methodology which could be used for a
qualitative inhalation exposure assessment and should only be considered preliminary. The
approaches selected for this initial methodology were documented in the associated exposure
assessment plan and the exposure assessment QAPP. The exposure assessment plan will
guide the future evaluation of calculation of EPCs using the BOTE Project data. The evaluation
will also look at sensitivity around the assumptions that were selected, and alternative
approaches will be considered to revise the exposure assessment plan. Potentially "better"
decisions and alternatives may be identified for use in future studies or real world events.
368
-------
Table 4-35. Data availability by medium and exposure unit per decontamination round.
Medium
Exposure Unit
Exposure
Setting
Data Availability
Pre-Decontamination
Post-Decontamination
Indoor Air
Rooms 101A; 102; and 101A and 102
combined
Office
Concentration of viable Bg spores (CPU)
measured by culture, collected by SKC at 4
time points*
Concentration of viable Bg spores
(CPU) measured by culture, as
collected by SKC at one time point
Indoor
Surfaces
Rooms 105; 107; 109; 209; 211; 213;
and all residential settings combined; all
first floor rooms combined; and all
second floor rooms combined
Residential
Quantity of viable Bg spores (CPU) measured
by culture, as collected by sponge, swab,
vacuum, and wipe (rooms 101A and 102 only)
Quantity of viable Bg spores (CPU) recovered
from RMC and settling plate (second floor
rooms only) samples and measured by culture,
which served as quality control samples
confirming Bg spore dissemination
Quantity of viable Bg spores (CPU)
measured by culture, as collected
by sponge, swab, vacuum, and
wipe (rooms 101A and 102 only)
Indoor
Surfaces
Rooms 101; 101A; 102; 103; 104; 106;
108; 110; 201; 201 A; 202; 203; 203A;
204; 205; 206; 207; 208; 210; 212;
copier room (floor 2); corridor + lobby
(floor 1); hallway (floor 2); janitor closet
(floor 2); mechanical room (floor 1);
mechanical room (floor 2); men's
bathroom (floor 1); men's bathroom
(floor 2); stairwell (floor 2); women's
bathroom (floor 1); women's bathroom
(floor 2); and all office, mailroom,
industrial settings combined
Office,
mailroom, or
industrial
Quantity of viable Bg spores (CPU) measured
by culture, as collected by sponge, swab,
vacuum, and wipe (rooms 101A and 102 only)
Quantity of viable Bg spores (CPU) recovered
from RMC and settling plate (second floor
rooms only) samples and measured by culture,
which served as quality control samples
confirming Bg spore dissemination
Quantity of viable Bg spores (CPU)
measured by culture, as collected
by sponge, swab, vacuum, and
wipe (rooms 101A and 102 only)
* Pre-decontamination air sampling was conducted at four different times including: before Bg spore dissemination, during Bg spore dissemination, before pre-
decontamination surface sampling, and during pre-decontamination surface sampling (intended to capture reaerosolization of Bg spores associated with human
activity).
369
-------
5. STATISTICAL ANALYSIS OF PRE- AND POST-
DECONTAMINATION SAMPLING RESULTS, DECONTAMINATION
EFFICACY, AND COST ASSESSMENT
5.1. Introduction and scope
The purpose of this analysis was to evaluate different decontamination methods and to assess
the relationship between these decontamination methods and other variables, like sampling
method and room type.
Statistical analyses were performed and visual representations created to provide insight into
these key issues. Pre-decontamination analyses were also performed to study how well
contamination was distributed across the building for each of the three events. The other test
factors, like sampling method, were also investigated to look for significant effects in the
recovery of the contamination. Post-decontamination analyses were performed to study the
effectiveness of the three decontamination methods, as well as how the other factors affected
the decontamination and recovery process. The effectiveness results will be contrasted with the
cost analysis results to help explain the overall differences between the three decontamination
methods.
5.2. Summary of Key Findings
The key results found within the statistical analyses are listed in this summary.
• Spatial analyses showed that there was no significant spatial correlation after the
contamination occurred (pre-decontamination). Usually sample results taken close to
one another were no more alike than samples that were taken far apart, suggesting that
the dissemination technique effectively dispersed the spores within the building.
• For the post-decontamination VHP® results, the spatial analyses did indicate some
correlation between sample results that were closely located (within 5 to 20 in), possibly
due to small areas that were not decontaminated as effectively as other areas.
• The statistical analysis confirmed that the contamination was applied so that the first
floor was significantly more contaminated than the second floor for each of the three
events (Figure 5-4).
• Sampling of the contamination prior to decontamination showed that the vacuum socks
reported much less contaminant than swabs and sponge-sticks (Figure 5-5). There were
also differences in the amount of contaminant found on the sampled objects (floor, desk,
vent, etc.) (Figure 5-7); however, these differences may be due in part to the different
sampling methods (see Appendix C for the details on the surface sampling protocols).
• There were no significant differences found in contamination for the different room types
(Figure 5-6). The amount of contaminant sampled in each room type was not always
consistent across the three rounds. The amount of contaminant sampled prior to the
VHP® decontamination was higher than the amount of contaminant sampled prior to the
370
-------
other two decontamination methods for each of the room types except for the residential
rooms, in which the amount of contaminant sampled prior to the VHP® decontamination
was the lowest amount.
• After decontamination, VHP® had significantly fewer ND (clean) samples (83.8% clean)
than pH-adjusted bleach and CIO2 (both just over 99.6% clean) (Table 5-2).
• After the VHP® decontamination, the clearance samples showed that the residential
room decontamination efficacy was significantly lower than the other room types (Table
5-6).
• After the VHP® decontamination, the clearance samples showed that the
decontamination efficacy according to the swab samples was significantly lower than for
vacuum socks and sponge-sticks.
• The VHP® and CIO2 methods were very similar in cost (between $800,000 and
$900,000), while the cost of the pH-adjusted bleach was nearly $1,200,000. The cost
differential between the VHP® and CIO2 methods was due mostly to the difference in the
fumigation contractor costs; the cost differential between the fumigations and the pH-
adjusted bleach method was largely due to waste management costs.
• CIO2 was the least expensive of the most effective decontamination methods (Figure
5-10).
5.3. Spatial Distribution Analyses
To evaluate whether the general spatial dissemination and post-decontamination residuals were
similar between the three events, a normalized spatial modeling analysis was performed.
Because the sample results are so dependent on the sampling technology used and on other
factors, the data were normalized to allow all results to be used in this spatial distribution
analysis. Further detail concerning how the data were normalized can be found in Appendix L.
Although building structures (walls, hallways, doorways) and contaminant dissemination
pathways (primarily via vents and open doorways) affect the spatial correlation of sample
results, kriging was used to explore the gross spatial nature of the contamination for each of the
three events. Further detail about how kriging was applied to the data can be found in Appendix
L. The spatial kriging estimates are graphically displayed in plots produced using VSP (Visual
Sample Plan)'1051. These plots were produced for each of the three events, as well as each floor,
for a total of six plots. Figure 5-1 shows the spatial kriged estimates for the first floor prior to the
pH-adjusted bleach decontamination. The full set of six plots showing the spatial nature prior to
each of the three contaminations on the first and second floors is found in Appendix L (Figures
L.1 -L.6).
Each spatial distribution plot is accompanied by a plot showing the variation associated with
those estimates, called a variogram plot. A variogram plot shows the variability (y-axis) of
sample values that are a certain distance apart (plus/minus a tolerance). This distance is shown
as increasing on the x-axis. The points on the plot show the lag distances that were selected to
make empirical measurements of the variability. The line represents the modeled relationship
between distance and variability. Figure 5-2 shows the accompanying variogram plot for the first
floor contamination prior to the Round 2 (pH-adjusted bleach) decontamination. This plot shows
that there is no spatial correlation. In other words, sample results taken close to one another are
371
-------
no more alike than samples that are taken very far apart. These results are very similar for the
second floor and the other decontamination events. The full set of six plots showing the
variogram plots for the data prior to each of the three contaminations on the first and second
floors is found in Appendix L (Figures L.7 - L.12). For Ba, Bg and similar contaminants, these
results are not unexpected, suggesting that any hotspot-like deposition model would not be
appropriate for a biological release.
Spatial distribution analyses were also performed for the VHP® post-decontamination data.
Figures L.13 and L.14 in Appendix L show the spatial nature of the remaining contamination on
the first and second floors. Figures L.15 and L.16 in Appendix L show the variogram plots for
each floor. For the post-decontamination VHP® results, there may be some spatial correlation
between sample results located very close to one another (within 5-20 in of each other). This
correlation could be feasible if the decontamination efficacy is somewhat patchy over the
building, or certain small patches were not decontaminated as effectively as others.
Figure 5-1. Kriged estimates (scale explained in Appendix L) of the contamination on the
first floor prior to the pH-adjusted bleach decontamination (square = vacuum sock,
diamond = swab, circle = sponge-stick).
372
-------
IfUK-
160K-
150K-
14J"IK
130K-
120K-
,. 110K-
2 100K-
Ł 90K-
1 80K-
/n 70K-
fidk
An u
JDK
inif
20 K
10K-
OK
(
•
*
X-" • • *
/
/
/
/ -
100 200 300 400 500 SOO 700 800 900 1000 11
Distance
DO
Figure 5-2. Variogram for the first floor contamination prior to the pH-adjusted bleach
decontamination.
5.3.1. Pre-Decontamination Analyses
The pre-decontamination data were analyzed to determine which factors had a significant effect
on the amount of contamination that was recovered and analyzed. The factors investigated
included:
• Sampling round with a particular decontamination method (VHP®, pH-adjusted bleach,
and CIO2);
• Floor (1st Floor and 2nd Floor);
• Room type (commercial, mailroom, residential, shop, and not assigned);
• Sampling method (vacuum sock, swab, and sponge-stick);
• Sampled object (bed, cabinet, ceiling, chair, couch, countertop, desk, file cabinet, floor,
mail slot, monitor, nightstand, return vent, shelves, sink, stove, supply vent, table, wall,
and workbench); and
• Laboratory (eight unidentified laboratories were used).
ANOVA was performed to determine if there were significant differences between the levels of
each of the listed factors and any possible interactions between them. A key assumption with
ANOVA is that the data are symmetrically distributed; however, contamination values, measured
in CFU/cm2, were positively skewed. Two different transformations were made to the data so
that analyses would be performed on more symmetrically distributed data. Analyses were
performed on the ranks of the data (a non-parametric approach) and the log (CFU/cm2). As
expected, results were generally similar between the two analysis methods. When performing
ANOVA, p-values less than 0.05 indicate that there are significant differences in sample results
between the levels of the factor (with 95% confidence). In the case of interactions, p-values less
than 0.05 indicate that the sampled contamination level changes inconsistently when looking at
two of the factors (with 95% confidence). Only factors that were significant, nearly significant, or
373
-------
in a significant interaction are included in the reported analyses, below. Plots will be used to
show the differences in the levels of each factor, as well as the interactions between factors.
The ANOVA results are found in Table 5-1. Analyses performed on the ranks and on the
log(CFU/cm2) showed similar results. The p-values from the ranks analysis are reported here.
The following conclusions were drawn:
• There were no significant differences in the sampled contamination levels between the
sampling events (p-value = 0.5721). The box plots in Figure 5-3 show that the means
and medians between the three sampling events were similar.
• As expected, the first floor was significantly more contaminated than the second floor (p-
value < 0.0001). Figure 5-4 confirms the differences between the floors.
• There was a significant difference in the amount of contamination found in the five
different room types (p-value < 0.0001). Figure 5-6 shows that the amount of
contamination found in the residential room type was smaller than the others.
• There was a significant difference in the amount of contamination observed by each of
the three sampling methods (p-value < 0.0001). The vacuum sock reported much less
contaminant than the other two methods, as shown in Figure 5-5.
• The amount of contaminant observed on each of the objects was significantly different
(p-value < 0.0001). The box plots in Figure 5-7 show how the many objects differed.
Objects were usually sampled using the method that was most appropriate, so only a
few objects were sampled using more than one sampling method. This difference in
sampling can confound the object effect, meaning that some, or possibly even most, of
these differences may be due more to the differences observed in sampling method
results. The sampling was performed to be similar to an actual event, so it was not
feasible to design an experiment that would separate this confounded effect.
• The interaction between sampling event and sampling method was significant (p-value =
0.0144). An interaction plot of the means is shown in Figure 5-8. The vacuum sock
results were consistent across the three events; however, the sponge-stick results were
much higher for the VHP® pre-decontamination event, while the swab results were much
higher for the CIO2 pre-decontamination event.
374
-------
Table 5-1. ANOVA results for the pre-decontamination data. Due to non-normality of the
data, analyses were performed on ranks and log (CFU/cm2). (Only factors that were
significant or in a significant interaction were included in the final analysis.)
Factor / Interaction
Sampling Event
Floor
Room Type
Method
Object
Sampling Event x Method
p-Value Based on Ranks
0.5721
< 0.0001
< 0.0001
< 0.0001
< 0.0001
0.0144
p-Value Based on log (CFU/cm2)
0.4024
< 0.0001
0.0350
< 0.0001
< 0.0001
0.0042
375
-------
o
o
o
o
o
m
i
o
o
OJ
I
LL
O
O .
o .
T
T
Amended Bleach Pre-Decon CIO2 Pre-Decon
VHP Pre-Decon
Figure 5-3. Box plots of extracted contamination (CFU/cm2 in log scale) for each pre-
decontamination round (middle box represents middle 50% of data, middle line in box
represents median, red square represents mean).
376
-------
o
o
CM
LJ_
O
O
O
o ,
o .
§
8
I
Floor 1
Floor 2
Figure 5-4. Box plots of extracted contamination (CFU/cm2 in log scale) for each floor
across all three pre-decontamination rounds (boxes represent middle 50% of data, middle
line in box represents median, red square represents mean).
377
-------
o
8
8 -
1
I
o
o
o
o
o
s
U-
O
o .
tf> -
Sock Vacuum
Sponge Stick
Swab
Figure 5-5. Box plots of extracted contamination (CFU/cm2 in log scale) for each sampling
method across all three pre-decontamination rounds (boxes represent middle 50% of
data, middle line in box represents median, red square represents mean).
378
-------
0
o
0
0
o -
o
o -
M 10 ~
u.
0
0 _
10 -
u
800 _
8
i
l
f
1 §
^ 0
1 °
J_
8
0
t
1
1
1 l
1
1
Commercial Mailroom Residential Shop Unassigned
Figure 5-6. Box plots of extracted contamination (CFU/cm2 in log scale) for each room
type across all three pre-decontamination rounds (boxes represent middle 50% of data,
middle line in box represents median, red square represents mean).
379
-------
CFU/cm2
Bed -
Blank -
Ceiling -
,-, _ , | ,
Couch —
Countetlop —
Desk Drawer —
Fife cabinel —
Lighl Fixture Inside —
Mailslol -
Monitor —
NighteUnd —
Return \fen1 —
Return \fent Exterior —
Stove —
_
TV -
Workbench -
• | H O
0 O C
X |
V I--
• 1
IB 1- - JO
• |- - 1 U
1- - --I
• |- - - H
1. H
\----i | BOO
1 !•
h-m-H
H • h--H
1 J • 1 1
1 "II
H
1 • 1
| f---H 0 0
*
I | • 0 0 0
If • - . O
I • H
•1 h H
h— | | • \--l
I • I
| • I'
1 • 1
J I H L
11 • r
1 ; n
T 1 • H O
*
Figure 5-7. Box plots of extracted contamination (CFU/cm2 in log scale) for each sampled
object across all three pre-decontamination rounds (boxes represent middle 50% of data,
middle line in box represents median, red square represents mean).
380
-------
CNJ
o
m
8
CM
O
ID
O
O
o —
Amertd&d Bleach Pre-Decon
CIO2 Pre-Decon
VHP Pre-Decon
I
.0
Figure 5-8. Interaction Plot of Sampling Method and Pre-Decontamination Rounds
Measured in CFU/cm2.
5.4. Post-Decontamination Analyses
Based on sample results after decontamination, the pH-adjusted bleach and CIO2
decontamination methods were obviously more effective than the VHP® method. No formal
statistical tests were required or performed to support this conclusion. A simple data summary is
provided in Table 5-2 to summarize the effectiveness of the three decontamination methods.
However, several statistical analyses were performed on the VHP® test results to explore the
effects of various factors on the VHP® decontamination effectiveness.
381
-------
Table 5-2 summarizes the effectiveness of each decontamination method by listing the number
of samples that reported contamination (positive) and the number of samples that reported no
contamination (ND). VHP® had significantly more positive samples, with 83.8% of the samples
coming back clean (ND), compared to over 99.6% for pH-adjusted bleach and CIO2. Samples
taken during the pH-adjusted bleach and CIO2 decontamination methods were ND in all cases,
except for one positive sample each. Table 5-3 lists the information about each of the positive
samples for pH-adjusted and CIO2.
Table 5-2. The decontamination effectiveness for each decontamination method.
Decontamination
Method
Amended Bleach
CIO2
VHP®
# of Samples Not
Clean (Positive)
1
1
52
# of Samples
Clean (ND)
268
311
269
% of Clean
Samples
99.63%
99.68%
83.80%
Decontamination
Efficiency
0.999998
0.999972
0.982778
Table 5-3. The sample characteristics of the pH-adjusted bleach and CIO2 samples that
showed positive growth.
Decon Method
pH-Adjusted
Bleach
CI02
Barcode
3085
4285
Floor
1st
«nd
Room
Men's Bathroom
Room 21 3
Method
Sponge Stick
Vacuum Sock
CPU
16
17
The VHP® post-decontamination data were analyzed further to study the effects of the other
factors during the testing. Three different response measures of decontamination efficacy were
analyzed. These response measures are defined as follows:
• DE = Decontamination efficiency = pre~post where the post values (measured in
CFU/cm2) are the post-VHP® decontamination values matched to the mean of those pre
values taken during the pre-VHP® decontamination event that were within 1 ft of the post
sample location using the same sampling method. In the rare occurrence that the post
value was higher than the pre value, the resulting negative value was set to zero. Only
those post values with a nearby pre value were included in this part of the analysis. The
decontamination efficiency measures the proportion of contamination in a sampling
location that was removed during decontamination.
• Log(DE) = Log(decontamination efficiency) = log rre~po j. In the few cases where
the pre~post value was zero, the value 0.0001 was substituted so that the logarithm
pre
could be calculated.
• Percentage clean = - where c is the number of samples that were clean (ND) and n is
the total number of samples.
382
-------
The first two response measures, DE and log (DE), are measured on a continuous scale, so
ANOVA was used to evaluate the effects of various factors. Each factor, previously listed in the
pre-decontamination analysis section, was included in the analyses, as well as interactions
between factors. Only those factors and interactions that were significant or nearly significant
are included in the reported results below. The decontamination efficiency data were not
normally distributed. Non-normality can have an adverse effect on the results using ANOVA. For
this reason, the log decontamination efficiency values were also analyzed and the results
reported. Taking the logarithm of skewed data is a common way of removing skewness from the
data distribution, resulting in a more symmetrically distributed data set.
During the analysis of DE, it became apparent that there was a single outlying sample that was
having a heavy influence on the analyses. This sample was a swab, taken on the 2nd floor, in a
room configured as a commercial facility. The value for this outlying sample was 9.7E3 CPU with
the next largest swab sample at 1.7E2 CPU. ANOVA was performed on the data with and
without this outlying value. Both sets of results are shown in Table 5-4.
The third response measure, percentage clean, was formed by the binomial response of
whether or not the samples returned clean (ND). Fisher's Exact Tests were performed to look for
significant differences in the percent of samples that returned clean for each of the factors of
interest. Interactions were not studied for this response measure.
The analyses were performed to determine which factors and interactions between factors had a
significant effect on decontamination. The p-values from these analyses are shown in Table 5-5.
p-Values less than 0.05 indicate that the factor or interaction has a significant effect on
decontamination with 95% confidence. The following conclusions were made about each of the
factors:
• Floor
o Fisher's Exact Test - These results showed a strong difference between floors when
looking at percentage of samples clean (ND) (p-value < 0.0001). Table 5-5 shows
that the 1st floor had only 75% samples clean after the VHP® decontamination, while
the 2nd floor had 95% clean.
o ANOVA (including outlier) - These tests showed a significant difference between
floors when looking at DE (p-values of 0.0094 from the DE ANOVA and 0.0125 when
analyzing the log(DE)). Table 5-5 shows DE was actually higher for the 1st floor,
which had received a much larger amount of contamination.
o ANOVA (excluding outlier) - When the outlier on the 2nd floor is removed, the DE
becomes a little higher for the 2nd floor, as should be expected, and the difference
between floors becomes non-significant (p-values of 0.7993 and 0.4211).
• Room Type
o Fisher's Exact Test - No statistically significant differences were found in the
percentage of samples clean among the different room types (p-value = 0.1166).
o ANOVA (including outlier) - The ANOVA tests showed no statistically significant
differences between the different room types with a significance level of 0.05 (p-
values of 0.0956 and 0.1869 when analyzing the logarithm). Some of the room types
did not have a lot of samples (mailroom and shop) and, therefore, the statistical
power to find differences was lower.
383
-------
o ANOVA (excluding outlier) - When the outlier was removed, the ANOVA on DE
showed a significant difference (p-value=0.0228), while the ANOVA of the log(DE)
showed a nearly significant difference (p-value=0.0547). Table 5-6 displays the mean
DE and percent of samples clean for each room when VHP® decontamination was
used. The mean DE for the commercial rooms increased from 0.979 to 0.995 when
the outlier was removed. The residential rooms had the lowest DE mean (0.944) and
percent clean value (76.67%), while the other four room types had very similar DE
means near 1.000 (with the outlier removed).
Method
o Fisher's Exact Test - When analyzing the percent clean, Table 5-7 shows that the
vacuum sock (78.57% clean) was lower than sponge-sticks (86.59% clean) and
swabs (86.67% clean), but this difference was not statistically significant according to
the Fisher's Exact Test (p-value=0.1851).
o ANOVA (including outlier) - The ANOVA tests showed a highly significant difference
between the sampling methods with respect to DE (p-value < 0.0001). Table 5-7
shows that the vacuum socks and sponge-sticks were very similar, while the mean
DE for the swabs was significantly lower.
o ANOVA (excluding outlier) - The swab mean DE increased from 0.874 to 0.932
when the outlier was removed. This difference was still significant when analyzing
the log (DE) (p-value=0.0226).
Floor x Method Interaction
o ANOVA (including outlier) - The ANOVA tests showed a significant interaction
between floor and method (p-values of 0.0182 and 0.0117). Figure 5-9 shows a plot
of this interaction. The plot shows that the Swab-2nd floor mean DE was heavily
influenced by the outlier (it is much lower than the others).
o ANOVA (excluding outlier) - When the outlier was removed, this interaction was no
longer significant (p-values of 0.3697 and 0.5143). The outlier was solely responsible
for the significant interaction that had been revealed. The dotted line on Figure 5-9
represents the swab means for both floors without the outlier, showing how much
influence the single outlier has.
As can be seen from the results, the swab-second floor outlier data point is highly influential.
There were no explanations for this data point and the data around it did not confirm this point
as a hotspot of contamination. With this data point included, the mean DE for swabs on the
second floor was unusually low. This result was counter-intuitive. The expectation was that
decontamination efficiency should be lower (or at least similar) for the first floor, because the
first floor was more heavily contaminated. This pattern was also observed for the vacuum socks
and sponge-stick samples. Also, analyses including this data point did not conclude that there
were significant differences between the room types, although the mean DE values seemed to
show that the mean residential DE value was lower than the others. Once the outlying data point
was removed, this difference was determined to be significant. For these reasons, the analyses
were performed with and without this data point, and the conclusions mentioned refer only to the
analyses without this data point.
384
-------
Table 5-4. ANOVA and Fisher's Exact Test results of the VHP® post-decontamination
data. (Only factors that were significant or close to significant were included in the final
model.)
Factor /
Interaction
Floor
Room Type
Method
Floor x Method
DE
(Decontamination
Efficiency)
ANOVA
p-value
0.0094
0.0956
<0.0001
0.0182
DE (outlier
removed)
ANOVA
p-value
0.79931
0.0228
0.0947
0.36971
Log (DE)
ANOVA
p-value
0.0125
0.1869
<0.0001
0.0117
Log (DE)
(outlier
removed)
ANOVA
p-value
0.421 11
0.0547
0.0226
0.51431
% Clean
Fisher's
Exact
p-value
<0.0001
0.1166
0.1851
1 This factor or interaction was highly nonsignificant for this analysis, so therefore this factor was not included in the
final model.
Table 5-5. VHP® post-decontamination results for each floor.
Floor
1st Floor
2nd Floor
Mean
Decontamination
Efficiency
0.985
0.979
Mean
Decontamination
Efficiency
(outlier removed)
0.985
0.990
# of Samples
Not Clean
45
7
# of Samples
Clean
132
137
% of Clean
Samples
74.58%
95.14%
Table 5-6. VHP® post-decontamination results for each room type.
Room Type
Commercial
Mailroom
Residential
Shop
Unassigned
Mean
Decontamination
Efficiency
0.979
0.999
0.944
1.000
0.998
Mean
Decontamination
Efficiency
(outlier
removed)
0.995
0.999
0.944
1.000
0.998
# of Samples
Not Clean
11
3
14
0
24
# of Samples
Clean
70
21
46
20
112
% of Clean
Samples
86.42%
87.50%
76.67%
100%
82.35%
385
-------
Table 5-7. VHP® post-decontamination results for each sample method.
Sample
Method
Vacuum Sock
Sponge-Stick
Swab
Mean
Decontamination
Efficiency
0.987
0.992
0.874
Mean
Decontamination
Efficiency
(outlier removed)
0.987
0.992
0.932
# of Samples
Not Clean
24
24
4
# of Samples
Clean
88
155
26
% of Clean
Samples
78.57%
86.59%
86.67%
o
o
in
o
o
01
CM
E
O
10
CD
O
CO
-•*•• Swab (outlier removed)
• Sock Vacuum
• Sponge Stick
* Swab
I
r-j
I
Figure 5-9. Interaction plot of sampling method and floor for the VHP® events (y-axis is
the mean DE for each combination of the factors).
386
-------
5.5. Conclusions from Statistical Analysis
The post decontamination data showed a clear difference between the three decontamination
methods. Both pH-adjusted bleach and CIO2 performed well, with only one sample each
showing positive growth (percent clean rates above 99.6%). VHP® had 52 samples showing
positive growth (83.8% clean rate), resulting in a much lower percent clean rate than the other
two methods. Figure 5-10 shows a summary of the estimated overall costs for each of the
decontamination methods (note that the y-axes in Figure 5-10 have been truncated). The VHP®
and CIO2 methods were very similar in cost (between $800,000 and $900,000), while pH-
adjusted bleach cost nearly $1,200,000. However, these overall cost estimates include an
unrealistically significant amount of characterization and clearance sampling due to the research
nature of BOTE Phase 1 study. Figure 5-10 also summarizes the performance of each
decontamination method using percentage of samples clean (ND) and decontamination
efficiency. With respect to performance, CIO2 and pH-adjusted bleach performed well in both
measured aspects, while VHP® performed significantly worse.
VHP
dOZ Amended
Bleach
% Clean Samples
CIO2
Amended VHP
Bleach
Decon Efficiency
1.0000
0.9950
0.9900
09850
0.9800
Amended CIO2 VHP
Bleach
Figure 5-10. Overall decontamination method cost and performance.
387
-------
6. QUALITY ASSURANCE AND QUALITY CONTROL
The purpose of this section is to describe QA and QC activities that were implemented during
the BOTE Project, Phase 1 (the subject of this report). Specifically, the research areas that were
EPA's responsibility were carried out in accordance with a series of QAPPs approved by EPA
prior to the start of testing. In addition, EPA QA personnel conducted an assessment of various
research components that were to have been conducted in accordance with a specific QAPP.
6.1. Background
As previously discussed, the BOTE Project was a field-level decontamination assessment
managed by the EPA and DHS with the DOD/DTRA serving as the interagency coordinating
study directorate. Three decontamination methods were assessed: (1) fumigation with VHP®, (2)
a treatment process including the use of pH-adjusted bleach, and (3) fumigation with CIO2.
The four principal objectives of the BOTE Project Phase 1 decontamination assessment were:
• Conduct and evaluate field-level studies of three decontamination technologies/protocols
from initial discovery to final environmental remediation.
• Demonstrate that biological sampling and analysis methods from previous studies
provide accurate characterization of Ba simulant concentration challenges for
detection/identification purposes.
• Collect and analyze the results from the decontamination study and perform a cost
analysis of all aspects of the remediation approaches.
• Determine the exposures associated with reentry into a building that has been
contaminated with surrogate Ba spores and subsequently decontaminated.
6.2. Quality Assurance Objectives
To ensure that data produced were defensible and reproducible, the EPA approved QAPPs
prior to the commencement of data collection and conducted assessments during the various
stages of data collection.
During the course of a project, the three types of audits that may be performed include: (1)
technical systems audits; (2) performance evaluation audits; and (3) data quality audits.
Technical systems audits are generally conducted prior to or during the early stages of a project.
The Technical Systems Audit is a qualitative on-site evaluation that determines whether or not a
project or analysis is being performed as described in existing test plans, QAPPs, or standard
methods.
Quantitative evaluations are made using performance evaluation audits. A performance
evaluation audit is an evaluation of a measurement system using a reference material with a
known value or composition. EPA often provides performance evaluation audit samples to
contractors so that those contractors can demonstrate their ability to perform a specific analysis
adequately.
388
-------
Data quality audits evaluate methods used to collect, interpret, and report project results.
Selected samples are tracked through laboratory analysis, data processing, and statistical
analysis procedures to determine whether all data modifications, and the reasons for those
modifications, were adequately documented.
These assessments are an integral part of any quality assurance program.
6.3. Key QA and QC Activities and Reporting
Through systematic checking, audits confirm that appropriate QA procedures are being followed
and that project performance meets specified standards. The on-site QA-related activities
pertinent to the BOTE Project included the following: a confirmation and evaluation of the test
organism, spore dissemination and reference testing, sample collection, tracking and handling,
cross-contamination reduction, field blanks, and sample analysis methods. The results of these
QA activities are discussed in this section.
6.3.1. Test Organism
To meet the objectives of the project, a non-pathogenic surrogate (Bg, ATCC 9372; also known
as B. atrophaeus and B. subtilis var. niger) was used. The Bg spore preparation was obtained
from DOD's Critical Reagents Program Antigen Repository.
Prior to its use, the Bg spore preparation was evaluated by EPA at the EPA's Aerosol Testing
Facility in Research Triangle Park, NC, and the EPA's BSL-2 Biocontaminant Suite at AWBERC
in Cincinnati, OH.
A technical systems audit was conducted during the evaluation of the spore preparation. There
were no audit findings that would impact the outcome of the project.
6.3.2. Spore Dissemination and Reference Testing
Three reference methods were used to indicate that the dissemination process was successful
at meeting the target surface loading criteria for the first and second floors of the test facility.
The reference methods were as follows: real-time particle measurements were taken at 20
locations throughout the facility during dissemination using the FLIR Systems, Inc. IBACs, and
surface loading (CPU per ft2) indications were provided for by using stainless steel reference
material coupons (RMCs) and TSA settling plates. Each of the reference methods is shown in
Table 2-4.
In Round 2, the majority of the RMCs were reported as being ND for viable Bg spores after
dissemination. Only four of 28 samples had non-zero CPU values reported. The mean of these
non-zero samples was consistent with the Round 1 results for Floor RMCs. It is unknown why
Bg was not detected on the majority of the RMCs in this round. The measured surface loading
on Floor 1 was within the target criteria of 1E4 to 1E6 CFU/ft2. This measured loading was
consistent with that estimated from the four Floor 1 RMCs with Bg detected (see Section
3.2.1.1); there was no statistically significant difference between the surface sampling and RMC
average values (t-test, two-tailed p-value = 0.4838). Because the surface sampling was the
ultimate method for determination of the pre- and post-decontamination Bg spore loading, these
389
-------
Floor 1 RMC values were not investigated further. No corrective action was deemed necessary
or taken.
Also in Round 2, all quantified RMC samples associated with the second floor rooms were
higher than the target spore loading of 1E2 to 2E2 CFU/ft2. One RMC on the second floor was
ND for Bg. Excluding the inclusion of this value as 0 CPU, the mean loading for second floor
RMCs was 7.3E3 (±4.7E3) CFU/ft2. When the unexplained NDs are included, the results did not
indicate a significant difference (f-test, p =0.3722)[811 between first and second floor surface
loadings. Excluding the NDs, the results did indicate a significant difference (Mest, p <0.0001)[811
between first and second floor surface loadings. These results were meant to provide an
indication of surface loading; the surface sampling results were the critical criteria for the
decontamination assessment. The Floor 2 loadings determined by surface sampling were within
the target criteria and well indicated by the RMCs. No corrective action was deemed necessary
or taken.
6.3.3. Sample Collection, Tracking and Handling
The BROOM1601 PDA was used by sample collection teams to track sample location, sample
types, sample matrices, date, time, samplers, and other pertinent data. Chain of custody forms
generated by the BROOM system were included with each shipment and verified by a second
person.
The sampling methods used in the BOTE Project are listed in Table 2-5. For surface sampling,
these methods were: cellulose sponge-stick wipes, macrofoam swabs, vacuum socks, and
Versalon wipes®. For air sampling, the methods were: SKC BioSamplers®, UV-APS, Dycor
XMX/2L-MIL Aerosol Collection System, and Mattson-Garvin Model 220 slit-to-agar. Sand and
water samples were also analyzed. Petri plates filled with sand were used as the sand samples.
The EPA water pathogen concentration was used for water sampling.
For the RV-PCR testing, sampling was done using the Versalon® wipes, with samples shipped
to either LLNL or EPA-OPP-MLB (Ft. Meade) for the prescribed analyses. As mentioned in
Section 3.2.2.1, due to the complexity and difficulties with the sample shipment, the desired
sample distribution plan as recommended by the statisticians could not be followed for some
events. No corrective action was possible on-site to improve sample shipping procedures. The
result was a reduced number of samples, hence, lower statistical power. However, no significant
impact on the results and conclusions is noted.
For the sand samples, during the placement and collection process for Round 1, sampling
personnel noted that the sample dishes were not individually bagged. Additionally, notes were
made regarding breakage of sampling dishes following collection during shipment to the
laboratory. These notes by the sampling personnel and the detected signal from the blank
samples were the basis for considering all Round 1 samples as contaminated through sampler
handling. Therefore, all Round 1 sample results were eliminated from the data analysis
conducted for this report. Corrective action for Rounds 2 and 3 were to ensure that all sample
dishes were individually bagged to prevent contamination. All other trip and site blanks collected
during the project were ND.
390
-------
During AAS, three one-hour samples were collected by each XMX and STA sampler during
each sampling event. However, in Round 1, the STA sampler located in the Hallway did not
function due to equipment failure. This equipment failure was corrected prior to AAS in
subsequent rounds.
6.3.4. Cross-Contamination Reduction Methods
During the on-site technical systems audit, the methods used to prevent cross-contamination
were observed by the QA team. The team observed the secondary enclosure of the facility,
personnel entry methods, and decontamination of both the samples and the personnel leaving
the building.
6.3.5. Sample Analysis Methods
Surface samples were analyzed for viable Bg by either the LRN or INL, in accordance with
standardized procedures. All surface samples intended for use in the assessment of
decontamination effectiveness were analyzed via the LRN. Surface samples (Versalon® wipes)
used for assessment of RV-PCR analyzed by LLNL and EPA-OPP-MLB (Ft. Meade). Surface
and air samples for the reaerosolization study were analyzed by INL. Water samples were also
analyzed by INL, as were all reference samples (RMCs and settling plates). All samples were
analyzed for quantifiable, viable Bg using dilution plating methods. Surface samples with less
than 30 CPU present at the lowest dilution were to be filter plated to attain lower detection limits.
Sand samples were sent to EPA AWBERC for further processing. The, samples were split for
analysis at that facility and for shipment to USGS for analysis.
6.3.5.1. Analysis of Samples by the LRN
The LRN is a network of federal, state, and local laboratories. A total of eight LRN laboratories
participated in the BOTE Project Phase 1, analyzing 1,937 samples. LRN analyzed samples by
culture method only (no PCR), because previous contamination events conducted at the INL
facility (PBF-632) used in the BOTE Project result in residual Bg DNA in the environment.
All samples on Floor 2 or with less than 30 CPU present from the lowest dilution plate were to be
filter plated to attain lower detection limits and more accurate data at the lower range.
In Round 2, one surface sample collected from the first floor with detectable Bg (#3085)
collected after decontamination yielded 16 CPU from the spread plate method, yet ND CPU from
the filter plate method. This result was unexpected because the filter plate method theoretically
provides a lower limit of detection. We are unsure of the cause of this anomaly. Since the value
of 16 CPU was below the quantitation limit of 30 CPU, this result was not expected to impact the
analysis or conclusions.
Additionally for Round 2, forty-eight samples resulting in less than 30 CPU during the spread
plate procedures were not subjected to the prescribed filter plating, a method with a lower limit
of detection. While it is unlikely that obtaining the filter plate data from these samples would
greatly change the efficacy results, more post-decontamination samples detectable for Bg may
have been discovered if the additional analysis had been conducted.
391
-------
6.3.5.2. Analysis of Samples by INL
The EPA QA staff conducted an audit of the INL laboratory procedures included the analysis for
the ISA settling plates and RMCs collected during pre-decontamination surface sampling. In
addition, the INL also analyzed wipes from the reaerosolization study, taken in Rooms 101A and
102.
All CPU counts and calculations were recorded in the appropriate laboratory notebook and the
viable count worksheet and archived. All quantitative results were recorded in Excel
spreadsheets according to sample identification number and sent to the EPA project officer after
analysis and quality control checks.
6.3.5.3. Analysis of Samples for the Rapid Viability-Polymerase Chain Reaction
Study
Only two Round 1 pre-decontamination samples were received and analyzed. These samples
represented QC (field blank) samples rather than wipe samples from surface sampling. For
these samples, only one of two (50%) showed agreement between methods due to a technical
issue with one of the RV-PCR samples (the filter cup leaked during incubation so spore
outgrowth and subsequent cell growth were compromised in this sample). Based on this issue, a
change was instituted in the protocol to cap the filter cup bottom before adding growth medium,
rather than after. Because capping is performed in the BSC, there is little risk for the additional
handling of the filter cups containing dry spores. After the protocol change was made, no filter
cup leakage was observed.
In Round 3, some negative control samples were positive by culture analysis possibly due to
cross-contamination (see Table 3-33). Protocol modifications including extra glove changes
were incorporated to prevent this error in the culture sample analysis that followed.
For samples analyzed by the EPA-OPP-MLB Laboratory, seven samples were determined to
show a difference between the culture and RV-PCR analysis methods. A likely contributing
factor to RV-PCR performance involved inexperience with the magnetic bead-based DNA
extraction procedure. Additional experience and ongoing research on this method will address
the problems with the reproducibility of performance of the DNA extraction and purification
protocol.
6.3.5.4. Analysis of Samples for the Sand Study
Sand samples were analyzed by EPA and USGS. One deviation to the QAPP should be noted:
the processing protocol incorporated an additional suspension and centrifugation of the pellet.
No QA issues were noted with the analysis. However, due to sample collection issues noted in
Section 6.3.3, data from Round 1 were not considered in the statistical analysis.
6.3.6. Collected Blank Samples
Contamination could occur during sample handling in the field or in the laboratory during sample
processing. Field blank samples were collected during each sampling campaign to determine
the potential for background contamination of sampling media.
392
-------
Surface sampling field blanks submitted for LRN analysis (swabs, sponge-stick wipes, and
vacuum socks) were included in the samples taken for each round of testing in the facility. Blank
samples were submitted from sampling teams designated for sampling on the first and second
floors. For Round 1, a total of 52 field blanks were taken pre-decontamination. Of the field
blanks collected, four samples from the second floor came back with detectable Bg counts from
the spread plate analysis. All four samples with detectable viable Bg were from the second floor.
Quantified CPU were 7.0EO, 3.4E1, 1.43E3, and 4.1E3. An additional six samples had
detectable viable Bg, via filter plate analysis (with non-detects from the spread plate analysis).
Four of these samples were from sample teams on the first floor and two were from teams
sampling on the second floor. Detected values ranged from 1 to 13 CPU. A total of 45 field blank
samples were collected during post-decontamination sampling for Round 1; one sample was
found to have detectable Bg counts via spread plating with a value of 17 CPU. Three additional
samples had Bg detected via filter plate analysis (with no detectable Bg via spread plating);
values ranges from 2 to 5 CPU. All blank samples with detectable Bg were from the first floor
sampling teams.
A total of 41 field blank samples were collected during pre-decontamination sampling in Round
2. Two samples had detectable Bg, one via spread plate analysis (67 CPU) and one via filter
plate analysis (3 CPU). Both samples were from Floor 1 sample teams. A total of 25 field blank
samples were collected in Round 2 during post-decontamination sampling. One sample from a
Floor 2 sample team had detectable Bg via filter plate analysis (6 CFU).
In Round 3, 52 field blank samples were collected during pre-decontamination sampling. Four
samples had detectable Bg, two via spread plate analysis (3.3E1 and 1.7E4 CFU) and two via
filter plate analysis (3 and 74 CFU). All four samples were from Floor 1 sample teams. A total of
47 field blank samples were collected during post-decontamination sampling. No samples had
detectable Bg either by spread or filter plate analysis.
For the sand samples, site blank and trip blank QA samples were collected during each round of
the BOTE Project. The purpose of the site blanks was to determine the potential for background
contamination of sampling media at the site. The site blanks were opened on site and then
immediately closed and re-bagged for shipment to the laboratory for analysis. The purpose of
the trip blanks was to determine the potential for sample contamination over the course of an
entire sampling round. Trip blanks were shipped out to the site with the sampling media, held in
sample kit boxes during sample collection (but never opened) and then shipped with the
samples to the laboratory for analysis. All site and trip negative controls were reported as ND
with the exception of two collected during Round 1 for which corrective action was taken.
Prior to use in AAS testing, XMX and STA samplers were tested for background contamination.
Sampling of the STA samplers revealed that four of the nine were contaminated with Bg prior to
sampling. All STA samplers were decontaminated with pH-adjusted bleach and placed in the
facility during the VHP® fumigation in Round 1 to further sterilize the STA samplers.
During the reaerosolization study, 54 background samples were collected prior to Bg
dissemination. Of these samples, three yielded detectable Bg. This was possibly due to residual
393
-------
contamination in this facility form prior use as a test bed. These results were determined to have
no impact on the testing, since they were very low levels compared to the amount of Bg that
would be released during dissemination. No corrective action was warranted, in accordance with
prior planning for the BOTE Project based upon MFP results.
6.4. Technical Systems Audit
The on-site technical systems audit lasted for approximately three weeks during the BOTE
Project testing. This audit was initiated at the start of the MFP (April 14, 2011) and went into
Round 2 (April 29, 2011). The first day was spent preparing and conducting quality control
checks of the sampling kits and observing the second part of the sampler training. The samplers
performed sampling exercises using the prepared sampling kits. During the mock run,
observation of the samplers via the closed-circuit camera system was conducted. In addition, at
the end of the sampling period, the removal of air samples was observed via the closed-circuit
camera system.
6.4.1. Summary of Observation and Findings
No significant findings were noted.
6.4.1.1. Sample Handling
Initially, the sample handling process was very cumbersome. The issue was resolved during a
discussion with the on-site EPA Project (Program) Manager, INL sample handler and EPA QA
personnel.
6.4.1.2. Sample Storage
The EPA water samples were not stored under refrigeration. Samples were left out on the
laboratory bench in the microbiology laboratory and table in the sample trailer, respectively. The
SOP stipulates that samples should be stored in a refrigerator.
6.4.1.3. Water Concentrator
One of the two pipettors used for the water analysis was out of calibration The P20 was
scheduled to be calibrated in September 2009.
6.4.1.4. Waste Removal Process
There was a deviation from the SOP for preparing the building prior to low tech decontamination
procedures using pH amended bleach. Some of the deviations noted in handling the waste
might have created possible safety hazards and cross-contamination issues. These issues
included the incomplete bagging of some materials, e.g., mattress, and not spraying some
materials prior to cutting.
6.5. Performance Evaluation Audit
Performance evaluation audit samples were prepared at EPA and were shipped via UPS
overnight to the receiving laboratories. The purpose of the performance evaluation audit
samples was to evaluate the efficiency and repeatability of sample processing across the
laboratories and sample types.
An aliquot of the Critical Reagents Program Bg utilized in the BOTE Project was subjected to
dilution plating to determine the starting titer. Working solutions with Bg concentrations of 5E3
394
-------
and 5E6 CFU/mL were prepared in PBST. For all performance evaluation audit samples, 0.2 ml_
of the working solution was used to inoculate sampling media. Samples were inoculated with a
micropipette, taking care to dispense the liquid slowly so that the liquid soaked completely into
the sample. Targeted final spike concentrations were 1E3 and 1E6 total CPU per sample.
Two replicate spike samples were randomly chosen and sent to each laboratory for each spike
concentration (blank, 1E3, and 1E6 CPU), and each sample type (swab, vacuum sock, sponge
stick). Seven of the eight LRN labs that participated in BOTE Project sample processing agreed
to receive, process, and report data from samples spiked with a known amount of Bg spores.
Samples were shipped to the laboratories in collection kits exactly as they would be received
from the BOTE Project field exercise. Sample identification was achieved through barcodes. The
sample type, inoculum amount, and laboratory used for analysis were all tracked by EPA, but
these data were not available to the LRN labs. During the performance evaluation audit, each
laboratory received only those types of samples that it received during actual BOTE Project
sample analysis.
Chains of custody were maintained for each change in sample possession.
Each laboratory received, processed, and analyzed samples using the exact protocols and
procedures used for actual BOTE Project samples. All titer determinations indicated that the
working solutions were within the acceptable range outlined by the QAPP (± 0.5 Iog10 of the
target concentration). Results from the titer determinations are presented in Table 6-1.
Results obtained from the LRN laboratories upon processing the performance evaluation audit
samples indicated that extraction of spores from sponge wipes and vacuum socks was more
efficient than extraction from swabs, especially for the samples spiked with 1E6 CPU (Table 6-2
through Table 6-6). The mean recovery from swabs spiked with 1E6 CPU was no different than
recoveries from swabs spiked with 1E3 CPU (p = 0.68, t-test). These data suggest that recovery
from swabs may demonstrate a negative bias as the concentration of spores on the swab
increases. Spores collected by swabs from surfaces may not demonstrate the same bias
observed from swabs spiked with a liquid inoculum.
Recoveries from sponge sticks were typically higher than the other two sample types, and best
approximated spike concentrations. Mean recoveries for sponge samples were of the same
order of magnitude (± <1 log) as the spiked amount and were within 50% of the target
concentration for both 1E3 and 1E5 CPU spiked samples (Table 6-5 and Table 6-6)
Considering only swabs and vacuum socks, swabs were better at approximating contamination
at the 1E3 CPU level, while vacuum socks more accurately predicted spike concentrations for
the 1E6 CPU spiked samples.
Nearly all blank samples (spiked with buffer only) were reported as no growth (zero CPU)
following analysis. The exceptions included two sponge wipe samples (3057, Lab #5 and 3235,
Lab #5) and one vacuum sock sample (5039, Lab #6).
395
-------
Recovery of no detectable viable spores was reported for several samples spiked with 1E3 and
1E6 CPU (Table 6-2 through Table 6-4). Laboratory #3 reported zero recovered CPU from all
1E3 spiked samples and reported recoveries 1 log lower than all other laboratories for 1E6
spiked vacuum socks. These data suggest that laboratory technique and proficiency can be a
source of bias.
In summary, the results of the performance evaluation audit indicate that extraction efficiencies
may vary between sampling devices. Sponge sticks demonstrated the closest approximation of
spike concentrations. Swabs demonstrated the worst approximation of spike concentration when
spiked with 1E6 CPU. Variability between laboratories can evidently be high. Further, one
laboratory systematically reported results lower than the known spike amount and lower than all
the other laboratories. Performance evaluation audits are an important tool for assessing the
quality of data received in a multi-laboratory study such as the BOTE Project.
Table 6-1. Aliquots of the stock spore solution used to spike performance evaluation
audit samples were subjected to tenfold dilution plating to determine the actual number
of spores in the inoculum.
Titer
Check
1
2
3
4
5
6
Target
CFU/mL
5E6
5E6
5E3
5E3
5E3
OEO
Achieved
CFU/mL
4.6E6
3.8E6
4.4E3
4.8E3
5.9E3
OEO
Inoculum
Volume
(my
0.2
0.2
0.2
0.2
0.2
0.2
Target Inoculum
(CFU)
1E6
1E6
1E3
1E3
1E3
OEO
Actual Inoculum
(CFU)
9.3E5
7.7E5
8.7E2
9.7E2
1.2E3
OEO
396
-------
Table 6-2. Recovery results from LRN performance evaluation audit swab samples sent to
seven of the eight participating LRN laboratories.
Sample
ID
4822
4815
4829
4825
4832
4835
4818
4821
4827
4830
4831
4834
4819
4820
4826
4828
4833
4837
4824
4836
4817
4816
4838
4823
Spike
(CPU)
OEO
OEO
OEO
OEO
OEO
OEO
OEO
OEO
1E3
1E3
1E3
1E3
1E3
1E3
1E3
1E3
1E6
1E6
1E6
1E6
1E6
1E6
1E6
1E6
Laboratory
Result
(CPU)
OEO
OEO
OEO
OEO
OEO
OEO
OEO
OEO
4.6E2
6.6E2
OEO
OEO
4.7E2
4.2E2
5.8E2
6.3E2
4.6E2
7.3E2
OEO
OEO
5.2E2
6.1E2
7.1E2
6.6E2
Percent of
Spike
Recovered
45.8
66.3
0.0
0.0
47.0
41.8
58.3
63.3
0.0
0.1
0.0
0.0
0.1
0.1
0.1
0.1
Lab
2
2
3
3
5
5
6
6
2
2
3
3
5
5
6
6
2
2
3
3
5
5
6
6
397
-------
Table 6-3. Recovery results from LRN performance evaluation audit vacuum sock
samples sent to seven of the eight participating LRN laboratories.
Sample
ID
5044
5048
5041
5043
5036
5047
5046
5038
5035
5045
5039
5040
5042
5037
5020
5029
5027
5028
5032
5034
5026
5033
5023
5030
5019
5031
5025
5024
5005
5004
5003
5016
5006
5011
5017
5009
5012
5018
5007
5015
5014
5013
Spike
(CPU)
OEO
OEO
OEO
OEO
OEO
OEO
OEO
OEO
OEO
OEO
OEO
OEO
OEO
OEO
1E3
1E3
1E3
1E3
1E3
1E3
1E3
1E3
1E3
1E3
1E3
1E3
1E3
1E3
1E6
1E6
1E6
1E6
1E6
1E6
1E6
1E6
1E6
1E6
1E6
1E6
1E6
1E6
Laboratory
Result
(CPU)
OEO
OEO
OEO
OEO
OEO
OEO
OEO
OEO
OEO
OEO
5.0EO
OEO
OEO
OEO
4.7E1
3.9E1
1.0E2
2.0E2
OEO
OEO
2.3E2
5.7E2
1.0E2
2.7E2
3.2E2
2.8E2
3.8E2
3.0E2
2.8E5
2.8E5
1.7E5
2.1E5
1.6E4
1.1 E4
2.9E5
6.0E5
2.6E5
1.8E5
2.1E5
3.7E5
3.5E5
3.4E5
Percent of
Spike
Recovered
4.7
3.9
10.0
19.5
0.0
0.0
23.0
57.0
10.0
26.8
32.3
27.5
38.3
29.8
28.8
27.7
16.8
21.0
1.6
1.1
29.0
60.2
25.5
18.0
21.2
37.0
35.0
33.8
Lab
1
1
2
2
3
3
4
4
5
5
6
6
7
7
1
1
2
2
3
3
4
4
5
5
6
6
7
7
1
1
2
2
3
3
4
4
5
5
6
6
7
7
398
-------
Table 6-4. Recovery results from LRN performance evaluation audit sponge-stick wipe
samples sent to seven of the eight participating LRN laboratories.
Sample
ID
3264
3046
3071
2050
3075
3267
3000
3081
3057
3235
2937
3266
2106
2100
3272
3270
3224
3225
2132
2911
3053
3037
2897
3040
2695
3033
3032
2451
2866
2912
2894
2393
2450
2874
2694
3034
2495
3257
3222
3036
3269
2891
Spike
(CPU)
OEO
OEO
OEO
OEO
OEO
OEO
OEO
OEO
OEO
OEO
OEO
OEO
OEO
OEO
1E3
1E3
1E3
1E3
1E3
1E3
1E3
1E3
1E3
1E3
1E3
1E3
1E3
1E3
1E6
1E6
1E6
1E6
1E6
1E6
1E6
1E6
1E6
1E6
1E6
1E6
1E6
1E6
Laboratory
Result
(CPU)
OEO
OEO
OEO
OEO
OEO
OEO
OEO
OEO
3.2EO
5.9EO
OEO
OEO
OEO
OEO
1.5E2
1.5E2
8.2E2
1.2E3
OEO
OEO
2.5E3
6.1E2
5.3E2
8.2E2
1.2E3
4.0E3
8.2E2
1.8E3
1.5E6
8.9E5
1.6E6
1.2E6
1.3E6
1.5E5
1.8E6
1.5E6
1.1 E6
1.0E6
1.6E6
1.9E6
1.4E6
1.4E6
Percent of
Spike
Recovered
14.5
15.0
82.4
116.4
0.0
0.0
248.2
60.6
53.4
81.9
120.2
402.0
81.5
179.2
148.9
89.4
163.3
122.7
124.7
14.6
178.5
146.0
111.8
103.8
161.2
193.6
141.5
141.1
Lab
1
1
2
2
3
3
4
4
5
5
6
6
7
7
1
1
2
2
3
3
4
4
5
5
6
6
7
7
1
1
2
2
3
3
4
4
5
5
6
6
7
7
399
-------
Table 6-5. Mean and percent recovery values (all data) by sample type pooled across
laboratories.
Sample Type
Swab
Vacuum Sock
Sponge Wipe
Recovered CPU (% of spike)
Blank
0
3.6E-1
6.5E-1
1E3
4.0E2 (40%)
2.0E2 (20%)
1.0E3(100%)
1E6
4.6E2 (0.05%)
2.6E5 (26%)
1.3E5(130%)
Table 6-6. Mean and percent recovery values (outliers excluded) by sample type pooled
across laboratories.
Sample Type
Swab
Vacuum Sock
Sponge Wipe
Recovered CPU (% of spike)
Blank
OEO
OEO
OEO
1E3
5.4E2 (54%)
3.1E2(31%)
1.4E3(140%)
1E6
6.2E2 (0.06%)
3.0E5 (30%)
1.4E6(140%)
Samples considered outliers include sample numbers: 4831, 4834, 4824, 4836, 5039, 5020, 5029, 5032, 5034, 5006,
5011, 3057, 3235, 3272, 3270, 2132, 2911, and 2874.
6.6. Data Quality Assessment
All LRN data from BROOM spreadsheets were checked and verified by EPA researchers. The
process included matching up the sample number, sample type and laboratory results from the
LRN Excel sheets to the BROOM spreadsheet. All discrepancies were corrected, based upon
investigation into the sample. Most issues dealt with incorrect manual barcode reading at the
laboratories. Future use of the labeling systems compatible with BROOM should consider larger
labels with longer barcodes.
A total of 1,972 samples were logged into the BROOM database during collection, targeted for
analysis by the LRN. In total, these samples were sent to eight different LRN laboratories in
accordance with the number and types of samples that they could accommodate. LRN data
were then imported into the BROOM database, cross-referenced by the sample number and
checked against sample type recorded in BROOM and on the LRN data sheet (MS Excel file).
Some errors occurred where samples were either not able to be located in BROOM, more than
one result was reported for the same sample number, or sample results were not returned. The
reconciliation process included cross referencing with the COC documentation (with regard to
where samples were sent) and checking with the LRN laboratories to verify sample identification
numbers that were reported. In summary, 1,966 samples were reconciled and their sampling
information and results verified. The loss of 0.3% of the surface sampling data analyzed by the
LRN did not impact the BOTE Project results, data analysis, or conclusions.
The sample analysis protocols for all surface and air samples analyzed by the LRN or at INL
were to include filter plating of a portion of the extract if spread plating results at the lowest
dilution factor were below the quantitation limit (30 CPU). All samples from the second floor
(lower starting load and all post-decontamination surface samples meeting these requirements)
400
-------
were to be filter plated. However, not all samples analyzed by the LRN that met that criteria were
indeed filter plated.
For the MFP, two samples were missing filter plate data; however, neither of these samples had
non-detectable levels of spores on the spread plate (i.e., results were still available). Therefore,
the results and conclusions were unaffected.
For the pre-decontamination samples for Round 1,19 samples meeting the criteria were not
filter plated. For the post-decontamination samples for Round 1, nine samples meeting the
criteria were not filter plated. However, all of these samples had spread plate results with
detectable CFU. Therefore, the results and conclusions were unaffected.
For the pre-decontamination samples for Round 2, ten samples meeting the criteria were not
filter plated. For the post-decontamination samples for Round 2, no filter plating data were
missing. The results and conclusions were unaffected by the missing pre-decontamination filter
plate data.
For the pre-decontamination samples for Round 3, nine samples meeting the criteria were not
filter plated. For the post-decontamination samples for Round 3, no filter plating data were
missing. The results and conclusions were unaffected by the missing pre-decontamination filter
plate data.
401
-------
7. CONCLUSIONS
The intent of Phase 1 of the BOTE Project was to develop an improved understanding of
response strategies for a single building, ultimately to extrapolate for use in wide area
remediation. This project was the first field level evaluation of decontamination technologies
under similar conditions outside a laboratory-controlled environment. The decontamination
efficacy, cost, labor, and waste analyses provide invaluable information to decision-makers
regarding time and resources required for each decontamination approach. Furthermore, the
BOTE Project provided an opportunity for improving the readiness for mitigating the effects of a
release of a bioagent over a wide area by allowing for:
EPA cross-regional training and biosampling experience;
Collaboration across regions and government agencies; and
Real-world experience with biological agent decontamination.
Phase 1 of the project consisted primarily of a decontamination assessment (Objective 1) and
included the demonstration and evaluation of sampling methods (Objective 2), the development
of a remediation cost analysis (Objective 3), and the development an exposure assessment plan
(Objective 4).
Three decontamination methods showing effectiveness against Bacillus anthracis (Ba) spores in
laboratory and/or field use were tested under field-relevant conditions from April 11 to May 19,
2011. The three decontamination methods utilized independently, in three separate testing
events (referred to as rounds) were: Round 1, fumigation with H2O2 using the STERIS VHP®
technology; Round 2, a decontamination process incorporating the spraying of surfaces with pH-
adjusted (or amended) bleach; and Round 3, fumigation with CIO2 by Sabre Technical Services,
LLC. Test parameters for each round included the decontamination method, level of
contamination, and contaminated environment (e.g., office setting, residential area, HVAC
system).
Outcomes of the project objectives and sub-objectives are described in the subsections that
follow.
7.1. Assessment of Decontamination Methods
The decontamination contractor or performer was responsible for the development of a
remediation plan. This plan included identification of materials or items to remove from the
facility prior to decontamination, materials or items to decontaminate in place and then remove
as waste, methods for decontaminant application, and procedures for returning the facility to the
EPA for post-decontamination sampling. A requirement of three days for facility remediation was
put in place for all rounds (from setup to aeration and return of the facility to the EPA); three
402
-------
additional drying days were allotted for the second round (after spraying of surfaces with pH-
adjusted bleach). All decontamination methods were able to function within this time constraint.
In summary, fumigation with CIO2 resulted in the fewest positive samples, followed by the
decontamination process incorporating the spraying of surfaces with pH-adjusted bleach.
Fumigation with VHP® resulted in the highest number of positive samples following treatment.
The pH-adjusted bleach process resulted in the highest relative cost, followed by fumigation with
CIO2, and lastly by fumigation with VHP®. Each decontamination method was performed a single
time in the BOTE Project; the results and conclusions should be considered based upon the
implementation as described. Potential variance in effectiveness due to differences in
implementation of each method on subsequent uses was not determined in this project. The
outcome of each decontamination process with respect to the assessment of effectiveness is
reported below.
Fumigation by Vaporous Hydrogen Peroxide (VHP®)
The fumigation contractor, STERIS, decided to leave all materials in the facility in place during
the decontamination process (i.e., in situ decontamination). Although fumigation of materials in
the laboratory with H2O2 has shown the process to be efficacious for inactivation of Bacillus
spores on some surfaces, the results from this field study indicate that the H2O2 exposure was
not sufficient to inactivate all the spores at the high and low challenge/test levels (surface
loading). Roughly a third of the samples on the first and second floor resulted in detectable Bg
after decontamination. A significantly greater percentage of samples with detected Bg were from
the residential room types, compared to the other room types.
The temperature was maintained above the desired setpoint throughout the testing. The main
condition that was not achieved was the target H2O2 concentration throughout the facility for the
specified amount of time, possibly a result of breakdown or adsorption of the H2O2 that was
being injected into the facility or simply not enough H2O2 was injected. These results indicate
that the H2O2 exposure was not sufficient to inactivate all the spores at either challenge level
(surface loading). One improvement may have been to increase the amount of H2O2 that was
introduced on each floor by doubling the number of T4 generators or using a higher-capacity
generator. The two generators could have been connected to one floor at a time as well, but this
would have required additional fumigation time. The contractor could also have removed some
of the porous materials to reduce H2O2 adsorption into the materials.
Upon post-decontamination inspection of the building, no damage to the building contents was
observed from exposure to the VHP® process.
Waste generated during fumigation with VHP® included only solid waste such as PPE and
sampling waste (e.g., packaging) from both building decontamination and sampling. A total of
1,350 Ib of waste was generated from the sampling and decontamination activities. Liquid waste
was also generated during this round (e.g., during sampling), from rinsate recovered from the
Decontamination Line.
403
-------
Surface Decontamination Approach using pH-Adjusted Bleach
The Round 2 decontamination process involved creating negative pressure on the facility using
NAMs, removing all porous materials and HVAC system supply-side for treatment on-site and
disposal, and then spraying all remaining surfaces in the facility with pH-adjusted bleach. The
results suggest that the decontamination process was highly efficacious on the first floor and
moderately efficacious on the second floor. Overall, only 3.2% of samples collected following
decontamination resulted in viable spores being recovered. Further, few viable Bg spores were
recovered from these samples, suggesting that even in these areas with detectable Bg,
contamination was significantly reduced by the complete decontamination process. HVAC
system (return side) decontamination procedures were also effective at removing contamination,
as all HVAC system samples post-decontamination were ND.
Because all porous materials were removed from the facility, decontamination efficacy was not
likely to be impacted by the room type.
Upon post-decontamination inspection of the building after drying, the laminated and wood
flooring demonstrated noticeable swelling (sufficient to require replacement if it were in an actual
residence). Bleach residue was noticeable on horizontal surfaces, but no appreciable damage
was indicated by the residue.
The largest amount of solid and liquid waste generated occurred during the application of the
pH-adjusted bleach decontamination process. In the decontamination that was used in the
BOTE Project, all porous surfaces were removed, bagged, decontaminated ex situ, and treated
as solid waste. The total weight of material removed prior to the pH-adjusted bleach spraying of
the facility was -7,100 Ib. The total volume of waste removed from the building was estimated
to be 68 cubic yards, based on having 3.25 dumpsters of material (21 cubic yards per
dumpster).
Fumigation with Chlorine Dioxide
Prior to fumigation with CIO2, the fumigation contractor (Sabre) elected to remove (treat ex situ
and dispose) a few porous materials due to concerns with longer aeration requirements if left in
place. The facility and all remaining contents were fumigated under conditions shown to be
effective for the inactivation of Ba spores in laboratory studies and past facility remediation
actions. Overall, this process was successful in the decontamination of this facility. Of 344 post-
decontamination surface samples, only one sample resulted in detectable Bg via spread plating
and an additional five with filter plating. The samples with detected Bg were from both porous
and nonporous material types. The Bis on the bottom floor were inactivated, with the exception
of one that was located inside a filing cabinet. Despite a 4 log reduction in detectable spores on
the second floor based upon surface sampling results, 31 of the 45 Bis came back positive. The
RH on the second floor was lower than the target and the RH on the first floor. In this case, the
conditions were insufficient to inactivate the 6 log Bis.
A post-test inspection of the building was completed after clearance sampling. Because this
building had been used in previous decontamination studies, it was difficult to assess whether
any new oxidation had occurred as a result of these tests. Materials that were placed in the
404
-------
building for these tests were inspected for damage. The only objects that showed any damage
were ring stand clamps and quick connects on gas sample lines. All other surfaces and
materials retained their original condition and color.
Sabre elected to have the heavier foam items removed because the foam would absorb the
CIO2 and extend the time required for aeration (to achieve the three-day decontamination
requirement). The items removed included two queen-size mattresses and the thicker foam
cushions from the couches and chairs. The total amount of material that was removed from the
building was 452 Ib and took two personnel 30 minutes to remove. These items were notionally
decontaminated with liquid CIO2 (i.e., included in the cost analysis as if they were treated on
site, but treatment was not actually performed in this study) and treated as solid waste. Liquid
waste was also generated during this round (e.g., during sampling), from rinsate recovered from
the Decontamination Line. A total of 877 Ib of solid waste was generated, from the sampling and
decontamination activities.
Decontamination Line and Decontamination Process Wash Water Treatment
Due to the small number of spores present in the personnel Decontamination Line wash water,
evaluation of the efficacy of the bleach treatment procedure for the collected waters was not
possible. However, a greater than three log inactivation (i.e., log reduction) was achieved using
the proposed protocol when the wash water was spiked with the Bg spores.
Results from the spiked wash water were similar to those obtained from laboratory experiments
using artificially generated wash water with similar water quality characteristics. These findings
suggest that the proposed inactivation procedure would be applicable for wash water derived
from similar PPE decontamination activities.
Use of the ultrafiltration concentrator allowed collection of concentrated samples. However, the
high turbidity of the wash water under the conditions experienced made the operation of the
ultrafiltration concentrator difficult due to filter clogging. For future wash water studies using the
ultrafiltration concentrator, improvements should be made so that turbid water is concentrated
more effectively.
7.2. Demonstration and assessment of biological sampling
methods
Previously evaluated biological sampling methods were evaluated. These methods included
surface sampling, RV-PCR, and aggressive air sampling. The outcome of each assessment is
reported below.
Surface Sampling
Surface sampling was conducted for four primary purposes: (1) assessment of decontamination
efficacy, (2) reaerosolization study, (3) correlation to AAS results, and (4) RV-PCR method
evaluation. For the assessment of decontamination efficacy, a total of 1,937 swab, sponge-stick
wipes, and vacuum sock samples were collected and analyzed by the LRN. In addition, 138
Versalon®wipe samples were collected for analysis by INL for use in the reaerosolization
405
-------
assessment. An additional 264 Versalon®wipe samples were collected for analysis by LLNL and
EPA (Ft. Meade) for the assessment of RV-PCR.
For the decontamination efficacy assessment, the surface loading of viable Bg spores pre-
decontamination was desired to be approximately 1E4 to 1E6 and 1E1 to 1E2 CFU per ft2 in the
rooms on the first and second floor, respectively.
The measured surface loading for all rounds on Floor 1 was within the target range of 1E4 to
1E6 CFU/ft2. The measured surface loading on for all rounds on Floor 2 was an order of
magnitude higher than the target range of 1E2 to 2E2 CFU/ft2. Although the surface loading on
Floor 2 was above the target range, there was still a significant difference between the
measured surface loadings on the two floors. Additional statistical analysis confirmed that the
contamination was applied so that the first floor was significantly more contaminated than the
second floor for each of the three events.
Rapid Viability -Polymerase Chain Reaction
A total of 264 BOTE Project samples were analyzed using both the RV-PCR and the traditional
microbiological culture methods to detect the presence of viable Bg spores (214 were processed
at LLNL and 50 were processed at the Ft. Meade EPA-MLB Laboratory to practice and gain
more familiarity with the method). The RV-PCR method was shown to work well for the
surrogate B. atrophaeus spores exposed to decontaminants at real-world application levels, and
with wipe samples containing background debris and indigenous microbial populations. At LLNL
a > 97% agreement was observed between RV-PCR and culture results (positive/negative for
Bg) for the field test that included samples with low spore levels (at or below the detection limit
of the plating method) after treatment with fumigants and surface disinfectants. The Ft. Meade
MLB Laboratory showed a lower percent agreement of 86%. The T9 endpoint appeared to be
sufficient to detect any spores that might have been delayed in germination due to
decontaminant exposure. Upon completion of this study, additional research has been ongoing
for RV-PCR method for Ba spores exposed to decontaminants to confirm the robustness of the
method for post-decontamination scenarios including the method endpoint (T9) for maintaining
the 10-CFU level LOD. Specifically, both disinfectant exposure effects and the influence of any
residual disinfectant from pH-adjusted bleach, H2O2 and CIO2 treatments on performance of the
RV-PCR method will be determined using Ba spores.
Aggressive Air Sampling
The main objective of AAS in the BOTE Project was to determine if, after application of
decontamination technology, disturbing indoor surfaces resulted in the detection of re-
aerosolized residual spores via air sampling. This procedure was being tested as a
supplemental measurement for the determination of effectiveness of the decontamination
process. The operation was conducted successfully after all three decontamination rounds and
AAS sample results were similar to surface sample results. AAS results after Round 1
(fumigation with VHP®) showed the highest concentrations of spores detected in the air; the
lowest were detected for Round 3 (fumigation with CIO^). The ability to employ AAS was
demonstrated successfully and it was shown that spores could be detected by this sampling
406
-------
method. Additional assessment of the method is needed to develop AAS as a viable option to
reduce the post-decontamination sampling burden.
7.3. Overall Cost Analysis
The main purpose of the cost analysis in the BOTE Project was to estimate the overall cost of
the application of various decontamination technologies as a function of materials, time
(including labor hours), and other resources.
Based on subsequent analysis of the cost data, the following major cost-related observations
were noted:
• Sampling and analysis were the largest contributors to the overall cost. This statement
must add the caveat that this was a research operational testing and evaluation project.
In a real incident for a building this size, fewer samples would most likely be taken.
However, sampling and analysis costs are still anticipated to be a major cost factor to
consider.
• The costs of the decontamination processes alone (the actual fumigation or surface
decontamination) were roughly equivalent for all three rounds. Overall costs for the
fumigation methods (VHP® and CIO2) were very similar (between $800,000 and
$900,000), while the pH-adjusted bleach cost nearly $1,200,000. The pH-adjusted
bleach decontamination process employed in this effort was more expensive to apply
than either of the fumigation technologies, largely due to waste management costs.
These costs are specific to the processes as they were employed in the BOTE Project.
• Waste management costs were a significant component of all three technologies,
particularly for the pH-adjusted bleach decontamination process; waste characterization
sampling was the largest single component of waste management costs. Almost all of
the waste generated during the fumigations was a result of personnel decontamination
operations. In addition, waste management costs could be reduced significantly if the
State allows disposal of treated and/or decontaminated items in a RCRA Subtitle D
landfill or allows the wastewater to be sent to a POTW.
• The cost of personnel decontamination was also a significant contribution, due to the
need for the Decontamination Line personnel being on site during any time when entries
into the building are considered, whether or not those Decontamination Line operations
staff are actually performing any personnel decontaminations.
7.4. Assessment of Potential Exposure
Exposure to Ba spores during a release may be due to the primary release or secondary due to
contact with reaerosolized spores or a contaminated surface. Spread of contamination (e.g., via
reaerosolization and dispersion, inside to outside facilities [or vice versa]) may further increase
the potential for exposure. The BOTE Project provided an opportunity to investigate some
aspects of exposure in a field setting:
• assess Bacillus spore migration from inside to outside a contaminated building;
407
-------
• investigate potential spore reaerosolization inside a contaminated building; and
• develop the concept of an exposure assessment plan.
7.4.1. Assessment of Reaerosolization
Reaerosolized spores were measured at both high (pre-decontamination) and low (post-
decontamination) levels of Bg spore surface contamination. A lower concentration of
reaerosolized Bg spores was observed at high levels of contamination than at low levels of
surface contamination. There were no significant differences in Bg spore concentrations
associated with sampling heights or locations within the rooms.
7.4.2. Assessment of Bacillus Spore Migration from Inside to Outside a
Contaminated Building
The detection of Bg genetic material in previously uncontaminated sand samples outside the
building suggests that spores have the potential to migrate out of a contaminated building and
settle into the surrounding environment. This migration was outside the facility but within the
secondary enclosure. No samples were taken outside the secondary enclosure. The study did
not differentiate when ex filtration occurred from the facility (i.e., during dissemination or
subsequent remediation activities).
7.4.3. Exposure Assessment Plan
A methodology to qualitatively characterize inhalational exposure associated with an adult's re-
entry into the BOTE facility contaminated with Bg spores, before and after decontamination, was
developed. The current methodology takes into account the specific site and the utilization of
both indoor air and surface sample analytical data.
During development of the methodology, several areas of uncertainty and variability were
acknowledged and included: lack of knowledge of recovery efficiencies of sampling methods;
analysis of ND data; choice of statistical software and data distributions used to calculate
exposure point concentrations; use of reaerosolization factors from the literature; and choice of
surrogate used. These areas of uncertainty and variability need to be examined to limit the
potential bias in the final exposure calculation. While the exposures calculated using this initial
methodology will be limited by uncertainties and should be considered qualitative, the lessons
learned from development of the methodology are critical to moving the science forward and
determining gaps/needs for quantitative exposure assessments.
7.4.4. Summary
The information and experience obtained during Phase 1 was used in Phase 2, the interagency
response and remediation exercise, to aid in the development of sampling, risk mitigation,
decontamination, and waste management plans. Phase 2 involved the interagency response to
a covert release of Ba (simulant); the exercise initiated with public health and federal law
enforcement notification and completed through facility remediation. Information on Phase 2 can
be found in the Exercise After Action Report'31. Overall, the BOTE Project provided the
opportunity to assess the interagency's current response and remediation capabilities and areas
408
-------
of need for future capability enhancements. It provided the opportunity to assess current
sampling and decontamination options in a standardized, field-scale test bed to understand the
scalability of laboratory efficacy testing to actual field-scale application. In total, field-scale
efficacy based upon the actual conditions achieved in the application of each in decontamination
method within the BOTE Project facility were well indicated by prior laboratory-based efficacy
testing data. However, the effort require or ability to achieve the target decontamination
conditions and the impact of not achieving those conditions (e.g., with fumigation with VHP®)
were captured during the BOTE Project Phase 1. Such information was also captured during
BOTE Phase 2, e.g.,for fumigation with methyl bromide. The field-scale opportunity offered an
assessment of the decontamination technologies, to provide added confidence to the
relationship to laboratory testing and ideas for future research needs to enhance response and
remediation capabilities.
409
-------
8. REFERENCES
1. U.S. Environmental Protection Agency Region 1 New England. 2008. After Action Report
- Danbury Anthrax Incident.
2. U. S. Environmental Protection Agency. 2005. Compilation of data on building
decontamination alternatives Washington, D.C. EPA/600/R-05/036.
3. Bio-Response Operational Testing and Evaluation (BOTE) Project Phase 2 After Action
Report. Washington, DC.
4. Amerithrax Investigation [Online]. Available:
http://www.fbi.gov/anthrax/amerithraxlinks.htm [Accessed January 26, 2010].
5. Jernigan, J. A., Stephens, D. S., Ashford, D. A., Omenaca, C., Topiel, M. S., Galbraith,
M., Tapper, M., Fisk, T. L, Zaki, S., Popovic, T., Meyer, R. F., Quinn, C. P., Harper, S.
A., Fridkin, S. K., Sejvar, J. J., Shepard, C. W., McConnell, M., Guarner, J., Shieh, W. J.,
Malecki, J. M., Gerberding, J. L, Hughes, J. M. and Perkins, B. A. 2001. Bioterrorism-
related inhalational anthrax: The first 10 cases reported in the United States. Emerging
Infectious Diseases, 7, 933-944.
6. Sharp, R. J. and Roberts, A. G. 2006. Anthrax: the challenges for decontamination.
Journal of Chemical Technology and Biotechnology, 81, 1612-1625.
7. Lengel, A. 2005. Little progress in FBI probe of anthrax attacks: internal report compiled
as agents hope for a break. The Washington Post, September 16, 2005.
8. Schmitt, K. and Zacchia, N. 2012. Total decontamination cost of the anthrax letter
attacks. Biosecur Bioterror, 10, 98-107.
9. Spotts Whitney, E. A., Beatty, M. E., Taylor, T. H., Weyant, R., Sobel, J., Arduino, M. J.
and Ashford, D. A. 2003. Inactivation of Bacillus anthracis spores. Emerging Infectious
Diseases, 9, 623-637.
10. U.S. Government Accountability Office. 2003 Capitol hill anthrax incident: EPA's cleanup
was successful; opportunities exist to enhance contract oversight. Washington, D.C.
GAO-03-686.
11. U.S. Government Accountability Office. 2004. U.S. Postal Service: Better Guidance Is
Needed to Ensure an Appropriate Response to Anthrax Contamination. Washington,
D.C. GAO-04-239.
12. U.S. Government Accountability Office. 2005. Anthrax detection: agencies need to
validate sampling activities in order to increase confidence in negative results.
Washington, D.C. GAO-05-251.
13. U.S. Department of Homeland Security. 2008. September 2007: Indoor Field Evaluation
of Sample Collection Methods and Strategies at Idaho National Laboratory.
14. U.S. Department of Homeland Security. 2008. September 2008: Indoor Field Evaluation
of Sample Collection Methods and Strategies at Idaho National Laboratory II. NSTD-09-
0163.
15. Ryan, S. P., Calfee, M. W., Wood, J. P., Attwood, B. C. and Frank W. Schaeffer, I. 2010.
Research to support the decontamination of surfaces and buildings contaminated with
biothreat agents. In: Rutala, W. A. (ed.) Disinfection, Sterilization, and Antisepsis.
Washington, DC: APIC.
16. Rastogi, V. K., Wallace, L., Smith, L. S., Ryan, S. P. and Martin, B. 2009. Quantitative
method to determine sporicidal decontamination of building surfaces by gaseous
fumigants, and issues related to laboratory-scale studies. Applied and Environmental
Microbiology, 75, 3688-3694.
410
-------
17. U.S. Environmental Protection Agency. 2009. Evaluation of liquid and foam technologies
for the decontamination of B. anthracis and B. subtilis spores on building and outdoor
materials. U.S. Environmental Protection Agency; Washington, D.C. EPA/600/R-09/150.
18. U.S. Environmental Protection Agency. 2011. Systematic investigation of liquid and
fumigant decontamination efficay against biological agents deposited on test coupons of
common indoor materials. U.S. Environmental Protection Agency; Washington, D.C.
EPA/600/R-11/076.
19. U.S. Environmental Protection Agency. 2012. Assessment of liquid and physical
decontamination methods for environmental surfaces contaminated with bacterial
spores: development and evaluation of the decontamination procedural steps. U.S.
Environmental Protection Agency,; Washingtion, DC. EPA/600/R-12/025.
20. U.S. Environmental Protection Agency. 2008. Material demand studies: interaction of
chlorine dioxide gas with building materials. U.S. Environmental Protection Agency;
Washington, D.C. EPA/600/R-08/091.
21. U.S. Environmental Protection Agency. 2010. Material demand studies: materials
sorption of vaporized hydrogen peroxide. U.S. Environmental Protection Agency;
Washington, DC. EPA/600/R-10/002.
22. U.S. Environmental Protection Agency. 2010. Compatibility of material and electronic
equipment with chlorine dioxide fumigation. U.S. Environmental Protection Agency;
Washington, DC. EPA/600/R-10/037.
23. U.S. Environmental Protection Agency. 2010. Compatibility of material and electronic
equipment with hydrogen peroxide and chlorine dioxide fumigation U.S. Environmental
Protection Agency; Washington, DC. EPA/600/R-10/169.
24. U.S. Environmental Protection Agency. 1989. Risk assessment guidance for Superfund
Volume I Human Health Evaluation Manual (Part A) Interim Final. U.S. Environmental
Protection Agency, Office of Emergency and Remedial Response; Washington, D.C.
EPA/540/1-89/002.
25. U.S. Environmental Protection Agency. 1992. Framework for ecological risk assessment.
U.S. Environmental Protection Agency, Risk Assessment Forum; Washington, D.C.
EPA/630/R-92/001.
26. NRC. 1983. Risk Assessment in the Federal Government: Managing the Process.
National Research Council; Washington, DC.
27. NRC. 2009. Science and decisions, advancing risk assessment. National Research
Council; Washington, D.C.
28. NRC. 2012. Exposure Science in the 21st Century: A Vision and A Strategy. National
Research Council; Washington, D.C.
29. International Life Sciences Institute (ILSI). 2000. Revised framework for microbial risk
assessment, an ILSI Risk Science Institute sorkshop report., Washington, D.C., ILSI
Press.
30. Rogers, J. V., Sabourin, C. L K., Choi, Y. W., Richter, W. R., Rudnicki, D. C., Riggs, K.
B., Taylor, M. L. and Chang, J. 2005. Decontamination assessment of Bacillus anthracis,
Bacillus subtilis, and Geobacillus stearothermophilus spores on indoor surfaces using a
hydrogen peroxide gas generator. Journal of Applied Microbiology, 99, 739-748.
31. Verce, M. F., Jayaraman, B., Ford, T. D., Fisher, S. E., Gadgil, A. J. and Carlsen, T. M.
2008. Minimizing decomposition of vaporized hydrogen peroxide for biological
decontamination of galvanized steel ducting. Environmental Science and Technology,
42,5765-5771.
32. U.S. Environmental Protection Agency. 2008. Effects of vapor-based decontamination
systems on selected building interior materials: chlorine dioxide. U.S. Environmental
Protection Agency; Washington, DC. EPA/600/R-08/054.
411
-------
33. U.S. Environmental Protection Agency. 2008. Effects of vapor-based decontamination
systems on selected building interior materials: vaporized hydrogen peroxide. U.S.
Environmental Protection Agency; Washington, DC. EPA/600/R-08/074.
34. U.S. Environmental Protection Agency. 2002. Federal on-scene coordinator's report for
the Capitol Hill site - Washington, D.C. U.S. Environmental Protection Agency;
Washington, DC. GS-10F-0076K.
35. Kane, S. R., Letant, S. E., Murphy, G. A., Alfaro, T. M., Krauter, P. W., Mahnke, R.,
Legler, T. C. and Raber, E. 2009. Rapid, high-throughput, culture-based PCR methods
to analyze samples for viable spores of Bacillus anthracis and its surrogates. Journal of
Microbiological Methods, 76, 278-284.
36. Letant, S. E., Kane, S. R., Murphy, G. A., Alfaro, T. M., Hodges, L R., Rose, L J. and
Raber, E. 2010. Most-probable-number rapid viability PCR method to detect viable
spores of Bacillus anthracis in swab sample. . Journal of Microbiological Methods, 81,
200-202.
37. Letant, S. E., Murphy, G. A., Alfaro, T. M., Avila, J. R., Kane, S. R., Raber, E., Bunt, B.
and Shah, S. 2011. Rapid Viability Polymerase Chain Reaction method for detection of
virulent Bacillus anthracis from environmental samples. Applied and Environmental
Microbiology 77, 6570-6578.
38. U.S. Code of Federal Regulations. 40 CFR Part 763, Subpart E, Appendix A.
39. OSHA. 2006. Anthrax e-Tool, Protecting the Worksite from Terrorism [Online]. Available:
http://www.osha.gov/SLTC/etools/anthrax/transition program.html [Accessed January 3,
2011].
40. Sinclair, R., Boone, S. A., Greenberg, D., Keim, P. and Gerba, C. P. 2008. Persistence
of category A select agents in the environment. Applied Environmental Microbiology, 74,
555-563.
41. Manchee, R. J., Broster, M. G., Melling, J., Henstridge, R. M. and Stagg, A. J. 1981.
Bacillus anthracis on Gruinard Island. Nature, 294, 254-255.
42. Lewis, J. C. 1969. Dormancy. In: Hurst, A. and Gould, G. W. (eds.) The bacterial spore.
London, United Kingdom: The Academic Press.
43. Turnbull, P. (ed.) 2008. Anthrax in humans: World Health Organization.
44. Ibraham, K. H., Brown, G., Wright, D. H. and Rotschafer, J. C. 1999. Bacillus anthracis:
medical issues of biologic warfare. Pharmacotherapy, 19, 690-701.
45. Sextro, R. G. and Lorenzetti, D. M. Modeling the spread of anthrax in buildings. Indoor
Air 2002 Conference, 2002 Monterey, California.
46. Doolan, D. L, Freilich, D. A., Brice, G. T., Burgess, T. H., Berzins, M. P., Bull, R. L,
Graber, N. L., Dabbs, J. L., Shatney, L. L, Blazes, D. L., Bebris, L. M., Malone, M. F.,
Eisold, J. F., Mateczun, A. J. and Martin, G. J. 2007. The US capitol bioterrorism anthrax
exposures: clinical epidemiological and immunological characteristics. Journal of
Infectious Disease, 195, 174-84.
47. Franco, C. and Bouri, N. 2010. Environmental decontamination following a large-scale
bioterrorism attack: federal progress and remaining gaps. Biosecurity and Bioterrorism,
8, 107-17.
48. Meselson, M., Guillemin, J., Hugh-Jones, M., Langmuir, A., Popova, I., Shelokov, A. and
Yampolskaya, O. 1994. The Sverdlovsk anthrax outbreak of 1979. Science, 266, 1202-
1208.
49. Weis, C. P., Intrepido, A. J., Miller, A. K., Cowim, P. G., Durno, M. A., Gennardt, J. S.
and Bull, R. 2002. Secondary aerosolization of viable Bacillus anthracis spores in a
contaminated U.S. Senate office. Journal of the American Medical Association, 288,
2853-2858.
412
-------
50. Price, P. N., Sohn, M. D., Lacommare, K. S. and McWilliams, J. A. 2009. Framework for
evaluating anthrax risk in buildings. Environmental Science and Technology, 43, 1783-7.
51. Inglesby, T. V., O'Toole, T., Henderson, D. A., Bartlett, J. G., Ascher, M. S., Eitzen, E.,
Friedlander, A. M., Gerberding, J., Hauer, J., Hughes, J., McDade, J., Osterholm, M. T.,
Parker, G., Perl, T. M., Russell, P. K. and Tonat, K. 2002. Anthrax as a biological
weapon, 2002: updated recommendations for management Journal of the American
Medical Association, 287, 2236-2252.
52. Thatcher, T. and Layton, D. 1995. Deposition, resuspension, and penetration of particles
within a residence. Atmospheric Environment, 29, 1487-1497.
53. Ferro, A. R., Kopperud, R. J. and Hildemann, L. M. 2004. Source strengths for indoor
human activities that resuspend particulate matter. Environmental Science and
Technology, 38, 1759-64.
54. Gomes, C., Freihaut, J. and Bahnfleth, W. 2007. Resuspension of allergen-containing
particles under mechanical and aerodynamic disturbances from human walking.
Atmospheric Environment, 41, 5257-5270.
55. Oberoi, R. C., Choi, J. I., Edwards, J. R., Rosati, J. A., Thornburg, J. and Rodes, C. E.
2010. Human-induced particle re-suspension in a room. Aerosol Science and
Technology, 44, 216-229.
56. Buttner, M. P., Cruz-Perez, P., Stetzenbach, L. D., Garrett, P. J. and Luedtke, A. E.
2002. Measurement of airborne fungal spore dispersal from three types of flooring
materials. . Aerobiologia, 18, 1-11.
57. Krauter, P. and Biermann, A. 2007. Reaerosolization of fluidized spores in ventilation
systems. Applied and Environmental Microbiology, 73, 2165-2172.
58. U.S. Centers for Disease Control and Prevention. 2004. Responding to detection of
aerosolized Bacillus anthracis by autonomous detection systems in the workplace.
Morbidity and Mortality Weekly Report, 53, 1-11.
59. Defense Threat Reduction Agency. 2011. Bio-Response Operational Testing and
Evaluation (BOTE), final test plan. Defense Threat Reduction Agency, Counter WMD
Technologies Test Support Division; Kirtland AFB, N.M.
60. Sandia National Laboratories. 2006. BROOM (Building Restoration Operations
Optimization Model) Beta Version 1.0. Sandia National Laboraties.
61. Brown, G. S., Betty, R. G., Brockmann, J. E., Lucero, D. A., Souza, C. A., Walsh, K. S.,
Boucher, R. M., Tezak, M. S. and Wilson., M. C. 2007. Evaluation of vacuum filter sock
surface sample collection method for Bacillus spores from porous and non-porous
surfaces. Journal of Environmental Monitoring, 9, 666-671.
62. Hodges, L. R., Rose, L. J., O'Connell, H. and Arduino, M. J. 2010. National validation
study of a swab protocol for the recovery of Bacillus anthracis spores from surfaces.
Journal of Microbiological Methods, 81, 141-146.
63. Rose, L. J., Hodges, L, O'Connell, H. and Noble-Wang, J. 2011. National validation
study of a cellulose sponge wipe-processing method for use after sampling Bacillus
anthracis spores from surfaces. Applied and Environmental Microbiology, 77, 8355-8359.
64. Brown, G. S., Betty, R. G., Brockmann, J. E., Lucero, D. A., Souza, C. A., Walsh, K. S.,
Boucher, R. M., Tezak, M., Wilson, M. C. and Rudolph., T. 2007. Evaluation of a wipe
surface sample method for collection of Bacillus spores from nonporous surfaces.
Applied and Environmental Microbiology, 73, 706-710.
65. Brown, G. S., Betty, R. G., Brockmann, J. E., Lucero, D. A., Souza, C. A., Walsh, K. S.,
Boucher, R. M., Tezak, M. S. and Wilson, M. C. 2007. Evaluation of vacuum filter sock
surface sample collection method for Bacillus spores from porous and non-porous
surfaces. Journal of Environmental Monitoring, 9, 666-671.
413
-------
66. U.S. Environmental Protection Agency and U.S. Centers for Disease Control and
Prevention. 2011. Comparison of ultrafiltration techniques for recovering biothreat agents
in water. U.S. Environmental Protection Agency Cincinnnati, OH. EPA 600/R-11/103.
67. Markoulatos, P., Siafakas, N. and Moncany, M. 2002. Multiplex polymerase chain
reaction: a practical approach. Journal of Clinical Laboratory Analysis, 16, 47-51.
68. Bessetti, J. 2007. An introduction to PCR inhibitors. Profiles in DNA, 10, 9-10.
69. Opel, K. L, Chung, D. and McCord, B. R. 2010. A study of PCR inhibition mechanisms
using real time PCR. Journal of Forensic Sciences, 55, 25-33.
70. Tsai, Y. L. and Olson, B. H. 1992. Rapid method for separation of bacterial DNA from
humic substances in sediments for polymerase chain reaction. Applied and
Environmental Microbiology, 58, 2292-2295.
71. Wilson, K. H., Wilson, W. J., Radosevich, J. L, DeSantis, T. Z., Viswanathan, V. S.,
Kuczmarski, T. A. and Andersen, G. L. 2002. High-density microarray of small-subunit
ribosomal DNA probes. Applied and Environmental Microbiology, 68, 2535-2541.
72. Bradley, M. D., Rose, L., Noble-Wang, J. and Arduino, M. 2011. Biological sample
preparation collaboration project: Detection of Bacillus anthracis spores in soil. U.S.
Environmental Protection Agency; Cincinnati, OH. EPA 600/R-10/177.
73. Chosewood, L. C. and Wilson, D. E. (eds.) 2009. Biosafety in microbiological and
biomedical laboratories, Washington, D.C.: U.S. Government Printing Office.
74. ASTM. 1998. D5465-93 Standard Practice for Determining Microbial Colony Counts from
Waters Analyzed by Plating Methods.
75. Promega Corporation. 2009. MagneSil blood genomic, max yield system. Madison, W.I.
Technical Bulletin TB312.
76. Saikaly, P. E., Barlaz, M. A. and Reyes III, F. 2007. Development of quantitative real-
time PCR assays for detection and quantification of surrogate biological warfare agents
in building debris and leachate. Applied and Environmental Microbiology, 73, 6557-6565.
77. Rice, E. W., Adcock, N. J., Sivaganesan, M. and Rose, L. J. 2005. Inactivation of
Bacillus anthracis Sterne, Bacillus cereus, and Bacillus thuringiensis subsp. israelensis
by chlorination. Applied and Environmental Microbiology, 71, 5587-5589.
78. Rose, L. J., Rice, E. W., Jensen, B., Murga, R., Peterson, A., Donlan, R. M. and Arduino,
M. J. 2005. Chlorine inactivation of bacterial bioterrorism agents. Applied and
Environmental Microbiology, 71, 566-568.
79. Sivaganesan, M., Adcock, N. J. and Rice, E. W. 2006. Inactivation of Bacillus globigii by
chlorination: A Bayesian model. Journal of Water Supply Research and Technology -
AQUA, 55, 33-43.
80. Federal National Response Team. 2012. NRT quick reference guide: Bacillus anthracis
PPE wash water decontamination [Online]. Available: www.epaosc.net/qrg [Accessed
June 15, 2012].
81. GraphPad QuickCalcs web site [Online]. Available:
http://qraphpad.com/quickcalcs/ttest1.cfm [Accessed July 2012].
82. Herzog, A. B., McLennan, S. D., Pandey, A. K., Gerba, C. P., Haas, C. N., Rose, J. B.
and Hashsham, S. A. 2009. Implications of limits of detection of various methods for
Bacillus anthracis in computing risks to human health. Applied and Environmental
Microbiology, 75, 6331-9.
83. Fleiss, J. L. 1981. Statistical methods for rates and proportions, Second Edition, New
York, John Wiley.
84. Mason, J. Y. 2010. Methods of using chlorine dioxide as a fumigant. United States patent
application US 11/270,973.
85. American Public Health Association, Water Environment Federation and American Water
Works Association. 1995. 4500-CIO2 Chlorine Dioxide, E. Amperometric Method II. In:
414
-------
Eaton, A. D., Clesceri, L. S., Rice, E. W. and Greenberg, A. E. (eds.) Standard Methods
for the Examination of Water and Waste Water. 20th ed. Baltimore, MD: American Water
Works Association.
86. Rastogi, V. K., Ryan, S. P., Wallace, L, Smith, L. S., Shah, S. S. and Martin, G. B. 2010.
Systematic evaluation of chlorine dioxide efficacy in decontamination of building interior
surfaces contaminated with anthrax spores Applied and Environmental Microbiology, 76,
3343-3351.
87. Canter, D. A. Remediating sites with anthrax contamination: building on experience.
AWMA/EPA Indoor air quality problems and engineering solutions specialty conference
and exhibition, July 21-23 2003 Research Triangle Park, N.C.
88. Juergensmeyer, M. A., Gingras, B. A., Scheffrahn, R. H. and Weinberg, M. J. 2007.
Methyl bromide fumigant lethal to Bacillus anthracis spores. Journal of Environmental
Health, 69, 24-26.
89. U. S. Environmental Protection Agency. 2008. Report on the 2007 workshop on
decontamination, cleanup, and associated issues for sites contaminated with chemical,
biological, or radiological materials. Washington, D.C. EPA/600/R-08/059.
90. U.S. Environmental Protection Agency. 2007. FIFRA SAP Meeting No. 2007-50 - A set
of scientific issues being considered by the Environmental Protection Agency regarding:
guidance on test methods for demonstrating the efficacy of antimicrobial products for
inactivating Bacillus anthracis spores on environmental surfaces [Online]. Available:
http://www.epa.gov/scipoly/sap/meetings/2007/071707 mtg.htm [Accessed January 26,
2010].
91. Baron, P. A., Estill, C. F., Deye, G. J., Hein, M. J., Beard, J. K., Larsen, L. D. and
Dahlstrom, G. E. 2008. Development of an aerosol system for uniformly depositing
Bacillus anthracis spore particles on surfaces. Aerosol Science and Technology, 42,
159-172.
92. Whitfield, W. 1979. A study of the effects of relative humidity on small particle adhesion
to surfaces. In: Mittal, M. (ed.) Surface contamination: genesis, detection and control.
New York: Plenum Press.
93. U.S. Statute. 1976. Resource Conservation and Recovery Act.
94. CareerMedia.com. 2011. 2011 Salary Table [Online]. Available:
http://careermedia.salary.com [Accessed November 18 2011].
95. U.S. Office of Personnel Management. 2011. 2011 General Schedule (GS) Locality Pay
Tables [Online]. Available: http://www.opm.gov/oca/11tables/indexgs.asp [Accessed
November 182011].
96. Meltzer, M. August 8, 2011 2011. RE: (personal communication). Type to Lemiuex, P. M.
97. HomeWyse.com. 2011. Cost to install duct: 2011 cost calculator (zip code based)
[Online]. Available: http://www.homewyse.com/services/cost to install duct.html
[Accessed November 18, 2011].
98. Bell, A. (ed.) 2007. HVAC equations, data, and rules of thumb, 2nd edition: McGraw-Hill.
99. Agency, U. S. E. P. 2001. Waste transfer stations: a manual for decision-making. Office
of Solid Waste and Emergency Response; Washington, D.C. E PA/530-D-01-001.
100. Procell, L. R., Hess, Z. A., Gehring, D. A., Lynn, J. T., Bartram, P. W., Brickhouse, M. D.,
Lalain, T. A., Ryan, S. P., Attwood, B. C. and Martin, G. B. 2010. Material demand
studies: materials sorption of vaporized hydrogen peroxide. U.S. Environmental
Protection Agency; Washington, DC. EPA/600/R-10/002.
101. Ryu, C., Lee, K., Yoo, C., Seong, W. K. and Oh, H. B. 2003. Sensitive and rapid
quantitative detection of anthrax spores isolated from soil samples by real-time PCR.
Microbiology and Immunology, 47, 693-9.
415
-------
102. Van Cuyk, S., Deshpande, A., Hollander, A., Franco, D. O., Teclemariam, N. P.,
Layshock, J. A., Ticknor, L. O., Brown, M. J. and Omberg., K. M. 2012. Transport of
Bacillus thuringiensis var. kurstaki from an outdoor release into buildings: pathways of
infiltration and a rapid method to identify contaminated buildings. Biosecurity and
Bioterrorism, 10, 215-227.
103. Read, S. J. 2001. Recovery efficiencies on nucleic acid extraction kits as measured by
quantitative LightCycler PCR. Molecular Pathology, 54, 86-90.
104. U.S. Environmental Protection Agency. 2012. ProUCL Software [Online]. Available:
http://www.epa.gov/osp/hstl/tsc/software.htm [Accessed January 29, 2013].
105. Pacific Northwest National Laboratory. VSP (Visual Sampling Plan) [Online]. Available:
http://vsp.pnnl.gov/ [Accessed July 31, 2013].
416
-------
Appendix A
INL Facility PBF-632 Test Room Photos and Inventory of
Contents
-------
Commercial Setting
(Rooms 106, 108, 110, 208, 210, and 212)
FigureA-1. Room 106
Figure A-2. Room 108
Figure A-3. Room 110
A-2
-------
Figure A-4. Room 208
Figure A-5. Room 210
Figure A-6. Room 212
A-3
-------
Residential Setting
(Rooms 105, 107, 109, 209, 211, and 213)
Figure A-7. Room 105
Figure A-8. Room 107
Figure A-9. Room 109
A-4
-------
F ig u re A-10. Room 209
Figure A-11. Room 211
Figure A-12. Room 213
A-5
-------
Figure A-13. Room 106
Figure A-14. Room 108
Figure A-15. Room 110
A-6
-------
F ig u re A-16. Room 208
Figure A-17. Room 210
Figure A-18. Room 212
A-7
-------
Industrial Setting
(Rooms 104 and 206)
Figure A-19. Room 104
F ig u re A-20. Room 206
A-8
-------
Office Building Mailroom Setting
(Rooms 103 and 207)
Figure A-21. Room 103
F ig u re A-22. Room 207
A-9
-------
Reaerosolization Study Rooms
(Rooms 101A and 102)
Figure A-23. Room 101A
Figure A-24. Room 102
A-10
-------
Table 1. Room Configuration and Inventory of Contents
Configuration
Commercial (Office) Setting
Commercial (Office) Setting
(Reaerosolization Study
Rooms)
Industrial Setting
Residential (Kitchen) Setting
Residential (Living Room)
Setting
Residential (Bed Room)
Setting
Mailroom Setting
Room
106
108
110
208
210
212
101A
102
104
206
105
209
107
211
109
213
103
207
Ceiling, Wall, and
Floor Type
a>
F
D)
.g
'a>
O
S
S
S
Q
1
1
1
1
1
1
in
^
o
o
CD
20
20
20
20
20
20
20
20
20
Binders
10
10
10
10
10
10
™-t>
to =
- §
3
C D)
I!
20
20
Bed with
2 Night Stands
1
1
&
o
w
1
1
8
ra
O
^
o
o
m
1
1
1
1
1
1
1
1
1
D)
C -^
= (D
LI- C
— J2
ro ro
^ O
2
1
1
1
1
1
1
4
3
-------
Appendix B
Bg Spore Characterization Study Results
-------
MORPHOLOGY, TITER, AND PHYSICAL CHARACTERISTICS
Summary: All morphological characteristics were consistent with previously documented
descriptions of Bacillus globigii. Escherichia coli, the negative control, did not fit any of the
morphological or physical characteristics consistent with B. globigii spores and vegetative cells.
1.1. Gram Stain
Summary: The Gram staining procedure was used to describe the test and negative control
organisms. Results indicated vegetative B. globigii cells were purple or Gram positive as
expected, while the negative control, E. coli, DH5a was pink or Gram negative, as expected.
1.2. Colony Morphology
Colonies of the test organism, and controls were described according to Methods for General
and Molecular Microbiology, 3rd ed.1. Representative photographs of these organisms on Petri
plates are on page 3.
Colonial growth of Critical Reagents Program (CRP) B. globigii on Tryptic Soy Agar Petri plates
(ISA) was characterized as:
• Orange/peach/salmon color
• Rough texture
• 1-3 mm diameter individual colonies
• Round form
• Undulate margin
• Flat (slightly convex) elevation
Colonial growth of B. globigii (positive control) on ISA was characterized as:
• Orange/peach/salmon color
• Rough texture
• 1-2 mm diameter individual colonies
• Round form
• Undulate margin
• Flat (slightly convex) elevation
Colonial growth of E. coli (negative control) on ISA was characterized as:
• Pearl white (translucent)
• Smooth texture
• 0.75 - 1 mm diameter individual colonies
• Round form
• Entire margin
• Convex elevation
1 Reddy, C.A., Beveridge, T.J., Breznak, J.A., Marzluf, G.A., and Schmidt, T.M. 2007. Methods for
General and Molecular Bacteriology, 3rd ed. ASM Press, Washington, D.C.
-------
1.3. liter Determination
Three dry aliquots (aliquots 2, 4, and 5) of B. globigii spores, supplied by the Critical Reagent
Program, each was weighed and cultured to determine the viable number of cells per gram.
liter determinations for each of the aliquots were as follows:
• Aliquot 2-1.34 X1011 Colony Forming Unit (CPU) gram'1 (11.13 Log10)
• Aliquot 4-2.68 X1011 CPU gram'1 (11.43 Log™)
• Aliquot 5-1.22 X1011 CPU gram'1 (11.09 Log™)
• Average titer = 1.74 X 1011 CPU gram'1 (± 8.1 X 1010 CPU gram'1)
The QA/QC requirement of having a minimum 1.0X 1010 CPU gram"1 titer was met. In addition,
abundance estimates from triplicate samples were within precision criteria of 0.5 Log of one
another, thus meeting the QA/QC criteria.
B-3
-------
Bacillus globigii
CRP-BOTE strain
Bacillus globigii
Positive Control
Escherichia coli
Negative Control
B-4
-------
1 1.4. Heat-Shock Test
2
3 Spore viability of the CRP B. globigii and the positive control B. globigii was determined for both
4 heat-shocked spores (80°C for 20 minutes) and non-heat-shocked spores. The heat-shocked
5 and the non-heat shocked samples were tested in triplicate and the percent survival was
6 determined.
7 Results were as follows:
8 B. Qlobigii Positive Control Strain
9 • Non-heat-shocked-1.12 X106 CPU (5.75 Log10)
10 • Heat-shocked - 9.58 X105 CPU (6.11 Log10)
11 • Log Difference = 0.07
12 CRP 6. alobigii Test Strain
13 • Non-heat-shocked - 5.65 X 105 CPU (5.75 Log10)
14 • Heat-shocked-1.28 X106 CPU (6.11 Log10)
15 • Log Difference = 0.36
16 The QA/QC requirement was for the Log difference before and after heat-shock to be no greater
17 than 0.5. This requirement was met.
18 1.5. Acid Resistance Test
19
20 Spore viability of the CRP B. globigii spores and the positive control B. globigii spores was
21 determined for hydrochloric acid (HCI)-exposures of 2, 5, 10, and 20 minutes. Non-HCI-
22 exposure control determinations were also conducted. Both the HCI exposed and the non-HCI
23 exposed samples were tested in duplicate. Spore viability was determined by the development
24 of turbidity in fluid thioglycollate culture tubes after 21 days incubation at 35°C. Growth in either
25 the aerobic or anaerobic fraction of the fluid thioglycollate medium was considered a positive
26 response for the sample. Spore growth in the fluid thioglycollate medium was confirmed or
27 refuted by plating each positive broth tube. An aliquot of 0.1 ml was plated onto TSA and
28 incubating overnight at 35°C. The results were as follows:
29 Negative control (no inoculum): No growth in any tube or on any plate
30 Positive control B. Qlobigii: Growth in all tubes and on plates for HCI exposures of 0, 2, 5, 10
31 minutes, 50% of samples viable (tubes, confirmed by plating) after 20 minutes HCI exposure.
32 CRP 6. QlobiQii: Growth in all tubes HCI exposed for 0 and 2 minutes HCI. Growth in 50% of
33 tubes exposed for 10 and 20 minutes to HCI. Only 25 % growth occurred in tubes exposed for
34 5 minutes to HCI. In all cases the growth in fluid thioglycollate broth tubes was confirmed by
35 plating on TSA.
B-5
-------
1
2
3
4
5
6
7
8
9
10
The QA/QC requirement was for the B. globigii spores to survive HCI exposure for a minimum of
2 minutes. This criterion was met by both the positive control B. globigii spores and the CRP B.
globigii spores.
1.6.
Microscopic Observation
Both the CRP B. globigii spore and positive control B. globigii spore preparations were
suspended in diluent to produce a monolayer under a 22 mm2 cover glass on a glass
microscope slide. These preparations were examined at 1,000 X using phase contrast optics.
Representative photographs appear below.
Bacillus globigii positive control
spore preparation; phase 1,000 X
oil immersion, note the preparation
is mono-dispersed. (2/1/2011)
CRP Bacillus globigii spore
preparation; phase 1,000 X oil
immersion, note there are
clumps to which spores appear
attached. (2/14/2011)
11
12
13
14
15
The B. globigii positive control spore preparation contained numerous spores that were of
consistent size and shape. The spores were mono-dispersed and showing no evidence of
clumping.
B-6
-------
1 The CRP B. globigii spore preparation exhibited clumps of various sizes. While these clumps
2 did not appear to be composed uniformly of spores, signs of spore adherence were evident.
3 Some of the smaller clumps appeared to be composed entirely of spores associations.
B-7
-------
1 QPCR AND SEQUENCING
2
3 2.1. qPCR
4
5 Both the CRP B. globigii spores and the positive control B. globigii spores had their DMA
6 extracted and amplified. The Surface Spore Protein (SSP) gene and the recF gene (DMA repair
7 gene) were the target of separate qPCR amplifications. As a negative control, E. coli
8 vegetative cell DMA was extracted and amplified. The results were that the negative control did
9 not amplify, but both the CRP spores as well as the positive control spores amplified in a dose
10 response manner producing low cycle threshold values.
11 The QA/QC requirement for the B. globigii spore molecular analysis was met for both the
12 positive control B. globigii spores, and the CRP B. globigii spores. The negative control reacted
13 as expected.
14
15 2.2. DNA Sequencing
16
17 For DNA sequencing analysis, PCR product from multiple B. globigii SSP gene and 16S
18 ribosomal gene were prepared and combined by ethanol precipitation. This material was
19 submitted to the CORE Molecular Genetics Laboratory at Cincinnati Children's Hospital Medical
20 Center for DNA sequence analysis. The results of the SSP gene sequence analysis were
21 inconclusive due to the small size of the amplified PCR product2. On the other hand, the 16S
22 sequencing results confirmed both the CRP spores and the positive control spores were greater
23 than 99% similar to known B. atrophaeus (syn. for B. globigii) strains in the NIH-BLAST
24 Database. The negative control E. coli DNA was 99% similar to known E. coli strains in the
25 NIH-BLAST Database.
26 The QA/QC requirement for the B. globigii spore sequence analysis was met for both the
27 positive control B. globigii prep, and the CRP B. globigii strain. The negative control reacted as
28 expected.
29
2 personal communication, David Fletcher, CORE Laboratory Manager
B-8
-------
1 AEROSOL PARTICLE SIZE
2
3 Aerosol particle size distribution analysis indicated an overall particle size of 3.38 urn, when the
4 CRP spore preparation was suspended in ethanol. The table and figure below indicate that
5 there was great agreement between the various determinations. When, however, the CRP
6 spore preparation was suspended in water, the determinations (data not shown) were not tight
7 and suggested a great deal of clumping. Rather than having one distinct peak as in the case
8 of the ethanol suspension, a number of broad based peaks were observed.
B-9
-------
Section III: Aerosol Particle Sizer Determination
BG Spore Size Distribution Overall Average
MMAD = 3.38
a. = 1.29
B SB U
Particle Aerodynamic Diameter (|im)
18
J tnctaOiH onfy t
•*•
zo
B-10
-------
BG Spore Size Distribution Testing
approx. 1 mg BG spores in 100 mL of 200 proof ethyl alcohol
Aerosolized with a 3-jet Collison nebulizer
Tests performed at 25 ± 3 °C and 40% ± 10% RH
Particle size distribution measured with UV-APS
9 consecutive 60-s samples in each of 3 runs
Spore density used =
Wet spore p=
Dry spore p=
*Run # 1 includes only samples 4-9
Overall Average
"= 0.25*pwet+0.75*pclry"
1.201
1.45
Particle aerodynamic
diameter
(urn)
0.523
0.542
0.583
0.626
0.673
0.723
0.777
0.835
0.898
0.965
1.037
1.114
1.197
1.286
1.382
1.486
1.596
1.715
1.843
1.981
2.129
2.288
2.458
2.642
2.839
3.051
3.278
3.523
3.786
4.068
4.371
4.698
5.048
5.425
5.829
6.264
6.732
7.234
7.774
8.354
8.977
9.647
10.37
11.14
11.97
12.86
13.82
14.86
15.96
17.15
18.43
19.81
Mass concentration w/ fluorescence >1
Average of
*Run#l Run #2
(mg/ms) (mg/ms)
0 0
0 0
0 0
0 0
0 0
0 0
0 2.31801E-08
0 0
5.35435E-08 3.56957E-08
6.6444E-08 0
8.2453E-08 5.49687E-08
3.06957E-07 1.36425E-07
6.34858E-07 7.6183E-07
2.36345E-06 1.89076E-06
3.91053E-06 3.51948E-06
9.46283E-06 7.76437E-06
2.61954E-05 1.18431E-05
4.5958E-05 3.1386E-05
9.7370 IE-OS 7.29502E-05
0.000191027 0.000146531
0.000334158 0.000269421
0.000515678 0.000454246
0.00074548 0.000682441
0.001041072 0.000892347
0.001227565 0.001116378
0.001521229 0.001374149
0.001647871 0.00147926
0.001640455 0.001589764
0.00167835 0.001675673
0.00151471 0.001697406
0.001317 0.001310817
0.000828666 0.001063967
0.00043799 0.000622072
0.000200865 0.000354468
2.93249E-05 0.000273699
0 7.27807E-05
0 6.02107E-05
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
Run #3
(mg/ms)
0
0
0
0
1.50527E-08
0
0
5.753E-08
3.56957E-08
4.4296E-08
0
1.36425E-07
3.38591E-07
1.57564E-06
1.95527E-06
6.95558E-06
1.44526E-05
3.56206E-05
6.86227E-05
0.000148065
0.000263233
0.000444204
0.000702232
0.000932371
0.001178462
0.00148621
0.001519239
0.001628591
0.001747946
0.001650901
0.00121601
0.000930971
0.000641115
0.000370223
0.000156399
8.49108E-05
1.50527E-05
3.73589E-05
4.63602E-05
0
0
0
0
0
0
0
0
0
0
0
0
0
Average
(mg/ms)
0
0
0
0
5.02E-09
0
7.73E-09
1.92E-08
4.16E-08
3.69E-08
4.58E-08
1.93E-07
5.78E-07
1.94E-06
3.13E-06
8.06E-06
1.75E-05
3.77E-05
7.96E-05
0.000162
0.000289
0.000471
0.00071
0.000955
0.001174
0.001461
0.001549
0.00162
0.001701
0.001621
0.001281
0.000941
0.000567
0.000309
0.000153
5.26E-05
2.51E-05
1.25E-05
1.55E-05
0
0
0
0
0
0
0
0
0
0
0
0
0
-------
Appendix C
Surface Sampling Protocols
-------
Entry Procedures
Entering the Building:
1) Persons A, B, and C: Don proper personal protection equipment (PPE) as stated in the
Sample Collection Plan in designated sample staging area.
i) Tyvek suit
ii) Tyvek booties
iii) M40 Pro-Mask or equivalent full face air purifying respirator
iv) Nitrile gloves
(1) Put on one pair of nitrile gloves and tape to suit
(2) Place four pairs of XX-large gloves onto gloved hands.
2) Person A (BROOM Operator): Obtain a BROOM PDA from table in designated sample
staging area and turn it on.
3) Person B (Supplier): Retrieve cart, sample collection supply bin and sample collection bin in
designated sample staging area. Clearly mark in large identifiable letters on the backs of the
team member's Tyvek® suits either an A, B or C to designate their assignment.
4) Person C (Collector): Open door to enter building.
5) Person B (Supplier): Move cart and supplies into building and position outside of designated
room for sample collection.
6) Person A (BROOM Operator): Carry BROOM PDA into building and direct team to
appropriate sample collection rooms in the order outlined by Sampling Lead, and direct the
samplers on the type and the location of samples to be collected as indicated on the PDA
and in the Sample Plan. Direct sample collection team to collect either an environmental or
negative (field blank) control sample and track on the PDA.
I
IMPORTANT NOTE: DO NOT TOUCH
ANYTHING ELSE OTHER THAN WHAT IS
STATED WITHIN THIS PROCEDURE
BEFORE SAMPLING WITHOUT
CHANGING GLOVES
C-1
-------
Sample Collection Protocols
Collecting Reference Material Coupon (RMC):
1) Persons A, B and C: Discard existing top pair of gloves.
Note: After each person has discarded 4 pairs of gloves, open a new Ziploc® bag with
clean gloves and place another set of 4 gloves over last pair on your hands, while inside
Ziploc® bag.
2) Person B (Supplier):
a) Open sample collection bin.
b) Remove RMC specimen kit from bin.
c) Hold specimen kit barcode label out for Person A to scan.
3) Person A (BROOM Operator): Scan sample barcode label located on outside of sample
Ziploc® bag into BROOM and enter in required fields.
4) Person B (Supplier):
a) Open Ziploc® bag and remove package of disposable forceps.
b) Open package of disposable forceps without touching them for Person C to remove from
packaging.
c) Discard forceps packaging into waste.
d) Move 50 ml_ conical tube to end of Ziploc® bag, and loosen cap. Once sample has
been collected, remove cap so Person C call place sample in tube.
C-2
-------
5) Person C (Collector):
a) Remove disposable forceps from packaging and transfer RMC into 50 ml_ conical tube
that Person B is holding, being careful not to touch surface of RMC, the 50 ml_ conical
tube or the plastic Ziploc® bag.
b) Discard disposable forceps into waste.
6) Person B (Supplier):
a) Immediately close and tighten cap to 50 ml_ conical tube and slide tube back into sample
collection Ziploc® bag.
b) Place sample into larger, clean Ziploc® bag.
7) Persons B and C: Remove gloves and discard in waste.
8) Repeat Steps 1-5 as necessary.
Collecting Settle Plates:
1) Persons A, B and C: Discard existing top pair of gloves.
Note: After each person has discarded 4 pairs of gloves, open a new Ziploc® bag with
clean gloves and place another set of 4 gloves over last pair on your hands, while inside
Ziploc® bag.
2) Person B (Supplier):
a. Open sample collection bin.
b. Remove a settle plate kit.
c. Hold settle plate barcode label out for Person A to scan.
3) Person A (BROOM Operator): Scan sample barcode label located on outside sample Ziploc11
bag into BROOM and enter in required fields.
4) Person B (Supplier): Open bag and maneuver lid towards top of bag without touching it.
C-3
-------
5) Person C (Collector):
a. Take lid out of Ziploc bag and place on agar plate.
b. Hold agar plate out for Person B to wrap in Parafilm and then place back in Ziploc® bag.
6) Person B (Supplier): Once collected, wrap the covered agar dish with Parafilm, and open
Ziploc® bag. Once Person C places plate into bag, seal it.
7) Persons B and C: Remove gloves and discard in waste.
8) Repeat steps 1 through 6, as necessary.
Collecting Swab Samples:
1) Persons A, B and C: Discard existing top pair of gloves. Note: After each person has
discarded 4 pairs of gloves, open a new Ziploc® bag with clean gloves and place another set
of 4 gloves over last pair on your hands, while inside Ziploc® bag.
2) Person B (Supplier):
a) Open sample collection bin.
b) Remove swab specimen kit from bin.
c) Hold specimen kit barcode label out for Person A to scan.
3) Person A (BROOM Operator): Scan sample barcode label located on outside of sample
Ziploc® bag into BROOM and enter in required fields.
4) Person B (Supplier):
a) Open outer Ziploc® bag containing prepackaged swab.
b) Open package of swab without touching it for Person C to remove from packaging.
c) Discard packaging in waste once Person C has removed swab.
d) Move 2 ml vial containing neutralizing buffer to top of bag for Person C.
e) After Person C removes swab and vial from bag, open template packaging in bin and
remove a 2" x 2" in template.
f) Hand template to Person C.
5) Person C (Collector):
a) Carefully remove swab from bag without touching bag or tip of swab.
b) Remove 2 ml vial and open with thumb.
c) Place tip of swab into vial to wet swab. Gently press swab on inside of vial to remove
excess solution from swab. Discard remaining solution and vial in waste.
d) Gently place sampling template in proper sampling location to minimize disruption of
settled aerosol.
C-4
-------
e) Wipe sample surface horizontally using S-strokes to cover entire sample area within
template using a consistent amount of pressure.
f) Turn swab over and wipe entire sample surface area
using vertical S-strokes within template using a
consistent amount of pressure.
g) Turn swab over and wipe
entire sample surface area
using diagonal S-strokes
within template using a
consistent amount of
pressure.
6) Person B (Supplier)
a) Open inner Ziploc®, move sterile 15 mL centrifuge
tube to end of bag and unscrew cap. Once sample has been collected, remove cap so
Person C call place sample in tube.
7) Person C (Collector)
a) Carefully place swab head into sterile centrifuge tube.
b) Break off head of swab by bending handle. The end of swab handle, touched by
Person C (Collector), should not touch or enter inside of tube.
c) Dispose of sampling template and remaining stick from swab in waste.
C-5
-------
8) Person B (Supplier):
a) Immediately close and tighten cap to centrifuge tube, place tube in inner Ziploc® bag.
b) Seal inner and outer Ziploc® bag.
c) Place sample into larger, clean Ziploc® bag.
9) Persons B and C: Remove gloves and discard in waste.
10) Repeat Steps 1-5 as necessary.
Collecting Wipe Samples (both sponge-stick and Versalon® wipes):
1) Persons A, B & C: Discard existing top pair of gloves. Note: After each person has
discarded 4 pairs of gloves, open a new Ziploc® bag with clean gloves and place another set
of 4 gloves over last pair on your hands, while inside Ziploc® bag.
2) Person B (Supplier):
a) Open sample collection bin.
b) Open template packaging in bin and remove a 10"x 10" template.
c) Hand template to Person C.
d) Remove one wipe specimen kit from bin.
e) Hold specimen kit barcode label out for Person A to scan.
3) Person A (BROOM Operator): Scan sample barcode label located on outside sample Ziploc®
bag into BROOM and enter in required fields.
4) Person B (Supplier):
a) Open outer Ziploc® bag containing prepackaged sponge-stick (or moistened Versalon®
wipes).
b) Open package without touching sponge-stick for Person C to remove from packaging.
i) For Versalon® wipe open Ziploc® bag containing pre-moistened wipe in a 50 mL
tube.
ii) Hold tube in bag and flick downward so wipe slides to cap.
iii) Carefully open cap which wipe should be stuck to. Be careful not to drop wipe.
iv) Once Person C removed Versalon® wipe, place cap back on tube.
c) Discard packaging into waste.
d) After sample is collected move inner Ziploc® bag to end of outer Ziploc® bag and open.
i) For wipe move 50 mL tube to end of Ziploc® bag and unscrew cap.
5) Person C (Collector):
a) Carefully remove sponge-stick from bag without touching bag.
C-6
-------
b) Gently place sampling template in proper sampling area to minimize disruption of settled
aerosol.
c) Wipe sample surface horizontally using S-strokes to
cover entire sample area within template using a
consistent amount of pressure.
d) Turn sponge-stick over and wipe same surface
vertically using the same technique within
template
i) For Versalon® wipes fold exposed side in.
e) Turn sponge-stick on edge (narrow side) and
wipe same surface diagonally using the same
technique within the template
i) For Versalon® wipes fold exposed side in.
f) With sponge-stick tip wipe perimeter of
sampling area once.
g) Carefully place sponge-stick into inner Ziploc®
bag that Person B is holding being careful not to
touch surface of bag.
i) For Versalon® wipes place wipe into 50 mL tube.
h) Break handle of sponge-stick off only allowing sampled
sponge portion to remain in bag.
i) Dispose of sampling template and remainder of sponge-
stick in waste.
6) Person B (Supplier):
a) Immediately seal inner Ziploc® bag containing sponge-stick sample and slide back into
outer Ziploc® bag and seal.
i) For wipe immediately close and tighten cap of 50 mL tube and slide back into Ziploc®
bag and seal.
b) Place sample into larger, clean Ziploc® bag.
7) Persons B and C: Remove gloves and discard in waste.
C-7
-------
8) Repeat Steps 1-5 as necessary.
Collecting HEPA Vacuum Sock Samples:
1) Persons A, B, and C: Discard existing top pair of gloves. Note: After each person has
discarded 4 pairs of gloves, open a new Ziploc® bag with clean gloves and place another set
of 4 gloves over last pair on your hands, while inside Ziploc® bag.
2) Person C (Collector):
a) Plug in HEPA vacuum power cord, place HEPA vacuum hose over shoulder, and be
sure not touch anything or drag on floor.
b) Remove plastic cover from nozzle without touching it in preparation for Person B to place
vacuum sock assembly onto nozzle.
3) Person B (Supplier):
a) Open sample supply bin.
b) Remove vacuum sock specimen kit from bin.
c) Hold specimen kit barcode label out for Person A to scan.
4) Person A (BROOM Operator): Scan sample barcode label located on outside sample Ziploc®
bag into BROOM and enter in required fields.
5) Person B (Supplier):
a) Open outer Ziploc® bag containing inner Ziploc® bag with vacuum sock assembly.
b) Open inner Ziploc® bag within outer Ziploc bag and push vacuum sock assembly from
bottom to expose cardboard applicator tube opening.
c) Using Ziploc® bag to handle vacuum sock assembly, place vacuum sock assembly onto
nozzle of vacuum tube while Person C holds vacuum nozzle. Once assembly is on,
grasp nozzle with Ziploc® so Person C can change gloves.
6) Person C (Collector): Dispose of top layer gloves into waste.
C-8
-------
7) Person B (Supplier)
a) Open template packaging in bin and remove a 24"x 24" template
b) Hand template to Person C.
c) Turn on vacuum.
8) Person C (Collector)
a) Gently place sampling template in proper sampling area to minimize disruption of settled
aerosol.
b) With vacuum nozzle perpendicular to sample surface, vacuum horizontally using S-
strokes to cover sample area within sampling template.
c) Vacuum same area vertically using same technique within template.
9) Person B (Supplier)
a) Turn off vacuum when sampling is completed.
b) Using inner Ziploc® bag, remove vacuum sock assembly from vacuum nozzle.
c) Seal inner Ziploc® bag and slide back into outer Ziploc® bag and seal.
d) Place sample into larger, clean Ziploc® bag.
10) Person C (Collector): Wipe down nozzle (in and out) and end of tubing with alcohol wipe and
place dirty wipe in waste. Allow nozzle to air dry and wrap nozzle with a clean Ziploc® bag.
C-9
-------
11) Persons B & C: Remove gloves and discard in waste.
12) Repeat Steps 1-5 as necessary.
Example of sampling kits. A kit will be made for each room of study rooms and another kit will
be made for remaining rooms. I will provide the breakdown of rooms.
Swab kits
C-10
-------
QC Sample Collection Protocols
QC FIELD NEGATIVE CONTROL (Field Blanks) - are to be performed when sampling teams
approximately reach center of each room following normal procedures for swab, wipe, vacuum
sock sample collection without actually sampling a surface.
Collecting QC Swab Field Negative Control:
1) Persons A, B and C: Discard existing top pair of gloves. Note: After each person has
discarded 4 pairs of gloves, open a new Ziploc® bag with clean gloves and place another set
of 4 gloves over last pair on your hands, while inside Ziploc® bag.
2) Person B (Supplier):
a) Open sample supply bin.
b) Remove swab specimen kit from bin.
c) Hold specimen kit barcode label out for Person A to scan.
3) Person A (BROOM Operator): Scan sample barcode label located on outside sample Ziploc®
bag into BROOM and enter in required fields.
4) Person B (Supplier):
a) Open outer Ziploc® bag containing prepackaged swab.
b) Open package of swab without touching it for Person C to remove from packaging.
c) Discard packaging in waste once Person C has removed swab.
d) Move 2 ml vial containing neutralizing buffer to top of bag for Person C.
5) Person C (Collector):
a) Carefully remove swab from bag without touching bag or tip of swab.
b) Remove 2 ml vial and open with thumb.
c) Place tip of swab into vial to wet swab. Gently press swab on inside of vial to remove
excess solution from swab. Discard remaining solution and vial in waste.
d) DO NOT TOUCH ANYTHING WITH SAMPLE, ESPECIALLY A CONTAMINATED
SURFACE.
6) Person B (Supplier)
a) Open inner Ziploc®, move sterile 15 mL centrifuge tube to end of bag and unscrew cap.
Remove cap so Person C call place sample in tube.
7) Person C (Collector)
C-11
-------
a) Carefully place swab head into centrifuge tube.
b) Break off head of swab by bending handle. The end of swab handle, touched by
Person C, should not touch or enter inside of tube.
8) Person B (Supplier):
a) Once swab sample is placed in conical tube, replace screw cap and keep conical tube in
Ziploc® bag.
b) Seal Ziploc® bag.
c) Place sample into larger, clean Ziploc® bag.
9) Persons B and C: Remove gloves and discard in waste.
10) Persons A, B and C: Continue with normal sampling operations.
Collecting QC Wipe Field Negative Control:
1) Persons A, B & C: Discard existing top pair of gloves. Note: After each person has
discarded 4 pairs of gloves, open a new Ziploc® bag with clean gloves and place another set
of 4 gloves over last pair on your hands, while inside Ziploc® bag.
2) Person B (Supplier):
a) Open sample supply bin.
b) Remove wipe specimen kit from bin.
c) Hold specimen kit barcode label out for Person A to scan.
3) Person A (BROOM Operator): Scan sample barcode label located on outside sample
Ziploc® bag into BROOM and enter in required fields.
4) Person B (Supplier):
a) Open outer Ziploc® bag containing prepackaged sponge-stick (or moistened Versalon*1
wipes).
b) Open package without touching sponge-stick for Person C to remove from packaging.
i) For Versalon® wipe open Ziploc® bag containing pre-moistened wipe in a 50 mL
tube.
ii) Hold tube in bag and flick downward so wipe slides to cap.
iii) Carefully open cap which wipe should be stuck to. Be careful not to drop wipe.
iv) Once Person C removed wipe, place cap back on tube.
c) Discard packaging into waste.
5) Person C (Collector):
a) Carefully remove sponge-stick without touching bag.
C-12
-------
b) DO NOT TOUCH ANYTHING WITH SAMPLE, ESPECIALLY A CONTAMINATED
SURFACE.
6) Person B (Supplier):
a) Move inner Ziploc® bag to end of outer Ziploc® bag and open.
i) For wipe move 50 mL tube to end of Ziploc® bag and unscrew cap.
7) Person C (Collector):
a) Carefully place sponge-stick into inner Ziploc® bag Person B is holding being careful not
to touch surface of bag.
i) For wipes place wipe into 50 mL tube.
b) Break handle of sponge-stick off only allowing sampled sponge portion to remain in bag.
Dispose of sampling template and remainder of sponge-stick in waste.
8) Person B (Supplier):
a) Immediately seal inner Ziploc® bag containing sponge-stick sample and slide back into
outer Ziploc® bag and seal.
i) For wipe immediately close and tighten cap of 50 mL tube and slide back into Ziploc®
bag and seal.
b) Place sample into larger, clean Ziploc® bag.
9) Persons B and C: Remove gloves and discard in waste.
10) Persons A, B and C: Continue with normal sampling operations.
Collecting QC Vacuum Sock Field Negative Control:
1) Persons A, B, and C: Discard existing top pair of gloves. Note: After each person has
discarded 4 pairs of gloves, open a new Ziploc® bag with clean gloves and place another set
of 4 gloves over last pair on your hands, while inside Ziploc® bag.
2) Person C (Collector):
a) Place HEPA vacuum hose over shoulder, and be sure not touch anything or drag on
floor.
b) Remove plastic cover from nozzle without touching it in preparation for Person B to place
vacuum sock assembly onto nozzle.
3) Person B (Supplier):
a) Open sample supply bin.
b) Remove vacuum sock specimen kit from bin.
c) Hold specimen kit barcode label out for Person A to scan.
C-13
-------
Person A (BROOM Operator): Scan sample barcode label located on the outside of the
Ziploc® bag containing the vacuum sock specimen kit into BROOM and enter in required
fields.
4) Person B (Supplier):
a) Open outer Ziploc® bag containing inner Ziploc® bag with vacuum sock assembly.
b) Open inner Ziploc® bag within outer Ziploc bag and push vacuum sock assembly from
bottom to expose cardboard applicator tube opening.
c) Using Ziploc® bag to handle vacuum sock assembly, place vacuum sock assembly onto
nozzle of vacuum tube while Person C holds vacuum nozzle. Once assembly is on,
grasp nozzle with Ziploc® so Person C can change gloves.
5) Person C (Collector)
a) Remove vacuum nozzle from Ziploc® bag.
b) DO NOT TURN ON VACUUM.
6) Person B (Supplier)
a) Using inner Ziploc® bag, remove vacuum sock assembly from vacuum nozzle.
b) Seal inner Ziploc® bag and slide back into outer Ziploc® bag and seal.
c) Place sample into larger, clean Ziploc® bag.
7) Person C (Collector): Wipe down nozzle (in and out) and end of tubing with alcohol wipe.
Allow nozzle to air dry and wrap nozzle with a clean Ziploc® bag.
8) Persons B and C: Remove gloves and discard in waste.
9) Persons A, B and C: Continue with normal sampling operations.
C-14
-------
Exit Procedures
Procedures for Exiting Rooms:
Upon Exiting:
1. Persons A, B and C: Remove gloves and discard before leaving a room.
Exiting Building and Decontamination Procedures:
1) Persons A, B, and C: At exit door remove booties and place in trash container.
2) Person C (Collector):
a) Open door to exit building.
b) Take cart from Person B and proceed to cart and HEPA vacuum decon table.
c) Remove HEPA vacuum hose and place in amended bleach bucket.
d) Extensively wipe down HEPA vacuum with Hype-Wipe bleach towelette.
e) Dispose of Hype-Wipe bleach towelette.
f) Tie up trash bag from cart and wipe down outside with Hype-Wipe bleach towelette.
g) Take decontaminated HEPA vacuum and trash bag to decon wash line tent and hand to
decon line personnel.
h) Wipe down flashlight and marker with Hype-Wipe bleach towelette left on cart.
i) Place flashlight and marker inside a clean Ziploc® bag and place back onto cart.
j) Proceed with cart to decon wash line tent.
k) Place cart on right side of tent.
I) Extensively wipe down and wash all parts of cart and Ziploc® bag containing flashlight
and marker.
m) Leave cart and Ziploc® bag behind and proceed to left side of decon line tent.
n) Follow normal SOP for self-decon.
o) Exit left side of decon wash line tent.
p) Retrieve cart from right side of decon wash line tent.
q) Perform any additional decon of personal masks.
r) Leave Ziploc® bag containing flashlight and marker on cart.
s) Decon line personnel will place decontaminated cart into appropriate conex box.
t) Disrobe and dispose of PPE into trash container.
u) Proceed to temper tent for medical checks, rest and rehydration.
v) Stay in localized designated area until leaving the INL Testing Site.
3) Person B (Supplier):
a) Push cart out of building.
b) Leave cart and HEPA vacuum for Person C to decon.
c) Take sample collection Ziploc® bags to designated sample decon table.
d) Open large Ziploc® bag and remove smaller double-bagged Ziploc® containing samples.
e) Dispose of large Ziploc® in trash container.
C-15
-------
f) Wipe down outside Ziploc® with Hype-Wipe bleach towelette.
g) Dispose of Hype-Wipe bleach towelette in trash container.
h) Change gloves.
i) Place all decontaminated double-bagged Ziploc® bags into new pre-labeled large Ziploc®
bags.
j) Take decontaminated sampling bags to decon wash line tent, rinse of bags, and hand to
designated sample shipment personnel.
k) Follow normal SOP for self-decon.
I) Exit left side of decon wash line tent.
m) Perform any additional decontamination of personal masks.
n) Disrobe and dispose of PPE into trash container.
o) Proceed to temper tent for medical checks, rest and rehydration.
p) Stay in localized designated area until leaving INL Testing Site.
4) Person A (BROOM Operator):
a) Upload data from PDA as described in training.
b) Carry BROOM PDA out of building.
c) Take sample supply bins to designated BROOM PDA decon table and bin remnant
(unused specimen kits and supplies) disposal area.
C-16
-------
d) Place remnants of sample supply bin (e.g. unused samples, gloves, trash bags) into
trash container.
e) Break down cardboard bins and dispose of in trash container.
f) Proceed to left side of decon wash line tent.
g) Follow normal SOP for self-decon.
h) Exit left side of decon wash line tent.
i) Perform any additional decon of personal masks.
j) Proceed to temper tent for medical checks, rest and rehydration.
k) Stay in localized designated area until leaving the INL Testing Site.
C-17
-------
Appendix D
Aggressive Air Sampling (AAS) Protocol
-------
A-1. Sample Collection Materials
1. U.S. EPA pre-labeled and barcoded sample kits:
a. Gloves, nitrile
b. 150 mm x 15 mm, disposable petri plate containing 70 ml_ trypticase soy agar
with lid and sealed with paraffin tape
c. Dry filter cartridges (37 mm, 3-piece cassettes with 0.8 |o,m pore mixed cellulose
ester filter)
d. Paraffin tape
e. Sample labels
f. Re-sealable plastic bags, 1-quart (individual sample bag)
g. Re-sealable plastic bags, 1-gallon or larger (sample transport bag)
2. Checklist and sampling map
3. Slot screwdriver
4. Hand-held PDA with BROOM software
5. Wet test meter (air flow calibrator)
6. Mattson-Garvin STA samplers with 60-min drive motors
7. Dycor XMX samplers with the dry impingement modules
8. Forced air equipment (such as a one-horse power mechanical leaf blower)
9. Oscillating floor fan (at least 20 inches in diameter)
10. ICxIBAC™ Sensors
11. Isopropyl alcohol
12. Soft cloth
13. STA sampler lubricant
Note: Prepare STA sample media (if petri plates were procured without media):
1. Place the 150 mm disposable petri plates on a level surface and pour the media (70 ml)
hot.
2. Media should be approximately 5.75mm deep in the plate.
A-2. Sample Collection Procedures
1. Ensure all sampler flow rates have current (within one year of sampling date) factory or
professional calibration certificates.
a. Ensure that the Mattson-Garvin STA sampler states the sampling rate as 1.00
cubic feet per minute on the Certificate of Calibration furnished with the unit.
2. Designate the two rooms to perform aggressive air sampling on the first floor, from areas
known to be previously least contaminated, and move to those known to be more
contaminated.
3. Isolate the room being sampled to the degree feasible by closing doors, hanging plastic
sheeting, etc., in order to prevent air from within the room from moving into other parts of
the facility during sampling.
4. Establish and maintain a negative pressure environment in the room or area being
sampled relative to outside air.
a. Place two HEPA-filtered portable ventilation unit (negative air unit) at one end of
the room enclosure. Note: Only one negative air unit will be operated and the
other kept as backup. NOTE: Only during the low-tech decontamination event
D-1
-------
will the entire BOTE Testing Facility be placed under negative pressure using
negative air units.
b. Seal the negative air units into the plastic barrier of the containment.
c. Exhaust filtered air outside of the enclosure through flexible duct hoses.
d. Determine the actual flow rate through each unit by measuring the average
velocity of the air entering the unit duct with a velometer and multiplying by the
duct area.
e. Ensure the air handling unit (AHU) system is operating for the entire aggressive
air sampling procedure.
f. Seal any AHU system vents that supply air into the room or area being sampled.
g. Ensure HEPA-filtered "make-up air" comes mainly from an airlock at the opposite
end of the room enclosure.
h. Measure and record pressure differences.
i. Ensure the pressure differential is at least 0.02 inches (0.5 mm) of water
and room air exchanges are kept at a minimum.
5. Ensure that the Dycor XMX sampler liquid impingement modules (LIM) are switched out
with the dry impingement modules (DIM) for each sampler.
a. If DIM is not installed, follow instructions in Dycor XMX/2L-MIL Operator's
Manual, Version 1.8, Section 3.3.
6. Perform functional test on each XMX by following instructions in Dycor XMX/2L-MIL
Operator's Manual, Version 1.8, Section 3.4.
7. Verify that the STA samplers have the correct drive motors (60-min) installed.
8. Prior to use, field calibrate each XMX sampler flow rate with a 37 mm, 3-piece cassette
with a 0.8 |o,m pore mixed cellulose ester (MCE) filter designated for calibration prior to
use.
9. Calibrate and adjust the airflow of the STA sampler:
a. Carefully inspect the air intake and the calibrated slit in the dome assembly to
assure that they are free of particulate matter.
b. Inspect the gasket. Wipe clean with damp cloth if necessary.
c. Place the dome assembly in position on the gasket and tighten down.
d. Turn the air control valve above the flowmeter, which is located on top of the
housing, to the "OFF" position (to the right until it no longer moves).
e. Set the desired revolution time on the Artisan Timer to 60 minutes.
f. Caution: Do not place your hand (or any object) over the throat of the air intake
while the vacuum pump is on.
g. Start the STA sampler by turning on the main power switch and then push the
START button on the Artisan timer.
h. Adjust the airflow to 60 cubic feet/hour by means of the airflow control valve on
the top of the flowmeter so the ball is centered at the red line (approx. 60 SCFH).
i. Attach a wet test meter and run three (3), one-minute checks.
i. If all three tests are between 59.70 and 60.30,
place red line flag in position.
ii. If not, adjust and repeat.
j. Shut off the sampler by turning off the main power switch.
k. Remove the dome assembly and wipe the air intake, calibrated slit and the brass
slit-to-agar distance gauge with a cloth, dampened with isopropyl alcohol.
10. Provide the Aggressive Air Sampling Team with a checklist and a map that outlines
where to place sampling equipment and fans.
11. Place ICx IBAC™ Sensors in close proximity to aerosol samplers and exhaust vents that
keep the rooms under negative pressure.
D-2
-------
a. Monitor and control the sensors remotely within the Admin Trailer.
b. Beginning 10 minutes prior to the entry that commences the forced air on
surfaces event, and throughout the aggressive air sampling procedure, measure
aerosol concentrations using the sensors.
12. Place one oscillating floor fan (at least 20 inches in diameter) near the center of the
room.
a. Position fan strategically to both direct airflow towards the ceiling and create
turbulence that will keep particulate matter in suspension.
b. The fan shall remain operational throughout the period of sample collection.
13. Place one XMX sampler and one STA sampler outside of the room or floor containment
in an adjacent zone.
a. Ensure that samplers are not in corners or near obstructions.
b. Ensure that one sampler is not placed within the air movement influence of
another sampler.
c. Collect samples using pre-labeled and barcoded sample kits provided by U.S.
EPA.
d. Note: Each kit will include all supplies needed to collect, label and package each
sample.
14. Collect field blanks.
a. Collect field blanks in the same areas as the corresponding air samples.
b. Do not let the field blanks come in contact with potentially contaminated surfaces.
c. Don a clean pair of disposable nitrile gloves over existing gloves and collect one
field blank for the XMX sampler.
i. While the XMX sampler is in the OFF position, open re-sealable plastic
bag containing the dry filter cartridge.
ii. Secure the dry filter cartridge into the XMX sampler.
iii. Remove the dry filter cartridge from the XMX sampler.
iv. Place the dry filter cartridge in a re-sealable 1-quart plastic bag and
securely seal the bag and label it as "field blank."
v. Note: Remove excessive air from the re-sealable plastic bags to increase
the number of samples that can be shipped in one container.
d. Don a clean pair of disposable nitrile gloves over existing gloves and collect one
field blank for the STA sampler.
i. While the STA sampler is in the OFF position, open re-sealable plastic
bag containing the petri plate.
ii. Remove the lid and load the bottom plate (containing the agar) into the
STA sampler.
iii. Remove the petri plate from the STA sampler and seal the with paraffin
tape.
iv. Place each petri plate in a re-sealable 1-quart plastic bag and securely
seal the bag and label it as "field blank."
v. Note: Remove excessive air from the re-sealable plastic bags to increase
the number of samples that can be shipped in one container.
e. Process the samples along with the other air samples.
15. Collect three, one-hour air samples with each sampler during the entire time aggressive
air sampling is conducted inside containment.
a. Don a clean pair of disposable nitrile gloves over existing gloves, for each sample
collected.
b. Place each sample in a re-sealable 1-quart plastic bag and securely seal the bag.
D-3
-------
c. Note: Remove excessive air from the re-sealable plastic bags to increase the
number of samples that can be shipped in one container.
d. Enter all the data into the hand-held PDA and scan the bar code on the sample
bag for each sample (to include blanks).
e. Decontaminate and package sample bags appropriately prior to being
transported to INL laboratories.
16. Place the three aerosol samplers (2 XMX/2L-MIL and 1 slit-to-agar) at central points
within each room or area to be sampled.
a. Ensure that samplers are not in corners or near obstructions.
b. Ensure that one sampler is not placed within the air movement influence of
another sampler.
c. Close all doors and keep closed until vacating the room.
d. Collect samples using pre-labeled and barcoded sample kits provided by U.S.
EPA.
e. Note: Each kit will include all supplies needed to collect, label and package each
sample.
f. Collect field blanks as described in step 14.
17. Prepare the XMX for sampling:
a. Plug the XMX into an outlet using the supplied power cord.
i. Check that power is on by turning the power dial and checking that the
timer display is lit and set at zero.
b. Remove the red filter cap from the dry filter cartridge.
i. Use a screwdriver to help take the dry filter cartridge apart into two pieces.
ii. Be careful not the touch the filter.
c. Unscrew the dry impingement module (DIM) nut and push the open end of the
dry filter cartridge (bottom half) onto the DIM.
i. Screw the DIM nut back on securely.
ii. Attach the quick connect fitting onto the bottom of the DIM nut.
d. Check all fittings and connects and close the DIM door.
e. Set the sampler for manual control.
i. Set the mode of the timer in the first dial to "H".
ii. Set the last dial on the timer (unit of time) to minutes.
iii. Set the middle dials to 060, the length of time to sample for in minutes.
iv. Note: The dials should read "H 0 6 0 m"
f. Collect a total of 19 samples (15 one-hour samples and 4 blanks) for each
sampling event
i. 11 XMX samples; 6 inside room, 3 outside room, 2 blanks
ii. 8 slit-to-agar samples; 3 inside room, 3 outside room, 2 blanks
iii. Don a clean pair of disposable nitrile gloves over existing gloves, for each
sample collected.
iv. Place each sample in a re-sealable 1-quart plastic bag and securely seal
the bag.
v. Note: Remove excessive air from the re-sealable plastic bags to increase
the number of samples that can be shipped in one container.
g. Enter all the data into the hand-held PDA and scan the bar code on the sample
bag for each sample (to include blanks).
h. Decontaminate and package sample bags appropriately prior to being
transported to INL laboratories.
18. Prepare the STA for sampling:
D-4
-------
a. Don a clean pair of disposable nitrile gloves over existing gloves and remove the
petri plate lid and place the bottom plate (containing the agar) on the petri plate
holder.
b. Replace dome assembly.
c. Adjust the height of the petri plate by fully depressing the slit-to-agar distance
gauge at the top of the dome assembly and then adjusting the elevation on the
right side of the unit so that the media surface touches the gauge.
d. Lock the elevation arm and release the distance gauge. The STA sampler is now
ready to operate.
e. Set the timer to the 60-minute interval (corresponds to the 60-minute installed
drive motor).
19. The sampling process will begin with the simultaneous activation of the five aerosol
samplers (three in the room or area being sampled and two outside the enclosure).
a. Start the XMX sampler by pressing the sampling button. Sampling will continue
for the time selected on the timer (60 minutes).
b. Start the STA sampler by turning on the main power switch and pressing the
START button on the timer. The unit will shut off after one revolution of the petri
dish (60 minutes).
c. Replace the 37 mm, 0.8 |o,m pore MCE filters in the three XMX samplers and agar
plates in the two STA samplers after the first 60 minutes of sampling.
i. Don a clean pair of disposable nitrile gloves over existing gloves, for each
sample collected.
d. Replace the filters and agar plates after the second 60 minutes of sampling.
i. Don a clean pair of disposable nitrile gloves over existing gloves, for each
sample collected.
e. Note: Sample for a total of 180 minutes (3 hours) for each sampler, resulting in
three air samples from each of the five samplers.
f. Note: Replace filter sample media as needed in the event that dust and debris
from the rooms cause the filters to become sufficiently plugged as to reduce the
sampler's volumetric sample collection flow rate to a level that is 20 percent
below its initial value.
20. Activate the oscillating floor fans at their lowest speed setting.
21. Use forced air equipment (such as a one-horse power mechanical leaf blower) to direct a
jet of air towards all surfaces in the room to dislodge and re-suspend any surviving
spores that might be present following the decontamination.
a. Ensure the tip of the leaf blower is at a less than 45-degree angle from the
surface.
b. Ensure that the tip of the leaf blower is as close to the surface as possible.
c. Ensure that a sweeping motion from side to side across the surface is maintained
as the leaf blower moves forward.
d. Agitate all such surfaces (walls, ceilings, floors, ledges, etc.).
i. Conduct forced air agitation for at least 20 minutes in the room.
1. Conduct forced air agitation for at least 15 minutes on all
horizontal surfaces, re-doing surfaces as time permits.
2. Conduct forced air agitation for at least 5 minutes on all vertical
surfaces and the ceiling, re-doing surfaces as time permits.
e. Vacate the room being sampled once the surface agitation phase has been
completed.
f. Close all doors and keep closed until reentry to collect samples.
D-5
-------
Figure A-1. An example of a 1 hp leaf blower.
22. Reenter the room when the aerosol samplers have run for a total of 60 minutes, and
deactivate each aerosol sampler and collect and replace the sampling media from each.
a. Don a clean pair of disposable nitrile gloves over existing gloves, for each sample
collected.
b. Ensure that the XMX/2L-MIL aerosol samplers collect three times the volume of
each area (room) onto the filters (approximately 150 minutes sampling with two
XMX/2L-MIL samplers should provide acceptable volumes).
c. Un-connect the filter cartridge from the XMX.
d. Re-connect the two halves of the filter (DIM).
e. Note: If extended sampling is recommended, deactivate the aerosol samplers,
replace the sampling media with new media and activate the aerosol samplers
and allow them to run for the specified period of time.
f. Place each sample in a re-sealable 1-quart plastic bag and securely seal the bag.
g. Note: Remove excessive air from the re-sealable plastic bags to increase the
number of samples that can be shipped in one container.
23. Deactivate the XMX and STA samplers outside the room being sampled, and collect and
replace the sampling media as described in step 19 above.
24. Prepare media blanks:
a. Provide one unopened dry filter cassette per lot used, as a media blank to the
processing laboratory.
b. Provide one unopened trypticase soy ager plate per lot used, as a media blank to
the processing laboratory.
c. Place each media blank in a re-sealable 1-quart plastic bag and securely seal the
bag.
d. Note: Remove excessive air from the re-sealable plastic bags to increase the
number of samples that can be shipped in one container.
e. Enter all the data into the hand-held PDA and scan the bar code on the sample
bag for each sample.
f. Package sample bags appropriately prior to being transported to INL laboratories.
25. Field calibrate each aerosol sampler flow rate with sampling media designated for
calibration after each sampling event.
a. Note: There are several O-rings in the XMX. These should be monitored and
replaced as required.
26. Clean the XMX samplers by air purging.
D-6
-------
a. Run the XMX samplers for 5 minutes as a precaution against cross-
contamination between sampling events (not between samples within the same
room).
27. Disassemble sampler components and decontaminate all parts.
a. Partially disassemble the XMX concentrator and spray it down with bleach and
water to decontaminate the internal components of the XMX.
b. Note: If positive surface or air sample results are discovered post-
decontamination, swab key parts of the XMX after decontamination to test
effectiveness.
28. Perform any needed maintenance on the XMX and STA samplers:
a. Clean the STA samplers with isopropyl alcohol and a soft cloth.
b. Lubricate the STA samplers with the lubricant provided with the sampling units
after each of the three sampling events.
i. Lubricate the two shafts: the drive shaft above the drive motor and the
elevating mechanism shaft below the drive motor.
29. Provide an equipment blank for each sampling device used during the sampling event:
a. In an area known to be free of contamination, prepare a filter cartridge in the
same manner as actual samples.
i. Operate the each XMX sampler for 60 minutes with a filter cartridge
designated as an equipment blank.
b. In an area known to be free of contamination, prepare a in the same manner as
actual samples.
i. Operate the each STA sampler for 60 minutes with a trypticase soy ager
plate designated as an equipment blank.
c. Place each equipment blank in a re-sealable 1-quart plastic bag and securely
seal the bag.
d. Note: Remove excessive air from the re-sealable plastic bags to increase the
number of samples that can be shipped in one container.
e. Enter all the data into the hand-held PDA and scan the bar code on the sample
bag for each sample.
f. Package sample bags appropriately prior to being transported to INL laboratories.
A-3. Sample Decontamination and Shipment
1. Place multiples of the re-sealable 1-quart plastic bags into a 1-gallon re-sealable plastic
bag.
2. Securely seal the 1-gallon re-sealable plastic bag and label the bag (e.g., identify
samples contained in the re-sealable plastic bag, sample locations, date and time
samples were collected, and name of individual collecting the samples).
3. Decontaminate the outer surface of the larger re-sealable plastic bag using a fresh pH-
adjusted bleach solution (household bleach diluted 1:9; pH-adjusted to 6.8-8.0) with a
10-minute contact time before the re-sealable plastic bag leaves the contaminated area.
4. Thoroughly dry the outside of the re-sealable plastic bag.
5. Complete a chain of custody form.
6. Note: Once the outer re-sealable plastic bag is decontaminated, it should not be opened
outside of appropriate containment in a laboratory.
7. Place the larger re-sealable plastic bag into an appropriate container for shipping.
8. Transport all samples to the processing laboratory on wet ice or on cold packs.
D-7
-------
9. Note: Samples may be stored at 2°C-8°C prior to processing and should be processed
within 48 hours of collection.
10. Send appropriate chain of custody forms and analytical request forms with each
shipment sent to the processing laboratory.
11. Note: The shipper is responsible for ensuring adherence to the most current and
appropriate regulations.
12. Note: Do not transport contaminated equipment/supplies in the same container as the
samples.
D-8
-------
Attachment B. Sample Analysis Materials and Procedures
B-1. Sample Analysis Materials
1. Slot screwdriver
2. Sterile tweezers or forceps that have flattened ends specifically for handling filters
3. 50 ml_ conical centrifuge tubes
4. Sterile extraction fluid, e.g. deionized water, phosphate buffer saline solution
5. Wrist action shaker
6. Trypticase soy agar plates
7. Cell spreader
B-2. Sample Procedures
1. Process the XMX samples under aseptic conditions, typically inside a Biological Safety
Cabinet (BSC) or at the INL laboratory:
a. Note: If a BSC is not available the technician can carry out the filter extraction on
the bench top by first swabbing the bench top area with 10 % bleach solution,
keep the working area to a minimum, that would be directly in front of the
technician. It would also be advantageous to have a Bunsen burner running
towards the back edge of the work area as this will provide convection currents
towards the flame and reduce any possible airborne contaminants.
b. Open the re-assembled filter cartridge again, containing the exposed filter.
c. To separate the cartridge, select a blunt instrument such as a slot screwdriver
and pry apart the lower joint between the lower and middle part of the cartridge
that is holding the filter in place.
d. Use a pair of sterile tweezers or forceps that have flattened ends specifically for
handling filters to carefully lift the filter from the cartridge and off of the supporting
cardboard backing.
e. Place the filter into a 50 ml conical centrifuge tube, which contains 20ml of sterile
extraction fluid, e.g. deionized water, phosphate buffer saline (PBS) solution, etc.
f. Seal the lid of the 50 ml conical centrifuge tube securely and allow soaking for 10
minutes.
g. Vortex the sample for 2 minutes to insure the filter is fully wetted.
h. Place the sample tube on a wrist action shaker for 15 minutes.
i. Perform heat shocking of the sample if it enhances the Bg analysis.
j. Decant extraction fluid into a new centrifuge tube to remove filter.
i. Leave enough of the liquid to archive for an additional sample if needed
k. Record the amount of remaining liquid and archived liquid to the nearest |oJ.
I. Aseptically dispense the remaining liquid unto a 100 mm trypticase soy agar
(ISA) plate and spread with a cell spreader.
m. Note: Only a small amount of liquid can be plated. Plate must remain agar side
up until all sample fluid is absorbed by the agar. Typical sample volume applied
is 100uL. This volume can be increased to 200 |o,L if the plates are pre-dried a
bit by leaving them out at room temperature overnight. Multiple replicates are
plated out depending on what total sample volume is being analyzed.
n. Analyze via the method of filter plating and leave the XMX filter inside the
extraction fluid.
D-9
-------
2. Analyze the first sample for Bg by Realtime-PCR and culture, morphology and
enumeration.
a. Note: Since we are conducting clearance sampling, we don't anticipate any
detectable concentrations. However, if we have an incomplete decontamination,
then we could see concentrations.
b. Two XMX samplers are simultaneously collecting air samples in each room
sampled. Therefore, do not analyze the second filter until results of the first filter
ensure we do not need to dilute.
3. Analyze the second sample for Bg by Realtime-PCR and culture, morphology and
enumeration.
a. Depending on the microbial concentration in the air, this filter sample may have to
be diluted with a series of 10-fold dilutions and then the dilutions all plated out.
b. It will be up to the end user to determine what range of dilutions are necessary
and usually only 2 dilutions need to be plated to bracket the correct cell
concentration such that the counts are between 30 and 300 on the plates.
D-10
-------
Appendix E
LRN Bg Analysis Protocols for the BOTE Project
-------
1.1. Materials
The following materials and supplies were necessary to complete the analysis procedures:
• Freshly prepared 10% bleach solution
• Sample extracts (from Swabs, Sponge Wipes, and Vacuum socks)
• PBST (Technova Brand, Part* P0201; Fisher Part* 50842946 or equivalent)
• Disposable polystyrene serological pipets (5 ml_ and 10 ml_)
• Tryptic Soy Agar (ISA) culture plates (such as BD, Part* 236950)
• MicroFunnel Disposable Filter Funnels, Pall Life Sciences (VWR P/N 55095-060)
• Disposable Sterile Forceps
• Disposable sterile 10 ul loops
• Laboratory tissue wipes
• Disposable protective clothing/aprons
• Disposable gloves
• Cell spreaders (such as Lazy-L, Fisher Part* NC9417825)
• Deionized water
• Racks for 15 ml and 50 ml centrifuge tubes
• Sterile, plastic, screw-cap 50 ml centrifuge tubes (such as BD, Part# 352070)
• Sterile, plastic, screw-cap 15 ml centrifuge tubes (such as BD, Part# 352097)
• Pipette tips with aerosol filter for 1 ml & 100 ul (similar to Rainin; Part# SR-L200F and
SR-L1000F)
1.2. Equipment
The following equipment was prescribed to complete the analysis procedures. (An important
note is that the initially recommended Barnant Portable Air/Pressure station provided
insufficient vacuum to allow the filters in conjunction with the filter manifold to operate
adequately. As a result, labs were forced to find alternate vacuum equipment to complete
the filter-plate analyses):
• Vortex Mixer (such as Daigger Vortex Genie 2, Daigger Part# EF3030A)
• Portable Pipet-Aid (Eppendorf Easypet Pipet, Fisher Part* 13-688-177 or Rainin
equivalent)
• Pipettors for 1 ml and 100 ul volumes (similar to Rainin Light touch LT1000 and LT100 or
Eppendorf equivalent)
• Vacuum tubing (Nalgene 180 Clear PVC Vacuum tubing, VWR Part* 63013-763)
• Vacuum pump or vacuum line with vacuum gauge (Cole Parmer; gauge catalog #07380-
62; connector kit catalog* 07395-20; and bushing catalog* 08539-83)
• Nalgene Heavy Duty Polypropylene Vacuum Bottles (Fisher Scientific, Part# 02-923-11)
• Quick Filling Venting Closure, Two Port (Fisher Scientific, Part* 02-923-19)
• Filter Funnel Manifold (Pall Corporation, 6 place, aluminum, Part# 15403, or Fisher Part#
xx2504735)
• Incubator (set to 35°C)
E-2
-------
• Biological Safety Cabinet (BSC)
• 40 kHz Sonicator bath (such as Branson Ultrasonic Cleaner Model 1510, Process
Equipment and Supply, Inc.; Part* 952-116)
• Centrifuge with rotors and sealable centrifuge buckets to hold 50 ml conical tubes
• Seward Stomacher® 400 Circulator (Seward; Part* 0400/001/AJ) with closure bags
(Part* BA6141/CLR) and rack (Part* BA6090)
1.3. Procedures
1.3.1. Sponge Wipe Processing and Plating Procedure
A. Preparation
1. Personnel must be familiar with this procedure
2. Equipment preparation
a) Assemble equipment in BSC as needed: Stomacher, vortex, filtration manifold,
automatic pipettors, racks, etc.
b) Assemble extra supplies and reagents near BSC.
3. Supply preparation
a) Unpack shipping containers directly into a biological safety cabinet.
b) If wipes are not in Stomacher® bags, label one 1 Stomacher® bag for each wipe
and place in a bag rack.
c) Label one specimen cup for each wipe sample.
d) Label two sterile 50 ml centrifuge tubes for each wipe sample and place in tube
rack.
e) For each sample, label 14 ISA plates on the agar side of the plate with the
sample number and the following:
Label 3 each as follows (for spread-plates):
• 1C'1
• 10'2
• ID'3
• 1C'4
Label 2 each as follows (for filter-plate):
. 10°
B. Perform sample processing, spore elution, and culture procedure
1. Dislodge spores from the sample wipes.
a) Don gloves and disposable protective clothing. All subsequent procedures
involving manipulation of wipes or spore suspensions must be carried out in a
BSC.
b) If the wipes are not in Stomacher bags, transfer each wipe to a Stomacher® bag
using sterile forceps. Change forceps between samples.
c) Add 90 ml of PBST to each bag that contains a wipe.
d) Stomach wipes in the PBST
E-3
-------
• Set the Stomacher® to 260 RPM.
• Place one bag containing wipe into the Stomacher® so the wipe rests evenly
between the homogenizer paddles.
• Stomach each wipe for 1 min.
• Open the door of the Stomacher® and remove the bag containing the wipe.
Grab the wipe on the outside of the bag with your hands. Move the wipe to
the top of the bag while using your hands to squeeze excess liquid from the
wipe.
• Remove and discard the wipe using sterile forceps.
e) Repeat steps (b) through (d) for all samples.
f) Allow bags to sit for 10 min to allow elution suspension foam to settle.
2. Concentrate wipe elution suspension.
a) Gently mix elution suspension up and down with a 50 ml pipette three times.
b) Split elution suspension volume equally
• Remove half of the suspension volume (-45 ml) with a sterile 50 ml
pipette and place it in a 50 ml screw capped centrifuge tube.
• Place remaining suspension (-45 ml) into a second 50 ml tube.
c) Record suspension volumes on tubes and data sheet.
d) Repeat steps (a) through (c) for all samples.
e) Centrifuge 50 ml centrifuge tubes
• Place tubes into sealing centrifuge buckets.
• Decontaminate centrifuge buckets before removing from the BSC.
• Centrifuge tubes at 3500 x g for 15 minutes. Do not use the brake option
on the centrifuge to slow the rotor, as re-suspension of pellet may occur.
f) Remove supernatant with a 50 ml pipette and discard to leave approximately 3 ml
in each tube. The pellet may be easily disturbed and not visible, so place pipette
tip away from the tube bottom.
g) Vortex and sonicate tubes
• Set vortexer to high intensity level and touch activation.
• Set sonicator water bath to high and turn on.
• Vortex tubes for 30 sec.
• Transfer tubes to sonicator bath and sonicate for 30 sec.
• Repeat vortex and sonication cycles two times.
h) Remove suspension from one tube with a sterile 5 ml pipette and place it in the
other tube of the same sample.
i) Measure final volume of suspension with 5 ml pipette and record on tube and
data sheet.
j) Repeat steps (e) through (i) for all samples.
3. Serially dilute the spore elution suspension in PBST.
a) Vortex elution suspension on high for 30 sec
b) Remove 0.1 ml of spore elution suspension (10°) and place in one tube (0.9 ml)
of PBST. This is the 10"1 suspension. Recap the 10"1 tube and vortex on high for
30 sec.
c) Open cap of the 10"1 suspension and remove 0.1 ml of this suspension and place
in a new 0.9 ml tube of PBST. This is the 10"2 suspension. Recap the PBST tube
and vortex on high for 30 sec.
E-4
-------
d) Open cap of the 10"2 suspension and remove 0.1 ml of this suspension and place
in a new 0.9 ml tube of PBST. This is the 10"3 suspension. Recap the PBST tube
and vortex on high for 30 sec.
e) You will have four spore suspensions: the initial wipe elution suspension (no
dilution=10°) and three serial dilutions of this suspension in PBST (10~1, 10"2, and
10'3).
f) Repeat steps (a) through (d) for all samples.
4. Culture diluted spore suspensions on TSA
a) After vortexing tubes well, remove 100 ul from the 10"3 suspension with the P100
pipette and place on to a plate of TSA labeled 10'4.
NOTE: the plating of 100 ul is an additional 1:10 dilution of the 10"3
suspension resulting in a 10"4 dilution on the plate. Repeat 2 more times
for a total of three inoculated plates.
b) Spread the inoculum on each of the three 10"4-labeled TSA plates with one Lazy-
L cell spreader. Discard spreader.
c) After vortexing tubes well, remove 100 ul from the 10"2 suspension with the P100
pipette and place on to a plate of TSA labeled 10'3.
NOTE: the plating of 100 ul is an additional 1:10 dilution of the 10"2
suspension resulting in a 10"3 dilution on the plate. Repeat 2 more times
for a total of three inoculated plates.
d) Spread the inoculum on each of the three 10"3-labeled TSA plates with one Lazy-
L cell spreader. Discard spreader.
e) After vortexing tubes well, remove 100 ul from the 10"1 suspension with the P100
pipette and place on to a plate of TSA labeled 10'2.
NOTE: the plating of 100 ul is an additional 1:10 dilution of the 10"1
suspension resulting in a 10"2 dilution on the plate. Repeat 2 more times
for a total of three inoculated plates.
f) Spread the inoculum on each of the three 10"2-labeled TSA plates with one Lazy-
L cell spreader. Discard spreader.
g) After vortexing tubes well, remove 100 ul from the initial wipe elution suspension
(10°) with the P100 pipette and place on to a plate of TSA labeled 10'1.
NOTE: the plating of 100 ul is an additional 1:10 dilution of the initial wipe
elution suspension (10°) resulting in a 10"1 dilution on the plate. Repeat 2
more times for a total of three inoculated plates.
h) Spread the inoculum on each of the three 10"1-labeled TSA plates with one Lazy-
L cell spreader. Discard spreader.
i) Place all plates in an incubator set at 35 ± 2 °C for a maximum of 3 days. Plates
should be examined within 18-24 hours after start of incubation and within 72
hours of sample collection. Count CPU of each suspect BG colony (orange in
color) and record on the viable count worksheet.
E-5
-------
• If the CPU is <300/plate, record actual number.
• If the CPU is >300/plate, record as "too numerous to count" (TNTC)
• If no growth of suspect colonies is observed, record as "None detected"
5. Capture spores on Microfunnel membranes and culture on TSA.
a) Place two 0.45 urn (pore-size) Microfunnels on the vacuum manifold.
b) Moisten Microfunnel membranes with 5 ml PBST, open vacuum and vacuum
through the filter. All filtering should be done with a vacuum pressure <20 cm Hg.
c) With the vacuum valve closed, place 10 ml of PBST into each filter cup.
d) Add 1.0 ml of 10° wipe elution suspension from 3(a) to each filter cup.
e) Open valves and vacuum the suspension through the filter.
f) Rinse the walls of each Microfunnel cup with 10 ml of PBST and vacuum through
the filter.
g) Squeeze the walls of the Microfunnel cup gently and separate the walls from the
base holding the filter. Remove each filter membrane with sterile forceps and
place grid-side up on a TSA plate. Make sure that the filter is in good contact with
the surface of the agar. If an air pocket occurs under the filter, use the sterile
forceps to lift the edge of the filter to release the air pocket for better contact with
the agar.
h) Record exact volume of the 10° wipe elution suspension filtered on each plate. It
should be 1 ml.
i) Repeat steps (a) through (i) for all each sample.
j) Incubate TSA plates with filter membranes at 35 ± 2 °C for a maximum of 3 days.
Plates should be examined within 18-24 hours after start of incubation and within
72 hours of sample collection. Count CPU of each suspect BG colony (orange in
color) and record on the viable count worksheet.
• If the CPU is <300/plate, record actual number.
• If the CPU is >300/plate, record as "too numerous to count" (TNTC)
• If no growth of suspect colonies is observed, record as "None detected"
1.3.2. Swab Processing and Plating Procedure
A. Preparation
1. Personnel must be familiar with this procedure.
2. Equipment preparation
a) Assemble equipment in BSC as needed: vortex, filtration manifold, automatic
pipettors, racks, etc.
b) Assemble extra supplies and reagents near BSC.
3. Supply preparation
a) Unpack shipping containers directly into a biological safety cabinet.
b) If swabs are not in sterile, plastic 15 ml screw cap centrifuge tubes, label one
15 ml tube for each swab and place in a tube rack.
c) For each sample, label 14 TSA plates with the sample number.
Label 3 each as follows (for spread-plates):
• 1C'1
• 10'2
E-6
-------
• 10'3
• 1C'4
Label 2 each as follows (for filter-plate):
. 10°
B. Perform sample processing, spore elution, and culture procedure
1. Dislodge spores from the sample swabs.
a) Don gloves and disposable protective clothing. All subsequent procedures
involving manipulation of swabs or spore suspensions must be carried out in a
BSC.
b) If the swabs are not in screw cap centrifuge tubes, transfer each swab to sterile,
plastic 15 ml screw cap centrifuge tube using sterile forceps.
If necessary, cut the handle of swab to fit into the tube using sterile scissors.
Change forceps and scissors between samples.
c) Add 5 ml of PBST to each tube that contains a swab.
d) Vortex swabs in the PBST.
• Set the vortex mixer to the highest intensity level and 'touch' activation.
• Vortex each swab in ten sec bursts for 2 min to dislodge spores from swab.
• Open the cap of the 15 ml centrifuge tube containing swab and spore elution
suspension. Using sterile forceps, lift the swab and use the forceps to press
the tip of the swab against the inside of the tube to remove extra liquid from
the foam tip.
e) Repeat steps (b) through (d) for all samples.
2. Serially dilute the spore elution suspension in PBST.
a) Vortex elution suspension on high for 30 sec
b) Remove 0.1 ml of spore elution suspension (10°) and place in one tube (0.9 ml)
of PBST. This is the 10"1 suspension. Recap the 10"1 tube and vortex on high for
30 sec.
c) Open cap of the 10"1 suspension and remove 0.1 ml of this suspension and place
in a new 0.9 ml tube of PBST. This is the 10"2 suspension. Recap the PBST tube
and vortex on high for 30 sec.
d) Open cap of the 10"2 suspension and remove 0.1 ml of this suspension and place
in a new 0.9 ml tube of PBST. This is the 10"3 suspension. Recap the PBST tube
and vortex on high for 30 sec.
e) You will have four spore suspensions: the initial wipe elution suspension (no
dilution=10°) and three serial dilutions of this suspension in PBST (10~1, 10"2, and
ID'3).
f) Repeat steps (a) through (d) for all samples.
3. Culture diluted spore suspensions on TSA.
ul from th
pipette and place on to a plate of TSA labeled 10'4
a) After vortexing tubes well, remove 100 ul from the 10"3 suspension with the P100
E-7
-------
NOTE: the plating of 100 |jl is an additional 1:10 dilution of the 10"3
suspension resulting in a 10"4 dilution on the plate. Repeat 2 more times
for a total of three inoculated plates.
b) Spread the inoculum on each of the three 10"4-labeled ISA plates with one Lazy-
L cell spreader. Discard spreader.
c) After vortexing tubes well, remove 100 ul from the 10"2 suspension with the P100
pipette and place on to a plate of ISA labeled 10'3.
NOTE: the plating of 100 ul is an additional 1:10 dilution of the 10"2
suspension resulting in a 10"3 dilution on the plate. Repeat 2 more times
for a total of three inoculated plates.
d) Spread the inoculum on each of the three 10"3-labeled ISA plates with one Lazy-
L cell spreader. Discard spreader.
e) After vortexing tubes well, remove 100 ul from the 10"1 suspension with a P100
pipette and place on to a plate of ISA labeled 10'2.
NOTE: the plating of 100 ul is an additional 1:10 dilution of the 10"1
suspension resulting in a 10"2 dilution on the plate. Repeat 2 more times
for a total of three inoculated plates.
f) Spread the inoculum on each of the three 10"2-labeled ISA plates with one Lazy-
L cell spreader. Discard spreader.
g) After vortexing tubes well, remove 100 ul from the initial wipe elution suspension
(10°) with the P100 pipette and place on to a plate of ISA labeled 10'1.
NOTE: the plating of 100 ul is an additional 1:10 dilution of the initial wipe
elution suspension (10°) resulting in a 10"1 dilution on the plate. Repeat 2
more times for a total of three inoculated plates.
h) Spread the inoculum on each of the three 10"1-labeled ISA plates with one Lazy-
L cell spreader. Discard spreader.
i) Place all plates in an incubator set at 35 ± 2 °C for a maximum of 3 days. Plates
should be examined within 18-24 hours after start of incubation and within 72
hours of sample collection. Count CPU of each suspect BG colony (orange in
color) and record on the viable count worksheet.
• If the CPU is <300/plate, record actual number.
• If the CPU is >300/plate, record as "too numerous to count" (TNTC)
• If no growth of suspect colonies is observed, record as "None detected"
4. Capture spores on Microfunnel membranes and culture on TSA
a) Place two 0.45 urn (pore-size) Microfunnels on the vacuum manifold.
b) Moisten Microfunnel membranes with 5 ml PBST, open vacuum and vacuum
through the filter. All filtering should be done with a vacuum pressure <20 cm Hg.
c) With the vacuum valve closed, place 10 ml of PBST into each filter cup.
d) Add 1.0 ml of 10° wipe elution suspension from 2(a) to each filter cup.
e) Open valves and vacuum the suspension through the filter.
E-8
-------
f) Rinse the walls of each Microfunnel cup with 10 ml of PBST and vacuum through
the filter.
g) Squeeze the walls of the Microfunnel cup gently and separate the walls from the
base holding the filter. Remove each filter membrane with sterile forceps and
place grid-side up on a ISA plate. Make sure that the filter is in good contact with
the surface of the agar. If an air pocket occurs under the filter, use the sterile
forceps to lift the edge of the filter to release the air pocket for better contact with
the agar.
h) Record exact volume of the 10° wipe elution suspension filtered on each plate. It
should be 1 ml.
i) Repeat steps (a) through (i) for all each sample.
j) Incubate ISA plates with filter membranes at 35 ± 2 °C for a maximum of 3 days.
Plates should be examined within 18-24 hours after start of incubation and within
72 hours of sample collection. Count CPU of each suspect BG colony (orange in
color) and record on the worksheet titled BG Spore Environmental Sample
Results Form - Manual Dilution and Filter Plating.
• If the CFU is <300/plate, record actual number.
• If the CFU is >300/plate, record as "too numerous to count" (TNTC)
• If no growth of suspect colonies is observed, record as "None detected"
1.3.3. Vacuum Sock Processing and Plating Procedure
A. Preparation
1. Personnel must be familiar with this procedure.
2. Equipment preparation
a) Assemble equipment in BSC: vortex, filtration manifold, automatic pipettors,
racks, etc.
b) Assemble extra supplies and reagents near BSC.
3. Supply preparation
a) Unpack shipping containers directly into a biological safety cabinet.
b) If vacuum socks are not in sterile, plastic specimen cup, label one 4 oz sterile
specimen cup for each vacuum sample.
c) For each sample, label 14 ISA plates with the sample number.
Label 3 each as follows (for spread-plates):
• 10'1
• 10'2
• ID'3
• 10'4
Label 2 each as follows (for filter-plate):
. 10°
B. Perform sample processing, spore elution, and culture procedure
1. Dislodge spores from the vacuum socks and concentrate elution suspension.
E-9
-------
a) Place 50 ml PBST into sterile, leak-proof, screw-cap wide-mouth plastic
container.
b) Remove the sock from the bag by holding onto upper blue plastic material.
Wet the sock by dipping the lower 1 inch of the vacuum sock into the liquid in
the container.
c) With disposable scissors or individually sterilized scissors, and while holding
the sock over the cup, cut lower edge of sock as close to the lower edge
seam as possible. (Flaming scissors with an alcohol lamp will not assure
sterilization when Bacillus spores are involved)
d) Submerge the sock in the PBST so that the liquid is allowed to enter the
opening and wet the contents inside.
e) When the liquid appears to have wet the sock beyond about 1 inch from the
bottom, cut a 1-inch vertical slit up the center from the bottom of the sock.
Then cut horizontally from side to side, about 1 inch from the bottom, allowing
two pieces to fall into the cup with PBST.
f) Submerge the lower edge of the sock again to allow wetting of the contents
inside. Again cut a 1-inch vertical slit up the center and horizontally from
side to side to allow another two sections to fall into the container with the
PBST.
g) Continue to submerge and cut the sock until all of the white filter part of the
sock is in pieces in the jar.
h) Discard the upper blue portion of the vacuum sock.
i) Change gloves.
j) Tightly close the container, seal with parafilm, and place on a platform
shaker/rotator with lock bars. Agitate samples at 300 rpm for 30 min.
NOTE: If shaker/rotator is outside of the BSC, the containers should be
enclosed in ziplock bags and a sealed biotransport box (Fisher Scientific;
catalog #15-251-2)
k) Remove transport container from the shaker and place in the BSC. Allow
settling for 1 min, then pipette off 30 ml of supernatant into 50-ml sterile,
screw-cap, conical tubes.
I) Discard the settled material.
m) Place conical tubes into sealing centrifuge buckets within the BSC. Transport
to centrifuge and place on swinging bucket rotor.
n) Centrifuge the supernatant at 3500 X g for 15 min. Do not use brake to slow
the rotor, as resuspension may occur.
o) After centrifugation, move the sealed centrifuge buckets back to the BSC.
p) Carefully pipette off 25 ml of the supernatant and re-suspend the pellet in the
remaining 5 ml by vortexing the 5 ml sample for 1 min with 10 sec bursts.
2. Serially dilute the spore elution suspension in PBST.
a) Vortex elution suspension on high for 30 sec
b) Remove 0.1 ml of spore elution suspension (10°) and place in one tube (0.9 ml)
of PBST. This is the 10"1 suspension. Recap the 10"1 tube and vortex on high for
30 sec.
E-10
-------
c) Open cap of the 10"1 suspension and remove 0.1 ml of this suspension and place
in a new 0.9 ml tube of PBST. This is the 10"2 suspension. Recap the PBST tube
and vortex on high for 30 sec.
d) Open cap of the 10"2 suspension and remove 0.1 ml of this suspension and place
in a new 0.9 ml tube of PBST. This is the 10"3 suspension. Recap the PBST tube
and vortex on high for 30 sec.
e) You will have four spore suspensions: the initial wipe elution suspension (no
dilution=10°) and three serial dilutions of this suspension in PBST (10~1, 10"2, and
10'3).
f) Repeat steps (a) through (d) for all samples.
3. Culture diluted spore suspensions on TSA.
a) After vortexing tubes well, remove 100 ul from the 10"3 suspension with the P100
pipette and place on to a plate of TSA labeled 10'4.
NOTE: the plating of 100 ul is an additional 1:10 dilution of the 10"3
suspension resulting in a 10"4 dilution on the plate. Repeat 2 more times
for a total of three inoculated plates.
b) Spread the inoculum on each of the three 10"4-labeled TSA plates with one Lazy-
L cell spreader. Discard spreader.
c) After vortexing tubes well, remove 100 ul from the 10"2 suspension with the P100
pipette and place on to a plate of TSA labeled 10'3.
NOTE: the plating of 100 ul is an additional 1:10 dilution of the 10"2
suspension resulting in a 10"3 dilution on the plate. Repeat 2 more times
for a total of three inoculated plates.
d) Spread the inoculum on each of the three 10"3-labeled TSA plates with
one Lazy-L cell spreader. Discard spreader.
e) After vortexing tubes well, remove 100 ul from the 10"1 suspension with
the P100 pipette and place on to a plate of TSA labeled 10'2.
NOTE: the plating of 100 ul is an additional 1:10 dilution of the 10"1
suspension resulting in a 10"2 dilution on the plate. Repeat 2 more times
for a total of three inoculated plates.
f) Spread the inoculum on each of the three 10"2-labeled TSA plates with one Lazy-
L cell spreader. Discard spreader.
g) After vortexing tubes well, remove 100 ul from the initial wipe elution suspension
(10°) with the P100 pipette and place on to a plate of TSA labeled 10'1.
NOTE: the plating of 100 ul is an additional 1:10 dilution of the initial wipe
elution suspension (10°) resulting in a 10"1 dilution on the plate. Repeat 2
more times for a total of three inoculated plates.
h) Spread the inoculum on each of the three 10"1-labeled TSA plates with one Lazy-
L cell spreader. Discard spreader.
E-11
-------
i) Place all plates in an incubator set at 35 ± 2 °C for a maximum of 3 days. Plates
should be examined within 18-24 hours after start of incubation and within 72
hours of sample collection. Count CPU of each suspect BG colony (orange in
color) and record on the worksheet titled BG Spore Environmental Sample
Results Form - Manual Dilution and Filter Plating..
• If the CFU is <300/plate, record actual number.
• If the CFU is >300/plate, record as "too numerous to count" (TNTC)
• If no growth of suspect colonies is observed, record as "None detected."
4. Capture spores on Microfunnel membranes and culture on ISA
a) Place two 0.45 urn (pore-size) Microfunnels on the vacuum manifold.
b) Moisten Microfunnel membranes with 5 ml PBST, open vacuum and vacuum
through the filter. All filtering should be done with a vacuum pressure <20 cm Hg.
c) With the vacuum valve closed, place 10 ml of PBST into each filter cup.
d) Add 1.0 ml of 10° wipe elution suspension from 2(a) to each filter cup.
e) Open valves and vacuum the suspension through the filter.
f) Rinse the walls of each Microfunnel cup with 10 ml of PBST and vacuum through
the filter.
g) Squeeze the walls of the Microfunnel cup gently and separate the walls from the
base holding the filter. Remove each filter membrane with sterile forceps and
place grid-side up on a TSA plate. Make sure that the filter is in good contact with
the surface of the agar. If an air pocket occurs under the filter, use the sterile
forceps to lift the edge of the filter to release the air pocket for better contact with
the agar.
h) Record exact volume of the 10° wipe elution suspension filtered on each plate. It
should be 1 ml.
i) Repeat steps (a) through (i) for all each sample.
j) Incubate TSA plates with filter membranes at 35 ± 2 °C for a maximum of 3 days.
Plates should be examined within 18-24 hours after start of incubation and within
72 hours of sample collection. Count CFU of each suspect BG colony (orange in
color) and record on the worksheet titled BG Spore Environmental Sample
Results Form - Manual Dilution and Filter Plating.
• If the CFU is <300/plate, record actual number.
• If the CFU is >300/plate, record as "too numerous to count" (TNTC)
• If no growth of suspect colonies is observed, record as "None detected"
1.3.4. Documentation of Results
Data were manually entered into Microsoft Excel Spreadsheets.
E-12
-------
Appendix F
Sand Sample Preparation, Collection, and
Extraction/Analysis Protocols
-------
-1: Dry Heat Sterilizing Sand Aliquots Conducted by EPA
-2: Placement and Collection Protocols for Sand Dish Study
-3: Division of Samples between the EPA and USGS, and Concentration of Bacillus
atrophaeus subsp. globigii (Bg) Spores
-4: Vacuum Based Protocol for the PowerSoil® DMA Isolation Kit
-5: qPCR Analysis of Isolated DMA from BOTE Samples
-6: USGS Soil DMA Extraction Protocol for the Detection of Bacillus atrophaeus subsp. globigii
F-2
-------
Appendix 1-1: Dry Heat Sterilization of Sand Aliquots Conducted by EPA
A. Consumables and Equipment
1. Aluminum weighing boats; 75 ml_, Fisher Cat #08-732-107
2. Aluminum foil
3. Sand, silica, Ace Hardware, Cat # 4315024 or equivalent
4. Parafilm®(Pechiney Plastic Packaging Company, Chicago, IL)
5. Office tape
6. Digital top loader balance
7. Forma Quick-Dry Oven, model 3096 (Forma Scientific, Inc., Marietta, OH) or equivalent
8. Plastic sterile Petri dishes, 150 by 15 mm (BD Biosciences, San Jose, CA; Falcon Cat #
25373-187, or equivalent)
B. Procedure:
1. Tare the weight of a single aluminum weigh boat.
2. Add to 50 g of sand to each of 250 weigh boats.
3. Cover individual weigh boats with aluminum foil and place, in a Forma-Quick Dry Oven
at 250 °C 10 hours. (Oven reached 250 °C after two hours and reached a high
temperature of 290 °C for two hours.)
4. Remove sterilize sand aliquots from the oven and allow to cool.
5. In a biological safety cabinet, aseptically transfer a sterile sand aliquot to a sterile 150
mm Petri dish and seal with a 14 in (35.6 cm) x 2 in (5.1 cm) piece of Parafilm® and two
pieces of office tape on either side of the diameter of the sealed Petri dish.
6. Place filled sand samples into secondary plastic bag and box for shipment to Idaho
National Laboratory, BOTE site. Store under ambient conditions until usage.
F-3
-------
Appendix 1-2: Placement and Collection Protocols for Sand Dish Study
For placement and collection start at tent site 1 and move in sequential order 1-10. Collect all
required sample blanks at site 5. Collect the two control positive building samples (B1-B2) while
in the building collecting other samples.
A. Placement of Trays
1. Person A (Supplier):
Remove pre-labeled orange painted holder from the kit and hand to Person B
(Collector).
2. Person B (Collector):
Place orange painted holder in specified site.
3. BROOM Operator:
Pre-scan all trays to mark coordinates in BROOM.
B. Placing Sand Dish Samples
Persons A and B: Discard existing top pair of gloves. Note: After each person has discarded all
but their base pairs of gloves, open the bag with clean gloves and place another set of 3 gloves
over the last pair on hands, while inside the zip-top bag. Each sampler will have their own bag of
fresh gloves.
1. Person A (Supplier):
a) Remove a sample supply kit. Kit contains sealed sand sample in an individual bag.
b) Open sample supply kit.
c) Remove pre-filled sand sample and check label on the bag to ensure correct.
d) Pass the labeled sand sample to Person B.
e) Discard top layer of gloves before placing next sample.
f) Repeat steps c through f until all samples are placed.
2. Person B (Collector):
a) Double check laboratory prepared sand sample labeling to ensure correct site
placement.
b) Remove packaging material from the prepared sand sample and dispose in waste
bag.
c) Place laboratory prepared sand sample on holder in specified site.
d) Remove lid from the sand sample and dispose in waste bag.
e) Remove exterior gloves.
f) Repeat steps b-f for all samples being placed.
C. Collecting Sand Dish Samples
Persons A and B: Discard existing top pair of gloves. Note: After each person has discarded all
but their base pairs of gloves, open the bag with clean gloves and place another set of 3 gloves
F-4
-------
over the last pair on hands, while inside the zip-top bag. Each sampler will have their own bag of
fresh gloves. Person C: replace gloves as needed.
1. Person A (Supplier):
a) Remove a sample collection kit. Kit contains clean Petri dish and pre-cut piece of
Parafilm®.
b) Slide Petri dish and Parafilm® up in bag without touching inside the bag or the clean
Petri dish/ Parafilm® and allow Person B to remove items from the bag.
c) Open bag and hold open for Person B to insert the sealed sand sample.
d) Immediately seal zip-top bag after the sand sample has been inserted.
e) Place into sample collection box.
f) Remove exterior layer of gloves and repeat steps a-e for duplicate samples.
2. Person B (Collector):
a) Take clean Petri dish from Person A.
b) Throw base into the waste bag and place lid on sand sample.
c) Take a piece of Parafilm® from Person A.
d) Wrap sand sample dish with Parafilm®.
e) Place two pieces of office tape on either side of the sand sample.
f) Double check sand sample label and hold for Person C to scan.
g) Place in sample collection zip-top bag.
h) Remove outer pair of gloves and collect duplicate sample following steps a-g.
i) After all samples are collected, transport samples to decontamination line.
j) Wipe exterior of zip-top sample bags with bleach wipe.
k) Discard wipe into waste bag.
I) Hand samples off to INL for packaging and shipping.
3. Person C (BROOM Operator)
a) Scan barcode on exterior of collected sand sample, record collection time, and
location.
b) Scan tray barcode with BROOM tool.
c) Record extra observations in Notes dialog box.
F-5
-------
Appendix 1-3: Division of Samples Between the EPA and USGS, and Concentration of
Bacillus atrophaeus subspecies globigii (Bg) Spores
Introduction
This document describes a method for storing and processing sand samples presumed to
contain Bg spores and extracting DMA from them. The sample processing consists of:
• dividing the sand samples between the EPA and USGS (D. 1-12, below)
• concentrating the spores from the remaining sand (D. 13-35, below)
• extracting DMA from the sand concentrate (D. 36, below).
• shipping samples to USGS (D. 37, below)
• cleanup is also described (D. 38-40, below).
Approximately 5 g aliquots from each of the BOTE sand samples will be sent to Dale Griffin
(USGS) by overnight delivery for analysis for the presence of Bg spores. Aliquots will be placed
aseptically in individual labeled, sterile 50 ml_ conical screw cap centrifuge tubes. The caps of
each tube will be sealed with Parafilm® for added security. The tubes will be placed in a large
bag before being boxed for shipment to the USGS.
The remaining sand sample (approximately 45 g) will be accurately weighted before spore
isolation. The sand matrix will be washed with phosphate buffered saline supplemented with
TWEEN®-20 (TWEEN®, Sigma-Aldrich, St. Louis, MO). After vigorous shaking, the sand will be
allowed to settle and the supernatant will be transferred to a centrifuge bottle. On completion of
centrifugation, the supernatant will be aspirated and discarded. DMA will be extracted from the
pellet using the MO BIO PowerSoil® DMA Isolation Kit. Extracted DMA will be assayed using the
qPCR Analysis of Isolated DMA, Appendix I-5.
A. Sample Storage:
Upon arrival of the samples from the field operation to EPA will be stored at 4 °C in the 150 mm
Petri dishes in which they were shipped until they are processed.
B. Sample Processing, Consumables:
1. 50 mL sterile conical centrifuge tubes in racks; Fisher Brand Cat #430043, or equivalent
2. 250 mL centrifuge tubes; Corning Cat # 430776, or equivalent
3. Sterile scoopulas, Fisher Brand Cat# 14-357Q, or equivalent
4. Sterile powder funnels, Fisher Brand Cat 10-371 D, or equivalent
5. Aluminum foil and assorted autoclave bags
6. Parafilm®
7. Paper towels
8. Permanent marker
9. Shipping box and XL zip-top bag, GSA Cat GS07F9232S, or equivalent
10. Dispatch® towels, 7" x 8", 50/box, Cat # 69101, or equivalent
11. 70 % Ethanol
12. Nitrile gloves, assorted sizes
13. Phosphate Buffered Saline supplemented with TWEEN®20 (PBST) (Table 13-1 and I3-2)
F-6
-------
Table 13-1: Components of 10X Phosphate Buffered Saline
Comp
NaCI
KH2PO4
Na2HPO4 12..,_
KCI
Reagent water
Adjust pH to 7.4 with 0.1 N HCI or 0.1 N NaOH, as
filter sterilize.
80 g
2g
29 g
2g
to 1 000 ml_
160 g
4g
58 g
4g
to 2 000 ml_
Autoclave or positive pressure
Table 13- 2: Phosphate Buffered Saline with 1% Tween 20
10 mL
1,OOOmL
Compone
Tween® 20 v,
(20) sorbitan m
Reagent water
Autoclave or positive pressure filter sterilize.
To make working PBST (0.01% Tween® 20): Mix 100 mL 10X PBS, 10 mL Tween® 20 with 800
mL reagent water; adjust the pH to 7.4 with 0.1 N HCI or 0.1 N NaOH, as necessary. Adjust the
volume to 1,000 mL with reagent water. Autoclave or positive pressure filter sterilize.
Sterile Pasteur, 10 mL and 25 mL serological pipettes
C. Sample Processing, Equipment:
1 . Single pan top loader balance
Sterile scoopulas, Fisher Cat # 14-357Q, or equivalent
Sterile powder funnels, Fisher Cat # 1 0-37D, or equivalent
Pipette aid
Son/all® Evolution RC centrifuge (Fisher Scientific, Pittsburg, PA)
Sorvall®, SLA fixed angle rotor
Sorvall®, 250 mL polypropylene Oak Ridge Centrifuge Bottles, centrifuge bottles, sterile,
Cat # 03937
Sorvall®, HS-4 swinging bucket rotor
Sorvall® 50 mL conical centrifuge insert, Cat # 03072, for use in Sorvall® HS-4 swinging
bucket rotor
2.
3.
4.
5.
6.
7.
8.
9.
D. Sample Processing, Procedure:
Safety: Minimal personal protective equipment to be worn by the operator(s) includes a
laboratory coat, double gloves, and safety glasses. All sample handling will be conducted in a
biological safety cabinet.
Divide the sand samples between EPA and USGS
1. The 50 mL and 250 mL tubes will be labeled with pre-printed labels.
2. After transferring the top loader balance to the biosafety cabinet, Check to make sure it
is level.
3. A 50 mL tube will be weighed and the weight recorded on the data log provided at the
end of this SOP.
4. Sterile scoopulas and powder funnels will be autoclaved prior to work and placed in the
biological safety cabinet along with precut pieces of Parafilm®.
F-7
-------
5. An individual paper towel will be laid out in the biosafety cabinet to serve as a work
surface for each sample division.
6. The paper towel will be sprayed with 70% ethanol to keep down dust and debris.
7. The appropriate 150 mm Petri dish and pre-labeled tubes will be set in the work area.
8. The contents of the 150 mm Petri dish will be mixed by a gentle shaking back and forth.
Then the Petri dish will be tapped upside down on the bench top to get the contents
deposited to the lid side of the Petri dish.
9. The sample container weight will be recorded on the sample log.
10. After opening the 150 mm Petri dish, using a sterile scoopula, aseptically transfer
approximately 5 g of the sand to a sterile 50 ml_ conical centrifuge tube.
11. Screw the cap on the centrifuge tube and seal the cap with a piece Parafilm® wrapped
around the cap.
12. Set the sample aside for shipment to the USGS. For shipment instructions skip to step
37.
Concentrate Spores from the Remaining Sand
13. Determine the weight of the sample remaining in the lid and record the result on sample
log.
14. Open and place a pre-labeled 250 ml_ centrifuge tube within a holder on a paper towel
sprayed with 70% ethanol.
15. Holding a sterile powder funnel in the neck of the centrifuge tube, aseptically transfer the
remaining sand from the Petri dish lid to 250 ml_ centrifuge tube.
16. Determine the weight of the empty Petri dish lid and record the result on sample log.
17. Using a pipette or wash bottle, aseptically transfer 25 ml_ of PBST into the lid of the 150
mm Petri dish, gently swirl it around the lid and pour it through funnel into the 250 ml_
centrifuge tube.
18. Using a pipette or wash bottle, aseptically transfer 25 ml_ of PBST into the bottom of the
150 mm Petri dish, gently swirled it around, and pour it through funnel into the 250 ml_
centrifuge tube.
19. Using a pipette or wash bottle, aseptically transfer another 25 ml_ of PBST to rinse the
funnel.
20. Adjust the volume in 250 ml_ centrifuge tube with PBST to 125 ml_ and recap it.
21. Appropriately dispose of the empty 150 mm Petri dish, scoopula, pipettes, and filter
funnel.
22. Shake the 250 ml_ centrifuge tube containing 125 ml_ PBST and sample back and forth
vigorously for 3 minutes.
23. Allow sand particles to sediment for 5 minutes
24. Using a sterile 25 ml_ pipette, transfer the supernatant to a sterile, pre-labeled 250ml_
Sorvall centrifuge bottle.
25. Repeat steps 1 through 24 six times. This is the number of samples that can be
processed in the Sorvall Evolution Refrigerated Centrifuge using a SLA fixed angle rotor.
26. Carefully dispose of the paper towel ensuring that any lost sand remains in the towel.
27. Wipe the work surface of the biosafety cabinet with a Dispatch® towel and then with 70%
ethanol.
28. Remove the outer pair of gloves and discard them. Put on a fresh pair of outer gloves
before processing the next sample
29. After balancing the bottles, centrifuge them at 5,900 x g for 20 minutes using a Sorvall
Evolution Refrigerated Centrifuge equipped with a SLA fixed angle rotor. The Sorvall
F-8
-------
Evolution Refrigerated Centrifuge brake should be set to 1 and the temperature should
be set to 4°C.
30. Aseptically aspirate the supernatant from the centrifuge bottle, using a sterile Pasteur
pipette connected to a suction flask and discard it.
31. Aseptically resuspend the pellet in the 250 ml_ centrifuge bottle in 25 ml_ PBST and
transfer the pellet resuspension to a sterile 50 ml_ conical centrifuge bottle.
32. Repeat step 31 for each of the 250 ml_ centrifuge bottles.
33. Transfer up to 4 of the 50 ml_ conical centrifuge tubes to the Sorvall HS-4 rotor equipped
with a Sorvall 50 ml_ insert in each cup.
34. Centrifuge the tubes at 5,900 x g for 20 minutes using a Sorvall Evolution Refrigerated
Centrifuge equipped with the HS-4 swinging bucket rotor. The Sorvall Evolution
Refrigerated Centrifuge brake should be set to 1 and the temperature should be set to
4°C.
35. Aseptically aspirate the supernatant carefully from the 50 ml_ centrifuge tube, using a
sterile Pasteur pipette connected to a suction flask and discard it. Save the pellet for
DMA extraction.
DNA Extraction
36. Extract the DNA from the individual pellets following the vacuum based manufacturer's
protocol from the PowerSoil® DNA Isolation Kit (MO BIO Laboratories, Inc., Carlsbad,
CA).
Shipment of the USGS Samples
37. Wipe the outsides of the USGS aliquots (samples) with a Dispatch towel. Then place the
USGS aliquots in a XL Ziploc® bag, wipe the outside of the Ziploc® bag with a Dispatch®
towel, and load it into a box for overnight shipment at ambient conditions. Seal and label
the box following the Safety, Health, and Environmental Management (SHEM) guidance.
If shipment will occur on a future date, store the samples in 4 °C. The shipping address
is as follows:
Dr. Dale Griffin
United States Geological Survey
2639 North Monroe Street, Suite A-200
Tallahassee, FL 32303
Office phone # 850-553-3675
Clean Up
38. Clean the biosafety cabinet according to the laboratory's SOP.
39. Sterilize, clean and re-autoclave the reusable scoopulas and powder funnels.
40. Fill out a request for shipping form and get it authorized by Mary Sullivan (U.S.
Environmental Protection Agency, Cincinnati, OH). Make a copy of the request for
shipping form for Mary Sullivan's records and deliver it with Pat Tapp (U.S.
Environmental Protection Agency, Cincinnati, OH). Before 4:00 PM take the package(s)
to the mailroom in the basement of EPA's Andrew W. Breidenbach Environmental
Research Center.
F-9
-------
Appendix 1-4: Vacuum Based Protocol for the PowerSoil® DNA Isolation Kit
A. Reagents:
PowerSoil® DNA Isolation Kit (MO Bio Cat. # 12888, or equivalent)
B. Equipment:
MO BIO Vortex Adapter tube holder for the vortex (MO BIO Catalog No. 13000-V1)
PowerVAC™ vacuum manifold (MO BIO Catalog #11991, or equivalent)
PowerVAC™ Mini System (MO BIO Catalog #11992)
PowerVAC™ Mini Spin Filter Adaptors (MO BIO Catalog #11992-20)
C. Procedure:
This procedure is based on the MO BIO PowerSoil® DNA Isolation Kit# 12888 instructions. The
Operator must wear gloves at all times.
1. Weigh out -0.25 g of the sand sample. Record the weight in the lab book and place the
sample in the MoBio garnet beating tube (PowerBead Tubes). Label the tube.
2. Gently vortex to mix.
3. Check Solution C1. If Solution C1 is precipitated, heat the solution to 60 °C until it
dissolves before using it.
4. Add 60 uL of Solution C1 and invert the tube several times or vortex briefly.
5. Secure PowerBead Tubes horizontally using the MO BIO Vortex Adapter tube holder for
the vortex (MO BIO Catalog No. 13000-V1) or secure tubes horizontally on a flat-bed
vortex pad with tape. Vortex at maximum speed for 10 minutes.
6. Make sure the PowerBead Tubes rotate freely in the centrifuge without rubbing.
Centrifuge tubes at 10,000 x g for 30 seconds at room temperature.
7. CAUTION: Be sure not to exceed 10,000 x g or the tubes could break.
8. Transfer the supernatant to a clean 2 ml_ Collection Tube (provided in the kit).
9. Note: Expect between 400 to 500 uL of supernatant. Supernatant could still contain
some sand particles.
10. Add 250 uL of Solution C2 and vortex for 5 seconds. Incubate at 4 °C for 5 minutes.
11. Centrifuge the tubes at room temperature for 1 minute at 10,000 x g.
12. Avoiding the pellet, transfer up to, but no more than, 600 uL of supernatant to a clean 2
ml_ Collection Tube (provided in the kit).
13. Add 200 uL of Solution C3 and vortex briefly. Incubate at 4 °C for 5 minutes.
14. Centrifuge the tubes at room temperature for 1 minute at 10,000 x g.
15. Avoiding the pellet, transfer up to, but no more than, 750 uL of supernatant into a clean 2
ml_ Collection Tube (provided in the kit).
16. Add 1,200 uL of Solution C4 to the supernatant and vortex for 5 seconds.
17. For each preparation, attach one aluminum PowerVac™ Mini Spin Filter Adapter (MO
BIO Catalog#11992-10 or 11992-20) into the Luer-Lok® fitting of one port in the manifold.
Gently press a Spin Filter column into the PowerVac™ Mini Spin Filter Adapter until
snugly in place. Ensure that all unused ports of the vacuum manifold are closed.
18. Note: Aluminum PowerVac™ Mini Spin Filter Adapters are reusable.
19. Transfer 650 uL of prepared sample lysate (from step 14) to the Spin Filter column.
20. Turn on the vacuum source and open the stopcock of the port. Hold the tube in place,
when opening the stopcock, to keep the spin filter steady. Allow the lysate to pass
through the Spin Filter column. After the lysate has passed through the column
completely, load again with the next 650 uL of lysate. Continue until all of the lysate has
F-10
-------
been loaded onto the Spin Filter column. Close the one-way Luer-Lok® stopcock of that
port.
21. Note: If Spin Filter Columns are filtering slowly, close the ports to samples that have
completed filtering to increase the pressure to the other columns.
22. Load 800 uL of 100% ethanol into the Spin Filter so that it completely fills the column.
Open the stopcock, while holding the column steady. Allow the ethanol to pass through
the column completely. Close the stopcock.
23. Add 500 uL of Solution C5 to each Spin Filter. Open the Luer-Lok® stopcock and apply a
vacuum until Solution C5 has passed through the Spin Filter completely. Continue to pull
a vacuum for another minute to dry the membrane. Close each port.
24. Turn off the vacuum source and open an unused port to vent the manifold. If all 20 ports
are in use, break the vacuum at the source. Make certain that all vacuum pressure is
released before performing the next step. It is important to turn off the vacuum at the
source to prevent backflow into the columns.
25. Remove the Spin Filter column and place in the original labeled 2 mL Collection Tube.
Place into the centrifuge and spin at 13,000 x g for 1 minute to completely dry the
membrane.
26. Transfer the Spin Filter column to a new 2 mL Collection Tube and add 100 uL of
Solution C6 to the center of the white filter membrane. Alternatively, sterile DMA-Free
PCR Grade Water could be used for elution from the silica Spin Filter membrane at this
step (MO BIO Catalog # 17000-10).
27. Centrifuge at room temperature for 30 seconds at 10,000 x g.
28. Discard the Spin Filter column. The DMA in the tube is now ready for any downstream
application. No further steps are required. MO BIO recommends storing DMA frozen (-20
°C to -80 °C). Solution C6 contains no EDTA.
29. Store the sample at -70 °C until ready to run qPCR analysis.
F-11
-------
Appendix 1-5: qPCR Analysis of Isolated DNA from BOTE Samples
Introduction:
Quantitative PCR (qPCR) is an adaptation of the basic PCR procedure to allow specific
quantification of copy numbers in original samples, rather than the plus/minus detection of
conventional PCR. One of the most commonly used methods for qPCR is known as Taqman™,
developed by Applied Biosystems. The method is based on the detection of fluorescence
increase from an oligonucleotide probe molecule when degraded by the 5' exonuclease activity
of the polymerase enzyme during the extension or elongation step of PCR. The oligonucleotide
probe molecule contains a fluorescent "Reporter" moiety covalently attached to one end of the
molecule, and a "Quencher" moiety attached to the other. The quencher moiety prevents
emission of light by the reporter moiety, when the probe is intact. During each PCR cycle,
double-stranded DNA is denatured at 95 °C, followed by specific binding of the PCR primers
and Taqman oligonucleotide probe to complementary regions of the denatured DNA strands
during the annealing step of PCR. During the extension or elongation step of PCR, the
polymerase enzyme traverses the template from the 3' end of each primer, degrading bound
probe with its 5' exonuclease activity, which separates the Reporter from the Quencher moieties.
This results in a light emission increase in proportion to the exponential increase in DNA copies
during PCR amplification. A threshold cycle (Ct) is then determined corresponding to the point at
which fluorescence begins to increase in a linear fashion. Samples with higher target cell
numbers will have a lower Ct, while those with lower target cell numbers will have a higher Ct.
A. Reagents Needed:
DNA Molecular Grade Water (AccuGENE, Cambrex # 51200, or equivalent)
qPCR Taq Polymerase (Applied Biosystems, TaqMan Universal PCR Master Mix, no Amp
Erase, cat. #4364341)
Primers and Probes specific for the recF gene of Bg DNA (Table A5-1)
Table 15-1: qPCR Primers and Probe (Sigma Aldrich, or Equivalent, HPLC Purified)
Target
Organism
B. globigii
quence (5' to 3')
6g42F CGC GCC CGA GGA CTT AA
6g104R ATG TCA AGA AAC CGC CGT C
Bg60FT FAM-TCT CGT AAA GGG CAG CCC GCA AG -TAMRA
Reference
Kane et. al
(2009)
B. Equipment Needed:
Applied Biosystems 7900HT Prism, or equivalent.
C. Procedure:
1. For each DNA sample isolated, perform two additional ten-fold dilutions1 of the isolated
DNA, (1:10 and 1:100 dilutions; 20 uL plus 180 uL molecular grade water per dilution).
Between experiments, store DNA dilutions at 20 °C to prevent degradation. To avoid excessive freeze
thaw cycle, which can degrade DNA, make multiple aliquots of the DNA dilutions before freezing.
F-12
-------
Note: Analysis of multiple dilutions of isolated DMA samples should provide a dose-response
with dilution, indicative of positive PCR amplification. (Background or non-specific PCR
amplification would give similar Ct values for all dilutions). Comparison of undiluted DMA and
1:10 and 1:1000 dilutions could be indicative of PCR inhibitors in the DMA sample (if undiluted
samples showed negative amplification, and diluted ones were positive and dose-responsive).
2. Prepare enough PCR "master mix" to analyze all samples. Enough master mix is
prepared to divide equally among all samples, plus approximately 4% (v/v) extra to allow
for loss due to pipetting etc. (Table I5-2).
Table I5-2: Master Mix Preparation Table
Reagent
Reaction
Volume (uL)
Master Mix Concentration
(ML1
Taqman Universal PCR Master Mix,
2X
Probe (10 pmol/ul_)
Forward Primer (10 pmol/uL)
Reverse Primer (10 pmol/uL)
Sample Template Volume
Water
Total Reaction Volume
Number of Samples
10
0.8
1
1
2
5.2
20
65
650
52
65
65
130
338
1300
0.4
0.5
0.5
Quantity Master
Mix + 4% (|JL)
676
54.08
67.6
67.6
135.2
351.52
1352
3. Figure 15-1 below shows a typical setup of a 96-well PCR plate for analysis of BOTE
DMA samples on the Prism 7900HT. Three dilutions (undiluted, 1:10 and 1:100) are
analyzed for each DMA sample, with triplicate PCR reactions per dilution. In addition,
previously prepared and characterized positive and negative DMA detection controls are
analyzed with each PCR 96-well run. In this case, Bg DMA (BOTE strain) and Bg DMA (+
strain) are used as positive (pos) controls (A1-A6), and E. coli DMA is used as a negative
(neg) detection control (A7-A9). Furthermore, no-template PCR controls (NTC) in which
water is substituted for sample template are used (A10-A12).
A
B
C
D
E
F
G
H
1
2
3
Bg BOTE DMA Pos
Control
DNA#1 Undiluted
DMA #2 Diluted 1:10
DMA #3 Diluted
1:100
DMA #5 Undiluted
DMA #6 Diluted 1:10
DMA #7 Diluted
1:100
DMA #9 Undiluted
4
5
6
Bg + DMA Pos Control
1:10
DNA#1 Diluted 1:10
DMA #2 Diluted
1:100
DMA #4 Undiluted
DMA #5 Diluted 1:10
DMA #6 Diluted
1:100
DMA #8 Undiluted
DMA #9 Diluted 1:10
7
8
9
E. coli DMA Neg
Control
DNA#1 Diluted 1:100
DMA #3 Undiluted
DMA #4 Diluted 1:10
DMA #5 Diluted 1:100
DMA #7 Undiluted
DMA #8 Diluted 1:10
DMA #9 Diluted 1:100
10
11
12
No Template Control
(NTC)
DNA #2 Undiluted
DNA #3 Diluted 1:10
DNA #4 Diluted
1:100
DNA #6 Undiluted
DNA #7 Diluted 1:10
DNA #8 Diluted
Empty
Empty
1:100
Empty
Figure 15-1: Typical qPCR 96 well plate set-up for analysis of triplicate BOTE extracted
DNA samples.
F-13
-------
Note: Use of an Excel® spreadsheet to enter sample information speeds up the process, as
entries can be copied and pasted into the Excel® spreadsheet. Data entry into the Applied
Biosystems 7900HT Prism software requires typing each entry individually.
4. Perform the PCR with a quantitative PCR unit (like Applied Biosystems 7900HT Prism),
using the PCR conditions listed in Table 15-3:
Table 15- 3: Thermocycler Program Specifications
95 10 minutes 1
95 15 seconds ._
60 1 minute
D. Data Analysis: Determination of Ct values by Prism SDS Software
In order to accurately compare Ct values between qPCR experimental runs, it is necessary to
consistently set Baseline and Threshold values in Prism SDS software when analyzing results
for an individual run. The software will automatically calculate both Baseline (number of cycles
corresponding to background noise preceding exponential signal, usually between cycles 3-15)
and Threshold settings (setting that determines actual Ct value).
Normally, an automatic baseline setting of 3-15 cycles is appropriate for most runs, and does
not need to be adjusted. However, the use of an automatic threshold setting can result in
differential calculation of Ct values, depending upon the threshold value determined by the
software. In order to accurately calculate Ct values, the threshold setting should be set manually
so that it corresponds to the midpoint in the exponential region of signal increase.
Procedure for setting proper threshold:
1. After completion of PCR run, select "Analyze" under Analysis Heading of SDS software.
Note: Once a qPCR run on the Applied Biosystems 7900HT Prism is completed, the primary
data is saved and stored automatically by the unit. The baseline and threshold values can be
changed and adjusted at any time, without loss of the primary data.
2. Open the results tab, select all data (Ctrl-a) and examine the amplification patterns. At
this point, the unit has automatically determined both the Baseline and Threshold values.
Leave the baseline setting on "Automatic Baseline" corresponding to cycles 3-15.
3. Next, choose Manual Ct, and adjust the threshold (green line) so that it is approximately
halfway up the exponential portion of the curve (Figure I5-2). For most primer and probe
systems, this corresponds to a setting of a 0.2 threshold value. Once the threshold is
properly set, resave the data (Ctrl-S).
F-14
-------
Figure 15-2: Representative qPCR amplification plot.
4. Using the Prism SDS Software, export the calculated Ct values by choosing File > Export
and select location for saving the file (saved as a .txt file).
5. Open the exported txt file using the Excel program, and Sort the data by choosing Data >
Sort > Sort by Well > Ascending. The Data should now be sorted so that Sample Names
(column B) ascend from A1-A12, B1-B12 etc.
6. Select and copy Ct values (column F) and Paste Special (values only) into Excel
spreadsheet containing sample information. Average Ct and SD values will then be
calculated. Analysis of DMA samples should result in a nearly linear Ct vs. dilution plot
(at least R2 > 0.95). E. coli controls should be negative (40 or greater Ct), and prior Bg
DMA control within 5% of prior determined Ct values.
References
ABI Prism 7900HT Sequence Detection System User Guide, Part Number 4317596 Rev. B
12/2002
AOAC (2002). Sporicidal activity of disinfectants. Official Method 966.04, AOAC International.
Brown, G. S., R. G. Betty, et al. (2007). Evaluation of a wipe surface sample method for
collection of Bacillus spores from nonporous surfaces. App Environ Microbio 73(3):
706-710.
Kane, S.R., Letant, S.E., Murphy, G.A., Alfaro, T.M., Krauter, P.W., Mahnke, R., Legler, T.C.,
and Raber, E. (2009). Rapid, high-throughput, culture-based PCR methods to analyze
samples for viable spores of Bacillus anthracis and its surrogates. J Microbio Methods
76(3): 278-284.
F-15
-------
Attachment 1-6: USGS Sand DNA Extraction Protocol for the Detection of Bacillus
atrophaeus subs p. globigii
Introduction
This document describes a method for the qPCR detection of DNA from Bacillus atrophaeus
subsp. globigii (Bg) spores. It consists of two primary steps: DNA isolation from spores of Bg
and qPCR analysis of the DNA. Specificity of qPCR detection is determined by comparison of
results to non-target DNA (for example Escherichia coli) and positive control DNA from formerly
characterized Bg spore preparations.
Quantitative PCR (qPCR) is an adaptation of the basic PCR procedure to allow specific
quantification of copy numbers in original samples, rather than the plus/minus detection of
conventional PCR. One of the most commonly used methods for qPCR is known as Taqman™,
developed by Applied Biosystems. The method is based on the detection of fluorescence
increase from an oligonucleotide probe molecule when degraded by the 5' exonuclease activity
of the polymerase enzyme during the extension or elongation step of PCR. The oligonucleotide
probe molecule contains a fluorescent "Reporter" moiety covalently attached to one end of the
molecule, and a "Quencher" moiety attached to the other. The quencher moiety prevents
emission of light by the reporter moiety, when the probe is intact (e.g. free in solution). During
each PCR cycle, double-stranded DNA is denatured at 95 °C, followed by specific binding of the
PCR primers and Taqman oligonucleotide probe to complementary regions of the denatured
DNA strands during the annealing step of PCR. Then during the extension or elongation step of
PCR, the polymerase enzyme traverses the template from the 3' end of each primer, degrading
bound probe with its 5' exonuclease activity, which separates the Reporter from the Quencher
moieties. This results in a light emission increase in proportion to the exponential increase in
DNA copies during PCR amplification. A threshold cycle (Ct) is then determined corresponding
to the point at which fluorescence begins to increase in a linear fashion. Samples with higher
target cell numbers will have a lower Ct, while those with lower target cell numbers will have a
higher Ct.
A. Reagents:
PowerSoil® DNA Isolation Kit (MO Bio cat. 12888, or equivalent)
DNA Molecular Grade Water (MO BIO Catalog # 17000-10), or equivalent)
qPCR Taq Polymerase (Applied Biosystems, TaqMan Universal PCR Master Mix, no Amp
Erase, catalog #4364341)
qPCR primers and probe (Eurofins MWG Operon, Table 16-1)
Table 16-1: qPCR Primers and Probe
Tar
Orga
B. globigii
6g42F CGC GCC CGA GGA CTT AA Kane et. al
6g104R ATG TCA AGA AAC CGC CGT C (2009)
Bg60FT FAM-TCT CGT AAA GGG CAG CCC GCA AG -TAMRA
B. Equipment:
MO BIO Vortex Adapter tube holder for the vortex (MO BIO Catalog No. 13000-V1)
PowerVAC™ vacuum manifold (MO BIO Catalog #11991, or equivalent)
PowerVAC™ Mini System (MO BIO Catalog #11992)
PowerVAC™ Mini Spin Filter Adaptors (MO BIO Catalog #11992-20)
F-16
-------
Applied BioSystems StepOne 48-well Real-Time PCR system (Applied Biosystems, Catalog
#4376373)
C. DNA Isolation
This procedure is based on the MO BIO PowerSoil® DNA Isolation Kit #12888 instructions. The
Operator must wear gloves at all times.
1. Weigh out -0.25 gram of the sand sample. Record the weight in the lab book and place
the sample in the MO BIO garnet beating tube (PowerBead Tubes). Label the tube.
2. Gently vortex to mix.
3. Check Solution C1. If Solution C1 is precipitated, heat solution to 60 °C until dissolved
before use.
4. Add 60 uL of Solution C1 and invert several times or vortex briefly.
5. Secure PowerBead Tubes horizontally using the MO BIO Vortex Adapter tube holder for
the vortex (MO BIO Catalog No. 13000-V1) or secure tubes horizontally on a flat-bed
vortex pad with tape. Vortex at maximum speed for 10 minutes.
6. Make sure the PowerBead Tubes rotate freely in the centrifuge without rubbing.
Centrifuge tubes at 10,000 x g for 30 seconds at room temperature. CAUTION: Be sure
not to exceed 10,000 x g or tubes might break.
7. Transfer the supernatant to a clean 2 ml_ Collection Tube (provided). Note: Expect
between 400 to 500 uL of supernatant. Supernatant could still contain some sand
particles.
8. Add 250 uL of Solution C2 and vortex for 5 seconds. Incubate at 4 °C for 5 minutes.
9. Centrifuge the tubes at room temperature for 1 minute at 10,000 x g.
10. Avoiding the pellet, transfer up to, but no more than, 600 uL of supernatant to a clean 2
ml_ Collection Tube (provided).
11. Add 200 uL of Solution C3 and vortex briefly. Incubate at 4 °C for 5 minutes.
12. Centrifuge the tubes at room temperature for 1 minute at 10,000 x g.
13. Avoiding the pellet, transfer up to, but no more than, 750 uL of supernatant into a clean
2ml_ Collection Tube (provided).
14. Add 1200 uL of Solution C4 to the supernatant and vortex for 5 seconds.
15. For each preparation, attach one aluminum PowerVac™ Mini Spin Filter Adapter (MO
BIO Catalog#11992-10 or 11992-20) into the Luer-Lok® fitting of one port in the manifold.
Gently press a Spin Filter column into the PowerVac™ Mini Spin Filter Adapter until
snugly in place. Ensure that all unused ports of the vacuum manifold are closed. Note:
Aluminum PowerVac™ Mini Spin Filter Adapters are reusable.
16. Transfer 650 uL of prepared sample lysate (from step 14) to the Spin Filter column.
17. Turn on the vacuum source and open the stopcock of the port. Hold the tube in place
when opening the stopcock to keep the spin filter steady. Allow the lysate to pass
through the Spin Filter column. After the lysate has passed through the column
completely, load again with the next 650 uL of lysate. Continue until all of the lysate has
been loaded onto the Spin Filter column. Close the one-way Luer-Lok® stopcock of that
port. Note: If Spin Filter Columns are filtering slowly, close the ports to samples that have
completed filtering to increase the pressure to the other columns.
18. Load 800 uL of 100% ethanol into the Spin Filter so that it completely fills the column.
Open the stopcock while holding the column steady. Allow the ethanol to pass through
the column completely. Close the stopcock.
19. Add 500 uL of Solution C5 to each Spin Filter. Open the Luer-Lok® stopcock and apply a
vacuum until Solution C5 has passed through the Spin Filter completely. Continue to pull
a vacuum for another minute to dry the membrane. Close each port.
F-17
-------
20. Turn off the vacuum source and open an unused port to vent the manifold. If all 20 ports
are in use, break the vacuum at the source. Make certain that all vacuum pressure is
released before performing the next step. It is important to turn off the vacuum at the
source to prevent backflow into the columns.
21. Remove the Spin Filter column and place in the original labeled 2 ml_ Collection Tube.
Place into the centrifuge and spin at 13,000 x g for 1 minute to completely dry the
membrane.
22. Transfer the Spin Filter column to a new 2 ml_ Collection Tube and add 100 uL of
Solution C6 to the center of the white filter membrane. Alternatively, sterile DMA-Free
PCR Grade Water could be used for elution from the silica Spin Filter membrane at this
step (MO BIO Catalog # 17000-10).
23. Centrifuge at room temperature for 30 seconds at 10,000 x g.
24. Discard the Spin Filter column. The DMA in the tube is now ready for any downstream
application. No further steps are required. It is recommend to store DMA frozen (-20 °C
to -80 °C). Solution C6 contains no EDTA.
25. Store the sample at -70 °C until ready to run qPCR analysis.
D. qPCR Analysis of Isolated DNA
1. Prepare enough PCR "master mix" to analyze all samples. Enough master mix is
prepared to divide equally among all samples, plus approximately 4% extra to allow for
loss due to pipetting etc. Use the embedded spreadsheet below to calculate master mix
volumes.
Table I6-2: Master Mix Preparation Table
Reagent
Number of Sai
Taqman Universal
Master Mix, 2X
Extracted Sample
Template vol (uL)
Total Reaction Volume
Reaction Quantity Master Final Concentration
Volume (uL) Mix (uL) (uM)
Quantity Master
Mix + 4%
10
12.5
2.0
2.0
2.0
4.5
2.0
25
125
20
20
20
45
1.0
1.0
1.0
130
20.8
20.8
20.8
46.8
2. 2 uL of DNA extracts are used directly from the DNA Isolation procedure previously
detailed.
3. Enter samples to be analyzed in the Applied BioSystems StepOne™ software package.
10% of the environmental sand samples will be run in duplicate.
In addition to the environmental samples, previously prepared and characterized positive
and negative DNA detection controls are analyzed, using previously prepared DNA
samples. In this case, E. coli DNA is used as a negative detection control, and B. globigii
F-18
-------
DMA is used as a positive control. Furthermore, no-template PCR controls which are
water substituted for sample template are used.
4. Perform PCR with an Applied BioSystems StepOne™ 48-well Real-Time PCR system
(Carlsbad, CA).
Table 16- 3: Thermocycler Program Specifications
Cycle
DNA denaturation
PCR amplification
Temperature (°C)
50
95
95
60
2 minutes
10 minutes
15 seconds
15 seconds
Number of Cycles
1
45
5. Analyze data and paste Ct values into sample spreadsheet, to determine average Ct and
SD values. E. coli controls should be negative (40 or greater Ct), and prior Bg DNA
control within 5% of prior determined Ct values.
The Applied BioSystems StepOne™ software package will give a visual representation of
the collected data similar to that below (Figure 16-1).
Figure 16-1: Applied BioSystems StepOne software screen shot
References
Kane, SR, Letant, SE, Murphy, GA, Alfaro, TM, Krauter, PW, Mahnke, R, Legler, TC, and
Raber, E (2009). Rapid, high-throughput, culture-based PCR methods to analyze samples for
viable spores of Bacillus anthracis and its surrogates. Journal of Microbiology Methods 76(3):
278-284.
MO BIO Laboratories, Inc. PowerSoil® DNA Isolation Kit Instruction Manual. Version 08202010.
F-19
-------
Appendix G
Report of Sample Results - LRN Samples
-------
Barcode
4160
2462
4211
4153
2934
3211
4193
2861
4166
3226
3227
4202
3230
Round
MFP
MFP
MFP
MFP
MFP
MFP
MFP
MFP
MFP
MFP
MFP
MFP
MFP
Floor
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Room
Corridor+Lobby
Corridor +Lobby
Room 101A
Room 101A
Room 101A
Room 101A
Room 102
Room 102
Room 103
Room 103
Room 103
Room 104
Room 104
X
15.077
5.781
19.523
19.831
19.033
19.489
17.810
19.499
13.493
15.321
13.093
13.793
15.033
y
16.656
13.165
7.327
8.009
6.827
8.313
15.708
17.171
6.414
8.714
11.021
18.848
20.344
z
2.500
1.894
0.500
0.000
0.792
0.000
0.000
1.000
2.500
0.688
0.280
0.480
0.420
Method
Vacuum Sock
Sponge Wipe
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Vacuum Sock
Sponge Wipe
Vacuum Sock
Sponge Wipe
Sponge Wipe
Vacuum Sock
Sponge Wipe
Area
(sqft)
4.000
0.694
4.000
4.000
0.694
0.694
4.000
0.694
4.000
0.694
0.694
4.000
0.694
Operator
Martinez
Terrill
Oudejans
Oudejans
Oudejans
Oudejans
Oudejans
Oudejans
Martinez
Martinez
Martinez
Martinez
Martinez
Acquisition
Date
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
Acquisition
Time
12:13:00 PM
10:56:00 AM
11:55:00 AM
12:19:00 PM
11:48:00 AM
12:16:00 PM
12:49:00 PM
12:34:00 PM
12:02:00 PM
11:48:00 AM
11:43:00 AM
12:36:00 PM
12:26:00 PM
Object
Ceiling
Return Vent
Chair
Floor
Desk
Floor
Floor
Desk
Ceiling
Countertop
Wall
Countertop
Countertop
Texture
Porous
Metal
Cloth
Carpet
Plastic
Carpet
Carpet
Plastic
Porous
Smooth
Textured
Metal
Porous
Orientation
Horizontal
Downward
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Upward
Vertical
Horizontal
Downward
Downward
Blank
FALSE
FALSE
FALSE
TRUE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
BROOM Notes
chair not in correct
aosition; chair neat
desk in front of
window is samplet
vacuum ;vertical
sampled
first.safety hazard
on power cord
blank vacuum
slight wet surface
vertical sampled
first
manual position;
sampled closer to
the outside wall
than
indicated.;socket
came off; air
sampling off
before vacuum.
restart sampling
after reassemling
unit.
vertical first; dirty
desk; carryover on
template
blank
Lab ID
1
2
2
2
2
2
3
2
2
2
2
2
2
Detected
Yes
No
Yes
No
No
No
Yes
No
No
No
No
No
No
Spread Plate Results
(CFU/sq ft)
2.3
ND
ND
ND
ND
ND
16.7
ND
ND
ND
ND
ND
ND
Filter Plate Results
(CFU/sq ft)
NA
ND
0.6
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
G-l
-------
Barcode
3231
4161
2935
4196
4154
2936
1693
4197
4232
4183
4207
2418
2931
1582
1513
4150
2855
1590
1592
4224
Round
MFP
MFP
MFP
MFP
MFP
MFP
MFP
MFP
MFP
MFP
MFP
MFP
MFP
MFP
MFP
MFP
MFP
MFP
MFP
MFP
Floor
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Room
Room 104
Room 105
Room 105
Room 106
Room 106
Room 106
Room 106
Room 107
Room 107
Room 107
Room 107
Room 107
Room 107
Room 107
Room 107
Room 108
Room 108
Room 108
Room 108
Room 109
X
13.774
10.902
11.836
11.285
10.994
11.581
10.312
8.307
8.426
8.313
8.913
8.021
7.094
8.378
7.236
8.000
7.326
8.716
7.201
5.673
y
18.886
10.524
12.052
20.380
18.447
15.620
18.006
10.778
8.328
11.931
9.014
11.087
6.901
11.373
9.732
18.439
18.010
19.910
18.978
8.714
z
0.480
0.000
0.762
0.350
0.400
1.000
0.400
1.200
1.300
2.199
0.200
1.200
1.300
1.200
0.800
0.486
0.814
2.325
0.811
0.381
Method
Sponge Wipe
Vacuum Sock
Sponge Wipe
Vacuum Sock
Vacuum Sock
Sponge Wipe
Swab
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Swab
Swab
Vacuum Sock
Sponge Wipe
Swab
Swab
Vacuum Sock
Area
(sqrt)
0.694
4.000
0.694
4.000
4.000
0.694
0.028
4.000
4.000
4.000
4.000
0.694
0.694
0.028
0.028
4.000
0.694
0.028
0.028
4.000
Operator
Martinez
Martinez
Martinez
Martinez
Martinez
Martinez
Martinez
Terrill
Terrill
Terrill
Terrill
Terrill
Terrill
Terrill
Terrill
Anaya
Anaya
Anaya
Anaya
Terrill
Acquisition
Date
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
Acquisition
Time
12:34:00 PM
11:27:00 AM
11:19:00 AM
10:59:00 AM
10:47:00 AM
10:35:00 AM
10:53:00 AM
12:36:00 PM
12:23:00 PM
11:48:00 AM
11:59:00 AM
12:30:00 PM
12:24:00 PM
12:31:00 PM
12:26:00 PM
12:28:00 PM
12:22:00 PM
12:45:00 PM
12:39:00 PM
11:24:00 AM
Object
Countertop
Floor
Countertop
Chair
Chair
Countertop
Computer
Countertop
Chair
Ceiling
Chair
Countertop
Shelves
Countertop
Desk
Chair
Desk
Ceiling
Computer
Chair
Texture
Porous
Carpet
Smooth
Cloth
Cloth
Smooth
Glass
Plastic
Leather
Porous
Porous
Plastic
Smooth
Plastic
Smooth
Porous
Smooth
Smooth
Smooth
Cloth
Orientation
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Vertical
Horizontal
Upward
Blank
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
TRUE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
blank
formica and
sampled on left
side
chair under
window, note fr
previous swab
sample, screen was
on opposite side ol
desk.
computer screen
correction to
sample. 2nd entry
of this sample.
ceiling tile was
taken down from
ceiling before
sampling for safetj
reasons
sample was taken
from couch.
sample object
menu didni have
that description.
need option for
blanks in drop
down menu for
sample object
2nd entry of
sample
2nd of this sample
Swab of the
diffuser on the
ceiling
Lower right hand
corner of the
computer monitor
sample taken from
bed. bed was not
an option in drop
down menu for
sample object.
Lab ID
2
1
2
2
3
2
4
1
2
2
2
2
2
4
4
2
2
4
4
3
Detected
No
Yes
No
Yes
Yes
No
No
No
No
No
Yes
No
No
No
No
Yes
No
No
No
Yes
Spread Plate Results
(CFU/sq ft)
ND
2.4
ND
8.3
8.3
ND
ND
ND
ND
ND
4.2
ND
ND
ND
ND
8.3
ND
ND
ND
12.5
Filter Plate Results
(CFU/sq ft)
ND
NA
ND
12.7
ND
ND
NA
NA
ND
ND
ND
ND
ND
NA
NA
ND
ND
NA
NA
ND
G-2
-------
Barcode
4140
2093
2417
4209
3213
2859
1688
3233
3201
3200
1691
3207
4181
3205
1692
3975
4138
3902
3210
Round
MFP
MFP
MFP
MFP
MFP
MFP
MFP
MFP
MFP
MFP
MFP
MFP
MFP
MFP
MFP
MFP
MFP
MFP
MFP
Floor
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Room
Room 109
Room 109
Room 109
Room 110
Room 110
Room 110
Room 110
Hallway
Room 206
Room 206
Room 206
Room 207
Room 208
Room 208
Room 208
Room 209
Room 209
Room 209
Room 209
X
5.873
5.680
6.221
5.212
4.373
4.917
4.211
6.493
16.142
17.385
17.321
16.200
13.571
13.302
12.849
14.356
13.371
14.371
13.271
y
12.014
6.290
10.431
20.467
19.996
14.457
18.851
12.668
20.154
14.420
20.254
6.250
18.427
15.079
18.469
8.540
10.869
12.140
11.869
z
0.000
0.200
1.743
0.496
0.697
0.000
0.892
3.000
3.789
3.395
3.000
5.291
3.000
3.000
3.000
3.000
5.266
3.000
3.000
Method
Vacuum Sock
Sponge Wipe
Sponge Wipe
Vacuum Sock
Sponge Wipe
Sponge Wipe
Swab
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Sponge Wipe
Vacuum Sock
Sponge Wipe
Swab
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Area
(sqft)
4.000
0.694
0.694
4.000
0.694
0.694
0.028
0.694
0.694
0.694
0.028
0.694
4.000
0.694
0.028
4.000
4.000
4.000
0.694
Operator
Terrill
Terrill
Terrill
Anaya
Anaya
Anaya
Anaya
Inman
Stephanie
Stephanie
Stephanie
Stephanie
Stephanie
Stephanie
Stephanie
Stephanie
Stephanie
Stephanie
Stephanie
Acquisition
Date
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
Acquisition
Time
11:09:00 AM
11:30:00 AM
11:12:00 AM
12:00:00 PM
11:54:00 AM
11:38:00 AM
11:44:00 AM
10:54:00 AM
12:07:00 PM
11:57:00 AM
12:13:00 PM
12:19:00 PM
11:37:00 AM
11:22:00 AM
11:32:00 AM
11:13:00 AM
11:03:00 AM
10:43:00 AM
10:55:00 AM
Object
Floor
Wall
Shelves
Chair
Shelves
Wall
Computer
Return Vent
Countertop
Wall
Supply Vent
Countertop
Chair
Desk
Computer
Floor
Ceiling
Floor
Countertop
Texture
Carpet
Textured
Metal
Porous
Smooth
Smooth
Smooth
Smooth
Textured
Textured
Metal
Smooth
Porous
Smooth
Smooth
Carpet
Porous
Carpet
Smooth
Orientation
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Vertical
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Blank
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
Sample was on the
front of the top of
the filing cabinet.
Sample was put in
the wrong bag that
bag was placed in
the correct one.
sample was taken
on bottom right
corner of the
computer monitor
horizontal vent
10x10 surface area
left side as viewed
by sampler
spongestick on
wood table top.
vertical
spongestick on
wall
swab sample from
diffuser on ceiling
spongestick on
tabletop. left uppei
corner when facing
table.
vacuum sample
from seat of desk
chair, no template
used due to size ol
area.
spongestick on
table.
swab sample from
computer monitor
upper left corner.
vacuum floor near
stove.
vac ceiling tile
sample of
threshold carpet.
spongestick on
Countertop.
Lab ID
7
2
2
2
2
2
4
2
5
5
4
5
5
5
4
5
5
5
5
Detected
No
No
Yes
Yes
No
No
No
No
No
No
No
Yes
Yes
Yes
No
Yes
No
Yes
Yes
Spread Plate Results
(CFU/sq ft)
ND
ND
268.8
4.2
ND
ND
ND
ND
ND
ND
ND
83.5
12.5
66.2
ND
ND
ND
ND
208.3
Filter Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
NA
ND
ND
ND
ND
7.8
ND
18.1
ND
2.9
ND
17.3
292.3
G-3
-------
Barcode
4174
2956
3212
1694
4213
4186
4185
4128
4177
3206
3234
1686
1522
4141
4201
4203
2320
3214
1577
1587
4170
4167
3496
3209
3109
2569
2073
Round
MFP
MFP
MFP
MFP
MFP
MFP
MFP
MFP
MFP
MFP
MFP
MFP
MFP
MFP
MFP
MFP
MFP
MFP
MFP
MFP
MFP
MFP
MFP
MFP
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
Floor
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 1
Floor 1
Floor 1
Room
Room 210
Room 210
Room 210
Room 210
Room 211
Room 211
Room 211
Room 211
Room 211
Room 211
Room 211
Room 211
Room 211
Room 212
Room 212
Room 212
Room 212
Room 212
Room 212
Room 212
Room 213
Room 213
Room 213
Room 213
X
10.637
12.150
12.102
9.799
10.302
11.611
11.599
10.850
10.850
10.326
9.600
10.540
9.600
7.279
6.879
7.179
6.890
7.179
6.779
6.879
8.118
7.998
6.735
7.710
0.247
1.339
0.637
y
20.379
19.962
16.945
18.845
10.940
12.224
9.440
8.276
10.630
11.701
6.940
11.463
9.459
18.455
14.555
16.120
19.871
15.555
18.955
15.355
8.740
11.696
10.416
6.373
3.131
3.131
3.131
z
3.000
3.000
3.000
3.000
3.000
3.000
4.000
3.396
3.000
3.000
4.500
3.000
3.494
3.000
3.000
3.000
3.000
3.000
3.000
3.000
3.489
3.000
4.768
3.296
0.000
0.000
0.000
Method
Vacuum Sock
Sponge Wipe
Sponge Wipe
Swab
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Swab
Swab
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Swab
Swab
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqft)
4.000
0.694
0.694
0.028
4.000
4.000
4.000
4.000
4.000
0.694
0.694
0.028
0.028
4.000
4.000
4.000
0.694
0.694
0.028
0.028
4.000
4.000
0.694
0.694
0.694
0.694
0.694
Operator
Inman
Inman
Inman
Inman
Cavada
Cavada
Cavada
Cavada
Cavada
Cavada
Cavada
Cavada
Cavada
Inman
Inman
Inman
Inman
Inman
Inman
Inman
Cavada
Cavada
Cavada
Cavada
Acquisition
Date
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/15/2011
4/17/2011
4/17/2011
4/17/2011
Acquisition
Time
11:46:00 AM
11:49:00 AM
11:53:00 AM
11:42:00 AM
11:37:00 AM
11:27:00 AM
12:06:00 PM
12:12:00 PM
11:49:00 AM
11:39:00 AM
12:25:00 PM
11:44:00 AM
12:20:00 PM
11:28:00 AM
11:24:00 AM
11:11:00 AM
11:36:00 AM
11:16:00 AM
11:31:00 AM
11:19:00 AM
11:02:00 AM
10:43:00 AM
11:21:00 AM
11:10:00 AM
3:26:00 PM
4:36:00 PM
4:34:00 PM
Object
Chair
Countertop
Wall
Computer
Floor
Floor
Chair
Chair
Floor
Floor
Shelves
Floor
Shelves
Chair
Floor
Ceiling
Countertop
Floor
Computer
Floor
Countertop
Floor
Shelves
Wall
Prep table
Floor
Countertop
Texture
Cloth
Metal
Textured
Glass
Carpet
Carpet
Cloth
Cloth
Carpet
Carpet
Smooth
Carpet
Smooth
Carpet
Smooth
Porous
Metal
Smooth
Glass
Smooth
Cloth
Carpet
Smooth
Smooth
Smooth
Smooth
Smooth
Orientation
Vertical
Vertical
Vertical
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Vertical
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Downward
Horizontal
Upward
Vertical
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Blank
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
TRUE
FALSE
FALSE
TRUE
FALSE
FALSE
TRUE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
BROOM Notes
chair far back wall
vacuum of seat
cushion
upper left corner o
top of 4 drawer
metel cabinet
lOinx lOin sample
above electrical
outlet on right wal
upper left corner o
computer monitor
couch
vacuum sample of
chair 2ft x 2ft area
blank vacuum
sample taken left
corner 2ft x 2ft
area
upper half closest
to wall
blank air
upper left corner
screen
blank wipe
on bed
on wall locker
field blank air
trailer
counter next to slit
to agar
Lab ID
2
5
5
4
7
5
5
5
5
5
5
4
4
2
2
2
5
2
4
4
5
5
5
5
4
4
4
Detected
Yes
Yes
Yes
No
No
Yes
Yes
Yes
Yes
Yes
Yes
No
No
Yes
No
No
No
No
No
No
Yes
Yes
Yes
Yes
No
No
Yes
Spread Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
25.0
12.5
12.5
ND
74.9
ND
ND
66.7
ND
ND
ND
ND
ND
ND
ND
ND
306.0
ND
ND
ND
1747.1
Filter Plate Results
(CFU/sq ft)
0.6
TNTC
2.7
ND
ND
21.9
14.4
0.6
ND
2.9
ND
ND
ND
31.6
ND
ND
ND
ND
ND
ND
5.2
27.9
35.0
6.9
NA
NA
NA
G-4
-------
Barcode
2458
1738
3866
2955
2979
2977
2953
3865
2976
2947
2940
2952
3800
3888
4099
4026
3794
3864
3534
2185
2951
2975
2933
2958
2954
2973
2944
Round
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
Floor
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Room
Bathroom M
Bathroom M
Bathroom M
Bathroom M
Bathroom M
Bathroom W
Bathroom W
Bathroom W
Bathroom W
Bathroom W
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
X
1.027
-0.117
26.645
26.184
26.645
25.839
24.572
25.679
24.209
27.109
25.189
27.068
22.820
26.210
26.212
24.527
16.863
15.612
9.982
24.897
26.580
22.820
27.436
24.985
23.637
27.315
22.841
y
3.053
3.105
19.709
17.943
20.631
19.479
20.362
16.287
15.298
16.455
16.782
14.535
15.384
12.439
9.027
8.380
12.978
12.954
13.267
8.565
6.531
15.829
11.908
11.418
13.088
9.635
9.455
z
0.000
0.000
2.469
0.000
0.000
0.000
0.000
2.478
0.000
0.000
0.000
0.498
2.478
2.392
2.365
0.000
0.000
2.374
2.397
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
Method
Sponge Wipe
Swab
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqft)
0.694
0.028
4.000
0.694
0.694
0.694
0.694
4.000
0.694
0.694
0.694
0.694
4.000
4.000
4.000
4.000
4.000
4.000
4.000
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
Operator
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Acquisition
Date
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
Acquisition
Time
4:35:00 PM
3:25:00 PM
3:17:00 PM
3:15:00 PM
3:12:00 PM
3:11:00 PM
3:08:00 PM
3:02:00 PM
2:43:00 PM
2:58:00 PM
2:55:00 PM
2:57:00 PM
2:48:00 PM
2:29:00 PM
2:12:00 PM
1:51:00 PM
12:35:00 PM
12:19:00 PM
11:45:00 AM
1:39:00 PM
1:55:00 PM
2:35:00 PM
2:25:00 PM
2:23:00 PM
2:18:00 PM
2:02:00 PM
1:33:00 PM
Object
Countertop
Respirator
Ceiling
Floor
Floor
Floor
Floor
Ceiling
Floor
Floor
Wall
Wall
Ceiling
Ceiling
Ceiling
Floor
Floor
Ceiling
Ceiling
Floor
Floor
Floor
Floor
Floor
Floor
Floor
Floor
Texture
Smooth
Smooth
Carpet
Smooth
Smooth
Smooth
Smooth
Carpet
Smooth
Smooth
Smooth
Smooth
Carpet
Smooth
Smooth
Smooth
Smooth
Carpet
Carpet
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Orientation
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Horizontal
Upward
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Blank
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
TRUE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
slit to agars air
trailer
under where the
sink was
in stall
under the soap
dispensers
ceiling tile was
marked
ceiling tile that is
marked
blank
blank
Sample was taken
on left half of the
ceiling tile.
Sample was taken
on the left half of
the ceiling tile.
blank
done on the floor
in front of the left
door
to the left when
going out the dooi
taken in front of
tray
left side of the
floor in front of thi
door
Lab ID
4
4
6
6
1
6
1
6
1
1
1
1
6
1
1
6
6
6
6
1
6
6
6
1
1
1
6
Detected
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Spread Plate Results
(CFU/sq ft)
357490.5
ND
52919.7
203973.1
618024.7
374481.9
496061.3
48752.8
542571.6
536235.8
678454.4
221.8
112923.1
99822.4
55036.5
ND
ND
20334.5
57086.6
2051.9
613608.9
276469.6
517420.5
492029.5
568394.6
469422.3
842368.3
Filter Plate Results
(CFU/sq ft)
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
G-5
-------
Barcode
2946
2091
2690
2667
2726
2412
1982
2948
2532
2689
2791
2790
Round
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
Floor
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Room
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
X
26.294
18.677
9.107
18.302
17.426
17.239
15.008
13.548
12.985
6.980
3.886
5.378
y
10.252
13.924
14.080
13.666
14.229
13.354
15.035
16.520
12.892
13.955
13.392
13.403
z
0.395
2.081
0.000
0.000
0.000
0.000
0.000
0.000
0.000
1.967
0.000
0.000
Method
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqft)
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
Operator
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Acquisition
Date
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
Acquisition
Time
2:07:00 PM
12:52:00 PM
11:52:00 AM
12:43:00 PM
12:40:00 PM
12:32:00 PM
12:14:00 PM
12:07:00 PM
11:57:00 AM
11:34:00 AM
11:26:00 AM
11:19:00 AM
Object
Countertop
Ceiling
Floor
Floor
Wall
Floor
Floor
Floor
Floor
Ceiling
Wall
Floor
Texture
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Orientation
Vertical
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Vertical
Horizontal
Upward
Blank
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
sample was taken
on the wooden sidi
of the counntertop
sample was taken
on hvac
Sample was taken
just before the
third door on the
left.
sample was taken
1 foot off the
ground
blank
Sample was taken
up against the left
hand wall.
Sample was taken
on the right side o]
the hallway in
between the 3rd
and 4th door.
Sample was done
on leftside of the
hvac.
Sample was taken
on the left side of
the door as facing
it.
Pre was done on
the left side of the
hallway.
Lab ID
6
6
6
1
1
6
6
6
6
6
6
6
Detected
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Spread Plate Results
(CFU/sq ft)
6086.2
ND
330707.5
460302.7
2829.5
49.0
50758.1
114302.9
450991.0
23.5
1689.5
338675.2
Filter Plate Results
(CFU/sq ft)
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
G-6
-------
Barcode
2797
1583
1689
4142
2960
2945
4238
2702
2949
2950
2727
3701
4175
4243
3857
2832
2090
2893
2926
2746
Round
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
Floor
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Room
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Mechanical Room
Mechanical Room
Mechanical Room
Room 101
Room 101
Room 101
Room 101
Room 101
Room 101A
Room 101A
Room 101A
Room 101A
Room 101A
Room 101A
Room 101A
Room 101A
Room 101A
X
14.048
24.066
16.175
17.623
17.162
19.274
19.955
16.601
21.758
18.403
16.100
19.432
19.523
16.574
21.356
19.193
18.082
21.497
19.214
20.177
y
13.955
8.657
13.330
19.364
18.365
20.285
10.760
11.811
11.211
10.159
11.060
8.527
6.914
9.504
8.756
8.331
6.215
6.425
6.551
6.509
z
0.000
0.000
0.000
1.298
0.000
0.000
1.996
0.000
0.000
0.000
0.397
0.000
0.500
0.000
0.000
0.000
1.000
1.000
1.000
1.000
Method
Sponge Wipe
Swab
Swab
Vacuum Sock
Sponge Wipe
Sponge Wipe
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqft)
0.694
0.028
0.028
4.000
0.694
0.694
4.000
0.694
0.694
0.694
0.694
4.000
4.000
4.000
4.000
0.694
0.694
0.694
0.694
0.694
Operator
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Oudejans
Oudejans
Oudejans
Oudejans
Oudejans
Oudejans
Oudejans
Oudejans
Oudejans
Acquisition
Date
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
Acquisition
Time
12:02:00 PM
1:42:00 PM
12:28:00 PM
3:28:00 PM
3:24:00 PM
3:23:00 PM
1:15:00 PM
1:00:00 PM
1:11:00 PM
1:08:00 PM
1:03:00 PM
12:51:00 PM
12:43:00 PM
12:20:00 PM
2:01:OOPM
12:53:00 PM
12:35:00 PM
1:28:00 PM
1:01:OOPM
1:18:00 PM
Object
Floor
Floor
Floor
Supply Vent
Floor
Floor
Ceiling
Floor
Floor
Floor
Wall
Floor
Chair
Floor
Floor
Floor
File cabinet
Desk
Desk
Desk
Texture
Smooth
Smooth
Smooth
Porous
Smooth
Smooth
Carpet
Smooth
Smooth
Smooth
Smooth
Carpet
Textured
Carpet
Carpet
Carpet
Metal
Plastic
Plastic
Plastic
Orientation
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Inclined
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Blank
FALSE
TRUE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
sample was taken
at the hallways left
corner where it
opens up and
becomes wilder.
blank
sample was a
blank
there were 4 filters
we used the left
side filters
ceiling tile sample
it was a half tile so
for post they will
have to take the
tile next to it. the
used tile was
marked on the
bottom
taken in doorway
ofroom 101
vac blank for
chair moved out
slighty from under
desk
left side of
doorway sampled
vacuum carpet; in
between stand,
cabinet , and black
box
blank sponge
sample in back of
top surface;cabinet
has now been
sampled across
near whole surface
[this and previous
sample]
sponge;front right
on desk;
front right;wet
surface
left front corner of
desk top;
Lab ID
1
4
4
1
1
1
6
6
1
6
6
4
4
4
4
1
1
4
4
4
Detected
Yes
No
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Spread Plate Results
(CFU/sq ft)
364306.3
ND
ND
TNTC
553371.1
504365.0
2412.6
7727.7
441487.4
426799.9
360.0
ND
9042.2
11375.6
5042.0
ND
461742.6
677254.5
334499.4
948444.3
Filter Plate Results
(CFU/sq ft)
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
TNTC
TNTC
TNTC
G-7
-------
Barcode
2793
2054
2353
2923
2877
2899
4165
3859
3862
4192
2180
2085
2058
2701
2081
2179
2917
2457
2745
2071
Round
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
Floor
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Room
Room 101A
Room 101A
Room 101A
Room 101A
Room 101A
Room 101A
Room 102
Room 102
Room 102
Room 102
Room 102
Room 102
Room 102
Room 102
Room 102
Room 102
Room 102
Room 102
Room 102
Room 102
X
18.215
18.237
20.087
21.539
21.581
21.874
19.943
19.284
18.279
18.793
19.318
18.234
16.905
17.754
18.256
18.703
19.351
19.887
20.222
18.145
y
9.523
8.930
9.143
7.870
7.263
6.278
16.553
16.050
16.687
15.424
14.654
14.609
15.670
17.290
16.999
16.899
16.117
17.223
16.307
15.089
z
1.300
1.300
1.000
1.000
1.000
1.000
0.000
0.000
0.000
0.000
1.300
1.300
1.000
1.000
0.000
1.000
0.000
1.000
1.000
1.000
Method
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqft)
0.694
0.694
0.694
0.694
0.694
0.694
4.000
4.000
4.000
4.000
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
Operator
Oudejans
Oudejans
Oudejans
Oudejans
Oudejans
Oudejans
Oudejans
Oudejans
Oudejans
Oudejans
Oudejans
Oudejans
Oudejans
Oudejans
Oudejans
Oudejans
Oudejans
Oudejans
Oudejans
Oudejans
Acquisition
Date
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
Acquisition
Time
2:31:00 PM
2:23:00 PM
2:06:00 PM
1:48:00 PM
1:43:00 PM
1:34:00 PM
2:51:00 PM
3:36:00 PM
3:30:00 PM
3:24:00 PM
4:45:00 PM
4:39:00 PM
4:26:00 PM
4:16:00 PM
3:54:00 PM
3:44:00 PM
3:39:00 PM
3:03:00 PM
2:44:00 PM
4:32:00 PM
Object
Workbench
Workbench
File cabinet
File cabinet
Desk
Desk
Floor
Floor
Floor
Floor
Workbench
Workbench
Cabinet
Cabinet
Cabinet
Desk
Floor
Desk
File cabinet
Desk
Texture
Plastic
Plastic
Metal
Metal
Plastic
Plastic
Carpet
Carpet
Carpet
Carpet
Plastic
Plastic
Metal
Metal
Metal
Plastic
Carpet
Plastic
Metal
Plastic
Orientation
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Blank
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
BROOM Notes
top surface center
uv aps against
back side;
vertical on uv aps
system, difficult
surface for
sponge; sample in
front and center
front of top of
cabinet
front of cabinet tof
surface;
left front of desk
surface;
desk back right
position
vacuum in front o]
stand
vacuum blank
in front of cabinet
and part of
desk;wobblely
template
in front of desk
with uv APS
left back on top of
uv APS;dirty
sample
top right corner in
IhebackofuvAPf
back of top cabinet
cabinet rotated 90
degrees wrt to
drawing and
Broom drawing.
Front of top
cabinet sampled
top front of cabine
front of desk left
side corner
dropped closed
sample bag before
sampling; wiped
clean
front right behind
the location of the
rmc
front / right side;
on desk to right ol
uv APS; weird
black substance on
desk
Lab ID
1
6
4
1
1
1
4
4
4
4
1
1
1
4
4
1
6
1
1
4
Detected
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Spread Plate Results
(CFU/sq ft)
2591902.3
364066.3
453438.9
385905.5
403184.8
730052.5
10125.6
ND
9333.9
14000.8
253670.4
4091845.8
679654.4
476910.0
232211.7
509740.8
5898.0
472302.2
393105.2
430447.8
Filter Plate Results
(CFU/sq ft)
NA
NA
TNTC
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
G-8
-------
Barcode
2352
4236
4162
2413
2531
2691
2182
2798
2553
2056
2107
1510
1690
4230
4242
2391
2088
2648
2390
2087
2086
Round
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
Floor
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Room
Room 102
Room 103
Room 103
Room 103
Room 103
Room 103
Room 103
Room 103
Room 103
Room 103
Room 103
Room 103
Room 103
Room 104
Room 104
Room 104
Room 104
Room 104
Room 104
Room 104
Room 104
X
19.128
14.842
13.699
15.586
14.842
13.191
13.736
13.682
13.258
15.897
14.531
14.648
14.842
16.196
14.020
15.611
15.634
14.277
15.294
14.699
14.067
y
16.620
11.307
10.690
12.194
11.307
7.715
8.429
6.602
6.219
6.794
6.052
6.682
11.307
19.514
18.368
20.661
20.052
20.006
18.244
19.444
18.321
z
1.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
2.274
1.193
1.298
0.899
0.894
0.798
0.000
1.400
Method
Sponge Wipe
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Swab
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqft)
0.694
4.000
4.000
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.028
0.028
4.000
4.000
0.694
0.694
0.694
0.694
0.694
0.694
Operator
Oudejans
Pasquerella
Pasquerella
Pasquerella
Pasquerella
Pasquerella
Pasquerella
Pasquerella
Pasquerella
Pasquerella
Pasquerella
Pasquerella
Pasquerella
Fetzer
Fetzer
Fetzer
Fetzer
Fetzer
Fetzer
Fetzer
Fetzer
Acquisition
Date
4/17/2011
4/18/2011
4/18/2011
4/18/2011
4/18/2011
4/18/2011
4/18/2011
4/18/2011
4/18/2011
4/18/2011
4/18/2011
4/18/2011
4/18/2011
4/18/2011
4/18/2011
4/18/2011
4/18/2011
4/18/2011
4/18/2011
4/18/2011
4/18/2011
Acquisition
Time
3:08:00 PM
11:49:00 AM
11:45:00 AM
11:03:00 AM
11:51:00 AM
11:35:00 AM
11:31:00 AM
11:29:00 AM
11:23:00 AM
11:16:00 AM
11:21:00 AM
11:38:00 AM
11:54:00 AM
11:45:00 AM
11:54:00 AM
11:35:00 AM
11:24:00 AM
11:20:00 AM
11:08:00 AM
11:05:00 AM
12:01:00 PM
Object
Desk
Table
Ceiling
Floor
Table
Mail slot
Table
Table
Floor
Floor
Wall
Supply Vent
Table
Ceiling
Ceiling
Wall
Table
Table
Table
Floor
Ceiling
Texture
Metal
Smooth
Textured
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Textured
Metal
Smooth
Porous
Porous
Smooth
Smooth
Smooth
Smooth
Smooth
Porous
Orientation
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Horizontal
Downward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Blank
FALSE
TRUE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
BROOM Notes
sample taken on
inside of drawer o]
desk;right side
location
tile was placed on
table on the left
side of room for
easier access to
sample, left side ol
tile was sampled.
blank sponge
on top of mail
slots, mid to right
side.
in front of mail
slots, right bottom
corner.
on table in front o]
mail slots, left
corner.
closer to corner of
room than previous
spongestick
sample.
previous
spongestick samph
location was a
horizontal sample.
may have been
marked vertical.
corner of bench
map wrong
sample taken on
top of bench map
wrong
Lab ID
1
4
1
2
5
3
3
3
5
2
5
1
7
4
4
5
2
2
5
5
2
Detected
Yes
No
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Spread Plate Results
(CFU/sq ft)
218391.8
ND
59253.4
378993.7
ND
491501.5
501101.1
690406.0
417584.2
479501.9
4079.8
738201.0
ND
16500.9
ND
1176.0
135700.5
203896.3
460302.7
403184.8
ND
Filter Plate Results
(CFU/sq ft)
NA
ND
NA
TNTC
ND
NA
NA
NA
TNTC
TNTC
ND
ND
ND
TNTC
0.6
501.7
TNTC
TNTC
TNTC
TNTC
ND
G-9
-------
Barcode
1651
1518
4250
4231
3909
4252
3533
2530
2444
2387
2057
1683
1594
4235
3858
3851
3852
4155
Round
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
Floor
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Room
Room 104
Room 104
Room 105
Room 105
Room 105
Room 105
Room 105
Room 105
Room 105
Room 105
Room 105
Room 105
Room 105
Room 106
Room 106
Room 106
Room 106
Room 106
X
14.067
15.094
11.747
10.513
10.864
10.446
10.895
12.519
12.038
10.141
9.990
11.294
10.132
10.995
10.902
11.229
12.750
12.312
y
18.321
19.344
10.990
12.137
8.923
6.405
9.673
11.350
6.756
9.575
11.004
6.527
8.634
18.779
19.832
20.581
19.856
19.806
z
1.397
2.599
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
2.284
0.583
0.397
1.491
0.000
Method
Swab
Swab
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Swab
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Area
(sqft)
0.028
0.028
4.000
4.000
4.000
4.000
4.000
0.694
0.694
0.694
0.694
0.028
0.028
4.000
4.000
4.000
4.000
4.000
Operator
Fetzer
Fetzer
Pasquerella
Pasquerella
Pasquerella
Pasquerella
Pasquerella
Pasquerella
Pasquerella
Pasquerella
Pasquerella
Pasquerella
Pasquerella
Fetzer
Fetzer
Fetzer
Fetzer
Fetzer
Acquisition
Date
4/18/2011
4/18/2011
4/18/2011
4/18/2011
4/18/2011
4/18/2011
4/18/2011
4/18/2011
4/18/2011
4/18/2011
4/18/2011
4/18/2011
4/18/2011
4/18/2011
4/18/2011
4/18/2011
4/18/2011
4/18/2011
Acquisition
Time
11:56:00 AM
11:39:00 AM
9:48:00 AM
9:16:00 AM
10:51:00 AM
10:39:00 AM
10:04:00 AM
9:38:00 AM
10:27:00 AM
10:16:00 AM
9:57:00 AM
10:59:00 AM
10:21:00 AM
10:36:00 AM
10:20:00 AM
10:10:00 AM
10:01:00 AM
9:52:00 AM
Object
Ceiling
Supply Vent
Floor
Floor
Ceiling
Floor
Floor
Sink
Table
Stove
Wall
Return Vent
Supply Vent
Ceiling
Wall
Chair
File cabinet
Floor
Texture
Porous
Metal
Carpet
Carpet
Textured
Carpet
Carpet
Metal
Smooth
Metal
Textured
Metal
Metal
Porous
Textured
Textured
Smooth
Smooth
Orientation
Horizontal
Downward
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Downward
Horizontal
Downward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Blank
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
left side of tile
sampled, placed or
stove for easier
access to sample.
far right corner of
room.
right of center in
front of stove
left side of sink.
far left corner of
table, closest to the
wall and corner of
room.
apprx one foot
from floor
additional sample
location ceiling.
previous swab
sample was taken
at an incorrect
location, this
sample location
corresponds to
map.
diffuser on floor to
the left of the
stove, not in
middle of room as
it is placed on
map.
previous sample at
wrong location.
this sample
relocated
vacuum sample 2fl
x 2ft area on
partition
books located on
top of metal
cabinet
Lab ID
7
7
6
2
4
1
1
2
2
2
2
1
7
1
1
1
1
6
Detected
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Spread Plate Results
(CFU/sq ft)
ND
1944529.5
10050.6
14750.8
15250.9
7012.9
5573.7
373953.9
239415.0
462990.6
698.9
ND
46212.6
11310.6
4900.3
45302.6
9548.9
12042.4
Filter Plate Results
(CFU/sq ft)
82.8
TNTC
NA
TNTC
TNTC
NA
NA
TNTC
TNTC
TNTC
479.2
ND
ND
NA
ND
NA
NA
NA
G-10
-------
Barcode
3855
2059
2055
2460
2698
2459
1699
1620
3699
4156
3854
4178
4003
2184
2079
2080
2082
1523
1574
1570
4182
4245
4176
4172
Round
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
Floor
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Room
Room 106
Room 106
Room 106
Room 106
Room 106
Room 106
Room 106
Room 106
Room 107
Room 107
Room 107
Room 107
Room 107
Room 107
Room 107
Room 107
Room 107
Room 107
Room 107
Room 107
Room 108
Room 108
Room 108
Room 108
X
10.878
10.481
10.504
10.574
10.106
11.912
11.042
10.411
8.431
9.386
9.255
8.775
7.817
8.489
7.713
7.113
8.326
7.132
8.041
8.367
7.806
9.599
9.651
8.213
y
18.429
19.926
17.914
15.107
16.791
15.920
19.505
18.452
8.314
10.140
12.048
7.370
11.770
9.305
12.331
6.614
7.125
9.991
7.431
6.616
19.617
19.957
17.807
20.517
z
0.394
0.692
0.783
0.000
0.590
0.597
2.197
0.991
0.293
0.294
0.000
0.000
2.281
0.296
0.691
1.284
0.000
0.575
0.000
2.160
0.595
1.483
2.270
0.396
Method
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Swab
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Swab
Swab
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Area
(sqft)
4.000
0.694
0.694
0.694
0.694
0.694
0.028
0.028
4.000
4.000
4.000
4.000
4.000
0.694
0.694
0.694
0.694
0.028
0.028
0.028
4.000
4.000
4.000
4.000
Operator
Fetzer
Fetzer
Fetzer
Fetzer
Fetzer
Fetzer
Fetzer
Fetzer
Perry
Perry
Perry
Perry
Perry
Perry
Perry
Perry
Perry
Perry
Perry
Perry
Perry
Perry
Perry
Perry
Acquisition
Date
4/18/2011
4/18/2011
4/18/2011
4/18/2011
4/18/2011
4/18/2011
4/18/2011
4/18/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
Acquisition
Time
9:38:00 AM
10:55:00 AM
9:27:00 AM
9:03:00 AM
9:21:00 AM
9:12:00 AM
10:47:00 AM
9:33:00 AM
4:45:00 PM
4:38:00 PM
4:00:00 PM
4:59:00 PM
5:14:00 PM
4:30:00 PM
4:06:00 PM
4:54:00 PM
5:02:00 PM
4:09:00 PM
4:57:00 PM
5:05:00 PM
6:21:OOPM
6:11:00 PM
6:34:00 PM
6:15:00 PM
Object
Chair
File cabinet
Desk
Floor
Wall
Table
Supply Vent
Monitor
Chair
Couch
Floor
Floor
Ceiling
Table
Wall
Shelves
Floor
Monitor
Floor
Supply Vent
Wall
File cabinet
Ceiling
Chair
Texture
Textured
Metal
Smooth
Smooth
Smooth
Smooth
Metal
Smooth
Leather
Cloth
Carpet
Carpet
Textured
Smooth
Paint
Smooth
Carpet
Glass
Carpet
Metal
Cloth
Metal
Textured
Cloth
Orientation
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Downward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Downward
Vertical
Horizontal
Upward
Horizontal
Downward
Horizontal
Upward
Blank
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
disregard last
sample location in
rm 108 picture ot
last is correct
upper left corner o
monitor
coffee table
TV SCREEN
vacuum sample on
cubicle partition
vacuumed around
and on top of the
books on the file
cabinet. The book
on the far left fell
over.
Lab ID
6
5
5
5
5
2
7
7
4
1
1
1
4
3
3
7
3
7
7
7
4
4
2
4
Detected
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
No
Yes
Yes
Yes
Yes
Yes
Spread Plate Results
(CFU/sq ft)
17401.0
486509.7
365314.2
422384.1
432.0
737252.2
2820768.1
167445.6
887.6
16684.3
3729.4
ND
2179.3
149754.4
22.1
306804.4
ND
64817.7
ND
106228.9
654.2
179.2
1020.9
7958.8
Filter Plate Results
(CFU/sq ft)
NA
TNTC
TNTC
TNTC
455.2
TNTC
TNTC
TNTC
TNTC
ND
ND
0.1
TNTC
NA
105.8
TNTC
ND
ND
ND
TNTC
TNTC
92.6
TNTC
TNTC
G-ll
-------
Barcode
4169
4171
2472
2415
2696
2700
2699
2195
1684
1586
1578
4253
3535
4248
4233
3850
3856
2416
2061
2388
2194
2812
Round
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
Floor
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Room
Room 108
Room 108
Room 108
Room 108
Room 108
Room 108
Room 108
Room 108
Room 108
Room 108
Room 108
Room 109
Room 109
Room 109
Room 109
Room 109
Room 109
Room 109
Room 109
Room 109
Room 109
Room 109
X
7.908
8.621
9.251
9.551
8.906
9.451
7.459
7.513
8.987
7.276
8.437
5.373
6.267
4.119
5.621
4.773
5.973
5.153
5.421
4.195
4.195
3.915
y
18.449
15.414
17.517
20.317
15.801
14.530
15.006
17.917
19.672
18.673
15.882
8.723
6.567
7.914
9.614
11.131
11.914
6.053
9.497
10.951
12.304
6.485
z
0.495
0.000
0.000
1.273
0.000
0.786
0.000
0.797
2.141
1.086
0.000
0.000
2.100
0.000
0.000
0.000
0.000
0.000
1.197
0.000
0.000
1.300
Method
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Swab
Swab
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqft)
4.000
4.000
0.694
0.694
0.694
0.694
0.694
0.694
0.028
0.028
0.028
4.000
4.000
4.000
4.000
4.000
4.000
0.694
0.694
0.694
0.694
0.694
Operator
Perry
Perry
Perry
Perry
Perry
Perry
Perry
Perry
Perry
Perry
Perry
Inman
Inman
Inman
Inman
Inman
Inman
Inman
Inman
Inman
Inman
Inman
Acquisition
Date
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
Acquisition
Time
5:46:00 PM
5:36:00 PM
5:40:00 PM
6:07:00 PM
5:32:00 PM
5:26:00 PM
5:21:00 PM
5:51:00 PM
6:26:00 PM
5:55:00 PM
5:34:00 PM
5:30:00 PM
5:47:00 PM
5:32:00 PM
5:06:00 PM
4:47:00 PM
4:42:00 PM
5:42:00 PM
4:59:00 PM
4:56:00 PM
4:53:00 PM
5:37:00 PM
Object
Chair
Floor
Floor
File cabinet
Floor
Wall
Floor
Desk
Supply Vent
Monitor
Floor
Bed
Ceiling
Bed
Floor
Floor
Floor
Wall
Floor
Countertop
Countertop
Wall
Texture
Cloth
Smooth
Smooth
Metal
Smooth
Paint
Smooth
Smooth
Metal
Plastic
Smooth
Cloth
Porous
Cloth
Carpet
Carpet
Carpet
Textured
Carpet
Smooth
Smooth
Textured
Orientation
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Vertical
Horizontal
Upward
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Horizontal
Upward
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Horizontal
Upward
Horizontal
Downward
Horizontal
Downward
Vertical
Blank
FALSE
TRUE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
BROOM Notes
right corner edge
ofbed
2ft x 2ft area
vacuum of ceiling
tile taken from
back half of room
top left corner of
bed 2ft x 2ft
vacuum blank
2ft x 2ft area
vacuum inside to
right against sink
edge along floor
2ft x 2ft area
vacuum just inside
doorway
sample area along
window seal of
back wall
horizontal swab
blank sponge
lOinx lOin spongi
swipe along
Countertop left sidi
of sink
lOin x lOin sponge
swipe along
Countertop of sink,
right side of sink
lOin x lOin sponge
swipe along wall
in averticle
manner map
called for
horizontal
Lab ID
4
1
7
6
7
3
3
7
7
7
7
3
3
3
7
3
7
1
6
6
1
1
Detected
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
No
Yes
Yes
Yes
No
Yes
Yes
Yes
Spread Plate Results
(CFU/sq ft)
7417.1
ND
364306.3
398721.0
ND
3340.7
368050.1
239991.0
810220.6
62417.0
ND
2387.6
6375.4
8708.8
ND
9750.6
15834.2
388785.4
ND
354802.6
485741.7
686.4
Filter Plate Results
(CFU/sq ft)
TNTC
0.5
TNTC
NA
ND
NA
NA
TNTC
TNTC
ND
ND
NA
NA
NA
ND
NA
TNTC
NA
NA
NA
NA
NA
G-12
-------
Barcode
1484
4148
4173
4244
4210
4158
3849
4014
3853
3203
3208
3202
Round
VHP Pre-
Decon
VHP Pre-
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
econ
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
Floor
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Room
Room 109
Room 110
Room 110
Room 110
Room 110
Room 110
Room 110
Room 110
Room 110
Room 110
Room 110
Room 110
x
5.921
4.911
5.073
6.673
4.773
6.575
4.173
5.173
5.873
6.011
4.173
6.575
y
9.914
19.695
20.596
19.896
18.491
20.549
17.151
16.251
15.651
17.196
19.896
20.193
z
0.000
1.296
0.000
0.000
0.000
0.000
0.000
0.000
2.050
0.000
0.000
0.000
Method
Swab
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqft)
0.028
4.000
4.000
4.000
4.000
4.000
4.000
4.000
4.000
0.694
0.694
0.694
Operator
Inman
Inman
Inman
Inman
Inman
Inman
Inman
Inman
Inman
Inman
Inman
Acquisition
Date
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
Acquisition
Time
5:03:00 PM
4:15:00 PM
4:10:00 PM
3:58:00 PM
3:49:00 PM
4:04:00 PM
3:40:00 PM
3:30:00 PM
4:33:00 PM
3:33:00 PM
4:20:00 PM
4:01:OOPM
Object
Floor
Wall
Chair
File cabinet
Chair
File cabinet
Floor
Floor
Ceiling
Floor
File cabinet
File cabinet
Texture
Carpet
Cloth
Cloth
Leather
Cloth
Metal
Smooth
Smooth
Porous
Smooth
Metal
Metal
Orientation
Horizontal
Upward
Vertical
Horizontal
Horizontal
Upward
Horizontal
Downward
Horizontal
Upward
Horizontal
ownwar
Horizontal
Downward
Horizontal
Upward
Horizontal
Downward
Horizontal
Downward
Horizontal
Upward
Blank
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
swab blank
vacuum sample 2fl
x 2ft area of
partition wall
opposite side wall
than entrance dooi
taken apprx 4ft
from floor
vacuum sample 2fl
along back wall
vacuum of books
located on right
side top of metal
cabinet that sits on
wall to right of
door towards back
vacuum of chair
that sits at desk on
left wall apprx
area of 2 ft x 2 ft
vacuum of metal
cabinet to far left
side of top sutface
against 2 walls
vacuum of apprx
2ft x 2ft area along
left wall next to
desk and garbage
can
vacuum blank
apprx 3 ft in air
2ft x 2ft area of
ceiling vacuumed,
tile removed apprx
4ft from door way
placed on floor
immediately to
left of entrance
lOin x lOin sponge
swipe offfloor
apprx halfway
inside room
lOin x lOin sponge
swipe of area on
top of file cabinet
that sits on
opposite side of
partition
sponge sample
lOinx lOin on top
surface of metal
cabinet back right
wall
Lab ID
4
3
3
7
3
3
3
7
3
6
4
4
Detected
No
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Spread Plate Results
(CFU/sq ft)
ND
1200.1
23959.7
1616.8
8083.8
733.4
6250.4
ND
1275.1
346354.9
382305.6
347026.9
Filter Plate Results
(CFU/sq ft)
NA
NA
NA
TNTC
NA
NA
NA
ND
NA
NA
TNTC
TNTC
G-13
-------
Barcode
2595
2872
3369
3199
1687
1649
1685
2943
3698
2992
2990
2974
4109
4218
2993
2972
2970
Round
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
Decon
VHP Pre-
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
Floor
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Room
Room 110
Room 110
Room 110
Room 110
Room 110
Room 110
Room 110
Bathroom M
Bathroom M
Bathroom M
Bathroom M
Bathroom W
Bathroom W
Bathroom W
Bathroom W
Bathroom W
x
5.373
5.673
4.459
4.373
4.311
4.973
5.411
13.272
27.919
28.753
29.615
26.807
28.364
27.780
26.613
29.003
27.808
y
15.951
14.596
17.822
20.705
18.251
16.051
20.008
12.457
19.558
20.503
20.086
20.142
17.166
17.528
16.166
17.889
17.166
z
0.000
0.000
0.000
1.698
0.000
0.000
2.080
3.391
5.317
3.566
3.000
3.000
3.996
5.250
3.000
3.000
3.985
Method
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Swab
Swab
Sponge Wipe
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqft)
0.694
0.694
0.694
0.694
0.028
0.028
0.028
0.694
4.000
0.694
0.694
0.694
4.000
4.000
0.694
0.694
0.694
Operator
Inman
Inman
Inman
Inman
Inman
Inman
Terrill
Terrill
Terrill
Terrill
Terrill
Terrill
Terrill
Terrill
Terrill
Terrill
Acquisition
Date
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
Acquisition
Time
3:24:00 PM
3:12:00 PM
3:45:00 PM
4:24:00 PM
3:53:00 PM
3:25:00 PM
4:28:00 PM
11:13:00 AM
5:28:00 PM
5:22:00 PM
5:18:00 PM
5:12:00 PM
5:00:00 PM
5:03:00 PM
4:44:00 PM
4:50:00 PM
4:55:00 PM
Object
Floor
Floor
Desk
Wall
Monitor
Supply Vent
Wall
Ceiling
Wall
Floor
Floor
Floor
Ceiling
Floor
Floor
Floor
Texture
Smooth
Smooth
Smooth
Textured
Glass
Smooth
Metal
Smooth
Porous
Textured
Smooth
Smooth
Porous
Smooth
Smooth
Smooth
Orientation
Horizontal
Downward
Horizontal
Upward
Horizontal
Downward
Vertical
Horizontal
Downward
Horizontal
Downward
Horizontal
Upward
Vertical
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Blank
TRUE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
TRUE
BROOM Notes
sponge blank
lOin x lOin sponge
swab just inside
door and to left on
floor
lOin x lOin sponge
off left corner of
desk that sits along
the left wall
sponge swipe area
lOin x lOin on
back wall apprx
5ftoffflloor
swab upper left
hand corner
computer monitor
that sits in middle
of desk along left
wall
swab blank apprx
swab of area along
vent duct in ceiling
along back half of
room
had to back track
to this sample
because identifier
was missed
ceiling tile was
placed on floor foi
easier access to
sample location
then placed back ir
original location
ceiling tile was
placed on floor foi
easier access to
sample location
then placed back it
original location
Lab ID
1
6
6
1
7
1
4
8
7
4
4
4
6
7
4
4
4
Detected
No
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
No
Spread Plate Results
(CFU/sq ft)
ND
327347.7
231543.3
4031.8
16804.6
ND
774210.8
ND
545.9
52.8
11441.8
10559.6
ND
558.4
11591.6
15095.4
ND
Filter Plate Results
(CFU/sq ft)
NA
NA
NA
NA
12506.2
ND
NA
13.2
TNTC
NA
NA
NA
NA
TNTC
NA
NA
ND
G-14
-------
Barcode
1579
2967
2984
2989
3896
3916
3915
3965
3015
3016
3008
3014
3003
2963
Round
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
Floor
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Room
Copier Room
Copier Room
Copier Room
Hallway
Hallway
Hallway
Hallway
Hallway
Hallway
Hallway
Hallway
Hallway
Hallway
X
28.169
29.463
28.768
27.795
16.908
15.727
8.917
25.287
26.023
25.532
23.718
22.320
16.399
15.836
y
16.805
13.351
13.962
13.212
13.190
12.749
12.836
16.078
17.647
15.097
13.405
13.356
13.144
13.375
z
3.995
3.000
3.295
3.000
4.096
5.233
5.283
5.282
3.290
3.000
4.898
3.000
4.096
3.000
Method
Swab
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqft)
0.028
0.694
0.694
0.694
4.000
4.000
4.000
4.000
0.694
0.694
0.694
0.694
0.694
0.694
Operator
Terrill
Terrill
Terrill
Terrill
Terrill
Terrill
Terrill
Terrill
Terrill
Terrill
Terrill
Terrill
Terrill
Terrill
Acquisition
Date
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
Acquisition
Time
4:54:00 PM
4:18:00 PM
4:17:00 PM
4:07:00 PM
10:26:00 AM
10:13:00 AM
9:48:00 AM
11:48:00 AM
11:37:00 AM
11:30:00 AM
11:24:00 AM
11:19:00 AM
10:21:00 AM
10:01:00 AM
Object
Floor
Wall
Floor
Ceiling
Ceiling
Ceiling
Wall
Wall
Floor
Return Vent
Floor
Ceiling
Floor
Texture
Smooth
Smooth
Smooth
Smooth
Porous
Porous
Porous
Porous
Smooth
Smooth
Metal
Smooth
Porous
Smooth
Orientation
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Blank
TRUE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
BROOM Notes
this is a correction
entry for this
sample, other
sample location
was placed outside
building
ceiling tile taken
down from ceiling
before sampling
for easier access
and then placed
back in original
position after
sampling
sample was taken
from ceiling tile
placed on the floor
for easier access
ceiling tile taken
down and placed
on the floor for
easier access to
sample location
then placed back
in original location
filter was taken
down from vent
before sampling
and replaced after
sampling, sample
was taken from
inside duct work.
Lab ID
4
4
4
4
7
4
1
6
8
3
3
8
3
3
Detected
No
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Spread Plate Results
(CFU/sq ft)
ND
5380.6
240.0
2399.9
ND
370.9
1045.6
355.9
ND
14112.4
1928.6
11271.9
ND
9004.5
Filter Plate Results
(CFU/sq ft)
NA
NA
NA
NA
ND
NA
NA
NA
18.0
NA
NA
TNTC
ND
NA
G-15
-------
Barcode
2964
2985
3021
1603
3863
2965
2983
3891
3064
3030
4163
2966
2986
3860
2994
2962
Round
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
Floor
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Room
Hallway
Hallway
Hallway
Hallway
Janitor Closet
Janitor Closet
Janitor Closet
Mechanical Room
Mechanical Room
Room 201
Room 201
Room 201
Room 201 A
Room 201 A
Room 201 A
X
8.775
8.765
25.394
16.700
29.073
27.016
28.378
19.215
19.019
22.255
27.795
28.990
26.405
27.906
26.294
29.185
y
13.772
13.466
18.662
13.445
15.352
14.879
15.074
19.275
20.084
19.741
11.766
10.849
11.711
7.930
6.957
5.957
z
4.889
3.000
3.000
4.096
5.280
3.000
3.000
3.598
3.000
3.000
5.265
3.000
3.000
5.270
3.000
3.497
Method
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Sponge Wipe
Sponge Wipe
Vacuum Sock
Sponge Wipe
Sponge Wipe
Area
(sqft)
0.694
0.694
0.694
0.028
4.000
0.694
0.694
4.000
0.694
0.694
4.000
0.694
0.694
4.000
0.694
0.694
Operator
Terrill
Terrill
Terrill
Terrill
Terrill
Terrill
Terrill
Terrill
Terrill
Terrill
Terrill
Terrill
Terrill
Terrill
Terrill
Terrill
Acquisition
Date
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
Acquisition
Time
9:37:00 AM
9:26:00 AM
11:54:00 AM
10:23:00 AM
4:42:00 PM
4:25:00 PM
4:32:00 PM
12:20:00 PM
12:14:00 PM
12:11:00 PM
3:36:00 PM
3:31:00 PM
3:25:00 PM
4:01:OOPM
3:47:00 PM
3:50:00 PM
Object
Return Vent
Floor
Floor
Ceiling
Ceiling
Floor
Floor
Floor
Floor
Ceiling
Floor
Floor
Ceiling
Floor
Wall
Texture
Metal
Smooth
Smooth
Porous
Porous
Smooth
Smooth
Smooth
Smooth
Porous
Smooth
Smooth
Porous
Smooth
Smooth
Orientation
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Blank
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
filter was taken
down from vent
before sampling
and replaced after
sampling
sample was taken
directly outside
airlock door
ceiling tile was
placed on floor for
easier access to
sample location
then placed back ir
original location
sample was taken
from filter and
was placed on the
floor for easier
access then placed
back in original
location, sample
location was
smaller than
vacuum templet.
ceiling tile was
placed on floor for
easier access to
sample location
then placed back ir
original location
ceiling tile was
placed on floor for
easier access to
sample location
then placed back ir
original location
Lab ID
3
3
3
4
7
1
1
7
3
3
6
4
4
7
4
4
Detected
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Spread Plate Results
(CFU/sq ft)
4276.6
14279.5
14716.2
ND
287.5
14393.7
15623.4
1064.1
47911.8
60213.7
237.5
13777.4
9309.2
562.5
13168.3
ND
Filter Plate Results
(CFU/sq ft)
NA
NA
NA
NA
173.1
NA
NA
TNTC
NA
NA
NA
TNTC
TNTC
ND
TNTC
NA
G-16
-------
Barcode
2982
2959
3009
3006
4025
3004
3002
3923
2991
2971
3910
3007
3010
3012
3861
3001
2999
2998
Round
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
Floor
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Room
Room 201 A
Room 201 A
Room 202
Room 202
Room 203
Room 203
Room 203
Room 203A
Room 203A
Room 203A
Room 204
Room 204
Room 204
Room 204
Room 205
Room 205
Room 205
Room 205
X
26.322
28.715
21.857
21.487
23.807
21.802
24.034
23.727
24.749
24.934
19.523
20.356
18.528
19.500
19.025
19.870
20.225
20.260
y
9.014
8.267
17.590
14.814
12.000
10.734
10.025
7.377
6.940
8.814
16.360
14.856
16.661
17.471
8.069
6.189
7.450
11.595
z
3.000
3.000
3.000
3.000
5.500
3.000
3.200
5.500
3.000
3.000
5.281
3.000
3.296
3.000
5.500
3.200
3.000
3.000
Method
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Sponge Wipe
Sponge Wipe
Vacuum Sock
Sponge Wipe
Sponge Wipe
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqrt)
0.694
0.694
0.694
0.694
4.000
0.694
0.694
4.000
0.694
0.694
4.000
0.694
0.694
0.694
4.000
0.694
0.694
0.694
Operator
Terrill
Terrill
Terrill
Terrill
Martinez
Martinez
Martinez
Martinez
Martinez
Martinez
Terrill
Terrill
Terrill
Terrill
Martinez
Martinez
Martinez
Martinez
Acquisition
Date
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
Acquisition
Time
3:43:00 PM
3:56:00 PM
11:06:00 AM
11:01:00 AM
3:04:00 PM
2:41:OOPM
2:38:00 PM
2:56:00 PM
2:51:00 PM
2:46:00 PM
10:58:00 AM
10:34:00 AM
10:42:00 AM
10:45:00 AM
2:24:00 PM
2:20:00 PM
2:15:00 PM
2:09:00 PM
Object
Floor
Floor
Floor
Floor
Ceiling
Floor
Wall
Ceiling
Floor
Floor
Ceiling
Ceiling
Wall
Floor
Ceiling
Wall
Floor
Floor
Texture
Smooth
Smooth
Smooth
Smooth
Textured
Textured
Textured
Textured
Textured
Textured
Porous
Smooth
Smooth
Smooth
Textured
Textured
Textured
Textured
Orientation
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Horizontal
Upward
Vertical
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Downward
Vertical
Horizontal
Upward
Horizontal
Upward
Blank
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
sample was taken
from ceiling befon
sampling for easier
access then tile
was replaced after
sampling
previous sample
location was
nearest to the door
entrance but
seemed to jump
with broom to next
room, it was inrm
205
-------
Barcode
3908
3918
3025
3024
3023
3018
3022
3020
3017
1652
1581
4114
4159
2995
2987
2938
2997
2939
2996
2981
2988
1739
1596
3917
Round
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
Floor
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Room
Room 206
Room 206
Room 206
Room 206
Room 206
Room 206
Room 206
Room 206
Room 206
Room 206
Room 206
Room 207
Room 207
Room 207
Room 207
Room 207
Room 207
Room 207
Room 207
Room 207
Room 207
Room 207
Room 207
Room 208
X
17.321
17.621
16.148
17.712
17.664
16.742
17.750
18.018
15.742
17.872
16.942
17.753
16.155
15.665
16.477
17.753
15.596
15.968
17.753
16.053
15.544
16.353
15.568
13.802
y
15.445
16.845
14.996
18.699
15.436
20.154
20.100
19.308
17.054
15.417
20.154
11.359
8.846
6.904
8.876
7.150
7.544
6.950
9.250
12.350
8.029
8.650
10.250
19.854
z
3.000
3.000
3.000
3.493
3.000
3.793
3.786
3.000
3.000
3.000
5.279
5.500
3.780
3.780
3.000
3.200
3.780
3.871
3.780
3.000
3.780
3.780
3.200
3.990
Method
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Swab
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Swab
Vacuum Sock
Area
(sqft)
4.000
4.000
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.028
0.028
4.000
4.000
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.028
0.028
4.000
Operator
Wenning
Wenning
Wenning
Wenning
Wenning
Wenning
Wenning
Wenning
Wenning
Wenning
Wenning
Martinez
Martinez
Martinez
Martinez
Martinez
Martinez
Martinez
Martinez
Martinez
Martinez
Martinez
Martinez
Wenning
Acquisition
Date
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
Acquisition
Time
4:41:OOPM
4:32:00 PM
3:21:00 PM
4:19:00 PM
4:45:00 PM
3:55:00 PM
4:12:00 PM
4:04:00 PM
3:49:00 PM
4:49:00 PM
4:25:00 PM
1:57:00 PM
1:46:00 PM
1:29:00 PM
1:54:00 PM
1:41:OOPM
1:33:00 PM
1:25:00 PM
1:13:00 PM
12:53:00 PM
1:36:00 PM
1:49:00 PM
1:05:00 PM
11:56:00 AM
Object
Ceiling
Ceiling
Floor
Desk
Floor
Table
Workbench
Floor
Floor
Floor
Ceiling
Ceiling
Floor
Table
Floor
Table
Table
Table
Table
Floor
Table
Floor
Supply Vent
Wall
Texture
Smooth
Smooth
Smooth
Smooth
Smooth
Textured
Textured
Textured
Smooth
Smooth
Smooth
Textured
Textured
Smooth
Textured
Textured
Smooth
Smooth
Smooth
Textured
Smooth
Textured
Metal
Textured
Orientation
Horizontal
Downward
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Blank
TRUE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
TRUE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
BROOM Notes
vacuum blank
vacuum sample
from ceiling tile
spongestick on
entrance threshold
floor.
spongestick on
worktable. tools
covering table.
spongestick on
floor, woodchips
present on floor,
sample collected
over area.
spongestick on
floor in front of
outlet
swab from ceiling
diffuser
blank and we had
no wipe sample
kit/supplies so we
made it a sponge
blank
this and last
sample were
blanks
vac sample from
vertical partition in
room, sample
collected from sidi
facing window.
Lab ID
1
2
3
6
7
7
3
7
6
7
7
7
6
7
7
7
7
7
7
7
7
4
4
1
Detected
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
No
Yes
Yes
No
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Spread Plate Results
(CFU/sq ft)
1.8
829.2
16597.8
18239.3
ND
5299.0
5032.6
48.0
15012.9
ND
107429.3
704.0
ND
12277.0
ND
2087.9
13992.7
11047.3
10857.2
16303.1
12302.9
ND
2400.7
36.0
Filter Plate Results
(CFU/sq ft)
ND
ND
NA
NA
ND
TNTC
NA
960.4
NA
ND
TNTC
TNTC
NA
TNTC
ND
ND
TNTC
TNTC
TNTC
ND
TNTC
ND
NA
10.9
G-18
-------
Barcode
3914
3889
4168
4130
4093
3029
3027
3028
1598
1575
3947
3893
3925
Round
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
Floor
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Room
Room 208
Room 208
Room 208
Room 208
Room 208
Room 208
Room 208
Room 208
Room 208
Room 208
Room 209
Room 209
Room 209
X
13.843
14.619
14.695
13.302
14.678
15.322
12.936
13.302
13.826
12.902
14.056
14.056
13.856
y
20.297
20.429
19.554
18.245
17.395
18.304
19.914
15.245
20.065
18.460
10.940
8.240
10.440
z
3.000
3.000
3.000
3.696
3.000
3.694
3.593
3.693
4.636
3.000
3.000
3.000
5.500
Method
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Swab
Vacuum Sock
Vacuum Sock
Vacuum Sock
Area
(sqft)
4.000
4.000
4.000
4.000
4.000
0.694
0.694
0.694
0.028
0.028
4.000
4.000
4.000
Operator
Wenning
Wenning
Wenning
Wenning
Wenning
Wenning
Wenning
Wenning
Wenning
Wenning
Martinez
Martinez
Martinez
Acquisition
Date
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
Acquisition
Time
11:47:00 AM
11:36:00 AM
11:28:00 AM
11:17:00 AM
12:07:00 PM
12:27:00 PM
12:20:00 PM
10:45:00 AM
12:32:00 PM
11:09:00 AM
11:33:00 AM
12:06:00 PM
12:39:00 PM
Object
Chair
Floor
Floor
Chair
Ceiling
Wall
File cabinet
Table
Ceiling
Monitor
Floor
Floor
Ceiling
Texture
Textured
Smooth
Smooth
Textured
Textured
Textured
Smooth
Smooth
Smooth
Smooth
Carpet
Carpet
Porous
Orientation
Horizontal
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Vertical
Vertical
Horizontal
Upward
Horizontal
Downward
Blank
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
DISREGARD
PREVIOUS VAC
SAMPLE
LOCATION. This
is correct location
for vac sample
3914. previously
placed in wrong
room.
vacuum in front o]
file cabinet.
immediately next
sample, no
template used due
to small space.
sample area
approx. 1ft x 2 ft
vacuum from flooi
in front of file
cabinet.
vacuum on seat of
chair.
vacuum sample
taken from ceiling
tile.
spongestick on file
cabinet.
spongestick on
table.
swab sample from
ceiling diffuser.
computer monitor
upper left corner
skipped previous
sample swab on
coil of fridge due
to no wetting agen
Lab ID
1
2
2
2
1
7
7
7
7
7
7
7
6
Detected
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Spread Plate Results
(CFU/sq ft)
1620.1
362.5
275.0
333.4
175.0
33.6
18277.7
20519.2
105628.8
2400.7
104.2
153.8
400.0
Filter Plate Results
(CFU/sq ft)
ND
ND
138.6
ND
ND
42.4
TNTC
TNTC
TNTC
3064.4
94.3
62.9
-12.1
G-19
-------
Barcode
2905
2092
2848
2850
2875
1643
3931
3936
4112
4116
3906
Round
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
Floor
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Room
Room 209
Room 209
Room 209
Room 209
Room 209
Room 209
Room 210
Room 210
Room 210
Room 210
Room 210
X
14.928
14.370
12.764
13.776
12.412
14.456
10.402
10.299
10.709
11.771
11.782
y
9.983
12.069
11.291
7.023
6.652
10.540
19.604
15.537
20.298
20.154
19.516
z
3.890
3.894
3.884
3.780
3.300
3.200
4.195
5.292
3.000
3.000
3.000
Method
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Area
(sqft)
0.694
0.694
0.694
0.694
0.694
0.028
4.000
4.000
4.000
4.000
4.000
Operator
Martinez
Martinez
Martinez
Martinez
Martinez
Martinez
Inman
Inman
Inman
Inman
Inman
Acquisition
Date
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
Acquisition
Time
11:49:00 AM
11:20:00 AM
11:44:00 AM
12:30:00 PM
12:23:00 PM
12:19:00 PM
12:25:00 PM
12:38:00 PM
12:19:00 PM
12:10:00 PM
12:05:00 PM
Object
Stove
Countertop
Countertop
Table
Wall
Supply Vent
Wall
Ceiling
Chair
Floor
Floor
Texture
Metal
Smooth
Smooth
Smooth
Textured
Metal
Cloth
Porous
Cloth
Smooth
Smooth
Orientation
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Blank
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
taken under the lip
2ft x 2ft vacuum
sample of cloth
partition sample
taken apprx 4 foot
off the ground
vacuum sample of
ceiling tile
removed and
placed on floor
immediately to
right of entrance
vacuum of chair
seat that is
againstfar back
wall surface area
of sample apprx
2ft x 2ft
vacuum of floor
inside room
towards back
against wall and
against metal
cabinet
vacuum of floor
inside towards
back wall against
metal cadinet
Lab ID
7
7
7
7
7
4
7
1
6
1
1
Detected
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Spread Plate Results
(CFU/sq ft)
5260.6
6177.4
13401.6
9775.8
290.4
1200.3
33.3
277.1
210.0
254.3
164.7
Filter Plate Results
(CFU/sq ft)
TNTC
ND
TNTC
TNTC
183.6
1656.5
29.9
NA
NA
NA
NA
G-20
-------
Barcode
4094
2344
2343
2342
2846
2851
2904
1642
1611
1580
4098
3964
4089
4105
4115
3746
2852
3258
3278
3271
1635
1640
Round
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
Floor
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Room
Room 210
Room 210
Room 210
Room 210
Room 210
Room 210
Room 210
Room 210
Room 210
Room 210
Room 211
Room 211
Room 211
Room 211
Room 211
Room 211
Room 211
Room 211
Room 211
Room 211
Room 211
Room 211
X
11.799
9.799
9.784
12.099
11.699
11.899
10.402
11.699
10.899
9.799
10.493
10.769
10.833
11.749
10.918
10.514
10.514
9.513
10.897
9.400
10.961
9.492
y
17.754
17.355
19.799
20.054
17.854
14.360
14.826
17.559
20.527
18.332
7.424
11.577
8.276
9.469
12.003
6.440
6.913
6.317
9.107
10.768
7.254
9.660
z
3.000
3.000
4.000
3.000
3.000
4.197
3.000
3.000
5.298
3.000
3.000
5.271
3.392
3.392
3.000
3.000
3.000
4.198
3.395
3.593
3.000
4.066
Method
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Swab
Swab
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Swab
Area
(sqft)
4.000
0.694
0.694
0.694
0.694
0.694
0.694
0.028
0.028
0.028
4.000
4.000
4.000
4.000
4.000
4.000
0.694
0.694
0.694
0.694
0.028
0.028
Operator
Inman
Inman
Inman
Inman
Inman
Inman
Inman
Inman
Inman
Inman
perry
perry
perry
perry
perry
perry
perry
perry
perry
perry
perry
perry
Acquisition
Date
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
Acquisition
Time
11:58:00 AM
11:39:00 AM
12:28:00 PM
12:15:00 PM
11:53:00 AM
11:33:00 AM
11:24:00 AM
11:52:00 AM
12:31:00 PM
11:48:00 AM
1:12:00 PM
1:32:00 PM
12:58:00 PM
12:52:00 PM
12:17:00 PM
1:24:00 PM
1:16:00 PM
1:07:00 PM
12:45:00 PM
12:27:00 PM
1:11:00 PM
12:35:00 PM
Object
Floor
Floor
File cabinet
File cabinet
Floor
Wall
Floor
Floor
Supply Vent
Monitor
Floor
Ceiling
Chair
Couch
Floor
Floor
Floor
Shelves
Table
Wall
Floor
Wall
Texture
Smooth
Smooth
Metal
Metal
Smooth
Textured
Smooth
Smooth
Metal
Glass
Carpet
Textured
Cloth
Cloth
Carpet
Carpet
Carpet
Smooth
Smooth
Textured
Carpet
Textured
Orientation
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Horizontal
Upward
Horizontal
Downward
Vertical
Horizontal
Downward
Horizontal
Downward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Vertical
Blank
TRUE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
TRUE
FALSE
BROOM Notes
blank vacuum
sample air
lOinx lOin swab
of floor inside next
to wall and corner
of garbage can
spongestick on
metal cabinet top
rear half against
wall
spongestick on
surface of metal
cabinet upper left
corner
blank sponge air
lOinx lOin swab
on wall
immediately to
right of entrance
apprx 4ft off
ground
lOinx lOin swab
of floor inside doo
immediately to
right of door
opening
blank swab inside
air
swab of vent in
ceiling towards far
back wall
2in x 2in swab
upper left corner
monitor
TV
Lab ID
6
5
5
5
5
5
5
4
4
4
3
3
3
3
8
8
3
3
3
3
4
4
Detected
No
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
No
Yes
Spread Plate Results
(CFU/sq ft)
ND
8323.8
13814.8
11335.3
ND
79.2
10817.8
ND
24006.5
ND
ND
166.7
362.5
312.5
445.9
108.3
ND
14398.5
5850.5
19.7
ND
ND
Filter Plate Results
(CFU/sq ft)
NA
TNTC
1319.2
TNTC
19.0
40.4
TNTC
NA
NA
745.4
ND
81.7
NA
NA
NA
NA
ND
NA
NA
ND
ND
1159.5
G-21
-------
Barcode
3602
3907
4095
4117
4288
4097
3934
3941
2345
2880
2346
2847
1589
1681
1639
4092
3966
Round
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
VHP Pre-
Decon
Floor
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Room
Room 212
Room 212
Room 212
Room 212
Room 212
Room 212
Room 212
Room 212
Room 212
Room 212
Room 212
Room 212
Room 212
Room 212
Room 212
Room 213
Room 213
x
7.490
8.379
8.579
7.208
7.697
8.290
8.779
7.379
6.482
8.179
6.479
8.090
7.979
8.389
6.879
8.194
7.661
y
19.854
15.876
18.554
20.054
20.337
20.354
19.554
18.378
19.764
16.954
16.172
14.576
19.946
16.206
18.424
7.676
7.920
z
3.984
3.000
5.286
3.000
3.000
3.000
3.000
3.000
3.000
3.000
3.000
3.000
5.170
3.000
3.000
3.387
3.580
Method
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Swab
Swab
Area
(sqft)
4.000
4.000
4.000
4.000
4.000
4.000
4.000
4.000
0.694
0.694
0.694
0.694
0.028
0.028
0.028
4.000
4.000
Operator
Inman
Inman
Inman
Inman
Inman
Inman
Inman
Inman
Inman
Inman
Acquisition
Date
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
Acquisition
Time
3:07:00 PM
10:29:00 AM
11:17:00 AM
11:04:00 AM
10:59:00 AM
10:55:00 AM
10:51:00 AM
10:37:00 AM
11:08:00 AM
10:24:00 AM
10:21:00 AM
10:11:00 AM
11:12:00 AM
10:26:00 AM
10:42:00 AM
11:46:00 AM
11:32:00 AM
Object
Wall
Floor
Ceiling
Floor
Chair
Floor
Floor
Cabinet
Floor
Wall
Floor
Supply Vent
Floor
Bed
Bed
Texture
Textured
Porous
Smooth
Cloth
Smooth
Smooth
Cloth
Metal
Smooth
Textured
Smooth
Metal
Glass
Cloth
Cloth
Orientation
Vertical
Horizontal
Upward
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Upward
Horizontal
Horizontal
Upward
Blank
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
BROOM Notes
vacuum sample
collected from
vertical partition
surface.
2ft x 2ft vacuum o:
ceiling tile
removed from
ceiling and placed
on floor in middle
ofroom
2ft x 2ft vacuum o:
floor in front of
cabinet on back
left wall
apprx 2ft x 2ft
surface area of
chair seat against
back wall ofroom
2ft x 2ft vacuum o:
floor against wall
and edge of metal
cabinet
2ft x 2ft vacuum o:
floor
vacuum of chair
lOin x lOin swab
back half of
cabinet top closest
to wall
blank sponge air
lOinx lOin swab
apprx 5ft up wall
surface
lOinx lOin apprx
6in off left door
jam
swab of vent shaft
in ceiling
upper left corner
area
true bed sample
Lab ID
1
4
1
7
6
6
1
1
5
5
5
8
4
4
4
8
3
Detected
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
No
No
Yes
Yes
Spread Plate Results
(CFU/sq ft)
1125
ND
177.8
495.9
ND
163.3
312.7
1017.7
4910.2
ND
97.9
7814.1
16804.6
ND
ND
325.0
112.5
Filter Plate Results
(CFU/sq ft)
ND
1.2
NA
ND
NA
NA
NA
NA
ND
ND
21.6
TNTC
NA
NA
NA
NA
134.0
G-22
-------
Barcode
3924
3905
4083
4059
2738
2878
2394
2739
3291
1641
2197
3031
3568
2233
2234
2236
2235
2916
2240
2582
2914
3594
3672
3629
3558
3585
3644
3549
3550
2606
Round
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Pre-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
Floor
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Room
Room 213
Room 213
Room 213
Room 213
Room 213
Room 213
Room 213
Room 213
Room 213
Room 213
Stairwell
Stairwell
Bathroom M
Bathroom M
Bathroom M
Bathroom M
Bathroom M
Bathroom W
Bathroom W
Bathroom W
Bathroom W
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
X
8.949
7.526
7.927
7.433
6.594
7.490
8.867
8.883
7.208
7.909
23.530
23.874
25.910
24.644
26.297
24.855
26.684
24.012
27.400
26.960
25.156
23.055
24.537
25.992
25.904
22.780
15.461
8.383
16.536
25.332
y
8.742
10.158
7.032
11.920
11.097
9.463
11.225
6.943
6.040
9.671
19.814
14.935
19.709
20.412
18.056
19.287
20.483
15.023
14.539
16.519
17.179
11.002
7.784
8.428
12.564
15.188
13.157
12.854
13.891
11.376
z
3.586
3.000
5.288
3.000
4.782
3.000
3.489
3.000
3.486
3.000
3.000
3.000
0.000
0.000
0.000
0.000
0.000
0.000
0.597
0.000
0.794
2.459
0.000
2.436
2.356
2.392
2.392
2.387
0.000
0.000
Method
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Sponge Wipe
Sponge Wipe
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Area
(sqft)
4.000
4.000
4.000
4.000
0.694
0.694
0.694
0.694
0.694
0.028
0.694
0.694
4.000
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
4.000
4.000
4.000
4.000
4.000
4.000
4.000
4.000
0.694
Operator
perry
perry
perry
Perry
perry
perry
Perry
perry
perry
perry
Terrill
Terrill
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Acquisition
Date
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/17/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
Acquisition
Time
11:21:00 AM
11:00:00 AM
12:07:00 PM
10:15:00 AM
11:14:00 AM
10:57:00 AM
10:22:00 AM
11:49:00 AM
11:55:00 AM
11:02:00 AM
12:04:00 PM
12:25:00 PM
1:26:00 PM
1:10:00 PM
1:16:00 PM
1:12:00 PM
1:14:00 PM
12:59:00 PM
1:02:00 PM
1:05:00 PM
1:07:00 PM
11:49:00 AM
11:59:00 AM
12:25:00 PM
12:49:00 PM
12:56:00 PM
10:23:00 AM
9:58:00 AM
10:25:00 AM
12:39:00 PM
Object
Bed
Floor
Ceiling
Floor
File cabinet
Floor
Sink
Bed
Wall
Floor
Floor
Floor
Ceiling
Floor
Floor
Floor
Floor
Floor
Wall
Floor
Wall
Ceiling
Floor
Ceiling
Ceiling
Ceiling
Ceiling
Ceiling
Ceiling
Floor
Texture
Cloth
Carpet
Textured
Carpet
Metal
Carpet
Smooth
Cloth
Smooth
Carpet
Smooth
Smooth
Porous
Smooth
Smooth
Smooth
Smooth
Smooth
Paint
Smooth
Paint
Porous
Smooth
Porous
Porous
Porous
Porous
Smooth
Porous
Smooth
Orientation
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Vertical
Horizontal
Downward
Vertical
Horizontal
Upward
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Blank
FALSE
TRUE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
BROOM Notes
lost laser, no
height
sample was taken
on first floor at
bottom of stairs
under the sink
ceiling tile
ceiling tile
ceiling tile
ceiling tile sample
Lab ID
3
8
3
8
7
6
7
3
3
4
3
3
5
2
2
2
2
2
2
1
2
2
2
2
3
5
5
5
2
5
Detected
Yes
No
Yes
Yes
Yes
No
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
No
Yes
No
Yes
No
No
No
Yes
No
Yes
No
Yes
Yes
No
No
No
Spread Plate Results
(CFU/sq ft)
420.9
ND
183.3
191.7
3019.1
ND
10823.6
8668.5
46.1
ND
233156.0
514348.6
104.2
921.6
ND
ND
ND
ND
ND
ND
ND
8.3
ND
16.7
ND
12.5
41.7
ND
ND
ND
Filter Plate Results
(CFU/sq ft)
NA
NA
125.4
NA
ND
ND
ND
ND
3.2
ND
NA
NA
58.1
203.0
ND
19.3
ND
4.0
ND
ND
ND
ND
ND
ND
ND
0.6
ND
ND
ND
ND
G-23
-------
Barcode
2601
2266
2267
2072
2070
2749
2599
2741
2725
2724
2721
2722
2652
2566
2653
2200
2742
2743
2241
2577
2436
3670
2679
2231
3621
2654
2825
2827
Round
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
Floor
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Room
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Mechanical Room
Mechanical Room
Mechanical Room
Room 101
Room 101
Room 101
Room 101
X
23.470
22.516
24.935
26.608
27.444
27.356
26.564
5.254
3.939
6.574
9.116
12.636
13.858
22.666
14.532
16.928
17.514
17.905
18.785
13.222
20.342
17.539
18.488
17.117
20.288
21.445
16.546
16.057
y
12.743
9.467
8.249
6.616
9.404
11.552
10.240
13.157
13.358
13.988
13.881
13.294
13.978
15.460
15.308
13.597
14.331
13.695
14.063
16.520
13.335
19.357
20.518
18.161
10.231
10.834
12.281
11.234
z
0.000
0.000
0.000
0.000
0.000
0.000
0.596
0.000
0.685
2.068
0.000
0.000
0.000
0.000
0.000
0.000
0.397
0.000
2.074
0.000
0.000
1.081
0.000
0.000
2.393
0.000
0.000
0.491
Method
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Sponge Wipe
Sponge Wipe
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqft)
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
4.000
0.694
0.694
4.000
0.694
0.694
0.694
Operator
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Johnson
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Anaya
Acquisition
Date
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
Acquisition
Time
11:37:00 AM
11:43:00 AM
12:00:00 PM
12:10:00 PM
12:18:00 PM
12:44:00 PM
12:23:00 PM
9:39:00 AM
9:44:00 AM
9:50:00 AM
10:02:00 AM
10:09:00 AM
10:14:00 AM
11:26:00 AM
10:17:00 AM
10:26:00 AM
10:34:00 AM
10:35:00 AM
10:38:00 AM
10:16:00 AM
2:53:00 PM
1:25:00 PM
1:21:OOPM
1:22:00 PM
11:12:00 AM
11:08:00 AM
10:48:00 AM
10:52:00 AM
Object
Floor
Floor
Floor
Floor
Floor
Floor
Wall
Floor
Wall
Ceiling
Floor
Floor
Floor
Floor
Floor
Floor
Wall
Floor
Ceiling
Floor
Floor
Supply Vent
Floor
Floor
Ceiling
Floor
Floor
Wall
Texture
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Porous
Smooth
Smooth
Porous
Smooth
Smooth
Paint
Orientation
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Vertical
Horizontal
Downward
Vertical
Horizontal
Upward
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Vertical
Horizontal
Downward
Horizontal
Upward
Horizontal
Downward
Horizontal
Downward
Inclined
Horizontal
Downward
Horizontal
Downward
Horizontal
Upward
Horizontal
Downward
Horizontal
Downward
Vertical
Blank
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
sample was on the
side of the counter
Sample was taken
on entrance door tc
the first floor
hvac sample
furnace vent
ceiling tile was
labeled for post
decon
Lab ID
5
5
2
2
1
1
1
5
5
2
1
2
2
5
2
2
5
5
5
2
5
5
5
5
5
5
5
5
Detected
No
No
No
Yes
No
No
No
No
No
No
No
Yes
No
Yes
Yes
No
No
No
No
No
No
Yes
Yes
Yes
Yes
No
No
No
Spread Plate Results
(CFU/sq ft)
ND
ND
ND
29.3
ND
ND
ND
ND
ND
ND
ND
56.6
ND
21.6
82.1
ND
ND
ND
ND
ND
ND
404.2
345.6
496.8
370.9
ND
ND
ND
Filter Plate Results
(CFU/sq ft)
ND
ND
ND
53.3
ND
ND
ND
ND
ND
ND
ND
31.7
ND
ND
38.2
ND
ND
ND
ND
ND
ND
TNTC
ND
ND
120.8
ND
ND
ND
G-24
-------
Barcode
4157
3961
3626
4064
2794
2942
2900
2463
3248
2723
2734
2957
Round
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
Floor
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Room
Room 101A
Room 101A
Room 101A
Room 101A
Room 101A
Room 101A
Room 101A
Room 101A
Room 101A
Room 101A
Room 101A
Room 101A
X
21.532
19.009
19.610
16.441
18.014
20.032
18.279
20.193
21.604
21.872
21.872
21.409
y
8.927
7.781
6.993
9.473
9.249
6.414
8.955
9.207
7.886
7.348
6.228
6.192
z
0.000
0.000
0.596
0.000
1.200
0.800
1.200
0.700
0.694
0.800
0.800
0.797
Method
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqft)
4.000
4.000
4.000
4.000
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
Operator
Oudejans
Oudejans
Oudejans
Oudejans
Oudejans
Oudejans
Oudejans
Oudejans
Oudejans
Oudejans
Oudejans
Oudejans
Acquisition
Date
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
Acquisition
Time
2:19:00 PM
1:45:00 PM
1:21:OOPM
1:06:00 PM
2:44:00 PM
1:38:00 PM
2:35:00 PM
2:23:00 PM
2:09:00 PM
2:04:00 PM
1:55:00 PM
1:52:00 PM
Object
Floor
Floor
Chair
Floor
Workbench
Desk
Workbench
Cabinet
Cabinet
Desk
Desk
Desk
Texture
Carpet
Carpet
Cloth
Carpet
Plastic
Plastic
Plastic
Metal
Metal
Plastic
Plastic
Plastic
Orientation
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Blank
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
vacuum to left of
cabinet and to righ
of black box on
floor; no marking
on floor from pre
decon sample; maj
have been in
overlap with pre
decon sample;
blank vacuum
chair;horizontal
surface; overlap
with previous
sample
vacuum sample to
right of previous
pre decon sample;
vacuum hose
extension works
great.
top of uv aps
system; front
center; horizontal
surface
25 percent overlap
with previous
sample pre decon
sample; left side in
middle of top
surface;
vertical surface of
uv aps system;
right side
front right corner
60 percent overlap
with pre decon
sample;
front right corner
of cabinet with 50
percentage overlap
with pre decon
sample;
left corner of desk
in back ;no overlaf
with pre decon
sample
right corner of
against back; no
overlap with pre
decon sample;
dropped bag befon
sampling, no
overlap with pre
decon sample;
front right corner
of desk
Lab ID
6
6
6
6
7
3
7
3
3
7
3
7
Detected
Yes
No
No
Yes
No
Yes
Yes
No
Yes
No
Yes
Yes
Spread Plate Results
(CFU/sq ft)
27.9
ND
ND
157.9
ND
ND
ND
ND
ND
ND
17.8
ND
Filter Plate Results
(CFU/sq ft)
NA
NA
NA
NA
ND
3.6
2.9
ND
19.2
ND
11.9
9.5
G-25
-------
Barcode
2844
1973
2354
3541
3660
3608
3609
3243
3282
3238
3509
3240
3506
3273
Round
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
Floor
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Room
Room 101A
Room 101A
Room 101A
Room 102
Room 102
Room 102
Room 102
Room 102
Room 102
Room 102
Room 102
Room 102
Room 102
Room 102
X
19.102
18.053
19.158
18.194
17.339
18.463
19.895
19.225
18.440
16.942
18.234
18.385
19.361
18.749
y
6.502
6.215
7.996
16.030
16.633
15.397
16.529
15.049
15.039
15.683
17.006
15.909
16.583
16.938
z
0.798
0.000
0.000
0.000
0.000
0.000
0.000
1.200
1.200
0.700
0.700
0.000
0.800
0.800
Method
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqft)
0.694
0.694
0.694
4.000
4.000
4.000
4.000
0.694
0.694
0.694
0.694
0.694
0.694
0.694
Operator
Oudejans
Oudejans
Oudejans
Oudejans
Oudejans
Oudejans
Oudejans
Oudejans
Oudejans
Oudejans
Oudejans
Oudejans
Oudejans
Oudejans
Acquisition
Date
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
Acquisition
Time
1:26:00 PM
1:15:00 PM
1:48:00 PM
3:42:00 PM
3:37:00 PM
3:12:00 PM
2:59:00 PM
4:25:00 PM
4:20:00 PM
4:09:00 PM
3:48:00 PM
3:45:00 PM
3:33:00 PM
3:28:00 PM
Object
Desk
Cabinet
Floor
Floor
Floor
Floor
Floor
Workbench
Workbench
Cabinet
Cabinet
Floor
Desk
Desk
Texture
Plastic
Metal
Carpet
Carpet
Carpet
Carpet
Carpet
Plastic
Plastic
Metal
Metal
Carpet
Metal
Plastic
Orientation
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Blank
FALSE
FALSE
TRUE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
BROOM Notes
sticky surface;
sample to the right
of previous pre
decon sample;
front right corner
of desk
2/3 overlap with
previous sample;
back on top of
metal cabinet;
blank sponge
blank vacuum
vacuum floor in
front of wooden
panel; some
overlap with pre
decon sample;
Air sampling
started in
preparing this
vacuum
sample;sample to
right of pre decon
sample with 25
percent overlap
vacuum in front o]
stand;
top of uv aps
system; left side ol
top surface
top of uv aps
system; right side
front
backside of top ot
metal cabinet
cabinet; 80
percentage overlap
with pre decon
sample;
80 percentage
overlap with pre
decon sample;
front of top of
metal cabinet
sponge blank
pulled drawer out.
sample inside
drawer; to left of
pre decon sample;
no overlap, closed
drawer.
front; left of the
middle; no overlap
with pre decon
sample; very dirty
Lab ID
7
3
3
7
7
6
6
7
7
7
7
7
7
7
Detected
No
Yes
No
No
Yes
Yes
No
Yes
No
Yes
Yes
No
Yes
Yes
Spread Plate Results
(CFU/sq ft)
ND
ND
ND
ND
29.2
27.9
ND
135.4
ND
27.8
64.8
ND
ND
105.6
Filter Plate Results
(CFU/sq ft)
ND
3.9
ND
ND
14.4
NA
NA
46.4
ND
3.6
ND
ND
5.3
179.9
G-26
-------
Barcode
3242
3244
3239
3218
3676
3571
3077
3013
3513
3530
3529
3528
3076
3525
1597
1654
3605
3714
3245
3495
3287
3277
3283
Round
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
Floor
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Room
Room 102
Room 102
Room 102
Room 102
Room 103
Room 103
Room 103
Room 103
Room 103
Room 103
Room 103
Room 103
Room 103
Room 103
Room 103
Room 103
Room 104
Room 104
Room 104
Room 104
Room 104
Room 104
Room 104
X
19.895
18.269
20.154
17.721
13.793
13.793
13.556
13.765
13.765
14.678
15.321
15.093
13.234
13.793
14.421
13.793
13.823
16.297
13.752
16.027
15.629
15.245
15.031
y
17.388
14.627
16.406
17.317
10.721
10.021
8.429
6.814
6.163
6.230
6.914
11.614
7.320
10.021
6.814
10.021
18.783
19.181
18.883
20.702
20.063
20.077
18.769
z
0.800
0.800
0.800
0.700
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
2.700
0.000
1.500
0.900
0.900
0.800
Method
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Swab
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqft)
0.694
0.694
0.694
0.694
4.000
4.000
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.028
0.028
4.000
4.000
0.694
0.694
0.694
0.694
0.694
Operator
Oudejans
Oudejans
Oudejans
Oudejans
Schuette
Schuette
Schuette
Schuette
Schuette
Schuette
Schuette
Schuette
Schuette
Schuette
Schuette
Schuette
Patureau
Patureau
Patureau
Patureau
Patureau
Patureau
Patureau
Acquisition
Date
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/23/2011
4/23/2011
4/23/2011
4/23/2011
4/23/2011
4/23/2011
4/23/2011
4/23/2011
4/23/2011
4/23/2011
4/23/2011
4/23/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
Acquisition
Time
3:17:00 PM
4:12:00 PM
2:53:00 PM
3:59:00 PM
11:19:00 AM
11:24:00 AM
11:03:00 AM
10:57:00 AM
10:53:00 AM
10:48:00 AM
10:39:00 AM
10:27:00 AM
11:10:00 AM
11:26:00 AM
11:13:00 AM
11:29:00 AM
5:03:00 PM
5:00:00 PM
5:07:00 PM
4:54:00 PM
4:47:00 PM
4:45:00 PM
4:38:00 PM
Object
Desk
Desk
Cabinet
Cabinet
Ceiling
Floor
Table
Table
Floor
Wall
Floor
Floor
Mail slot
Floor
Supply Vent
Floor
Ceiling
Ceiling
Ceiling
Wall
Table
Table
Table
Texture
Metal
Plastic
Metal
Metal
Porous
Smooth
Smooth
Smooth
Smooth
Textured
Smooth
Smooth
Metal
Smooth
Metal
Smooth
Textured
Textured
Textured
Textured
Textured
Textured
Smooth
Orientation
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Vertical
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Blank
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
TRUE
TRUE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
right corner of
desk in the back.nc
overlap with pre
decon sample;
sample on desk to
right of uvaps
system; no overlap
with pre decon
sample;
overlap with pre
decon sample; 80
percent; cabinet
front
front of top of
metal cabinet;
overlap with pre
decon sample;
ceiling tile plenum
side
top of mail
slot.. .previous
sample taken on
top of mail slot
also
air diffuser on
ceiling
interior bag ripped
vac blank
sponge blank
above previous
sample outline
right of previous
sample outline
tools on table also
sampled
left of previous
sample outline
when looking fron
entrance
Lab ID
7
7
7
7
4
4
1
1
1
1
1
1
1
1
1
1
2
4
7
7
7
7
7
Detected
Yes
No
Yes
No
Yes
No
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
No
No
Yes
No
Yes
Yes
No
Yes
Spread Plate Results
(CFU/sq ft)
ND
ND
25.9
ND
4.2
ND
ND
ND
120.0
ND
ND
ND
ND
ND
6001.6
ND
ND
ND
ND
ND
ND
ND
ND
Filter Plate Results
(CFU/sq ft)
2.1
ND
32.4
ND
ND
ND
14.7
35.7
13.2
ND
38.0
20.2
8.1
3.3
270.1
ND
ND
2.3
ND
5.2
6.6
ND
2.6
G-27
-------
Barcode
3252
1519
1481
3686
3718
3569
3981
3595
3074
3502
3501
3066
1676
3674
3631
Round
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
Floor
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Room
Room 104
Room 104
Room 104
Room 105
Room 105
Room 105
Room 105
Room 105
Room 105
Room 105
Room 105
Room 105
Room 105
Room 106
Room 106
X
14.832
13.624
15.515
11.682
10.654
10.606
10.102
10.831
9.983
10.445
12.519
11.731
11.302
10.195
12.646
y
19.579
18.940
19.395
10.966
12.186
9.575
7.024
9.415
11.262
9.685
10.434
6.667
6.824
19.661
19.784
z
0.000
0.000
2.700
0.000
0.000
0.000
0.000
0.000
0.300
0.800
0.800
0.000
0.000
1.200
1.400
Method
Sponge Wipe
Swab
Swab
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Vacuum Sock
Vacuum Sock
Area
(sqft)
0.694
0.028
0.028
4.000
4.000
4.000
4.000
4.000
0.694
0.694
0.694
0.694
0.028
4.000
4.000
Operator
Patureau
Patureau
Patureau
Schmelzer
Schmelzer
Schuette
Schuette
Schuette
Schmelzer
Schmelzer
Schmelzer
Schuette
Schuette
Patureau
Patureau
Acquisition
Date
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/23/2011
4/23/2011
4/23/2011
4/22/2011
4/22/2011
4/22/2011
4/23/2011
4/23/2011
4/22/2011
4/22/2011
Acquisition
Time
4:36:00 PM
5:06:00 PM
4:56:00 PM
5:08:00 PM
4:52:00 PM
10:13:00 AM
10:00:00 AM
9:45:00 AM
5:32:00 PM
5:29:00 PM
5:21:00 PM
9:56:00 AM
10:06:00 AM
4:08:00 PM
4:14:00 PM
Object
Floor
Ceiling
Supply Vent
Floor
Floor
Ceiling
Floor
Floor
Wall
Stove
Sink
Table
Supply Vent
Wall
Shelves
Texture
Smooth
Textured
Metal
Carpet
Carpet
Porous
Carpet
Carpet
Smooth
Smooth
Metal
Smooth
Metal
Cloth
Smooth
Orientation
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Downward
Blank
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
left of previous
sample outline
swab blank
sample was taken
from in front of
sink [B. Melton
moved to room
105]
sample was taken
from the center of
doorway on carpet
[B. Melton moved
to room 105]
ceiling tile plenum
side. ..outside bag
of sample droppec
on floor.. .wiped
outside of bag off
with alcohol wipe
sampler may have
stepped in part of
sample location
sample was taken
from the right on
the previous
sample [B.Melton
moved to room
105]
sample was taken
from the front top
of the stove
[B. Melton moved
to room 105]
sample was taken
from right side of
sink [B. Melton
moved to room
105]
air diffuser on
ceiling
on backside of
partition in top
right, some overla[
with previous
sample since
outline was in
center of wall.
around and on
books
Lab ID
7
1
1
2
2
4
4
4
6
6
6
1
1
5
2
Detected
No
No
No
Yes
Yes
Yes
Yes
Yes
No
No
No
Yes
Yes
No
Yes
Spread Plate Results
(CFU/sq ft)
ND
ND
ND
ND
4.2
4.2
ND
ND
ND
ND
ND
144.5
3601.0
ND
8.3
Filter Plate Results
(CFU/sq ft)
ND
ND
ND
1.7
2.3
1.7
1.2
1.2
NA
NA
NA
143.2
2970.8
ND
0.6
G-28
-------
Barcode
3616
3725
3663
3668
3254
2840
2839
3247
3246
1682
1482
3578
3720
3598
3599
3593
3508
3167
3067
3158
Round
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
Floor
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Room
Room 106
Room 106
Room 106
Room 106
Room 106
Room 106
Room 106
Room 106
Room 106
Room 106
Room 106
Room 107
Room 107
Room 107
Room 107
Room 107
Room 107
Room 107
Room 107
Room 107
X
12.168
10.763
11.243
10.994
11.786
10.180
10.512
9.981
10.781
11.333
10.212
8.400
8.531
9.018
7.760
9.213
7.111
8.380
8.413
7.542
y
20.524
18.418
20.447
18.433
15.772
19.892
18.406
16.206
14.796
19.589
18.707
7.190
8.414
11.935
11.750
9.714
6.730
7.210
9.414
12.317
z
0.000
2.700
0.500
0.500
0.800
0.700
0.797
1.088
0.000
2.700
0.997
0.000
0.000
0.000
2.300
0.000
1.100
0.000
0.000
0.000
Method
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Swab
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqft)
4.000
4.000
4.000
4.000
0.694
0.694
0.694
0.694
0.694
0.028
0.028
4.000
4.000
4.000
4.000
4.000
0.694
0.694
0.694
0.694
Operator
Patureau
Patureau
Patureau
Patureau
Patureau
Patureau
Patureau
Patureau
Patureau
Patureau
Patureau
Schmelzer
Schmelzer
Schmelzer
Schmelzer
Schmelzer
Schmelzer
Schmelzer
Schmelzer
Schmelzer
Acquisition
Date
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
Acquisition
Time
3:57:00 PM
3:51:00 PM
4:03:00 PM
3:44:00 PM
4:28:00 PM
4:18:00 PM
3:28:00 PM
3:25:00 PM
3:16:00 PM
3:38:00 PM
3:32:00 PM
3:38:00 PM
3:23:00 PM
2:53:00 PM
4:26:00 PM
3:00:00 PM
3:45:00 PM
3:36:00 PM
3:18:00 PM
4:06:00 PM
Object
Floor
Ceiling
Chair
Chair
Table
File cabinet
Desk
Wall
Floor
Supply Vent
Monitor
Couch
Chair
Floor
Ceiling
Couch
Shelves
Couch
Table
Wall
Texture
Smooth
Textured
Cloth
Cloth
Smooth
Metal
Smooth
Textured
Smooth
Metal
Smooth
Leather
Leather
Carpet
Textured
Cloth
Smooth
Leather
Smooth
Smooth
Orientation
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Vertical
Horizontal
Downward
Horizontal
Downward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Blank
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
BROOM Notes
floor adjacent to
wall and shelf
ceiling tile above
desk
grey chair by
window
red desk chair seat
cushion
to right of previous
sample if facing
door
adjacent to wall
right of previous
sample outline
above previous
sample outline
right of previous
sample outline
taken in upper
right corner as
instructed,
although previous
sample marked in
upper left corner
blank
sample was taken
from chair cushion
adjacent to right
by light switch
[B. Melton moved
to room 107,
moved to door]
inner bag fell on
the floor
[B. Melton moved
to room 107,
moved to correct
spot]
sample from
middle cushion
sample was taken
from center of top
surface [B. Melton
moved to shelves]
blank
sample was taken
from adjacent sides
of the previous
sample
sample was taken
from left side of
the previous
sample
Lab ID
4
2
4
4
7
7
7
7
7
1
1
2
2
2
2
2
6
6
6
6
Detected
Yes
Yes
No
No
No
Yes
Yes
No
Yes
Yes
No
Yes
No
No
Yes
No
No
No
Yes
No
Spread Plate Results
(CFU/sq ft)
12.5
16.7
ND
ND
ND
23.5
91.2
ND
ND
ND
ND
ND
ND
ND
41.7
ND
ND
ND
1970324.9
ND
Filter Plate Results
(CFU/sq ft)
2.9
8.6
ND
ND
ND
25.9
51.4
ND
17.6
180.0
ND
1.2
ND
ND
20.1
ND
NA
NA
NA
NA
G-29
-------
Barcode
1647
1695
1605
3977
3719
3633
3592
3658
3978
3520
3524
3522
3505
3065
3063
1576
1637
1520
3627
3669
Round
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
Floor
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Room
Room 107
Room 107
Room 107
Room 108
Room 108
Room 108
Room 108
Room 108
Room 108
Room 108
Room 108
Room 108
Room 108
Room 108
Room 108
Room 108
Room 108
Room 108
Room 109
Room 109
X
7.124
8.380
8.300
8.513
9.413
8.013
9.413
9.413
7.713
9.451
8.513
9.151
7.151
7.413
9.413
8.613
8.813
7.151
4.854
5.674
y
9.537
7.190
6.900
16.430
19.717
20.617
19.917
17.230
18.917
20.017
16.230
14.717
19.317
18.017
16.730
16.530
19.717
18.717
7.707
7.723
z
0.800
0.000
2.300
1.184
1.299
0.498
1.198
2.700
0.495
1.298
1.095
0.794
0.798
0.000
0.000
1.187
2.700
0.897
0.000
0.000
Method
Swab
Swab
Swab
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Swab
Swab
Vacuum Sock
Vacuum Sock
Area
(sqft)
0.028
0.028
0.028
4.000
4.000
4.000
4.000
4.000
4.000
0.694
0.694
0.694
0.694
0.694
0.694
0.028
0.028
0.028
4.000
4.000
Operator
Schmelzer
Schmelzer
Schmelzer
Patureau
Patureau
Patureau
Patureau
Patureau
Patureau
Patureau
Patureau
Patureau
Patureau
Patureau
Patureau
Patureau
Patureau
Patureau
Schuette
Schuette
Acquisition
Date
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
Acquisition
Time
3:55:00 PM
3:34:00 PM
4:13:00 PM
3:08:00 PM
2:41:OOPM
2:36:00 PM
2:31:00 PM
2:21:OOPM
2:12:00 PM
2:46:00 PM
3:05:00 PM
3:00:00 PM
1:58:00 PM
1:47:00 PM
2:57:00 PM
3:10:00 PM
2:53:00 PM
2:04:00 PM
3:51:00 PM
3:45:00 PM
Object
Monitor
Couch
Supply Vent
Wall
Shelves
Chair
Wall
Ceiling
Chair
Shelves
Wall
Wall
Desk
Floor
Floor
Wall
Supply Vent
Monitor
Bed
Bed
Texture
Glass
Leather
Textured
Textured
Metal
Cloth
Cloth
Textured
Cloth
Metal
Textured
Textured
Smooth
Smooth
Smooth
Textured
Metal
Smooth
Porous
Porous
Orientation
Vertical
Horizontal
Upward
Inclined
Vertical
Horizontal
Downward
Horizontal
Downward
Vertical
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Vertical
Vertical
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Vertical
Horizontal
Downward
Vertical
Horizontal
Upward
Horizontal
Upward
Blank
FALSE
TRUE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
sample was taken
from upper left of
the monitor
[B. Melton moved
to room 107,
moved across
room]
blank
vent surface is
rusty [B. Melton
moved to room
107]
vacuum blank
top of bookshelf,
on and around
books
grey chair by
window
above previous
sample outline
seat of chair
sample taken to
right of previous
sample outline.
book was blocking
top 2 inches of
template, spine of
book sponged.
blank sponge
above previous
sample outline
sample taken in far
right corner of
desk near wall, du(
to obstruction by
ceiling tile
above previous
sample outline
right of previous
sample outline,
adjacent to wall
swab blank
sample taken to
right of previous
sample outline
Lab ID
1
1
1
4
2
4
4
4
4
6
6
6
7
7
7
1
1
1
3
3
Detected
No
Yes
No
No
Yes
No
No
No
Yes
Yes
No
Yes
No
Yes
No
No
No
No
Yes
Yes
Spread Plate Results
(CFU/sq ft)
ND
600.2
ND
ND
12.5
ND
ND
ND
ND
27.9
ND
18.6
ND
ND
ND
ND
ND
ND
ND
ND
Filter Plate Results
(CFU/sq ft)
ND
90.0
ND
ND
1.7
ND
ND
ND
1.2
NA
NA
NA
ND
2.4
ND
ND
ND
ND
0.6
0.6
G-30
-------
Barcode
3576
3563
3681
3689
2604
2660
2657
2658
2659
2429
3716
3655
3612
3648
3722
3584
3868
3280
Round
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
Floor
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Room
Room 109
Room 109
Room 109
Room 109
Room 109
Room 109
Room 109
Room 109
Room 109
Room 109
Room 110
Room 110
Room 110
Room 110
Room 110
Room 110
Room 110
Room 110
X
5.221
4.709
5.921
5.821
3.874
5.221
4.227
4.340
4.324
4.855
5.711
4.932
5.811
4.145
4.973
6.619
6.619
4.546
y
10.531
10.728
11.414
6.914
6.485
10.531
9.539
10.921
12.239
6.038
16.651
18.452
16.551
19.576
19.251
20.572
19.817
17.922
z
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
Method
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Area
(sqft)
4.000
4.000
4.000
4.000
0.694
0.694
0.694
0.694
0.694
0.694
4.000
4.000
4.000
4.000
4.000
4.000
4.000
0.694
Operator
Schuette
Schuette
Schuette
Schuette
Schuette
Schuette
Schuette
Schuette
Schuette
Schuette
Schuette
Schuette
Schuette
Schuette
Schuette
Schuette
Schuette
Schuette
Acquisition
Date
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
Acquisition
Time
3:36:00 PM
3:08:00 PM
3:01:00 PM
4:08:00 PM
3:58:00 PM
3:37:00 PM
3:25:00 PM
3:21:00 PM
3:18:00 PM
4:01:OOPM
1:20:00 PM
1:54:00 PM
2:51:00 PM
2:42:00 PM
2:34:00 PM
2:14:00 PM
2:10:00 PM
1:38:00 PM
Object
Floor
Floor
Floor
Ceiling
Wall
Floor
Nightstand
Countertop
Countertop
Countertop
Floor
Chair
Ceiling
Wall
Chair
File cabinet
File cabinet
Desk
Texture
Carpet
Carpet
Carpet
Porous
Textured
Carpet
Smooth
Smooth
Smooth
Paint
Smooth
Cloth
Porous
Porous
Cloth
Smooth
Textured
Smooth
Orientation
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Blank
TRUE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
had to move heatei
to hallway in ordei
to access sample
location
no marking as to
which side was
samped pre-
VHP... ceiling tile
already down on
bed
pre-VHP sample
marked approx 3.5
ftup... took post
sample
immediately below
pre
no marking of
template from pre-
VHP sampling
Countertop left of
sink
Countertop right o]
sink
sample on
windowsill...no
marking from pre-
VHP sampling
ceiling tile on
plenum
side. ..ceiling tile
already on floor
partition
current chair
location not the
same as on the
map [on the map
chair is under far
window]. ..BROO
M location is
chair's current
location
vacuum on and
around books on
top of file cabinet
Lab ID
7
3
3
7
3
3
3
3
3
3
7
7
7
7
7
3
7
3
Detected
No
Yes
No
Yes
No
No
No
Yes
No
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
No
Spread Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
153.6
ND
19.2
ND
ND
ND
4.2
ND
ND
ND
ND
Filter Plate Results
(CFU/sq ft)
ND
0.6
ND
0.6
ND
ND
ND
ND
ND
ND
0.6
0.6
4.0
1.2
ND
0.6
0.6
ND
G-31
-------
Barcode
3279
3275
3281
3284
3286
3285
1633
1653
1634
3684
2674
2646
2682
3580
3666
Round
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
Decon
VHP Post-
VHP Post-
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Floor
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Room
Room 110
Room 110
Room 110
Room 110
Room 110
Room 110
Room 110
Room 110
Room 110
Bathroom M
Bathroom M
Bathroom M
Bathroom M
Bathroom W
Bathroom W
X
5.711
5.373
6.011
4.386
4.530
6.619
5.711
5.311
4.177
27.804
29.782
27.050
28.728
27.734
28.146
y
16.651
14.751
17.496
19.865
20.700
20.234
16.651
19.551
18.517
19.731
20.193
20.108
20.452
17.404
17.777
z
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
3.000
3.000
3.000
3.700
3.000
3.000
Method
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Swab
Swab
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Vacuum Sock
Area
(sqft)
0.694
0.694
0.694
0.694
0.694
0.694
0.028
0.028
0.028
4.000
0.694
0.694
0.694
4.000
4.000
Operator
Schuette
Schuette
Schuette
Schuette
Schuette
Schuette
Schuette
Schuette
Schuette
Patel
Patel
Patel
Patel
Patel
Patel
Acquisition
Date
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
Acquisition
Time
1:28:00 PM
1:11:00 PM
1:46:00 PM
2:27:00 PM
2:22:00 PM
2:17:00 PM
1:26:00 PM
2:45:00 PM
1:41:OOPM
5:40:00 PM
5:33:00 PM
5:31:00 PM
5:36:00 PM
5:28:00 PM
5:24:00 PM
Object
Floor
Floor
Floor
File cabinet
Wall
File cabinet
Floor
Supply Vent
Monitor
Ceiling
Floor
Floor
Wall
Ceiling
Ceiling
Texture
Smooth
Smooth
Smooth
Smooth
Textured
Smooth
Metal
Smooth
Textured
Smooth
Smooth
Textured
Textured
Textured
Orientation
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Horizontal
Horizontal
Downward
Vertical
Horizontal
Downward
Horizontal
Blank
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
BROOM Notes
pre-VHP sample
was taken approx
5 ft from
ground.. .took post
sample directly
under pre sample
diffuser on ceiling
upper right
ceiling tile in
center of room
sampled, team
could not find
marked tile, tile
was placed on
floor outside
bathroom since no
electrical outlet
was available.
point manually
picked, light is
adequate.
sample taken on
floor or rear stall
entrance.
sample taken on
floor of entrance tc
men's bathroom.
template used.
adequate light
available.
sample taken on
left wall of men's
bathroom near rear
stall entrance.
blank vacuum
sample taken of
ceiling tile, ceiling
bathroom mirrors
on left side of
room, manually
picked point.
Lab ID
3
3
3
3
3
3
1
1
1
6
4
4
4
6
6
Detected
No
No
No
Yes
No
No
No
No
No
No
No
No
No
No
No
Spread Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Filter Plate Results
(CFU/sq ft)
ND
ND
ND
4.6
ND
ND
ND
ND
ND
NA
ND
ND
ND
NA
NA
G-32
-------
Barcode
2673
1984
2230
2678
2608
2237
3607
3589
3886
3583
2053
3142
2437
2048
2030
3170
3159
Round
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
Floor
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Room
Bathroom W
Bathroom W
Bathroom W
Copier Room
Copier Room
Copier Room
Hallway
Hallway
Hallway
Hallway
Hallway
Hallway
Hallway
Hallway
Hallway
Hallway
X
28.739
26.672
28.146
30.005
29.405
27.505
7.593
8.993
7.693
24.537
25 013
26.537
26.437
25.883
24.805
13.193
25.437
y
17.740
16.797
17.365
13.363
14.131
13.563
12.668
13.068
12.768
12.768
15 730
13.490
13.677
18.850
13.068
13.828
12.968
z
3.000
3.000
3.000
3.000
3.682
3.000
3.000
3.000
3.000
3.000
3 000
3.000
3.000
3.000
3.000
3.495
3.000
Method
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqft)
0.694
0.694
0.694
0.694
0.694
0.694
4.000
4.000
4.000
4.000
0694
0.694
0.694
0.694
0.694
0.694
0.694
Operator
Patel
Patel
Patel
Patel
Patel
Patel
Johnson
Johnson
Johnson
Johnson
Johnson
Johnson
Johnson
Johnson
Johnson
Johnson
Acquisition
Date
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
Acquisition
Time
5:19:00 PM
5:16:00 PM
5:21:00 PM
5:00:00 PM
3:15:00 PM
3:12:00 PM
12:13:00 PM
2:28:00 PM
11:00:00 AM
1:21:OOPM
2-16-00 PM
1:54:00 PM
2:08:00 PM
2:20:00 PM
2:23:00 PM
12:22:00 PM
12:27:00 PM
Object
Floor
Floor
Floor
Floor
Wall
Floor
Ceiling
Ceiling
Ceiling
Ceiling
Floor
Ceiling
Wall
Floor
Floor
Floor
Texture
Smooth
Smooth
Smooth
Porous
Porous
Porous
Porous
Porous
Porous
Porous
Smooth
Porous
Textured
Smooth
Smooth
Smooth
Orientation
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Vertical
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Horizontal
Upward
Vertical
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Blank
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
sample taken on
floor near
bathroom stall, no
light available.
manually picked
point.
sample taken on
floor of bathroom
entrance, no light
available.
manually picked
point since laser
could not read.
blank in center of
room, manually
picked point.
sample taken on
floor at very end o:
room, no light
available, no
furniture in room.
sample on left wal
taken, no light
available for
picture, sample
taken just above ar
electrical outlet.
floor sample taken
near entrance of
copier room, room
has no light or
furniture, room is
also numbered 25
on doorway.
previous sample
taken in the
middle, current
taken on the left
side.
Lab ID
4
4
4
4
4
4
5
3
3
3
5
3
3
3
5
3
5
Detected
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
Spread Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Filter Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
G-33
-------
Barcode
3169
2046
2045
2044
2043
3713
2677
2676
3640
2049
2052
3551
2680
2750
Round
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
Floor
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Room
Hallway
Hallway
Hallway
Hallway
Hallway
Janitor Closet
Janitor Closet
Janitor Closet
Mechanical Room
Mechanical Room
Room 201
Room 201
Room 201
X
17.027
8.174
8.693
7.693
13.293
29.164
27.601
28.596
22.021
22.193
18.893
29.015
26.715
29.015
y
12.768
13.829
12.868
13.868
12.668
15.978
14.552
15.678
18.602
19.854
19.854
11.469
12.069
11.369
z
3.000
3.000
3.000
3.000
3.000
3.000
3.000
3.000
3.000
3.000
3.000
3.000
3.000
3.000
Method
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Sponge Wipe
Sponge Wipe
Area
(sqft)
0.694
0.694
0.694
0.694
0.694
4.000
0.694
0.694
4.000
0.694
0.694
4.000
0.694
0.694
Operator
Johnson
Johnson
Johnson
Johnson
Johnson
Patel
Patel
Patel
Johnson
Johnson
Patel
Patel
Patel
Acquisition
Date
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
Acquisition
Time
1:20:00 PM
10:33:00 AM
10:42:00 AM
11:52:00 AM
12:03:00 PM
5:12:00 PM
5:02:00 PM
5:04:00 PM
2:46:00 PM
2:38:00 PM
2:43:00 PM
2:43:00 PM
2:36:00 PM
2:39:00 PM
Object
Ceiling
Return Vent
Floor
Floor
Wall
Ceiling
Floor
Floor
Floor
Floor
Ceiling
Floor
Floor
Texture
Porous
Porous
Smooth
Smooth
Textured
Textured
Porous
Smooth
Smooth
Smooth
Textured
Smooth
Smooth
Orientation
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Horizontal
Horizontal
Downward
Horizontal
Horizontal
Downward
Horizontal
Downward
Horizontal
Horizontal
Downward
Horizontal
Downward
Blank
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
No marking,
completed far left
side.
No marking, took
sample under
second red arrow
Center wall
beneath previous
sample taken.
sample taken on
ceiling tile, tile
was placed on
access, tile
sampled is located
towards far end of
room, no light
available.
sample on floor at
entrance of
janitorial room.
room is numbered
24 on door way. nc
light available, no
furniture in room.
sample taken on
floor towards the
left wall of room.
no light in room.
Sample taken from
air exchange.
sample of ceiling
tile taken, ceiling
tile was placed on
floor for easier
access, right side
of tile was
sampled.
sample taken of
floor at entrance o]
room 201.
template was used
floor sample near
window with duct
work taken.
template was used
Lab ID
3
3
3
3
3
6
4
4
3
5
5
7
1
4
Detected
No
No
No
No
No
No
No
No
Yes
Yes
No
Yes
No
No
Spread Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Filter Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
NA
ND
ND
1.2
22.0
ND
0.6
ND
ND
G-34
-------
Barcode
3574
2748
2675
2238
2684
2047
2051
3695
Round
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
Floor
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Room
Room 201 A
Room 201 A
Room 201 A
Room 201 A
Room 201 A
Room 202
Room 202
Room 203
X
28.715
26.815
29.415
29.015
29.015
22.084
22.178
24.949
y
7.667
8.967
6.067
6.940
8.940
16.045
16.045
10.849
z
3.000
3.000
3.697
3.000
3.000
4.194
3.000
3.000
Method
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Area
(sqft)
4.000
0.694
0.694
0.694
0.694
0.694
0.694
4.000
Operator
Patel
Patel
Patel
Patel
Patel
Johnson
Johnson
Patel
Acquisition
Date
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
Acquisition
Time
3:01:00 PM
2:51:00 PM
3:07:00 PM
2:57:00 PM
2:53:00 PM
1:59:00 PM
2:02:00 PM
2:07:00 PM
Object
Ceiling
Floor
Wall
Floor
Floor
Floor
Floor
Ceiling
Texture
Textured
Smooth
Textured
Smooth
Smooth
Smooth
Smooth
Textured
Orientation
Horizontal
Downward
Horizontal
Downward
Vertical
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Blank
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
sample of ceiling
taken, left side of
tile was sampled.
black particles
noticeable, tile is
located near left
window.
floor sample taken
on entrance of
room 201 A.
template was used
sample taken on
wall near window
opposite of
taken on lower len
of window.
template was used
no furniture in
room.
floor sample taken
to right of window
opposite entrance.
sample is near
right far corner of
room oppositte
entrance. ofroom.
floor sample taken
on left side of
room near left
window, template
was used.
sample taken on
ceiling tile, side to
right of entrance
was sampled.
minimal light
available, ceiling
tile was placed on
floor for easier
access, this room
has no furniture.
Lab ID
6
4
4
4
1
5
5
7
Detected
No
No
No
No
No
Yes
No
No
Spread Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
ND
Filter Plate Results
(CFU/sq ft)
NA
ND
ND
ND
ND
3.2
ND
ND
G-35
-------
Barcode
2712
2587
2709
3570
2710
2711
4065
Round
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
Floor
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Room
Room 203
Room 203
Room 203
Room 203A
Room 203A
Room 203A
Room 204
X
23.750
24.949
24.949
23.449
25.049
24.008
20.293
y
10.050
10.149
12.049
8.240
8.840
9.540
17.290
z
3.493
3.000
3.000
3.000
3.000
3.000
3.000
Method
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Sponge Wipe
Sponge Wipe
Vacuum Sock
Area
(sqft)
0.694
0.694
0.694
4.000
0.694
0.694
4.000
Operator
Patel
Patel
Patel
Patel
Patel
Patel
Johnson
Acquisition
Date
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
Acquisition
Time
2:15:00 PM
2:12:00 PM
2:01:OOPM
2:29:00 PM
2:24:00 PM
2:22:00 PM
1:51:00 PM
Object
Wall
Floor
Floor
Ceiling
Floor
Floor
Ceiling
Texture
Textured
Smooth
Smooth
Textured
Smooth
Smooth
Porous
Orientation
Vertical
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Horizontal
Upward
Blank
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
sample taken on
wall near door waj
of room 204 A.
minimal light
available.
-------
Barcode
3140
3139
3168
3688
2707
2192
2814
3573
3545
3521
3183
3138
3141
3157
3130
Round
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
VHP Post-
Decon
Decon
VHP Post-
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
Floor
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Room
Room 204
Room 204
Room 204
Room 205
Room 205
Room 205
Room 205
Room 206
Room 206
Room 206
Room 206
Room 206
Room 206
Room 206
Room 206
X
19.293
19.693
20.293
19.625
19.625
19.625
20.425
17.477
17.790
17.710
17.844
17.647
17.065
15.830
17.396
y
16.214
16.214
14.590
8.750
7.850
6.269
11.650
16.565
17.433
18.596
19.626
20.350
20.368
16.807
16.744
z
3.000
3.000
3.000
3.000
3.000
3.594
3.000
3.000
3.000
3.800
3.000
3.000
3.296
3.997
3.000
Method
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqft)
0.694
0.694
0.694
4.000
0.694
0.694
0.694
4.000
4.000
0.694
0.694
0.694
0.694
0.694
0.694
Operator
Johnson
Johnson
Johnson
Patel
Patel
Patel
Patel
Johnson
Johnson
Johnson
Johnson
Johnson
Johnson
Johnson
Johnson
Acquisition
Date
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
Acquisition
Time
1:37:00 PM
1:33:00 PM
1:28:00 PM
1:54:00 PM
1:47:00 PM
1:49:00 PM
1:45:00 PM
1:05:00 PM
1:15:00 PM
1:00:00 PM
12:55:00 PM
12:46:00 PM
12:41:00 PM
12:35:00 PM
1:08:00 PM
Object
Floor
Wall
Floor
Ceiling
Floor
Wall
Floor
Table
Ceiling
Table
Floor
Workbench
Workbench
Floor
Table
Texture
Smooth
Textured
Smooth
Porous
Smooth
Textured
Smooth
Smooth
Porous
Smooth
Porous
Porous
Porous
Smooth
Smooth
Orientation
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Horizontal
Downward
Vertical
Horizontal
Horizontal
Upward
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Blank
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
BROOM Notes
No lighting in
room.
sample taken of
ceiling tile, tile
was placed on
floor for easier
access, sample
taken on side
closest to entrance
sample taken on
room, sample is
closer to window
than entrance.
sample taken on
wall, template
used.
sampl taken on
floor next to
entrance of room
205. unexpected
error with broom
occurred, restarted
and appears to
work fine, template
was used .
[B. Melton moved
from 208 to 206]
[B. Melton moved
from 208 to 206]
[B. Melton moved
from 208 to 206]
Sawdust on floor
forced to tap
sample taken.
[B. Melton moved
from 208 to 206
and moved positor
per notes]
Tools moved for
sampling.
[B. Melton moved
from 208 to 206
and moved to
workbench]
[B. Melton moved
from 208 to 206
and moved to
workbench]
[B. Melton moved
from 208 to 206]
[B. Melton moved
from 208 to 206]
Lab ID
5
5
3
7
1
1
4
5
5
3
5
5
5
5
5
Detected
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
Spread Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Filter Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
G-37
-------
Barcode
1494
1628
3587
3556
2818
2823
2713
2704
2708
2829
2816
2744
Round
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Floor
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Room
Room 206
Room 206
Room 207
Room 207
Room 207
Room 207
Room 207
Room 207
Room 207
Room 207
Room 207
Room 207
X
17.593
16.582
17.553
17.353
17.353
17.353
15.653
17.968
15.653
15.653
15.653
15.653
y
16.744
16.816
10.259
11.259
11.259
9.159
6.550
6.859
8.750
6.550
8.150
8.150
z
3.000
5.800
3.000
3.000
3.000
3.796
3.697
3.596
3.798
3.772
3.000
3.698
Method
Swab
Swab
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqft)
0.028
0.028
4.000
4.000
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
Operator
Johnson
Johnson
Patel
Patel
Patel
Patel
Patel
Patel
Patel
Patel
Patel
Patel
Acquisition
Date
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
Acquisition
Time
1:03:00 PM
12:50:00 PM
1:31:00 PM
1:26:00 PM
1:23:00 PM
1:19:00 PM
1:09:00 PM
1:17:00 PM
1:07:00 PM
1:02:00 PM
12:57:00 PM
1:13:00 PM
Object
Table
Supply Vent
Floor
Floor
Table
Table
Wall
Table
Table
Floor
Table
Texture
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Textured
Smooth
Smooth
Smooth
Smooth
Orientation
Horizontal
Upward
Vertical
Horizontal
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Horizontal
Vertical
Horizontal
Downward
Horizontal
Horizontal
Downward
Horizontal
Blank
TRUE
FALSE
FALSE
TRUE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
[B. Melton moved
from 208 to 206]
[B. Melton moved
from 208 to 206]
sample taken on
top side of ceiling
tile farthest away
from entrance.
black particles
noticeable, no
template used, tile
placed on floor foi
easier access.
blank
sample taken on
table surface with
mail, sample
location is near
center of table.
template used
sample taken on
table surface near
center of table.
sample is adjacent
to previous sample
sample taken on
left wall in front ol
a table with mail.
sample top surface
of table adjacent to
previous sample.
template used.
sample taken near
center of table.
template used.
sampled left cornel
nearest entrance.
sample taken on
floor at entrance o]
room 207.
template used.
sample taken on
table surface on
nearest window-
farthest point from
room entrance.
template used.
Lab ID
1
1
6
7
4
4
4
4
4
4
4
4
Detected
No
Yes
No
No
No
No
No
No
No
No
No
No
Spread Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Filter Plate Results
(CFU/sq ft)
ND
89.9
NA
ND
ND
ND
ND
ND
ND
ND
ND
ND
G-38
-------
Barcode
1650
1606
3603
3548
3717
Round
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
Floor
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Room
Room 207
Room 207
Room 208
Room 208
Room 208
x
16.370
17.353
13.755
13.200
13.512
y
7.150
11.259
20.389
16.870
18.456
z
3.000
3.000
3.594
5.800
3.595
Method
Swab
Swab
Vacuum Sock
Vacuum Sock
Vacuum Sock
Area
(sqft)
0.028
0.028
4.000
4.000
4.000
Operator
Patel
Patel
Patel
Patel
Patel
Acquisition
Date
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
Acquisition
Time
1:36:00 PM
1:24:00 PM
12:20:00 PM
12:51:00 PM
12:45:00 PM
Object
Ceiling
Floor
Chair
Ceiling
Chair
Texture
Metal
Smooth
Smooth
Porous
Carpet
Orientation
Horizontal
Upward
Horizontal
Downward
Horizontal
Upward
Horizontal
Downward
Horizontal
Downward
Blank
FALSE
TRUE
FALSE
FALSE
FALSE
BROOM Notes
ceiling vent
sampled with
swab, swab is
noticeably brown
after sampling.
vent is near center
of room.
-------
Barcode
3667
3552
3604
3498
3093
3126
1630
Round
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
Floor
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Room
Room 208
Room 208
Room 208
Room 208
Room 208
Room 208
Room 208
X
13.738
14.726
14.936
12.876
15.294
13.602
12.792
y
19.484
20.390
19.303
19.961
18.225
15.145
18.600
z
4.490
3.000
3.000
3.698
3.697
3.798
4.096
Method
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Area
(sqft)
4.000
4.000
4.000
0.694
0.694
0.694
0.028
Operator
Patel
Patel
Patel
Patel
Patel
Patel
Patel
Acquisition
Date
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
Acquisition
Time
12:26:00 PM
12:17:00 PM
12:03:00 PM
12:38:00 PM
11:58:00 AM
11:38:00 AM
12:41:00 PM
Object
Floor
Floor
Floor
File cabinet
Floor
Floor
Monitor
Texture
Smooth
Smooth
Smooth
Metal
Smooth
Smooth
Smooth
Orientation
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Vertical
Blank
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
when stading with
back towards
entrance, sample
taken on far left,
upper corner of
partition facing
window, template
was not used.
[B. Melton moved
to partition from
near doorway per
photo]
vacuum sample on
far right corner of
room away from
entrance and
adjacent to front
side of filing
cabinet, template
was used.
[B. Melton moved
from doorway per
notes]
Vacuum sample ol
floor adjacent to
right wall and next
Template was
used. [B. Melton
moved from same
location in room
206 to 208]
sample on top of
filing cabinet on
left side of room.
template was used
[B. Melton moved
slightly to top of
cabinet]
sample on right
wall adjacent to
filing cabinet.
[B. Melton nudged
through wall from
room 206 to 208]
sample taken on
table closest to
door entrance.
sample of
computer monitor
on upper right side
when facing
monitor, template
was used
[B. Melton moved
to monitor from
round table per
notes]
Lab ID
7
7
6
4
4
1
1
Detected
No
Yes
No
No
No
No
No
Spread Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
Filter Plate Results
(CFU/sq ft)
ND
0.6
NA
ND
ND
ND
ND
G-40
-------
Barcode
1624
3636
3708
3682
2600
1975
2655
2596
2603
3557
3712
4075
3606
3613
Round
VHP Post-
VHP Post-
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
VHP Post-
VHP Post-
Floor
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Room
Room 208
Room 209
Room 209
Room 209
Room 209
Room 209
Room 209
Room 209
Room 209
Room 210
Room 210
Room 210
Room 210
Room 210
X
13.871
14.756
13.778
14.730
13.573
12.600
13.828
15.021
12.763
10.731
10.155
10.411
10.646
11.564
y
20.045
10.552
8.096
10.877
12.017
6.868
6.893
9.373
11.456
16.288
15.605
19.854
20.324
19.513
z
5.800
3.000
3.000
3.000
4.000
3.400
3.800
4.000
4.000
3.000
5.182
4.391
3 394
3.000
Method
Swab
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Vacuum Sock
Area
(sqft)
0.028
4.000
4.000
4.000
0.694
0.694
0.694
0.694
0.694
4.000
4.000
4.000
4.000
4.000
Operator
Patel
Patel
Patel
Patel
Patel
Patel
Patel
Patel
Patel
Mclntyre
Mclntyre
Acquisition
Date
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
Acquisition
Time
12:32:00 PM
6:14:00 PM
6:08:00 PM
6:02:00 PM
5:48:00 PM
5:59:00 PM
5:56:00 PM
5:53:00 PM
5:50:00 PM
1:50:00 PM
1:46:00 PM
1:25:00 PM
1:20:00 PM
1:16:00 PM
Object
Ceiling
Ceiling
Floor
Floor
Table
Wall
Table
Table
Table
Floor
Ceiling
Wall
Texture
Metal
Textured
Carpet
Carpet
Smooth
Textured
Smooth
Smooth
Smooth
Textured
Textured
Cloth
Cloth
Smooth
Orientation
Horizontal
Horizontal
Horizontal
Downward
Horizontal
Downward
Horizontal
Vertical
Horizontal
Downward
Horizontal
Downward
Horizontal
Vertical
Vertical
Horizontal
Downward
Horizontal
Downward
Blank
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
sample of ceiling
vent on far side of
room near window
sample taken of
ceiling tile near
209. tile was
placed on floor foi
easier access, point
manually picked.
sample taken on
floor in front of
round table, point
manually picked.
sample taken on
floor near entrance
of room 209. point
manually picked.
sample taken on
table surface to
right of entrance o:
room 209. point
manually picked.
sample of wall
taken to right of
round table.
adequate light
available, point
manually picked.
sample taken on
center of table
surface located at
far end of room.
table located near
window.
sample taken on
stove top- far right
corner when facing
it. template used.
sample taken on
table surface to
right of sink.
1ST of 3
red
Lab ID
1
7
7
6
1
1
6
4
1
3
5
5
3
5
Detected
No
No
No
No
Yes
No
NA
No
No
No
No
No
Yes
No
Spread Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
NA
ND
ND
ND
ND
ND
29.2
ND
Filter Plate Results
(CFU/sq ft)
ND
ND
ND
NA
5.9
ND
NA
ND
ND
ND
ND
ND
10.9
ND
G-41
-------
Barcode
4073
3527
3128
3526
3136
3137
3127
1696
1670
1524
3630
3643
3540
3702
3665
4076
2918
2207
2276
2903
1609
1584
3601
3614
Round
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
Floor
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Room
Room 210
Room 210
Room 210
Room 210
Room 210
Room 210
Room 210
Room 210
Room 210
Room 210
Room 211
Room 211
Room 211
Room 211
Room 211
Room 211
Room 211
Room 211
Room 211
Room 211
Room 211
Room 211
Room 212
Room 212
X
11.399
10.924
11.158
11.543
11.948
9.941
9.557
10.902
10.134
11.585
10.198
11.234
10.443
10.817
11.881
10.256
10.888
10.543
9.551
9.637
10.917
9.623
8.379
6.379
y
19.054
14.922
16.331
14.687
20.260
19.790
17.228
19.875
18.338
16.182
10.715
11.276
6.185
8.075
9.413
11.959
9.470
10.744
6.717
11.412
10.715
9.427
16.054
16.455
z
3.000
3.000
3.000
4.296
4.270
3.997
3.000
5.238
3.991
3.000
3.000
5.800
3.000
4.000
4.000
3.000
4.000
3.000
5.000
4.000
3.000
5.000
3.000
5.157
Method
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Swab
Swab
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Swab
Vacuum Sock
Vacuum Sock
Area
(sqft)
4.000
0.694
0.694
0.694
0.694
0.694
0.694
0.028
0.028
0.028
4.000
4.000
4.000
4.000
4.000
4.000
0.694
0.694
0.694
0.694
0.028
0.028
4.000
4.000
Operator
Mclntyre
Mclntyre
Mclntyre
Mclntyre
Mclntyre
Mclntyre
Mclntyre
Mclntyre
Mclntyre
Mclntyre
Mclntyre
Mclntyre
Mclntyre
Mclntyre
Mclntyre
Mclntyre
Mclntyre
Mclntyre
Mclntyre
Mclntyre
Mclntyre
Mclntyre
Mclntyre
Mclntyre
Acquisition
Date
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
Acquisition
Time
1:11:00 PM
1:00:00 PM
1:51:00 PM
1:42:00 PM
1:34:00 PM
1:32:00 PM
1:05:00 PM
1:39:00 PM
1:08:00 PM
1:52:00 PM
4:51:00 PM
4:50:00 PM
4:38:00 PM
4:30:00 PM
4:21:OOPM
4:05:00 PM
4:15:00 PM
4:58:00 PM
4:43:00 PM
4:13:00 PM
5:00:00 PM
4:19:00 PM
12:51:00 PM
12:45:00 PM
Object
Floor
Floor
Floor
Wall
File cabinet
Desk
Floor
Ceiling
Desk
Floor
Floor
Ceiling
Floor
Chair
Couch
Floor
Countertop
Floor
File cabinet
Wall
Floor
Table
Floor
Ceiling
Texture
Smooth
Smooth
Textured
Textured
Metal
Metal
Smooth
Metal
Smooth
Textured
Porous
Porous
Porous
Porous
Porous
Porous
Smooth
Porous
Smooth
Textured
Porous
Smooth
Smooth
Porous
Orientation
Horizontal
Downward
Horizontal
Downward
Vertical
Vertical
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Vertical
Horizontal
Downward
Vertical
Horizontal
Upward
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Horizontal
Upward
Horizontal
Downward
Vertical
Horizontal
Upward
Vertical
Horizontal
Downward
Vertical
Blank
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
TRUE
FALSE
TRUE
FALSE
BROOM Notes
On the floor as yoi
walk in.
2
next to trash can
computer
3
coffee table
book shelf
tv
blank
celing tile
Lab ID
3
6
6
6
6
6
6
1
1
1
6
6
3
3
3
5
6
6
6
6
1
1
3
5
Detected
No
NA
No
NA
No
No
No
Yes
No
No
No
Yes
No
Yes
No
No
NA
No
NA
No
No
No
No
No
Spread Plate Results
(CFU/sq ft)
ND
NA
ND
NA
ND
ND
ND
349295.1
ND
ND
ND
27.9
ND
4.2
ND
ND
NA
ND
NA
ND
ND
ND
ND
ND
Filter Plate Results
(CFU/sq ft)
ND
NA
NA
NA
NA
NA
NA
ND
ND
ND
NA
NA
ND
ND
ND
ND
NA
NA
NA
NA
ND
ND
ND
ND
G-42
-------
Barcode
3610
3564
3664
3532
3659
3590
3026
3068
3070
3089
1629
1595
1680
3600
3645
3546
3547
3565
Round
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
Floor
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Room
Room 212
Room 212
Room 212
Room 212
Room 212
Room 212
Room 212
Room 212
Room 212
Room 212
Room 212
Room 212
Room 212
Room 213
Room 213
Room 213
Room 213
Room 213
X
8.879
7.352
8.379
8.729
8.579
7.879
8.679
7.321
7.258
8.379
8.379
6.679
8.379
7.342
8.185
8.618
6.937
7.718
y
15.155
19.926
19.554
18.862
15.755
18.643
15.655
15.763
18.154
15.555
20.156
15.155
15.555
10.403
7.065
7.667
8.346
11.841
z
4.189
3.000
3.993
3.295
3.896
3.596
3.589
4.083
4.296
3.000
5.196
3.295
3.000
3.000
5.800
3.000
3.000
3.000
Method
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Swab
Swab
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Area
(sqft)
0.014
4.000
4.000
4.000
4.000
4.000
0.694
0.694
0.694
0.694
0.028
0.028
0.028
4.000
4.000
4.000
4.000
4.000
Operator
Mclntyre
Mclntyre
Mclntyre
Mclntyre
Mclntyre
Mclntyre
Mclntyre
Mclntyre
Mclntyre
Mclntyre
Mclntyre
Mclntyre
Mclntyre
Mclntyre
Mclntyre
Mclntyre
Mclntyre
Mclntyre
Acquisition
Date
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
Acquisition
Time
12:35:00 PM
12:16:00 PM
12:10:00 PM
12:01:00 PM
11:56:00 AM
11:39:00 AM
12:24:00 PM
11:21:00 AM
11:29:00 AM
12:53:00 PM
12:39:00 PM
11:45:00 AM
12:54:00 PM
3:54:00 PM
3:42:00 PM
2:30:00 PM
2:21:OOPM
1:57:00 PM
Object
Wall
Floor
Floor
Floor
Chair
Chair
File cabinet
Floor
Wall
Floor
Ceiling
Desk
Floor
Floor
Ceiling
Bed
Floor
Floor
Texture
Metal
Smooth
Smooth
Smooth
Cloth
Cloth
Metal
Smooth
Textured
Smooth
Metal
Glass
Smooth
Porous
Porous
Porous
Carpet
Carpet
Orientation
Vertical
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Horizontal
Upward
Vertical
Horizontal
Downward
Vertical
Vertical
Horizontal
Downward
Vertical
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Blank
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
TRUE
TRUE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
Partisan wasn't
able to line up.
There might be to
exstra cordinates.
Floor ,next to
filling cabinet .
Next to the wall.
The red chair was
moved around ,so
we took the sampli
. In the order they
were laid out.
The sample was 01
the wall next to a
desk.
blank
computer
The last 3 samples
Z cordinates have
been put in
manualy
In front of bed
when you 1st walk
in.
Lab ID
5
3
6
5
3
5
6
6
6
6
1
1
1
3
5
6
4
5
Detected
No
No
No
No
No
No
NA
NA
NA
No
No
No
No
No
No
Yes
Yes
No
Spread Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
NA
NA
NA
ND
ND
ND
ND
ND
ND
92.9
ND
ND
Filter Plate Results
(CFU/sq ft)
ND
ND
NA
ND
ND
ND
NA
NA
NA
NA
ND
ND
ND
ND
ND
NA
0.6
ND
G-43
-------
Barcode
3696
2672
2609
2610
2602
2347
2435
1971
2248
2259
2084
2279
2594
2210
2278
3581
2083
2494
2930
2270
2271
Round
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
VHP Post-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Floor
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Room
Room 213
Room 213
Room 213
Room 213
Room 213
Room 213
Stairwell
Bathroom M
Bathroom M
Bathroom M
Bathroom M
Bathroom W
Bathroom W
Bathroom W
Bathroom W
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
X
8.588
7.715
6.624
8.353
8.962
8.528
23.777
26.044
26.059
25.088
24.693
27.373
24.914
24.122
27.251
25.146
22.978
5.481
3.915
6.150
9.481
y
8.622
10.403
10.698
6.082
6.863
11.727
19.425
20.556
17.980
19.291
20.525
14.559
17.058
15.315
16.799
8.683
17.630
13.436
13.332
14.013
14.137
z
3.000
3.000
5.800
5.000
5.000
3.798
3.000
0.000
0.000
0.000
0.000
0.100
0.000
0.000
0.000
0.000
0.000
0.000
0.000
2.800
0.000
Method
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqft)
4.000
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
4.000
0.694
0.694
0.694
0.694
0.694
Operator
Mclntyre
Mclntyre
Mclntyre
Mclntyre
Mclntyre
Mclntyre
Johnson
Thomas
Thomas
Thomas
Thomas
Thomas
Thomas
Thomas
Thomas
Thomas
Thomas
Thomas
Thomas
Thomas
Thomas
Acquisition
Date
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/22/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
Acquisition
Time
2:13:00 PM
3:55:00 PM
3:50:00 PM
3:37:00 PM
3:33:00 PM
2:02:00 PM
2:34:00 PM
11:19:00 AM
11:18:00 AM
11:16:00 AM
11:14:00 AM
10:59:00 AM
11:05:00 AM
10:54:00 AM
11:03:00 AM
10:12:00 AM
11:10:00 AM
8:29:00 AM
8:33:00 AM
8:38:00 AM
8:49:00 AM
Object
Bed
Floor
File cabinet
Wall
Cabinet
Sink
Floor
Floor
Floor
Floor
Floor
Wall
Floor
Floor
Floor
Floor
Ceiling
Floor
Wall
Supply Vent
Floor
Texture
Porous
Porous
Metal
Textured
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Orientation
Horizontal
Downward
Vertical
Horizontal
Downward
Vertical
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Blank
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
bed
Under window .
Floor sponge stick
Floor sponge stick
Floor sponge stick
Floor sponge stick
Vertical sponge
stick.
Floor sponge stick
Floor sponge stick
Floor sponge stick
Vac blank.
Ceiling sponge
stick of overhead
light ballast.
Sample taken at
end furthest from
door.
Horizontal floor
sponge. Delay due
to suit breach.
Sponge stick wipe
of inside of hall
door.
Sample taken on
inside of air
supply.
Floor sponge wipe
Sample collected
to left.
Lab ID
3
6
6
6
6
6
5
5
5
5
5
7
5
7
5
5
7
1
7
1
1
Detected
No
NA
NA
No
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Spread Plate Results
(CFU/sq ft)
ND
NA
NA
ND
334.4
418.0
ND
91580.5
161561.9
131755.0
168804.8
710.4
186641.0
290149.1
266869.9
ND
118478.7
268309.9
2227.1
291589.0
333395.4
Filter Plate Results
(CFU/sq ft)
ND
NA
NA
NA
NA
NA
ND
ND
TNTC
TNTC
TNTC
261.4
TNTC
TNTC
TNTC
ND
TNTC
ND
ND
NA
NA
G-44
-------
Barcode
2272
2273
2356
2906
2513
2512
2490
2800
2547
2269
2209
2885
2902
2400
2392
2419
3293
3294
3295
Round
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Floor
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Room
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
X
13.186
13.643
13.091
16.525
17.748
17.094
21.290
14.934
26.337
23.598
22.485
24.789
27.348
25.295
26.188
27.438
25.146
22.676
22.200
y
12.875
14.451
16.140
13.478
13.450
14.317
13.958
15.552
6.501
12.917
16.481
11.348
11.558
11.378
9.326
9.685
8.683
8.234
9.749
z
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
2.800
0.000
0.000
2.800
0.000
0.000
0.000
0.000
Method
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqft)
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
Operator
Thomas
Thomas
Thomas
Thomas
Thomas
Thomas
Thomas
Thomas
Thomas
Thomas
Thomas
Thomas
Thomas
Thomas
Thomas
Thomas
Thomas
Thomas
Thomas
Acquisition
Date
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
Acquisition
Time
8:54:00 AM
8:57:00 AM
9:01:00 AM
9:08:00 AM
9:42:00 AM
9:45:00 AM
9:50:00 AM
9:05:00 AM
10:21:00 AM
9:56:00 AM
10:48:00 AM
10:43:00 AM
10:40:00 AM
10:38:00 AM
10:30:00 AM
10:23:00 AM
10:17:00 AM
10:04:00 AM
10:02:00 AM
Object
Floor
Floor
Floor
Floor
Floor
Wall
Return Vent
Floor
Floor
Floor
Floor
Ceiling
Floor
Floor
Ceiling
Floor
Floor
Floor
Wall
Texture
Smooth
Smooth
Smooth
Smooth
Smooth
Textured
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Textured
Orientation
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Blank
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
BROOM Notes
Floor sponge wipe
Floor sponge wipe
Floor sponge wipe
Sponge stick
blank.
Floor sponge wipe
Vertical wall
sponge wipe.
Horizontal air duct
side opposite
stairwell.
Floor sponge wipe
Floor sponge wipe
Floor sponge wipe
Floor sponge stick
Sponge stick of
overhead light
ballast left side
closest to door.
Floor sponge stick
Floor sponge stick
Sponge stick of
ceiling light ballasl
furthest from
northeast door.
Floor sponge wipe
Sponge blank.
Floor sponge wipe
Wall sponge wipe.
Lab ID
1
1
1
5
1
1
1
7
1
1
7
1
1
7
1
1
2
5
1
Detected
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Spread Plate Results
(CFU/sq ft)
166649.7
319188.0
268789.9
ND
280789.4
1679.9
457182.8
221175.7
329267.6
339347.2
244214.8
163193.9
198568.5
261734.1
222423.6
235191.1
ND
47604.6
2183.9
Filter Plate Results
(CFU/sq ft)
NA
NA
NA
ND
NA
NA
NA
TNTC
NA
NA
TNTC
NA
NA
ND
NA
NA
ND
18.6
NA
G-45
-------
Barcode
2559
3683
2370
2249
2250
2557
2492
2533
2151
2915
3572
3544
3566
3560
2334
2333
2421
2280
2202
2505
Round
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Floor
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Room
Corridor+Lobby
Mechanical Room
Mechanical Room
Mechanical Room
Mechanical Room
Room 101
Room 101
Room 101
Room 101
Room 101
Room 101 A
Room 101 A
Room 101 A
Room 101 A
Room 101 A
Room 101 A
Room 101 A
Room 101 A
Room 101 A
Room 101 A
X
26.217
17.729
16.742
16.970
18.837
16.793
16.064
17.626
21.766
19.742
19.314
19.419
17.232
21.608
19.075
20.232
21.532
21.532
21.521
21.486
y
10.193
19.169
19.230
18.087
20.465
12.073
11.230
10.161
11.269
10.941
7.834
6.814
9.114
9.047
6.596
6.614
7.027
6.214
7.306
7.851
z
0.200
1.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.583
0.500
0.586
0.591
0.500
0.500
Method
Sponge Wipe
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqft)
0.694
4.000
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
4.000
4.000
4.000
4.000
0.694
0.694
0.694
0.694
0.694
0.694
Operator
Thomas
Thomas
Thomas
Thomas
Thomas
Thomas
Thomas
Thomas
Thomas
Thomas
Nickel
Nickel
Nickel
Nickel
Nickel
Nickel
Nickel
Nickel
Nickel
Nickel
Acquisition
Date
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
Acquisition
Time
10:27:00 AM
11:34:00 AM
11:25:00 AM
11:23:00 AM
11:26:00 AM
9:15:00 AM
9:18:00 AM
9:24:00 AM
9:31:00 AM
9:37:00 AM
10:55:00 AM
10:03:00 AM
9:41:00 AM
10:53:00 AM
9:58:00 AM
10:19:00 AM
10:28:00 AM
10:31:00 AM
10:36:00 AM
10:41:00 AM
Object
Wall
Return Vent
Wall
Floor
Floor
Floor
Wall
Floor
Floor
Ceiling
Floor
Chair
Floor
Cabinet
Desk
Desk
Desk
Desk
Desk
Cabinet
Texture
Porous
Smooth
Smooth
Smooth
Smooth
Smooth
Textured
Textured
Smooth
Smooth
Carpet
Porous
Carpet
Metal
Smooth
Smooth
Smooth
Smooth
Smooth
Metal
Orientation
Vertical
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Blank
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
Vertical sponge
stick.
Vac sample of
furnace filter.
Wall sponge stick.
Floor sponge stick
Floor sponge stick
Floor sponge wipe
Wall sponge stick
wipe.
Floor sponge wipe
Floor sponge wipe
Sample of half of
ballast closest to
101 A.
last samplle
iincorrectly cited
onn wrong desk
Lab ID
1
2
5
5
5
1
1
1
5
5
5
4
4
4
2
2
2
2
2
2
Detected
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Spread Plate Results
(CFU/sq ft)
2721.5
1950111.2
3628.7
398625.0
318228.0
240230.9
571.2
385185.5
258710.3
221751.6
ND
241.7
21917.9
9083.9
103095.3
87548.7
114043.7
117960.4
124747.3
295668.9
Filter Plate Results
(CFU/sq ft)
NA
TNTC
1690.8
TNTC
TNTC
NA
453.6
NA
TNTC
TNTC
ND
NA
NA
NA
-1438.5
-1438.5
-1438.5
-1438.5
-1438.5
-1438.5
G-46
-------
Barcode
2357
2336
2568
2420
2534
3553
3597
3586
3697
2651
2340
2671
2670
2337
2511
2489
2485
2650
2257
Round
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Floor
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Room
Room 101A
Room 101A
Room 101A
Room 101A
Room 101A
Room 102
Room 102
Room 102
Room 102
Room 102
Room 102
Room 102
Room 102
Room 102
Room 102
Room 102
Room 102
Room 102
Room 102
X
18.232
19.371
17.879
18.001
19.227
19.860
18.783
18.402
19.221
20.195
19.846
18.698
19.175
18.791
18.299
17.663
16.900
19.299
18.356
y
6.214
9.240
9.363
8.924
8.238
16.536
15.381
16.554
16.072
16.437
17.166
16.896
14.622
16.032
16.989
17.237
15.665
16.581
14.630
z
0.578
0.500
0.750
0.000
0.000
0.000
0.000
0.000
0.000
1.000
1.500
1.500
0.000
0.000
1.000
1.000
1.000
1.000
0.000
Method
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqft)
0.694
0.694
0.694
0.694
0.694
4.000
4.000
4.000
4.000
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
Operator
Nickel
Nickel
Nickel
Nickel
Nickel
yoder
yoder
yoder
yoder
yoder
yoder
yoder
yoder
yoder
yoder
yoder
yoder
yoder
yoder
Acquisition
Date
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
Acquisition
Time
9:52:00 AM
11:03:00 AM
11:23:00 AM
11:14:00 AM
10:59:00 AM
12:09:00 PM
12:15:00 PM
12:42:00 PM
12:57:00 PM
12:21:00 PM
12:29:00 PM
12:38:00 PM
1:38:00 PM
12:55:00 PM
1:01:OOPM
1:11:00 PM
1:20:00 PM
12:51:00 PM
1:31:00 PM
Object
Cabinet
Cabinet
Desk
Desk
Floor
Floor
Floor
Floor
Floor
File cabinet
Desk
Desk
Desk
Floor
File cabinet
File cabinet
File cabinet
Desk
Desk
Texture
Metal
Metal
Plastic
Plastic
Smooth
Carpet
Carpet
Carpet
Metal
Metal
Smooth
Smooth
Plastic
Metal
Metal
Metal
Metal
Smooth
Plastic
Orientation
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Blank
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
on uv aps
blank
top back left of
uvaps
blank
sticker residue on
surface
top front of file
cabinet; cabinet is
rotated 90 degrees
from broom
diagram
in center desk
drawer
top back right of
uvaps
Lab ID
2
4
2
4
4
5
5
5
5
2
2
4
4
4
4
4
4
4
2
Detected
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Spread Plate Results
(CFU/sq ft)
123326.6
262022.1
48334.2
62776.8
ND
14625.8
10500.6
3666.9
ND
327347.7
149567.2
184313.1
145751.3
ND
223191.6
190303.2
271189.8
97993.1
165917.7
Filter Plate Results
(CFU/sq ft)
-1438.5
NA
-1438.5
NA
NA
TNTC
TNTC
TNTC
ND
-1438.5
-1438.5
NA
NA
NA
NA
NA
NA
NA
-1438.5
G-47
-------
Barcode
2486
2109
2570
2405
2108
2927
2510
2535
2845
2841
1675
1601
2849
2540
2882
2478
2473
2772
2898
Round
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Floor
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Room
Room 102
Room 103
Room 103
Room 103
Room 103
Room 103
Room 103
Room 103
Room 103
Room 103
Room 103
Room 103
Room 104
Room 104
Room 104
Room 104
Room 104
Room 104
Room 104
X
17.990
14.633
14.666
15.568
15.510
14.432
13.179
13.371
13.766
13.269
14.698
13.165
13.836
13.660
15.159
15.470
15.565
16.389
14.970
y
14.822
10.902
11.557
11.841
7.035
6.217
6.145
6.044
6.591
7.026
11.928
8.186
19.134
18.564
20.354
20.354
20.761
19.351
18.225
z
0.500
2.600
0.000
0.000
0.000
2.600
0.000
0.300
1.000
2.000
0.000
2.000
0.000
0.000
1.000
1.000
1.200
2.600
1.000
Method
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Swab
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqft)
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.028
0.028
0.694
0.694
0.694
0.694
0.694
0.694
0.694
Operator
yoder
McCormick
McCormick
McCormick
McCormick
McCormick
McCormick
McCormick
McCormick
McCormick
McCormick
McCormick
McCormick
McCormick
McCormick
McCormick
McCormick
McCormick
McCormick
Acquisition
Date
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
Acquisition
Time
1:23:00 PM
1:47:00 PM
1:56:00 PM
1:43:00 PM
2:09:00 PM
2:13:00 PM
2:16:00 PM
2:19:00 PM
2:22:00 PM
2:34:00 PM
1:58:00 PM
2:38:00 PM
12:50:00 PM
12:43:00 PM
1:00:00 PM
1:05:00 PM
1:20:00 PM
1:27:00 PM
1:34:00 PM
Object
Desk
Ceiling
Floor
Floor
Floor
Ceiling
Floor
Wall
Table
Shelves
Floor
Shelves
Floor
Floor
Table
Table
Wall
Ceiling
Table
Texture
Metal
Metal
Metal
Smooth
Smooth
Metal
Smooth
Smooth
Smooth
Smooth
Metal
Metal
Smooth
Smooth
Porous
Porous
Paint
Metal
Smooth
Orientation
Vertical
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Blank
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
uvaps was moved
no room for
sample on desk.
sample taken on
vertical right side
of desk instead
left side nearest
door
[B. Melton moved
across room]
included pipecuttei
included monkey
wrench
side nearest the
door opposite side
of vent
Lab ID
2
2
2
1
2
2
2
2
2
2
7
7
2
2
1
1
1
1
1
Detected
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Spread Plate Results
(CFU/sq ft)
2131.1
14284.3
ND
357682.5
418688.2
128827.1
692037.9
8927.7
282373.4
530380.0
ND
163244.5
238023.0
ND
8232.6
7948.5
7159.4
60717.7
461982.6
Filter Plate Results
(CFU/sq ft)
1132.9
1439942.9
ND
NA
-1438.5
-1438.5
-1438.5
TNTC
-1438.5
-1438.5
ND
TNTC
-1438.5
ND
NA
NA
NA
NA
NA
G-48
-------
Barcode
1533
1667
3671
3542
3653
3691
2120
2503
2541
2544
2907
2341
2246
1495
3652
3588
Round
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Floor
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Room
Room 104
Room 104
Room 105
Room 105
Room 105
Room 105
Room 105
Room 105
Room 105
Room 105
Room 105
Room 105
Room 105
Room 105
Room 106
Room 106
X
15.268
14.038
10.439
11.150
10.389
10.841
11.344
12.474
9.921
10.332
11.003
12.011
11.018
11.154
10.969
12.064
y
19.676
18.836
12.236
10.871
6.478
10.295
8.099
11.153
10.860
9.632
9.003
6.761
9.493
6.619
18.401
19.756
z
2.400
0.000
0.000
0.000
0.000
0.000
2.800
1.000
0.500
1.000
2.800
1.000
2.200
2.800
0.250
0.000
Method
Swab
Swab
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Vacuum Sock
Vacuum Sock
Area
(sqft)
0.028
0.028
4.000
4.000
4.000
4.000
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.028
4.000
4.000
Operator
McCormick
McCormick
England
England
England
England
England
England
England
England
England
England
Steve Merritt
England
England
England
Acquisition
Date
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
Acquisition
Time
12:54:00 PM
12:37:00 PM
12:37:00 PM
12:45:00 PM
1:24:00 PM
12:57:00 PM
2:25:00 PM
1:03:00 PM
1:00:00 PM
1:07:00 PM
1:13:00 PM
1:20:00 PM
5:18:00 PM
1:27:00 PM
1:55:00 PM
2:00:00 PM
Object
Ceiling
Floor
Floor
Floor
Floor
Floor
Ceiling
Sink
Wall
Stove
Ceiling
Table
Ceiling
Supply Vent
Chair
Floor
Texture
Metal
Smooth
Carpet
Carpet
Carpet
Carpet
Metal
Metal
Plastic
Metal
Porous
Smooth
Smooth
Metal
Cloth
Smooth
Orientation
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Blank
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
[discard BJM]
sample taken from
ceiling light
(inside)- half
completed and
marked
[discard BJM]
sample taken on
top of ceiling tile
with sponge stick-
sponge loses
wetness for
diagonal
replacement
sample taken on
top of metal
ballast, sample
taken from next to
left side of X.
sample taken from
top of chair-
dimensions are
approximately 1 .5
ft by 2 ft
sample taken from
next to cabinet-
overlaid on
previous sample
Lab ID
7
7
6
1
1
1
4
7
7
7
7
7
2
7
1
1
Detected
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Spread Plate Results
(CFU/sq ft)
348695.0
ND
11667.3
10258.1
2392.6
10118.9
1216.8
450991.0
1857.1
675622.5
23759.1
335987.3
137913.2
1566426.6
570.9
6332.0
Filter Plate Results
(CFU/sq ft)
TNTC
ND
NA
NA
NA
NA
NA
TNTC
ND
TNTC
TNTC
TNTC
-1438.5
TNTC
NA
NA
G-49
-------
Barcode
3680
2355
2496
2268
2919
2198
2196
2252
1645
1666
3715
3723
3654
3567
2830
2335
2826
2554
Round
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Floor
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Room
Room 106
Room 106
Room 106
Room 106
Room 106
Room 106
Room 106
Room 106
Room 106
Room 106
Room 107
Room 107
Room 107
Room 107
Room 107
Room 107
Room 107
Room 107
X
11.213
11.290
10.374
9.938
10.515
10.209
12.069
10.869
11.276
10.270
8.713
9.369
8.505
8.113
8.431
8.731
7.931
7.713
y
20.442
15.824
19.899
16.510
17.946
14.748
15.895
16.296
19.739
18.552
12.014
10.232
8.350
6.731
9.414
6.414
10.631
12.231
z
0.250
2.800
0.250
0.250
0.500
0.000
1.500
2.200
2.800
0.750
0.000
0.000
0.000
1.194
0.000
1.189
0.000
0.396
Method
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Swab
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqft)
4.000
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.028
0.028
4.000
4.000
4.000
4.000
0.694
0.694
0.694
0.694
Operator
England
England
England
England
England
England
England
Steve Merritt
England
England
Pobiedzinski
Pobiedzinski
Pobiedzinski
Pobiedzinski
Pobiedzinski
Pobiedzinski
Pobiedzinski
Pobiedzinski
Acquisition
Date
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
Acquisition
Time
2:18:00 PM
2:05:00 PM
2:16:00 PM
1:41:OOPM
1:46:00 PM
1:32:00 PM
1:37:00 PM
5:14:00 PM
2:09:00 PM
1:49:00 PM
1:46:00 PM
1:52:00 PM
2:11:00 PM
2:26:00 PM
2:04:00 PM
2:21:OOPM
2:43:00 PM
2:50:00 PM
Object
Chair
Ceiling
File cabinet
Wall
Desk
Floor
Table
Ceiling
Supply Vent
Monitor
Floor
Couch
Chair
File cabinet
Table
File cabinet
Ceiling
Wall
Texture
Cloth
Metal
Metal
Plastic
Smooth
Smooth
Smooth
Smooth
Metal
Smooth
Carpet
Cloth
Leather
Metal
Porous
Metal
Metal
Textured
Orientation
Horizontal
Upward
Horizontal
Downward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Inclined
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Vertical
Blank
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
TRUE
FALSE
FALSE
BROOM Notes
sample taken on
horizontal upward
facing surface-
approx 1.5ft by 2
ft
[discard, inside
light BJM]
sponge stick
sample colocated
with rmc coupon
on table
replacement
sample taken on
top of metal
ballast, replaces
sample taken
inside light.
sample taken in
middle of monitor
floor in front of
door
first seat cushion
blank
back table left
facing tv
blank
above light fixture
wall left of door
Lab ID
1
7
4
4
4
4
4
2
7
7
6
1
6
6
2
2
2
2
Detected
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
No
No
Yes
Yes
Spread Plate Results
(CFU/sq ft)
3960.2
168.0
292117.0
3492.3
312948.2
378993.7
335219.4
48046.2
257470.1
100227.3
11959.0
12867.4
2179.3
ND
ND
ND
126715.2
1614.7
Filter Plate Results
(CFU/sq ft)
NA
160.7
NA
NA
NA
NA
NA
-1438.5
TNTC
TNTC
NA
NA
NA
NA
ND
ND
-1438.5
975.9
G-50
-------
Barcode
2484
1665
1527
3646
3692
3721
3945
2892
2153
2152
2466
2471
2165
2908
1668
1531
1631
3634
3538
3635
Round
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Floor
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Room
Room 107
Room 107
Room 107
Room 108
Room 108
Room 108
Room 108
Room 108
Room 108
Room 108
Room 108
Room 108
Room 108
Room 108
Room 108
Room 108
Room 108
Room 109
Room 109
Room 109
X
7.017
8.431
8.831
9.051
7.899
8.226
9.113
9.151
9.651
7.551
9.151
7.651
9.536
8.613
8.851
7.363
9.151
5.334
4.160
4.673
y
6.587
6.814
6.314
15.830
18.465
20.437
20.017
14.630
17.030
17.917
15.830
16.017
20.527
18.517
15.730
18.435
19.617
10.214
7.817
11.631
z
0.000
0.000
1.187
1.270
0.000
0.000
0.000
0.677
0.000
0.000
1.496
0.000
0.000
0.000
1.391
0.000
0.000
0.000
0.000
0.000
Method
Sponge Wipe
Swab
Swab
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Swab
Swab
Vacuum Sock
Vacuum Sock
Vacuum Sock
Area
(sqft)
0.694
0.028
0.028
4.000
4.000
4.000
4.000
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.028
0.028
0.028
4.000
4.000
4.000
Operator
Pobiedzinski
Pobiedzinski
Pobiedzinski
Pobiedzinski
Pobiedzinski
Pobiedzinski
Pobiedzinski
Pobiedzinski
Pobiedzinski
Pobiedzinski
Pobiedzinski
Pobiedzinski
Pobiedzinski
Pobiedzinski
Pobiedzinski
Pobiedzinski
Pobiedzinski
Carnahan
Carnahan
Carnahan
Acquisition
Date
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
Acquisition
Time
2:33:00 PM
2:48:00 PM
2:19:00 PM
1:33:00 PM
12:39:00 PM
12:49:00 PM
1:10:00 PM
1:37:00 PM
1:24:00 PM
12:28:00 PM
1:31:00 PM
12:18:00 PM
1:01:OOPM
1:21:OOPM
1:29:00 PM
12:31:00 PM
1:16:00 PM
12:45:00 PM
1:04:00 PM
1:14:00 PM
Object
Shelves
Supply Vent
File cabinet
Floor
Chair
Chair
Floor
Wall
Floor
Desk
Floor
Floor
File cabinet
Ceiling
Floor
Monitor
Return Vent
Floor
Bed
Floor
Texture
Smooth
Metal
Metal
Smooth
Cloth
Cloth
Smooth
Textured
Smooth
Smooth
Smooth
Smooth
Metal
Metal
Smooth
Smooth
Smooth
Cloth
Cloth
Carpet
Orientation
Horizontal
Upward
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Horizontal
Upward
Inclined
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Blank
FALSE
FALSE
TRUE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
TRUE
FALSE
FALSE
BROOM Notes
left of shelf
blank
blank
whole chair seat
back room chair
front middle of fih
cabinet
wall
floor middle of
room by wall
front left
blank
floor in front of
door
front left
above light fixture
blank
bottom left corner
ventilation system;
blank vacuum
sample
vacuum sample
taken on bed.
sample taken on
floor
Lab ID
2
6
6
6
6
1
1
2
2
2
2
2
2
2
7
6
7
7
7
4
Detected
Yes
Yes
No
No
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
No
Yes
Yes
No
Yes
Yes
Spread Plate Results
(CFU/sq ft)
167840.1
214858.5
ND
ND
18042.7
5467.0
9743.1
752.6
205926.6
193874.3
ND
379041.7
648695.6
107098.4
ND
33609.2
1446393.9
ND
17292.7
14709.2
Filter Plate Results
(CFU/sq ft)
ND
NA
NA
NA
NA
NA
NA
641.5
-1438.5
-1438.5
ND
-1438.5
-1438.5
-1438.5
ND
NA
TNTC
ND
TNTC
NA
G-51
-------
Barcode
3591
3703
2873
2555
2507
2502
2371
2504
3693
3948
3575
3679
2539
2515
2506
2556
2291
2381
2360
Round
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Floor
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Room
Room 109
Room 109
Room 109
Room 109
Room 109
Room 109
Room 109
Room 109
Room 110
Room 110
Room 110
Room 110
Room 110
Room 110
Room 110
Room 110
Room 110
Room 110
Room 110
X
5.504
6.249
5.576
5.921
4.015
4.069
4.146
4.175
6.158
5.195
5.453
4.951
6.640
4.187
4.431
4.873
5.659
5.973
4.539
y
8.622
12.027
10.080
8.331
6.489
9.566
12.160
11.013
20.168
20.413
16.254
18.452
20.228
19.887
20.758
16.051
14.783
17.296
17.936
z
0.000
0.000
0.000
2.178
1.082
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
2.236
0.000
0.000
0.000
Method
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqft)
4.000
4.000
0.694
0.694
0.694
0.694
0.694
0.694
4.000
4.000
4.000
4.000
0.694
0.694
0.694
0.694
0.694
0.694
0.694
Operator
Carnahan
Carnahan
Carnahan
Carnahan
Carnahan
Carnahan
Carnahan
Carnahan
Carnahan
Carnahan
Carnahan
Carnahan
Carnahan
Carnahan
Carnahan
Carnahan
Carnahan
Carnahan
Carnahan
Acquisition
Date
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
Acquisition
Time
12:37:00 PM
12:18:00 PM
12:44:00 PM
12:52:00 PM
12:58:00 PM
1:10:00 PM
1:20:00 PM
1:18:00 PM
1:55:00 PM
1:57:00 PM
1:37:00 PM
1:43:00 PM
1:59:00 PM
2:00:00 PM
2:03:00 PM
2:08:00 PM
1:25:00 PM
1:32:00 PM
1:45:00 PM
Object
Bed
Floor
Floor
Ceiling
Wall
Table
Sink
Sink
Floor
Chair
Floor
Chair
File cabinet
File cabinet
Wall
Ceiling
Floor
Floor
Desk
Texture
Cloth
Carpet
Cloth
Metal
Textured
Smooth
Smooth
Smooth
Smooth
Cloth
Smooth
Cloth
Metal
Metal
Paint
Metal
Smooth
Smooth
Smooth
Orientation
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Inclined
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Blank
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
vacuum sample on
top of bed
sample taken on
floor in door way
ofroom 109
blank sponge
sample location
sample taken in
ceiling tile on of
light closest to
entrance
sample taken on
wall.
sample taken on
table
sample taken on
sink counter right
of sink
sample taken on
counter top next to
sink on left side
sample taken on
floor in front of
filing cabinet
sample taken on
chair seat
blank vacuum
sample taken on
chair seat
sample taken on
top of filing
cabinet
sample taken on
wall 5ft up from
wall,
sample taken on
top of light in
ceiling . light
closest to entrance
sample taken on
floor at entrance o]
room
sample taken on
floor
sample taken on
desk
Lab ID
7
7
1
4
4
1
4
1
4
4
7
7
1
1
1
4
4
1
4
Detected
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Spread Plate Results
(CFU/sq ft)
42085.7
25834.8
ND
266438.0
2350.0
256790.3
239751.0
293077.0
9625.5
8792.2
16.7
8458.8
410672.5
249974.6
6277.7
39430.5
357682.5
248966.6
277189.6
Filter Plate Results
(CFU/sq ft)
TNTC
TNTC
4.3
NA
NA
NA
NA
NA
NA
NA
10.4
TNTC
NA
NA
NA
NA
NA
NA
NA
G-52
-------
Barcode
1976
1553
1636
1571
2118
2597
2141
2135
2379
2222
2127
2123
2125
3677
2375
2122
2115
2251
2784
2378
Round
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Floor
Floor 1
Floor 1
Floor 1
Floor 1
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Room
Room 110
Room 110
Room 110
Room 110
Bathroom M
Bathroom M
Bathroom M
Bathroom W
Bathroom W
Bathroom W
Copier Room
Copier Room
Copier Room
Hallway
Hallway
Hallway
Hallway
Hallway
Hallway
Hallway
X
5.338
4.951
5.286
4.270
29.559
28.951
26.484
28.684
28.835
26.714
27.558
29.735
28.657
16.996
11.293
8.693
6.693
9.837
18.027
23.253
y
16.086
19.806
16.306
18.465
20.517
20.675
19.906
17.572
17.810
15.974
13.860
13.781
14.177
13.534
13.458
13.668
12.858
12.558
13.858
13.812
z
0.000
0.000
0.000
0.000
3.000
3.000
3.000
3.000
3.000
3.000
3.000
3.000
3.250
3.000
3.000
5.000
3.000
3.250
3.000
5.000
Method
Sponge Wipe
Swab
Swab
Swab
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqft)
0.694
0.028
0.028
0.028
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
4.000
0.694
0.694
0.694
0.694
0.694
0.694
Operator
Carnahan
Carnahan
Carnahan
Carnahan
England
England
England
England
England
England
England
England
England
England
England
England
England
England
England
England
Acquisition
Date
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
Acquisition
Time
1:36:00 PM
1:52:00 PM
1:34:00 PM
1:50:00 PM
10:18:00 AM
10:21:00 AM
10:15:00 AM
10:10:00 AM
10:11:00 AM
10:07:00 AM
9:54:00 AM
9:57:00 AM
9:59:00 AM
8:46:00 AM
8:24:00 AM
8:20:00 AM
8:12:00 AM
8:36:00 AM
8:39:00 AM
8:56:00 AM
Object
Floor
Supply Vent
Floor
Monitor
Floor
Wall
Floor
Floor
Floor
Floor
Floor
Floor
Wall
Floor
Floor
Supply Vent
Floor
Wall
Floor
Return Vent
Texture
Smooth
Metal
Smooth
Smooth
Smooth
Plastic
Smooth
Smooth
Smooth
Smooth
Smooth
Textured
Plastic
Smooth
Smooth
Metal
Smooth
Plastic
Smooth
Metal
Orientation
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Blank
TRUE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
blank sponge
sample taken from
supply vent
blank swab
sample taken on
upper left corner o
screen
blank
[B. Melton vent
exterior]
sample taken from
inside ductwork
Lab ID
4
7
7
7
5
5
5
5
5
5
5
5
5
1
5
2
5
2
2
2
Detected
No
Yes
No
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
No
Yes
No
Yes
Yes
Yes
Yes
Spread Plate Results
(CFU/sq ft)
ND
1182322.0
ND
4201.1
21706.5
193.0
24594.3
ND
21023.2
29374.9
5955.6
4084.2
292.8
ND
18555.6
ND
13031.5
ND
22578.3
5828.9
Filter Plate Results
(CFU/sq ft)
NA
TNTC
ND
3975.5
TNTC
ND
TNTC
ND
TNTC
TNTC
167.2
TNTC
TNTC
ND
ND
ND
ND
11.7
TNTC
ND
G-53
-------
Barcode
2589
2453
2614
2668
2374
2822
1486
2124
2126
3946
2644
2116
2428
1986
2482
2245
2550
2361
Round
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Floor
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Room
Hallway
Hallway
Hallway
Hallway
Hallway
Hallway
Hallway
Janitor Closet
Janitor Closet
Mechanical Room
Mechanical Room
Mechanical Room
Room 201
Room 201
Room 201
Room 201
Room 201 A
Room 201 A
X
25.331
26.016
25.409
6.793
16.996
20.869
16.996
26.828
28.286
18.857
18.896
22.659
26.150
29.430
27.804
27.140
26.812
29.487
y
14.336
17.339
18.814
13.768
13.534
13.782
13.534
15.058
14.852
19.269
19.993
19.612
11.975
10.286
10.661
10.507
9.289
6.565
z
3.000
3.000
3.000
3.000
3.000
3.000
3.000
3.000
3.000
3.000
3.000
3.000
3.000
3.000
5.500
5.200
3.000
3.000
Method
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Sponge Wipe
Sponge Wipe
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqft)
0.694
0.694
0.694
0.694
0.694
0.694
0.028
0.694
0.694
4.000
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
Operator
England
England
England
England
England
England
England
England
England
England
England
England
England
England
England
Steve Merritt
England
England
Acquisition
Date
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
Acquisition
Time
8:59:00 AM
9:03:00 AM
9:06:00 AM
8:32:00 AM
8:43:00 AM
8:50:00 AM
8:42:00 AM
10:02:00 AM
10:05:00 AM
10:39:00 AM
10:33:00 AM
10:31:00 AM
9:27:00 AM
9:29:00 AM
9:34:00 AM
5:03:00 PM
9:37:00 AM
9:40:00 AM
Object
Floor
Wall
Floor
Return Vent
Floor
Floor
Floor
Floor
Floor
Return Vent
Floor
Floor
Floor
Floor
Ceiling
Ceiling
Floor
Floor
Texture
Smooth
Plastic
Smooth
Cloth
Smooth
Smooth
Smooth
Smooth
Smooth
Porous
Smooth
Smooth
Smooth
Smooth
Metal
Smooth
Smooth
Smooth
Orientation
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Blank
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
sample taken from
inside ducrwork-
duplucating
previously taken
sample from
exterior.
sample taken from
inside ductwork-
filter
[discard BJM]
sample taken from
half of light fixtun
Sampled top of
ballast on the side
marked with X.
Replaces previous
sample taken
inside light.
sample taken from
inside room 201a
Lab ID
2
2
2
2
2
2
7
5
5
1
5
5
2
2
2
2
2
5
Detected
Yes
Yes
Yes
Yes
No
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Spread Plate Results
(CFU/sq ft)
29029.3
ND
16005.0
18604.1
ND
28866.1
ND
20932.0
27359.0
8083.8
40558.5
57789.8
20274.4
20628.7
ND
12564.0
19957.6
26639.0
Filter Plate Results
(CFU/sq ft)
TNTC
9.4
ND
ND
ND
TNTC
ND
TNTC
TNTC
NA
TNTC
TNTC
TNTC
TNTC
20.3
1266530.4
TNTC
92.9
G-54
-------
Barcode
2640
2493
2895
2521
2605
2669
2616
2645
2643
2129
2130
2128
2838
2114
2376
2134
2133
Round
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Floor
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Room
Room 201 A
Room 201 A
Room 201 A
Room 201 A
Room 202
Room 202
Room 203
Room 203
Room 203
Room 203
Room 203A
Room 203A
Room 204
Room 204
Room 204
Room 205
Room 205
X
29.252
29.015
27.778
27.021
22.334
21.476
25.083
22.849
21.749
24.149
24.339
25.282
20.234
19.350
18.615
19.161
20.139
y
5.995
7.788
6.720
6.549
17.490
14.494
12.169
10.549
10.649
9.849
6.492
9.380
14.605
17.504
17.285
7.969
8.096
z
3.000
3.000
5.500
5.200
3.000
3.000
3.000
5.500
3.000
3.390
3.000
3.000
3.000
3.000
3.000
5.500
3.000
Method
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqft)
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
Operator
England
England
England
Steve Merritt
England
England
Philpot
Philpot
Philpot
Philpot
Philpot
Philpot
England
England
England
Philpot
Philpot
Acquisition
Date
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
Acquisition
Time
9:45:00 AM
9:43:00 AM
9:50:00 AM
5:06:00 PM
9:23:00 AM
9:20:00 AM
11:09:00 AM
11:12:00 AM
11:14:00 AM
11:16:00 AM
11:21:00 AM
11:18:00 AM
9:10:00 AM
9:14:00 AM
9:17:00 AM
11:04:00 AM
10:59:00 AM
Object
Wall
Floor
Wall
Ceiling
Floor
Floor
Floor
Ceiling
Floor
Wall
Floor
Floor
Floor
Floor
Wall
Ceiling
Floor
Texture
Plastic
Smooth
Plastic
Smooth
Smooth
Smooth
Smooth
Metal
Smooth
Textured
Smooth
Smooth
Smooth
Smooth
Plastic
Metal
Smooth
Orientation
Vertical
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Downward
Horizontal
Upward
Blank
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
previous sample-
2361 incorrectly
placed, belongs in
corner directly
across from door
in room 201a
[discard BJM]
sample taken from
inside light fixture
Replacement
sample for
previous sample
taken inside light.
Collected on top o:
ballast.
light balast plenun
side, removed
adjacent tile to
collect sample
plenum side balast
sample was
collected by
removing adjacent
tile
Lab ID
5
5
5
2
2
2
3
3
3
3
6
3
2
2
2
3
3
Detected
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Spread Plate Results
(CFU/sq ft)
ND
15666.6
106.6
8109.8
24479.1
22213.6
34102.7
6335.8
30094.9
100.8
25297.0
25199.1
18143.3
19799.3
26.9
10818.8
33238.7
Filter Plate Results
(CFU/sq ft)
185.8
TNTC
TNTC
792742.7
TNTC
TNTC
NA
ND
NA
40.2
NA
NA
ND
TNTC
44.9
NA
NA
G-55
-------
Barcode
2598
2119
2143
2307
2137
2136
2140
2588
2145
2142
1656
2639
2117
2131
2615
2508
2383
2369
2623
Round
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Floor
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Room
Room 205
Room 205
Room 206
Room 206
Room 206
Room 206
Room 206
Room 206
Room 206
Room 206
Room 206
Room 207
Room 207
Room 207
Room 207
Room 207
Room 207
Room 207
Room 207
X
20.534
20.783
18.001
16.169
15.742
17.464
17.822
16.794
16.642
17.330
16.642
15.571
15.547
15.547
16.053
17.467
15.571
16.353
17.220
y
6.050
12.036
20.123
14.739
17.454
18.636
19.540
20.123
16.345
18.209
16.345
6.897
7.478
8.155
9.968
10.196
6.390
11.984
11.282
z
3.496
3.000
3.778
3.000
3.000
3.694
3.000
3.784
3.000
5.800
3.000
3.700
3.700
3.700
3.000
3.700
3.700
3.000
5.500
Method
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqft)
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.028
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
Operator
Philpot
Philpot
Schuette
Schuette
Schuette
Schuette
Schuette
Schuette
Schuette
Schuette
Schuette
Philpot
Philpot
Philpot
Philpot
Philpot
Philpot
Philpot
Philpot
Acquisition
Date
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
Acquisition
Time
11:05:00 AM
10:57:00 AM
11:25:00 AM
11:00:00 AM
11:06:00 AM
11:13:00 AM
11:19:00 AM
11:28:00 AM
11:40:00 AM
11:36:00 AM
11:39:00 AM
10:42:00 AM
10:44:00 AM
10:46:00 AM
10:50:00 AM
10:54:00 AM
10:40:00 AM
10:16:00 AM
10:28:00 AM
Object
Wall
Floor
Table
Floor
Floor
Table
Floor
Table
Floor
Ceiling
Floor
Table
Table
Table
Supply Vent
Desk
Table
Floor
Ceiling
Texture
Textured
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Metal
Smooth
Smooth
Smooth
Smooth
Metal
Smooth
Smooth
Smooth
Textured
Orientation
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Blank
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
TRUE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
moved tools out o]
way to take samph
sample on floor
with woodchips
moved tools out o]
way to take samph
sponge on top of
light
outside barcoded
sample bag
dropped on floor
sponge stick blank
light balast above
light plenum side,
removed adjacent
ceiling tile to
collect sample
Lab ID
6
3
4
4
4
4
6
6
4
4
7
3
3
3
3
3
3
3
6
Detected
No
Yes
Yes
Yes
Yes
Yes
Yes
No
No
Yes
No
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Spread Plate Results
(CFU/sq ft)
ND
25727.0
1751.9
18082.8
20387.7
13958.8
6963.6
ND
ND
3543.7
ND
13343.5
14639.4
11457.2
ND
1296.0
5207.8
20879.2
9863.6
Filter Plate Results
(CFU/sq ft)
NA
NA
660.7
TNTC
TNTC
TNTC
NA
NA
ND
ND
ND
NA
NA
NA
ND
ND
ND
NA
NA
G-56
-------
Barcode
2300
1593
1737
4102
3958
2146
2138
2147
2311
2309
1564
1625
4104
3929
2836
2833
2308
2647
2144
Round
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Floor
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Room
Room 207
Room 207
Room 207
Room 208
Room 208
Room 208
Room 208
Room 208
Room 208
Room 208
Room 208
Room 208
Room 209
Room 209
Room 209
Room 209
Room 209
Room 209
Room 209
X
17.956
16.559
16.077
13.485
13.768
12.651
13.560
15.168
13.857
15.317
12.726
14.006
13.902
14.756
14.456
15.018
13.967
12.456
12.722
y
7.695
8.421
9.775
18.403
20.383
19.845
14.934
20.562
17.632
18.433
18.284
19.964
10.769
8.240
10.640
9.806
7.114
6.840
10.151
z
3.400
5.500
3.000
3.490
3.394
3.691
3.689
4.292
5.800
3.294
4.275
5.800
3.000
3.000
5.474
3.900
3.700
3.300
3.798
Method
Sponge Wipe
Swab
Swab
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Swab
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqft)
0.694
0.028
0.028
4.000
4.000
0.694
0.694
0.694
0.694
0.694
0.028
0.028
4.000
4.000
0.694
0.694
0.694
0.694
0.694
Operator
Philpot
Philpot
Philpot
Schuette
Schuette
Schuette
Schuette
Schuette
Schuette
Schuette
Schuette
Schuette
Philpot
Philpot
Philpot
Philpot
Philpot
Philpot
Philpot
Acquisition
Date
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
Acquisition
Time
10:35:00 AM
10:48:00 AM
10:51:00 AM
10:18:00 AM
10:42:00 AM
10:45:00 AM
10:09:00 AM
10:48:00 AM
10:56:00 AM
10:27:00 AM
10:22:00 AM
10:51:00 AM
9:20:00 AM
9:27:00 AM
9:46:00 AM
9:40:00 AM
9:58:00 AM
10:01:00 AM
10:03:00 AM
Object
Wall
Supply Vent
Bed
Chair
Chair
File cabinet
Table
File cabinet
Ceiling
Wall
Monitor
Supply Vent
Floor
Floor
Ceiling
Stove
Table
Wall
Countertop
Texture
Textured
Metal
Metal
Porous
Porous
Metal
Smooth
Metal
Metal
Textured
Smooth
Metal
Carpet
Carpet
Porous
Metal
Smooth
Textured
Smooth
Orientation
Vertical
Horizontal
Downward
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Blank
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
diffuser swab
sample
swab stick blank
pulled in different
place than pre-
VHP b/c RMC on
top of previous
sample
top of light
upper left of
monitor
air diffuser on
ceiling
right of stove on
floor
area is 2x2 feet,
larger than
template for the
sponge
burner and smooth
metal surface
table surface
Lab ID
6
7
7
2
2
6
4
4
4
4
7
7
1
1
3
3
3
3
6
Detected
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
No
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Spread Plate Results
(CFU/sq ft)
132.0
86423.5
ND
25.0
16.7
5740.6
7737.8
7479.1
9739.8
ND
ND
65417.8
269.9
20.0
1267.2
8495.7
7895.7
336.0
3621.9
Filter Plate Results
(CFU/sq ft)
NA
TNTC
ND
1.2
2.9
NA
TNTC
ND
TNTC
ND
ND
ND
NA
37.5
NA
NA
NA
270.1
NA
G-57
-------
Barcode
2139
2253
4054
3531
4227
2104
2113
2110
2112
2621
2837
2111
1534
1526
1617
3937
4134
4061
3939
4249
Round
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Floor
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Room
Room 209
Room 209
Room 210
Room 210
Room 210
Room 210
Room 210
Room 210
Room 210
Room 210
Room 210
Room 210
Room 210
Room 210
Room 210
Room 211
Room 211
Room 211
Room 211
Room 211
X
13.856
13.955
11.485
11.402
10.662
11.199
11.208
12.117
9.535
10.730
11.907
9.608
11.498
10.823
9.677
10.765
11.865
10.700
10.133
10.895
y
12.069
8.834
17.638
19.554
20.345
16.045
17.596
20.456
19.799
14.844
14.399
17.223
17.471
19.827
18.335
11.858
9.465
6.240
7.420
8.172
z
3.797
3.000
3.000
3.000
3.500
5.500
3.000
4.295
3.766
3.000
3.330
3.000
3.000
5.500
4.098
3.000
3.395
3.000
3.000
3.397
Method
Sponge Wipe
Sponge Wipe
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Swab
Swab
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Area
(sqft)
0.694
0.694
4.000
4.000
4.000
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.028
0.028
0.028
4.000
4.000
4.000
4.000
4.000
Operator
Philpot
J. Murray
Myers
Myers
Myers
Myers
Myers
Myers
Myers
Myers
Myers
Myers
Myers
Myers
Myers
Myers
Myers
Myers
Myers
Myers
Acquisition
Date
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
Acquisition
Time
10:07:00 AM
3:24:00 PM
11:27:00 AM
11:04:00 AM
11:08:00 AM
11:22:00 AM
11:29:00 AM
11:13:00 AM
11:16:00 AM
10:36:00 AM
10:45:00 AM
10:52:00 AM
11:30:00 AM
11:19:00 AM
10:57:00 AM
9:34:00 AM
10:22:00 AM
10:03:00 AM
10:08:00 AM
10:15:00 AM
Object
Countertop
Ceiling
Floor
Floor
Chair
Ceiling
Floor
Shelves
File cabinet
Floor
Wall
Floor
Floor
Supply Vent
Monitor
Floor
Couch
Floor
Floor
Chair
Texture
Smooth
Metal
Carpet
Smooth
Cloth
Metal
Carpet
Metal
Metal
Smooth
Textured
Smooth
Carpet
Metal
Smooth
Carpet
Cloth
Carpet
Carpet
Porous
Orientation
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Inclined
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Blank
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
BROOM Notes
taken from last
light ballast right
side
vacuum blank
wood/laminate
floor, 210
red office chair,
210
light ballast, 210
sponge stick blank
210
file cabinet, 210
wood floor
wipe, wall, 210
hvacvent, 210
monitor swab,
upper right corner
vacuum sample
211
couch, 211
vacuum, 211
chair, 211
Lab ID
3
2
2
1
1
6
6
6
6
6
6
6
7
7
7
2
1
2
2
1
Detected
Yes
Yes
No
Yes
Yes
Yes
No
Yes
Yes
Yes
No
Yes
No
Yes
Yes
Yes
Yes
Yes
No
Yes
Spread Plate Results
(CFU/sq ft)
6971.7
16230.1
ND
182.8
3.6
16499.4
ND
8344.0
5831.8
12095.5
ND
12339.9
ND
67218.3
ND
275.0
17.5
41.7
ND
11.3
Filter Plate Results
(CFU/sq ft)
NA
1805347.7
ND
89.8
2.7
NA
NA
NA
NA
NA
NA
NA
ND
TNTC
993.9
153.5
10.9
0.6
ND
4.3
G-58
-------
Barcode
2382
2824
1979
2380
2579
1731
1663
1677
3951
3989
4118
3938
2312
2149
2224
2835
1599
Round
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Floor
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Room
Room 211
Room 211
Room 211
Room 211
Room 211
Room 211
Room 211
Room 211
Room 212
Room 212
Room 212
Room 212
Room 212
Room 212
Room 212
Room 212
Room 212
X
10.000
9.524
10.937
10.499
10.092
10.499
9.462
10.133
7.404
7.669
8.468
8.591
6.515
8.176
6.375
8.041
7.588
y
11.659
6.238
9.131
10.959
7.420
7.911
9.716
7.393
18.397
20.352
16.220
20.441
19.774
16.220
16.616
14.630
20.509
z
3.491
4.294
3.590
5.500
3.000
5.500
3.995
3.000
3.700
3.300
3.000
3.000
3.750
3.000
4.500
3.000
5.400
Method
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Swab
Swab
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Area
(sqft)
0.694
0.694
0.694
0.694
0.694
0.028
0.028
0.028
4.000
4.000
4.000
4.000
0.694
0.694
0.694
0.694
0.028
Operator
Myers
Myers
Myers
Myers
Myers
Philpot
Myers
Myers
McCormack
McCormack
McCormack
McCormack
McCormack
McCormack
McCormack
McCormack
McCormack
Acquisition
Date
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
Acquisition
Time
9:47:00 AM
10:01:00 AM
10:20:00 AM
10:32:00 AM
10:09:00 AM
9:55:00 AM
9:52:00 AM
10:12:00 AM
10:11:00 AM
10:31:00 AM
10:00:00 AM
10:25:00 AM
10:40:00 AM
9:59:00 AM
9:55:00 AM
9:46:00 AM
10:37:00 AM
Object
Wall
Shelves
Table
Ceiling
Table
Ceiling
Monitor
Table
Chair
Chair
Cabinet
Floor
File cabinet
Cabinet
Wall
Floor
Supply Vent
Texture
Textured
Smooth
Smooth
Metal
Smooth
Metal
Smooth
Smooth
Porous
Porous
Paint
Smooth
Metal
Paint
Paint
Smooth
Metal
Orientation
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Vertical
Horizontal
Upward
Vertical
Blank
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
TRUE
FALSE
FALSE
TRUE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
BROOM Notes
sponge wipe,
shelves, 211
sponge stick, 211
light ballast,
sample area
indicated by
sharpie marks on
ceiling grid,from
door: right side on
window end
honey comb
grating patially
removed before
sampling, was
aggressively
handled and or
disturbed prior too
swab, upper right,
monitor, existing
template
Lab ID
6
6
6
6
6
7
7
7
4
4
4
1
7
7
7
7
7
Detected
Yes
Yes
Yes
Yes
No
Yes
Yes
No
Yes
Yes
No
Yes
Yes
No
No
Yes
Yes
Spread Plate Results
(CFU/sq ft)
230.4
241.2
10549.0
16741.8
ND
ND
1800.5
ND
8.3
16.7
ND
41.3
11558.0
ND
ND
14135.0
19805.4
Filter Plate Results
(CFU/sq ft)
NA
NA
NA
NA
NA
82.8
579.8
ND
17.3
19.6
ND
51.2
TNTC
ND
ND
TNTC
13831.4
G-59
-------
Barcode
1644
1787
3926
3700
3706
4111
3935
2638
2402
2820
2310
2618
2221
2121
2148
3485
3085
3087
3095
2870
Round
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Pre-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Floor
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Room
Room 212
Room 212
Room 213
Room 213
Room 213
Room 213
Room 213
Room 213
Room 213
Room 213
Room 213
Room 213
Room 213
Stairwell
Stairwell
Bathroom M
Bathroom M
Bathroom M
Bathroom M
Bathroom W
X
6.774
8.210
6.854
,857
7.772
8.311
7.239
7.264
7.633
8.941
8.899
7.726
6.782
23.867
24.262
25.574
26.192
25.807
24.808
24.467
y
18.350
15.928
8.126
8.604
10.681
7.695
12.075
7.640
5.945
6.744
10.843
11.055
10.978
14.722
19.974
18.678
19.703
20.571
20.162
15.055
z
4.200
3.000
3.000
3.700
3.000
3.700
3.000
5.700
3.500
3.750
4.000
3.000
5.000
3.000
3.000
0.000
0.000
0.000
0.000
0.000
Method
Swab
Swab
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqft)
0.028
0.028
4.000
4.000
4.000
4.000
4.000
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
Operator
McCormack
McCormack
McCormack
McCormack
McCormack
McCormack
McCormack
McCormack
McCormack
McCormack
McCormack
McCormack
McCormack
England
England
Siegmund
Siegmund
Siegmund
Siegmund
Siegmund
Acquisition
Date
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
4/26/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
Acquisition
Time
10:18:00 AM
10:04:00 AM
9:16:00 AM
9:09:00 AM
8:55:00 AM
9:22:00 AM
8:35:00 AM
9:42:00 AM
9:36:00 AM
9:33:00 AM
9:00:00 AM
8:56:00 AM
8:46:00 AM
10:43:00 AM
10:25:00 AM
3:05:00 PM
3:02:00 PM
2:58:00 PM
2:48:00 PM
2:26:00 PM
Object
Monitor
Cabinet
Floor
Bed
Floor
Bed
Floor
Ceiling
Wall
Nightstand
Sink
Floor
File cabinet
Floor
Floor
Floor
Floor
Floor
Floor
Floor
Texture
Plastic
Paint
Carpet
Porous
Metal
Porous
Carpet
Metal
Textured
Plastic
Plastic
Metal
Metal
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Orientation
Inclined
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Blank
FALSE
TRUE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
sample taken on
top of lighting unit
closest to entrance
sample was taken
on the floor in
front of the heater
sample was taken
at entrance of
bathroom
sample was taken
2ft from entrance.
Lab ID
7
7
4
4
1
1
4
7
7
7
7
7
7
5
2
2
2
2
2
5
Detected
No
No
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
No
Yes
No
No
No
Spread Plate Results
(CFU/sq ft)
ND
ND
145.8
625.0
ND
260.0
516.7
15758.8
ND
8725.6
6451.0
ND
2855.9
287269.2
325619.7
ND
23.5
ND
ND
ND
Filter Plate Results
(CFU/sq ft)
ND
ND
142.6
TNTC
ND
NA
263.4
TNTC
36.4
TNTC
TNTC
ND
ND
TNTC
TNTC
ND
ND
ND
ND
ND
G-60
-------
Barcode
2212
2869
2913
4133
2783
2732
2636
2792
3129
3132
3307
3135
3147
3146
3366
2762
3149
Round
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Floor
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Room
Bathroom W
Bathroom W
Bathroom W
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
X
26.365
25.281
27.100
25.604
22.678
20.813
17.458
17.155
14.630
15.756
26.587
13.936
13.138
13.939
26.318
22.857
8.977
y
14.503
16.970
16.204
8.283
16.495
13.984
13.978
14.306
15.615
14.039
10.487
16.639
13.057
14.522
9.416
7.619
14.179
z
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.396
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
Method
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqft)
0.694
0.694
0.694
4.000
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
Operator
Siegmund
Siegmund
Siegmund
Siegmund
Siegmund
Siegmund
Siegmund
Siegmund
Siegmund
Siegmund
Siegmund
Siegmund
Siegmund
Siegmund
Siegmund
Siegmund
Siegmund
Acquisition
Date
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
Acquisition
Time
2:34:00 PM
2:42:00 PM
2:39:00 PM
12:39:00 PM
11:23:00 AM
11:11:00 AM
11:02:00 AM
10:57:00 AM
10:08:00 AM
10:14:00 AM
12:22:00 PM
10:05:00 AM
9:45:00 AM
9:52:00 AM
12:27:00 PM
12:48:00 PM
9:36:00 AM
Object
Wall
Floor
Floor
Floor
Floor
Supply Vent
Floor
Wall
Floor
Floor
Countertop
Floor
Floor
Floor
Ceiling
Floor
Floor
Texture
Smooth
Smooth
Smooth
Smooth
Smooth
Metal
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Orientation
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Blank
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
sample was taken
above heater
sample was taken
on the floor where
sinks used to be.
blank
sample was taken
from the right side
of the previous
sample.
sample was taken
in vent before
stairway.
sample was taken
on the right side o]
the previous
sample.
sample was taken
on the floor 3ft
from sinks.
blank
sample was taken
in front of rm 104
doorway.
sample was taken
14in from right
wall.
sample was taken
taken from left sidi
looking down at
the previous
sample, space
heater on previous
sample.
sample was taken
on the rightside of
the previous
sample.
sample was taken
next to the left
wall before rm
106
Lab ID
5
2
2
2
5
5
5
5
5
2
5
5
5
5
5
5
5
Detected
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
Spread Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Filter Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
G-61
-------
Barcode
2842
3304
2929
3303
3313
2922
3316
3319
3323
2803
3302
3156
3154
3166
2440
4032
Round
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Floor
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Room
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Mechanical Room
X
24.162
26.179
22.975
27.411
26.012
23.610
27.210
27.440
25.753
24.746
25.125
3.900
4.395
6.078
21.406
17.563
y
11.033
6.341
16.898
9.317
10.468
12.919
12.931
11.608
11.493
11.325
8.029
13.478
13.169
13.992
14.289
19.386
z
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
Method
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Area
(sqft)
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
4.000
Operator
Siegmund
Siegmund
Siegmund
Siegmund
Siegmund
Siegmund
Siegmund
Siegmund
Siegmund
Siegmund
Siegmund
Siegmund
Siegmund
Siegmund
Bartos
Siegmund
Acquisition
Date
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
Acquisition
Time
12:09:00 PM
12:34:00 PM
11:27:00 AM
12:24:00 PM
12:18:00 PM
12:06:00 PM
12:02:00 PM
11:51:00 AM
11:47:00 AM
11:42:00 AM
12:35:00 PM
9:14:00 AM
9:11:00 AM
9:21:00 AM
12:31:00 PM
3:29:00 PM
Object
Countertop
Floor
Ceiling
Floor
Countertop
Floor
Floor
Floor
Floor
Ceiling
Floor
Wall
Floor
Supply Vent
Floor
Supply Vent
Texture
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Metal
Smooth
Smooth
Smooth
Metal
Smooth
Cloth
Orientation
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Inclined
Blank
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
sample was taken
on edge of bar.
sample was taken
in center of
doorway.
sample was taken
on light fixture
sample was taken
in center before
doorway.
sample was taken
center of bar top.
bleach residue was
in perimeter of the
sample location.
bleach residue was
perimeter of
sample location.
blank
sample was taken
on door below
window
bleach residue
forward of sample
sample was taken
in the opening ove
rm 1 1 0 doorway
sample collected at
bottom of stairs on
floor one
immediately
against the riser
and centered on
stairwell, very
dirty sample.
sample was taken
on the filter for the
furnace, looking at
the furnace, it was
the filter in the
lower right corner
Lab ID
5
5
5
5
2
5
5
5
5
2
5
5
5
5
2
2
Detected
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
Spread Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Filter Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
G-62
-------
Barcode
3483
3487
3484
2633
3312
2910
3123
3118
3078
3434
2613
2755
Round
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Bleach Post-
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Bleach Post-
Bleach Post-
Amended
Bleach Post-
Floor
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Room
Mechanical Room
Mechanical Room
Mechanical Room
Room 101
Room 101
Room 101
Room 101
Room 101
Room 101
Room 101A
Room 101A
Room 101A
X
16.821
17.492
18.702
16.645
21.380
19.632
16.050
17.646
22.013
18.292
19.038
20.388
y
19.218
18.764
20.399
12.169
11.154
10.609
11.226
10.987
10.187
9.323
6.479
7.581
z
1.180
0.000
0.000
0.000
0.000
0.000
1.588
0.000
1.398
0.000
0.000
0.000
Method
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqft)
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
Operator
Siegmund
Siegmund
Siegmund
Siegmund
Siegmund
Siegmund
Siegmund
Siegmund
Siegmund
Willison
Willison
Willison
Acquisition
Date
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
Acquisition
Time
3:19:00 PM
3:21:00 PM
3:15:00 PM
10:18:00 AM
10:51:00 AM
10:43:00 AM
10:25:00 AM
10:38:00 AM
12:53:00 PM
12:10:00 PM
11:07:00 AM
11:50:00 AM
Object
Wall
Floor
Floor
Floor
Floor
Ceiling
Wall
Floor
Floor
Desk
Desk
Floor
Texture
Smooth
Smooth
Smooth
Smooth
Smooth
Metal
Smooth
Smooth
Smooth
Plastic
Smooth
Smooth
Orientation
Vertical
Horizontal
Horizontal
P
.
Horizontal
Horizontal
Upward
Vertical
P
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Blank
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
BROOM Notes
sample was taken
in front of double
doors.
sample was taken
in center of rm 101
doorway.
sample was taken
1ft behind previous
sample when
looking at the
doorway.
sample was taken
right side of the
previous sample 01
light fixture.
sample was taken
2 l/2ft above first
location.
sample was taken
next to venilation
equipment.
sample was taken
1 l/2ft from the
left of doorway
middle front of uv
aps unit, white
amm ended bleach
in sample square.
previous sample
should be floor
sample in front of
desk on opposite
wall . current
sample moved
backwards slightly
from front off
desk due hobo box
on front rt corners.
black stuff on
sample spot
not actually on
floor . taken
where standing at
5 ft height, same
goes for previous
sample blank .
Lab ID
2
5
2
5
2
2
2
2
5
1
1
4
Detected
No
No
No
No
No
No
No
No
No
No
No
No
Spread Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Filter Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
G-63
-------
Barcode
2377
2277
2756
2642
2215
3250
2223
2220
2641
2218
2747
3306
Round
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Bleach Post-
Amended
Bleach Post-
Amended
Bleach Post-
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Dec on
Amended
Bleach Post-
Dec on
Amended
Bleach Post-
Decon
Floor
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Room
Room 101 A
Room 101 A
Room 101 A
Room 101 A
Room 101 A
Room 101 A
Room 101 A
Room 101 A
Room 101 A
Room 101 A
Room 101 A
Room 102
X
21.687
21.555
21.643
21.806
18.588
20.300
19.494
18.157
16.323
21.407
20.093
20.404
y
8.319
7.773
7.375
6.282
9.087
6.592
7.089
6.150
9.314
6.297
9.131
16.259
z
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
Method
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqft)
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
Operator
Willison
Willison
Willison
Willison
Willison
Willison
Willison
Willison
Willison
Willison
Willison
Willison
Acquisition
Date
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
Acquisition
Time
11:46:00 AM
11:39:00 AM
11:36:00 AM
11:30:00 AM
12:02:00 PM
11:18:00 AM
11:03:00 AM
10:59:00 AM
10:49:00 AM
11:25:00 AM
11:53:00 AM
12:21:00 PM
Object
Floor
File cabinet
Desk
Desk
Desk
Desk
Floor
File cabinet
Floor
Desk
File cabinet
File cabinet
Texture
Smooth
Smooth
Smooth
Smooth
Plastic
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Orientation
Horizontal
u'ward
Horizontal
Upward
.
Horizontal
Upward
.
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
.
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Blank
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
floor sample
square is in
somewhat debris
free area, no
debris on floor.
overlap from pre-
work on sample
square, black
debris on file
cabinet .
white ammended
bleach residual in
sample square .
sticky surface in
sample square.
residual white
ammended bleach
in sample square.
surface dirty.
black debris...
vertical upward on
plastic on front rt
of uv-apsunit.
rust inside sample
square . moved
slightly backwards
from front of
desL.will have
overlap
approximately 25
percent .
floor sample,
ammended residua
bleach, floor looks
painted
overlap from
previos
sample., approxima
tely 60 percent.
1ft inside door to
the rt side. ..up nex
to door.
white ammended
bleach spots withir
sample square.
dirty surface.
dirt and rust in
sample square.
overlap from pre
work as well.
approximately 80
percent .
90 percent overlap
from pre work.
white ammended
bleach in sample
square.
Lab ID
4
4
4
4
4
1
1
6
7
4
4
4
Detected
No
No
No
No
No
No
No
No
No
No
No
No
Spread Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Filter Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
NA
ND
ND
ND
ND
G-64
-------
Barcode
3478
3519
3072
3151
3134
3150
3143
3181
3153
3144
3256
3299
3290
3131
Round
Amended
Bleach Post-
Decon
Bleach Post-
Bleach Post-
Bleach Post-
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Bleach Post-
Amended
Bleach Post-
Decon
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Floor
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Room
Room 102
Room 102
Room 102
Room 102
Room 102
Room 102
Room 102
Room 102
Room 102
Room 102
Room 102
Room 102
Room 102
Room 102
X
19.936
18.160
19.288
18.628
18.340
18.088
17.896
19.144
19.264
18.976
19.936
17.932
18.868
16.877
y
16.535
16.955
14.819
14.831
14.759
14.807
17.171
16.223
16.715
16.883
17.327
16.571
15.527
15.791
z
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
Method
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqft)
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
Operator
Willison
Willison
Willison
Willison
Willison
Willison
Willison
Willison
Willison
Willison
Willison
Willison
Willison
Willison
Acquisition
Date
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
Acquisition
Time
12:17:00 PM
12:58:00 PM
1:35:00 PM
1:31:00 PM
1:24:00 PM
1:19:00 PM
1:06:00 PM
12:54:00 PM
12:48:00 PM
12:41:00 PM
12:36:00 PM
12:31:00 PM
12:27:00 PM
1:16:00 PM
Object
Floor
File cabinet
Desk
Desk
Desk
Desk
File cabinet
Floor
Desk
Desk
Desk
Floor
Floor
File cabinet
Texture
Smooth
Smooth
Plastic
Plastic
Smooth
Metal
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Orientation
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
HOnZ°°'
pwar
Vertical
Horizontal
P
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
.
pwar
Horizontal
pwar
Horizontal
Upward
Blank
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
rust in sample
square on floor.
80 percent overlap
from pre work, rus
in sample square
white ammended
bleach in sample
square on top of m
aps unit, ceiling
tile debris in
sample square.
lots ofwhite
ammended bleach
in sample square.
on top of uvaps
unit, top rt. red caf
in sample square
ceiling tile debris
and white
ammended bleach
in sample square.
on top of desk to
thertof uv aps
unit.
vertical sponge on
70 percent overlap
from pre work.
ammended bleach
in sample square.
and rust.
blank in middle ot
room 5 ft height.
center drawer
sample inside desk
drawer, no visible
debris present.
50 percent
ammended bleach
in sample square.
ammended bleach
and ceiling tile
debris in sample
square.
rust and ceiling tih
debris in sample
square.
ceiling tile debris
in sample square
and rust.
75 percent overlap
from pre work anc
rust in sample
square.
Lab ID
4
6
6
6
6
4
4
6
4
6
1
4
1
4
Detected
No
No
No
No
No
No
No
No
No
No
No
No
No
No
Spread Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Filter Plate Results
(CFU/sq ft)
ND
NA
NA
NA
NA
ND
ND
NA
ND
NA
ND
ND
ND
ND
G-65
-------
Barcode
3162
3160
3314
3317
3310
3311
3265
2779
1714
1610
3120
3119
3116
3125
3124
3050
3114
Round
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Floor
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Room
Room 103
Room 103
Room 103
Room 103
Room 103
Room 103
Room 103
Room 103
Room 103
Room 103
Room 104
Room 104
Room 104
Room 104
Room 104
Room 104
Room 104
X
13.524
14.636
13.236
13.721
13.921
15.438
13.943
15.671
13.160
13.617
16.514
15.097
13.552
14.452
15.680
15.593
15.036
y
6.264
6.114
7.050
7.121
10.221
11.876
9.805
6.916
8.296
10.227
20.782
18.393
18.444
18.944
19.529
20.245
20.383
z
0.000
0.300
2.000
0.587
2.276
0.000
0.000
0.000
2.000
0.000
1.000
0.690
0.000
0.000
2.500
1.000
1.000
Method
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Swab
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqft)
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.028
0.028
0.694
0.694
0.694
0.694
0.694
0.694
0.694
Operator
Schmaedick
Schmaedick
Schmaedick
Schmaedick
Schmaedick
Schmaedick
Schmaedick
Schmaedick
Schmaedick
Schmaedick
Schmaedick
Schmaedick
Schmaedick
Schmaedick
Schmaedick
Schmaedick
Schmaedick
Acquisition
Date
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
Acquisition
Time
12:52:00 PM
12:57:00 PM
12:49:00 PM
12:43:00 PM
12:26:00 PM
12:22:00 PM
12:29:00 PM
1:02:00 PM
12:46:00 PM
12:30:00 PM
1:22:00 PM
1:40:00 PM
1:33:00 PM
1:30:00 PM
1:27:00 PM
1:17:00 PM
1:09:00 PM
Object
Floor
Wall
Shelves
Table
Ceiling
Floor
Floor
Floor
Shelves
Floor
Wall
Table
Floor
Floor
Ceiling
Table
Table
Texture
Smooth
Textured
Smooth
Smooth
Smooth
Smooth
Smooth
Textured
Smooth
Smooth
Textured
Smooth
Smooth
Smooth
Metal
Smooth
Textured
Orientation
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Blank
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
TRUE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
to right of square
box marked on
floor
vert wall sample
textured
song template was
slight;y large fot
shelf top
tabletop has some
white and dust on
it
top of light top of
light dusty sig.
dust
little dusty
blank blank
large liquid stain
in are of sample
dusty
dust and grime
smooth surface top
of shelves ditting
ontop of desk
blank swab
textured wall, no
visual, next to
template markings
table top no visible
staining- photo out
of mem
blank
smooth, light dust
no photo out of
mem
some insulation
fallen on top of
light ballast, dusty,
covered with
parti culatr
previous sttempt at
this sample
crashed computer,
sample info was re
entered no more
pics will be tsken
due to full memoiy
no major visible
dust present
Lab ID
4
4
4
4
4
7
7
4
4
4
4
7
4
4
4
4
4
Detected
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
Spread Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Filter Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
G-66
-------
Barcode
2247
2581
2781
2780
3441
3333
3268
3289
3493
4031
3115
2447
3292
3482
3112
3117
Round
Bleach Post-
Bleach Post-
Amended
Bleach Post-
Decon
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Bleach Post-
Bleach Post-
Bleach Post-
Amended
Bleach Post-
Decon
A
Bleach Post-
Floor
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Room
Room 105
Room 105
Room 105
Room 105
Room 105
Room 105
Room 105
Room 105
Room 105
Room 106
Room 106
Room 106
Room 106
Room 106
Room 106
Room 106
X
9.920
11.881
12.533
10.501
10.271
11.096
11.517
10.252
11.287
12.081
10.192
11.481
10.099
10.853
12.125
10.676
y
11.153
10.923
10.923
11.652
9.409
9.102
6.782
6.437
9.869
20.220
16.148
16.420
19.988
20.362
15.780
14.675
z
0.250
0.000
0.000
0.000
1.000
2.800
0.593
0.000
0.000
0.000
0.000
2.700
1.000
0.000
0.000
0.000
Method
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqft)
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
4.000
0.694
0.694
0.694
0.694
0.694
0.694
Operator
Schmaedick
Schmaedick
Schmaedick
Schmaedick
Schmaedick
Schmaedick
Schmaedick
Schmaedick
Schmaedick
Owen
Owen
Owen
Owen
Owen
Owen
Acquisition
Date
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
Acquisition
Time
11:46:00 AM
11:41:00 AM
11:36:00 AM
11:28:00 AM
11:54:00 AM
12:00:00 PM
12:10:00 PM
12:15:00 PM
12:02:00 PM
3:28:00 PM
3:01:00 PM
3:52:00 PM
3:45:OOPM
3:37:00 PM
3:09:00 PM
2:58:00 PM
Object
Wall
Floor
Sink
Floor
Stove
Ceiling
Table
Floor
Floor
Wall
Ceiling
File cabinet
Floor
Table
Floor
Texture
Smooth
Smooth
Smooth
Smooth
Metal
Metal
Smooth
Smooth
Smooth
Smooth
Textured
Metal
Smooth
Smooth
Smooth
Smooth
Orientation
Vertical
Horizontal
Upward
.
TT
pwar
TT
pwar
Horizontal
Upward
Horizontal
Downward
Horizontal
Upward
TT
pwar
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Downward
Upward
Upward
orizon
pwar
Horizontal
Upward
Blank
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
wall sample next
laser location is ofl
when compared to
map, likely due to
map sccale
template size area
on right sibe of
sink
no coloring
right over top of
burner
on ballast above
light dusty powdei
some white hazing
color
no color
no visible white
color
sample taken on
floor against wall
sample taken on
wall, left of
previous sample
sample taken on
top of light closest
to 2nd light, top o
light real dirty
sample taken on
leftside of room
again the wall
sample taken on
floor to left side o]
window Ifoot out
residual bleach
stains on floor
between this and
previous sample
sample taken on
table surface in
front of last samph
sample taken Ifoot
inside the door to
the right of old
sample
Lab ID
1
7
1
1
1
7
1
7
1
2
6
7
6
6
6
6
Detected
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
Spread Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Filter Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
NA
ND
NA
NA
NA
NA
G-67
-------
Barcode
2810
3110
2968
2089
2183
3113
3486
2389
3322
4113
3963
3297
3296
3262
3473
3413
3301
2941
3308
3337
Round
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Floor
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Room
Room 106
Room 107
Room 107
Room 107
Room 107
Room 107
Room 107
Room 107
Room 107
Room 108
Room 108
Room 108
Room 108
Room 108
Room 108
Room 108
Room 108
Room 108
Room 108
Room 109
X
10.538
8.413
8.470
7.113
9.631
7.665
7.646
8.605
8.531
8.651
9.286
8.213
9.543
7.473
7.913
7.113
9.551
9.451
8.651
5.073
y
18.311
9.214
6.882
6.514
9.214
10.602
12.400
7.975
11.331
16.730
20.098
20.617
14.554
14.771
19.117
18.217
17.930
20.117
16.930
10.931
z
1.000
0.000
0.000
1.191
0.000
2.800
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
2.247
0.779
0.000
1.387
0.000
0.000
Method
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqft)
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
4.000
4.000
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
Operator
Owen
Schmaedick
Schmaedick
Schmaedick
Schmaedick
Schmaedick
Schmaedick
Schmaedick
Schmaedick
Bechtal
Bechtal
Bechtal
Bechtal
Bechtal
Bechtal
Bechtal
Bechtal
Bechtal
Bechtal
Bechtal
Acquisition
Date
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
Acquisition
Time
3:17:00 PM
10:57:00 AM
11:22:00 AM
11:17:00 AM
11:01:00 AM
10:44:00 AM
10:38:00 AM
11:11:00 AM
10:34:00 AM
12:30:00 PM
12:26:00 PM
12:20:00 PM
12:47:00 PM
12:10:00 PM
12:54:00 PM
12:14:00 PM
12:43:00 PM
12:38:00 PM
12:34:00 PM
1:30:00 PM
Object
Desk
Floor
Floor
Cabinet
Floor
Ceiling
Wall
Floor
Floor
Floor
Floor
Floor
Wall
Floor
Ceiling
Desk
Floor
File cabinet
Floor
Floor
Texture
Smooth
Smooth
Smooth
Smooth
Smooth
Metal
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Textured
Smooth
Metal
Smooth
Smooth
Metal
Smooth
Smooth
Orientation
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Blank
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
TRUE
BROOM Notes
sample taken on
desk to right of
previous sample
smooth-no coloring
blank blank blank
really dusty white
particles
rough on outside o
sqr, dust
light fixture ballasl
next marked
square on wall
little bit of rust- no
other coloring
some white bleach
coloring
blank
templates don't lay
fiat, too dark for
photos in this roon
laminate flloor
left of previous
sample outline
laminate flloor
light ballast no
template used
desk with white
residue
laminate flloor
slight debris in
area
blank
blank
Lab ID
6
7
1
1
7
1
7
7
1
2
8
8
6
1
6
8
5
8
8
5
Detected
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
Spread Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Filter Plate Results
(CFU/sq ft)
NA
ND
ND
ND
ND
ND
ND
ND
ND
ND
NA
NA
NA
ND
NA
NA
ND
NA
NA
ND
G-68
-------
Barcode
3331
3260
2314
3318
3349
3298
3338
3253
3480
4035
3942
3164
3148
3516
3511
3163
3514
3305
3477
Round
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Floor
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Room
Room 109
Room 109
Room 109
Room 109
Room 109
Room 109
Room 109
Room 109
Room 109
Room 110
Room 110
Room 110
Room 110
Room 110
Room 110
Room 110
Room 110
Room 110
Room 110
X
5.721
5.145
4.573
5.621
4.312
4.205
4.213
4.515
6.251
5.873
5.024
4.373
5.111
6.582
6.550
5.173
4.293
4.473
5.973
y
6.314
6.107
7.414
8.414
9.662
12.237
11.011
11.567
12.042
20.396
16.568
19.996
16.451
16.306
20.100
16.749
20.744
18.396
14.796
z
2.348
0.000
0.000
0.000
1.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
2.381
0.000
1.291
0.000
1.298
0.000
0.000
Method
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqft)
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
4.000
4.000
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
Operator
Bechtal
Bechtal
Bechtal
Bechtal
Bechtal
Bechtal
Bechtal
Bechtal
Bechtal
Bechtal
Bechtal
Bechtal
Bechtal
Bechtal
Bechtal
Bechtal
Bechtal
Bechtal
Bechtal
Acquisition
Date
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
Acquisition
Time
1:28:00 PM
1:25:00 PM
1:20:00 PM
1:17:00 PM
1:15:00 PM
1:13:00 PM
1:10:00 PM
1:05:00 PM
12:57:00 PM
11:38:00 AM
11:42:00 AM
11:19:00 AM
12:05:00 PM
11:59:00 AM
11:51:00 AM
11:45:00 AM
11:24:00 AM
11:09:00 AM
10:59:00 AM
Object
Ceiling
Wall
Floor
Floor
Countertop
Sink
Sink
Floor
Floor
Floor
Floor
File cabinet
Ceiling
Floor
Cabinet
Floor
Wall
Desk
Floor
Texture
Metal
Textured
Textured
Textured
Smooth
Smooth
Smooth
Textured
Textured
Smooth
Smooth
Metal
Metal
Smooth
Metal
Smooth
Textured
Smooth
Smooth
Orientation
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Blank
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
BROOM Notes
light ballast
template not used
concrete flloor
white residue
concrete flloor
debris on surface
debris on top
concrete flloor
concrete flloor
floor in front of
file cabinet
file cabinet , no
anomolies
top of light fixtun
laminate flloor
file cabinet top
center
wall below
marking
desk
laminate floor
Lab ID
1
5
5
4
5
5
4
4
8
8
8
1
1
1
1
1
5
5
5
Detected
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
Spread Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Filter Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
ND
NA
NA
NA
ND
ND
ND
ND
ND
ND
ND
ND
G-69
-------
Barcode
3161
2439
2303
2302
2442
2443
2811
2630
2292
2299
3554
Round
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Dec on
Amended
Bleach Post-
Dec on
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Dec on
Amended
Bleach Post-
Dec on
Amended
Bleach Post-
Amended
Bleach Post-
Decon
Floor
Floor 1
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Room
Room 110
Bathroom M
Bathroom M
Bathroom M
Bathroom W
Bathroom W
Bathroom W
Copier Room
Copier Room
Copier Room
Hallway
X
5.373
26.330
29.825
28.197
28.900
29.078
26.955
28.742
29.776
27.374
19.637
y
20.496
20.071
20.250
20.621
17.811
17.029
16.470
14.208
13.342
13.314
12.868
z
0.000
3.000
3.000
3.000
3.000
3.000
3.000
3.000
3.000
3.000
3.983
Method
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Area
(sqft)
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
4.000
Operator
Bechtal
Owen
Owen
Owen
Bartos
Bartos
Bartos
Bartos
Bartos
Bartos
Bartos
Acquisition
Date
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
Acquisition
Time
11:28:00 AM
2:15:00 PM
2:28:00 PM
2:24:00 PM
12:14:00 PM
12:10:00 PM
12:07:00 PM
11:48:00 AM
11:40:00 AM
11:36:00 AM
9:38:00 AM
Object
Floor
Floor
Floor
Wall
Floor
Floor
Floor
Wall
Floor
Floor
Floor
Texture
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Textured
Textured
Textured
Smooth
Orientation
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Blank
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
TRUE
BROOM Notes
laminate flloor
inside door
touching thresholc
Ifoot inside of stal
on wall above
heater, left of old
sample
sample collected
immediately
adjacent to pre
sample on stall
side, no visible
bleach, but little
light .dirty
sample]
black collected at
corner of stall door
[towards entrance
to bath]
could not locate
pre sample.
sample collected
inside womens
bath centered
between where
wall 'juts' out
sample collected
immediately
adjacent to the
right of pre sample
. in between
power sockets.
sample collected
center of back
wall, 1 foot from
wall, tile like
surface, sample
very dirty, could
not locate previous
sample.
sample collected
immediately
adjacent to pre
sample to the right
when standing in
doorway, very
dirty sample, tile
like surface.
vacuum blank.
collected a .98
meter height.
Lab ID
5
6
6
6
7
7
7
7
7
7
2
Detected
No
No
No
No
No
No
No
No
No
No
No
Spread Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Filter Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
G-70
-------
Barcode
2815
2786
2785
2788
2761
2662
2663
2760
2666
Round
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Bleach Post-
Decon
Floor
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Room
Hallway
Hallway
Hallway
Hallway
Hallway
Hallway
Hallway
Hallway
Hallway
X
25.370
26.054
22.994
21.637
16.493
17.008
12.973
11.093
7.893
y
18.503
17.551
13.488
13.568
12.868
13.751
12.519
13.458
13.358
z
3.000
3.300
5.083
3.000
3.000
3.000
3.000
3.000
4.864
Method
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqft)
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
Operator
Bartos
Bartos
Bartos
Bartos
Bartos
Bartos
Bartos
Bartos
Bartos
Acquisition
Date
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
Acquisition
Time
10:08:00 AM
10:04:00 AM
9:55:00 AM
9:45:00 AM
9:34:00 AM
9:28:00 AM
9:23:00 AM
9:16:00 AM
9:09:00 AM
Object
Floor
Wall
Return Vent
Floor
Floor
Floor
Wall
Floor
Return Vent
Texture
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Orientation
Horizontal
Upward
Vertical
Upward
Horizontal
Upward
.
Upward
Horizontal
Upward
Vertical
.
Upward
Upward
Blank
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
sample collected 1
foot out [template
edge] from air lock
in center of hall.
no residue in area.
minor dirt in
sample area, no
photo taken
sample collected
immediately
adjacent [right
side] of pre
sample, no photo
taken
sample from insidi
return vent, no
photo taken.
sample collected
on floor
immediately
adjacent to pre
sample location
[far side from
entrance door]
anddirectly in fron
of rm 202.
sponge stick blank
sampled directly
under entrance
doorway side of
hazaed taped
ductwork at 5 '
elevation.
immediately
adjacent to pre
decon sample
[entrance doorway
side] some bleach
residue in area but
not in template.
wall 1 foot above
floor, immediately
adjacent [right
side] to #2 square
box on wall.
sample on floor.
no bleach residue
apparent
inside return vent.
Lab ID
7
7
7
5
7
7
7
7
Detected
No
No
No
No
No
No
No
No
No
Spread Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
ND
ND
Filter Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
ND
ND
G-71
-------
Barcode
2664
2759
2293
2612
4121
3079
2622
2295
Round
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Dec on
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Floor
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Room
Hallway
Hallway
Janitor Closet
Janitor Closet
Mechanical Room
Mechanical Room
Mechanical Room
Room 201
X
6.993
25.634
28.016
27.290
18.911
18.628
22.761
27.307
y
13.358
14.885
15.018
14.906
19.220
20.265
19.713
10.825
z
3.000
3.000
3.000
3.000
3.000
3.000
3.000
5.271
Method
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqft)
0.694
0.694
0.694
0.694
4.000
0.694
0.694
0.694
Operator
Bartos
Bartos
Bartos
Bartos
Owen
Owen
Owen
Bartos
Acquisition
Date
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
Acquisition
Time
9:00:00 AM
9:52:00 AM
12:00:00 PM
11:54:00 AM
2:42:00 PM
2:48:00 PM
2:37:00 PM
10:54:00 AM
Object
Floor
Floor
Floor
Floor
Floor
Floor
Floor
Ceiling
Texture
Smooth
Smooth
Smooth
Smooth
Textured
Smooth
Smooth
Smooth
Orientation
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Upward
Horizontal
Downward
Blank
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
inside
doorway.floor is
dirty, photo is verj
dark, photo taken
facing entrance
doorway .
laser pointer not
working, sample
collected center ol
rm 24 doorway 12
inches from
threshhold.
[sample template
edge is 12 inches
off threshold, glut
like residue in
sample area.
could not locate
pre sample .
sample collected
center of doorway
directly in front ol
sink, tile floor
[same as previous
sample] verydirtj
sample]
could not locate
pre sample.
sample collected
center of doorway
at top of door arc
[swing of door]
filter in back of
furnace
on back wall on
only grey tile
sampled from top
of light fixture.
fixture farthest
from exterior wall.
sampke collected
from the 1/2
towards exterior
wall, very dirty
surface, no photo
taken.
Lab ID
7
7
6
7
2
7
6
7
Detected
No
No
No
No
No
No
No
No
Spread Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
ND
Filter Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
ND
G-72
-------
Barcode
2294
2304
2359
2297
2305
2301
Round
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Dec on
Bleach Post-
Amended
Bleach Post-
Bleach Post-
Bleach Post-
Floor
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Room
Room 201
Room 201
Room 201 A
Room 201 A
Room 201 A
Room 201 A
X
29.095
26.231
28.420
27.631
28.664
26.731
y
11.369
11.300
6.019
7.866
8.340
6.840
z
3.000
3.000
3.300
3.000
3.000
3.000
Method
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqft)
0.694
0.694
0.694
0.694
0.694
0.694
Operator
Bartos
Bartos
Bartos
Bartos
Bartos
Bartos
Acquisition
Date
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
Acquisition
Time
10:43:00 AM
10:39:00 AM
11:21:00 AM
11:26:00 AM
11:13:00 AM
11:08:00 AM
Object
Floor
Floor
Wall
Ceiling
Floor
Floor
Texture
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Orientation
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Blank
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
sample collected
immediately
adjacent to pre on
side opposite
exterior wall.
increased dirt in
sample area
[compared to
previous
samples ]minimal
residue in area
sample collected
immediately
adjacent to pre
[side away from
doorway and
towards rm 201a]
sample collected
immediately
adjacent and to the
right of pre sample
[under window].
very clean.
sample collected
on top of light
fixture [when
standing in
doorway far and
right fixture.
sampled left top
half [towards
ceiling vent] very
dirty sample.
sample collected
immediately
adjacent towards
exterior wall [wall
to the left when
standing in
doorway ] bleach
residue present in
and around sample
area.
sample collected
immediately
adjacent to pre
sample [to the left
when standing in
doorway ]
residual bleach
visible, but
probably due to
available light]
top left of photo
shows residue]
Lab ID
7
6
7
7
7
7
Detected
No
No
No
No
Yes
No
Spread Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
Filter Plate Results
(CFU/sq ft)
ND
ND
ND
ND
4.0
ND
G-73
-------
Barcode
3479
2787
3251
2736
2737
2733
2692
2693
2799
2150
2296
Round
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Floor
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Room
Room 201 A
Room 202
Room 202
Room 203
Room 203
Room 203
Room 203
Room 203A
Room 203A
Room 204
Room 204
X
26.553
22.325
21.531
23.696
22.013
25.091
23.757
24.003
25.235
19.782
18.637
y
9.240
17.163
15.000
9.865
11.363
11.938
11.076
6.438
9.311
16.831
16.719
z
3.000
3.000
3.000
3.000
3.000
3.000
3.000
3.000
3.000
3.000
3.500
Method
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqft)
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
Operator
Bartos
Bartos
Bartos
Negron
Negron
Negron
Negron
Negron
Negron
Bartos
Bartos
Acquisition
Date
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
Acquisition
Time
11:00:00 AM
10:17:00 AM
10:14:00 AM
1:17:00 PM
1:10:00 PM
1:08:00 PM
1:06:00 PM
1:22:00 PM
1:19:00 PM
10:27:00 AM
10:31:00 AM
Object
Floor
Floor
Floor
Wall
Floor
Floor
Ceiling
Floor
Floor
Floor
Wall
Texture
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Orientation
Horizontal
d
pwar
Horizontal
Upward
Horizontal
Upward
Vertical
Upward
Upward
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Blank
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
sample collected
immediately
adjacent [to right
when standing in
doorway]
sample collected
immediately
adjacent to pre
sample [doorway
side of pre sample
] minor dirt . no
residue in area .
sample collected
immediately
adjacent to pre
sample [toward
back of room].
minor dirt on
sponge [consistant
with other
samples]
fluorescent light
closest to entrance
of room 203
sample taken on
side closest to
entrance
floor in front of
window
entrance of room
203A
sample collected
immediately
adjacent [on right
side when back to
doorway ] to pre
sample . no
residue in area no
photo taken
sample collected
immediately
adjacent towards
back wall
[approximately .5
metersoff ground.
Lab ID
7
7
7
6
7
6
5
5
5
7
6
Detected
No
No
No
No
No
Yes
No
No
No
No
No
Spread Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Filter Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
3.6
ND
ND
ND
ND
ND
G-74
-------
Barcode
2834
2735
2752
2715
2731
2529
2580
2527
3490
2632
3096
3489
2765
2764
2808
2805
2768
Round
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Floor
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Room
Room 204
Room 205
Room 205
Room 205
Room 205
Room 206
Room 206
Room 206
Room 206
Room 206
Room 206
Room 206
Room 206
Room 206
Room 206
Room 206
Room 206
X
19.993
18.944
19.878
20.705
18.742
15.742
18.221
16.042
16.921
15.842
16.121
16.521
16.574
17.980
17.621
16.621
17.321
y
15.090
9.092
6.110
11.875
6.779
17.154
20.454
18.354
15.654
15.445
15.354
15.654
19.497
19.520
20.554
20.454
18.954
z
3.000
3.000
3.000
3.000
3.000
3.000
3.000
3.497
3.000
3.000
3.000
3.000
3.000
3.000
3.000
3.000
3.000
Method
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqft)
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
Operator
Bartos
Negron
Negron
Negron
Negron
Negron
Negron
Negron
Negron
Negron
Negron
Negron
Negron
Negron
Negron
Negron
Negron
Acquisition
Date
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
Acquisition
Time
10:22:00 AM
12:59:00 PM
12:55:00 PM
12:49:00 PM
12:53:00 PM
11:53:00 AM
10:32:00 AM
10:19:00 AM
10:15:00 AM
10:28:00 AM
11:51:00 AM
10:11:00 AM
12:10:00 PM
12:07:00 PM
12:05:00 PM
12:01:00 PM
11:56:00 AM
Object
Floor
Floor
Wall
Floor
Ceiling
Floor
Floor
Floor
Floor
Floor
Floor
Floor
Floor
Floor
Table
Table
Table
Texture
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Orientation
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Blank
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
sample collected
immediately
adjacent to pre
sample [towards
backwall]. no
residue in area
very minimal dirt
sample taken on
floor under center
fluorescent light
fluorescent light
closest to window
on right side facing
window
sample collected it
room 206
product residue on
desk surface
sligth residue on
floor
entrance of room
206. sample
collected in room
206
residue on suface
table [B .Melton
changed to sponge
stick]
blank sponge
in room 206
in room 206
in room 206 table
in back of room
in room 206
Lab ID
7
6
5
6
5
6
7
6
5
2
6
2
6
6
7
7
2
Detected
No
No
No
No
No
Yes
No
No
No
No
No
No
No
No
No
No
No
Spread Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Filter Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
3.7
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
G-75
-------
Barcode
2789
2751
2688
2714
2729
2705
2730
2687
2686
2685
3539
2619
2524
2362
2774
2796
Round
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Floor
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Room
Room 206
Room 207
Room 207
Room 207
Room 207
Room 207
Room 207
Room 207
Room 207
Room 207
Room 208
Room 208
Room 208
Room 209
Room 209
Room 209
X
16.891
15.925
17.633
16.455
16.666
15.765
15.735
15.735
16.939
17.633
14.571
14.470
13.771
12.971
12.471
13.956
y
17.273
11.853
11.326
9.804
10.954
8.500
9.023
9.526
9.863
11.326
19.754
18.770
17.454
11.969
7.469
6.669
z
3.000
3.000
3.000
3.000
3.000
3.000
3.000
3.000
3.000
3.000
3.000
3.000
3.000
3.000
3.000
3.000
Method
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqft)
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
4.000
0.694
0.694
0.694
0.694
0.694
Operator
Negron
Negron
Negron
Negron
Negron
Negron
Negron
Negron
Negron
Negron
Negron
Negron
Negron
Negron
Negron
Negron
Acquisition
Date
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
Acquisition
Time
12:15:00 PM
12:20:00 PM
12:24:00 PM
12:44:00 PM
12:43:00 PM
12:38:00 PM
12:36:00 PM
12:35:00 PM
12:32:00 PM
12:27:00 PM
11:03:00 AM
10:38:00 AM
10:47:00 AM
11:10:00 AM
11:42:00 AM
11:40:00 AM
Object
Ceiling
Floor
Table
Countertop
Ceiling
Table
Table
Table
Table
Wall
Floor
Wall
Supply Vent
Countertop
Wall
Table
Texture
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Orientation
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Blank
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
middle fluorescent
light in room 206
entrance of room
207
table on left of
entrance of room
blank sponge
fluorescent light
closest to entrance
. sample taken
from right side
facing window
sample 4 of 4 on
table surface
sample 3 of 4 on
table surface
sample 2 of four
on table surface in
front of entrance
on top of table
surface on right
side of room far
end of room
window
facing window on
left wall
approximately 7
feet from window
wall
no template
available
room 208 not 206
on ceiling
fluorescent
significant residue
on suface
Lab ID
6
6
2
5
7
5
6
5
5
6
8
5
7
2
7
5
Detected
No
Yes
No
No
No
No
No
No
No
No
No
No
No
No
No
No
Spread Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Filter Plate Results
(CFU/sq ft)
ND
8.8
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
G-76
-------
Barcode
2526
2525
2773
2528
2561
3876
4122
2767
2637
2367
Round
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Dec on
A
Bleach Post-
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Bleach Post-
Floor
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Room
Room 209
Room 209
Room 209
Room 209
Room 209
Room 210
Room 210
Room 210
Room 210
Room 210
X
14.056
14.056
14.856
12.671
14.456
11.402
11.716
11.199
9.836
10.135
y
8.369
8.369
9.469
10.269
10.969
19.354
17.049
15.645
19.778
20.411
z
3.000
3.000
3.896
3.000
3.000
3.000
4.190
5.490
3.797
3.000
Method
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqft)
0.694
0.694
0.694
0.694
0.694
4.000
4.000
0.694
0.694
0.694
Operator
Negron
Negron
Negron
Negron
Negron
Huyser
Huyser
Huyser
Huyser
Huyser
Acquisition
Date
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
Acquisition
Time
11:37:00 AM
11:34:00 AM
11:25:00 AM
11:16:00 AM
11:19:00 AM
11:49:00 AM
11:39:00 AM
12:14:00 PM
12:08:00 PM
12:03:00 PM
Object
Ceiling
Floor
Stove
Countertop
Floor
Floor
Floor
Ceiling
Cabinet
Floor
Texture
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Metal
Metal
Smooth
Orientation
Horizontal
Downward
.
Upward
Horizontal
Upward
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Blank
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
BROOM Notes
fluorescent light
closest to entrance
of room . surface
extremely dirty
nO template used.
surface dirty
nO visible residue
Vacuum sample
taken on floor in
front of large file
cabinet. In grid,
thin gray line
approx 2 x 8" of
caked dust similar
to the footprints.
Rust and white
discoloration on
floor near grid but
not in grid."
Vacuum sample
blank taken.
Sample did not
touch surface.
Sponge sample on
top of ceiling light
taken on south ent
of light away from
black X. Observed
rust and dust in
small piles which
were collected by
sponge.
Sponge sample on
small file cabinet
in front of blank
marking. Only 4
small surface
scratches observec
in paint surface.
Sponge sample on
floor between
small file cabinet
and wall beneath
window. Lightly
caked dust similar
to footprints
observed in grid.
Lab ID
7
6
5
5
1
8
6
1
2
Detected
No
Yes
No
No
No
No
No
No
No
No
Spread Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Filter Plate Results
(CFU/sq ft)
ND
2.7
ND
ND
ND
ND
ND
ND
ND
ND
G-77
-------
Barcode
2414
2901
2611
2365
2593
3086
2777
2770
2771
Round
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Bleach Post-
Amended
Bleach Post-
Dec on
Amended
Bleach Post-
Decon
Bleach Post-
Amended
Bleach Post-
Decon
Bleach Post-
Amended
Bleach Post-
Decon
Floor
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Room
Room 210
Room 210
Room 210
Room 210
Room 210
Room 211
Room 211
Room 211
Room 211
X
11.699
10.202
9.999
11.999
11.391
11.099
11.799
10.899
9.400
y
17.045
16.954
14.854
19.754
14.378
8.940
8.940
11.159
11.259
z
4.496
3.000
3.000
4.398
3.600
3.394
3.000
5.497
3.397
Method
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqft)
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
Operator
Huyser
Huyser
Huyser
Huyser
Huyser
Huyser
Huyser
Huyser
Huyser
Acquisition
Date
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
Acquisition
Time
11:35:00 AM
11:26:00 AM
11:22:00 AM
12:00:00 PM
11:31:00 AM
12:35:00 PM
12:29:00 PM
12:26:00 PM
12:21:00 PM
Object
Floor
Floor
Floor
Cabinet
Wall
Table
Floor
Ceiling
Wall
Texture
Smooth
Smooth
Smooth
Metal
Paint
Smooth
Smooth
Metal
Paint
Orientation
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Upward
Upward
Vertical
Blank
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
Sponge sample
blank. Sample did
not touch surface.
Sponge sample on
floor near
southeast corner ol
desk. No unusual
features in grid.
Sponge sample on
floor near
doorway. Floor is
faux wood
laminate. Light
footprints are
observed on floor
and in grid.
Sponge sample on
large file cabinet.
Only 3-5 spots of
rust pitting
observed on
surface. Very light
gray dust observec
on surface.
Sponge sample on
wall to right of
existing taped grid
No unusual
features in grid.
Sponge sample on
stonne table
surface, in center
of table away from
black marking.
Very light dust
collected by
sponge in grid.
Sponge sample on
floor between low
table and wall.
Sponge sample on
top of ceiling light
at north end away
from black
Observed small
piles of rust and
dust which were
collected onto
sponge.
Sponge sample on
wall to left of
black marking. No
unusual features in
grid.
Lab ID
6
6
1
6
5
7
5
6
5
Detected
No
No
Yes
No
No
No
No
No
No
Spread Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
ND
ND
Filter Plate Results
(CFU/sq ft)
ND
ND
2.3
ND
ND
ND
ND
ND
ND
G-78
-------
Barcode
2776
3171
3488
3510
3080
4110
3949
3492
3517
Round
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Bleach Post-
Dec on
Bleach Post-
Dec on
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Dec on
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Floor
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Room
Room 211
Room 211
Room 211
Room 211
Room 211
Room 212
Room 212
Room 212
Room 212
X
11.200
11.099
10.815
9.600
10.899
8.390
8.606
8.390
8.490
y
11.840
6.240
7.181
6.659
7.940
16.355
20.405
16.355
15.054
z
3.000
3.000
4.475
4.398
3.000
4.493
3.000
4.463
3.000
Method
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Area
(sqft)
0.694
0.694
0.694
0.694
0.694
4.000
4.000
0.694
0.694
Operator
Huyser
Huyser
Huyser
Huyser
Huyser
Huyser
Huyser
Huyser
Huyser
Acquisition
Date
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
Acquisition
Time
12:17:00 PM
12:41:00 PM
12:48:00 PM
12:46:00 PM
12:38:00 PM
10:43:00 AM
11:06:00 AM
10:40:00 AM
10:35:00 AM
Object
Floor
Floor
Floor
Shelves
Floor
Floor
Floor
Floor
Floor
Texture
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Orientation
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Upward
Horizontal
Upward
Horizontal
Upward
.
Upward
Blank
FALSE
FALSE
TRUE
FALSE
FALSE
TRUE
FALSE
TRUE
FALSE
BROOM Notes
Sponge sample on
floor near
doorway. Floor is
smooth painted
concrete. Very
light dusted
footprints observec
in room .
Sponge sample on
floor near wall
below window to
left. No unusual
features in grid.
Sponge sample
blank taken.
Sample did not
touch surface.
Sponge sample on
top of shelves.
dust and a few
small black
particles on shelf
collected by
sponge.
Sponge sample on
floor south of
table. Light brown
dust in only small
parts of grid,
collected by
sponge.
Vacuum blank
taken. Sample did
not touch surface.
Vacuum sample
taken on floor
adjacent to large
file cabinet and
wall. Whit
discoloration
observed on floor
in spots approx 2 x
6". Some light
dust observed."
Sponge blank
taken. Sponge did
not touch surface.
Sponge sample on
floor 24 from
doorway. Floor is
faux wood
laminate.
Footprints
observed on floor
and some thin dust
from footprints
wiped up by
sponge."
Lab ID
1
6
2
1
2
1
1
7
5
Detected
No
No
No
No
No
Yes
Yes
No
No
Spread Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
ND
ND
Filter Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
1.6
0.6
ND
ND
G-79
-------
Barcode
2769
2775
3491
2766
2363
3481
3512
Round
Amended
Bleach Post-
Decon
Bleach Post-
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Floor
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Room
Room 212
Room 212
Room 212
Room 212
Room 213
Room 213
Room 213
X
6.290
6.838
7.390
6.990
6.990
7.918
8.818
y
16.070
19.857
17.454
20.354
7.540
6.040
6.840
z
3.696
3.800
3.000
3.000
3.000
3.594
3.000
Method
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqft)
0.694
0.694
0.694
0.694
0.694
0.694
0.694
Operator
Huyser
Huyser
Huyser
Huyser
Huyser
Huyser
Huyser
Acquisition
Date
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
Acquisition
Time
10:55:00 AM
10:59:00 AM
10:48:00 AM
11:15:00 AM
10:03:00 AM
10:28:00 AM
10:23:00 AM
Object
Wall
Cabinet
Floor
Floor
Floor
Wall
Floor
Texture
Paint
Metal
Smooth
Smooth
Smooth
Smooth
Smooth
Orientation
Vertical
Horizontal
Upward
Horizontal
Upward
.
Upward
.
Upward
Vertical
Horizontal
Upward
Blank
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
Sponge sample on
wall to left of I-
beam approx 12
and lower right of
window. A plastic
ceiling light cover
that was leaned
against the wall
was moved. It had
covered only part
of the grid area."
Sponge sample on
2-drawer cabinet
top. Some
scratches and worn
paint on surface in
grid.
Sponge sample on
floor near
southeast corner ol
desk. No
discoloration or
unusual features in
grid.
Sponge sample on
floor between wall
and small cabinet.
Rust and white
discoloration on
floor in straight
lines, partially
removed by
sponge.
Sponge sample on
floor near east
window, 2ft from
wall. Rust
discoloration near
location, not in
grid.
Sponge sample on
wall to right of
black marking.
Approx 1 8 from
floor. No markings
or unusual features
in or near grid."
Sponge sample on
floor near wall,
approx 7. No
discoloration or
dust/debris in grid
Dust/debris in
corner at wall/floor
edge outside of
grid."
Lab ID
7
7
7
1
7
5
7
Detected
No
No
No
No
No
No
No
Spread Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
Filter Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
G-80
-------
Barcode
1988
2366
2315
1981
2634
2635
2778
Round
Amended
Bleach Post-
Decon
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Amended
Bleach Post-
Decon
Floor
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Room
Room 213
Room 213
Room 213
Room 213
Room 213
Room 213
Room 213
X
8.118
8.918
6.518
9.018
8.118
7.518
8.018
y
7.540
8.340
10.840
11.259
10.859
12.159
8.159
z
3.000
3.000
4.697
3.897
3.000
3.000
5.487
Method
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqft)
0.694
0.694
0.694
0.694
0.694
0.694
0.694
Operator
Huyser
Huyser
Huyser
Huyser
Huyser
Huyser
Huyser
Acquisition
Date
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
5/5/2011
Acquisition
Time
10:10:00 AM
9:57:00 AM
9:52:00 AM
9:42:00 AM
9:36:00 AM
9:31:00 AM
10:16:00 AM
Object
Floor
Floor
Cabinet
Sink
Floor
Floor
Ceiling
Texture
Smooth
Smooth
Metal
Smooth
Smooth
Smooth
Metal
Orientation
.
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Blank
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
BROOM Notes
Sponge sample on
floor. Rust
discoloration on
floor in grid.
Approxhalf of rus
in grid was
transferred to the
sponge during
collection.
Sponge sample on
floor between bed
frames. Rust
discoloration on
floor, partially in
grid. Small grit
and debris in
corner near wall,
outside sample
grid.
Sponge sample on
metal cabinet top.
Surface is dusty
with white debris
and is damaged
with pitted rust
spots.
Sponge sample on
countertop
adjacent to sink,
not in sink. Roll ol
duct tape andmisc
piece of small
plastic moved fron
sample area to
place template -
gloves changed
prior to collection.
Blank sponge. Did
not touch surface.
Sponge on floor.
White stains on
floor between
sample grid and
south wall. 3 stains
of 3x12" each."
Sponge sample on
top of ceiling light
near north end,
opposite side of
black X mark.
Surface is rusted
and contains
darkened
dust/debris.
Lab ID
1
6
1
7
5
7
Detected
No
No
No
No
No
No
No
Spread Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
Filter Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
G-81
-------
Barcode
2441
2858
2399
2886
4123
2078
2075
2263
2373
3956
2258
2404
2500
2499
4229
3959
4088
4028
3960
2548
2166
2518
2519
2586
Round
Amended
Bleach Post-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
Floor
Floor 2
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Room
Stairwell
Bathroom M
Bathroom M
Bathroom M
Bathroom M
Bathroom M
Bathroom W
Bathroom W
Bathroom W
Bathroom W
Bathroom W
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
X
23.882
1.851
1.212
0.574
27.038
26.390
25.473
24.783
25.707
27.062
25.119
27.110
24.915
24.415
7.481
7.481
22.781
15.110
15.110
13.708
13.708
13.431
13.431
14.537
y
18.677
3.672
3.672
3.672
20.080
18.448
19.389
20.224
20.689
15.209
16.841
16.217
14.513
14.649
12.760
12.760
14.419
13.259
13.259
13.841
13.841
16.041
16.041
15.616
z
3.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.993
2.900
0.000
0.000
0.595
0.000
0.000
2.900
2.900
2.900
2.900
0.000
0.000
0.000
0.000
0.000
Method
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqft)
0.694
0.694
0.694
0.694
4.000
0.694
0.694
0.694
0.694
4.000
0.694
0.694
0.694
0.694
4.000
4.000
4.000
4.000
4.000
0.694
0.694
0.694
0.694
0.694
Operator
Bartos
Mattorano
Mattorano
Mattorano
Mingolla
Mingolla
Mingolla
Mingolla
Mingolla
Mingolla
Mingolla
Mingolla
Mingolla
Mingolla
Mingolla
Mingolla
Mingolla
Mingolla
Mingolla
Mingolla
Mingolla
Mingolla
Mingolla
Mingolla
Acquisition
Date
5/5/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
Acquisition
Time
12:24:00 PM
3:33:00 PM
3:32:00 PM
3:31:00 PM
12:32:00 PM
12:24:00 PM
12:20:00 PM
12:12:00 PM
12:17:00 PM
12:09:00 PM
12:01:00 PM
11:58:00 AM
11:53:00 AM
11:49:00 AM
9:32:00 AM
9:31:00 AM
11:41:00 AM
10:29:00 AM
10:26:00 AM
9:49:00 AM
9:53:00 AM
9:57:00 AM
9:59:00 AM
10:01:00 AM
Object
Floor
Floor
Floor
Floor
Ceiling
Floor
Floor
Floor
Wall
Ceiling
Floor
Floor
Wall
Floor
Ceiling
Ceiling
Ceiling
Floor
Floor
Floor
Floor
Floor
Floor
Floor
Texture
Textured
Smooth
Smooth
Smooth
Porous
Smooth
Smooth
Smooth
Smooth
Porous
Smooth
Smooth
Smooth
Smooth
Porous
Porous
Porous
Textured
Textured
Smooth
Smooth
Smooth
Smooth
Smooth
Orientation
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Blank
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
TRUE
TRUE
FALSE
TRUE
FALSE
TRUE
BROOM Notes
sample collected at
center of top of
stairs [just off
therubberized
tread], sample
collected on
tilelike
surface.sample
very dirty.
metal roof,
adjacent to stacks
on roof, inside ven
cap
on roof, inside
chimney stack
ceiling tile was
placed on floor in
front of the stall
sponge sample
taken on floor just
left of the sink
sponge sample
taken on floor
sponge sample
taken on floor
sponge sample
taken on wall
above vent
ceiling tile was
placed on floor
sponge sample
taken on floor
sponge sample
taken on floor just
inside of the stall
wall sample taken
above vent
sponge sample
taken on floor
ceiling tile blank
vacuum
ceiling tile, left
side
ceiling tile was
placed on floor
vacuum sample on
top of tile
blank vacuum
sample
sponge blank
sponge sample
taken on floor
sponge blank
sponge sample
taken on floor
sponge sample
blank
Lab ID
2
6
6
6
6
1
4
1
1
6
1
1
1
1
6
6
6
4
4
4
4
6
6
6
Detected
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
No
Yes
No
Yes
No
Spread Plate Results
(CFU/sq ft)
ND
6279.1
380.6
1171.2
2671.0
279445.5
309588.3
273589.7
2682.1
1137.6
237591.0
184697.0
6527.8
243350.8
8.3
8875.5
3654390.0
12709.1
ND
ND
232359.2
ND
196485.4
ND
Filter Plate Results
(CFU/sq ft)
ND
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
G-82
-------
Barcode
2517
2201
2426
2205
1974
2239
2592
2607
2843
2206
2424
2455
2501
2856
2358
2828
3962
2542
2397
2909
3772
2497
2349
2350
2498
4087
4100
Round
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
Floor
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Room
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Mechanical Room
Mechanical Room
Mechanical Room
Mechanical Room
Room 101
Room 101
Room 101
Room 101
Room 101
Room 101A
Room 101A
X
14.537
12.481
3.951
12.481
9.707
9.707
6.042
5.284
3.951
5.281
6.042
21.521
22.877
21.508
18.210
16.907
17.686
16.835
17.818
18.801
19.633
18.332
21.233
16.133
16.669
19.132
16.632
y
15.616
13.341
13.371
13.341
14.081
14.081
14.216
13.614
13.371
13.636
14.216
12.982
15.897
13.915
13.025
14.230
19.186
19.016
18.467
20.604
10.609
10.821
10.909
11.621
11.830
8.127
9.414
z
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.285
0.000
1.874
2.800
0.000
1.977
0.000
0.497
0.000
0.500
0.000
0.000
0.000
0.000
0.000
0.695
0.000
0.000
0.000
Method
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Vacuum Sock
Area
(sqft)
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
4.000
0.694
0.694
0.694
4.000
0.694
0.694
0.694
0.694
4.000
4.000
Operator
Mingolla
Mingolla
Mingolla
Mingolla
Mingolla
Mingolla
Mingolla
Mingolla
Mingolla
Mingolla
Mingolla
schademann
Mingolla
Mingolla
Mingolla
Mingolla
Mingolla
Mingolla
Mingolla
Mingolla
Mingolla
Mingolla
Mingolla
Mingolla
Mingolla
lee
lee
Acquisition
Date
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
Acquisition
Time
10:05:00 AM
9:46:00 AM
9:01:00 AM
9:41:00 AM
9:38:00 AM
9:35:00 AM
9:12:00 AM
9:04:00 AM
9:00:00 AM
8:53:00 AM
9:11:00 AM
3:23:00 PM
11:44:00 AM
11:02:00 AM
10:56:00 AM
10:51:00 AM
4:22:00 PM
4:18:00 PM
4:11:00 PM
4:07:00 PM
11:25:00 AM
11:15:00 AM
11:27:00 AM
11:11:00 AM
11:06:00 AM
12:11:00 PM
12:06:00 PM
Object
Floor
Floor
Wall
Floor
Floor
Floor
Supply Vent
Floor
Wall
Floor
Supply Vent
Floor
Floor
Return Vent
Floor
Wall
Return Vent
Wall
Floor
Floor
Ceiling
Floor
Floor
Wall
Floor
Floor
Floor
Texture
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Metal
Smooth
Smooth
Smooth
Metal
Smooth
Smooth
Metal
Smooth
Textured
Cloth
Textured
Smooth
Smooth
Porous
Smooth
Smooth
Textured
Smooth
Carpet
Carpet
Orientation
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Blank
FALSE
FALSE
TRUE
TRUE
FALSE
TRUE
TRUE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
BROOM Notes
sponge sample
taken on floor
sponge sample
taken on floor
entrance door
blank sponge
sponge blank taker
on floor
horizontal sponge
sample taken on
floor
horizontal sponge
blank
Isthvac sample
blank
floor blank for
sample 1
entrance door of
hallway
just before doors
first hvac sample
sponge sample
taken on floor
hvac sample
number 2.
sponge sample
taken on floor
vertical sample
taken on wall
filter in back of
furnace
sponge sample
taken on wall
sponge sample
taken on floor
sponge sample
taken on floor in
front of door way
ceiling tile was
placed on floor
sponge sample
taken on floor
sponge sample
taken on floor
sponge sample
taken on wall
behind door
sponge sample
taken on floor
loose carpet
strands in template
Lab ID
6
6
4
4
6
4
4
4
4
6
4
7
1
1
4
4
5
7
7
7
6
1
1
1
4
6
2
Detected
Yes
Yes
No
No
Yes
No
No
No
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
No
Yes
No
Yes
Spread Plate Results
(CFU/sq ft)
179777.2
166188.9
ND
ND
172179.1
ND
ND
ND
5254.8
144153.0
203032.3
169260.8
ND
8267.7
345971.0
1889.2
TNTC
3148.7
269481.0
47652.6
14042.5
ND
328307.6
ND
343379.1
ND
2137.6
Filter Plate Results
(CFU/sq ft)
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
-1438.5
NA
NA
NA
NA
TNTC
TNTC
-1438.5
-1438.5
NA
NA
NA
NA
NA
NA
TNTC
G-83
-------
Barcode
3678
4125
2427
2306
1977
1980
2384
2425
1987
1978
2216
1985
2368
4081
4063
3873
4039
2432
Round
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
Floor
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Room
Room 101A
Room 101A
Room 101A
Room 101A
Room 101A
Room 101A
Room 101A
Room 101A
Room 101A
Room 101A
Room 101A
Room 101A
Room 101A
Room 102
Room 102
Room 102
Room 102
Room 102
X
19.632
21.732
18.532
18.532
19.993
19.089
18.532
18.208
20.310
21.886
21.886
21.479
21.564
18.792
17.840
18.814
19.864
19.339
y
6.955
9.527
8.427
9.327
9.123
6.548
8.827
6.073
6.531
6.209
7.327
6.531
7.921
15.750
16.571
16.297
16.472
16.702
z
0.000
0.000
0.000
0.999
0.595
0.700
0.988
0.000
0.700
0.700
0.700
0.700
0.595
0.000
0.000
0.000
0.000
0.000
Method
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Area
(sqrt)
4.000
4.000
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
4.000
4.000
4.000
4.000
0.694
Operator
lee
lee
lee
lee
lee
lee
lee
lee
lee
lee
lee
lee
lee
lee
lee
lee
lee
lee
Acquisition
Date
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
Acquisition
Time
1:57:00 PM
1:17:00 PM
12:18:00 PM
12:37:00 PM
12:57:00 PM
2:22:00 PM
12:24:00 PM
2:30:00 PM
2:05:00 PM
1:46:00 PM
1:36:00 PM
1:32:00 PM
1:24:00 PM
4:53:00 PM
4:44:00 PM
4:36:00 PM
4:30:00 PM
5:52:00 PM
Object
Chair
Floor
Floor
Desk
File cabinet
Desk
Desk
File cabinet
Desk
Desk
Desk
Desk
File cabinet
Floor
Floor
Floor
Floor
Desk
Texture
Cloth
Carpet
Carpet
Plastic
Smooth
Smooth
Plastic
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Carpet
Carpet
Carpet
Carpet
Smooth
Orientation
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Blank
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
vacuumed entire
seat of chair, less
than surface area
of template, hard
to reach front of
chair since it was
pushed under desk
hard to vacuum
straight up and
down.
vacuum over
tubing on floor.
difficult to reach
corner.
uv APS on desk.
middle back.
sticky stuff on
front left corner
on top of filing
cabinet
sticky tape residue
uv APS on desk.
wiping over duct
tape, ridged
surface in places
liquid leaked out
when twisting
handle
extra chair not on
map between
desks.
front left on filing
cabinet . liquid
leaked out when
breaking handle o]
sponge
blank
electric cord covet
through middle of
template
cart hit filing
cabinet while
vacuuming sample
inside drawer of
desk, black crusty
stuff, sticky.
Lab ID
2
2
2
2
2
2
2
2
2
2
2
2
2
4
4
4
4
6
Detected
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Spread Plate Results
(CFU/sq ft)
22501.3
2162.6
ND
122491.4
601897.3
460446.7
20965.6
631272.2
553995.1
583658.0
663527.0
502301.1
518092.5
ND
2362.6
4666.9
6833.7
132815.8
Filter Plate Results
(CFU/sq ft)
TNTC
TNTC
ND
ND
-1438.5
-1438.5
TNTC
-1438.5
-1438.5
-1438.5
-1438.5
-1438.5
-1438.5
NA
NA
NA
NA
NA
G-84
-------
Barcode
2423
2523
2430
2422
2475
2464
2522
2431
2433
2706
2321
3649
3647
2243
2244
2327
2718
2326
2332
2476
1790
1733
Round
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
Floor
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Room
Room 102
Room 102
Room 102
Room 102
Room 102
Room 102
Room 102
Room 102
Room 102
Room 102
Room 103
Room 103
Room 103
Room 103
Room 103
Room 103
Room 103
Room 103
Room 103
Room 103
Room 103
Room 103
X
19.875
18.737
18.311
17.447
16.921
18.190
19.470
18.923
20.214
18.551
14.631
13.779
13.274
14.615
13.202
15.831
13.734
13.345
15.535
13.692
14.643
13.499
y
17.369
16.866
17.336
17.249
15.553
15.028
14.612
15.980
16.308
14.645
11.190
10.310
6.247
6.192
7.520
6.332
7.926
9.946
11.887
6.625
6.597
10.198
z
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
2.900
0.000
0.000
1.800
2.000
0.000
1.000
0.000
0.000
1.000
2.900
0.000
Method
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Swab
Area
(sqft)
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
4.000
4.000
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.028
0.028
Operator
lee
lee
lee
lee
lee
lee
lee
lee
lee
lee
Ball
Ball
Ball
Ball
Ball
Ball
Ball
Ball
Ball
Ball
Ball
Ball
Acquisition
Date
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
Acquisition
Time
5:49:00 PM
5:41:00 PM
5:35:00 PM
5:28:00 PM
5:19:00 PM
5:13:00 PM
5:02:00 PM
4:50:00 PM
4:14:00 PM
5:09:00 PM
4:08:00 PM
3:36:00 PM
4:12:00 PM
3:56:00 PM
3:53:00 PM
3:46:00 PM
3:51:00 PM
3:32:00 PM
3:28:00 PM
3:50:00 PM
3:59:00 PM
3:33:00 PM
Object
Desk
Desk
File cabinet
File cabinet
File cabinet
Desk
Desk
Floor
File cabinet
Desk
Ceiling
Floor
Floor
Wall
Mail slot
Floor
Table
Floor
Floor
Table
Supply Vent
Floor
Texture
Smooth
Smooth
Metal
Metal
Metal
Smooth
Plastic
Carpet
Smooth
Plastic
Porous
Smooth
Smooth
Textured
Metal
Smooth
Smooth
Smooth
Smooth
Smooth
Metal
Smooth
Orientation
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Horizontal
Upward
Blank
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
TRUE
BROOM Notes
rust on surface.
sticky tape residue
map diagram
wrong, this filing
cabinet faces
forward, not
sideways, sample
taken on top front
rust on surface
sticky tape residue
sticker decal.
ridges on uv APS
on desk.
blank
uv APS on desk.
ridges, missing
some area due to
handle depression
on box
[B. Melton barcode
is incorrect]
previous 3
samples(wipe,
sponge, swab) are
blanks as well.
forgot to check
blank box.
[B. Melton
addressed this]
Lab ID
4
6
6
6
6
4
6
6
4
6
5
5
5
5
5
5
5
5
5
5
1
1
Detected
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
No
Spread Plate Results
(CFU/sq ft)
268261.9
141498.7
209656.1
198515.7
375825.8
383889.5
113558.9
ND
387057.4
95554.8
6917.1
ND
3094923.4
5133.9
192712.7
215127.9
244790.8
ND
383601.5
390129.3
822223.9
ND
Filter Plate Results
(CFU/sq ft)
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
TNTC
18.6
TNTC
TNTC
TNTC
TNTC
TNTC
ND
TNTC
TNTC
NA
NA
G-85
-------
Barcode
3559
3596
2330
2331
2329
2325
2328
3173
1672
1673
3685
3562
3662
3705
3582
2322
2323
2228
2229
1517
3690
3927
Round
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
Floor
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Room
Room 104
Room 104
Room 104
Room 104
Room 104
Room 104
Room 104
Room 104
Room 104
Room 104
Room 105
Room 105
Room 105
Room 105
Room 105
Room 105
Room 105
Room 105
Room 105
Room 105
Room 106
Room 106
X
13.994
16.394
14.852
15.243
14.494
14.094
14.994
14.701
15.160
14.952
11.781
10.489
10.702
11.081
10.381
10.320
12.681
10.081
11.489
11.402
11.048
11.274
y
18.944
19.396
20.496
20.735
20.344
19.344
18.544
19.118
19.308
19.696
10.224
11.993
7.824
10.034
6.524
9.197
10.821
11.221
6.680
7.024
19.643
20.421
z
1.298
0.000
0.600
1.200
0.497
0.000
0.600
1.000
1.000
2.900
0.000
0.000
2.900
0.000
0.000
0.000
0.000
0.500
0.000
2.900
1.597
0.496
Method
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Swab
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Vacuum Sock
Vacuum Sock
Area
(sqft)
4.000
4.000
0.694
0.694
0.694
0.694
0.694
0.694
0.028
0.028
4.000
4.000
4.000
4.000
4.000
0.694
0.694
0.694
0.694
0.028
4.000
4.000
Operator
schademann
schademann
schademann
schademann
schademann
schademann
schademann
schademann
schademann
schademann
Mingolla
Mingolla
Mingolla
Mingolla
Mingolla
Mingolla
Mingolla
Mingolla
Mingolla
Mingolla
nash
nash
Acquisition
Date
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
Acquisition
Time
3:44:00 PM
3:42:00 PM
4:00:00 PM
4:06:00 PM
3:57:00 PM
3:52:00 PM
3:34:00 PM
4:10:00 PM
4:12:00 PM
3:48:00 PM
3:21:00 PM
2:51:00 PM
4:01:OOPM
3:27:00 PM
3:42:00 PM
3:33:00 PM
3:12:00 PM
2:57:00 PM
3:47:00 PM
3:52:00 PM
4:19:00 PM
4:12:00 PM
Object
Ceiling
Ceiling
Table
Wall
Table
Floor
Table
Wall
Wall
Ceiling
Floor
Floor
Ceiling
Floor
Floor
Stove
Sink
Wall
Table
Supply Vent
Wall
Chair
Texture
Porous
Porous
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Carpet
Carpet
Textured
Carpet
Carpet
Metal
Metal
Textured
Smooth
Metal
Cloth
Cloth
Orientation
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Vertical
Horizontal
Downward
Horizontal
Downward
Horizontal
Upward
Vertical
Vertical
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Horizontal
Upward
Horizontal
Downward
Horizontal
Upward
Inclined
Vertical
Horizontal
Downward
Horizontal
Upward
Vertical
Horizontal
Upward
Blank
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
overlaid previous
template.
swab of diffuser.
vacuum sample on
floor
carpet vacuum
sample just inside
doorway of room
top of tile in
ceiling
vacuum sample on
floor in front stove
vacuum sample on
floor in back of
room
sponge sample
taken on stove
stick wipe sample
taken in sink
105 wall sample
taken just inside
door
sponge sample
taken on table
surface
vent
vertical vacuum
sample on partisan
facing rear of
room, at top of
partisan between
desk and end of
partisan.
vacuum sample on
chair on back wall
chair is seated
onder right side ot
window
Lab ID
2
2
7
7
7
7
7
7
1
1
5
5
5
5
5
7
7
7
7
1
7
7
Detected
No
Yes
Yes
Yes
Yes
Yes
Yes
No
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Spread Plate Results
(CFU/sq ft)
ND
2116.8
72285.3
3124.7
111298.2
333299.4
ND
ND
ND
942256.6
966.7
2054.3
3583.5
833.4
1162.6
94306.8
134279.7
422.4
147570.4
828225.5
2421.0
11459.0
Filter Plate Results
(CFU/sq ft)
ND
TNTC
TNTC
TNTC
TNTC
TNTC
TNTC
ND
NA
NA
TNTC
TNTC
TNTC
TNTC
TNTC
TNTC
TNTC
297.4
TNTC
NA
TNTC
TNTC
G-86
-------
Barcode
3673
4052
3952
2255
2468
2262
2285
2467
1608
1602
3642
Round
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
Floor
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Room
Room 106
Room 106
Room 106
Room 106
Room 106
Room 106
Room 106
Room 106
Room 106
Room 106
Room 107
X
12.676
10.968
12.641
10.570
12.113
10.142
10.623
9.922
11.379
10.254
9.443
y
20.153
18.394
18.505
14.962
15.842
19.885
18.139
17.094
19.779
18.292
10.245
z
1.498
0.497
0.000
0.000
0.772
0.696
0.896
0.595
0.000
1.092
0.000
Method
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Swab
Vacuum Sock
Area
(sqft)
4.000
4.000
4.000
0.694
0.694
0.694
0.694
0.694
0.028
0.028
4.000
Operator
nash
nash
nash
nash
nash
nash
nash
nash
nash
nash
nash
Acquisition
Date
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
Acquisition
Time
4:07:00 PM
3:48:00 PM
4:36:00 PM
3:24:00 PM
3:31:00 PM
4:28:00 PM
3:55:00 PM
3:39:00 PM
4:30:00 PM
4:01:OOPM
2:22:00 PM
Object
File cabinet
Chair
Ceiling
Floor
Table
File cabinet
Desk
Wall
Supply Vent
Monitor
Couch
Texture
Smooth
Cloth
Textured
Smooth
Smooth
Smooth
Smooth
Textured
Smooth
Smooth
Cloth
Orientation
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Inclined
Inclined
Horizontal
Upward
Blank
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
vacuum sample
from the books on
top of the file
cabinet, books
were in center on
top of cabinet
vacuum sample on
chair near desk.
seat cushion was
vacuumed
top of ceiling tile
next to wall. 2nd
full tile from rear
wall, the half
sampled is toward
the entry door to
room
swab stick on floor
in entryway. about
1 foot in right
center.
sponge stick
sample on table,
far end when
standing in
doorway [straight
forward],
swab stick sample
back corner [left].
placed template
against wall.
swab stick sample
on table , in
front/left of
monitor due to
previous sampling
team, template on
edge of table
swab stick sample
on wall, about 2
feet left of desk.
diffuser on ceiling
sampled with a
swab. 2 feet from
centerline with
room.
swab sample on
monitor, upper left
corner of monitor.
vacuum sample,
couch, facing is
left cushion, sitting
is right cushion.
Lab ID
7
5
7
7
7
7
6
7
1
1
7
Detected
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Spread Plate Results
(CFU/sq ft)
1941.8
7458.8
1800.1
117619.6
173638.3
150051.9
146020.1
739.7
780212.5
68418.6
9833.9
Filter Plate Results
(CFU/sq ft)
TNTC
TNTC
TNTC
TNTC
TNTC
TNTC
NA
ND
NA
NA
TNTC
G-87
-------
Barcode
3740
3579
3796
3943
2313
1989
2578
2401
1492
1560
1552
3933
Round
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
Floor
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Room
Room 107
Room 107
Room 107
Room 107
Room 107
Room 107
Room 107
Room 107
Room 107
Room 107
Room 107
Room 108
X
8.480
9.431
9.131
7.831
8.115
7.043
9.127
8.559
7.406
8.331
9.127
7.936
y
8.381
12.131
7.214
11.477
12.362
6.423
7.213
9.250
9.724
6.814
7.213
18.379
z
0.000
0.000
0.000
0.000
0.497
1.289
1.176
0.000
0.794
0.000
0.891
0.000
Method
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Swab
Swab
Area
(sqft)
4.000
4.000
4.000
4.000
0.694
0.694
0.694
0.694
0.028
0.028
0.028
4.000
Operator
nash
nash
nash
nash
nash
nash
nash
nash
nash
nash
Griffin
Acquisition
Date
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
Acquisition
Time
2:29:00 PM
1:54:00 PM
2:32:00 PM
3:15:00 PM
3:07:00 PM
2:48:00 PM
2:35:00 PM
2:13:00 PM
3:01:00 PM
2:53:00 PM
2:40:00 PM
4:56:00 PM
Object
Chair
Floor
Floor
Ceiling
Wall
Shelves
Floor
Table
Monitor
Supply Vent
Floor
Texture
Cloth
Carpet
Carpet
Textured
Textured
Smooth
Carpet
Smooth
Smooth
Smooth
Carpet
Cloth
Orientation
Horizontal
Upward
Horizontal
Upward
Horizontal
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Inclined
Horizontal
Upward
Horizontal
Downward
Blank
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
TRUE
FALSE
BROOM Notes
vacuum sample, 01
chair cushion, onlj
chair in room. On
a general note, I
don't like vacuum
samples ;]
vacuum sample on
floor,
approximately 6
inches in room, lef
center of
doorway... debri
was
collected\noted
after sample was
taken.
blank sample ,
vacuum, center of
open area
top of ceiling tile..
left side if standing
at door, vacuum
sample ... noted
with marker also
2 feet from
doorway, vertical
swab stick .
sponge stick
sample on shelf,
back corner is
where template
placement is.
swab sample ,
blank, same height
as vacuum blank.
center of open
area, back part of
room
sponge stick
sample, on table,
centerline length,
template edge
width,
swab sample on tv
monitor, upper left
corner.
diffuser vent swab
rear center of roon
, on ceiling
swab sample
blank, same
location and heigh
as other blanks.
center area of back
part of room
Lab ID
5
7
7
7
7
6
7
7
1
1
1
6
Detected
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Spread Plate Results
(CFU/sq ft)
4208 6
2075.1
ND
7625.4
280.8
111317.4
ND
223167.6
696189.6
126034.3
ND
3916.9
Filter Plate Results
(CFU/sq ft)
TNTC
TNTC
ND
TNTC
262.7
NA
4.0
TNTC
NA
NA
NA
NA
G-8
-------
Barcode
3561
3704
3618
4120
4060
2227
2681
2281
2683
2264
1562
1566
1742
3900
4132
4091
4058
3638
3650
2286
2283
2282
2819
2284
3928
3656
4119
3628
3611
Round
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
Floor
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Room
Room 108
Room 108
Room 108
Room 108
Room 108
Room 108
Room 108
Room 108
Room 108
Room 108
Room 108
Room 108
Room 108
Room 109
Room 109
Room 109
Room 109
Room 109
Room 109
Room 109
Room 109
Room 109
Room 109
Room 109
Room 110
Room 110
Room 110
Room 110
Room 110
X
8.932
8.237
9.664
7.955
9.345
7.617
7.335
9.364
8.799
8.349
7.335
8.725
8.838
5.464
4.900
5.019
4.400
6.255
6.069
4.945
4.173
4.173
4.153
5.194
6.246
5.156
6.565
4.837
4.912
y
16.031
20.408
20.182
19.563
18.905
15.148
17.910
17.233
14.488
16.219
18.454
16.388
19.525
8.730
11.275
10.816
7.917
11.985
6.548
11.290
11.045
12.052
7.095
6.047
15.318
20.314
19.976
19.656
18.436
z
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
Method
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Swab
Swab
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Area
(sqft)
4.000
4.000
4.000
4.000
4.000
0.694
0.694
0.694
0.694
0.694
0.028
0.028
0.028
4.000
4.000
4.000
4.000
4.000
4.000
0.694
0.694
0.694
0.694
0.694
4.000
4.000
4.000
4.000
4.000
Operator
Griffin
Griffin
Griffin
Griffin
Griffin
Griffin
Griffin
Griffin
Griffin
Griffin
Griffin
Griffin
Griffin
Ventura
Ventura
Ventura
Ventura
Ventura
Ventura
Ventura
Ventura
Ventura
Ventura
Ventura
Griffin
Griffin
Griffin
Griffin
Griffin
Acquisition
Date
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
Acquisition
Time
4:46:00 PM
5:16:00 PM
5:19:00 PM
5:13:00 PM
5:29:00 PM
4:33:00 PM
5:00:00 PM
4:49:00 PM
4:39:00 PM
4:44:00 PM
5:02:00 PM
4:45:00 PM
5:23:00 PM
11:15:00 AM
10:46:00 AM
10:43:00 AM
11:27:00 AM
10:09:00 AM
11:54:00 AM
10:49:00 AM
10:25:00 AM
10:19:00 AM
11:36:00 AM
11:44:00 AM
4:25:00 PM
4:12:00 PM
4:06:00 PM
4:01:OOPM
3:37:00 PM
Object
Floor
Chair
File cabinet
Wall
Ceiling
Floor
Desk
Floor
Wall
Floor
Monitor
Floor
Return Vent
Bed
Floor
Floor
Bed
Floor
Ceiling
Floor
Sink
Sink
Countertop
Wall
Ceiling
Chair
File cabinet
Wall
Chair
Texture
Smooth
Cloth
Smooth
Cloth
Smooth
Smooth
Smooth
Smooth
Textured
Smooth
Smooth
Smooth
Metal
Porous
Carpet
Carpet
Porous
Carpet
Porous
Carpet
Smooth
Smooth
Porous
Textured
Smooth
Cloth
Smooth
Cloth
Cloth
Orientation
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Vertical
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Vertical
Horizontal
Downward
Vertical
Horizontal
Downward
Horizontal
Upward
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Vertical
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Vertical
Horizontal
Downward
Blank
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
TRUE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
blank taken
vacuum sample
taken on books
wall
divider/partition
sample taken from
back half of ceiling
tile
blank taken
bottom left corner
of computer
monitor
blank taken
Dottom right corne
ofbed
top left of bed
second tile from
left if looking at
window
blank
countertop left of
sink
left nightstand
vertical wall
sample below
window
books on cabinet
wall
divider/partition
Lab ID
6
6
7
4
7
6
6
6
6
6
1
1
1
4
6
4
6
4
6
6
6
6
6
6
6
4
6
6
6
Detected
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
No
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Spread Plate Results
(CFU/sq ft)
ND
11959.0
208.3
562.5
3362.7
168785.6
168152.1
126139.2
423.3
24191.1
28207.7
ND
2952804.1
1437.6
ND
983.4
533.4
2225.1
6208.7
ND
225154.7
183103.5
128827.1
2763.3
1766.8
6333.7
725.0
2196.0
1900.1
Filter Plate Results
(CFU/sq ft)
NA
NA
162.2
NA
TNTC
NA
NA
NA
NA
NA
33399.1
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
G-89
-------
Barcode
4086
2317
2318
2265
2289
2316
2226
2225
1563
1711
1546
3871
3174
3175
3176
4036
4234
3178
3179
3177
2795
2809
2728
3878
3957
4053
4084
Round
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
Floor
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Room
Room 110
Room 110
Room 110
Room 110
Room 110
Room 110
Room 110
Room 110
Room 110
Room 110
Room 110
Bathroom M
Bathroom M
Bathroom M
Bathroom M
Bathroom W
Bathroom W
Bathroom W
Bathroom W
Bathroom W
Copier Room
Copier Room
Copier Room
Hallway
Hallway
Hallway
Hallway
X
5.588
4.161
6.565
4.499
6.433
3.954
5.945
5.738
4.161
5.457
5.411
28.113
29.213
28.789
27.203
27.800
27.500
28.498
26.865
28.197
29.905
28.515
27.405
10.293
25.292
18.837
18.937
y
15.937
19.882
19.600
17.872
17.421
16.088
16.369
15.205
18.360
19.544
16.396
20.159
20.258
20.660
20.259
17.292
17.912
18.012
16.194
17.094
13.731
14.211
13.863
12.868
16.232
13.468
12.858
z
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
5.900
3.000
3.400
3.000
4.100
5.900
3.000
3.000
3.000
3.000
5.900
3.000
5.900
5.900
4.200
5.900
Method
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Swab
Swab
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Area
(sqft)
4.000
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.028
0.028
0.028
4.000
0.694
0.694
0.694
4.000
4.000
0.694
0.694
0.694
0.694
0.694
0.694
4.000
4.000
4.000
4.000
Operator
Griffin
Griffin
Griffin
Griffin
Griffin
Griffin
Griffin
Griffin
Griffin
Griffin
Griffin
schademann
schademann
schademann
schademann
schademann
schademann
schademann
schademann
schademann
schademann
schademann
schademann
schademann
schademann
schademann
schademann
Acquisition
Date
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
Acquisition
Time
3:17:00 PM
4:15:00 PM
4:09:00 PM
3:41:00 PM
3:28:00 PM
3:23:00 PM
3:12:00 PM
2:48:00 PM
3:48:00 PM
4:20:00 PM
3:05:00 PM
3:01:00 PM
2:54:00 PM
2:51:00 PM
2:47:00 PM
2:39:00 PM
2:32:00 PM
2:41:OOPM
2:26:00 PM
2:42:00 PM
12:22:00 PM
12:19:00 PM
12:17:00 PM
9:18:00 AM
10:33:00 AM
10:07:00 AM
10:01:00 AM
Object
Floor
File cabinet
File cabinet
Desk
Floor
Wall
Floor
Floor
Monitor
Return Vent
Floor
Ceiling
Floor
Wall
Floor
Ceiling
Ceiling
Floor
Floor
Floor
Floor
Wall
Floor
Ceiling
Ceiling
Ceiling
Ceiling
Texture
Smooth
Metal
Smooth
Smooth
Smooth
Textured
Smooth
Smooth
Smooth
Metal
Smooth
Porous
Smooth
Smooth
Smooth
Porous
Porous
Smooth
Smooth
Smooth
Textured
Smooth
Textured
Porous
Porous
Porous
Porous
Orientation
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Vertical
Horizontal
Downward
Horizontal
Downward
Inclined
Horizontal
Upward
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Blank
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
BROOM Notes
bottom left corner
had placed vac
blank sample in
bag with ceiling
tile sample from
rm26.
overlain with team
2 template.
overlain with team
2 template.
plenum side of
ceiling, middle hal
panel.
ceiling tile
between 205 and
207. on opposite
side from diagram
due to vent.
Lab ID
6
6
6
6
6
6
6
6
1
1
1
2
7
7
7
2
2
7
7
7
4
4
4
4
4
4
4
Detected
No
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
No
Yes
Yes
No
Yes
No
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
No
Yes
Spread Plate Results
(CFU/sq ft)
ND
185306.6
243350.8
202600.4
152922.2
1532.1
ND
141143.5
46212.6
450122.6
ND
1891.8
9497.9
ND
11768.2
ND
345.9
6479.8
7144.5
ND
22007.2
ND
8536.0
495.9
883.4
ND
437.5
Filter Plate Results
(CFU/sq ft)
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
TNTC
TNTC
ND
TNTC
ND
TNTC
ND
ND
ND
TNTC
10.2
TNTC
TNTC
TNTC
ND
TNTC
G-90
-------
Barcode
3196
3194
3198
3195
2831
3197
3192
2029
2028
2862
3187
3694
3184
3185
3687
3180
3172
4247
3182
3186
3911
2782
2867
Round
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
Floor
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Room
Hallway
Hallway
Hallway
Hallway
Hallway
Hallway
Hallway
Hallway
Hallway
Hallway
Hallway
Janitor Closet
Janitor Closet
Janitor Closet
Mechanical Room
Mechanical Room
Mechanical Room
Room 201
Room 201
Room 201
Room 201 A
Room 201 A
Room 201 A
X
7.693
7.310
13.193
17.593
25.312
11.193
25.275
25.354
21.637
23.137
17.737
28.800
26.898
28.598
19.070
22.131
19.021
27.231
29.415
26.612
28.531
26.415
29.231
y
13.468
13.437
13.068
14.058
19.643
13.358
18.829
14.898
13.868
13.668
13.168
15.349
14.954
15.278
19.284
19.519
20.554
11.249
11.699
11.699
7.867
9.040
7.667
z
5.500
3.000
3.300
3.000
3.300
3.000
3.000
3.000
3.000
5.096
4.200
5.900
3.000
3.000
3.500
3.000
3.000
5.900
3.000
3.000
5.900
3.000
3.000
Method
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Sponge Wipe
Sponge Wipe
Vacuum Sock
Sponge Wipe
Sponge Wipe
Vacuum Sock
Sponge Wipe
Sponge Wipe
Vacuum Sock
Sponge Wipe
Sponge Wipe
Area
(sqrt)
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
4.000
0.694
0.694
4.000
0.694
0.694
4.000
0.694
0.694
4.000
0.694
0.694
Operator
schademann
schademann
schademann
schademann
schademann
schademann
schademann
schademann
schademann
schademann
schademann
schademann
schademann
schademann
schademann
schademann
schademann
schademann
schademann
schademann
schademann
schademann
schademann
Acquisition
Date
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
Acquisition
Time
9:33:00 AM
9:01:00 AM
9:45:00 AM
9:51:00 AM
10:38:00 AM
9:40:00 AM
11:12:00 AM
10:27:00 AM
10:20:00 AM
10:19:00 AM
10:05:00 AM
12:34:00 PM
12:26:00 PM
12:27:00 PM
3:16:00 PM
3:11:00 PM
3:19:00 PM
11:52:00 AM
11:46:00 AM
11:44:00 AM
12:12:00 PM
11:57:00 AM
12:07:00 PM
Object
Return Vent
Floor
Wall
Floor
Wall
Floor
Floor
Floor
Floor
Return Vent
Ceiling
Ceiling
Floor
Floor
Return Vent
Floor
Floor
Ceiling
Floor
Floor
Ceiling
Floor
Floor
Texture
Metal
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Metal
Porous
Porous
Textured
Smooth
Porous
Smooth
Smooth
Porous
Smooth
Smooth
Porous
Smooth
Smooth
Orientation
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Blank
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
sampled verticle
on 212 side of
wall.
overlaw with team
2 template.
floor between 206
and 204 next wall.
covered team 2
template.
used range finder
for location.
appears off. actual
location is 0.3 m
outside of airlock.
[B. Melton moved
outside of airlock
from room 203]
return vent.
vertical side.
on clear spot on
floor.
vacuum from
return furnace
filter.
Lab ID
6
6
6
6
6
6
4
6
6
6
6
4
4
4
2
7
7
4
4
4
4
4
4
Detected
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Spread Plate Results
(CFU/sq ft)
148.8
18182.2
ND
15836.5
ND
18506.2
13218.7
10052.7
13780.3
28.8
ND
250.0
7645.2
17510.7
17459.3
53974.0
20980.0
416.7
16235.4
8950.7
1358.4
15543.7
17482.4
Filter Plate Results
(CFU/sq ft)
37.2
TNTC
ND
TNTC
9.3
TNTC
TNTC
TNTC
TNTC
46.5
ND
TNTC
TNTC
TNTC
TNTC
TNTC
-1438.5
TNTC
TNTC
ND
TNTC
TNTC
TNTC
G-91
-------
Barcode
2890
2865
3191
2920
4136
2203
2661
4034
2208
2204
3944
3188
3189
3190
3710
2199
2446
2881
3785
3754
2883
2854
Round
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
Floor
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Room
Room 201 A
Room 201 A
Room 202
Room 202
Room 203
Room 203
Room 203
Room 203A
Room 203A
Room 203A
Room 204
Room 204
Room 204
Room 204
Room 205
Room 205
Room 205
Room 205
Room 206
Room 206
Room 206
Room 206
X
29.031
26.515
21.964
21.534
24.349
22.947
22.947
24.046
24.046
24.960
19.993
19.693
18.756
20.193
20.234
20.134
19.115
20.325
17.821
16.221
16.321
17.642
y
5.967
6.840
17.597
14.786
10.149
10.957
9.849
6.240
6.640
9.081
16.290
17.490
16.990
14.990
8.850
6.150
9.609
11.450
16.845
18.754
15.055
15.249
z
3.300
3.000
3.000
3.000
5.900
3.000
4.000
5.900
3.000
3.000
5.900
3.000
3.396
3.000
5.900
4.000
3.000
3.000
5.900
4.000
3.000
3.000
Method
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Sponge Wipe
Sponge Wipe
Vacuum Sock
Sponge Wipe
Sponge Wipe
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Area
(sqft)
0.694
0.694
0.694
0.694
4.000
0.694
0.694
4.000
0.694
0.694
4.000
0.694
0.694
0.694
4.000
0.694
0.694
0.694
4.000
4.000
0.694
0.694
Operator
schademann
schademann
schademann
schademann
Kroone
Kroone
Kroone
Kroone
Kroone
Kroone
schademann
schademann
schademann
schademann
Kroone
Kroone
Kroone
Kroone
Kroone
Kroone
Kroone
Kroone
Acquisition
Date
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
Acquisition
Time
12:05:00 PM
12:00:00 PM
11:21:00 AM
11:18:00 AM
2:16:00 PM
2:02:00 PM
2:08:00 PM
2:35:00 PM
2:29:00 PM
2:24:00 PM
11:37:00 AM
11:32:00 AM
11:28:00 AM
11:26:00 AM
1:54:00 PM
1:46:00 PM
1:40:00 PM
1:34:00 PM
12:05:00 PM
11:32:00 AM
11:01:00 AM
11:13:00 AM
Object
Wall
Floor
Floor
Floor
Ceiling
Floor
Wall
Ceiling
Floor
Floor
Ceiling
Floor
Wall
Floor
Ceiling
Wall
Floor
Floor
Ceiling
Table
Floor
Floor
Texture
Smooth
Smooth
Smooth
Smooth
Textured
Smooth
Smooth
Textured
Smooth
Smooth
Porous
Smooth
Smooth
Smooth
Textured
Smooth
Smooth
Smooth
Textured
Smooth
Smooth
Smooth
Orientation
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Vertical
Horizontal
Upward
Horizontal
Downward
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Downward
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Blank
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
BROOM Notes
had put bag into
foam stick bag
prior to scan.
[incorrect barcode
for this sample,
look for sponge
stick in sample
trailer -B. Melton,
also changed
barcode to 2890]
manuel location as
range finder
appears to provide
inaccurate results.
manuel location as
previous one done
by range finder
was incorrect.
tile placed on fiooi
to ssample
on floor
placed tile on flooi
to sample
near door entrance
broke corner of tih
while removing.
see photo.
corrected entry.
ceilinbg tile
blank
on floor in swing
of door
8 feet into room 01
left wall
Lab ID
4
4
4
4
1
5
5
2
5
5
4
4
4
4
1
5
5
5
1
1
5
5
Detected
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Spread Plate Results
(CFU/sq ft)
18.7
21541.6
18863.3
12723.4
189.0
14002.0
ND
325.0
13559.5
15731.9
462.5
12747.4
ND
14089.9
243.3
ND
14663.4
8046.4
240.7
ND
13335.3
14946.6
Filter Plate Results
(CFU/sq ft)
80.6
TNTC
TNTC
TNTC
NA
TNTC
ND
115206.3
TNTC
TNTC
TNTC
TNTC
ND
TNTC
NA
46.4
ND
TNTC
NA
NA
TNTC
TNTC
G-92
-------
Barcode
3155
2348
2319
2887
2853
1588
1496
3709
4131
3241
2864
2448
2452
2717
2719
2461
2863
1567
1506
4126
3875
3932
4062
3940
2631
3193
2617
2620
Round
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
Floor
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Room
Room 206
Room 206
Room 206
Room 206
Room 206
Room 206
Room 206
Room 207
Room 207
Room 207
Room 207
Room 207
Room 207
Room 207
Room 207
Room 207
Room 207
Room 207
Room 207
Room 208
Room 208
Room 208
Room 208
Room 208
Room 208
Room 208
Room 208
Room 208
X
17.721
18.248
16.715
17.042
17.821
17.421
17.142
17.553
16.753
15.886
15.477
15.456
15.854
17.512
16.653
17.953
15.890
16.753
16.753
13.281
15.167
13.825
13.482
13.562
12.963
15.270
14.866
13.558
y
19.654
20.671
19.895
18.754
18.354
20.154
18.754
11.059
9.150
6.487
6.767
8.112
7.865
9.598
9.050
7.250
12.016
7.150
9.250
16.605
20.321
20.396
19.655
18.419
19.865
19.115
16.839
15.174
z
3.000
4.000
4.000
4.000
4.000
5.900
4.000
5.900
3.000
4.000
5.000
5.000
4.000
4.000
4.000
3.300
3.000
5.900
4.000
5.500
3.000
3.000
3.000
3.000
3.000
3.000
3.000
3.000
Method
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Swab
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Swab
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqft)
0.694
0.694
0.694
0.694
0.694
0.028
0.028
4.000
4.000
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.028
0.028
4.000
4.000
4.000
4.000
4.000
0.694
0.694
0.694
0.694
Operator
Kroone
Kroone
Kroone
Kroone
Kroone
Kroone
Kroone
Kroone
Kroone
Kroone
Kroone
Kroone
Kroone
Kroone
Kroone
Kroone
Kroone
Kroone
Kroone
Ball
Ball
Ball
Ball
Ball
Ball
Ball
Ball
Ball
Acquisition
Date
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
Acquisition
Time
11:55:00 AM
11:51:00 AM
11:43:00 AM
11:38:00 AM
11:28:00 AM
11:59:00 AM
11:36:00 AM
1:27:00 PM
12:28:00 PM
1:10:00 PM
1:19:00 PM
1:14:00 PM
1:06:00 PM
12:53:00 PM
12:32:00 PM
12:50:00 PM
12:16:00 PM
12:40:00 PM
12:36:00 PM
12:55:00 PM
12:44:00 PM
12:30:00 PM
12:26:00 PM
12:12:00 PM
12:38:00 PM
12:18:00 PM
12:02:00 PM
11:57:00 AM
Object
Floor
Table
Table
Table
Table
Ceiling
Table
Ceiling
Floor
Table
Shelves
Shelves
Table
Table
Table
Wall
Floor
Supply Vent
Table
Ceiling
File cabinet
Chair
Wall
Chair
File cabinet
Wall
Floor
Table
Texture
Smooth
Smooth
Smooth
Smooth
Smooth
Textured
Smooth
Textured
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Porous
Metal
Porous
Porous
Porous
Metal
Textured
Smooth
Smooth
Orientation
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Vertical
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Blank
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
TRUE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
wood shavings on
floor where sarnie
taken
inadverte tly did
tsable shouldhave
done floor
moved tools on
bench
sticks don't break
correcged this is
thecorrect [B.
Melton addressed]
ceiling ventstick
did notbreak
blank nevertrust
those who
packyyour sz piers
k
1 fOOt off floor
Lab ID
5
5
5
5
5
1
1
1
1
5
5
5
5
5
5
5
5
1
1
7
7
7
6
7
2
2
2
2
Detected
Yes
Yes
Yes
No
Yes
Yes
No
Yes
No
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Spread Plate Results
(CFU/sq ft)
234.2
3386.8
3686.3
ND
13209.1
86423.5
ND
235.2
ND
8942.1
12700.3
11746.1
14101.9
11413.5
ND
ND
14471.5
39010.6
ND
612.5
179.2
579.2
87.5
529.2
18717.9
100.8
3959.9
15004.2
Filter Plate Results
(CFU/sq ft)
-1438.5
TNTC
TNTC
ND
TNTC
NA
NA
NA
NA
TNTC
TNTC
TNTC
TNTC
TNTC
ND
9.3
TNTC
NA
NA
TNTC
131.7
ND
27.8
ND
TNTC
35.6
ND
TNTC
G-93
-------
Barcode
1783
1678
3954
4228
4037
2871
2763
3255
2339
2806
1658
3879
4225
3955
4040
3930
3472
3474
2978
3466
3470
3468
1549
1740
1544
4049
3637
3632
Round
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
Floor
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Room
Room 208
Room 208
Room 209
Room 209
Room 209
Room 209
Room 209
Room 209
Room 209
Room 209
Room 209
Room 210
Room 210
Room 210
Room 210
Room 210
Room 210
Room 210
Room 210
Room 210
Room 210
Room 210
Room 210
Room 210
Room 210
Room 211
Room 211
Room 211
X
12.794
13.816
14.703
13.403
14.611
12.453
13.836
14.761
12.812
14.082
14.402
10.047
12.098
10.680
10.425
11.798
12.098
9.701
10.153
10.153
11.912
11.888
10.836
9.663
11.655
10.472
10.822
11.171
y
18.799
20.093
8.642
10.933
10.995
6.781
7.385
9.588
10.895
11.964
7.749
15.751
19.960
20.329
19.634
15.579
20.542
19.833
14.986
16.745
15.864
14.404
19.974
18.910
15.865
11.885
7.183
10.626
z
3.000
3.000
3.000
3.000
5.500
5.000
4.000
4.500
4.000
4.500
5.500
3.000
3.000
3.000
3.000
3.000
3.000
3.000
3.000
3.000
3.000
4.000
3.000
3.000
3.000
3.000
3.000
4.000
Method
Swab
Swab
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Swab
Swab
Vacuum Sock
Vacuum Sock
Vacuum Sock
Area
(sqrt)
0.028
0.028
4.000
4.000
4.000
0.694
0.694
0.694
0.694
0.694
0.028
4.000
4.000
4.000
4.000
4.000
0.694
0.694
0.694
0.694
0.694
0.694
0.028
0.028
0.028
4.000
4.000
4.000
Operator
Ball
Ball
Ball
Ball
Ball
Ball
Ball
Ball
Ball
Ball
Ball
Ball
Ball
Ball
Ball
Ball
Ball
Ball
Ball
Ball
Ball
Ball
Ball
Ball
Ball
Hearn
Hearn
Hearn
Acquisition
Date
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
Acquisition
Time
12:05:00 PM
12:48:00 PM
1:20:00 PM
1:09:00 PM
1:36:00 PM
1:28:00 PM
1:26:00 PM
1:17:00 PM
1:12:00 PM
1:04:00 PM
1:31:00 PM
11:44:00 AM
11:28:00 AM
11:16:00 AM
11:03:00 AM
10:33:00 AM
11:21:00 AM
11:09:00 AM
9:55:00 AM
10:01:00 AM
10:28:00 AM
10:39:00 AM
11:32:00 AM
10:49:00 AM
10:24:00 AM
11:44:00 AM
12:14:00 PM
12:06:00 PM
Object
Monitor
Supply Vent
Floor
Floor
Ceiling
Wall
Table
Stove
Sink
Countertop
Supply Vent
Ceiling
File cabinet
Chair
Wall
Floor
File cabinet
File cabinet
Floor
Floor
Floor
Wall
Supply Vent
Monitor
Floor
Floor
Floor
Floor
Texture
Smooth
Metal
Porous
Carpet
Porous
Textured
Smooth
Paint
Metal
Smooth
Metal
Porous
Metal
Porous
Porous
Smooth
Metal
Metal
Smooth
Smooth
Smooth
Textured
Metal
Smooth
Smooth
Carpet
Carpet
Smooth
Orientation
Inclined
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Downward
Inclined
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Blank
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
TRUE
BROOM Notes
outter bag droppet
on floor
vacuum around
books and on top
ofbooks
some slats bent
and not swabbed
upper right corner
vacuum sample on
right side of door
inrm211.
vacuum sample 1
ft behind chair on
window side of
room
vacuum blank
Lab ID
1
1
7
6
7
1
2
1
1
2
1
7
7
6
7
7
2
2
1
2
1
1
1
1
1
5
5
2
Detected
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
No
Yes
Yes
No
Spread Plate Results
(CFU/sq ft)
3000.8
29408.0
133.3
62.5
962.6
18316.1
16578.6
11358.3
ND
15161.7
91825.0
725.0
241.7
591.7
166.7
ND
9879.9
16449.9
ND
19692.7
ND
17.3
40211.0
4801.3
ND
191.7
95.8
ND
Filter Plate Results
(CFU/sq ft)
1530.4
21876.0
108.7
46.5
TNTC
NA
TNTC
NA
NA
TNTC
NA
TNTC
190.3
ND
225.4
ND
TNTC
TNTC
2002.2
TNTC
NA
18.1
33309.1
1440.4
NA
102.9
67.3
ND
G-94
-------
Barcode
4030
3869
4056
2338
2060
2213
1730
1724
3880
4223
4027
4289
4127
4038
2274
2546
2807
Round
C1O2 Pre-
Decon
C1O2 Pre-
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
C1O2 Pre-
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
Decon
Floor
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Room
Room 211
Room 211
Room 211
Room 211
Room 211
Room 211
Room 211
Room 211
Room 212
Room 212
Room 212
Room 212
Room 212
Room 212
Room 212
Room 212
Room 212
X
10.909
11.835
10.839
9.493
11.084
9.458
11.294
9.913
7.734
8.363
9.115
7.717
7.297
7.350
7.402
9.132
6.441
y
8.232
9.403
11.728
6.256
10.364
11.378
10.487
9.525
15.502
18.963
20.309
20.413
19.539
18.368
15.467
14.750
16.008
z
3.300
3.400
5.900
5.000
4.000
3.300
4.000
3.800
3.100
5.500
4.800
3.400
5.000
3.600
3.100
3.000
3.200
Method
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Swab
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqft)
4.000
4.000
4.000
0.694
0.694
0.694
0.028
0.028
4.000
4.000
4.000
4.000
4.000
4.000
0.694
0.694
0.694
Operator
Hearn
Hearn
Hearn
Hearn
Hearn
Hearn
Hearn
Hearn
Hearn
Hearn
Hearn
Hearn
Acquisition
Date
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
Acquisition
Time
11:59:00 AM
11:55:00 AM
12:24:00 PM
12:20:00 PM
12:08:00 PM
11:47:00 AM
12:05:00 PM
11:50:00 AM
9:55:00 AM
10:36:00 AM
10:28:00 AM
10:23:00 AM
10:16:00 AM
10:08:00 AM
9:50:00 AM
9:47:00 AM
9:59:00 AM
Object
Chair
Couch
Ceiling
Shelves
Floor
Wall
Chair
Shelves
Ceiling
File cabinet
Chair
Shelves
Chair
Floor
Floor
Wall
Texture
Cloth
Cloth
Porous
Smooth
Smooth
Paint
Paint
Smooth
Porous
Cloth
Metal
Cloth
Smooth
Smooth
Paint
Orientation
Horizontal
Upward
Horizontal
Upward
Horizontal
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Horizontal
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Blank
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
TRUE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
BROOM Notes
vacuum sample
taken on cloth
chair cushion.
chair is on side of
room toward
window
vacuum sample
center cushion
topside of ceiling
tile vacuum samph
. photo taken from
window end of
room.
sponge sample on
far side of shelves
in rear of room
sponge blank
sponge sample on
wall . 2 ft from
wall on door side
of room, 1.5 ft
above floor
upper right corner
of screen.
vacuum top of
ceiling tile . pictun
doorway, sample
taken in rear half
of ceiling tile
vacuum top and
sides of
books/binders on
file cabinet
vacuum seat of
cloth chair
vacuum top of
partition
vacuum sample
taken on chair seat
sponge blank
directly next to
door in middle
vertical snonoe
directly left of wal
beam.
Lab ID
5
5
5
1
1
1
1
1
2
5
5
2
2
1
1
1
1
Detected
Yes
Yes
Yes
No
No
Yes
No
Yes
No
Yes
Yes
Yes
Yes
Yes
No
Yes
No
Spread Plate Results
(CFU/sq ft)
1570.9
1158.4
908.4
ND
ND
ND
ND
5401.5
ND
787.5
58.3
1300.1
75.0
265.0
ND
27704.6
ND
Filter Plate Results
(CFU/sq ft)
TNTC
TNTC
TNTC
NA
NA
2.3
NA
5851.6
ND
TNTC
35.1
TNTC
69.6
NA
NA
NA
NA
G-95
-------
Barcode
2275
3475
1550
1734
1525
4246
3881
4108
3950
3877
3537
2480
2395
2477
2543
Round
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
C1O2 Pre-
Decon
C1O2 Pre-
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
C1O2 Pre-
Decon
Floor
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Room
Room 212
Room 212
Room 212
Room 212
Room 212
Room 213
Room 213
Room 213
Room 213
Room 213
Room 213
Room 213
Room 213
Room 213
Room 213
X
8.853
6.790
7.542
6.790
7.769
7.822
8.569
8.123
7.267
6.812
6.829
6.583
7.092
8.910
6.812
y
17.652
19.539
15.519
18.438
19.959
8.128
8.204
11.412
10.713
11.534
8.204
6.861
5.993
10.888
11.534
z
3.000
3.600
3.100
4.200
5.500
3.300
3.000
3.000
5.900
3.000
3.300
3.800
3.500
4.000
3.100
Method
Sponge Wipe
Sponge Wipe
Swab
Swab
Swab
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqrt)
0.694
0.694
0.028
0.028
0.028
4.000
4.000
4.000
4.000
4.000
4.000
0.694
0.694
0.694
0.694
Operator
Hearn
Hearn
Hearn
Hearn
Hearn
Hearn
Hearn
Hearn
Hearn
Hearn
Acquisition
Date
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
5/11/2011
Acquisition
Time
10:42:00 AM
10:20:00 AM
9:53:00 AM
10:06:00 AM
10:13:00 AM
11:16:00 AM
11:01:00 AM
10:52:00 AM
11:33:00 AM
10:48:00 AM
11:20:00 AM
11:12:00 AM
11:08:00 AM
10:55:00 AM
10:51:00 AM
Object
Floor
File cabinet
Desk
Supply Vent
Bed
Floor
Floor
Ceiling
Floor
Bed
Cabinet
Wall
Countertop
Floor
Texture
Smooth
Metal
Smooth
Smooth
Metal
Cloth
Carpet
Smooth
Carpet
Cloth
Metal
Paint
Smooth
Smooth
Orientation
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Blank
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
BROOM Notes
sponge sample on
floor 1 ft from wal
and two ft left of
outlet
sponge top of file
cabinet behind
partition, location
of sample is front
half of file cabinet
see red markings
swab upper right
computer monitor
ceiling vent.
vacuum sample
foot of bed on side
closest to door
vacuum sample
taken at foot of
bed. room
backwards from
picture.
ceiling tile vacuun
. second tile in
from doorway and
pic taken from
doorway, vacuum
half of tile furthest
from door
vacuum sample
taken one ft in
from doorway
vacuum sample
pillow end of bed
on side of room
furthest from door
horizontal sponge
on cabinet next to
bed on far side of
room
vertical sponge 1 f
above floor
sponge sample on
countertop. right
side of sink
blank sponge
Lab ID
1
1
1
1
1
5
5
1
5
1
1
1
1
1
1
Detected
Yes
Yes
No
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
No
Yes
Yes
No
Spread Plate Results
(CFU/sq ft)
13804.3
357106.5
ND
ND
39010.6
750.0
154.2
ND
3187.7
256.5
609.2
ND
192.0
15119.4
ND
Filter Plate Results
(CFU/sq ft)
NA
NA
NA
2610.7
NA
TNTC
-249.8
NA
TNTC
NA
NA
NA
77.8
NA
NA
G-96
-------
Barcode
3497
4106
2456
2516
3448
2403
3867
3454
3471
3443
3455
4124
3625
4051
4085
4251
3442
3450
3437
3447
3452
3451
3438
3449
3439
3469
Round
C1O2 Pre-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
Floor
Floor 2
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Room
Stairwell
Bathroom M
Bathroom M
Bathroom M
Bathroom M
Bathroom M
Bathroom W
Bathroom W
Bathroom W
Bathroom W
Bathroom W
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
X
23.947
26.782
25.964
26.275
24.723
24.582
27.115
25.259
27.093
24.356
25.400
8.281
16.096
22.804
17.307
24.331
13.599
12.481
8.281
6.078
5.081
4.081
22.945
13.333
14.336
23.052
y
19.617
19.911
18.444
20.701
20.222
19.121
15.149
16.751
16.497
14.917
14.579
12.861
13.519
16.441
13.980
7.353
14.092
12.941
14.138
13.897
13.565
13.365
18.299
16.641
15.141
17.397
z
3.000
0.000
0.000
0.000
0.000
0.000
2.700
0.000
0.000
0.000
0.700
2.800
0.000
2.600
0.000
0.000
0.000
0.000
0.000
2.000
0.000
0.600
2.000
0.000
0.000
0.000
Method
Sponge Wipe
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqft)
0.694
4.000
0.694
0.694
0.694
0.694
4.000
0.694
0.694
0.694
0.694
4.000
4.000
4.000
4.000
4.000
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
Operator
schademann
Berendzen
Berendzen
Berendzen
Berendzen
Berendzen
Berendzen
Berendzen
Berendzen
Berendzen
Berendzen
Berendzen
Berendzen
Berendzen
Berendzen
Berendzen
Berendzen
Berendzen
Berendzen
Berendzen
Berendzen
Berendzen
Berendzen
Berendzen
Berendzen
Berendzen
Acquisition
Date
5/11/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
Acquisition
Time
3:09:00 PM
11:32:00 AM
11:42:00 AM
11:29:00 AM
11:26:00 AM
11:48:00 AM
11:12:00 AM
11:18:00 AM
11:16:00 AM
11:03:00 AM
11:06:00 AM
9:13:00 AM
9:58:00 AM
10:41:00 AM
10:09:00 AM
3:00:00 PM
9:39:00 AM
9:34:00 AM
9:19:00 AM
8:57:00 AM
8:48:00 AM
8:44:00 AM
10:50:00 AM
9:49:00 AM
9:53:00 AM
10:47:00 AM
Object
Floor
Ceiling
Floor
Wall
Floor
Floor
Ceiling
Floor
Floor
Floor
Wall
Ceiling
Ceiling
Ceiling
Floor
Floor
Floor
Floor
Floor
Supply Vent
Floor
Wall
Floor
Floor
Floor
Floor
Texture
Smooth
Porous
Smooth
Smooth
Smooth
Smooth
Porous
Smooth
Smooth
Smooth
Smooth
Porous
Porous
Porous
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Orientation
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Vertical
Horizontal
Upward
Horizontal
Downward
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Downward
Horizontal
Upward
Horizontal
Downward
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Blank
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
BROOM Notes
disregard previous
location for 3497
[B. Melton
addressed]
sample taken on
plenum side of the
ceiling tile
sample taken on
floor
sample taken on
floor
sample taken on
floor in front of
room 13
sample taken on
wall
sample was taken
on hidden surface
of ceiling tile
sample taken on
plenum side of
ceiling tile
blank
sample taken on
floor
sample taken on
floor
sample was taken
in hvac vent
sample taken on
floor
sample was taken
on door
blank
sample taken on
floor
Lab ID
7
5
7
7
8
7
5
7
7
7
7
5
5
5
5
4
7
8
7
7
1
7
8
7
7
8
Detected
Yes
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
Spread Plate Results
(CFU/sq ft)
218391.8
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Filter Plate Results
(CFU/sq ft)
-1438.5
ND
ND
ND
NA
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
NA
ND
ND
NA
ND
NA
ND
ND
NA
G-97
-------
Barcode
3446
3440
3433
3435
3359
3874
3465
2324
3467
3953
3463
3444
3445
3460
3735
3555
3872
3430
3428
Round
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
Floor
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Room
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Corridor+Lobby
Mechanical Room
Mechanical Room
Mechanical Room
Mechanical Room
Room 101
Room 101
Room 101
Room 101
Room 101
Room 101A
Room 101A
Room 101A
Room 101A
Room 101A
X
21.010
19.258
18.891
18.750
21.284
17.537
18.665
17.057
16.860
20.732
18.733
16.128
16.580
21.517
17.022
20.732
18.532
20.201
18.932
y
13.516
13.106
14.178
12.909
12.837
19.163
20.573
18.232
19.219
11.321
10.609
11.933
11.848
10.974
9.427
8.327
7.827
9.157
8.827
z
2.600
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
2.800
0.000
0.700
0.000
0.000
0.000
0.000
0.000
0.594
0.892
Method
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Area
(sqft)
0.694
0.694
0.694
0.694
0.694
4.000
0.694
0.694
0.694
4.000
0.694
0.694
0.694
0.694
4.000
4.000
4.000
0.694
0.694
Operator
Berendzen
Berendzen
Berendzen
Berendzen
Schaedemann
Berendzen
Berendzen
Berendzen
Berendzen
Berendzen
Berendzen
Berendzen
Berendzen
Berendzen
Lee
Lee
Lee
Lee
Lee
Acquisition
Date
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
Acquisition
Time
10:31:00 AM
10:19:00 AM
10:16:00 AM
10:13:00 AM
12:09:00 PM
12:07:00 PM
11:53:00 AM
12:00:00 PM
12:02:00 PM
2:18:00 PM
2:34:00 PM
2:31:00 PM
2:27:00 PM
2:02:00 PM
10:30:00 AM
11:08:00 AM
10:53:00 AM
11:02:00 AM
10:35:00 AM
Object
Supply Vent
Floor
Wall
Floor
Floor
Supply Vent
Floor
Floor
Wall
Ceiling
Floor
Wall
Floor
Floor
Floor
Floor
Floor
File cabinet
Desk
Texture
Smooth
Smooth
Smooth
Smooth
Smooth
Textured
Smooth
Smooth
Smooth
Porous
Smooth
Smooth
Smooth
Smooth
Carpet
Carpet
Carpet
Smooth
Plastic
Orientation
Horizontal
Downward
Horizontal
Downward
Vertical
Horizontal
Downward
Horizontal
Upward
Vertical
Horizontal
Downward
Horizontal
Downward
Vertical
Horizontal
Downward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Blank
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
BROOM Notes
sample taken in
hvac
randy collected
near wall adjacent
to stairs
to dark for photo
[B. Melton changec
barcode from 3465
to 3467]
sample taken on
plenum side of the
ceiling tile
sample taken on
floor
sample taken on
wall behind swing
of door
sample taken on
floor in front of
door of room 101
sample taken on
floor in door of
room 101
carpet fibers
caught in hose anc
sock, included
fiber in sample
bag.
closed sample bag
dropped on floor
before taking
sample, different
fron planned
location, skc in the
blank
about 80 %
overlap with
predecon
sample.tape on
corner.
vertical sample on
uv APS on desk.
ridges on box. han:
to put sponge flat
on surface.
Lab ID
5
7
7
7
5
5
7
7
8
4
2
2
2
2
1
4
4
4
4
Detected
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
Spread Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Filter Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
NA
ND
ND
ND
ND
G-98
-------
Barcode
3426
3423
3431
3432
3422
3415
3424
3420
3425
3736
3731
3742
3747
3405
3417
3410
3407
3427
3414
3419
Round
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
Floor
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Room
Room 101A
Room 101A
Room 101A
Room 101A
Room 101A
Room 101A
Room 101A
Room 101A
Room 101A
Room 102
Room 102
Room 102
Room 102
Room 102
Room 102
Room 102
Room 102
Room 102
Room 102
Room 102
X
18.333
21.452
18.332
21.332
21.524
20.273
19.258
17.898
21.887
19.217
19.606
18.117
18.632
18.938
17.989
19.992
20.583
18.811
19.149
19.507
y
9.171
7.325
7.714
7.914
6.256
6.147
6.546
6.219
6.763
15.530
16.483
15.518
16.034
16.857
17.142
16.899
16.224
15.929
16.688
15.106
z
1.097
0.688
0.000
0.696
0.686
0.694
0.700
0.700
0.692
0.000
0.000
0.000
0.000
0.700
0.700
0.700
0.000
0.000
0.600
1.100
Method
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqft)
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
4.000
4.000
4.000
4.000
0.694
0.694
0.694
0.694
0.694
0.694
0.694
Operator
Lee
Lee
Lee
Lee
Lee
Lee
Lee
Lee
Lee
Lee
Lee
Lee
Lee
Lee
Lee
Lee
Lee
Lee
Lee
Lee
Acquisition
Date
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
Acquisition
Time
10:45:00 AM
11:24:00 AM
10:58:00 AM
11:17:00 AM
11:34:00 AM
11:39:00 AM
11:50:00 AM
12:00:00 PM
11:32:00 AM
12:11:00 PM
12:19:00 PM
12:30:00 PM
12:33:00 PM
1:32:00 PM
1:19:00 PM
1:44:00 PM
12:44:00 PM
12:37:00 PM
1:46:00 PM
12:53:00 PM
Object
Desk
Desk
Floor
File cabinet
Desk
Desk
Desk
File cabinet
Desk
Floor
Floor
Floor
Floor
Desk
File cabinet
Desk
File cabinet
Floor
Desk
Desk
Texture
Plastic
Smooth
Carpet
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Carpet
Carpet
Carpet
Carpet
Smooth
Smooth
Smooth
Smooth
Carpet
Smooth
Plastic
Orientation
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Blank
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
BROOM Notes
sampling port in
corner of template
sticker on uv APS
on desk.
sand or something
on desk.
blank
about 80% overlap
with predecon
sample.
cord crossing
template area.
about 80% overlap
with predecon
sample, little bit ol
rust.
outer bag dropped
on floor.
black cord cover
crossing template.
black cord cover
crossing template.
blank, inner bag
folded sideways.
removed and
straightened.
about 80% overlap
with predecon
sample, lots of
rust.
about 80% overlap
with predecon
sample
blank
inside desk drawer
uv APS on desk.
ridges on box.
little white stuff ot
box.
Lab ID
4
4
4
4
4
4
4
4
4
1
1
1
1
4
4
4
4
4
4
4
Detected
No
No
No
Yes
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
Spread Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Filter Plate Results
(CFU/sq ft)
ND
ND
ND
3.2
ND
ND
ND
ND
ND
NA
NA
NA
NA
ND
ND
ND
ND
ND
ND
ND
G-99
-------
Barcode
3421
3418
3416
3412
4068
3750
3406
3401
3409
3400
3411
3403
3408
3402
1657
1514
3617
4067
3384
3368
3389
3387
3397
3379
1744
Round
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
Floor
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Room
Room 102
Room 102
Room 102
Room 102
Room 103
Room 103
Room 103
Room 103
Room 103
Room 103
Room 103
Room 103
Room 103
Room 103
Room 103
Room 103
Room 104
Room 104
Room 104
Room 104
Room 104
Room 104
Room 104
Room 104
Room 104
X
18.516
18.231
16.913
18.431
14.704
14.609
13.625
13.208
13.719
13.322
14.212
14.485
15.739
15.390
14.458
14.402
16.385
15.491
15.763
13.691
15.232
15.724
15.815
15.232
15.167
y
15.022
15.033
15.792
17.363
9.864
10.584
7.574
7.669
6.779
6.249
9.751
6.104
7.092
11.988
9.561
6.680
19.583
19.104
19.298
19.311
20.127
20.723
20.114
18.275
19.311
z
1.100
0.700
0.700
0.700
0.000
2.500
0.686
1.584
0.581
0.000
0.000
1.085
0.000
0.000
0.000
2.500
0.000
1.300
1.300
0.000
1.100
1.200
1.100
1.000
1.300
Method
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Swab
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Area
(sqft)
0.694
0.694
0.694
0.694
4.000
4.000
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.028
0.028
4.000
4.000
0.694
0.694
0.694
0.694
0.694
0.694
0.028
Operator
Lee
Lee
Lee
Lee
Chong
Chong
Chong
Chong
Chong
Chong
Chong
Chong
Chong
Chong
Chong
Chong
Jordan
Jordan
Jordan
Jordan
Jordan
Jordan
Jordan
Jordan
Jordan
Acquisition
Date
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
Acquisition
Time
12:59:00 PM
1:06:00 PM
1:11:00 PM
1:29:00 PM
3:12:00 PM
3:35:00 PM
3:24:00 PM
3:28:00 PM
3:22:00 PM
3:20:00 PM
3:08:00 PM
2:58:00 PM
2:53:00 PM
2:41:OOPM
3:10:00 PM
3:01:00 PM
2:29:00 PM
1:51:00 PM
1:53:00 PM
2:06:00 PM
2:13:00 PM
2:26:00 PM
2:17:00 PM
1:46:00 PM
1:57:00 PM
Object
Desk
Desk
File cabinet
File cabinet
Floor
Ceiling
Table
Mail slot
Table
Floor
Floor
Wall
Floor
Floor
Floor
Supply Vent
Ceiling
Desk
Desk
Floor
Desk
Wall
Desk
Desk
Desk
Texture
Plastic
Smooth
Smooth
Smooth
Smooth
Porous
Smooth
Metal
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Porous
Smooth
Smooth
Textured
Textured
Textured
Textured
Smooth
Smooth
Orientation
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Blank
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
TRUE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
BROOM Notes
uv APS on desk.
lots of white stuff.
ridges on box .
missing some area
because of handle
depression
sticker residue.
some white stuff
on desk.
about 80% overlap
with predecon
sample, little bit o]
rust.
about 80% overlap
with predecon
sample, moderate
rust.
top of mail slot
ceiling tile vacuun
blank taken
blank taken
sponge sample on
floor
sponge sample on
wooden table
sponge sample on
wall above
previous sample
sponge sample on
wooden table
sponge sample on
table
blank taken
Lab ID
4
4
4
4
1
1
1
1
1
1
1
1
1
1
7
7
4
4
2
2
2
2
2
2
7
Detected
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
Spread Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Filter Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
G-100
-------
Barcode
1541
3729
3745
3619
3904
4066
3395
3394
3398
3396
1674
3732
3846
3730
3885
3755
3365
3372
3370
3363
3373
1572
1613
3758
3615
3757
4016
3364
2211
Round
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
Floor
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Room
Room 104
Room 105
Room 105
Room 105
Room 105
Room 105
Room 105
Room 105
Room 105
Room 105
Room 105
Room 106
Room 106
Room 106
Room 106
Room 106
Room 106
Room 106
Room 106
Room 106
Room 106
Room 106
Room 106
Room 107
Room 107
Room 107
Room 107
Room 107
Room 107
X
14.934
10.565
10.917
11.786
10.477
10.958
11.967
10.345
12.481
9.981
11.731
12.556
11.058
11.272
12.648
11.058
9.988
10.477
12.388
10.171
10.691
11.257
10.340
7.921
8.465
9.104
9.345
8.656
7.733
y
19.635
6.857
9.044
11.059
12.070
7.238
6.846
9.638
10.947
11.059
6.408
18.865
19.660
20.470
20.119
18.468
16.175
14.876
15.365
19.905
18.422
19.889
18.437
11.970
8.382
6.428
12.170
9.754
12.345
z
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
Method
Swab
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Swab
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Area
(sqft)
0.028
4.000
4.000
4.000
4.000
4.000
0.694
0.694
0.694
0.694
0.028
4.000
4.000
4.000
4.000
4.000
0.694
0.694
0.694
0.694
0.694
0.028
0.028
4.000
4.000
4.000
4.000
0.694
0.694
Operator
Jordan
Kuberacki
Kuberacki
Kuberacki
Kuberacki
Kuberacki
Kuberacki
Kuberacki
Kuberacki
Kuberacki
Kuberacki
Kuberacki
Kuberacki
Kuberacki
Kuberacki
Kuberacki
Kuberacki
Kuberacki
Kuberacki
Kuberacki
Kuberacki
Kuberacki
Kuberacki
Kuberacki
Kuberacki
Kuberacki
Kuberacki
Kuberacki
Kuberacki
Acquisition
Date
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
Acquisition
Time
2:02:00 PM
4:13:00 PM
3:49:00 PM
3:35:00 PM
3:14:00 PM
4:27:00 PM
4:03:00 PM
3:56:00 PM
3:40:00 PM
3:28:00 PM
4:17:00 PM
1:16:00 PM
12:58:00 PM
12:52:00 PM
12:46:00 PM
12:31:00 PM
12:07:00 PM
11:53:00 AM
12:03:00 PM
1:04:00 PM
12:22:00 PM
1:07:00 PM
12:26:00 PM
11:41:00 AM
11:10:00 AM
11:01:00 AM
10:33:00 AM
10:46:00 AM
11:47:00 AM
Object
Ceiling
Floor
Floor
Floor
Floor
Ceiling
Table
Stove
Sink
Wall
Return Vent
Ceiling
Wall
Chair
File cabinet
Chair
Wall
Floor
Table
File cabinet
Desk
Return Vent
Monitor
Ceiling
Chair
Floor
Floor
Floor
Wall
Texture
Textured
Carpet
Carpet
Carpet
Carpet
Porous
Smooth
Metal
Metal
Textured
Metal
Porous
Cloth
Carpet
Porous
Cloth
Textured
Smooth
Smooth
Metal
Smooth
Metal
Glass
Porous
Cloth
Carpet
Carpet
Smooth
Textured
Orientation
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Inclined
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Inclined
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Blank
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
BROOM Notes
diffuser
sample taken on
diffuser
ceiling title, up
faceing side
partition wall on
window side.
lower middle .
books on desk
top of ceiling tile
only left and right
pattern in one
direction
completed
floor at door
entrance
center left on tabh
as you enter room.
Lab ID
7
1
2
4
1
1
4
4
1
4
7
1
1
1
1
1
1
1
1
4
1
7
7
1
1
1
1
1
1
Detected
No
No
No
Yes
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
Spread Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Filter Plate Results
(CFU/sq ft)
ND
ND
ND
0.6
ND
ND
ND
ND
ND
ND
ND
NA
NA
NA
NA
NA
NA
NA
NA
ND
NA
ND
ND
NA
NA
NA
NA
NA
NA
G-101
-------
Barcode
3391
3385
1568
1612
1569
3751
4070
4071
3887
3724
3739
3378
3380
3371
3390
3375
3361
1512
1488
1615
3657
3727
3624
4017
3733
3651
3367
Round
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
Floor
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Room
Room 107
Room 107
Room 107
Room 107
Room 107
Room 108
Room 108
Room 108
Room 108
Room 108
Room 108
Room 108
Room 108
Room 108
Room 108
Room 108
Room 108
Room 108
Room 108
Room 108
Room 109
Room 109
Room 109
Room 109
Room 109
Room 109
Room 109
X
7.113
9.160
8.221
7.395
9.254
8.949
9.653
8.270
7.985
7.998
8.641
9.653
9.420
8.528
8.917
7.509
7.245
8.873
7.409
8.415
6.105
5.537
4.110
5.437
4.957
6.351
5.102
y
6.729
6.616
6.522
9.772
6.729
18.917
20.136
20.475
19.508
18.490
15.987
20.513
17.370
16.251
14.567
14.705
18.225
19.570
18.678
15.837
11.964
7.901
8.715
9.674
11.139
6.624
6.133
z
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
Method
Sponge Wipe
Sponge Wipe
Swab
Swab
Swab
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Swab
Swab
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Area
(sqft)
0.694
0.694
0.028
0.028
0.028
4.000
4.000
4.000
4.000
4.000
4.000
0.694
0.694
0.694
0.694
0.694
0.694
0.028
0.028
0.028
4.000
4.000
4.000
4.000
4.000
4.000
0.694
Operator
Kuberacki
Kuberacki
Kuberacki
Kuberacki
Kuberacki
Ricks
Ricks
Ricks
Ricks
Ricks
Ricks
Ricks
Ricks
Ricks
Ricks
Ricks
Ricks
Ricks
Ricks
Ricks
Ricks
Ricks
Ricks
Ricks
Ricks
Ricks
Ricks
Acquisition
Date
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
Acquisition
Time
11:27:00 AM
10:50:00 AM
11:33:00 AM
11:14:00 AM
10:54:00 AM
3:52:00 PM
3:44:00 PM
3:34:00 PM
3:29:00 PM
3:21:00 PM
2:49:00 PM
3:37:00 PM
2:58:00 PM
2:48:00 PM
2:41:OOPM
2:32:00 PM
3:02:00 PM
3:41:00 PM
3:07:00 PM
2:45:00 PM
9:52:00 AM
10:57:00 AM
10:50:00 AM
10:32:00 AM
10:06:00 AM
11:19:00 AM
11:13:00 AM
Object
Shelves
Floor
Return Vent
Monitor
Floor
Ceiling
File cabinet
Chair
Wall
Chair
Floor
File cabinet
Floor
Floor
Wall
Floor
Desk
Return Vent
Monitor
Floor
Floor
Bed
Bed
Nightstand
Floor
Ceiling
Wall
Texture
Smooth
Carpet
Smooth
Glass
Carpet
Porous
Porous
Porous
Porous
Porous
Smooth
Metal
Smooth
Smooth
Textured
Smooth
Smooth
Metal
Smooth
Smooth
Carpet
Cloth
Cloth
Smooth
Carpet
Smooth
Smooth
Orientation
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Blank
FALSE
TRUE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
BROOM Notes
air vent diffuser
blades
upper middle of
screen
vacuum from
ceiling tile
vacuum from
books
vacuum from chaii
vacuum from
partition
vacuum from chaii
blank vacuum
sponge stick left
side of file cabinet
sponge stick from
floor
blank sponge stick
vertical sponge
stick
entrance
back middle of
desktop
swab from return
vent
bottom right of
monitor
blank swab
floor in front of
door
1 side top of
mattress at foot
end
blank vacuum
sample
floor in front of 1
side of sink
back side of
ceiling tile to left
of window.
window parallel to
bed
center of wall
under window
Lab ID
1
1
7
7
7
2
2
2
2
1
2
4
4
4
4
4
4
7
7
7
2
2
1
1
2
1
4
Detected
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
Spread Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Filter Plate Results
(CFU/sq ft)
NA
NA
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
G-102
-------
Barcode
3360
3393
3381
3377
3784
3543
3761
3756
4107
3987
2625
3343
3354
3351
2626
3336
1490
1741
1743
3726
3350
2932
Round
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
Floor
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 1
Floor 2
Floor 2
Floor 2
Room
Room 109
Room 109
Room 109
Room 109
Room 110
Room 110
Room 110
Room 110
Room 110
Room 110
Room 110
Room 110
Room 110
Room 110
Room 110
Room 110
Room 110
Room 110
Room 110
Bathroom M
Bathroom M
Bathroom M
X
4.154
5.570
4.344
4.288
5.111
5.226
6.093
6.551
4.981
4.964
5.897
4.457
6.240
3.934
5.291
6.600
5.618
4.261
4.735
27.896
28.832
28.795
y
6.914
9.964
12.265
10.961
15.259
20.479
15.162
20.577
19.628
18.483
14.507
17.927
16.504
16.455
15.472
19.971
19.595
18.548
15.226
19.554
20.727
20.487
z
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
5.800
3.200
3.000
Method
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Swab
Swab
Vacuum Sock
Sponge Wipe
Sponge Wipe
Area
(sqft)
0.694
0.694
0.694
0.694
4.000
4.000
4.000
0.070
4.000
4.000
0.694
0.694
0.694
0.694
0.694
0.694
0.028
0.028
0.028
4.000
0.694
0.694
Operator
Ricks
Ricks
Ricks
Ricks
Ricks
Ricks
Ricks
Ricks
Ricks
Ricks
Ricks
Ricks
Ricks
Ricks
Ricks
Ricks
Ricks
Ricks
Ricks
Schaedemann
Schaedemann
Schaedemann
Acquisition
Date
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
Acquisition
Time
11:08:00 AM
10:30:00 AM
9:58:00 AM
10:01:00 AM
11:35:00 AM
12:15:00 PM
12:35:00 PM
12:20:00 PM
12:11:00 PM
11:53:00 AM
11:27:00 AM
11:58:00 AM
11:47:00 AM
11:43:00 AM
11:40:00 AM
12:26:00 PM
12:29:00 PM
12:01:00 PM
11:37:00 AM
2:18:00 PM
2:10:00 PM
2:04:00 PM
Object
Nightstand
Nightstand
Countertop
Countertop
Floor
Chair
Ceiling
File cabinet
Wall
Chair
Floor
Desk
Floor
Wall
Floor
File cabinet
Supply Vent
Monitor
Floor
Ceiling
Wall
Floor
Texture
Smooth
Smooth
Smooth
Smooth
Smooth
Porous
Smooth
Textured
Porous
Porous
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Porous
Smooth
Smooth
Orientation
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Inclined
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Blank
TRUE
TRUE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
BROOM Notes
night stand
between bed and
window number 2
blank sponge stick
r side of sink on
counter surface
1 side of sink on
coounter surface
chair under
window at far
wall opposite of
entry
ceiling tile above
door
top and around
books on file
cabinet [B. Melton
changed to vac]
far side of partitior
facing window on
wall opposite of
entry
chair in front of
desk [B.Melton
changed barcode
from 3$87 to
3577] [B.Melton
changed again to
3987]
inside front of
room entry on
floor
1 side of desk in
front of printer
floor center of
room
wall under first
window on left
top r of file cabine
vent in ceiling
above partition
bottom right of
computer monitor
vacuum sample of
ceiling tile.
stick sample on
wall adjacent to
pre sample.
sponge stick
sample on floor.
could not locate
pre sample.
Lab ID
4
4
4
4
1
1
1
2
2
2
4
4
4
4
4
4
7
7
7
4
2
2
Detected
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
Yes
No
No
No
No
No
No
Spread Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Filter Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
3.4
ND
ND
ND
ND
ND
ND
G-103
-------
Barcode
3362
3743
3749
3392
3358
3347
2884
2703
3453
4069
3536
4055
3748
3328
2754
2757
3346
3340
3341
Round
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
Floor
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Room
Bathroom M
Bathroom W
Bathroom W
Bathroom W
Bathroom W
Bathroom W
Copier Room
Copier Room
Copier Room
Hallway
Hallway
Hallway
Hallway
Hallway
Hallway
Hallway
Hallway
Hallway
Hallway
X
26.534
28.600
28.339
29.006
28.890
26.309
28.736
30.005
27.699
25.379
21.407
10.793
19.337
20.748
26.040
25.397
25.422
22.623
21.764
y
19.583
16.490
17.346
17.433
17.810
16.331
14.205
13.459
13.993
16.089
13.467
13.158
12.958
13.310
17.860
18.267
14.643
13.543
13.627
z
3.000
3.000
5.800
3.000
3.000
3.000
3.250
3.000
3.000
5.800
4.000
5.800
5.800
4.000
3.200
3.000
3.000
5.800
3.000
Method
Sponge Wipe
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqft)
0.694
4.000
4.000
0.694
0.694
0.694
0.694
0.694
0.694
4.000
4.000
4.000
4.000
0.694
0.694
0.694
0.694
0.694
0.694
Operator
Schaedemann
Schaedemann
Schaedemann
Schaedemann
Schaedemann
Schaedemann
Schaedemann
Schaedemann
Schaedemann
Schaedemann
Schaedemann
Schaedemann
Schaedemann
Schaedemann
Schaedemann
Schaedemann
Schaedemann
Schaedemann
Schaedemann
Acquisition
Date
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
Acquisition
Time
2:00:00 PM
11:43:00 AM
11:55:00 AM
11:45:00 AM
11:49:00 AM
11:39:00 AM
11:18:00 AM
11:14:00 AM
11:11:00 AM
9:55:00 AM
9:28:00 AM
8:58:00 AM
9:21:00 AM
9:26:00 AM
9:50:00 AM
9:46:00 AM
9:42:00 AM
9:38:00 AM
9:33:00 AM
Object
Floor
Floor
Ceiling
Floor
Floor
Floor
Wall
Floor
Floor
Ceiling
Ceiling
Ceiling
Ceiling
Ceiling
Wall
Floor
Floor
Return Vent
Floor
Texture
Smooth
Smooth
Porous
Smooth
Smooth
Smooth
Textured
Textured
Textured
Porous
Porous
Porous
Porous
Porous
Smooth
Smooth
Smooth
Metal
Smooth
Orientation
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Blank
FALSE
TRUE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
sponge stick
sample on floor.
could not locate
pre sample.
vacume blank
taken by randy
randy collected
near center of
room
randy collected
blank sponge stick
randy collected
near stall wall
randy collected in
doorway on left
randy collected in
between sockets
randy collected
near back wall
center
randy collected
under door stop
vacuum sample on
ceiling tile on
opposite half of pn
sample.
vacuum sample
blank.
vacuum sample of
top side of ceiling
tile.
vacuum sample
from ceiling
adjacent to pre
sample.
sponge stick
sample, blank.
sponge stick
sample on wall
adjacent to pre
sample.
sponge stick
sample on floor
adjacent to pre
sample
immediately
adjacent to airlock
sponge stick
sample on floor
adjacent to pre
sample.
sponge stick
sample on return
vent on hallway
side.
sponge stick
sample on floor
adjacent to pre
sample.
Lab ID
2
4
4
2
2
1
2
2
2
2
2
2
2
2
1
1
1
1
1
Detected
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
Spread Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Filter Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
G-104
-------
Barcode
3329
3326
3330
3327
3335
4074
3383
3344
4135
2879
3334
3661
3459
3355
4072
3376
3374
3462
2821
Round
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
Floor
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Room
Hallway
Hallway
Hallway
Hallway
Hallway
Janitor Closet
Janitor Closet
Janitor Closet
Mechanical Room
Mechanical Room
Mechanical Room
Room 201
Room 201
Room 201
Room 201 A
Room 201 A
Room 201 A
Room 201 A
Room 201 A
X
6.993
10.993
7.593
17.893
13.565
29.155
28.633
26.965
19.074
19.090
22.521
26.931
29.364
26.329
28.658
26.336
26.540
29.133
29.787
y
13.376
13.558
13.158
13.768
12.474
15.076
14.974
14.829
19.178
20.157
19.002
11.249
10.720
12.008
7.476
6.626
9.341
6.053
7.346
z
3.000
3.000
5.800
3.000
3.200
5.800
3.000
3.000
3.796
3.000
3.000
5.800
3.000
3.000
5.800
3.000
3.000
3.250
3.000
Method
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Sponge Wipe
Sponge Wipe
Vacuum Sock
Sponge Wipe
Sponge Wipe
Vacuum Sock
Sponge Wipe
Sponge Wipe
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqrt)
0.694
0.694
0.694
0.694
0.694
4.000
0.694
0.694
4.000
0.694
0.694
4.000
0.694
0.694
4.000
0.694
0.694
0.694
0.694
Operator
Schaedemann
Schaedemann
Schaedemann
Schaedemann
Schaedemann
Schaedemann
Schaedemann
Schaedemann
Schaedemann
Schaedemann
Schaedemann
Schaedemann
Schaedemann
Schaedemann
Schaedemann
Schaedemann
Schaedemann
Schaedemann
Schaedemann
Acquisition
Date
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
Acquisition
Time
8:43:00 AM
8:49:00 AM
9:06:00 AM
9:15:00 AM
9:10:00 AM
11:36:00 AM
11:27:00 AM
11:22:00 AM
2:34:00 PM
2:28:00 PM
2:24:00 PM
10:41:00 AM
10:33:00 AM
10:28:00 AM
11:06:00 AM
10:50:00 AM
10:46:00 AM
10:58:00 AM
10:54:00 AM
Object
Floor
Floor
Return Vent
Floor
Wall
Ceiling
Floor
Floor
Return Vent
Floor
Floor
Ceiling
Floor
Floor
Ceiling
Floor
Floor
Wall
Floor
Texture
Smooth
Smooth
Metal
Smooth
Smooth
Porous
Smooth
Smooth
Porous
Smooth
Smooth
Porous
Smooth
Smooth
Porous
Smooth
Smooth
Textured
Smooth
Orientation
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Downward
Vertical
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Horizontal
Downward
Horizontal
Downward
Horizontal
Upward
Horizontal
Downward
Horizontal
Downward
Horizontal
Upward
Horizontal
Downward
Horizontal
Downward
Vertical
Horizontal
Downward
Blank
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
sponge stick
sample on floor.
sponge stick
sample on floor.
sponge stick
sample on return
vent on hallway
side.
sponge stick
sample on floor
adjacent to pre
sample.
sponge stick
sample on wall
adjacent to pre
sample.
randy collected
near center of
room
randy collected in
center of room .
trouble finding
sample location .
randy collected in
doorway rm 24
vacuum sample of
return furnace
filter, opposite hali
from pre sample.
sponge stick
sample on floor
adjacent to pre
sample.
sponge stick
sample on floor
adjacent to pre
sample.
randy collected
ceiling tile vacuun
sample
randy collected
near window
randy collected in
doorway
randy collected
from ceiling tile
near center of
room
randy collected in
far right corner
randy collected in
doorway 201a
randy collected
near window on
far side under
window
randy collected
near window on
left of room
Lab ID
1
1
1
1
1
2
1
2
2
1
1
2
1
1
4
2
2
2
2
Detected
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
Spread Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Filter Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
G-105
-------
Barcode
3342
3339
3890
3436
2813
3620
2753
2697
3762
3356
Round
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
°
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
Floor
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Room
Room 202
Room 202
Room 203
Room 203
Room 203
Room 203A
Room 203A
Room 203A
Room 204
Room 204
X
22.278
21.801
24.449
23.736
23.736
22.743
24.641
24.727
19.696
18.624
y
17.445
15.061
10.149
9.925
10.449
6.565
8.986
6.740
16.274
17.275
z
3.000
3.000
5.800
3.600
3.000
5.800
3.000
3.000
5.800
3.500
Method
Sponge Wipe
Sponge Wipe
Vacuum Sock
Sponge Wipe
Sponge Wipe
Vacuum Sock
Sponge Wipe
Sponge Wipe
Vacuum Sock
Sponge Wipe
Area
(sqft)
0.694
0.694
4.000
0.694
0.694
4.000
0.694
0.694
4.000
0.694
Operator
Schaedemann
Schaedemann
Harvey
Harvey
Harvey
Harvey
Harvey
Harvey
Schaedemann
Schaedemann
Acquisition
Date
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
Acquisition
Time
10:03:00 AM
10:00:00 AM
12:23:00 PM
12:12:00 PM
12:16:00 PM
12:41:00 PM
12:31:00 PM
12:34:00 PM
10:20:00 AM
10:13:00 AM
Object
Floor
Floor
Ceiling
Wall
Floor
Ceiling
Floor
Floor
Ceiling
Wall
Texture
Smooth
Smooth
Textured
Textured
Smooth
Textured
Smooth
Smooth
Porous
Smooth
Orientation
Horizontal
Downward
Horizontal
Downward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Blank
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
sponge stick
sample on floor
adjacent to pre
sample.
sponge stick
sample on floor
adjacent to pre
sample
vacuum sample on
top of tile, tile is to
right of entrance tt
room 203a. 1 tile
to left of door way
to 203 a. 3
sponge taken on
wall Sinches to
right of door to
203a. 1 foot off of
floor 1
sponge sample
taken on floor. 10
feet to right of
door. 5feet from
door wall and 2
feetfrom left wall.
2
vacuum sample on
top of tile . tile to
right of window. 8
inches from back
wall. 1 tile length
from right wall. 3.
sponge stick taken
inside door 18
inches in. 1 an
extra sponge stick
in bag. 2ndary
containment has a
whole in it,put in
glove bag with
large 1.
sponge sample
taken on floor to
left of window. 36
inches from back
wall with window
24 inches from left
wall. 2
vacuum sample of
ceiling tile
opposite of pre
sample.
sponge stick
sample on wall
adjacent to pre
sample.
Lab ID
1
1
7
4
1
5
1
1
2
1
Detected
No
No
No
No
No
No
No
No
No
No
Spread Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Filter Plate Results
(CFU/sq ft)
ND
ND
ND
ND
NA
ND
NA
NA
ND
ND
G-106
-------
Barcode
3353
3352
3763
2817
2469
2740
3983
3790
3107
3103
3106
2802
2449
3165
2801
1565
1539
3728
Round
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
Floor
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Room
Room 204
Room 204
Room 205
Room 205
Room 205
Room 205
Room 206
Room 206
Room 206
Room 206
Room 206
Room 206
Room 206
Room 206
Room 206
Room 206
Room 206
Room 207
X
19.993
19.704
20.034
20.054
20.525
18.725
17.442
15.921
16.944
17.742
17.821
16.121
15.921
15.921
17.842
17.742
15.921
16.948
y
14.790
17.218
9.450
6.064
11.450
9.669
18.254
17.554
20.298
20.154
18.845
15.454
17.554
17.554
19.654
20.154
17.554
9.341
z
3.000
3.000
5.800
3.600
3.000
3.000
3.000
3.000
3.900
3.897
3.696
3.000
3.000
3.000
3.000
3.000
3.000
4.000
Method
Sponge Wipe
Sponge Wipe
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Swab
Vacuum Sock
Area
(sqft)
0.694
0.694
4.000
0.694
0.694
0.694
4.000
4.000
0.694
0.694
0.694
0.694
0.694
0.694
0.694
0.028
0.028
4.000
Operator
Schaedemann
Schaedemann
Harvey
Harvey
Harvey
Harvey
Harvey
Harvey
Harvey
Harvey
Harvey
Harvey
Harvey
Harvey
Harvey
Harvey
Harvey
Harvey
Acquisition
Date
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
Acquisition
Time
10:07:00 AM
10:10:00 AM
12:01:00 PM
11:55:00 AM
11:46:00 AM
11:50:00 AM
10:38:00 AM
10:02:00 AM
10:17:00 AM
10:22:00 AM
10:13:00 AM
9:50:00 AM
9:55:00 AM
10:04:00 AM
10:08:00 AM
10:26:00 AM
9:59:00 AM
10:53:00 AM
Object
Floor
Floor
Ceiling
Wall
Floor
Floor
Ceiling
Ceiling
Workbench
Workbench
Table
Floor
Floor
Floor
Floor
Ceiling
Floor
Floor
Texture
Smooth
Smooth
Textured
Textured
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Orientation
Horizontal
Downward
Horizontal
Downward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Blank
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
TRUE
TRUE
BROOM Notes
sponge stick
sample on floor
adjacent to pre
sample.
sponge stick
sample on floor
adjacent to pre
sample.
vacuum sample on
top of ceiling tile.
3rdd ceiling tile
inside room from
door. Itile width
plus 8 inches from
left wall. 4
on wall opposite
door to left of
windw 3feet to left
of window. 3
inside door at
entrance. 3 feet in
from door. 24
inches from wall
behind door.
sponge sample. 1
sample taken on
floor 18 inches
wall. 11 feet on
right side of room.
11
4
8 (moved a tool to
take sample)
9 (moved tool to
take sample)
7
1
2
5
6
10
3
vacuum blank 3
Lab ID
1
1
7
1
4
1
7
7
2
2
2
2
2
2
2
7
7
7
Detected
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
Spread Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Filter Plate Results
(CFU/sq ft)
ND
ND
ND
NA
ND
NA
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
G-107
-------
Barcode
3753
3321
2372
3324
3276
3348
3325
2758
Round
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
Floor
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Room
Room 207
Room 207
Room 207
Room 207
Room 207
Room 207
Room 207
Room 207
X
17.453
16.157
16.053
16.825
16.124
15.519
15.551
17.956
y
11.059
6.637
11.659
9.267
8.273
6.751
8.060
7.733
z
5.800
3.080
3.000
4.000
3.080
4.900
4.800
3.500
Method
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqft)
4.000
0.694
0.694
0.694
0.694
0.694
0.694
0.694
Operator
Harvey
Harvey
Harvey
Harvey
Harvey
Harvey
Harvey
Harvey
Acquisition
Date
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
Acquisition
Time
11:38:00 AM
11:11:00 AM
10:47:00 AM
10:56:00 AM
11:05:00 AM
11:14:00 AM
11:18:00 AM
11:25:00 AM
Object
Ceiling
Desk
Floor
Floor
Desk
Cabinet
Cabinet
Wall
Texture
Textured
Smooth
Smooth
Smooth
Smooth
Smooth
Smooth
Textured
Orientation
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Blank
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
note forr previous
sample 1 1 . cap of
swab dropped on
floor, cap wiped
out with alcohol
wipe, this sampi
is vacuum sample
taken on top of
ceilingtile. tile on
left wall when you
enter room 6inches
from left wall and
1 ceiling tile lengtl
from wall that
sponge sample
taken on desk.2.5
feet from edge
facing door 4
inches in from
longest edge. 7
sponge stick by
doorl
sponge blank
sponge 4
[B .Melton: there
are two 3324. One
needs to be 3320.
Guessed that the
other one was
3320.]
sponge sample
taken on desk
corner near door.
moved little box 01
desk. 6
sponge on top of
mailbox 3 feet
from ceiling. 18
inches from door a
leading long edge
of mail box. 8
sponge on top of
mail cabinet at
corner from door.
this area partially
covers previos
sampled area. 9
on wall to right of
desk that is
opposite from
door . 6 inches
from desk 1 foot
up. 10
Lab ID
7
2
4
2
4
4
4
1
Detected
No
No
No
No
No
No
No
Yes
Spread Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
ND
Filter Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
4.7
G-108
-------
Barcode
3345
1516
1736
3982
3799
3738
3994
3996
3102
3099
3461
3105
1504
1669
3764
4011
3801
3094
3100
3104
3091
3097
1722
3787
3765
Round
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
Floor
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Room
Room 207
Room 207
Room 207
Room 208
Room 208
Room 208
Room 208
Room 208
Room 208
Room 208
Room 208
Room 208
Room 208
Room 208
Room 209
Room 209
Room 209
Room 209
Room 209
Room 209
Room 209
Room 209
Room 209
Room 210
Room 210
X
17.434
16.953
16.418
14.359
15.110
13.557
13.216
13.786
14.780
15.326
13.367
12.694
13.845
12.778
14.769
13.475
14.669
13.008
12.834
14.971
12.418
13.459
14.604
10.406
10.675
y
10.194
9.150
8.845
15.576
20.164
18.433
19.544
20.370
16.905
18.593
15.450
19.872
19.977
18.376
7.934
10.916
11.022
11.955
10.897
9.624
7.468
6.714
7.730
19.588
20.373
z
3.080
3.000
5.800
5.500
4.300
3.500
4.394
3.300
3.000
4.179
3.700
3.600
5.500
4.200
3.000
3.000
5.500
3.800
3.600
3.700
4.065
3.600
5.500
4.300
3.400
Method
Sponge Wipe
Swab
Swab
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Swab
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Vacuum Sock
Vacuum Sock
Area
(sqft)
0.694
0.028
0.028
4.000
4.000
4.000
4.000
4.000
0.694
0.694
0.694
0.694
0.028
0.028
4.000
4.000
4.000
0.694
0.694
0.694
0.694
0.694
0.028
4.000
4.000
Operator
Harvey
Harvey
Harvey
Chong
Chong
Chong
Chong
Chong
Chong
Chong
Chong
Chong
Chong
Chong
Chong
Chong
Chong
Chong
Chong
Chong
Chong
Chong
Chong
Chong
Chong
Acquisition
Date
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
Acquisition
Time
11:01:00 AM
10:51:00 AM
11:28:00 AM
11:47:00 AM
11:28:00 AM
11:05:00 AM
11:20:00 AM
11:24:00 AM
11:38:00 AM
11:35:00 AM
10:57:00 AM
11:09:00 AM
11:42:00 AM
11:01:00 AM
12:22:00 PM
12:03:00 PM
12:32:00 PM
11:57:00 AM
12:08:00 PM
12:11:00 PM
12:18:00 PM
12:15:00 PM
12:28:00 PM
9:57:00 AM
10:03:00 AM
Object
Desk
Floor
Supply Vent
Ceiling
File cabinet
Chair
Wall
Chair
Floor
Wall
Table
File cabinet
Return Vent
Monitor
Floor
Floor
Ceiling
Countertop
Sink
Stove
Wall
Table
Return Vent
Wall
Chair
Texture
Smooth
Smooth
Textured
Porous
Metal
Cloth
Porous
Cloth
Smooth
Smooth
Smooth
Metal
Smooth
Smooth
Carpet
Carpet
Porous
Smooth
Metal
Metal
Smooth
Smooth
Metal
Porous
Porous
Orientation
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Inclined
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Blank
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
sponge sample
taken on table
surface Ifoot in
from door entrana
at tablee edge 5
blank sponge 2
supply vent. swab.
11
2nd tile in from
door closer to right
wall
top of cabinet and
books consistent
w/last sample
event
partition next to
cabinet sample
side facing
window
ceiling vent
upper left on
screen
2nd tile in from
door
[B. Melton moved
to other side of
table]
partition next to
desk sample faces
window
Lab ID
4
7
7
7
1
7
1
7
1
1
1
1
1
1
7
7
1
1
1
1
1
1
1
1
7
Detected
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
Spread Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Filter Plate Results
(CFU/sq ft)
ND
ND
ND
ND
NA
ND
NA
ND
NA
NA
NA
NA
NA
NA
ND
ND
NA
NA
NA
NA
NA
NA
NA
NA
ND
G-109
-------
Barcode
3788
4013
3773
3088
3098
3090
3092
3083
3315
1735
1559
1604
4216
3734
4096
4282
3122
3121
3111
1508
1554
3992
4000
3737
3771
4018
Round
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
Floor
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Room
Room 210
Room 210
Room 210
Room 210
Room 210
Room 210
Room 210
Room 210
Room 210
Room 210
Room 210
Room 210
Room 211
Room 211
Room 211
Room 211
Room 211
Room 211
Room 211
Room 211
Room 211
Room 212
Room 212
Room 212
Room 212
Room 212
X
10.444
12.141
11.931
9.890
9.993
9.601
12.037
11.785
11.793
11.666
10.692
9.704
10.483
11.786
11.492
10.299
9.492
9.583
11.951
9.528
11.749
7.826
7.502
7.177
7.696
9.060
y
15.954
20.373
18.190
16.883
14.942
19.877
19.774
17.952
14.358
18.282
19.909
18.267
11.697
11.954
7.420
6.704
11.257
6.943
7.420
9.348
7.071
15.482
18.426
19.552
20.332
20.115
z
5.500
4.300
3.000
3.000
3.000
3.590
4.400
3.000
4.196
3.000
5.500
4.200
3.000
3.000
4.000
3.000
3.600
4.200
4.000
4.000
4.000
4.298
3.597
4.298
3.500
4.582
Method
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Swab
Swab
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Swab
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Area
(sqrt)
4.000
4.000
4.000
0.694
0.694
0.694
0.694
0.694
0.694
0.028
0.028
0.028
4.000
4.000
4.000
4.000
0.694
0.694
0.694
0.028
0.028
4.000
4.000
4.000
4.000
4.000
Operator
Chong
Chong
Chong
Chong
Chong
Chong
Chong
Chong
Chong
Chong
Chong
Chong
Jordan
Jordan
Jordan
Jordan
Jordan
Jordan
Jordan
Jordan
Jordan
Jordan
Jordan
Jordan
Jordan
Jordan
Acquisition
Date
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
Acquisition
Time
10:50:00 AM
10:20:00 AM
10:30:00 AM
9:41:00 AM
9:37:00 AM
10:10:00 AM
10:15:00 AM
10:29:00 AM
10:38:00 AM
10:25:00 AM
10:42:00 AM
9:45:00 AM
11:52:00 AM
11:50:00 AM
12:13:00 PM
12:24:00 PM
12:01:00 PM
12:11:00 PM
12:14:00 PM
12:07:00 PM
12:17:00 PM
9:42:00 AM
9:50:00 AM
10:14:00 AM
10:22:00 AM
10:24:00 AM
Object
Ceiling
File cabinet
Floor
Floor
Floor
File cabinet
File cabinet
Floor
Wall
Floor
Return Vent
Monitor
Ceiling
Floor
Countertop
Floor
Wall
Countertop
Countertop
Monitor
Countertop
Floor
Chair
Wall
Chair
File cabinet
Texture
Porous
Metal
Smooth
Smooth
Smooth
Metal
Metal
Smooth
Smooth
Smooth
Smooth
Smooth
Porous
Porous
Smooth
Porous
Textured
Smooth
Smooth
Smooth
Smooth
Textured
Porous
Smooth
Porous
Textured
Orientation
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Downward
Inclined
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Blank
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
TRUE
FALSE
TRUE
TRUE
FALSE
FALSE
FALSE
FALSE
BROOM Notes
2nd tile in from
door 2nd tile in
from left wall
vacuum only top o
cabinet and books
consistent w/last
sample event -did
not vacuum arounc
bottom of cabinet
top left
ceiling tile vacuun
sample
floor right inside o
door
blank taken
left of previous
sample taken
right side of
previous sample
taken
blank taken
upper left of screer
blank taken
blank
same spot as
previous sample
sample ofnon
porous surface to
repeat last
sampling point
aooks on top of fill
cabinet
Lab ID
7
7
1
1
1
1
1
1
1
1
1
1
8
8
7
7
7
7
7
7
1
8
8
8
7
7
Detected
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
Spread Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Filter Plate Results
(CFU/sq ft)
ND
ND
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
ND
ND
ND
ND
ND
ND
ND
ND
NA
ND
ND
ND
ND
ND
G-110
-------
Barcode
3741
3504
3320
3101
3386
3133
1498
1499
1664
3769
4022
4285
3984
3786
4012
3523
3152
3204
3309
3357
Round
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
C1O2 Post-
Decon
Floor
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Floor 2
Room
Room 212
Room 212
Room 212
Room 212
Room 212
Room 212
Room 212
Room 212
Room 212
Room 213
Room 213
Room 213
Room 213
Room 213
Room 213
Room 213
Room 213
Room 213
Room 213
Stairwell
X
8.541
8.541
6.484
8.064
6.722
8.476
6.614
7.610
7.826
8.896
7.743
6.741
7.166
7.954
7.620
7.499
6.377
8.106
8.925
23.868
y
18.535
14.919
16.023
15.937
19.855
17.149
18.275
15.937
19.725
7.832
7.863
8.197
10.868
10.291
12.113
6.102
6.679
10.656
11.961
19.419
z
3.000
3.000
3.500
4.198
3.893
3.000
4.193
4.500
3.000
3.000
3.300
3.300
3.000
4.096
3.000
3.596
3.694
4.184
3.793
3.000
Method
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Swab
Swab
Swab
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Vacuum Sock
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Sponge Wipe
Area
(sqft)
4.000
0.694
0.694
0.694
0.694
0.694
0.028
0.028
0.028
4.000
4.000
4.000
4.000
4.000
4.000
0.694
0.694
0.694
0.694
0.694
Operator
Jordan
Jordan
Jordan
Jordan
Jordan
Jordan
Jordan
Jordan
Jordan
Jordan
Jordan
Jordan
Jordan
Jordan
Jordan
Jordan
Jordan
Jordan
Jordan
Schaedemann
Acquisition
Date
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
5/16/2011
Acquisition
Time
10:32:00 AM
9:30:00 AM
9:35:00 AM
9:39:00 AM
10:09:00 AM
9:47:00 AM
10:01:00 AM
9:38:00 AM
10:04:00 AM
11:16:00 AM
11:24:00 AM
11:32:00 AM
11:41:00 AM
10:59:00 AM
10:51:00 AM
11:02:00 AM
11:08:00 AM
10:57:00 AM
10:55:00 AM
12:03:00 PM
Object
Ceiling
Floor
Floor
Floor
File cabinet
Floor
Monitor
Floor
Ceiling
Floor
Bed
Bed
Ceiling
Countertop
Floor
Wall
Nightstand
Countertop
Countertop
Floor
Texture
Porous
Smooth
Textured
Textured
Smooth
Smooth
Smooth
Textured
Smooth
Porous
Porous
Porous
Porous
Smooth
Porous
Textured
Smooth
Smooth
Smooth
Smooth
Orientation
Horizontal
Downward
Horizontal
Upward
Vertical
Vertical
Horizontal
Upward
Horizontal
Upward
Inclined
Vertical
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Downward
Horizontal
Upward
Horizontal
Upward
Vertical
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Horizontal
Upward
Blank
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
BROOM Notes
left of previous
sample [B.Melton:
there were two
3324, one needs to
be 3320. Guessing
this is the one.]
blank
file cabinet
under previous
sample
upper left of
monitor
ceiling diffuser
swab
actually on the
floor bed is on the
opposite wall
on bed
on bed
ceiling tile top
sidey
blank
left of the doorwaj
up an left of
previous sample
taken
blank
otherside of
Countertop
randy collected in
doorway of stair
well
Lab ID
8
7
7
7
7
7
1
1
1
8
7
7
8
8
8
7
7
7
7
5
Detected
No
No
No
No
No
No
No
No
No
No
No
Yes
No
No
No
No
No
No
No
Yes
Spread Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
4.2
ND
ND
ND
ND
ND
ND
ND
ND
Filter Plate Results
(CFU/sq ft)
ND
ND
ND
ND
ND
ND
NA
NA
NA
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
9.3
G-lll
-------
Appendix H
Waste Management and Cost Analysis Spreadsheet
-------
Start here
K-2
-------
MASTER BOTE Cost Analysis O61313.xlsx
Issue
Graph of Waste Activities
Graph of time by labor type for each decon technology
Graph of cost by labor type for each decon technology
Worksheet to Resolve
TBD
TBD
TBD
Status
not started
not started
not started
Outstanding Issues
6/13/2013
-------
MASTER BOTE Cost Analysis O61313.xlsx
I
Item Cost
per Unit
Unit
110
109
108
107
106
105
104
103
213
212
211
210
209
208
207
206
Floor 1
Floor 2
c
.2
1
_op
|
Office
Residential
Office
Residential
Office
Residential
Shop
Mail room
Residential
Office
Residential
Office
Residential
Office
Mail room
Shop
HVAC Duct
HVAC Duct
1
o
21
21
21
21
21
21
10
21
21
21
21
21
21
21
21
21
200
200
1
10
10
10
10
10
10
12
10
10
10
10
10
10
10
10
10
NA
NA
C
S
210
210
210
210
210
210
120
210
210
210
210
210
210
210
210
210
NA
NA
Laminate Floor
1
1
1
1
1
1
3270
S
e-
3
i
i
i
i
i
i
Cost
Ceiling Tiles
S 5
ea 2x4
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
S
S 25
ea
20
20
20
20
20
20
20
20
20
$4,500
Binders
S 25
ea
10
10
10
10
10
10
$1,500
$ -
ea
20
20
$ -
I
$ 500
ea
1
1
$1,000
$ 750
ea
1
1
$1,500
'ra
6
$ 100
ea
2
2
1
4
4
2
1
2
4
4
$2,600
Monitor
$ 200
ea
1
1
1
1
1
1
$1,200
?
$ 250
ea
1
1
$ 500
o
1
$ 250
ea
1
1
1
1
1
1
$1,500
Wall Divider
$ 800
ea
1
1
1
1
1
1
$4,800
Laminate Floor Sq. R
$ 3.70
Sqft
210
0
210
0
210
0
0
0
0
210
0
210
0
210
0
0
$4,662
Carpet Sq. R
$ 2.78
Sqft
0
210
0
210
0
210
0
0
210
0
210
0
210
0
0
0
$3,500
Ceiling Tile SqR
$ 0.63
Sqft
210
210
210
210
210
210
120
210
210
210
210
210
210
210
210
210
$2,044
HVAC Duct Refit Cost
(total for all linear
feet installed)
$ 44.07
ft
200
200
$ 17,628
Refit Cost (Fixed)
$ 2,200
$ 500
$ 2,200
$ 1,100
$ 2,400
$ 400
$ 500
$ 500
$ 500
$ 2,200
$ 1,100
$ 2,200
$ 400
$ 2,400
$ -
$ 500
$19,100
Refit Cost (per unit
area or linearfoot)
$ 4.33
$ 3.40
$ 4.33
$ 3.40
$ 4.33
$ 3.40
$ 0.63
$ 0.63
$ 3.40
$ 4.33
$ 3.40
$ 4.33
$ 3.40
$ 4.33
$ 0.63
$ 0.63
$ -
Time to Replace (hrs):
Labor Cost to replace:
10
$
7,854
Average Refit Cost
Office
Residential
Mail Room
Shop
Labor
Fixed
$2,267
$ 667
$ 250
$ 500
Cost/ft2
$ 4.33
$ 3.40
$ 0.63
$ 0.63
$ 2.40
Round 2 Replacement Costs:) $ 46,934 |
AB Building Refit
Page H-3
6/13/2013
-------
MASTER BOTE Cost Analysis O61313.xlsx
c
0
c
LO
LO
Q
VHP® - Characterization Samplingl
VHP®- Decon
VHP® - Clearance Sampling
AB- Characterization Sampling2
AB Decon - Removal
AB Decon - Spray
AB Decon - Dry
AB - Clearance Sampling
CIO2 - Characterization Samplings
CLO2- Decon
CIO2 - Clearance Sampling
AB- Building Reset
c
o
4-*
(D
M
t5
ro
o |2p
i Ł
> 00
c
0
u
QJ
0
Q_
I
;>
u
c
(D
(D
U |2P
I Ł
> 00
c
0
'4-*
(D
M
U
O
-------
MASTER BOTE Cost Analysis O61313.xlsx
Lab
UT
Mean
Std Dev
HEPA
Labor
Hours Per
Sample
1.00
S i.oo
Multiplier
for BSL-3
vs BSL-2
Analysis
1.5
Sponge
Stick
Analysis
Labor
Hours per
Sample
0.79
S 0.79
Swab
Analysis
Labor
Hours per
Sample
0.70
S 0.70
HEPA
Analysis
Labor
Cost Per
Sample
S 151
S 150.74
Sponge
Stick
Analysis
Labor
Cost per
Sample
S 118
S 118.27
Swabs
Labor
Cost per
Sample
S 105
S 105
LRN
Expendab
les Cost
Per
Sample
S 41.37
S 41.37
S -
LRN
Equipmen
t Cost Per
Sample
S 1.87
S 1.87
S -
LRN HEPA
Analysis
Cost Per
Sample
S 192.10
$288.16
S -
LRN
Sponge
Stick
Analysis
Cost Per
Sample
S 159.64
S 239.46
S -
LRN Swab
Analysis
Cost Per
Sample
S 146.18
S 219.27
S -
Waste
Sample
Analytical
Cost
Water
Sample
Analytical
Cost
Hggressro
eAir
Sample
Analysis
Labor +
Material
Hours per
163.05
S 245
S -
244.58
175.08
Wipes
(EPA)
Labor +
Material
Cost per
Sample
S 154
S 231
S -
average of
other
analysis
types
average of
other
analysis
types
Wipes
(LLNL)
Labor +
Material
Cost per
Sample
S 426
$ 640
S -
Note — Aggressive Air Samples, and Wipe
Samples are not calculated from LRN
Worksheets
LLNL Cost for LLNL Wipe Analysis:
Number of RV PCR Analyses:
Cost Per RV PCR Analysis:
INL Cost for EPA Wipe Analysis:
Number of RV PCR Analyses:
Cost Per RV PCR Analysis:
Lumped Analytical Costs:
Purchase Order LRN Expendables Costs:
Purchase Order LRN Equipment Costs:
S 145,000
340
S 426
S 27,000
175
S 154
S 31,104
S 77,897
S 49,283
Temperature 5 min; Free Cl 20 min; pH 5
min; Turbidity 10 min; TSS 10 min; COD 20
min
Analytical Costs
Page H-5
6/13/2013
-------
MASTER BOTE Cost Analysis 061313.x
€
3
•5
&
<
2
1
Mean
Std Deviation
•5
Ł•
<
2
1
VHP®
pH-Adjusted Bleach Pr
CIO2
Average
1
8
5
§
VHP® Mean
VHP® SD
AB Mean
ABSD
CLO2 Mean
CLO2 SD
t
Q.
€
3
%
o
VHP®
pH-Adjusted Bleach Pr
CIO2
Restoration Costs
VHP®
pH-Adjusted Bleach Pr
CIO2
3
5
(2
VHP®
pH-Adjusted Bleach Pr
CI02
1
|
E
1
"a.
E
I Ł
S Ł
S 252
S 121
Attributable Other
Sampling/Analytical
Costs
S 148,513
S 148,513
S 148,513
S 148,513
E
S
11
s 8-
3 i
CP
$ 271
S 129
S 345
S 165
$ 271
S 129
1
1 i
H. "Ł
1 5
S &
S 2,863
S 10,378
S 1,431
Ł
Ł 5
« Ł
*i«
III
Crla
S
S 7,854
S
~i 1C Cost
S 46,737
S 54,907
S 46,737
1
§
O
E
1
•a.
E
I Ł
S Ł
S 697
S 78
N on -Attributable
Other
Sampling/Analytical
Related Costs
S 95,138
S 95,138
S 95,138
S 95,138
Ł
i!
u
Cp
S 271
S 129
S 345
S 165
S 271
S 129
If
s i
3 S
$
S 17,939
$
Cost of Replacing
Removed Items
Cu
$
S 46,934
$
IS
m
S 665,188
S 665,188
S 665,188
Ł
S
a.
E
1
•ft.
E
|
S 720
S 328
|
3
§
O
S 10,020
S 10,736
S 10,736
S 10,497
If
I;
« S
3 Q
Cpd
S 722
S 81
S 822
S 92
S 722
S 81
Cost of Removal Teams
Entering
S
S 30,497
S
Restoration Cost
Cr
S
S 54,788
S
L Decon Cost
S 147,627
S 388,142
S 176,590
Cost of Aggressive Air Sampling
Team Prep per Entry
S 273
S 130
Sampling Labor
S 37,049
S 39,564
S 39,673
S 38,762
Ł
it
I!
II
Crw
S 722
S 81
S 822
S 92
S 722
S 81
Decon Contractor Fixed
Costs
S 105,493
S 66,831
S 169,093
"Ł
ll
$
$ 54,788
$
Cost of Aggressive Air Sampling
- Team Decon per Entry
$ 725
$ 82
•z
Ł
i
$ 23,894
$ 23,894
$ 23,961
S 23,916
E
§
Ł Ł
Jj
Cdl
S 773
S 352
S 985
S 449
S 773
S 352
J2
•z
€
i
I
S 1,587
S 31,976
S 1,587
i Total Cost
S 859,551
S 1,163,024
S 888,514
Cost of Aggressive Air Sampling
• Team per Entry
S 779
S 355
1
S 313,490
S 313,490
S 314,649
S 313,877
l!
i »
II
Crw
S 773
S 352
S 985
S 449
S 773
S 352
Waste Management Costs
S 37,684
S 230,521
S 4,478
. Labor Cost Per HEPA Sample
S 53
S 18
s
i
S 42,166
S 25,725
S 67,053
S 44,981
!
ii
§1
Cdc
S 105,493
S
S 66,831
S
S 169,093
S
Mean Decon Cost
Cd
S 147,627
S 388,142
S 176,590
Material Cost Per HEPA Sample
S 29
S
Total Sampling Cost
S 660,251
S 646,324
S 688,987
$ 665,188
al Cost for
Team
11
Cdm
S 1,587
S
S 31,976
S
S 1,587
S
Decon Cost Std Dev
S
S 19,062
S
Analysis Cost Per HEPA Sample
S 288
S
Sampling Cost Std Dev
S 27,398
S 28,412
S 28,489
$ 28,099
I
O u
Cdw
S 37,684
S
$230,521
S
S 4,478
S
Additional
Decon line
Additional
Labor Cost Per Sponge Stick
Sample
S 34
S 10
non-attribu
non-attribu
non-attribu
Level C
Level B
Level C
Decon lineo
ops cost inc
Decon lineo
! ! i 1 i i I l i ! ! i i i
1 f 1 I I J I I 1 1 1 1 ! !
i • ? i • ? i • 1 i • ! i •
S 3 « S 3 « S 3 « S 3 « S 3
.= . .= . 5 . • . .= . 5 . • . .= . 5 • .= 5 . • . .= .
1! f!l!l!f!l!l!f! 1 1 f Illlf!
S 20 S 239 S 30 S 19 S 231 S 30 S 19 S 640 S 29 S 21 S 219 S 57 S 18 S 245
S - S- S9S- S- S9S- S- S9S- S- S- S- S-
able costs get applied to all rounds
able costs get applied to all rounds
able costs get applied to all rounds
ps costs incudedfordecon contractor and safety entres
uded for safety entries
ps costs incudedfordecon contractor and safety entres
Cost Equation
Page H-6
-------
MASTER BOTE Cost Analysis O61313.xlsx
Date
4/16/11
4/17/11
4/18/11
4/19/11
4/20/11
4/21/11
4/22/11
4/23/11
4/24/11
4/25/11
4/26/11
4/27/11
4/28/11
4/29/11
4/30/11
5/1/11
5/2/11
5/3/11
5/4/11
5/5/11
5/6/11
5/7/11
5/8/11
5/9/11
5/10/11
5/11/11
5/12/11
5/13/11
5/14/11
5/15/11
5/16/11
5/17/11
Activity
Dissemination
VHP® - Characterization Samplingl
VHP® - Characterization Samplingl
VHP®- Decon
VHP®- Decon
VHP®- Decon
VHP® - Clearance Sampling
VHP® - Clearance Sampling
DAY OFF
Dissemination
AB - Characterization Sampling2
AB - Characterization Sampling2
AB Decon - Removal
AB Decon - Removal
AB Decon - Spray
AB Decon - Dry
DAY OFF
AB Decon - Dry
AB Decon - Dry
AB - Clearance Sampling
AB - Clearance Sampling
DAY OFF
DAY OFF
AB - Building Reset
Dissemination
CIO2 - Characterization Samplings
CIO2 - Characterization Samplings
CLO2 - Decon
CLO2 - Decon
CLO2 - Decon
CIO2 - Clearance Sampling
CIO2 - Clearance Sampling
Daily Activity List
Page H-7
6/13/2013
-------
MASTER BOTE Cost Analysis O61313.xlsx
Date
16-Apr
17-Apr
18-Apr
19-Apr
20-Apr
21-Apr
22-Apr
23 -Apr
24-Apr
25 -Apr
26-Apr
27-Apr
28-Apr
29-Apr
30-Apr
1-May
2-May
3-May
4-May
5-May
6-May
7-May
8-May
9-May
10-May
11-May
12-May
13-May
14-May
15-May
16-May
17-May
Mass (Ib)
18
147.2
80.2
14.2
59.8
380.6
171.4
36.8
0
0
261.6
6.2
65
10236.62
2339.4
0
0
0
0
157
887.7
0
0
0
52
171.4
0
0
0
0
0
451.6
Volume (gal)
38
0
211
0
0
55
36
38
0
28
84
0
26
0
633
0
0
0
0
63
38
0
0
0
0
137
0
0
0
0
0
38
Activity
Dissemination
VHP® - Characterization Samplingl
VHP® - Characterization Samplingl
VHP®- Decon
VHP®- Decon
VHP®- Decon
VHP® - Clearance Sampling
VHP® - Clearance Sampling
DAY OFF
Dissemination
AB - Characterization Sampling2
AB - Characterization Sampling2
AB Decon - Removal
AB Decon - Removal
AB Decon - Spray
AB Decon - Dry
DAY OFF
AB Decon - Dry
AB Decon - Dry
AB - Clearance Sampling
AB - Clearance Sampling
DAY OFF
DAY OFF
AB - Building Reset
Dissemination
CIO2 - Characterization Samplings
CIO2 - Characterization Samplings
CLO2 - Decon
CLO2 - Decon
CLO2 - Decon
CIO2 - Clearance Sampling
CIO2 - Clearance Sampling
Daily Waste Generation
Page H-8
6/13/2013
-------
MASTER BOTE Cost Analysis O61313.xlsx
01
4J
CD
Q
4/17/11
4/18/11
4/19/11
4/20/11
4/21/11
4/22/11
4/23/11
4/24/11
4/25/11
4/26/11
4/27/11
4/28/11
4/29/11
4/30/11
5/1/11
5/2/11
5/3/11
5/4/11
5/5/11
5/11/11
Number of Staff
6
6
4
2
4
7
6
0
0
6
0
4
4
4
4
0
0
0
6
7
E
E
.2?
(75
+j
CD
01
E
E
7:00
7:30
7:30
7:58
12:00
7:00
7:45
na
na
7:00
na
7:00
7:00
7:05
7:00
na
na
na
7:00
7:00
4-1
O
E
OD
(75
+j
CD
01
E
E
20:00
14:24
19:02
11:37
19:30
19:30
17:00
na
na
18:00
na
19:00
18:30
17:55
18:00
na
na
na
16:50
18:30
Decon Line Time (min)
780
414
692
240
450
750
555
na
na
660
na
720
690
650
660
na
na
na
590
690
01
4-1
O
z
In addition 2 Officers, 1 hr 45 min
EMT'sOnly
All day, decon of STERIS equipment
4 decon line 1 paramedic 1 captain
ditto
No Deon Line
No Deon Line
1 lead, 1EMT, 4 personnel
No Decon Line
In Addition 1 person 11:50-13:30
In Addition 1 person 15:55-17:30
1 lead, 1 emt, 2 personnel
2 Additional People 0730-1000
No Deon Line
No Deon Line
No Deon Line
1 lead, 1 EMT, 4 personnel
4 Decom, 1 EMT, 1 Captain, 1 additional
Decon Line Ops
Page H-9
6/13/2013
-------
MASTER BOTE Cost Analysis O61313.xlsx
0)
+•*
1
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/18/11
4/18/11
4/18/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/23/11
4/26/11
4/26/11
4/26/11
4/26/11
4/26/11
4/26/11
4/26/11
4/26/11
4/26/11
4/26/11
4/26/11
4/26/11
E
s
7
6
5
8
4
9
3
12
6
5
7
8
6
4
3
4
12
3
5
4
7
9
8
5
13
6
3
3
5
6
4
8
9
7
8
3
6
5
4
Ł•
4-»
Ł
LLJ
4-»
ra
Ol
P
12:27
12:43
12:51
13:35
15:16
15:32
16:47
18:28
17:02
17:51
17:35
18:35
12:04
11:57
12:41
11:08
12:42
13:28
14:37
14:56
16:10
16:29
17:09
17:20
17:38
18:30
11:32
10:45
11:10
11:30
11:40
11:25
12:05
11:52
13:40
14:10
14:28
14:40
14:53
±i
X
LLJ
4-»
ra
Ol
t
12:47
12:58
13:14
13:52
15:35
15:50
16:57
18:48
17:16
18:02
17:48
18:55
12:20
12:09
12:47
11:15
12:48
13:45
14:51
15:09
16:29
16:43
17:33
17:40
17:55
18:42
11:49
11:03
11:20
11:45
11:52
11:40
12:20
12:04
13:55
14:25
14:35
14:52
15:00
"c"
|E
Ol
E
i-
c
o
u
Ol
Q
E
8
Ol
Q.
E
as
ifi
20
15
23
17
19
18
10
20
14
11
13
20
16
12
6
7
6
17
14
13
19
14
24
20
17
12
17
18
10
15
12
15
15
12
15
15
7
12
7
Sample Team Recovery Time (min)
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
U)
01
4-»
O
3 personnel 27 samples
3 personnel no samples
4 personnel 42 samples
3 personnel 37 samples
3 personnel 43 samples
3 personnel 50 samples
3 personnel 58 samples
2 personnel 72 samples
3 personnel 36 samples
4 personnel and 41 samples
3 personnel and 24 sample
3 personnel 38 samples
4 personnel and 33 samples
3 personnel and 32 samples
2 personnel and 24 samples
3 personnel 0 samples
Equipment, personnel Honeywell
Samples, 3 personnel, equipment
Left to get more gloves
37 Samples, 3 personnel
4 personnel, 37 samples
52 samples 3 people
3 personnel 53 samples
3 people 53 samples
3 people 0 samples
3 people 65 samples
3 people, 23 samples
3 people, 43 samples
3 people, 34 samples
3 people, 42 samples
3 people, 51 samples
0 samples (one sampler feeling sick); need number of people
3 people, 32 samples
3 people, 37 samples
3 people, 58 samples
3 people, 38 samples
3 people, 31 samples
3 people, 32 samples
3 people, 37 samples
Personnel
3
3
4
3
3
3
3
2
3
4
3
3
4
3
2
3
3
3
4
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
Samples
27
0
42
37
43
50
58
72
36
41
24
38
33
32
24
0
0
37
37
52
53
53
0
65
23
43
34
42
51
0
32
37
58
38
31
32
37
Entry Team Decon Line Time
Page H-10
6/13/2013
-------
MASTER BOTE Cost Analysis O61313.xlsx
0)
+•*
1
5/5/11
5/5/11
5/5/11
5/5/11
5/5/11
5/5/11
5/5/11
5/5/11
5/5/11
5/5/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
E
s
1
3
2
4
5
6
7
8
1
2
1
1
2
2
3
4
4
5
5
6
7
7
8
Ł•
4-»
Ł
LLJ
4-»
ra
Ol
P
12:31
13:00
12:54
13:30
14:00
13:42
13:48
11:47
15:52
15:33
12:40
16:05
12:40
16:30
12:30
13:40
16:10
12:07
17:35
14:45
14:35
17:55
16:45
±i
X
LLJ
4-»
ra
Ol
t
12:47
13:17
13:03
13:44
14:05
13:58
14:02
11:55
16:05
15:47
13:05
16:41
12:58
16:49
12:50
14:01
16:34
12:21
17:51
15:03
14:50
18:09
17:11
"c"
|E
Ol
E
i-
c
o
u
Ol
Q
E
8
Ol
Q.
E
as
ifi
16
17
9
14
5
16
14
8
13
14
25
36
18
19
20
21
24
14
16
18
15
14
26
Sample Team Recovery Time (min)
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
U)
01
4-»
O
Personnel
4
3
3
3
3
3
3
1
3
3
3
2
3
3
3
3
3
3
3
3
3
3
3
Samples
2
37
0
42
48
48
51
0
Entry Team Decon Line Time
Page H-ll
6/13/2013
-------
MASTER BOTE Cost Analysis O61313.xlsx
Ol
4-1
CD
Q
4/17/11
4/18/11
4/18/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
5/5/11
5/5/11
5/5/11
5/5/11
5/5/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
E
CD
e
7
6
4
3
4
5
6
4
9
7
8
3
9
1
4
5
7
6
1
2
3
4
5
6
7
Time at Beginning of Donning
8:00
8:00
9:00
12:22
8:15
8:07
8:07
8:45
8:45
8:50
9:57
11:08
Time at Last Taping
8:53
8:53
9:30
10:14
11:13
11:31
11:44
12:45
8:51
8:45
8:41
9:32
9:24
9:43
10:46
11:53
"c"
1
Ol
E
P
Q.
01
(X
E
CD
01
66
53
53
30
23
10
14
12
16
32
34
47
39
53
49
45
Ol
*j
o
z
Clearance Sampling; Exit bldg 13:28
Entered bldg 12:58
Entered 13:05, sharna wteam7
Entered bldg 13:28
Entered 14:31
Timed by Lukas
vitals until entry
donning only; 3 people
donning only; 3 people
donning only; 3 people
donning only; 3 people
donning, sample briefing, mask test
donning, sample briefing, mask test
donning, sample briefing, mask test
donning, sample briefing, mask test
donning, sample briefing, mask test
donning, sample briefing, mask test
donning, sample briefing, mask test
Entry Team Prep Time
Page H-12
6/13/2013
-------
MASTER BOTE Cost Analysis O61313.xlsx
Waste Knob
Value:
1 = If Deconned Waste is treated as MSW
2 = If Deconned Waste has Premium Charge
3 = If Deconned Waste is treated as Contaminated
2
Purchased LRN Equipment Variables
Equipment Amortization Period (months):
BOTE Amortization Period (months):
60
6
set these numbers equal to each other to consider expendable equpment
[Multiplier for LRN BSL-3 vs BSL-2 Analysis
[Average Length of Day (hrs)
12
[Post-Entry Rest Period (hrs)
Travel Variables
Airfare to Site (S/person):
Rental Car (1 perteam) (S/week/team):
Lodging (S/day/person):
Meals and Incidental Expenses (S/day/person):
s
s
s
s
450
450
150
35
travel = airfare + M&IE lodging + rental car + labor/M&IE for 2 travel days
Decon Round Variables
Round 1 Characterization Sampling Days
Round 1 Decon Days
Round 1 Clearance Sampling Days
Round 2 Characterization Sampling Days
Round 2 Decon Days
Round 2 Clearance Sampling Days
Round 3 Characterization Sampling Days
Round 3 Decon Days
Round 3 Clearance Sampling Days
2
3
2
2
5
2
2
3
2
Building Info
Number of Square Feet on Each Floor (ft2)
Number of Rooms on Each Floor
Number of Floors
Height of Story (ft)
Average Area Per Room (ft2)
Building Volume (ft3)
Average Volume Per Room (ft3)
4025
12
2
10
335
80500
3354
from test plan - not used yet
from test plan - average between floor land floor 2
not used yet
used in RoundX
not used yet
not used yet
Waste Sampling Knobs
Number of Waste Samples Per 100 Ib
Number of Water Samples Per 55 gal
3
1
based on Phase 2
Knobs
PageH-13
6/13/2013
-------
MASTER BOTE Cost Analysis O61313.xlsx
1
Contract with Steris
Contract with Sabre
teaters for Bottom Floor
"ravel - pH-Adjusted Bleach Process
)econ Team
'egasus soil sampling kit prep
NL lab anlysis of samples
USGS lab analysis of soil samples
'egasus analysis of water samples
NL water sampling tasks
Sandia tasks for aggresive sampling
RV-PCR - LLNL Sampling Kit Prep
SKC Honeywell Sampling Kit Prep
Slit to agar - SNL Sampling Kit Prep
Water - INL Sampling Kit Prep
nstrumentation - HOBOs for in-room
monitoring
nstrumentation - HOBOs for sample
shipment
HeaterforbldgAllmand MH-100
NL-Purchasing
N L Sample Packaging Team
.evel B Support
Round 1 Incident Command Costs
IC/safety/OSC labor + travel)
Round 2 Incident Command Costs
IC/safety/OSC labor + travel)
Round 3 Incident Command Costs
IC/safety/OSC labor + travel)
"ravel - Round 1 Characterization
SamplingTeams
"ravel - Round 1 Clearance Sampling
"earns
"ravel - Round 2 Characterization
SamplingTeams
"ravel - Round 2 Clearance Sampling
"earns
"ravel - Round 3 Characterization
SamplingTeams
"ravel - Round 3 Clearance Sampling
"earns
5ROOM Support - Round 1
BROOM Support - Round 2
5ROOM Support - Round 3
BROOM Team Travel - Round 1
Characterization Sampling
BROOM Team Travel - Round 2
Characterization Sampling
BROOM Team Travel - Round 3
Characterization Sampling
BROOM Team Travel - Round 1
Clearance Sampling
BROOM Team Travel - Round 2
Clearance Sampling
BROOM Team Travel - Round 3
Clearance Sampling
Time Preparing Sample Boxes - Round
. Characterization Sampling
"ime Preparing Sample Boxes - Round
. Clearance Sampling
Time Preparing Sample Boxes - Round
2 Characterization Sampling
"ime Preparing Sample Boxes - Round
2 Clearance Sampling
1
S 99,000
S 165,000
S 2,400
S 47,692
S 10,000
S 27,000
S 20,000
S 6,125
S 10,500
S 4,050
S 7,613
S 17,161
S 2,700
S 46,737
S 54,907
S 46,737
S 60,803
S 60,803
S 60,803
S 60,803
S 60,803
S 60,803
S 12,26
S 12,26
S 12,26
S 6,319
S 6,319
S 6,319
S 6,319
S 6,319
S 6,319
S 1,003
S 1,003
S 1,003
S 1,003
1
expenses + 2 travel days
NOT USED - From email from Erin on
2/9/11: $7500-$10,000 for Pegasus to
sterilize the sand, fill soil containers,
and set up lab spikes for the soil
project. Probably another $500-$1000
TCAD will spend on supplies for the
sampling kits.
Based on IA wINL
NOT USED
DCMDPR
DCMDPR
70 hours labor
50 hours labor * Team Rate per
round
$900/day x3 days
airfare + rental car + lodging +
expenses (over round + 2 extra days) +
2 travel days
expenses (over round + 2 extra days) +
2 travel days
airfare + rental car + lodging +
expenses (over round + 2 extra days) +
2 travel days
expenses + 2 travel days
expenses + 2 travel days
expenses + 2 travel days
expenses + 2 travel days
expenses + 2 travel days
expenses + 2 travel days
per day charge
per day charge
per day charge
airfare + rental car + lodging +
expenses + 2 travel days
airfare + rental car + lodging +
expenses + 2 travel days
airfare + rental car + lodging +
expenses + 2 travel days
airfare + rental car + lodging +
expenses + 2 travel days
airfare + rental car + lodging +
expenses + 2 travel days
airfare + rental car + lodging +
expenses + 2 travel days
based on "Room Sample Box Prep
Time" worksheet
based on "Room Sample Box Prep
Time" worksheet
based on "Room Sample Box Prep
Time" worksheet
based on "Room Sample Box Prep
Time" worksheet
Analytical
X
Sampling -RMC
Sampling- Swab
_a
00
c
iL
S>
I
00
c
iL
Sampling -HEPA
Sampling -Agg. Air
Sampling - Round 1 General
X
X
X
X
X
X
X
Sampling - Round 2 General
X
X
X
X
X
X
X
Sampling - Round 3 General
X
X
X
X
X
Sampling - General
X
X
1
>
X
X
S
X
X
0
u
X
"E
X
X
Incident Command Costs (VHP®)
X
Incident Command Costs (AB)
X
Incident Command Costs (CLO2)
X
Lumped Costs
Page H-14
6/13/2013
-------
MASTER BOTE Cost Analysis O61313.xlsx
E
±i
I
1
Ime Preparing Sample Boxes - Round
3 Characterization Sampling
Ime Preparing Sample Boxes - Round
3 Clearance Sampling
PA Personnel Purchasing Equipment
and Expendables for Phase 1
'raining of Sampling Crews
"ost Analytical Results Data Analysis
START Contractor Post-Deployment
Activities
PNNLVSP Sample Planning
Motional Labor for Removing HVAC
)uct During pH-Adjusted Bleach
"rocess Decon
)econ Line Setup and Takedown
PNNL Statistical Data Analysis
|
$ 1,003
$ 1,003
$ 6,398
$ 10,076
$ 23,490
$ 7,752
$ 17,001
$ 4,594
$ 701
$ 34,002
B
Z
based on "Room Sample Box Prep
Time" worksheet
based on "Room Sample Box Prep
Time" worksheet
100 hours per MWC 8/4/11 - divided
into 3 rounds
All teams trained for 4 hours per
round
Assumed 40 hours
Assumed 4.5 hours per Dave Rees
8/9/11
60 Hours Per Brett; not divided per
round
8 hours for Removal team in Level B;
Shannons, email 11/16/11
2 guys, 2 hours setup; 2 hours
takedown (Mike Carpenter)
200 hours per Brett A.
T5
|
X
1
QŁ
DO
1.
1
•M
1
DO
lL
&
_a
00
lL
&
*
DO
1.
1
1
00
iL
&
1
DO
1.
1
5
Sj
ID
c
DO
1.
1
5
Sj
ID
c
10
1.
1
5
Si
ID
|
00
iL
&
X
X
c
ID
00
=5.
1
X
X
X
X
s.
>
g
D
X
<
g
D
X
X
X
0
u
g
D
X
"E
u
g
D
1
>
fi
a
c
I
u
c
1
Ł1
-------
MASTER BOTE Cost Analysis O61313.xlsx
Worksheet
All
All
All
All
All
Analytical Costs
Cost Equations
Cost Equations
Cost Equations
Cost Equations
Lumped Costs
Lumped Costs
Lumped Costs
Lumped Costs
Lumped Costs
Numbers of Samples
QC
Room Sample Time
Time Per Sample
Summary
Cost Equations
Waste
Waste Summary
Waste
Analytical Costs
Waste
AB Building Refit
AB Building Refit
Analytical Costs
Analytical Costs
Waste Cost
Notes and Assumptions
Cells with a GREEN color have user inputs associated with them
Cells with a YELLOW color have outstanding issues associated with their data
Cells with a WHITE color are calculated based on formulas
Cells with a BLUE color are used in other worksheets
Used a single average time per entry based on TGD data
There is a knob that has the multiplier for BSL-3 analysis versus BSL-2 Analysis
Sampling cost does not include RMC, soil, or SKC samples
Average material cost per sample type = total materials for that sample type + total lumped costs for that sample
type + general sample costs distributed among number of HEPA, wipe, swab, air, sponge
Decon contractor fixed costs = the sum of all lumped costs + purchase order costs for each round, plus 1/3 of the
general decontamination costs that are not attributed to any given round
Purchase orders for sampling supplies and HOBOs are equally distributed among all samples of all main types
(wipes, swabs, sponge sticks, aggressive air)
505 gal of AB used; Remaining Stock - Vinegar 72 cases, 4-1 gal bottles per case (2.37 ea Walmart)
505 gal of AB used; Remaining Stock - Bleach 62 cases, 6 3-qt bottles per case (1.98 ea Walmart)
Assume sampling and BROOM teams fly in, rent 1 car per team, stay duration of sampling, fly out; 1 day travel
each way
Assume decon teams fly in, rent 1 car per team, stay duration of decon, fly out; 1 day travel each way
Assumed that only travel being paid for samplers, decon, safety, OSC, command. Other travel (e.g., decon line ops,
sample kit box prep, other helpers, from local labor)
Total number of samples for each round was estimated by dividing the total number of each type of sample by 3
Recuperation time after entries was estimated by adjusting to minimize the difference between calculating the
decon line time and the decon team time using either total days or based on entries
Assumed 15 aggressive air samples per entry; 3 hours per team
Assumed 30 aggressive air samples per round; 3 hours per team
To calculate waste distribution between sampling and decon, liquid and solid were combined
Cost of Safety Team is included in 1C costs. Cost of decon from safety team entering building is in decon cost.
Laminate Flooring = 21 ft2/carton; 35 Ib/carton = 1.67 Ib/ft2
Laminate Flooring was notionally added to Removal Operations
Did not include porous materials removed after CL02 clearance sampling because it didn't have anything to do
with the decon or clearance process, and wouldn't have applied to reset
The UT Lab Costs are what the costs are - no additional labs responded with tracking data
HVAC material = 24 gauge; 7.71 Ib/linear foot; HVAC equations, data, and rules of thumb By Arthur A. Bell
Laminate floor cost: 3.70/ft2 installed; email from Martin 8/8/11
Cost to install HVAC Duct: from http://www.homewyse.com/services/cost_to_install_duct.html
Analytical Cost of Aggressive Air Samples = average of analytical costs of HEPA vac, sponge stick, swab, and EPA
wipes
Analytical Cost of Waste Samples = average of analytical costs of HEPA vac, sponge stick, swab, and EPA wipes
MSW Transportation Cost = $3/mile in 2002; assumed it is $5/mile in 2012
Notes and Assumptions
Page H-16
6/13/2013
-------
MASTER BOTE Cost Analysis O61313.xlsx
HEPAVac
Sponge Sticks
Wipes (EPA Prep)
Wipes (LLNL Prep)
Swabs
RMC
Aggressive Air
Round 1- VHP®
253
520
58
113
105
54
30
Round 2- AB
253
520
58
113
105
54
30
Round 3 - CLO2
254
520
59
114
105
54
30
Total w/o RMC
Total
760
1560
175
340
315
162
90
3240
Numbers of Samples
Page H-17
6/13/2013
-------
MASTER BOTE Cost Analysis O61313.xlsx
1
•D
S
•5
1
Stomachers (4)
Rainin Pipette Tips for LRN
Orbi Shaker
Magic Clamp Platform
Magic Clamps
Containers
Scissors
50 ml conical Tubes
50 ml pipettes
Forceps
15 ml Conical tubes
Cups
5 ml pipettes
Pressure Station
Vacuum bottles
Venting Closure
Vaccuum Tubing
Funnel Manifold
10 ml pipettes
Cell Spreaders
200uLpipettips, 960/pack
ImL pi pet tipss, 768/pack
Filter Funnels (extra from VWR)
Forceps
15ml conical
50ml con ical(RMC)
50ml conical (Wipe)
Fisher twirl-em bag (24oz)
whirl pak bag (24 oz) (RMC)
whirl pak bag (24 oz) (Swab)
small baggie (4x6) (RMC)
small baggie (4x6) (Swab)
small baggie (4x6) (Wipe)
baggie (6x9) for sponges
ziploc sandwith bags for templates
large baggie (12x15)
10x13" 2milziplock bags
8x10" 2mil ziplock bags (RMC)
8x10" 2mil ziplock bags (Swab)
8x10" 2mil ziplock bags (Wipe)
large fisher baggie (lOOoz) (Sponge)
large fisher baggie (lOOoz) (HEPA)
ziploc gallon bags for templates
2gal ziplock bags for gloves
sterile swab
microstein tubes (2ml)
dispatch wipes
dispatch wipe canisters
sponge sticks
HEPA vacuum kits
Kendal gauze wipe
Referee coupons
neutralizing buffer (Swab)
neutralizing buffer (Wipe)
gloves (L)
tt
3
$ 17,596.00
$ 703.50
$ 5,680.00
$ 1,408.00
$ 672.00
$ 1,072.00
$ 4,620.00
$ 4,073.30
$ 3,276.00
$ 3,697.50
$ 472.00
$ 868.68
$ 3,414.60
$ 1,936.32
$ 2,092.00
$ 804.00
$ 656.40
$ 13,818.48
$ 2,168.00
$ 2,322.18
$ 660.00
$ 819.00
$ 10,963.68
$ 102.00
$ 75.48
$ 48.32
$ 52.20
$ 324.00
$ 52.72
$ 102.50
$ 41.02
$ 79.77
$ 44.32
$ 273.64
$ 77.12
$ 279.00
$ 135.66
$ 31.29
$ 31.29
$ 31.29
$ 235.02
$ 156.68
$ 158.76
$ 48.41
$ 415.61
$ 44.10
$ 362.36
$ 609.00
$ 2,224.00
$ 7,184.00
$ 11.20
$ 79.06
$ 109.94
$ 1,100.00
Ł
1
NOT USED
Analysis Expenables
X
X
X
X
X
X
X
X
X
X
X
X
X
Analysis Equipment
X
X
X
X
X
X
X
X
X
X
DO
c
ll
X
X
X
X
X
X
Sampling - Swab
X
X
X
X
X
X
X
Sampling -Wipe
X
X
X
X
X
Sampling - Sponge
X
X
X
X
<
I
DO
C
ll
X
X
X
'I
1
DO
C
ll
Sampling - General
X
X
X
X
X
X
X
1
>
c
CQ
<
c
°
u
c
i
ID
c
I
>
c
1
i
1
Waste Management - AB
Waste Management - CLO2
Purchase Orders
Page H-18
6/13/2013
-------
MASTER BOTE Cost Analysis O61313.xlsx
1
•D
S
•5
1
gloves (XL)
sampling carts
bar codes (RMC)
bar codes (Swab)
bar codes (Wipe)
bar codes (Sponge)
bar codes (HEPA)
Ecomony File storage box
Parts Bin with 4 dividers
sampling templates
ethanol wipes
sharpie markers
INL-Purchased ISA Plates, PBST, Pall Filters
ARCADIS-Purchased Supplies for Sampling Kits
300 gallons bleach (Clorox, 96 oz bottles)
350 gallons vinegar (Oasis Foods white vinegar 4% acidity)
RFID Reader and Tags
PPE for Sampling Purchased by DTRA
PPE for Decon purchased by DTRA
Equipment and Materials Purchased by START Contractors
PPE Purchased by Dino
Other Sampling Equipment Purchased by Dino
PPE Purchased byTCAD (cartridges)
tt
5
S 1,100.00
S 2,400.00
S 14.76
S 28.70
S 15.95
S 142.14
S 69.25
S 336.00
S 181.50
S 4,290.00
S 496.25
S 70.32
$43,386.72
S 23,665.00
S 792.00
S 829.50
S 3,775.99
S 21,864.07
S 4,760.73
S 28,768.00
S 678.50
S 1,863.72
S 303.30
S
I
Labor on "Lumped Costs" Worksheet
1.98/3 qt; 505 gal of AB used
2.37/gal; 505 gal of AB used
$28624.80 prorated by sampling &decon entries
$28624.80 prorated by sampling &decon entries
per Dave Rees 8/9/11
Per Erin 8/5/11
Per Erin 8/9/11 - bought 100 used 30
Analysis Expenables
X
Analysis Equipment
DO
C
ll
X
Sampling - Swab
X
Sampling -Wipe
X
Sampling - Sponge
X
<
I
DO
C
ll
X
'I
1
DO
C
ll
Sampling - General
X
X
X
X
X
X
X
X
X
X
X
X
1
>
c
CQ
<
c
X
X
X
°
u
c
i
ID
c
X
I
>
c
1
i
1
Waste Management - AB
X
Waste Management - CLO2
Purchase Orders
Page H-19
6/13/2013
-------
MASTER BOTE Cost Analysis O61313.xlsx
§
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/18/11
4/18/11
4/18/11
4/18/11
4/18/11
4/18/11
213
213
213
211
211
209
209
209
209
212
212
212
212
212
212
210
210
210
210
210
210
208
208
208
208
208
109
109
109
109
109
109
109
107
107
107
107
107
107
107
107
107
110
110
110
110
110
110
110
108
108
108
108
108
Other
Other
Other
Other
Other
Other
Other
Other
Other
Other
Other
105
105
105
105
105
105
o
Upstairs
Upstairs
Upstairs
Upstairs
Upstairs
Upstairs
Upstairs
Upstairs
Upstairs
Upstairs
Upstairs
Upstairs
Upstairs
Upstairs
Upstairs
Upstairs
Upstairs
Upstairs
Upstairs
Upstairs
Upstairs
Upstairs
Upstairs
Upstairs
Upstairs
Upstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Upstairs
Upstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Room Type
Residence
Residence
Residence
Residence
Residence
Residence
Residence
Residence
Residence
Commercia
Commercia
Commercia
Commercia
Commercia
Commercia
Commercia
Commercia
Commercia
Commercia
Commercia
Commercia
Commercia
Commercia
Commercia
Commercia
Commercia
Residence
Residence
Residence
Residence
Residence
Residence
Residence
Residence
Residence
Residence
Residence
Residence
Residence
Residence
Residence
Commercia
Commercia
Commercia
Commercia
Commercia
Commercia
Commercia
Commercia
Commercia
Commercia
Commercia
Commercia
Commercia
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Residence
Residence
Residence
Residence
Residence
Residence
|
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
-s
+J
t/>
TRUE
TRUE
FALSE
FALSE
TRUE
TRUE
FALSE
FALSE
TRUE
TRUE
FALSE
FALSE
FALSE
FALSE
TRUE
TRUE
TRUE
FALSE
TRUE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
TRUE
FALSE
TRUE
TRUE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
TRUE
FALSE
FALSE
TRUE
FALSE
TRUE
FALSE
FALSE
FALSE
TRUE
TRUE
FALSE
TRUE
FALSE
TRUE
TRUE
TRUE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
TRUE
FALSE
TRUE
FALSE
TRUE
TRUE
FALSE
FALSE
J2
V)
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
TRUE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
HI
Q.
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
TRUE
FALSE
FALSE
FALSE
TRUE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
TRUE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
1
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
TRUE
TRUE
FALSE
FALSE
FALSE
TRUE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
TRUE
TRUE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
TRUE
FALSE
FALSE
TRUE
FALSE
TRUE
FALSE
TRUE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
TRUE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
TRUE
FALSE
TRUE
FALSE
Start Time (m.s)
0.00
0.00
0.00
1.10
10.12
4.50
0.00
0.00
1.11
0.00
0.00
0.00
4.00
0.00
3.50
0.00
8.35
6.57
0.00
0.43
4.43
0.00
0.00
3.02
1.24
0.00
12.49
0.00
4.30
8.03
0.56
0.00
5.20
0.00
5.21
11.03
2.43
0.00
8.20
0.00
2.16
4.13
0.00
0.00
0.00
3.36
6.27
10.18
0.00
0.00
2.43
4.41
12.06
16.40
2.56
9.20
0.00
1.01
4.14
5.49
9.06
12.35
0.00
15.48
0.00
2.50
13.02
21.56
25.05
0.00
0.00
End Time (m.s)
5.49
3.34
6.41
4.44
15.15
8.02
7.52
10.04
7.56
4.45
2.37
4.58
8.58
3.46
5.35
3.42
12.26
9.53
3.02
2.25
10.14
4.44
6.25
9.42
10.06
6.23
17.19
4.21
8.11
11.02
3.53
5.32
8.12
2.40
8.18
17.11
10.47
4.12
9.37
1.20
4.01
13.31
2.00
5.48
3.43
7.00
10.28
13.55
4.23
4.45
4.02
9.56
16.23
21.31
8.12
14.35
4.50
2.53
8.55
8.39
11.38
15.38
1.57
18.27
1.58
8.09
21.56
25.05
28.05
10.56
2.39
LOE Sampler A [BROOM] (1-5)
3
3
2
3
3
4
3
2
3
5
4
4
3
4
4
4
4
3
3
3
2
4
3
3
2
3
3
3
3
3
3
3
3
3
3
3
3
2
4
3
3
2
5
3
3
2
3
2
3
3
3
3
3
2
3
4
3
3
3
3
3
3
2
3
2
3
2
0
3
2
3
LOE Sampler B [Prep] (1-5)
3
4
3
3
3
3
2
3
4
4
5
3
3
3
5
5
4
4
4
4
3
4
5
3
2
3
3
3
4
3
3
3
4
4
3
4
3
4
5
4
3
2
5
3
3
3
4
3
4
4
4
4
4
3
4
4
4
5
4
3
4
4
3
3
3
3
3
5
3
2
3
LOE Sampler C [Sample Acquisition] (1-
5)
4
3
4
4
3
4
4
5
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
5
4
4
4
4
4
4
5
4
4
4
5
4
3
5
4
4
4
4
4
4
4
4
4
4
4
3
3
4
4
3
4
5
4
4
4
5
4
5
4
5
4
5
5
4
5
4
Ł
I
Counter
Blank
Bed
TV
Table
Counter
Partition
Floor
Wall
Settling plate
Chair
Computer screen
Floor
Room 213 Blank
Floor
Wall
Computer screen
File cabinet
Ceiling vent
Ceiling tile
Table
Computer screen
Floor
Ceiling tile
File cabinet
Floor
Shelf
Cabinet
File cabinet
Nightstand
Bed
Wall
TV
Table
Table
Couch
Cabinet
Blank
Blank
Air vent
Ceiling tile
Blank
Floor
Desktop
Chair
Computer screen
Books
File cabinet
Floor
Blank
Floor
Chair
Desk
Wall
Floor
Room 102 floor
Room 102 Blank
Room 102 vertical file cabinet
Room 102 horizontal file cabinet
Room 102 Horizontal file cabinet
Room 102 File cabinet
Room 101A air sample rack
Room 102 Table
Room 101A
Wall
Floor
Time between vacuum and sponge sample
Stove
Floor
Air supply duct
Resource Tracker Sampling Data
Page H-20
6/13/2013
-------
MASTER BOTE Cost Analysis O61313.xlsx
§
4/18/11
4/18/11
4/18/11
4/18/11
4/18/11
4/18/11
4/18/11
4/18/11
4/18/11
4/18/11
4/18/11
4/18/11
4/18/11
4/18/11
4/18/11
4/18/11
4/18/11
4/18/11
4/18/11
4/18/11
4/18/11
4/18/11
4/18/11
4/18/11
4/18/11
4/18/11
4/18/11
4/18/11
4/18/11
4/18/11
4/18/11
4/18/11
4/18/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
106
106
106
106
106
106
106
103
103
103
103
103
103
103
103
103
103
103
103
103
103
104
104
104
104
104
104
104
104
104
104
104
104
108
108
108
108
108
108
108
108
108
108
109
109
109
109
109
109
107
107
107
107
107
107
107
107
107
110
110
110
110
110
110
110
110
110
110
110
101A
101A
o
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Room Type
Commercial
Commercial
Commercial
Commercial
Commercial
Commercial
Commercial
Mai room
Mai room
Mai room
Mai room
Mai room
Mai room
Mai room
Mai room
Mai room
Mai room
Mai room
Mai room
Mai room
Mai room
Shop
Shop
Shop
Shop
Shop
Shop
Shop
Shop
Shop
Shop
Shop
Shop
Commercial
Commercial
Commercial
Commercial
Commercial
Commercial
Commercial
Commercial
Commercial
Commercial
Residence
Residence
Res dence
Res dence
Residence
Residence
Res dence
Res dence
Residence
Residence
Res dence
Res dence
Residence
Residence
Res dence
Commercia
Commercia
Commercia
Commercia
Commercia
Commercia
Commercia
Commercia
Commercia
Commercia
Commercia
|
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
-s
+J
t/>
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
TRUE
TRUE
TRUE
TRUE
TRUE
TRUE
TRUE
FALSE
FALSE
FALSE
TRUE
TRUE
FALSE
FALSE
TRUE
FALSE
TRUE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
TRUE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
J2
V)
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
TRUE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
HI
Q.
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
TRUE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
TRUE
TRUE
FALSE
FALSE
FALSE
TRUE
TRUE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
TRUE
FALSE
TRUE
FALSE
TRUE
FALSE
TRUE
FALSE
TRUE
FALSE
FALSE
TRUE
TRUE
FALSE
FALSE
FALSE
TRUE
FALSE
1
TRUE
FALSE
TRUE
TRUE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
TRUE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
TRUE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
TRUE
TRUE
TRUE
TRUE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
TRUE
FALSE
TRUE
Start Time (m.s)
0.00
10.43
10.43
19.05
27.20
0.00
5.25
0.00
-0.40
4.50
0.00
0.00
3.33
0.00
3.25
5.20
14.01
15.03
16.22
17.36
18.40
3.14
1.30
0.00
4.46
0.00
5.00
8.43
20.53
0.00
1.32
2.47
3.42
13.58
0.34
12.23
22.03
38.49
0.00
2.46
7.33
15.40
16.56
22.29
0.00
7.32
10.56
18.35
3.26
18.37
21.18
24.15
31.24
1.00
3.39
18.32
24.50
0.00
0.00
5.53
0.00
0.00
0.00
2.01
19.16
22.45
1.43
13.47
29.40
2.02
8.12
End Time (m.s)
9.46
12.24
17.02
26.03
38.17
4.12
9.07
3.43
2.25
7.56
2.56
3.21
5.28
1.56
5.00
11.30
15.03
16.22
17.18
18.23
19.44
6.45
4.48
4.02
8.38
3.35
7.30
19.43
21.56
1.11
2.47
3.42
4.30
16.32
7.51
16.43
26.05
41.26
2.35
4.37
13.31
16.3
20.11
24.48
3.09
8.40
17.37
23.33
9.19
21.09
22.21
28.42
36.24
2.47
6.20
21.10
27.13
2.19
5.00
10.02
2.12
0.35
1.49
3.06
21.32
26.00
7.59
16.53
35.38
5.45
9.12
LOE Sampler A [BROOM] (1-5)
3
0
3
3
2
3
3
2
3
3
3
3
3
3
3
3
4
0
4
4
3
3
3
4
3
3
3
2
3
3
3
0
3
3
3
3
3
0
3
3
3
5
5
3
3
4
3
3
3
3
5
3
2
4
5
3
3
2
2
3
3
3
3
3
2
3
3
3
3
3
3
LOE Sampler B [Prep] (1-5)
3
5
3
2
2
4
3
2
3
4
3
3
4
4
4
4
5
5
5
5
5
3
4
3
3
3
4
2
5
5
5
5
5
4
3
4
3
0
3
3
3
5
5
4
4
5
3
3
3
4
4
3
3
5
5
4
5
3
4
3
5
5
5
5
3
3
3
3
3
2
5
LOE Sampler C [Sample Acquisition] (1-
5)
5
5
5
5
5
4
4
4
4
4
4
4
4
4
4
5
5
5
5
4
4
4
4
4
4
4
5
5
5
4
4
5
4
4
5
5
5
0
5
4
5
5
5
4
4
4
5
5
5
4
5
4
5
5
4
5
5
4
4
4
4
3
4
3
4
5
5
5
5
5
4
Ł
I
Floor
Time between vacuum samples
Books and file cabinet
Chair
Floor
Air supply duct
File cabinet
Floor
Desk
Floor
Wall
Work bench
Table
Top of mailbox
Air supply duct
Ceiling tile
Blank
Time between vacuum and sponge stick
Blank
Blank
Blank
Floor
Floor
Work bench
Work bench
Wall
Air supply duct
Ceiling tile
Blank
Blank
Blank
Time between blanks
Blank
108 Floor
108 Chair
Ceiling tile
Partition
File cabinet
File cabinet
Air supply vent
Wall
Blank
Blank
Wall
On top of cabinet
Blank
Bed
Bed
Ceiling tile
Table
Blank
Table
Chair
Blank
Blank
Bookshelf
TV
Table
Floor
Floor
Blank
Blank
Blank
Blank
Desk
Floor
Books
Wall
Partition
Ida Sponge
101 Blank
Resource Tracker Sampling Data
Page H-21
6/13/2013
-------
MASTER BOTE Cost Analysis O61313.xlsx
§
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/23/11
4/23/11
4/23/11
4/23/11
4/23/11
4/23/11
4/23/11
4/23/11
4/23/11
4/23/11
4/23/11
4/23/11
4/23/11
4/23/11
4/23/11
4/23/11
4/23/11
4/23/11
4/23/11
4/23/11
4/23/11
4/23/11
4/23/11
4/26/11
4/26/11
4/26/11
4/26/11
4/26/11
4/26/11
4/26/11
4/26/11
4/26/11
4/26/11
4/26/11
4/26/11
4/26/11
4/26/11
4/26/11
4/26/11
4/26/11
4/26/11
4/26/11
4/26/11
4/26/11
4/26/11
4/26/11
4/26/11
4/26/11
4/26/11
4/26/11
4/26/11
4/26/11
4/26/11
4/26/11
4/26/11
4/26/11
4/26/11
4/26/11
101A
101A
101A
101A
101A
101A
101A
101A
101A
101A
106
106
106
105
105
105
105
105
105
103
103
103
103
103
103
103
103
103
103
103
103
103
103
103
103
103
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
o
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Room Type
Commercial
Commercial
Commercial
Residence
Residence
Residence
Residence
Residence
Residence
Mai room
Mai room
Mai room
Mai room
Mai room
Mai room
Mai room
Mai room
Mai room
Mai room
Mai room
Mai room
Mai room
Mai room
Mai room
Mai room
Mai room
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
|
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
•B
+J
t/>
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
TRUE
FALSE
TRUE
FALSE
TRUE
TRUE
TRUE
TRUE
FALSE
TRUE
FALSE
FALSE
FALSE
TRUE
TRUE
FALSE
FALSE
TRUE
TRUE
TRUE
TRUE
TRUE
TRUE
TRUE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
TRUE
FALSE
TRUE
TRUE
TRUE
TRUE
TRUE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
TRUE
TRUE
FALSE
FALSE
TRUE
FALSE
TRUE
J2
V)
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
HI
Q.
FALSE
TRUE
TRUE
TRUE
FALSE
FALSE
TRUE
FALSE
TRUE
TRUE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
TRUE
FALSE
TRUE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
TRUE
FALSE
TRUE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
TRUE
FALSE
TRUE
FALSE
FALSE
TRUE
FALSE
FALSE
TRUE
TRUE
FALSE
TRUE
FALSE
1
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
TRUE
TRUE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
TRUE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
Start Time (m.s)
4.21
6.37
5.06
8.51
19.28
22.30
28.26
31.02
15.42
30.43
20.00
1.49
19.27
15.14
8.18
7.20
4.16
0.00
31.36
0.00
10.35
14.23
18.30
22.07
28.56
32.12
34.57
41.27
45.10
48.34
52.03
101.50
103.01
104.06
108.03
0.00
10.34
8.49
16.29
17.17
23.12
23.48
28.17
29.32
35.12
31.02
35.48
39.15
41.04
44.27
48.07
49.43
53.48
56.50
53.35
56.54
0.00
2.08
7.25
10.21
14.41
22.13
26.13
26.41
27.45
31.25
37.15
40.13
44.36
49.00
End Time (m.s)
3.15
6.04
7.14
8.03
14.19
20.03
27.16
30.44
33.34
16.01
33.26
24.28
4.34
23.34
18.17
12.36
8.12
7.18
3.55
33.36
4.28
13.33
16.39
21.13
25.46
30.52
34.43
37.39
44.06
46.40
51.28
56.59
102.30
104.00
107.12
109.01
9.39
15.36
13.45
20.05
20.33
25.32
27.17
30.30
34.48
38.53
32.57
37.15
40.43
43.59
47.44
50.41
51.48
57.35
59.08
55.44
59.58
3.56
4.23
10.17
14.04
21.53
23.20
26.31
27.31
31.09
33.32
39.57
43.43
47.41
52.03
LOE Sampler A [BROOM] (1-5)
3
3
5
2
3
5
3
3
4
5
3
3
3
3
3
2
0
3
3
3
3
3
3
3
3
2
3
3
3
2
3
2
4
4
4
4
3
4
3
3
3
3
3
3
3
3
3
3
4
3
3
3
3
3
3
3
3
3
3
3
3
3
4
4
3
3
3
3
3
3
3
LOE Sampler B [Prep] (1-5)
4
3
5
4
3
0
3
4
4
5
3
3
3
4
4
4
5
4
4
4
4
3
3
4
4
3
4
3
3
4
4
3
5
5
5
5
3
4
4
3
3
4
3
3
3
4
4
4
5
3
3
3
4
4
3
4
3
3
4
4
3
3
5
5
5
4
4
4
4
3
3
LOE Sampler C [Sample Acquisition] (1-
5)
4
4
4
5
0
5
5
5
5
4
5
5
5
4
4
5
5
4
4
4
4
4
4
4
4
4
4
5
4
4
5
4
4
4
4
4
4
4
5
5
4
4
4
5
4
4
5
4
4
5
4
4
4
5
4
4
4
4
4
4
5
5
5
4
5
5
4
4
4
5
Ł
I
101A Desk
101A Desk
lOla Blank
101A Desk
101A Floor
Glove change
101A Desk
Vertical on box
Horizontal on box
Blank
Floor
Ceiling tile
Chair
Ceiling tile
Air supply vent
Floor
Change gloves
Table
Table
Floor
Floor
Floor
Floor
Floor
Wall
Floor
Table
Desk
On top of mailbox
On top of mailbox
Air supply vent
Ceiling tile
Blank
Blank
Blank
Blank
101A Floor
101A File cabinet
Floor
101A File cabinet
Air duct
Floor
lOlAdesk
Lobby Wall
101A Chair
Lobby Floor
Lobby Floor
lOlAdesk
Lobby blank
lOlAdesk
lOlAdesk
lOlAdesk
Lobby Floor
lOlAdesk
Lobby ceiling light
Lobby wall
lOlAdesk
lOlAdesk
Lobby floor
101A file cabinet
101A file cabinet
101A floor
101A blank
101A blank
101A blank
101A file cabinet
101A file cabinet
lOlAdesk
101A vertical side of UVAPS
lOlAtopof UVAPS
101A top of UVAPS
Resource Tracker Sampling Data
Page H-22
6/13/2013
-------
MASTER BOTE Cost Analysis O61313.xlsx
§
4/26/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
Hall
109
109
109
109
109
109
109
109
109
109
109
109
109
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
105
105
105
105
105
105
110
110
110
108
108
108
108
108
108
108
108
108
108
106
106
106
106
106
106
106
106
104
104
104
104
o
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Downstairs
Room Type
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Hall
Commercia
Commercia
Commercia
Commercia
Commercia
Commercia
Commercia
Commercia
Commercia
Commercia
Commercia
Commercia
Commercia
Commercia
Commercia
Commercia
Commercia
Commercia
Shop
Shop
Shop
Shop
|
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
-s
+J
t/>
FALSE
FALSE
TRUE
TRUE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
TRUE
TRUE
FALSE
TRUE
TRUE
FALSE
TRUE
TRUE
TRUE
FALSE
TRUE
TRUE
TRUE
FALSE
FALSE
TRUE
FALSE
TRUE
FALSE
TRUE
FALSE
FALSE
TRUE
TRUE
FALSE
FALSE
TRUE
TRUE
FALSE
TRUE
FALSE
TRUE
FALSE
TRUE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
TRUE
FALSE
TRUE
FALSE
J2
V)
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
TRUE
FALSE
FALSE
FALSE
TRUE
HI
Q.
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
1
FALSE
TRUE
FALSE
FALSE
TRUE
TRUE
FALSE
FALSE
FALSE
TRUE
TRUE
FALSE
FALSE
TRUE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
TRUE
FALSE
TRUE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
TRUE
FALSE
FALSE
TRUE
TRUE
FALSE
FALSE
TRUE
FALSE
FALSE
FALSE
FALSE
FALSE
Start Time (m.s)
52.06
2.00
15.23
25.13
5.12
16.70
20.04
23.56
5.55
14.07
27.13
0.00
7.06
16.51
0.00
5.32
0.00
9.36
15.02
17.40
24.45
20.01
0.00
11.07
14.52
24.45
30.59
0.30
5.21
13.10
20.20
24.13
28.43
4.26
0.00
0.00
3.21
13.18
18.14
22.35
29.25
36.17
40.04
42.33
23.44
25.46
27.25
13.10
17.45
22.48
16.03
28.56
35.47
0.00
11.30
39.44
43.20
44.36
46.26
End Time (m.s)
55.29
11.04
22.38
29.19
13.31
18.40
21.31
25.06
10.07
21.33
34.57
5.55
12.51
22.35
2.36
8.03
5.45
11.50
16.57
21.34
28.18
22.46
3.19
12.38
17.19
29.45
33.30
5.24
8.40
18.33
23.52
28.08
36.20
8.21
3.50
2.01
8.30
15.12
21.04
23.30
31.28
38.34
41.58
44.15
25.18
27.08
28.31
16.07
20.40
26.24
22.04
32.27
38.10
5.53
13.44
41.57
43.43
45.22
47.54
LOE Sampler A [BROOM] (1-5)
3
3
3
3
3
3
4
3
3
3
3
3
3
3
3
3
3
2
3
4
0
3
4
3
4
3
3
3
3
3
0
3
3
3
3
3
3
3
4
5
3
3
3
3
5
5
4
3
3
3
3
3
3
4
3
3
5
5
4
LOE Sampler B [Prep] (1-5)
3
3
3
4
3
5
5
5
4
3
4
4
4
4
3
3
3
4
3
4
4
4
4
4
4
4
3
4
4
3
0
3
3
3
4
4
3
4
4
5
4
4
4
4
5
5
5
3
3
4
4
3
3
3
4
3
5
5
5
LOE Sampler C [Sample Acquisition] (1-
5)
5
5
5
4
5
5
5
5
4
5
5
4
4
5
4
4
5
4
4
4
4
4
4
4
4
5
4
4
4
5
0
4
5
4
4
4
5
4
4
5
4
4
4
4
4
4
4
4
4
4
5
4
4
4
4
4
5
5
4
Ł
I
lOlAtopof UVAPS
Floor
Counter
Counter
Floor
Blank
Blank
Blank
Side table
Bed
Bed
Side table
Wall
Ceiling tile
Floor
Floor
Ceiling tile
Floor
Bathroom Floor
Bathroom Wall
Blank no A person
Wall
Floor
Room 101 Floor
Room 101 Wall
Ceiling tile
Floor
Ceiling tile
Stove
Floor
Table
Air supply vent
Ceiling tile
Table
Floor
Air supply vent
Ceiling tile
Floor
Wall
Blank
Floor
Chair
Desk
Computer screen
Blank
Blank
Blank
Wall
Floor
Chair
Ceiling tile
Desk
Computer screen
Partition
Air supply vent
Wall
Blank
Blank
Blank
Resource Tracker Sampling Data
Page H-23
6/13/2013
-------
MASTER BOTE Cost Analysis 061313.xlsx
Date
7/26/2011
7/27/2011
7/27/2011
7/28/2011
7/28/2011
7/28/2011
7/28/2011
7/28/2011
8/1/2011
8/1/2011
8/1/2011
8/2/2011
8/2/2011
8/2/2011
8/2/2011
8/2/2011
8/2/2011
8/3/2011
8/3/2011
8/3/2011
8/3/2011
8/3/2011
8/3/2011
8/3/2011
8/3/2011
8/4/2011
8/4/2011
8/4/2011
8/4/2011
8/4/2011
8/4/2011
8/5/2011
8/5/2011
8/5/2011
8/5/2011
8/5/2011
8/5/2011
8/5/2011
8/5/2011
8/5/2011
8/10/2011
8/10/2011
8/10/2011
8/10/2011
8/10/2011
8/10/2011
8/10/2011
8/10/2011
8/10/2011
8/10/2011
Revisions
Baseline Spreadsheet
added analytical costs
added material costs for decontaminations
added cost contribution from 1C
adde bar charts with breakdown of costs to "Cost Equations" worksheet
improved estimation of travel costs for START contractors
color coded cells: Cells with a GREEN color have user inputs associated with them; Cells with a YELLOW color have
outstanding issues associated with their data; Cells with a WHITE color are calculated based on formulas; Cells with a BLUE
color are used in other worksheets
Added amortization table for LRN-procured equipment
Added multiplier for analysis to scale BSL-2 vs BSL-3 analytical costs; created "knob" to adjust
Added revision list worksheet
Created AB Building Refit worksheet
Added vinegar and bleach costs to Lumped Costs
Added labor costs and sample time for aggressive air samples
Added travel costs of sampling teams
Fixed error in travel cost of decon teams
Got rid of sample times for RMC, SKC
Added BROOM labor and travel support
Fixed error in room sample box prep time averaging
Added room sample box prep time into Lumped Costs
Added QC check to compare costs for decon line and AB decon team using total days versus entries
Put many quantities in hours not minutes
Added "names" to many cells for ease of debugging
Split up sampling/analytical cost components
Made plot to show sampling/analytical cost breakdown
Put plots on separate worksheets
Added ceiling tiles to AB Refit Worksheet (est $5 each, 2 foot x 4 foot)
Added laminate flooring (est $l/ft2 material, $l/ft2 installation)
Added EPA Labor for purchasing stuff
Added factor for time going between samples
Moved bleach and vinegar purchase to Purchases worksheet
Added books, binders, and mail to AB Refit Worksheet (estimated costs)
Changed Activity "AB Dry and Reset" to "AB Dry"
Calculated waste cost contribution due to sampling and decon
Included waste management in sampling and decon cost breakdowns
Included time training sampling crews
Included data analysis time
Added room and building dimensions on "Knobs" sheet
Added "Other" contribution to Waste Costs from Purchase Orders
Add cost of RFID tag reader and tags to Waste "Other" Costs
Added Charts for Waste Distribution by Category and Activity
Separated Decon Line Labor from Sampling Team Entry Labor
Separated Decon Line Labor from Decontamination Team Entry
Added Decon line labor for Round 1 and Round 3 decon contractor entries
Added Decon Line Labor for Safety Team Entries
Changed START contractor purchase to match note from Dave Rees 8/9/11
Added START contractor post-deployment reporting, cleanup, etc per Dave Rees 8/9/11
Added laminate floor replacement and installation cost per Martin Melzer ($3/ft2)
Added PPE purchased by Bruce Hinds
Added PPE and stuff purchased by Dino
Adjusted generation date for some waste generated from the spraying
Revisions
Page H-24
6/13/2013
-------
MASTER BOTE Cost Analysis 061313.xlsx
8/10/2011
8/10/2011
8/10/2011
8/10/2011
8/11/2011
8/11/2011
8/11/2011
8/11/2011
8/11/2011
8/11/2011
8/11/2011
8/11/2011
8/29/2011
8/29/2011
8/29/2011
11/10/2011
11/10/2011
11/10/2011
11/16/2011
11/16/2011
11/16/2011
11/16/2011
11/17/2011
11/17/2011
11/17/2011
11/17/2011
11/18/2011
11/18/2011
11/18/2011
5/29/2012
5/29/2012
8/1/2012
8/1/2012
9/21/2012
Added Liquid Waste Distribution Chart
Changed Solid wase Distribution Chart to be absolute numbers
Added TCAD-purchased cartridges
Updated Building Refit Team makeup and hours per Steve Reese 8/10/11
Update Building Refit Worksheet to calculate total refit costs
Created "RoundX" worksheet and graphs to run what if scenarios on all the other data
Changed Decon Line Ops Summary to have total time for each activity
Added waste sampling team and water sampling team
Added waste sampling time and water sampling time (need numbers)
Added waste analytical cost and water analytical cost (need numbers)
Added waste and water sampling and analysis to the Waste Cost worksheet
On Summary worksheet estimated amount of Sabre and Steris mob/demob vs cost per square foot
Changed Sabre and STERIS breakdown to 33% mobilization/demobilization
Put data into water analytical costs based on Scott Minamyer email 8/18/11
Changed water sampling time to reflect Scott Minamyer email 8/29/11
Added PNNL VSP Sample Planning
Went through the general sampling and general decon and divided by rounds if appropriate
Changed Waste Samples per 100 Ib from 1 to 3 to approximate the 1 sample per bag decision from
Phase II
Clarified a couple headings on the Cost Equations Worksheet
A few corrections on AB Refit Worksheet based on Joe's QC
Changed BSL2-BSL3 multiplier to 1.5 (based on email exchange between Worth, Joe, and Paul
Added PNNL Statistical Analysis Labor
Slightly modified Team Entry spreadsheet based on Erin's QC
Added HVAC replacement cost to AB Refit Worksheet
Added HVAC removal cost to Lumped Costs Worksheet
Added HVAC weight into waste from AB
Created Salary Table worksheet and based loaded labor rates on careermedia.salary.com & Govt Salary Table
Changed Waste Sampling Team to 3 people
Changed Waste Sample time to 10 minutes
Updated headers to reflect that this is Appendix K
Updated Graphs to add ® sign to VHP and to change Amended Bleach to "pH-Adjusted Bleach Process"
Updated waste generation days to reflect correct dates for materials removed the day after spraying
Updated daily waste generation graph with headings for the 3 rounds
Changed CLO2 to CIO2 in graphs
Revisions
Page H-25
6/13/2013
-------
MASTER BOTE Cost Analysis O61313.xlsx
Date
4/19/11
4/19/11
4/19/11
4/19/11
4/27/11
4/27/11
4/27/11
Number
of People
7
7
7
7
2
2
3
Activity
Folding 100 vacuum templates
Tabbing 95 medium size templates
Pre-bleach 22 sampling boxes packing 1st and 2nd floor
Adding templates to 22 pre-bleach boxes
preparing 5 sample kits
preparing templates (for 42, 10 x 10 kits)
assembled 20, pre CIO2 kits
Time
(min)
12
5
42
22
105
120
120
Number
of Boxes
22
5
20
Average
Box Prep
Time
(person -
min/box)
25.8
42.0
18.0
Room Sample Box Prep Time
Page H-26
6/13/2013
-------
MASTER BOTE Cost Analysis O61313.xlsx
0)
+J
&
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/18/11
4/18/11
4/18/11
4/18/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/23/11
4/23/11
4/23/11
4/23/11
4/26/11
4/26/11
4/26/11
o
o
ge
102
102
107
108
109
110
206
207
208
209
210
211
212
213
101A
Floor 1 Other
Floor 2 Other
Floor 2 Other
103
104
105
106
102
104
105
106
107
108
109
110
206
207
208
209
210
211
212
213
101A
Floor 1 Other
Floor 2 Other
Floor 2 Other
103
105
106
Floor 1 Other
102
103
104
E
IS
Ł•
3
12
8
8
5
5
6
4
6
4
5
8
5
8
3
9
7
7
4
6
4
6
9
8
13
8
13
8
7
7
6
6
6
6
6
5
5
5
9
3
4
4
3
3
8
5
5
Sample Start
14:36
17:17
15:50
17:16
16:40
15:08
14:59
12:36
10:21
11:12
11:20
12:13
10:05
10:04
12:20
11:12
9:18
15:15
11:02
10:56
8:59
8:53
14:46
16:32
16:39
15:12
14:43
13:36
14:52
13:06
17:45
12:53
11:34
16:53
13:57
15:58
11:15
13:53
13:01
9:34
10:14
11:43
10:18
9:44
9:00
13:00
12:05
13:45
12:35
•O
C
UJ
HI
a.
E
Ł
16:47
18:28
17:16
18:35
17:51
16:36
16:51
14:05
12:35
12:48
12:43
13:22
11:17
12:07
14:35
15:32
12:27
17:35
11:54
12:00
11:02
10:56
16:26
17:09
17:30
16:32
16:34
15:12
16:10
14:52
18:30
13:55
12:53
17:44
15:15
17:20
13:53
15:58
14:46
13:27
11:04
14:57
11:33
10:14
12:00
16:00
13:39
14:35
13:45
Time to Sample Room (min)
131
71
86
79
71
88
112
89
134
96
83
69
72
123
135
260
189
140
52
64
123
123
100
37
51
80
111
96
78
106
45
62
79
51
78
82
158
125
105
233
50
194
75
30
180
180
94
50
70
W
1
Sharna entered at 1520 and stayed with team 5
Total samples include both entries
Total samples include both entries
air samples 16:38 to 16:41?
105 Continued on 4/23/11
Sharna wt 7
EPA team, Char, Erin, Lukas
Stopped sampling for bio break; total samples for bot
Resumed sampling after bio break
105 Continued from 4/22/11
AAS 15 samples per entry; 3 hours (time is forced)
AAS 15 samples per entry; 3 hours (time is forced)
Room Sample Time
Page H-27
6/13/2013
-------
MASTER BOTE Cost Analysis O61313.xlsx
0)
+J
&
4/26/11
4/26/11
4/26/11
4/26/11
4/26/11
4/26/11
4/26/11
4/26/11
4/26/11
4/26/11
4/26/11
4/26/11
4/26/11
4/26/11
4/26/11
4/26/11
4/26/11
5/5/11
5/5/11
5/5/11
5/5/11
5/5/11
5/5/11
5/5/11
5/5/11
5/5/11
5/5/11
5/5/11
5/5/11
5/5/11
5/5/11
5/5/11
5/5/11
5/5/11
5/5/11
5/5/11
5/5/11
5/5/11
5/6/11
5/6/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
o
o
ge
105
105
106
107
108
109
110
206
207
208
209
210
211
212
213
101A
Floor 1 Other
101A
102
103
104
105
106
107
108
109
110
206
207
208
209
210
211
212
213
Floor 1 Other
Floor 2 Other
Floor 1 Other
105
106
101A
102
103
104
105
106
107
108
109
E
IS
Ł•
6
6
6
4
4
3
3
9
6
9
6
7
7
5
5
8
4
7
7
5
5
5
1
5
6
6
6
4
4
4
4
3
3
3
3
2
1
2
7
7
4
1
2
8
8
5
5
Sample Start
12:35
14:20
13:29
13:40
12:15
12:12
13:22
11:00
10:10
10:00
9:08
10:30
9:28
9:43
8:45
9:35
8:20
10:44
12:15
12:25
13:07
11:23
14:06
10:25
11:59
12:57
10:51
11:49
12:16
10:02
11:06
11:15
12:13
10:29
9:24
9:02
8:51
14:19
9:00
13:00
11:54
16:04
15:23
15:30
15:25
15:19
13:40
16:27
9:52
•O
C
UJ
HI
a.
E
Ł
13:28
14:25
14:20
14:51
13:38
13:22
14:10
11:42
10:55
10:58
10:10
11:32
10:30
10:43
9:42
11:30
11:00
12:15
13:35
13:06
13:58
12:24
15:52
11:23
12:56
13:33
11:58
12:15
12:45
11:05
11:48
12:13
12:46
11:15
10:28
12:54
10:19
15:31
12:00
16:00
14:30
17:53
16:10
16:03
16:25
16:40
15:18
17:32
12:05
Time to Sample Room (min)
53
5
51
71
83
70
48
42
45
58
62
62
62
60
57
115
160
91
80
41
51
61
106
58
57
36
67
26
29
63
42
58
33
46
64
232
88
72
180
180
156
109
47
33
60
81
98
65
133
W
1
T. 6 had to return to rm.105; combined number of sai
AAS 15 samples per entry; 3 hours (time is forced)
AAS 15 samples per entry; 3 hours (time is forced)
Room Sample Time
Page H-28
6/13/2013
-------
MASTER BOTE Cost Analysis O61313.xlsx
0)
+J
&
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/16/11
5/16/11
5/16/11
5/16/11
5/16/11
5/16/11
5/16/11
5/16/11
5/16/11
5/16/11
5/16/11
5/16/11
5/16/11
5/16/11
5/16/11
5/16/11
5/16/11
5/16/11
5/16/11
5/16/11
5/16/11
5/16/11
5/16/11
5/17/11
5/17/11
o
o
ge
110
Floor 1 Other
206
207
208
209
210
211
212
213
Floor 2 Other
101A
102
103
104
105
106
107
108
109
110
Floor 1 Other
Floor 1 Other
206
207
208
209
210
211
212
213
Floor 2 Other
Floor 2 Other
Floor 2 Other
105
106
E
IS
Ł•
5
2
6
6
4
4
4
4
3
3
1
7
7
4
3
8
8
8
5
5
5
2
2
6
6
4
4
4
3
3
3
1
6
1
Sample Start
14:40
8:42
10:53
11:45
12:26
12:59
9:48
11:36
9:38
10:41
8:46
10:30
12:11
14:41
13:46
15:14
11:53
10:33
14:32
9:52
11:27
8:44
14:02
9:50
10:47
10:57
11:57
9:37
11:50
9:30
10:51
8:43
14:00
11:46
9:00
13:00
•O
C
UJ
HI
a.
E
Ł
16:26
12:35
11:44
13:27
12:57
13:36
11:34
12:26
10:40
12:27
12:39
12:00
13:36
15:35
14:29
16:27
13:16
11:47
15:52
11:19
12:35
12:07
15:00
10:38
11:38
11:47
12:32
10:50
12:24
10:32
11:41
12:09
14:34
12:41
12:00
16:00
Time to Sample Room (min)
106
233
51
102
31
37
106
50
62
106
233
90
85
54
43
73
83
74
80
87
68
203
58
48
51
50
35
73
34
62
50
206
34
55
180
180
W
1
from BROOM sheet
from BROOM sheet
from BROOM sheet
from BROOM sheet
from BROOM sheet
from BROOM sheet
from BROOM sheet
from BROOM sheet
from BROOM sheet
from BROOM sheet
from BROOM sheet
from BROOM sheet
from BROOM sheet
from BROOM sheet
from BROOM sheet
from BROOM sheet
from BROOM sheet
from BROOM sheet
from BROOM sheet
from BROOM sheet
from BROOM sheet
from BROOM sheet; all floor 2 other combined
from BROOM sheet; all floor 2 other combined
AAS 15 samples per entry; 3 hours (time is forced)
AAS 15 samples per entry; 3 hours (time is forced)
Room Sample Time
Page H-29
6/13/2013
-------
MASTER BOTE Cost Analysis O61313.xlsx
Labor Category
PL1
PL2
PL3
PL4
TL1
TL2
TL3
EMT
OSC/Commander
Job Classification
Engineer 1
Engineer II
Engineer III
Engineer V
Engineering Aide 1
Engineering Aide II
Engineering Aide III
Paramedic
GS-13Step5
Annual
Salary
Median
including
Bonuses
55561
66560
80311
110450
43046
51549
56966
37594
95559
Hourly Rate
(Assuming
1949 hours
worked per
year)
S 29
S 34
S 41
S 57
S 22
S 26
S 29
S 19
S 49
Loaded
Hourly Rate
(Assuming 3x
Loading
Factor)
S 86
S 102
S 124
S 170
S 66
S 79
S 88
S 58
S 147
Source
http://careermedia.salary.com
http://careermedia.salarv.com
http://careermedia.salarv.com
http://careermedia.salarv.com
http://careermedia.salarv.com
http://careermedia.salarv.com
http://careermedia.salarv.com
http://careermedia.salarv.com
US Govt Salary Table; Locality Pay for Research Triangle Park, NC
Salary Table
Page H-30
6/13/2013
-------
MASTER BOTE Cost Ana lysis O61313.xl
Entry Team Prep Time (hr)
VHP® - Characterization Samplingl
VHP® - Clearance Sampling
AB- Clearance Sampling
CI02 - Characterization Samplings
Overall Entry Team Prep Time (hr)
Entry Team Decon LJne Time (hr)
VHP® - Characterization Samplingl
VHP® - Clearance Sampling
AB - Characterization SamplingZ
AB- Clearance Sampling
CI02 - Characterization Samplings
Average Entry Team Decon Line Time (hr)
Mean
0.96
0.44
0.22
0.71
0.60
Mean
0.81
0.80
0.76
0.76
0.89
O.S1
SD
7.5
4.9
2.6
7.9
0.29
SD
0.08
0.09
0.06
0.07
0.10
0.09
Notes
Used for all types of entry teams
Notes
Used for all types of entry teams
n Sample Box Prep Time (man hours/box)
Sample Times (hr)
Sponge Stick
HEPAVac
Swab
Wipe
Aggressive Air
Decon LJne Ops Time (min)
VHP® - Characterization Samplingl
VHP®- Decon
VHP® - Clearance Sampling
AB - Characterization Sampling2
AB Decon - Removal
Waste Generation (solids in Ib and liquids in gal)
Dissemination
VHP® - Characterization Samplingl
VHP®- Decon
VHP® - Clearance Sampling
AB - Characterization Sampling2
AB Decon - Removal
AB Decon - Spray
AB Decon - Dry
AB- Clearance Sampling
CI02 - Characterization Samplings
CL02- Decon
CI02- Clearance Sampling
AB - Building Reset
Breakdown by Round
VHP®
pH-Adjusted Bleach Process
CI02
Total
Breakdown by Activity
VHP® Sampling
VHP® Decontamination
AB Sampling
AB Decontamination
CI02 Sampling
CI02 Decontamination
Other
Distribution by Activity
VHP®
pH-Adjusted Bleach Process
CI02
Total Solid Waste (Ib)
Total Liquid Waste (gal)
Sampling Waste Generation by Building Size
VHP®
pH-Adjusted Bleach Process
CI02
Decon Waste Generation by Building Size
VHP®
pH-Adjusted Bleach Process
CI02
Decontamination Rate and Contract Cost
VHP®
pH-Adjusted Bleach Process
CI02
Mean
0.07
0.10
0.05
0.06
0.10
Mean
1194.0
1382.0
1305.0
660.0
1410.0
Category IS
Uncontamin
ated - Solid
70.0
17.4
7.2
19.8
20.8
5.6
4.2
0.0
15.8
10.6
0.0
0.0
0.0
114.4
46.4
10.6
171.4
37.2
7.2
36.6
9.8
10.6
0.0
70.0
Solid
Sampling
436
1313
427
15341
1516
Sol id Waste
(Ib/ft2)
0.05
0.16
0.05
Sol id Waste
(Ib/ft2)
0.11
0.06
Pre- Decon
Prep Time
(hr)
12.0
0.0
12.0
SD
0.02
0.04
0.02
0.02
0.00
SD
308.0
308.0
308.0
308.0
308.0
Category 2S
Contaminat
ed - Solid
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
Decon &
Other
914
13977
450
Liquid
Waste
(gal/ft>)
0.04
0.03
0.02
Liquid
Waste
(gal/ft>)
0.04
0.02
Decon and
Removal
Time (hr)
24.0
28.1
24.0
Notes
Category 3S
Decon tarn in
ated - Solid
0.0
0.0
0.0
0.0
0.0
10141.6
2157.4
0.0
703.5
0.0
0.0
315.0
0.0
0.0
13002.5
315.0
13317.5
0.0
0.0
703.5
12299.0
315.0
0.0
0.0
Liquid
Sampling
285
278
175
Category 4S
Decon
Waste -
Solid
0.0
210.0
447.4
188.4
247.0
154.4
177.8
0.0
325.4
101.2
0.0
0.0
0.0
845.8
904.6
101.2
1851.6
398.4
447.4
572.4
332.2
101.2
0.0
0.0
Decon &
Other
362
958
197
Sol id Waste
(Ib/ft3)
0.005
0.016
0.005
Sol id Waste
(Ib/ft3)
0.011
0.006
Post-Decon
Stabilization
Time (hr)
24
72
24
Liquid
Waste
(Ib/ft3)
0.004
0.003
0.002
Liquid
Waste
(gal/ft3)
0.004
0.002
Est.
Mob/Demo
b Fraction
of Fixed
Cost
0.33
0.33
Total Solid
(Ib)
70
227
455
208
268
10302
2339
0
1045
112
0
315
0
960
13954
427
15341
436
455
1313
12641
427
0
70
Solid + Liquid
Sampling
53%
10%
94%
Category 1L
Uncontamin
ated-
Liquid
66.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
66.0
0.0
0.0
66.0
0.0
0.0
0.0
0.0
0.0
0.0
66.0
Decon &
Other
47%
90%
6%
Cost
$ 99,000
$ 165,000
Mob/Demo
bCost
$ 32,670
$ 54,450
Category 2 L
Contaminat
ed - Liquid
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
Lumps solid E
Lumps solid E
Lumps solid E
Cost ($/Ft2
$ 8.24
$ 13.73
Category 3 L
Decon tarn in
ated-
Liquid
0.0
0.0
0.0
0.0
0.0
0.0
105.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
105.0
0.0
105.0
0.0
0.0
0.0
105.0
0.0
0.0
0.0
Category
4L- Decon
Waste -
Liquid
0.0
211.0
55.0
73.5
177.0
26.0
527.9
0.0
100.5
137.0
0.0
37.5
0.0
339.5
831.4
174.5
1345.4
284.5
55.0
277.5
553.9
174.5
0.0
0.0
Total Liquid
(gal)
66
211
55
74
177
26
633
0
101
137
0
38
0
406
936
175
1516
285
55
278
659
175
0
66
Notes
amount removed is doubled
nd liquid together; other costs evenly distributed among 3 rounds
nd liquid together; other costs evenly distributed among 3 rounds
nd liquid together; other costs evenly distributed among 3 rounds
-------
MASTER BOTE Cost Analysis O61313.xlsx
Date
4/16/11
4/17/11
4/18/11
4/19/11
4/20/11
4/21/11
4/22/11
4/23/11
4/24/11
4/25/11
4/26/11
4/27/11
4/28/11
4/29/11
4/30/11
5/1/11
5/2/11
5/3/11
5/4/11
5/5/11
5/6/11
5/7/11
5/8/11
5/9/11
5/10/11
5/11/11
5/12/11
5/13/11
5/14/11
5/15/11
5/16/11
5/17/11
Activity
Dissemination
VHP* - Characterization Samplingl
VHP* - Characterization Samplingl
VHP*- Decon
VHP*- Decon
VHP*- Decon
VHP* - Clearance Sampling
VHP* - Clearance Sampling
DAY OFF
Dissemination
AB - Characterization Sampling2
AB - Characterization Sampling2
AB Decon - Removal
AB Decon - Removal
AB Decon - Spray
AB Decon - Dry
DAY OFF
AB Decon - Dry
AB Decon - Dry
AB - Clearance Sampling
AB - Clearance Sampling
DAY OFF
DAY OFF
AB- Building Reset
Dissemination
CIO2 - Characterization Samplings
CIO2 - Characterization Samplings
CLO2 - Decon
CLO2 - Decon
CLO2 - Decon
CIO2 - Clearance Sampling
CIO2 - Clearance Sampling
Sample
Team
Entries
11
2
11
1
15
9
4
13
14
Decon
Team
Entries
4
6
Decon
Contractor
Entries
2
1
1
1
1
Removal
Team
Entries
9
8
Instrumentation
Team Entries
1
1
1
1
1
2
1
1
1
1
1
Aggressive Air
Sampling Team
Entries
0
3
2
3
Building
Upfit
Team
Entries
Health
and
Safety
Team
Entries
1
1
1
1
2
1
1
SKC
Biosampling
Team Entries
1
1
1
1
1
1
Soil
Sampling
Team
Entries
1
1
1
1
1
2
1
1
1
Average
Entry
Time (hrs)
3.05
2.15
2.10
0.18
1.37
2.42
1.73
0.57
1.83
1.72
2.13
2.17
1.13
2.73
1.28
0.40
2.65
1.62
1.00
2.07
Notes
Activity
Round 1-VHP*
Round 2- AB
Round3-CL02
Total
Total Entries
Total Sampling Team Entries
Total Decon/Removal Team Entries
Sample
Team
Entries
25
28
27
80
157
124
27
Decon
Team
Entries
0
10
0
10
Decon
Contractor
Entries
4
0
2
6
Removal
Team
Entries
0
17
0
17
Instrumentation
Team Entries
2
7
3
12
Aggressive Air
Sampling Team
Entries
3
2
3
8
Building
Upfit
Team
Entries
0
0
0
0
Health
and
Safety
Team
Entries
4
2
2
8
SKC
Biosampling
Team Entries
2
2
2
6
Soil
Sampling
Team
Entries
3
4
3
10
Average
Entry
Time (hrs)
1.72
1.72
1.72
SD Entry Time (hrs)
0.78
0.78
0.78
Max Entry
Time (hrs)
3.0
3.0
3.0
Team Entries
Page H-32
6/13/2013
-------
MASTER BOTE Cost Analysis O61313.xlsx
Labor Rates (S/hr Loaded)
Sampling Team
Decontamination Team (Level C)
Removal Team (Level B)
Removal Team (Level C)
Decontamination Team (Level B)
Decon Line Setup Team
Decon Line Ops Team
Instrumentation Team
Sample Packaging Team
Waste Handling Team
Lab Analyst Team
BROOM Team
PNNL Statistical Analysis
Neptune Statistical Analysis
Data Analysis Team
PNLLVSPTeam
ARCADIS Sample Kit Prep
Aggressive Air Sampling Team
Building UpfitTeam
Health and Safety Team
Documentation/Plan Writing Team
Command Team
OSC
Regulatory Coordination Team
INL Equipment Purchase Team
Room Sample Box Prep Team
EPA Purchasing Team
Waste Sampling Team
Water Sampling Team
OSC/Commander
$147
0.33
0.33
0.33
0.33
0.33
1.00
0.50
1.00
0.25
1.00
1.00
1.00
1.00
1.00
$ 58
1.00
3
$86
4.00
1.00
1.00
1.00
0.50
3.00
3.00
3
$102
0.25
1.00
3.00
3
$124
3.00
2.33
3.33
2.33
3.33
2.00
0.25
3
$170
0.67
0.67
0.67
0.67
1.00
1.00
1.00
1.00
2.00
1.00
2.00
0.25
1.00
*H
$66
3.00
1.00
1.00
g
$79
3.00
3.00
3.00
2
$88
2.00
1.00
1.00
3.00
3.00
0.25
# on Team
3.3
3.3
4.3
3.3
4.3
2.0
5.0
4.5
3.0
4.0
1.5
2.0
1.0
1.0
4.0
1.0
1.0
4.0
10.0
1.0
1.8
1.0
1.0
3.0
1.3
3.0
1.3
3.0
3.0
# of Teams
6
3
3
3
3
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
l/l
-^
Ł
20.0
10.0
13.0
10.0
13.0
2.0
5.0
4.5
3.0
4.0
1.5
2.0
1.0
1.0
4.0
1.0
1.0
4.0
10.0
1.0
1.8
1.0
1.0
3.0
1.3
3.0
1.3
3.0
3.0
1_
1
8
$420
$451
$574
$451
$574
$175
$443
$416
$343
$385
$150
$256
$170
$170
$587
$170
$102
$454
$785
$147
$140
$147
$147
$487
$109
$263
$192
$257
$257
l/l
|
AVERAGE(7,2,8,1,9,7,2,8,8)
2 monitors, 3 teams of 2 each
2 monitors, 3 teams of 2 each, 1 extra SCBA guy
2 monitors, 3 teams of 2 each
2 monitors, 3 teams of 2 each, 1 extra SCBA guy (4 hours spraying - 4
sprayers, 4 leaders, 2 folks outside)
2 guys for 1 day probably
NOT INCLUDED
50 hrs per round for Kara and Frank; Nik and Paul 50% time
240 hours total re Bob K
200 hours total re Brett A
40 hours total re Jacky R for aeroso
100 hours per Brett A; notional
105 hours total per Worth C
10 guys per Steve Reese 8/10/11
Notional
Assume part of a professional and a technician
Assume part of a professional to spec out purchases and purchasing
person to make purchases
Included Yet
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Team Makeup
Page H-33
6/13/2013
-------
MASTER BOTE Cost Analysis O61313.xlsx
Total Time Spent Sampling (min)
Time for Samples (min) [sponge, vac, swab, wipe]
Fraction of Room Sample Time Accounted For
Sample Type
Sponge Stick
HEPAVac
Swab
Wipe
Aggressive Air
11630
9276
0.798
Mean
Time Per
Sample
(hr)
0.065
0.101
0.055
0.057
0.100
used to adjust mean t
Std. Dev.
Per
Sample
(hr)
0.024
0.044
0.020
0.022
0.000
Adjusted
Time Per
Sample
(hr)
0.082
0.127
0.069
0.071
0.125
Time Per Sample
Page H-34
6/13/2013
-------
MASTER BOTE Cost Analysis O61313.xlsx
0)
1
4/16/11
4/16/11
4/16/11
4/16/11
4/16/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/17/11
4/18/11
4/18/11
4/18/11
4/18/11
4/18/11
4/18/11
4/18/11
4/18/11
4/18/11
4/19/11
4/19/11
4/20/11
4/20/11
4/20/11
4/20/11
4/20/11
4/20/11
4/21/11
4/21/11
4/21/11
4/21/11
4/21/11
4/21/11
4/21/11
4/21/11
4/21/11
4/21/11
4/21/11
4/21/11
4/21/11
4/21/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/22/11
4/23/11
4/23/11
4/23/11
4/23/11
1
i-
9:00
10:56
11:30
11:30
18:00
9:49
12:30
13:17
15:00
15:32
15:48
16:29
17:37
18:12
19:00
19:03
19:03
19:03
19:03
09:43
09:43
09:43
12:25
12:52
12:55
13:09
13:09
18:00
18:00
18:00
10:59
10:59
10:59
10:59
12:00
12:00
17:56
18:09
18:09
18:09
18:09
18:09
18:09
18:09
18:09
18:09
18:58
19:22
18:00
18:00
10:15
10:15
14:02
16:02
16:47
17:00
17:05
17:00
17:30
18:06
18:06
18:06
18:06
18:40
18:50
18:57
11:44
12:16
13:01
18:00
Source
Building
Building
Building
Building
Decontamination line
Donning Trailer
Donning Trailer
Decontamination line
Decontamination line
Decontamination line
Donning Trailer
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Donning trailer
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Donning Trailer
Donning Trailer
Donning trailer
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Donning trailer
Decontamination line
Decontamination line
Decontamination line
c
o
S.
1
Booties, Gloves, Parafilm, Petri Dish Lids
Trash from Sampling Kits
Composite of Rinsewater and Filtrate
Gloves, Booties, Box
Gloves, Booties
Glove boxes, gloves, donning trash
Glove boxes, gloves, donning trash
PPE and boxes
PPE and bags
PPE, boxes and bags
Boxes and PPE trash
PPE and bags
PPE and bags and boxes
PPE and bags and boxes
PPE and bags and boxes
PPE and bags and boxes
Boxes
Misc
Boxes
PPE
Boxes
Misc
PPE
PPE
Decontamination rinsewater
Boxes and PPE
Misc
Decontamination rinsewater
VHP® setup PPE waste
VHP® setup PPE waste
Misc VHP® setup waste
VHP® setup waste
VHP® setup waste
PPE, boxes, garbage
PPE
PPE
STERIS fans (5)
STERIS hoses
STERIS aerator
STERIS cables
STERIS cables
STERIS cables
STERIS sensors (3)
STERIS hoses and fan
STERIS hoses
STERIS cables
Heater
Misc
PPE
Decontamination wastewater
PPE packaging and trash
PPE packaging and boxes
PPE stuff
PPE stuff
PPE stuff
Bleach water
PPE stuff
Bleach water
PPE stuff
PPE stuff
PPE stuff
Sample kit boxes
Sample kit boxes
PPE, sample kit boxes
PPE stuff
PPE, sample kit boxes
PPE packaging
PPE
PPE
Decon Line Tent
Weight (Ib)
6.4
3.2
7.2
1.2
4.4
4.2
13.0
20.8
12.2
8. 8
7.6
10.8
14.2
16.6
11.6
6.4
5.6
11.0
26.4
6.0
5.4
10.8
13.6
10.4
7.6
6.2
8
7
5
9
7.2
30
1.6
29.6
18.4
100.6
21
22
30
41
26
12
48
10
4
IS
3.6
8
3.4
43
10.2
11
13.2
9
8. 8
4.6
7.6
14.6
23.8
10.6
4.8
10.6
21.4
s
$
38
110
101
55
IS
IS
38
s
Pre-dissemination
Pre-dissemination
Pre-dissemination
Pre-dissemination
Pre-dissemination PPE from Decon Tent; weighed next AM
Leftovers from 4-18; weighed next day at 9 AM
Leftovers from 4-19; weighed next day at 10:14 am
Leftovers from 4-19; weighed next day at 10:14 am
79 fans altogether
5 aerators altogether
6 altogether
Need to subtract fan weight
9 Altogether
Leftovers from 4/21; weighed at 9:25 AM
Leftovers from 4/21; Soapy water from day before; dumped at 11:22 AM
Notionally Added (575 Ib, 37.5 gal) - reuse tents
Waste
Page H-35
6/13/2013
-------
MASTER BOTE Cost Analysis O61313.xlsx
0)
1
4/25/11
4/26/11
4/26/11
4/26/11
4/26/11
4/26/11
4/26/11
4/26/11
4/26/11
4/26/11
4/26/11
4/26/11
4/27/11
4/27/11
4/28/11
4/28/11
4/28/11
4/28/11
4/28/11
4/28/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
1
i-
10:00
800
9:45
9:50
10:10
12:35
12:45
15:05
15:24
0:00
17:40
17:40
10:05
12:20
9:00
10:00
12:50
4:35
18:00
18:30
9:20
10:45
17:30
16:30
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
Source
Decontamination line
Decontamination line
Donning trailer
Decontamination line
Donning trailer
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Donning trailer
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Donning Trailer
Decontamination line
Decontamination line
Decontamination line
Room 109
Other
Room 104
Other
Room 105
Other
Other
Other
Other
Room 107
Other
Room 107
Room 106
Room 106
Room 109
Other
Room 105
Room 106
Room 105
Room 105
Other
Other
Other
Other
Room 108
Other
Room 103
Other
Other
Other
Room 103
Room 108
Room 103
Room 108
Other
Other
Other
Other
Other
Other
Other
Other
Other
Room 110
Room 104
Other
c
o
s.
1
Bleach water
Soapy water
PPE
PPE
PPE
PPE, sample boxes
Bleach water
Bleach water
PPE, sample boxes
PPE
PPE
PPE
Bleach water
Sample kit box waste
Bleach water
PPE waste
PPE waste
PPE waste
Bleach water
PPE
PPE packaging
PPE waste
PPE waste
PPE waste
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
Weight (Ib)
11
44
3.6
91
50
22.0
11.0
29.0
6.2
6.4
18.4
20.2
20.0
5.6
11.0
51.0
27.4
180.0
48.8
47.6
50.8
56.0
1S.S
90.8
48.8
42.4
71.2
38.8
80.4
24.8
78.4
93.6
64.0
50.0
62.4
40.4
50.8
56.8
68.8
34.4
35.2
80.0
19.2
42.8
91.2
70.8
53.2
49.2
62.4
49.6
51.2
48.8
60.8
68.8
34.4
47.2
49.2
52.0
70.4
43.2
67.6
38.8
79.6
s
$
28
28
28
28
93
5
21
s
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
Waste
Page H-36
6/13/2013
-------
MASTER BOTE Cost Analysis O61313.xlsx
0)
1
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/29/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
1
i-
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
8:30
10:40
11:37
14:55
17:00
17:45
10:45
11:30
13:37
15:50
17:10
17:12
18:00
18:00
18:00
18:00
18:00
18:00
13:43
12:05
12:05
12:05
12:05
12:05
18:00
18:00
18:00
18:00
18:00
18:00
Source
Room 109
Room 110
Room 104
Room 105
Other
Other
Other
Room 109
Other
Room 104
Other
Other
Other
Other
Other
Room 110
Other
Room 110
Room 110
Room 108
Room 110
Room 108
Other
Room 108
Room 110
Room 108
Room 110
Room 110
Room 108
Room 106
Room 106
Rooms lOlAand 102
Rooms lOlAand 102
Rooms lOlAand 102
Rooms lOlAand 102
Rooms lOlAand 102
Room 110
Room 108
Room 106
Building
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Building
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Donning trailer
Building
Decontamination line
Donning trailer
Decontamination line
Other
Other
Other
Room 105
Room 109
Room 108
c
o
s.
1
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
Laminate Floor that Should have Been Removed
Laminate Floor that Should have Been Removed
Laminate Floor that Should have Been Removed
HVAC Duct
Bleach water
ppe waste
Bleach water
Bleach water
Bleach water
ppe waste
ppe waste
ppe waste
ppe waste
ppe waste
bldg decon related waste
ppe waste
ppe waste
Residual Bleach water from Spray
Bleach water
PPE waste
boxes
Bleach water
PPE
PPE
3 Fans
PPE
Boxes
Misc
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
Weight (Ib)
94.0
45.6
38.4
49.2
75.6
79.6
45.2
aa.o
77.6
18.4
61.2
36.8
11.6
65.6
18.4
61.6
34.4
86.8
84.8
aa.o
65.2
69.2
45.6
48.4
79.2
60.8
61.2
120.0
126.0
60.0
119.2
53.2
82.4
61.2
49.2
35.2
700.1
700.1
700.1
3080.0
15.6
14.2
12.2
10.0
14.0
12.6
37.2
4.6
11.2
13.2
17.2
8.4
2.0
18.6
2.4
2.2
5.0
13.2
55.2
5.2
29.6
23.2
97.6
s
$
55
37
28
110
105
248
37
s
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
Notionally Added
Notionally Added
Notionally Added
Notional Assumed 24 gauge 7.7 Ib/ft
Collected 5/1; Leftovers from 4/30;
Collected 5/1; Leftovers from 4/30;
Collected 5/1; Leftovers from 4/30;
Collected 5/1; Leftovers from 4/30;
Collected 5/1; Leftovers from 4/30;
Collected 5/1; Leftovers from 4/30;
Collected 5/1; Leftovers from 4/30;
Collected 5/1; Leftovers from 4/30; Pumped from Building
Collected 5/1; Leftovers from 4/30;
Collected 5/1; Leftovers from 4/30; weighed 5/3 at 11 AM
Collected 5/1; Leftovers from 4/30; weighed 5/3 at 11 AM
Collected 5/1; Leftovers from 4/30; weighed 5/3 at 11 AM
Collected 5/3; Leftovers from 4/30; Entries resetting furniture locations
Collected 5/3; Leftovers from 4/30;
Collected 5/4; Leftovers from 4/30; 16 Fans Total from 1st and 2nd floors
Collected 5/4; Leftovers from 4/30;
Collected 5/4; Leftovers from 4/30;
Collected 5/4; Leftovers from 4/30;
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
Waste
Page H-37
6/13/2013
-------
MASTER BOTE Cost Analysis O61313.xlsx
0)
1
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
4/30/11
5/5/11
5/5/11
5/5/11
5/5/11
5/5/11
5/5/11
5/5/11
5/5/11
5/5/11
5/5/11
5/5/11
5/5/11
5/5/11
5/5/11
5/5/11
5/5/11
5/5/11
5/6/11
5/6/11
5/6/11
5/6/11
5/6/11
5/6/11
5/6/11
5/6/11
5/6/11
5/6/11
5/6/11
5/6/11
5/6/11
5/6/11
1
i-
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
8:00
11:26
12:34
14:40
14:40
14:40
14:40
14:40
14:40
14:40
14:40
15:52
18:00
18:00
18:00
18:00
18:00
18:00
12:30
13:30
14:06
16:00
16:00
8:00
8:03
8:05
8:10
8:14
8:17
8:22
8:27
8:35
Source
Room 106
Other
Room 107
Room 106
Room 107
Room 105
Room 106
Room 106
Room 105
Room 106
Room 106
Room 109
Room 107
Room 105
Room 107
Other
Other
Room 105
Other
Room 106
Room 105
Room 106
Other
Room 107
Other
Other
Other
Room 107
Room 107
Other
Other
Room 107
Room 107
Room 107
Room 104
Room 107
Other
Other
Decontamination line
Donning trailer
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Other
Building
Decontamination line
Decontamination line
Building
Building
Building
Building
Building
Building
Building
Decontamination line
Building
c
o
s.
1
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
porous stuff (ceiling tile, carpet, furniture, etc.)
Wastewater
PPE waste
PPE waste
PPE and boxes
PPE and boxes
PPE and boxes
PPE and boxes
PPE and boxes
PPE and boxes
PPE and boxes
PPE and boxes
PPE and boxes
PPE and misc
PPE and misc
PPE and misc
PPE and misc
PPE and misc
Wastewater
PPE and misc
PPE
Aggressive Air Sampling Fans (1)
PPE and bleach bottles
PPE
8 Chairs w/o Backs
porous material (ceiling tile & boxes)
porous material (ceilingtile)
porous material (ceilingtile)
porous material (ceilingtile)
porous material (ceilingtile boxes)
porous material (ceilingtile)
Empty Chlorox bottles & broken ceiling tiles
porous material (ceilingtile)
Weight (Ib)
28.0
51.2
55.2
64.8
48.4
36.4
83.2
57.6
35.2
43.2
83.6
48.8
48.4
40.4
45.2
109.6
40.0
59.6
79.6
9.2
62.0
15.6
103.2
96.0
27.6
22.8
8.4
39.6
61.2
54.8
46.0
74.4
42.4
49.2
10.0
43.6
48.8
41.6
5.2
6.6
7.6
4.2
12.4
25.8
17.4
5.8
6.8
12.0
11.8
16.0
5.6
6.6
10.0
3.2
11.0
13.8
7.8
9.4
16.4
8.0
14.0
20.0
20.8
23.2
16.8
18.4
22.0
17-8
s
$
14
63
s
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
first floor inside bldg - Qty is 2x
changed date to 4/30 - generated that day
5/6 from day before
5/6 from day before
5/6 from day before
5/6 from day before
5/6 from day before
5/6 from day before
Building Envelope Up till 5/6
4 fans total
8 Chairs from 5/6 (recorded on 5/9)
From 5/6 (recorded on 5/9)
From 5/6 (recorded on 5/9)
From 5/6 (recorded on 5/9)
From 5/6 (recorded on 5/9)
From 5/6 (recorded on 5/9)
From 5/6 (recorded on 5/9)
From 5/6 (recorded on 5/9)
From 5/6 (recorded on 5/9)
Waste
Page H-3S
6/13/2013
-------
MASTER BOTE Cost Analysis O61313.xlsx
0)
1
5/6/11
5/6/11
5/6/11
5/6/11
5/6/11
5/6/11
5/6/11
5/6/11
5/6/11
5/6/11
5/6/11
5/6/11
5/6/11
5/6/11
5/6/11
5/6/11
5/6/11
5/6/11
5/6/11
5/6/11
5/6/11
5/6/11
5/6/11
5/6/11
5/6/11
5/6/11
5/6/11
5/6/11
5/6/11
5/6/11
5/6/11
5/6/11
5/6/11
5/6/11
5/6/11
5/10/11
5/10/11
5/10/11
5/10/11
5/10/11
5/10/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/11/11
5/17/11
5/17/11
5/17/11
5/17/11
5/17/11
5/17/11
5/17/11
5/17/11
1
i-
8:43
8:50
8:53
8:56
9:00
9:04
9:08
9:09
9:12
9:15
9:18
9:21
9:23
9:25
9:28
9:30
9:33
9:35
9:38
9:41
9:45
9:48
9:50
9:54
9:58
10:00
10:04
10:08
10:12
10:15
10:18
10:22
10:25
10:30
18:00
14:30
10:45
10:45
10:45
10:45
10:45
10:59
11:10
11:11
11:12
11:23
13:04
14:04
14:05
14:24
14:25
15:04
15:06
15:20
16:50
16:51
17:10
17:13
17:14
17:15
18:22
18:23
18:00
18:00
18:00
18:00
18:00
18:00
18:00
18:00
Source
Building
Building
Decontamination line
Building
Decontamination line
Decontamination line
Decontamination line
Building
Decontamination line
Building
Building
Decontamination line
Decontamination line
Building
Building
Building
Building
Building
Building
Building
Building
Decontamination line
Building
Building
Building
Building
Building
Building
Building
Building
Building
Building
Building
Donning trailer
Decontamination line
Building
Decontamination line
Decontamination line
Building
Building
Building
Decontamination line
Donningtrailer
Donningtrailer
Decontamination line
Decontamination line
Decontamination line
Donningtrailer
Donningtrailer
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Decontamination line
Building
Building
Building
Building
Building
Building
Building
c
o
S.
1
porous material (ceilingtile)
porous material (ceilingtile)
Empty Chlorox bottles & boxes that they came in
Wooden Pallet
Empty vinegar bottles & boxes that they came in
Flexible Ducting &PPE
Empty Chlorox bottles & broken ceiling tiles
Pieces of Wooden Pallet
Empty Chlorox&vinegar bottles w/ boxes they came in
Ceiling tiles & metal pipe
porous material (ceilingtile)
Empty vinegar bottles & boxes that they came in
Empty Chlorox&vinegar bottles w/ boxes they came in
porous material (ceilingtile)
Vent, Screens, Ceiling Tiles, Wood Shelves
porous material (ceiling tiles & cardboard)
plastic & wire flexible ductwork
plastic & wire flexible ductwork
porous material (ceilingtile)
porous material (ceiling tiles & cardboard)
plastic & wire flexible ductwork
ductwork, Empty Chlorox bottles & boxes
porous material (ceilingtile)
Lighting fixture
porous material (ceilingtile & wood)
porous material (ceilingtile)
porous material (ceilingtile)
porous material (ceiling tiles & cardboard)
plastic & wire flexible ductwork
plastic & wire flexible ductwork
plastic & wire flexible ductwork
plastic & wire flexible ductwork
plastic & wire flexible ductwork
PPE waste
Decon Line Tent
Cardboard
PPE & Card board
PPE & Card board
Misc
Misc
Ceilingtile
Status of Liquid Collection
PPE
PPE
PPE
Status of Liquid Collection
PPE
PPE
Cardboard
Status of Liquid Collection
Status of Liquid Collection
PPE
PPE
PPE, Cardboard
PPE, Cardboard
PPE, Cardboard
PPE, Cardboard
PPE, Cardboard
PPE, Cardboard
PPE, Cardboard
PPE, Cardboard
PPE, Cardboard
PPE, Sampling Trash
Porous Materials Removed After CIO2 Fumigation
Porous Materials Removed After CIO2 Fumigation
Porous Materials Removed After CIO2 Fumigation
Porous Materials Removed After CIO2 Fumigation
Porous Materials Removed After CIO2 Fumigation
Porous Materials Removed After CIO2 Fumigation
Porous Materials Removed After CIO2 Fumigation
Weight (Ib)
40.6
10.0
15.4
35.4
19.6
12.0
9.2
3.0
10.4
25.0
24.4
14.4
14.6
36.1
58.4
18.4
21.4
21.2
23.4
16.4
16.6
19.2
23.8
21.0
29.0
36.0
18.6
8. 8
8.0
16.0
7.0
7.6
16.8
10.6
9.2
8.2
5.8
8.2
2.8
17.8
4.0
1.6
12.0
14.0
1.6
3.4
15.6
12.6
15.0
13.6
9.8
8.6
13.4
18.4
10.6
6.6
10.6
82.2
19.6
19.8
20.0
19.6
19.4
19.2
19.0
s
$
38
40
55
28
14
s
From 5/6 (recorded on 5/9)
From 5/6 (recorded on 5/9)
From AB Decon (recorded on 5/9)
From 5/6 (recorded on 5/9)
From AB Decon (recorded on 5/9)
From AB Decon (recorded on 5/9)
From AB Decon (recorded on 5/9)
From 5/6 (recorded on 5/9)
From AB Decon (recorded on 5/9)
From 5/6 (recorded on 5/9)
From 5/6 (recorded on 5/9)
From AB Decon (recorded on 5/9)
From AB Decon (recorded on 5/9)
From 5/6 (recorded on 5/9)
From 5/6 (recorded on 5/9)
From 5/6 (recorded on 5/9)
From 5/6 (recorded on 5/9)
From 5/6 (recorded on 5/9)
From 5/6 (recorded on 5/9)
From 5/6 (recorded on 5/9)
From 5/6 (recorded on 5/9)
From AB Decon (recorded on 5/9)
From 5/6 (recorded on 5/9)
From 5/6 (recorded on 5/9)
From 5/6 (recorded on 5/9)
From 5/6 (recorded on 5/9)
From 5/6 (recorded on 5/9)
From 5/6 (recorded on 5/9)
From 5/6 (recorded on 5/9)
From 5/6 (recorded on 5/9)
From 5/6 (recorded on 5/9)
From 5/6 (recorded on 5/9)
From 5/6 (recorded on 5/9)
From 5/6 (recorded on 5/9)
Notionally Added (575 Ib, 37.5 gal) - reuse tents
From 5/9 (recorded on 5/9)
From 5/10
From 5/10
From 5/10
From 5/10
From 5/10
BlueBarrelfll
Blue Barrel #1 now full
Blue Barrel #2 Half Full
Couch Cushion (didn't include)
Couch Cushion (didn't include)
Couch Cushion (didn't include)
Couch Cushion (didn't include)
Couch Cushion (didn't include)
Couch Cushion (didn't include)
Chair Cushions (didn't include)
Waste
Page H-39
6/13/2013
-------
MASTER BOTE Cost Analysis O61313.xlsx
0)
8
5/17/11
5/17/11
5/17/11
5/17/11
5/17/11
5/17/11
5/17/11
E
h
18:00
18:00
18:00
18:00
18:00
18:00
18:00
8
8
Building
Building
Building
Building
Building
Building
Decontamination line
o
X.
1
Porous Materials Removed After CIO2 Fumigation
Porous Materials Removed After CIO2 Fumigation
Porous Materials Removed After CIO2 Fumigation
Porous Materials Removed After CIO2 Fumigation
Porous Materials Removed After CIO2 Fumigation
Porous Materials Removed After CIO2 Fumigation
Decon Line Tent
_Q
Ł
.»
3
27.6
33.6
46.0
21.8
91.0
95.0
&
|
38
Ł
2
Chair (didn't include)
Chair (didn't include)
Chair (didn't include)
Chair Cushions (didn't include)
Queen Mattress (didn't include)
Queen Mattress (didn't include)
Notionally Added (575 Ib, 37.5 gal) - reuse tents
Waste
Page H-40
6/13/2013
-------
MASTER BOTE Cost Analysis O61313.xlsx
Waste Fixed Cost Elements
Waste Management Plan
Transportation Plan
Tracking and Reporting Plan
Health and Safety Plan and Oversight Costs
Contract Oversight Costs
Communications and Community Outreach
Plan and Costs
Coordination with Regulatory Agencies and
Facilities
Total Fixed Costs
Waste Variable Cost Elements
Standard MSW Disposal Fee (S/lb)
Standard POTW Disposal Fee (S/gal)
Standard Transportation Fee (S/mile)
Miles to Local Landfill
Miles to Secure Landfill
Miles to POTW
Multiplier for Premium Disposal
Multiplier for Contaminated Disposal
Truck Capacity (Ib)
Truck Capacity (gal)
Solid Waste Collection, Handling and
Segregation, Packaging, Labeling,
Containerization (S/lb)
Interim Storage
Hours
40
40
40
40
40
40
40
S 0.05
S 0.005
S 5
10
200
10
10
100
40000
5000
S 3.85
Loaded
Rate (S/hr)
S 140
S 140
S 140
S 140
S 147
S 140
S 487
Labor Cost
S 5,597
S 5,597
S 5,597
S 5,597
S 5,884
S 5,597
S 19,484
S 53,353
assumed SlOO/ton
assumed $50/10000 gal
scaleable?
scaleable?
scaleable?
scaleable?
Waste
Knob (1,
2,3) 2
1 = If Deconned Waste is treated as MSW
2 = If Deconned Waste has Premium Charge
3 = If Deconned Waste is treated as Contaminated
assumed rate of 100 Ib/hr or 100 gal/hr
not used
Disposal Cost for Different Waste
Categories
If Deconned Waste = Contaminated
If Deconned Waste = Premium Charge
If Deconned Waste = MSW
Transportation Costs for Different
Categories (S) [assuming 1 truckload to
landfill or POTW]
if Deconned Waste = MSW
if Deconned Waste = Premium Charge
if Deconned Waste = Contaminated
Category
1s-
Uncontami
nated -
Solid (S/lb)
S 0.05
S 0.05
S 0.05
Category
1s-
Uncontami
nated -
Solid
(S/truckloa
d)
S 50
S 50
S 50
Category
2s-
Contamina
ted - Solid
(S/lb)
S 5.00
S 5.00
S 5.00
Category
2s-
Contamina
ted - Solid
(S/truckloa
d)
S 100,000
S 100,000
S 100,000
Category 3s
-
Decontamin
ated - Solid
(S/lb)
S 5.00
S 0.50
S 0.05
Category 3s
-
Decontamin
ated - Solid
(S/truckloa
d)
S 1,000
S 10,000
S 100,000
Category
4s - Decon
Waste -
Solid (S/lb)
S 5.00
S 0.50
S 0.05
Category
4s - Decon
Waste -
Solid
(S/truckloa
d)
S 1,000
S 10,000
S 100,000
Category
1L-
Uncontam
inated -
Liquid
(S/gal)
S 0.005
S 0.005
S 0.005
Category
1L-
Uncontam
inated -
Liquid
(S/trucklo
ad)
S 50
S 50
S 50
Category
2L-
Contamina
ted - Liquid
(S/gal)
S 0.50
S 0.50
S 0.50
Category
2L-
Contamina
ted - Liquid
(S/truckloa
d)
S 5,000
S 5,000
S 5,000
Category
3L-
Decontamin
ated-
Liquid
(S/gal)
S 0.50
S 0.05
S o.oi
Category
3L-
Decontamin
ated-
Liquid
(S/truckloa
d)
S 50
S 500
S 5,000
Category
4L - Decon
Waste -
Liquid
(S/gal)
S 0.50
S 0.05
S o.oi
Category
4L - Decon
Waste -
Liquid
(S/truckloa
d)
S 50
S 500
S 5,000
Waste Cost
Page H-41
6/13/2013
-------
MASTER BOTE Cost Analysis O61313.xlsx
Quantities of Different Waste Categories
VHP*
pH-Adjusted Bleach Process
CI02
Category
1S-
Uncontami
nated -
Solid
114.4
46.4
10.6
Category
2S-
Contamina
ted - Solid
0.0
0.0
0.0
Category 3S
-
Decontamin
ated - Solid
0.0
13002.5
315.0
Category
4S - Decon
Waste -
Solid
845.8
904.6
101.2
Category
1L-
Uncontam
inated -
Liquid
66.0
0.0
0.0
Category
2L-
Contamina
ted - Liquid
0.0
0.0
0.0
Category 3L
-
Decontamin
ated-
Liquid
0.0
105.0
0.0
Category 4L
- Decon
Waste -
Liquid
339.5
831.4
174.5
Total Solid
(Ib)
960.2
13953.5
426.8
Total
Liquid (gal)
405.5
936.4
174.5
Calculated Disposal Costs (Tipping Fees)
Estimated Disposal Costs if Deconned
Waste = Contaminated
VHP®
pH-Adjusted Bleach Process
CI02
Estimated Disposal Costs if Deconned
Waste = Premium Charge
VHP*
pH-Adjusted Bleach Process
CI02
Estimated Disposal Costs if Deconned
Waste = MSW
VHP*
pH-Adjusted Bleach Process
CI02
Category
1s-
Uncontami
nated -
Solid
S 5.7
S 2.3
S 0.5
Category
1s-
Uncontami
nated -
Solid
S 5.72
S 2.32
S 0.53
Category
1s-
Uncontami
nated -
Solid
S 5.72
S 2.32
S 0.53
Category
2s-
Contamina
ted - Solid
S
S
S
Category
2s-
Contamina
ted - Solid
S
S
S
Category
2s-
Contamina
ted - Solid
S
S
S
Category 3s
Decontamin
ated - Solid
S
S 65,012.6
S 1,575.0
Category 3s
Decontamin
ated - Solid
S
S 6,501.26
S 157.50
Category 3s
Decontamin
ated - Solid
S
S 650.13
S 15.75
Category
4s - Decon
Waste -
Solid
S 4,229.0
S 4,523.0
S 506.0
Category
4s - Decon
Waste -
Solid
S 422.90
S 452.30
S 50.60
Category
4s - Decon
Waste -
Solid
S 42.29
S 45.23
S 5.06
Category
1L-
Uncontam
inated -
Liquid
S 0.3
S -
S -
Category
1L-
Uncontam
inated -
Liquid
S 0.33
S -
S -
Category
1L-
Uncontam
inated -
Liquid
S 0.33
S -
S -
Category
2L-
Contamina
ted - Liquid
S
S
S
Category
2L-
Contamina
ted - Liquid
S
S
S
Category
2L-
Contamina
ted - Liquid
S
S
S
Category
3L-
Decontamin
ated-
Liquid
S
S 52.5
S
Category
3L-
Decontamin
ated-
Liquid
S
S 5.25
S
Category
3L-
Decontamin
ated-
Liquid
S
S 0.53
S
Category
4L - Decon
Waste -
Liquid
S 169.8
S 415.7
S 87.3
Category
4L - Decon
Waste -
Liquid
S 16.98
S 41.57
S 8.73
Category
4L - Decon
Waste -
Liquid
S 1.70
S 4.16
S 0.87
Total
S 4,405
S 70,006
S 2,169
Total
S 446
S 7,003
S 217
Total
S 50
S 702
S 22
Transportation Costs
Estimated Transportation Costs if
Deconned Waste = MSW
VHP*
pH-Adjusted Bleach Process
CI02
Category
1s-
Uncontami
nated -
Solid
50
50
50
Category
2s-
Contamina
ted - Solid
S
S
S
Category 3s
-
Decontamin
ated - Solid
1000
1000
1000
Category
4s - Decon
Waste -
Solid
incl.
incl.
incl.
Category
1L-
Uncontam
inated -
Liquid
S 50
S -
S -
Category
2L-
Contamina
ted - Liquid
S
S
S
Category
3L-
Decontamin
ated-
Liquid
50
50
50
Category
4L - Decon
Waste -
Liquid
incl.
incl.
incl.
Total
S 1,150
S 1,100
S 1,100
Waste Cost
Page H-42
6/13/2013
-------
MASTER BOTE Cost Analysis O61313.xlsx
Estimated Transportation Costs if
Deconned Waste = Premium Charge
VHP®
pH-Adjusted Bleach Process
CI02
Estimated Transportation Costs if
Deconned Waste = Contaminated
VHP"
pH-Adjusted Bleach Process
CI02
Category
1s-
Uncontami
nated -
Solid
50
50
50
Category
1s-
Uncontami
nated -
Solid
50
50
50
Category
2s-
Contamina
ted - Solid
s
s
s
Category
2s-
Contamina
ted - Solid
S
s
s
Category 3s
-
Decontamin
ated - Solid
S 10,000
S 10,000
S 10,000
Category 3s
-
Decontamin
ated - Solid
S 100,000
S 100,000
S 100,000
Category
4s - Decon
Waste -
Solid
incl.
incl.
incl.
Category
4s - Decon
Waste -
Solid
incl.
incl.
incl.
Category
1L-
Uncontam
inated -
Liquid
S 50
S -
S -
Category
1L-
Uncontam
inated -
Liquid
S 50
S -
S -
Category
2L-
Contamina
ted - Liquid
S
s
s
Category
2L-
Contamina
ted - Liquid
S
s
s
Category
3L-
Decontamin
ated-
Liquid
S 500
S 500
S 500
Category
3L-
Decontamin
ated-
Liquid
S 5,000
S 5,000
S 5,000
Category
4L - Decon
Waste -
Liquid
incl.
incl.
incl.
Category
4L - Decon
Waste -
Liquid
incl.
incl.
incl.
Total
S 10,600
S 10,550
S 10,550
Total
S 105,100
S 105,050
S 105,050
Handling, Segregation, Packaging,
Labelling, and Other Costs
VHP"
pH-Adjusted Bleach Process
CIO2
Category
1s-
Uncontami
nated -
Solid
S 441
S 179
S 41
Category
2s-
Contamina
ted - Solid
S
s
s
Category 3s
-
Decontamin
ated - Solid
S
S 50,077
S 1,213
Category
4s - Decon
Waste -
Solid
S 3,257
S 3,484
S 390
Category
1L-
Uncontam
inated -
Liquid
S 254
S -
S -
Category
2L-
Contamina
ted - Liquid
S
s
s
Category
3L-
Decontamin
ated-
Liquid
S
S 404
S
Category
4L - Decon
Waste -
Liquid
S 1,308
S 3,202
S 672
Total
S 5,260
S 61,122
S 2,316
includes other costs frori
includes other costs frori
includes other costs frori
Total Waste Management Costs
VHP"
pH-Adjusted Bleach Process
CI02
Fixed Costs
S 53,353
S 53,353
S 53,353
Handling
Costs
S 5,260
S 61,122
S 2,316
Waste
Sampling
and
Analytical
Costs
S 10,192
S 124,218
S 5,096
Decontaminated Waste = MSW
Disposal
Costs
S 50
S 702
S 22
Transport
ation
Costs
S 1,150
S 1,100
S 1,100
Total
S 70,004
S 240,495
S 61,887
Decontaminated Waste = Premium
Disposal
Costs
S 446
S 7,003
S 217
Transportat
ion Costs
S 10,600
S 10,550
S 10,550
Total
S 79,850
S 256,245
S 71,532
Decontaminated !
Contaminat
Disposal
Costs
S 4,405
S 70,006
S 2,169
Transporta
tion Costs
S 105,100
S 105,050
S 105,050
Waste Management Costs (Based on
Waste Knob)
VHP"
pH-Adjusted Bleach Process
CIO2
Total
S 79,850
S 256,245
S 71532
Contributio
n From
Sampling
S 42,166
S 25,725
S 67 053
Contributio
n From
Decon &
Other
S 37,684
S 230,521
S 4478
Waste Cost
Page H-43
6/13/2013
-------
MASTER BOTE Cost Analysis O61313.xlsx
Waste Quantity (solids in Ib and liquids in gal)
Decon Line Solid (Ib)
Dissemination
VHP® - Characterization Samplingl
VHP" - Decon
VHP" - Clearance Sampling
AB - Characterization Sampling2
AB Decon - Removal
AB Decon -Spray
AB Decon - Dry
AB - Clearance Sampling
CIO2 - Characterization Samplings
CLO2 - Decon
CIO2 - Clearance Sampling
AB- Building Reset
Decon Line Liquid (gal)
Dissemination
VHP® - Characterization Samplingl
VHP® - Decon
VHP® - Clearance Sampling
AB - Characterization Sampling2
AB Decon - Removal
AB Decon -Spray
AB Decon - Dry
AB - Clearance Sampling
CIO2 - Characterization Samplings
CLO2 - Decon
CIO2 - Clearance Sampling
AB- Building Reset
Donning Trailer (Ib)
Dissemination
VHP® - Characterization Samplingl
VHP® - Decon
VHP® - Clearance Sampling
AB - Characterization Sampling2
AB Decon - Removal
AB Decon -Spray
AB Decon - Dry
AB - Clearance Sampling
CIO2 - Characterization Samplings
CLO2 - Decon
CIO2 - Clearance Sampling
AB- Building Reset
Building- Solid (Ib)
Dissemination
VHP® - Characterization Samplingl
VHP® - Decon
VHP® - Clearance Sampling
AB - Characterization Sampling2
AB Decon - Removal
AB Decon -Spray
AB Decon - Dry
AB - Clearance Sampling
CIO2 - Characterization Samplings
CLO2 - Decon
CIO2 - Clearance Sampling
AB- Building Reset
Building - Liquid (gal)
Dissemination
VHP® - Characterization Samplingl
VHP® - Decon
VHP® - Clearance Sampling
AB - Characterization Sampling2
AB Decon - Removal
AB Decon -Spray
AB Decon - Dry
AB - Clearance Sampling
CIO2 - Characterization Samplings
CLO2 - Decon
CIO2 - Clearance Sampling
AB- Building Reset
Room 110 (Ib)
Dissemination
VHP® - Characterization Samplingl
Total (Ib)
or (gal)
15.2
210.0
447.4
188.4
247.0
154.4
177.8
0.0
325.4
101.2
0.0
0.0
0.0
28.0
211.0
55.0
73.5
177.0
26.0
527.9
0.0
100.5
137.0
0.0
37.5
0.0
0.0
17.4
7.2
19.8
20.8
5.6
4.2
0.0
15.8
10.6
0.0
0.0
0.0
54.8
0.0
0.0
0.0
0.0
3080.0
18.6
0.0
689.7
0.0
0.0
315.0
0.0
38.0
0.0
0.0
0.0
0.0
0.0
105.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0 0
Waste
Category
IS
4S
4S
4S
4S
4S
4S
4S
4S
4S
4S
4S
IS
1L
4L
4L
4L
4L
4L
4L
4L
4L
4L
4L
4L
1L
IS
IS
IS
IS
IS
IS
IS
IS
IS
IS
IS
IS
IS
IS
2S
2S
3S
2S
3S
3S
3S
3S
2S
2S
3S
IS
1L
3L
3L
3L
3L
3L
3L
3L
3L
3L
3L
3L
1L
IS
2S
Category
1S-
Uncontam
inated -
Solid
15.2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
17.4
7.2
19.8
20.8
5.6
4.2
0
15.8
10.6
0
0
0
54.8
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Category
2S-
Contamin
ated-
Solid
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Category
3S-
Deconta
minated -
Solid
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
3080
18.6
0
689.7
0
0
315
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Category
4S-
Decon
Waste -
Solid
0
210
447.4
188.4
247
154.4
177.8
0
325.4
101.2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Category
1L-
Unconta
minated -
Liquid
0
0
0
0
0
0
0
0
0
0
0
0
0
28
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
38
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Category
2L-
Contamin
ated-
Liquid
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Category
3L-
Deconta
minated -
Liquid
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
105
0
0
0
0
0
0
0
0
Category
4L-
Decon
Waste -
Liquid
0
0
0
0
0
0
0
0
0
0
0
0
0
0
211
55
73.5
177
26
527.913
0
100.5
137
0
37.5
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Waste Summary
Page H-44
6/13/2013
-------
MASTER BOTE Cost Analysis O61313.xlsx
Waste Quantity (solids in Ib and liquids in gal)
VHP" - Decon
VHP® - Clearance Sampling
AB - Characterization Sampling2
AB Decon - Removal
AB Decon -Spray
AB Decon - Dry
AB - Clearance Sampling
CIO2 - Characterization Samplings
CLO2 - Decon
CIO2 - Clearance Sampling
AB- Building Reset
Room 109 (Ib)
Dissemination
VHP® - Characterization Samplingl
VHP® - Decon
VHP® - Clearance Sampling
AB - Characterization Sampling2
AB Decon - Removal
AB Decon -Spray
AB Decon - Dry
AB - Clearance Sampling
CIO2 - Characterization Samplings
CLO2 - Decon
CIO2 - Clearance Sampling
AB- Building Reset
Room 108 (Ib)
Dissemination
VHP® - Characterization Samplingl
VHP® - Decon
VHP® - Clearance Sampling
AB - Characterization Sampling2
AB Decon - Removal
AB Decon -Spray
AB Decon - Dry
AB - Clearance Sampling
CIO2 - Characterization Samplings
CLO2 - Decon
CIO2 - Clearance Sampling
AB- Building Reset
Room 107 (Ib)
Dissemination
VHP® - Characterization Samplingl
VHP® - Decon
VHP® - Clearance Sampling
AB - Characterization Sampling2
AB Decon - Removal
AB Decon -Spray
AB Decon - Dry
AB - Clearance Sampling
CIO2 - Characterization Samplings
CLO2 - Decon
CIO2 - Clearance Sampling
AB- Building Reset
Room 106 (Ib)
Dissemination
VHP® - Characterization Samplingl
VHP® - Decon
VHP® - Clearance Sampling
AB - Characterization Sampling2
AB Decon - Removal
AB Decon -Spray
AB Decon - Dry
AB - Clearance Sampling
CIO2 - Characterization Samplings
CLO2 - Decon
CIO2 - Clearance Sampling
AB- Building Reset
Room 105 (Ib)
Dissemination
VHP® - Characterization Samplingl
VHP® - Decon
VHP® - Clearance Sampling
AB - Characterization Sampling2
Total (Ib)
or (gal)
0.0
0.0
0.0
1372.1
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
455.6
72.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
1286.1
97.6
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
151.6
603.6
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
1044.9
385.2
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
Waste
Category
2S
3S
2S
3S
3S
3S
3S
2S
2S
3S
IS
IS
2S
2S
3S
2S
3S
3S
3S
3S
2S
2S
3S
IS
IS
2S
2S
3S
2S
3S
3S
3S
3S
2S
2S
3S
IS
IS
2S
2S
3S
2S
3S
3S
3S
3S
2S
2S
3S
IS
IS
2S
2S
3S
2S
3S
3S
3S
3S
2S
2S
3S
IS
IS
2S
2S
3S
2S
Category
1S-
Uncontam
inated -
Solid
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Category
2S-
Contamin
ated-
Solid
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Category
3S-
Deconta
minated -
Solid
0
0
0
1372.14
0
0
0
0
0
0
0
0
0
0
0
0
455.6
72
0
0
0
0
0
0
0
0
0
0
0
1286.14
97.6
0
0
0
0
0
0
0
0
0
0
0
151.6
603.6
0
0
0
0
0
0
0
0
0
0
0
1044.94
385.2
0
0
0
0
0
0
0
0
0
0
0
Category
4S-
Decon
Waste -
Solid
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Category
1L-
Unconta
minated -
Liquid
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Category
2L-
Contamin
ated-
Liquid
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Category
3L-
Deconta
minated -
Liquid
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Category
4L-
Decon
Waste -
Liquid
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Waste Summary
Page H-45
6/13/2013
-------
MASTER BOTE Cost Analysis O61313.xlsx
Waste Quantity (solids in Ib and liquids in gal)
AB Decon - Removal
AB Decon -Spray
AB Decon - Dry
AB - Clearance Sampling
CIO2 - Characterization Samplings
CLO2 - Decon
CIO2 - Clearance Sampling
AB- Building Reset
Room 104 (Ib)
Dissemination
VHP" - Characterization Samplingl
VHP" - Decon
VHP® - Clearance Sampling
AB - Characterization Sampling2
AB Decon - Removal
AB Decon -Spray
AB Decon - Dry
AB - Clearance Sampling
CIO2 - Characterization Samplings
CLO2 - Decon
CIO2 - Clearance Sampling
AB- Building Reset
Room 103 (Ib)
Dissemination
VHP® - Characterization Samplingl
VHP® - Decon
VHP® - Clearance Sampling
AB - Characterization Sampling2
AB Decon - Removal
AB Decon -Spray
AB Decon - Dry
AB - Clearance Sampling
CIO2 - Characterization Samplings
CLO2 - Decon
CIO2 - Clearance Sampling
AB- Building Reset
Rooms 101A and 102 (Ib)
Dissemination
VHP® - Characterization Samplingl
VHP® - Decon
VHP® - Clearance Sampling
AB - Characterization Sampling2
AB Decon - Removal
AB Decon -Spray
AB Decon - Dry
AB - Clearance Sampling
CIO2 - Characterization Samplings
CLO2 - Decon
CIO2 - Clearance Sampling
AB- Building Reset
Other (Ib)
Dissemination
VHP® - Characterization Samplingl
VHP® - Decon
VHP® - Clearance Sampling
AB - Characterization Sampling2
AB Decon - Removal
AB Decon -Spray
AB Decon - Dry
AB - Clearance Sampling
CIO2 - Characterization Samplings
CLO2 - Decon
CIO2 - Clearance Sampling
AB- Building Reset
Total (Ib)
or (gal)
246.4
263.2
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
143.2
10.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
141.6
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
281.2
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
1938.8
707.2
0.0
13.8
0.0
0.0
0.0
0.0
Waste
Category
3S
3S
3S
3S
2S
2S
3S
IS
IS
2S
2S
3S
2S
3S
3S
3S
3S
2S
2S
3S
IS
IS
2S
2S
3S
2S
3S
3S
3S
3S
2S
2S
3S
IS
IS
2S
2S
3S
2S
3S
3S
3S
3S
2S
2S
3S
IS
IS
2S
2S
3S
2S
3S
3S
3S
3S
2S
2S
3S
IS
Category
1S-
Uncontam
inated -
Solid
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Category
2S-
Contamin
ated-
Solid
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Category
3S-
Deconta
minated -
Solid
246.4
263.2
0
0
0
0
0
0
0
0
0
0
0
143.2
10
0
0
0
0
0
0
0
0
0
0
0
141.6
0
0
0
0
0
0
0
0
0
0
0
0
281.2
0
0
0
0
0
0
0
0
0
0
0
0
1938.8
707.2
0
13.8
0
0
0
0
Category
4S-
Decon
Waste -
Solid
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Category
1L-
Unconta
minated -
Liquid
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Category
2L-
Contamin
ated-
Liquid
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Category
3L-
Deconta
minated -
Liquid
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Category
4L-
Decon
Waste -
Liquid
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Total Solid Waste (Ib)
Total Liquid Waste (gal)
15341
1516
Total
171.4 |
0.0
| 13317.5 |
1851.6 |
66.0
0.0
| 105.0
1345.4
Waste Summary
Page H-46
6/13/2013
-------
Appendix I
I BAG Sensor Data
-------
IBAC Sensor Locations
Rounds 1-3
1st Floor Layout
(_) IBAC Sensor
Spore Release Point
IBAC Sensor Locations
Rounds 1-3
2nd Floor Layout
IBAC Sensor
Spore Release Point
>JlH -i--
HE OFFICE. I (
, \\ 30= • [i vJ'I
I-2
-------
1st Floor IBAC Data
Test E vent #1
April 16, 2011
Release Material: Bacillus atropheus
Location: 1 ^ Floor Air Supply
ReleaseTime:13:18
Amount:200mg (10 generators, 4 mg/mLsuspension,
5 mL solution pergenerator, 50 ml total)
•HVAC System shut off at 15:18 (+ 2 hours)
1
2
3
4
5
6
7
8
9
10
11
IBAC
SSN
069
064
034
133
040
218
041
224
043
147
055
Location
Office 101 A
Office 103
Office 104
Office 105
Office 106
Office 107
Office 108
Office 109
Office 110
HVAC Mechanical Room
Lobby
Time to Reach
Sensor
1 minute
5 minutes
1 minute
9 minutes
2 minutes
12 minutes
6 minutes
20 seconds
2 minutes
15 seconds
2 minutes
Peak Particles
(Particles/Liter)
207,660
225,900
287,100
179,925
252,200
111,230
198,850
233,200
118,400
465,840
354,080
Average Particles
2 hour duration
(Particles/Liter)
107,080
117,250
131,950
103,600
121,550
77,650
104,050
110,000
77,300
144,750
140,500
Average
240,000
112,500
Test Event #1 - IBAC Particle Plots
April 16, 2011 -13:00-22:00
21:00
I-3
-------
Test Event #1 - IBAC Particle Plots
April 16, 2011 -13:00-22:00
21:00
Test Event #1 - IBAC Particle Plots
April 16, 2011-13:00-22:00
21:00
I-4
-------
Test Event #1 - IBAC Particle Plots
April 16, 2011-13:00-22:00
21:00
19:00
21:00
Dissemination
Start time: 13:18
HVACoffat
15:18
I-5
-------
2nd Floor I BAG Data
Test Event #1
April 16,2011
Release Material: Bacillus atropheus
Location: 2nd Floor Air Supply
Release Time: 13:18
Amount: O.Smg (1 generator, 0.5 mg/mL suspension,
1 ml total)
• HVAC System shut off at 15:18 (+ 2 hours)
1
2
3
4
5
6
7
8
9
10
IBAC
SSN
028
014
018
010
020
007
017
011
024
016
Location
Office 201
Office 206
Office 207
Office 208
Office 209
Office 210
Office 211
Office 21 2
Office 21 3
Hallway near airlock
Time to Reach
Sensor
2 minutes
4 minutes
1 minute
5 minutes
2 minutes
2 minutes
2 minutes
5 minutes
1 minute
3 minutes
Peak Particles
(Particles/Liter)
11,200
14,900
11,725
10,980
8,730
8,000
14,060
10,050
13,575
6,350
Average Particles
2 hour duration
(Particles/Liter)
5,000
6,950
6,900
5,200
4,650
4,550
5,320
4,900
4,400
3,060
Average
10,950
5,100
Test Event #1 - IBAC Particle Plots
April 16, 2011 -13:00-05:00
I-6
-------
Test Event #1 - IBAC Particle Plots
April 16, 2011-13:00-05:00
Test Event #1 - IBAC Particle Plots
April 16, 2011 -13:00-05:00
I-7
-------
Test Event #1 - IBAC Particle Plots
April 16, 2011-13:00-05:00
1st Floor IBAC Data
Test Event #2
April 25, 2011
Release Mate rial: Bacillus atropheus
Location: 1st Floor Air Supply
Release Time: 14:16
Amount:200ma (10 generators, 4 mg/mLsuspension,
5 ml_ solution pergenerator, 50 ml total)
• HVAC System shut off at 16:16 (+ 2 hours)
1
2
3
4
5
6
7
8
9
10
11
IBAC
SSN
069
064
034
133
040
218
041
224
043
147
055
Location
Office 101 A
Office 103
Office 104
Office 105
Office 106
Office 107
Office 108
Office 109
Office 110
HVAC Mechanical Room
Lobby
Time to Reach
Sensor
1 minute
3 minutes
10 seconds
7 minutes
5 minutes
5 minutes
3 minutes
10 seconds
4 minutes
5 seconds
1 minute
Peak Particles
(Particles/Liter)
298,940
255,800
337,590
164,250
262,600
183,000
163,570
214,075
171,400
428,580
367,865
Average Particles
2 hour duration
(Particles/Liter)
95,400
92,950
112,700
84,350
106,800
82,500
83,775
88,650
71,000
112,600
129,640
Average
258,880
96,400
-------
Test Event #2 - IBAC Particle Plots
April 25, 2011-14:00-19:00
19:00
Test Event #2 - IBAC Particle Plots
April 25, 2011-14:00-19:00
400
19:00
I-9
-------
Test Event #2 - IBAC Particle Plots
April 25, 2011-14:00-19:00
19:00
Test Event #2 - IBAC Particle Plots
April 25, 2011 -14:00-19:00
1-10
-------
2nd Floor IBAC Data
Test Event #2
April 25, 2011
19:00
Release Material: Bacillus atropheus
Location: 2nd Floor Air Supply
ReleaseTime: 14:16
Amount: O.Smq (1 generator, 0.5 mg/mLsuspension,
1ml total)
• HVAC System shut off at 16:16 (+ 2 hours)
1
2
3
4
5
6
7
8
9
10
IBAC
SSN
028
014
018
010
020
007
017
011
024
016
Location
Office 201
Office 206
Office 207
Office 208
Office 209
Office 210
Office 211
Office 21 2
Office 21 3
Hallway near air lock
Time to Reach
Sensor
4 minutes
3 minutes
2 minutes
1 minute
1 minute
1 minute
1 minute
1 minute
1 minute
5 minutes
Peak Particles
(Particles/Liter)
10,615
11,700
12,570
11,600
11,580
9,400
21,220
10,730
14,580
8,530
Average Particles
2 hour duration
(Particles/Liter)
5,110
6,230
5,375
3,600
3,500
3,640
2,785
3,185
3,815
4,730
Average
12,250
4,200
1-11
-------
Test Event #2 - IBAC Particle Plots
April 25, 2011-14:00-19:00
19:00
Test Event #2 - IBAC Particle Plots
April 25, 2011-14:00-19:00
19:00
1-12
-------
Test Event #2 - IBAC Particle Plots
April 25, 2011-14:00-19:00
19:00
1-13
-------
1st Floor IBAC Data
Round #3
May 10, 2011
Release Mate rial: Bacillus atropheus
Location: 1st Floor Air Supply
Release!! me: 15:22
Amount:200mg (10 generators, 4 mg/mLsuspension,
5 ml_ solution pergenerator, 50 ml total)
• HVAC System shut off at 17:22 (+ 2 hours)
1
2
3
4
5
6
7
8
9
10
11
IBAC
SSN
069
064
034
133
040
218
041
224
043
147
055
Location
Office 101 A
Office 103
Office 104
Office 105
Office 106
Office 107
Office 108
Office 109
Office 110
HVAC Mechanical Room
Lobby
Time to Reach
Sensor
20 seconds
3 minutes
10 seconds
2 minutes
3 minutes
4 minutes
4 minutes
30 seconds
2 minutes
10 seconds
1 minute
Peak Particles
(Particles/Liter)
430,930
215,070
277,675
124,225
178,600
88,765
151,830
274,650
205,400
378,100
394,300
Average Particles
2 hour duration
(Particles/Liter)
112,800
81,950
95,950
64,100
75,100
59,685
68,315
77,335
61,175
102,915
108,200
Average | 247,230 | 82,500 |
Test Event #3 - IBAC Particle Plots
May 10, 2011 -15:00-20:00
500 i
400
O)fo
|| Ł5 300 •
>7 1? 200
"3
100
0
15
250
200 •
rafo"
ŁŁ, 150-
3~
'r'o 100
°s
50
0
15
00
00
Ap
:\P
>v^___^
16:00
17
16:00
Dissemination
Starttime: 15:22
17
00
-~— —
00
18
00
Office 101 A
069
19:00 20:00
1
18:00
HVAC off at
17:22
Off ice 103
064
1 1
19:00 20:00
1-14
-------
Test Event #3 - IBAC Particle Plots
May 10, 2011-15:00-20:00
20:00
Test Event #3 - IBAC Particle Plots
May 10, 2011-15:00-20:00
200
20:00
1-15
-------
Test Event #3 - IBAC Particle Plots
May 10, 2011-15:00-20:00
20:00
250
20:OC
1-16
-------
2nd Floor IBAC Data
Round #3
May 10, 2011
Release Material: Bacillus atropheus
Location: 2nd Floor Air Supply
ReleaseTime: 15:22
Amount: O.Sma (1 generator, 0.5 mg/mLsuspension,
1ml total)
• HVAC System shut off at 17:22 (+ 2 hours)
1
2
3
4
5
6
7
8
9
10
IBAC
SSN
028
014
018
010
020
007
017
011
024
016
Location
Office 201
Office 206
Office 207
Office 208
Office 209
Office 210
Office 211
Office 21 2
Office 21 3
Hallway near air lock
Time to Reach
Sensor
1 minute
1 minute
1 minute
30 seconds
1 minute
2 minutes
10 seconds
1 minute
10 seconds
30 seconds
Peak Particles
(Particles/Liter)
8,215
17,480
16,000
27,290
14,580
14,150
18,950
11,980
9,450
8,220
Average Particles
2 hour duration
(Particles/Liter)
6,350
7,360
12,500
7,430
8,050
9,450
7,730
7,360
5,500
6,340
Average I
14,630
7,800
Test Event #3 - IBAC Particle Plots
May 10, 2011 -15:00-22:00
21:00 22:00
1-17
-------
Test Event #3 - IBAC Particle Plots
May 10, 2011 -15:00-22:00
30
o« 25
-------
1-19
-------
Appendix J
Sampling Plan Analysis
-------
Sampling Plan Analysis for the Proposed BOTE Exercise
Brett Amidan & Brent Pulsipher, PNNL
October 2010
The following sample plan characteristics have been proposed:
• Three sampling events, one for each of three decon methods, where each event consists of
sampling from 16 study rooms and 12 other rooms;
• The study rooms for each event consist of 6 commercial rooms, 6 residential rooms, 2 industrial
rooms, and 2 mailrooms, where half of each type are on the lower level of the building (given
one concentration level of the contaminant) and the other half on the upper level (given a
different concentration level of the contaminant);
• Each study room will be sampled using 2 swabs, 2 vacuums, and 4 sponges;
• The other rooms consist of the non-study rooms on both floors;
• Each other room will be sampled using 1 swab, 1 vacuum, and 1 sponge sample;
• A sample will consist of a paired measurement, where a predefined area is sampled before
decon, and then a predefined adjacent area is sampled after decon;
• This results in 164 samples taken during each of the three events.
This analysis considers the estimated statistical power when making comparisons across this data based
upon proposed sample size estimates. Statistical power is defined as the probability that the test will
reject a false null hypothesis, or in other words, that if differences between means of different levels
actually exist, then they will be detected (statistically significant difference).
Only those comparisons that are related to the goals of the experiment will be included in the statistical
power and sample size calculations. Those comparisons used to determine sample size include:
• Determining differences between the three decon methods;
• Comparing decon effectiveness in the study rooms (commercial, residential, industrial, and
mail); and
• Comparing decon effectiveness with the sampling media (swab, vacuum, and sponge-wipe).
Other comparisons may be made during the analysis, but they will not be considered during the sample
size determination. The "other" rooms may provide useful information in comparing decon methods,
but they are not being considered in sample size calculations for the room type comparison. The
"other" room samples are included in all the other comparisons. Samples are also planned for inside the
air ducts, but those samples will also not be included in these calculations.
Surface types will be sampled with the most appropriate method, meaning that non-porous areas with
sponges, porous areas with vacuums, and small areas with swabs. Surface types will not be included in
the sample size calculations; however, it is important for the sampling plan to make sure each surface
type available is well represented.
J-2
-------
In order to calculate statistical power (Dean, 1999), the following items need to be defined:
1. The type I error rate (a),
2. The response variable,
3. The detectable difference, and
4. The estimated variability in the data (standard deviation).
The type I error rate (a) is the probability of rejecting a true null hypothesis. This is commonly called the
significance or confidence level of the experiment. For this study a will be held constant at 0.05, thus
allowing for a 95% confidence statement of the null hypothesis being rejected.
The response variable chosen for this exercise is dependent upon how well the decon methods work. If
the reduction in contamination is in the 75% to 100% range (or even 50% to 100%) range, then it won't
be necessary to take the log of the response. If the reduction in contamination is more like 98% to
100%, then it will be necessary to take the log of the response. Statistical power and sample size
calculations will be presented using each possibility.
Percentage of Contamination Removed (%CR) Response Variable
Under the assumption that the reduction in contamination is in the 75% to 100% range, a reasonable
response variable to be analyzed is the percentage of contamination that is removed: %CR = (Pre —
Post)/Pre, where %CR is the percentage of contamination removed, Pre is the pre-decon value, and
Post is the post-decon value. To calculate this, each Pre value should be paired with a Post value that is
taken from the same general location. Corrections may be necessary if Pre values are 0, or if a Pre value
is smaller than the paired Post value. This means that although there are 164 samples taken before
decon and 164 samples taken after decon, the analysis will combine these results into a new derived
variable (%CR) that has 164 values for each event. It should be noted that these power calculations
assume that we are comparing the mean %CR for each factor of interest.
The detectable difference is the amount of difference between levels of a given factor that would be
considered to be statistically significant. For example, suppose decon method 1 removes 85% of the
contamination and decon method 2 removes 90% of the contamination. Is this difference of 5%C7? of
practical significance; and how likely should the experiment be able to detect this amount of difference?
This 5%C7? difference is the detectable difference and if it is very important to detect differences of this
size, then the statistical power should be high (probably in the 90% to 99% range) for that difference.
The true %CR values, by definition, will range between 0 and 100. Knowing this, the variability can be
estimated. The measure of variability used in these calculations is the standard deviation (SD). If the
%CR values generally range from 50% to 100%, meaning that between half of the contamination to all
the contamination is being removed, then a conservative estimate of the SD would be 15%C7?. Likewise,
if the %CR values range from 0% to 100%, meaning that sometimes contamination is not removed and
sometimes it is all removed (an extreme case scenario), then the SD is estimated to be about 30%CR. If
J-3
-------
the %CR values generally range from 75% to 100%, meaning that more than % of the contamination is
removed, then the conservative estimate for SD goes down further to around 8%CR.
Tables 1 and 2 show the statistically detectable differences between the levels of each factor with a
15%C7? standard deviation (Table 1) and an 8%CR standard deviation (Table 2). These detectable
differences are calculated assuming statistical power of 95% and 90%. They are calculated for the
proposed sample sizes (n) and if the sample sizes were decreased by 10% or increased by 10%. For
cases where the standard deviation is 30%CR, the detectable differences in Table 1 would be doubled.
For example, with a standard deviation of 15%CR, there would be a 95% probability that differences of
6.5%C7? or more contamination removed between the three decon methods would be statistically
detected. For this same case, if the standard deviation was 8%CR, then the detectable difference
decreases to 3.5%C7?. If the standard deviation was 30%CR, then the detectable difference would
increase to 13.0%C7? (double the detectable difference using 15%C7? standard deviation).
The decon methods are expected to remove contamination and it wouldn't be surprising if they remove
at least 75% of the contamination, probably more. Under those assumptions, in most comparisons a
difference of 4%C7? or more should easily be detected given the proposed sample amounts (Table 2).
The only comparisons that may not be detected at that level are ones involving the industrial or mail
rooms, where fewer samples are being taken. If a decon method only removes about half to % of the
contamination, while the others remove nearly all, then differences of around 7%CR should be
detectable, except in comparisons involving the industrial or mailrooms (Table 1). This should still be ok
because the difference between Vi (50%C7?) and nearly one (<100%C7?) is easily bigger than 7%CR.
Table 3 shows how the detectable difference decreases for comparisons involving the industrial and
mailrooms when increasing the number of samples by 50% or 100%. Doubling the number of samples in
industrial and mailrooms will decrease the detectable difference by about 2%CR.
J-4
-------
Table 1. The Detectable Differences (reported here in %CR) for each statistical comparison between the different
levels of each factor given the following: 1) 95% or 90% statistical power, 2) 15 %CR standard deviation, and 3)
sample sizes of proposed size (n), n - 10%*n, and n + 10%*n.
Factor Levels
Proposed
n
Proposed n
95% Power
90% Power
n - 10%*n
95% Power
90% Power
n + 10%*n
95% Power
90% Power
Decon Methods
Chlor Dysis
Vapor Hydrogen peroxide
Amended Bleach
164
each
6.5
5.9
6.9
6.2
6.2
5.6
Room Type
Commercial
Residential
Industrial
Mailroom
144
each
48
each
7.4
12.8
6.7
11.7
7.7
13.6
7.0
12.3
7.0
12.2
6.4
11.1
Concentration Amount
Low
High
246
each
4.9
4.4
5.2
4.6
4.7
4.2
Sampling Method
Swabs
Vacuums
Sponges (Wipes)
132
each
228
7.3
5.5
6.6
5.0
7.7
5.8
6.9
5.3
6.8
5.3
6.2
4.8
Table 2. The Detectable Differences (reported here in %CR) for each statistical comparison between the different
levels of each factor given the following: 1) 95% or 90% statistical power, 2) 8 %CR standard deviation, and 3)
sample sizes of proposed size (n), n - 10%*n, and n + 10%*n.
Factors
Proposed
n
Proposed n
95% Power
90% Power
n - 10%*n
95% Power
90% Power
n + 10%*n
95% Power
90% Power
Decon Methods
Chlor Dysis
Vapor Hydrogen peroxide
Amended Bleach
164
each
3.5
3.2
3.7
3.3
3.3
3.0
Room Type
Commercial
Residential
Industrial
Mailroom
144
each
48
each
3.9
6.8
3.6
6.2
4.1
7.2
3.8
6.6
3.7
6.5
3.4
5.9
Concentration Amount
Low
High
246
each
2.6
2.3
2.8
2.5
2.5
2.2
Sampling Method
Swabs
Vacuums
Sponges (Wipes)
132
each
228
3.9
3.0
3.5
2.7
4.1
3.1
3.7
2.7
3.6
2.8
3.3
2.5
J-5
-------
Table 3. The Detectable Differences (reported here in %CR) for comparisons involving industrial and mail rooms
for the proposed sample amount (48 each) and 50% and 100% increases in the sample amount, given the
following: 1) 95% or 90% statistical power, and 2) 8 %CR or 15 %CR standard deviation.
Sample Amounts
n = 48 each (2 swab, 2 vac, 4 sponge per room)
(currently proposed)
n = 72 each (3 swab, 3 vac, 6 sponge per room)
(50% Increase)
n = 96 each (4 swab, 4 vac, 8 sponge per room)
(100% Increase)
8 %CR Standard Deviation
95% Power
6.8
5.6
4.8
90% Power
6.2
5.1
4.4
15 %CR Standard Deviation
95% Power
12.8
10.4
9.0
90% Power
11.7
9.5
8.2
log(%CR) Response Variable
Under the assumption that the reduction in contamination is in the 98% to 100% range, a reasonable
response variable to be analyzed is taking the log (Wolfe, 1999) of the percentage of contamination that
is removed: Iog10 (%CR) = Iog10 (
), where %CR is the percentage of contamination removed,
Pre is the pre-decon value, and Post is the post-decon value. The same methodology described in the
previous section will be used to calculate this value, except that the logic will be taken of the %CR
values.
Assuming that the data varies in the 98% to 100% contamination removed range; a conservative
estimate can be made of the standard deviation by taking the range of the data and dividing by four.
The logic estimate of standard deviation can then be estimated using the formula:
BSD = q°gio(D-i°gio(o.98)) = 0 002ig where ESD js the estimated standard deviation. In the case that
4
99% to 100% contamination is removed, the ESD value would be 0.00109. Both of these situations will
be investigated.
Table 4 shows the statistically detectable differences between the levels of each factor with an ESD of
0.00219 (assuming a 98%CR to 100%C7? range). Table 5 shows the statistically detectable differences
between the levels of each factor with an ESD of 0.00109 (assuming a 99%CR to 100%C7? range). These
detectable differences are calculated assuming statistical power of 95% and 90%. They are calculated
for the proposed sample sizes (n) and if the sample sizes were decreased by 10% or increased by 10%.
These tables can be used to determine what size of difference between characteristics would be
considered statistically significant. For example, if comparing the three decon methods, with 95%
statistical power and assuming a range of 98%CR to 100%C7?, a difference of 0.22%CR would be
detectable, given the current sampling plan (see Table 4). This means if decon method A has a mean
%CR of 98.78% and decon method B has a mean %CR of 99.00%, there is a 95% probability that this
difference would be considered statistically different. If the percent contamination removed is actually
J-6
-------
in the 99%CR to 100%CR range, then a difference of 0.11%CR would be detectable, with 95% statistical
power and the current sampling plan (see Table 5).
Assuming the larger standard deviation in which the percent contamination removed is between 98%
and 100%, nearly all characteristics (levels) can be compared with a detectable difference of 0.25%C7? or
less. The only comparisons with a larger detectable difference are ones involving the industrial and
mailrooms. These comparisons will require a difference of 0.43%C7? or more to have a 95% probability
of being considered statistically significant. Increasing the numbers of samples within industrial and
mailrooms would be the best way to lower this detectable difference.
Table 6 shows how the detectable difference decreases for comparisons involving the industrial and
mailrooms when increasing the number of samples by 50% or 100%. Doubling the number of samples in
industrial and mailrooms will decrease the detectable difference by about 0.13%CR.
Table 4. The Detectable Differences (reported here in %CR) when analyzing log10(%CR) for each statistical
comparison between the different levels of each factor given the following: 1) 95% or 90% statistical power, 2)
BSD = 0.00219, and 3) sample sizes of proposed size (n), n - 10%*n, and n + 10%*n.
Factor Levels
Proposed
n
Proposed n
95% Power
90% Power
n - 10%*n
95% Power
90% Power
n + 10%*n
95% Power
90% Power
Decon Methods
Chlor Dysis
Vapor Hydrogen peroxide
Amended Bleach
164
each
0.22
0.20
0.23
0.21
0.21
0.19
Room Type
Commercial
Residential
Industrial
Mailroom
144
each
48
each
0.25
0.43
0.22
0.39
0.26
0.45
0.24
0.41
0.24
0.41
0.21
0.37
Concentration Amount
Low
High
246
each
0.16
0.15
0.17
0.16
0.16
0.14
Sampling Method
Swabs
Vacuums
Sponges (Wipes)
132
each
228
0.24
0.19
0.22
0.17
0.26
0.20
0.23
0.18
0.23
0.18
0.21
0.16
J-7
-------
Table 5. The Detectable Differences (reported here in %CR) when analyzing log10(%CR) for each statistical
comparison between the different levels of each factor given the following: 1) 95% or 90% statistical power, 2)
BSD = 0.00109, and 3) sample sizes of proposed size (n), n - 10%*n, and n + 10%*n.
Factor Levels
Proposed
n
Proposed n
95% Power
90% Power
n - 10%*n
95% Power
90% Power
n + 10%*n
95% Power
90% Power
Decon Methods
Chlor Dysis
Vapor Hydrogen peroxide
Amended Bleach
164
each
0.11
0.10
0.12
0.10
0.10
0.09
Room Type
Commercial
Residential
Industrial
Mailroom
144
each
48
each
0.12
0.21
0.11
0.19
0.13
0.23
0.12
0.21
0.12
0.20
0.11
0.18
Concentration Amount
Low
High
246
each
0.08
0.07
0.09
0.08
0.08
0.07
Sampling Method
Swabs
Vacuums
Sponges (Wipes)
132
each
228
0.12
0.09
0.11
0.08
0.13
0.10
0.12
0.09
0.11
0.09
0.10
0.08
Table 6. The Detectable Differences (reported here in %CR) when analyzing log10(%CR) for comparisons involving
industrial and mail rooms for the proposed sample amount (48 each) and 50% and 100% increases in the sample
amount, given the following: 1) 95% or 90% statistical power, and 2) ESD = 0.00219 and 0.00109.
Sample Amounts
n = 48 each (2 swab, 2 vac, 4 sponge per room)
(currently proposed)
n = 72 each (3 swab, 3 vac, 6 sponge per room)
(50% Increase)
n = 96 each (4 swab, 4 vac, 8 sponge per room)
(100% Increase)
ESD = 0.00219
95% Power
0.43
0.35
0.30
90% Power
0.39
0.32
0.27
ESD = 0.00109
95% Power
0.21
0.17
0.15
90% Power
0.19
0.16
0.14
J-8
-------
Recommendations
In most cases, the proposed sample numbers should provide high statistical power to find reasonable
detectable differences for this exercise. The one case that could use improvement is comparisons
including the industrial and mail rooms. Tables 3 and 6 show how much the detectable differences
decrease when increasing the numbers of samples taken in each of these room types. If the numbers of
samples in each room are increased by 50% (1.5X) such that 3 swab, 3 vacuum, and 6 sponge samples
are taken per room, then the detectable difference will decrease by 1%CR to 2%CR when the data
ranges between 75%C7? and 100%CR, or decreases by 0.08%O? when the data ranges between 98%CR
and 100%CR. If the numbers of samples in each room are doubled (2X), then the detectable difference
will decrease by 2%CR to 4%C7? or by 0.13%C7?, considering the same ranges of the data. Ideally adding
another room of each type to the exercise would help alleviate these concerns. Because this is
logistically not possible, it is recommended to increase the numbers of samples in each of these rooms,
in order to increase the statistical power in detecting significant differences.
References
Dean, Angela and Daniel Voss. Design and Analysis of Experiments. Springer (1999).
Wolfe, Rory and John B. Carlin. "Sample-Size Calculation for a Log-Transformed Outcome Measure."
Controlled Clinical Trials 20:547-554 (1999).
J-9
-------
Appendix K
Standard Operating Procedure: Rapid-Viability Polymerase
Chain Reaction (RV-PCR) Method for the Bio-Response
-------
General Methods
Bacterial Strains, Spore Preparations, and Biological Safety
The surrogate strain B. atrophaeus deposited as Bacillus subtilis var. niger (American Type Culture
Collection, ATCC #9372), previously named Bacillus globigii (BG) was used as a positive control for
culture and RV-PCR analyses. Spore stocks of BG strain ATCC#9372 from Apex Laboratories, Inc. were
stored in 70% Milli-Q water and 30% ethanol solution at -20°C. The BG spore preparation from CRP was
obtained in dry form and was refrigerated upon receipt. The dry spores preparation was suspended in
phosphate-buffered saline solution with 0.05% Tween-20 (PBST), and dilutions were prepared in PBST
and stored at 4°C.
All procedures involving manipulation of BG spores and bacterial cultures were performed by trained
personnel wearing appropriate personal protective equipment including safety glasses, double-gloves,
and solid front laboratory gowns in an approved Class II biosafety cabinet (BSC). Bacterial stocks were
handled in BSCs to prevent laboratory contamination as part of good laboratory practices. The BSC and
equipment used in the BSC were decontaminated before and after work with bacteria following standard
procedures (freshly prepared 10% bleach solution followed by isopropanol treatment and finally deionized,
sterile water rinse). Sterile techniques were used for all sample handling and analysis.
Combined Culture and RV-PCR Protocols: Sample Processing and Analysis
This combined method allowed analysis of the same wipe sample by both culture and RV-PCR methods.
Two spore extraction steps were included with the combined extraction solution split into half for culture
analysis and half for RV-PCR analysis. The combined protocol was designed to provide the same
concentration factor for both culture and RV-PCR analyses.
Experimental controls consisted of one positive and one negative control per24-tube rack and 24-filter
cup manifold that were included with samples for each pre- or post-decontamination portion of each
event. Positive control samples were wipe samples spiked with BG spores (ATCC #9372) at a level of
approx. 500-1000 CFU/sample (actual CPU were determined by plating). Traditional viability analysis
was used to quantify the level of spores spiked onto positive control samples. Spiked control wipes were
stored at 4°C until use. The control samples were processed in parallel with the field samples (all samples
were processed following the same laboratory procedures). The sample processing and analysis steps
are outlined below, followed by the detailed protocol.
K-2
-------
Sample Processing
20 ml cold Extraction Buffer with Tween was added to the sample (wipe in a conical tube with
mesh support).
One negative wipe control and one positive wipe control were included per manifold [each manifold
holds up to 24 filter cups, allowing up to 22 samples per manifold with one positive control (PC)
and one negative control (NC)]. Control wipes were processed with same protocol steps as
sample wipes.
The sample was vortexed, and 14 ml were removed to a 50 ml tube.
14 ml Extraction Buffer without Tween was added to the original sample tube with wipe and
support.
The sample was vortexed, and 14 ml were removed and combined with the first 14 ml aliquot in
the appropriate 50 ml tube. The total expected volume from spore extraction was 28 ml.
Combined aliquots were mixed and after allowing the suspension to sit for 30 sec to let large
particles to settle 13 ml were transferred to a filter cup to collect spores for RV-PCR analysis.
From the remaining volume, 13 ml were used for culture analysis.
Culture Processing and Analysis Steps
Contents of the 50 ml tube were mixed by vortexing and 13 ml were transferred to another 50 ml
centrifuge tube.
After centrifugation, 9.5 ml of supernatant were removed to waste. The pellet was suspended in
the remaining 3.5 ml (referred to as the 10° suspension).
A 10-fold dilution of the 10° suspension was performed (0.5 ml added to 4.5 ml Butterfield Buffer)
yielding a 10~1 suspension; a 10-fold dilution was performed on the 10~1 suspension (0.5 ml
added to 4.5 ml buffer) yielding a 10~2 suspension.
Three replicates of 100 (oL each for each dilution (10°, 10"1, and 10"2; 9 plates total per sample)
were plated onto Tryptic Soy Agar (TSA) plates, followed by incubation at 35°C for 18-24 hr, such
that colony counts obtained represented 10~1, 10~2, and 10" dilutions of the original sample, when
expressed on a per ml basis.
Note: The 35°C incubation temperature was 35 ± 2°C.
An aliquot of 0.5 ml from the 10° suspension was filtered through a microfunnel filter, and the filter
was placed onto a TSA plate, followed by incubation at 35°C for 18-24 hr.
The remaining 10° suspension was used for an enrichment culture. The suspension was
transferred to a 15 ml conical tube, followed by centrifugation, removal of the supernatant and
resuspension in 5 ml Tryptic Soy Broth (TSB). Cultures were incubated with shaking at 35°C for
18-24 hr. If turbidity was observed, the suspension was used to streak onto TSA plates for
isolation of BG colonies.
Two colonies per sample were tested by real-time PCR to confirm BG colony counts to confirm
identification based on colony morphology and presence of orange pigmentation.
If no BG colonies were detected, the enrichment culture was analyzed by real-time PCR.
RV-PCR Processing and Analysis Steps
K-3
-------
A 13 ml aliquot was transferred from each sample tube to a filter cup, and spores in the extraction
buffer were collected on a 0.45 |j,m filter using a vacuum manifold and a vacuum pump (or similar
vacuum source).
Filters were then washed with 20 ml cold High-Salt Phosphate Buffer (210 mM; pH 6.0) followed by
7 ml of cold Phosphate Wash Buffer (25 mM; pH 7.4).
Filter cups are then sealed on the bottom, and 3.5 ml of cold TSB growth medium were added.
Top caps were added to the filter cups and cups were vortexed.
After mixing, a 1 ml aliquot was taken from each filter cup (TO aliquot), transferred to a 2 ml
Eppendorf tube and processed for DMA extraction and purification using a Promega Magnesil kit
as described below.
The cups were sealed on the top and incubated for 9 hr with shaking at 35°C.
After incubation, 1 ml of each sample was transferred from the filter cup into a 2 ml Eppendorf
tube (referred to as T9 aliquot).
For both TO and T9 aliquots (1 ml), 600 ^L of Promega Bead Mix (combined Lysis Buffer and Bead
Mix) were added, followed by addition of 360 (oL Promega Lysis Buffer.
Sample, buffer and bead mix were mixed by pipetting and tubes were mounted on a tube rack
interfaced with a magnet. Beads with attached DMA were attracted to the magnet and the
supernatant was removed by pipetting.
An additional lysis with 360 (oL of Lysis Buffer was conducted with mixing by pipetting and removal
of the supernatant.
Two washes with 360 (o,L of Promega Salt Wash solution were then performed, followed by mixing
by pipetting and removal of the supernatant.
Finally, three washes with 500 (oL of Promega Alcohol Wash solution were performed with mixing
and supernatant removal.
Beads were allowed to air-dry for 2 minutes, followed by transfer of the tube rack from the magnetic
support to a 2 mL tube heat block and heating for ~45 min. at 80°C, or until beads were dry.
Elution of DMA was then performed after removing tubes from heat block and allowing them to
reach room temperature. A 200 (oL aliquot of Promega Elution Buffer was added and the tube
contents were mixed by vortexing. The tubes were then transferred to the magnetic support, and
the eluent with DMA was recovered.
Although a cleaner DMA sample was generated after DMA extraction and purification, a 10-fold
dilution was conducted by adding 10 (oL to 90 (oL PCR-grade water prior to PCR for both TO and
T9 aliquots.
Three replicate PCR analyses were conducted for each sample and control wipe for both TO and
T9. Average Ct values and standard deviations were reported, and averages were used to
calculate the ACt from TO to T9.
Data Analysis
Initial and final cycle thresholds [Ct(TO) and Ct(T9)] from RV-PCR assays were used in the algorithm to
determine whether viable spores were present in the sample; the algorithm used Ct(TO), Ct(T9) and ACt
[Ct(TO) - Ct(T9)]. When the PCR yielded no Ct value for TO and/or forT9 (i.e., non-detect or
"Undetermined" on instrument software), the Ct value(s) was set to 45 for Delta Ct calculation and result.
The algorithm used for positive detection by RV-PCR was Ct(T9) < 39 and ACt > 6. Ct values at TO and
T9 as well as ACt were expressed as the average and standard deviation of three replicate analyses.
K-4
-------
The BG colony forming units (CPUs) were determined manually based on colony morphology and the
results were recorded the day following culture incubation. Since colony counts are most accurate when
between 25 and 250, any plates or filters contained more than 250 colonies were recorded as too
numerous to count (TNTC). Since the comparison with RV-PCR analysis is qualitative, presence of BG
colonies on any of the sample culture plates including those that were TNTC represented a positive result
(BG presence) for that sample. The CPU were corrected for dilution factor. Real-time PCR analysis on
selected colonies was also reported; Ct values < 35 were required to confirm the sample was positive for
BG by culture. Results from the sample enrichment cultures were reported if no BG colonies were
detected for serial dilution or filter membrane plates; in this case, attempts were made to isolate BG
colonies from the enrichment culture, followed by real-time PCR confirmation. If no presumptive BG
colonies were evident after re-streaking, the enrichment culture was extracted for DMA and analyzed by
real-time PCR. As for PCR analysis of colony DMA, if Ct values < 35 were obtained, the sample was
considered positive for viable BG by the culture method.
Both culture and RV-PCR data were obtained for all field samples, field blank samples, and laboratory
control samples for all three events (pre- and post-decontamination). Since the RV-PCR method is
qualitative, the comparison between RV-PCR and the culture method was performed in terms of
positive/negative (presence/absence) BG detection, although average values (with standard deviations)
for Ct values at TO and T9, ACt values, and plate count data were also recorded.
References
1. Kane, S. R., S. E. Letant, G. A. Murphy, T. M. Alfaro, P. W. Krauter, R. Mahnke, T. C. Legler and E.
Raber. 2009. Rapid, high-throughput, culture-based PCR methods to analyze samples for viable
spores of Bacillus anthracis and its surrogates. J. Microbiol. Methods 76(3): 278-284.
2. Letant, S. E., S. R. Kane, G. A. Murphy, T. M. Alfaro, L. R. Hodges, L. J. Rose and E. Raber. 2010.
Most-probable-number rapid viability PCR method to detect viable spores of Bacillus anthracis in
swab sample. J. Microbiol. Methods 81(2): 200-202.
3. Letant, S. E., G. A. Murphy, T. M. Alfaro, J. R. Avila, S. R. Kane, E. Raber, T. M. Bunt, and S. R.
Shah. 2011. Rapid Viability Polymerase Chain Reaction method for detection of virulent Bacillus
anthracis from environmental samples. Appl. Environ. Microbiol 77(18): 6570-6578.
K-5
-------
Protocol for Evaluation of the Rapid Viability Polymerase Chain Reaction (RV-PCR) Method in the
Bio-Response Operational Testing and Evaluation (BOTE) Phase-l
Qualitative detection of Bacillus atrophaeus subsp. globigii (BG) spores in wipe samples
General
The purpose of this procedure is to evaluate the manual Rapid Viability (RV)-PCR method for determining
the presence/absence of viable Bacillus atrophaeus subsp. globigii (BG) spores on wipe samples in the
Bio-Response Operational Testing and Evaluation (BOTE). The method uses processing steps to 1)
remove spores from wipes, 2) collect spores by filtration, and 3) culture spores for outgrowth and
subsequent DMA extraction and real-time PCR analysis. The RV-PCR method uses the change in PCR
response before and after high throughput culturing to determine if viable spores were present in the
sample; results are compared with performance criteria developed from testing of low levels of live target
spores in backgrounds containing high levels of killed target spores, high levels of non-target cells and
spores, or high levels of debris.
Equipment
• Biosafety Cabinet (BSC)
• PCR preparation hood (optional)
• Shaker incubator (Innova, Model KS 4000I Control, or similar)
• ABI 7500 Fast Real-Time PCR System (Applied Biosystems)
• Qubit™ fluorometer (Invitrogen)
• Refrigerated centrifuge (Eppendorf 581 OR or similar), with rotor adapters for 50 ml conical tubes
and 96-well plates
• Filter cup manifold (custom made) - see protocol Annex C
• Allen wrench for manifold
• Capping tray (custom made) - see protocol Annex C
• 30 ml tube rack (custom made)
• Vacuum pump (Cole Parmer, Model EW-07061-40, or similar), or vacuum source capable of <20
psi
• Vacuum pump filters for pump (Acrovent, Cat. No. 4249)
• Vacuum trap accessories - see protocol Annex C
• Platform vortexer (VWR, Model 58816-115) with Velcro straps
• Single-tube vortexer
• Heat block (for 2 ml Eppendorf tubes)
• Single-channel pipettors (1 ml, 200 (oL, 10 (oi. or 20 (oL)
• Eight-channel pipettor (100 |al_)
• Serological pipet aid
• Dynamag magnetic racks (Invitrogen)
• Plate turn-table
K-6
-------
Reagents
• Extraction Buffer with Tween (see preparation procedure in protocol Annex D)
• Extraction Buffer without Tween (see preparation procedure in protocol Annex D)
• High Salt Wash buffer (see preparation procedure in protocol Annex D)
• 1X Wash Buffer (see preparation procedure in protocol Annex D)
• Phosphate-buffered Saline Tween-20 (PBST) buffer (Teknova, Cat. No. P0201, see composition
in protocol Annex D)
• Butterfield Buffer (see preparation procedure in protocol Annex D)
• Tryptic Soy Broth (TSB) growth medium (VWR, Cat. No. 90000-378)
• Tryptic Soy Agar (TSA) (see preparation procedure in protocol Annex D)
• Promega reagents for DNA extraction and purification procedure:
o Magnesil Blood Genomic, Max Yield System, Kit (Promega, Cat. No., MD1360; VWR, Cat.
No. PAMD1360)
o Salt Wash (VWR, Cat. No. PAMD1401)
o Magnesil ParaMagnetic Particles (PMPs) (VWR, Cat. No. PAMD1441)
o Lysis Buffer (VWR, Cat. No. PAMD1392)
o Elution Buffer (VWR Cat. No. PAMD1421)
o Alcohol Wash, Blood (VWR Cat. No. PAMD1411)
o Anti-Foam Reagent (VWR, Cat. No. PAMD1431)
• TaqMan Universal PCR Master-Mix (Applied Biosystems, Cat. No. 4305719)
• PCR probe and primers for the BG chromosome (1 assay targeting the recFgene; Kane et al.,
2009)
• PCR water- Ultra Pure, Molecular Biology Grade (Quality Biological, Inc., Cat. No. 351-029-721)
• 1 L Filter System, PES, 0.2|am (Cat. No. 87006-066)
Strains
• Bacillus atrophaeus (ATCC#9372) ~10 /ml spore suspension (Apex Laboratories, Cat. No. RBC-
343-E8)
• Bacillus globigii, dry spore preparation, Critical Reagents Program
Supplies
General supplies:
• Gloves
• Bleach wipes
• 10% bleach (prepared daily) squeeze bottle
• Wipes
• Waste Coffin
• Zip lock bags (large ~20" x28", medium ~12"x 16", small ~7" x8")
• Sharps waste container
• Absorbent pad
• Medium and Large biohazard bag(s) and rubber band(s)
• Sterile scalpels
• Isopropyl alcohol squeeze bottle
• Deionized water squeeze bottle
• Autoclave tape
• Large photo-tray or similar tray for transport of racks
• Marker Pen
K-7
-------
• Timer
• Disposable pipette tips: 1 ml, 200 (oL, and 10
•
Serological pipets: 5 ml, 10 ml (with 13 ml graduation), 25 ml, and 50 ml
For culture analysis:
• ISA plates (see preparation procedure in protocol Annex D)
• Lazy-L spreaders (GSS, Cat. No. 101100-886)
• MicroFunnel Filter Funnels (0.45 urn MCE membrane) (Pall Sciences, Cat. No. 4800 or VWR, Cat.
No. 28143-544)
For RV-PCR analysis:
• 30 ml screw cap tubes (E&K Scientific, Cat. No. EK-T324S)
• Disposable nylon forceps (GSS, Cat. No. 12576-933)
• Monofilament polyester mesh disc (McMaster Carr, Cat. No. 93185 T17, or 2"x2" cut squares from
mesh sheets, McMaster Carr, Cat. No. 9218T13)
• Whatman Autocups (VWR, Cat. No. 1602-0465)
• Polyethylene caps, blue with pull-tabs (McMaster Carr, Cat. No. 94075K56) in beaker, for
vortexing and incubation steps
• Polyethylene caps, red, tapered (Caplugs, Cat. No. T-14, red), for covering filter cups during liquid
transfer steps
• Polyethylene quick turn tube fittings (Ark-Plas Products, Cat. No. 51525K365)
• 96-well 2 ml Bioblocks (E&K Scientific, Cat. No. 662000)
• Reagent reservoirs (Thermo-Fisher, Cat. No. 8086)
• Disposable serological pipettes: 25, 10, and 5 ml
• Single tube holder
• Screw cap tubes, 2 ml (GSS, Cat. No. 20170-237)
• 96 well rack(s) for 2 ml tubes (8x12 layout)
• Foil plate seals (E&K Scientific, Cat. No. T592100)
• Adhesive plate sealers (Edge Bio, Cat. No. 48461)
ForPCR:
• PCR plates (Applied Biosystems, Cat. No. 4346906)
• PCR plate seals (Applied Biosystems, Cat. No. 4311971)
• 96 well plate holders, Costar-black (GSS, Cat. No. 29442-922)
• Optical seals (Applied Biosystems, Cat. No. 4311971)
Laboratory set-up
Put PPE (personal protective equipment) on: lab coat, safety glasses, double gloves.
Prepare fresh bleach solution (1 volume bleach + 9 volumes water). Date and label with
initials.
Clean/bleach Biosafety Cabinet (BSC) and bench surfaces.
All sample manipulations are performed in the BSC.
K-8
-------
Preparation of spore dilution for positive wipe control samples
1. Place original stock spore suspension (approx. 108/ml_; check vial for measured concentration) in
rack in bag
2. Place bagged rack on the plate vortexer for 20 min on setting 7
3. After vortexing, in a 2 ml screw-cap tube, aliquot 100 uL of the 108/ml_ stock spore suspension into
900 uL of PBST to yield a working stock with concentration of 107/ml_
4. Vortex on single tube vortexer at 2,000 rpm or high speed for 1 min
5. Aliquot 500 uL of the working stock to a 15 ml conical tube containing 4.5 ml of PBST tube to yield
a spore concentration of 106/ml_
6. Vortex for 1 min
7. Aliquot 3 ml of the 106/ml_ spore stock, and add to 27 ml of PBST to a 50 ml conical tube to yield a
spore concentration of 105/ml_
8. Vortex for 1 min
9. Aliquot 1 ml of 105/ml_ stock to 9 ml of PBST to yield 104/ml_ or 103/100 uL
10. Vortex 104/ml_ stock for 1 min, and aliquot 1 ml to 9 ml of PBST to yield 103/ml_ or 102/100 uL
11. Vortex 103/ml_ stock for 1 min, and aliquot 1 ml to 9 ml of PBST to yield 102/ml_ or 101/100 uL
12. To determine actual spore concentrations added to samples, plate on TSA plates as follows:
3 replicates each with 100 uL of 101 spore stock
3 replicates each with 100 uL of 102 spore stock
3 replicates each with 50 uL of 103 spore stock
13. Invert plates and incubate at 35°C overnight. Determine plate counts for BG and take average of 3
replicates, corrected for dilution, to determine spore density of spiking solution.
K-9
-------
Preparation of laboratory positive and negative wipe control samples
Note: One positive and one negative control wipe should be included per manifold (up to 22 samples).
1. Set-up control wipe tubes as follows: Open wipe package and separate each wipe using disposable
forceps. Add support to wipe and transfer to 30 ml tube. Repeat steps until you have all
assigned control wipe tubes.
2. Place tubes in racks.
3. Pre-wet wipes by adding 1.5 ml of PBST buffer to each wipe.
4. For negative control wipes: Do not add spores. Add 100 ul_ of PBST buffer.
5. For positive control wipes: Add 100 uL of 104 spores/mL, following rack-manifold layout.
6. Store control wipe tubes at 4°C overnight and process next day.
Set up for RV-PCR analysis:
1. 50 ml tube rack: Fill tube rack with 50 ml screw cap conical tubes.
2. Manifold: In BSC, assemble manifold by connecting upper part (with 24 openings) to lower part
(with port) using six Allen screws. Add filter cups to manifold. Place red cap in each filter cup. Verify
all filter cups are completely pushed down in manifold such that the filter cup bottom is touching the
top surface of the manifold. Prepare 2 bags for red filter cup caps (25 caps each), and two beakers
each containing 25 blue filter cup caps (50 total for each 24-cup manifold).
3. Vacuum: Prepare vacuum pump or house vacuum source; connect vacuum source to in-line filter,
and to waste container with fresh 10% bleach; If using external vacuum pump, tape pump exhaust
tube to BSC to vent exhaust inside BSC if needed.
4. Capping Tray(s): Add bottom caps to capping tray(s).
5. For TO Samples: In the BSC, for each sample or control, set up a 2 ml Eppendorf tube.
6. Tape filter-cup layout on outside glass window of the BSC.
Manual sample processing (TO)
Note: Sample processing and analysis flowcharts are shown in Annex E.
1. If wipe sample in 30 ml tube does not already have an internal mesh support, add support by
holding the wipe to the side of the tube with a sterile forceps while introducing the mesh support
inside the tube. The support keeps the wipe to the side of the tube and clear of pipetting activities,
and also improves efficiency of spore extraction during vortexing.
2. In the BSC, add 20 ml of cold Extraction Buffer (including Tween) to wipe samples placed in 30 ml
tubes in tube rack (up to 24 tubes per rack). Uncap one tube at a time, add 20 ml extraction buffer,
close tube, and place it back in tube rack. Document rack and tube layout, and rack and tube labels
in lab notebook.
3. Cap tubes in tube rack. Place tube rack in plastic bag and seal it. Transfer bagged tube rack to
platform vortexer (outside BSC).
4. Vortex samples for 20 min on platform vortexer, position 7.
5. After vortexing, transfer sample tube rack to BSC. Remove tube rack from plastic bag.
6. Vortex one sample tube on single-tube vortexer for 3-5 seconds. Uncap tube, using 25 ml
serological pipette transfer 14 ml to appropriate 50 ml tube (same position in 50 ml tube rack as in
30 ml tube rack). Dispose pipette in waste container. Cap sample tube, and place tube back in rack.
Change gloves.
7. Repeat step 6 for each sample tube.
8. Change waste bag if needed.
K-10
-------
9. Perform second spore extraction. Uncap each sample tube individually.
10. Add 14 ml of cold Extraction Buffer (without Tween). Recap tube.
11. After all tubes received 14 ml Extraction Buffer, cap tubes in tube rack. Place rack in plastic bag
and seal it. Transfer bagged tube rack to platform vortexer (outside BSC).
12. Repeat steps 4-7 above, except only vortex for 10 min instead of 20 min. After the second spore
extraction and transfer, each 50 ml tube should contain 28 ml.
13. Cap the original sample tube, and store at 4°C.
K-11
-------
RV-PCR sample processing
1. Place into a 35°C incubator, blue filter cup caps (pull-ring caps) in a zip lock bag (one for each filter
cup). Caps are easier to place on filter cups when pre-warmed.
2. Place into the BSC: 10 ml serological pipettes, filter cup manifold, with filter cups and red caps.
Label filter cups following the same format as for the tube rack layout. Document filter cup layout and
labels in lab notebook. Cap all filter cups with red caps.
3. Uncap individual 50 ml tube and mix contents by pipetting liquid up and down five or more times with
a 10 ml serological pipette. Put pipet in waste and place tube cap loosely on top of tube. Let the
suspension sit for 30 sec to allow large particles to settle. Using new 10 ml serological pipet with
graduation up to 13 ml, transfer 13 ml to the filter cup (same position in filter cup manifold as in 50
ml tube rack). Take care to not pick up large settled particles. Dispose pipette in waste container.
Replace red cap on filter cup, and cap 50 ml tube, and place in rack. Change gloves. The remaining
liquid in the 50 ml tube will be used for traditional culture analysis (see below) that could occur in
parallel with RV-PCR analysis. If not possible to process in parallel, store the 50 ml tubes at 4°C
until samples are plated.
4. Repeat step 3 for each filter cup. After transfers are complete, remove red filter cup caps and dispose
to waste.
5. Turn on vacuum pump at 10 psi. Complete filtration of liquid through filter cups. Turn off vacuum.
Place a new red cap on each filter cup.
6. Place into the BSC: 25 ml serological pipettes, and cold High Salt Wash Buffer (pH 6.0).
7. To each filter cup, transfer 20 ml of cold High Salt Wash Buffer (pH 6.0) using a 25 ml serological
pipette. Use new pipette for each filter cup, lifting red cap and replacing after addition. After transfers
are complete, remove red filter cup caps and dispose to waste. Turn on vacuum. After filtration is
complete, turn off vacuum, place new red cap on each filter cup.
8. Place into the BSC: 10 ml serological pipettes and cold 1X Wash Buffer (pH 7.4).
9. Transfer 7 ml of cold 1X Wash Buffer (pH 7.4) to each filter-cup using a 10 ml serological pipette.
Use new pipette for each filter cup, lifting red cap and replacing after addition. After transfers are
complete, remove red filter cup caps and dispose to waste. Turn on vacuum.
10. Complete filtration of liquid through filter cups. Turn off vacuum pump. Change gloves. Place red cap
on each filter cup.
11. Unscrew manifold top using Allen wrench. Break seal on manifold to ensure there is no vacuum, by
inserting a plate sealer between manifold top and bottom. Change gloves. Lift top part of manifold and
transfer to capping tray fitted with bottom caps. Press down to cap bottom of filter-cups. Change
gloves.
12. Place into the BSC: 5 ml serological pipettes, 200-|aL pipettor, 200-|aL tips, cold TSB medium, sharps
container, and warm filter cup caps. Prepare TSB medium in 50 ml conical tubes and keep on ice
before addition.
13. Pipette 3.5 ml of cold TSB medium into each filter cup using a 5 ml serological pipette. Use new
pipette for each filter cup, lifting red cap and replacing after addition.
14. Firmly press red caps into filter cups prior to vortexing.
15. Place capped filter-cup manifold in plastic bag. Bleach bag.
16. Vortex filter cups for 10 min on platform vortexer, setting 7.
17. Place into the BSC, 2 ml Eppendorf tubes in a 96-tube rack (8 rows x 12 columns format).
18. Label 2 ml tubes as Time 0 (TO) samples following the same layout as the filter cups and place in
same 8 rows x 3 columns format as the filter cup manifold. Document rack and tube layout, and
labels in lab notebook.
19. After vortexing, transfer filter-cup manifold in capping tray to BSC. Remove bag.
20. Uncap one filter cup at a time, and open the corresponding 2 ml Eppendorf tube. Using a 1 ml
pipettor, swirl pipette tip gently in filter cup, while gently pipetting up and down 5 or more times to mix
sample (and to avoid aerosol generation). Aliquot 1 ml out of each cup, and transfer to corresponding
2 ml Eppendorf tube, cap tube and place on ice.
21. On same filter cup sample, following the same aliquoting procedure, remove 100 uL and transfer to
pre-chilled 1.5 ml Eppendorf tube. Add 900 uL of cold 10 mM Tris buffer (pH 8). Cap 1.5 ml tube
K-12
-------
and keep on ice (or cold block). If sample cannot be heat-lysed within 30 min, store at -20°C until
ready to process by heat lysis. Cap filter cup firmly with blue cap. Change gloves.
22. Repeat steps 20-21 for each filter cup.
23. After aliquoting from each filter cup, place capped filter-cup manifold in plastic bag. Bleach bag.
24. Transfer bagged filter-cup manifold in capping tray to shaker incubator at 35°C, speed 230 rpm.
Incubate for 9 hr.
25. Process 1 ml TO samples in 2 ml tubes using Manual DMA Extraction and Purification Protocol as
described below.
26. Process 100 uL aliquots added to Tris buffer (1 ml total volume in 1.5 ml tubes) using Modified Heat
Lysis Protocol described below. If aliquots cannot be processed immediately, store tubes at -20°C
and then bring to room temperature before processing.
Traditional culture analysis of TO samples
1. For 50 ml tubes containing remaining extract (from step 3 above), mix contents by pipetting up and
down 5 times with 10 ml serological pipet. Let tube sit for 30 sec to allow coarse particles to settle.
Take a new 10 ml serological pipet with graduation up to 13 ml and transfer 13 ml to a new 50 ml
conical tube, being careful to not pick up any large, settled particulates. Check that tube labels
match.
2. Place tubes containing 13 ml extract into adapters of swinging bucket rotor of refrigerated centrifuge
(Eppendorf 581 OR or similar), with rotor adapters for 50 ml conical tubes. Centrifuge at 4,000 rpm
(3,220 x g) for 30 min at 4°C. Set acceleration to 9 (or high) and brake to 0. After centrifugation,
place tubes back in BSC, and transfer to tube rack in original sample layout.
3. Open one tube at a time, remove 9.5 ml with a 10 ml serological pipette (taking care to keep pipette
clear of tube bottom to avoid dislodging the pellet), and transfer the liquid to 15 ml tube for archive or
to waste. (Note: Pellet might not be visible). Recap tube, and return tube to rack. Change gloves.
Note: This produces the same concentration factor for both RV-PCR and culture analysis.
4. Repeat step 3 for remaining tubes.
Note: Do not let tubes sit for extended time after centrifugation, to avoid resuspension of pelleted
material.
5. After removing 9.5 ml supernatant from each tube, recap tube, and vortex each 50 ml tube on
single-tube vortexer on low setting (to avoid foam generation) for 20 sec or until pellet is resuspended
completely. Place tube back in tube rack.
6. Resuspend pellets for all 50 ml tubes.
7. Perform serial dilution of the concentrated suspension in Butterfield Buffer (BB). Keep dilution series
tubes for a given sample together. Keep same sample layout with eight rows per column.
8. Uncap 50 ml tube; mix contents by vortexing at low speed for 10-20 sec. Remove 0.5 ml of spore
elution suspension (10°) and place in one 15 ml conical tube containing 4.5 ml BB, making a 10~1
suspension. Recap the 10~1 tube and vortex on high for 30 sec.
9. Open cap of the 10~1 suspension and remove 0.5 ml of this suspension and place in a new 15 ml
conical tube containing 4.5 ml BB, making a 10~2 suspension. Recap the BB tube and vortex on high
for 30 sec. Note: This produces three spore suspensions: the initial concentrated suspension (no
dilution=10°) and two serial dilutions of this suspension in BB (10~1 and 10~2).
10. Repeat steps 8-9 for all samples.
11. Prior to plating onto TSA plates, vortex each 10"1 and 10"2 suspension for 10 sec on a single tube
vortexer set to high. For the 10° suspension, vortex for 20 sec on low setting to avoid foam
generation.
12. Using a 200-uL pipettor, remove 100 uL from the 10~2 suspension and place on to a plate of TSA
labeled 10~3. Repeat 2 more times with a new tip each time for a total of three inoculated plates.
Note: Plating of 100 uL is an additional 1:10 dilution of the 10~2 suspension resulting in a 10~3 dilution
on the plate.
K-13
-------
13. Spread the inoculum on each of the three 10"3-labeled TSA plates with a sterile Lazy-L cell spreader
for each replicate plate. Discard each spreader after use.
14. Vortex the 10~1 tube from the same sample for 10 sec on high, remove 100 uL from the 10~1
suspension with the 200 uL pipettor and place on to a plate of ISA labeled 10~2. Repeat 2 more times
for a total of three inoculated plates.
Note: Plating
on the plate.
Note: Plating of 100 uL is an additional 1:10 dilution of the 10~1 suspension resulting in a 10~2 dilution
15. Spread the inoculum on each of the three 10"2-labeled ISA plates with a Lazy-L cell spreader for each
replicate plate. Discard each spreader after use.
16. Mix the contents from the 10° tube by vortexing; remove 100 uL from the 10° suspension with the
200-uL pipettor and place on to a plate of ISA labeled 10~1. Repeat 2 more times for a total of three
inoculated plates.
Note: Plating
on the plate.
Note: Plating of 100 uL is an additional 1:10 dilution of the 10° suspension resulting in a 10~1 dilution
17. Spread the inoculum on each of the three 10"1-labeled ISA plates with a Lazy-L cell spreader for each
replicate plate. Discard each spreader after use.
18. Invert plates and incubate at 35°C for 18-24 hrs.
19. Perform filtration of spore suspension through MicroFunnel filter funnel. Set up filter funnels in
vacuum filtration manifold.
20. Moisten MicroFunnel membrane with 5 mL PBST; turn on vacuum (< 5 psi) and vacuum through the
filter. Turn vacuum off.
21. Pipet 10 mL of PBST buffer to filter funnel. Do not turn on vacuum.
22. Pipet 0.5 mL of 10° spore suspension to filter funnel, turn on vacuum (< 5 psi) and vacuum through
the filter. Turn vacuum off.
23. Rinse filter with 10 mL of PBST; turn on vacuum (< 5 psi) and vacuum through the filter. Turn vacuum
off.
24. Squeeze the walls of the MicroFunnel cup gently and separate the walls from the base holding the
filter. Remove each filter membrane with sterile forceps and place grid-side up on a TSA plate. Make
sure that the filter is in good contact with the surface of the agar. If an air pocket occurs under the
filter, use the sterile forceps to lift the edge of the filter to release the air pocket for better contact with
the agar. Repeat filter funnel protocol for each sample.
25. Incubate TSA plate with filter membrane at 35°C for 18-24 hrs.
26. For preparation of enrichment sample, transfer the remaining 10° spore suspension to a 15 mL
conical tube. Centrifuge at 4,000 rpm (3,220 x g) for 30 min at 4°C. Set acceleration to 9 (or high)
and brake to 0. Note: Use higher centrifugation speeds if supported by available instrumentation but
do not exceed tube allowances.
27. Remove tubes from rotor and set up in tube rack in same layout as for filter-cup manifold. Open one
tube at a time, transfer supernatant to waste using a 1 mL pipettor (taking care to keep pipette clear of
tube bottom to avoid dislodging the pellet), and transfer the pipette tip containing liquid to waste (Note:
Pellet might not be visible). Recap tube, and return tube to rack. Change gloves.
28. Repeat step 27 for remaining tubes.
Note: Do not let tubes sit for extended time after centrifugation, to avoid resuspension of pelleted
material.
29. Using a 5 mL serological pipette, transfer 5 mL of TSB medium to each 15 mL tube, using a new
pipette for each sample.
30. After removing supernatant from each tube, recap tube, and vortex 15 mL tube on single-tube
vortexer on medium setting (~1,500 rpm) for 10 sec or until pellet is resuspended completely. Place
tube back in tube rack.
31. Resuspend pellets for all 15 mL tubes.
32. Incubate 15 mL tubes with media at 35°C, with shaking (200 rpm) for 18-24 hrs.
33. Follow laboratory cleanup protocol in Annex A.
K-14
-------
Culture Data Results
1. Record colony-forming units (CPUs) per plate that are consistent with BG colony morphology
(non-spreading, circular, flat colonies approximately 2 mm in size with orange color). Colony
counts are most accurate when between 25 and 250. If any serial dilution plates or filter
membrane plates contain more than 250 colonies, record number as too numerous to count
(TNTC). If plates for all serial dilutions contain BG colonies that are TNTC, record this
information, as well as that the sample is positive for BG. Qualitative results are sufficient for
comparison between RV-PCR and culture methods; therefore, no corrective action is required if
only TNTC culture results are obtained for one or more samples.
For plates with <250 colonies, determine the average and standard deviation of the three
replicates, then convert average CPU to CFU/mL by multiplying by the dilution factor (see
below). For the serial dilution plates, use the dilution that yields CFU/plate between 25 and
250, if more than one dilution yields BG colonies. If serial dilution plates have <25 CFU per
plate, examine plates with filter membranes.
a. For 10° suspension, the dilution factor is 10 (since 100 (oL plated).
b. For 10~1 suspension, the dilution factor is 10 (since 100 (oL plated) x 10 (for 1/10
dilution), or 100.
c. For 10~2 suspension, the dilution factor is 10 (since 100 ^L plated) x 100 (for 1/100
dilution), or 1000.
d. For filter membrane plate, the average plate CFU is already in units of CFU/mL
since 1 ml of spore extract was filtered.
e. Convert CFU/mL to CFU/sample by multiplying CFU/mL by the total volume of
spore extract added to the sample (i.e., 33 mL). Determine average and standard
deviations for plate count data. Record both CFU/mL and CFU/sample.
Note: CFU/sample is calculated based on the total extract volume; however, this
result was compared qualitatively with the split sample analyzed by RV-PCR (13
mL).
f. CFU counts based on colony morphology/pigmentation can be confirmed if
necessary (see Confirmation of CFU values by real-time PCR analysis, #3 below).
2. If no BG colonies are identified from either dilution series plates or filter plates, examine the TSB
enrichment sample. If broth is not turbid, incubate for an additional 24 hours at 35°C at 2,000
rpm. If broth is turbid, record as positive growth, and streak for isolation of BG colonies onto
TSA plates using a sterile, disposable loop (3 replicate plates) and incubate at 35°C for 18-24
hrs. Examine plates and record if BG colonies are present after incubation.
3. Confirmation of CFU values by real-time PCR analysis (optional).
a. If BG CFU values are inconclusive based on colony morphology and orange
pigmentation, real-time PCR can be used for confirmation. Select 2-5 colonies from
plates for each sample. If isolated colonies are present on serial dilution plates,
select colonies from these plates. If no colonies are present, select colonies from
filter plates; finally, if no colonies are present on dilution or filter plates, select
colonies from enrichment broth plates.
K-15
-------
b. Follow LRN procedure: "Rapid preparation of cell lysates from culture-grown Gram-
positive bacteria for subsequent testing by fluorogenic 5' nuclease assay" (Boil Prep
protocol). Use real-time PCR recipe and conditions as outlined in Annex B.
Extended Enrichment Culture Analysis
Note: Due to low spore levels and the splitting of extract between culture and RV-PCR analysis,
discrepancies might occur between method results. The following protocol involves PCR analysis of
concentrated, purified enrichment culture samples remaining after conducting the modified LRN protocol
(i.e., after dilution series/plating, filter funnel plating and enrichment culture re-streaking and PCR analysis
of small aliquot [5 uL] of enrichment culture). By performing this protocol, a more accurate assessment of
positive and negative culture results could be obtained. The protocol should only be performed for
samples that are positive by RV-PCR and negative by the modified LRN protocols listed above.
1. Briefly vortex 15 mL enrichment culture (EC) tube.
2. Aliquot 1 mLto labeled 2 mL Eppendorf tube.
3. Centrifuge at 4°C for 10-15 min at 14,000 rpm.
4. Remove supernatant.
5. Repeat steps 2-4 by taking another aliquot from same EC tube and combining pellets together the
remaining EC volume has been processed.
6. After centrifugation is complete, add 1 mL 10 mM Tris buffer (pH 8) and resuspend the pellet by
vortexing.
7. Process using the 1 mL Manual DNA Extraction and Purification Protocol described below.
Manual DNA Extraction and Purification Protocol
1. After incubation for 9 hr, take filter-cup manifold out of incubator.
2. Vortex filter cups for 10 min on platform vortexer, setting 7.
3. Transfer filter-cup manifold to BSC.
4. Set up 2 mL Eppendorf tubes in a 96-well tube rack (8x12) and verify that 2 mL tube labels match
the filter cup layout. Maintain the tube layout when transferring tubes between magnetic stand and
96-well tube rack. Do not use the 1.5 mL Eppendorf tubes.
5. Uncap one filter cup at a time, and open the corresponding 2 mL tube. Using a 1 mL pipettor, swirl
pipette tip gently in filter cup, while gently pipetting up and down 5 or more times to mix sample (and
to avoid aerosol generation). Aliquot 1 mL out of each cup, and transfer to corresponding 2 mL tube in
the 96-well tube rack (T9 samples); Cap tube and place on ice.
6. On same filter cup sample, following the same aliquoting procedure, remove 100 uL and transfer to
pre-chilled 1.5 mL Eppendorf tube containing cold 900 uL of 10 mM Tris buffer (pH 8). Cap 1.5 mL
tube and keep on ice. Cap filter cup firmly with blue cap. Change gloves.
7. Repeat steps 5-6 for each filter cup. Store 2 mL tubes at -20°C if the remaining steps of the protocol
cannot be conducted immediately, otherwise keep on ice.
Note: 1 mL TO and T9 samples are processed using the same protocol as follows:
K-16
-------
DMA extraction and purification phase:
Note: Prepare Lysis Buffer with Anti-Foam Reagent according to manufacturer's instructions. Prepare
Alcohol Wash solution by adding ethanol and isopropanol according to manufacturer's instructions.
8. If samples were stored at-20°C, thaw, and then centrifuge at 13,000-14,000 rpm (4°C)for5 min.
Remove 900 uL supernatant taking care to not disturb the pellet. Add 900 uL 10 mM Tris buffer (pH
8) and completely resuspend pellet. Process one tube at a time.
Note: For the following steps when processing 1-2 ml volumes in 2 ml tubes (during Lysis Buffer and
PMP addition steps and buffer removal steps), change gloves between each sample.
9. Vortex PMPs for on high setting (~2,500 rpm) for 30 to 60 sec, or until resuspended. During use,
verify that PMPs remain suspended, and repeat vortex step, if necessary.
10. 1st Lysis: Uncap one tube individually and add 600 (o,L of PMPs (lysis buffer + magnetic beads) to
each tube (containing 1 mL culture), and mix by pipetting up and down 5 times.
11. Take new pipet tip and add 360 (oL of Lysis Buffer to each tube. Cap tube and mix by vortexing on
high for 10 sec. Change gloves between samples.
12. Repeat steps 10 and 11 for all TO and T9 tubes.
13. Vortex each tube for 10 sec (on high) and place on magnetic stand. After all tubes are in the stand,
invert tubes 180 degrees (upside-down) turning away from you, then right side-up, then upside down
toward you, then to right side-up (caps up) position. This step allows all PMPs to contact the magnet.
Check if beads remain in the caps and if so, repeat the tube inversion cycle again. Let tubes sit for 5-
10 sec before opening.
14. Uncap each tube individually and using a 1 mL pipettor, place pipet tip in bottom of 2 mLtube, taking
care not to disturb PMPs. Withdraw all liquid and discard pipet tip with liquid to waste. Recap tube.
Change gloves.
15. 2nd Lysis: Uncap each tube individually and add 360 \JL of Lysis Buffer. Cap and vortex on high for
10 sec, and transfer to 96-well tube rack.
16. After adding Lysis Buffer solution to all tubes, vortex each tube for 10 sec (on high) and place on
magnetic stand. After all tubes are in the stand, follow tube inversion cycle as described in step 13.
17. Remove liquid as described in step 14. Recap tube.
18. 1st Salt Wash: Uncap each tube individually and add 360 (oL of Salt Wash solution. Cap and vortex
on high for 10 sec, and transfer to 96-well tube rack.
19. After adding Salt Wash solution to all tubes, vortex each tube for 10 sec (on high) and place on
magnetic stand. After all tubes are in the stand, follow tube inversion cycle as described in step 13.
20. Remove liquid as described in step 14. Recap tube.
21. 2nd Salt Wash: Repeat steps 18-20 for all tubes.
22. 1st Alcohol Wash: Uncap each tube individually and add 500 ^L of Alcohol Wash solution. Cap and
vortex on high for 10 sec, and transfer to 96-well tube rack.
23. After adding Alcohol Wash solution to all tubes, vortex each tube 10 sec (on high) and place on
magnetic stand. After all tubes are in the stand, follow tube inversion cycle as described in step 13.
24. Remove liquid as described in step 14. Recap tube.
25. 2nd Alcohol Wash: Repeat steps 22-24 for all tubes.
26. 3rd Alcohol Wash: Repeat steps 22-24 for all tubes.
27. Air dry for 2 min.
28. Heat dry on heat block at 80°C until samples are dry (30-60 min). Allow all alcohol solution to
evaporate since alcohol could interfere with real-time PCR analysis.
DNA elution phase:
K-17
-------
29. Move 2 ml Eppendorf tubes out of heat block and add 200 (oL of Elution Buffer to each tube in 96-well
tube rack. Mix briefly by vortex, place on magnet, collect liquid with a micropipette, and transfer to a
clean 2 ml tube (typically 80 (oL are collected) following the tube layout (check tube labels to keep
correct order). Visually verify absence of magnetic bead (PMP) carryover during final transfer. If
magnetic bead carryover occurred, place 2 ml tube on magnet, collect liquid, and transfer to a clean
2 ml tube (ensure correct tube labels during transfer).
30. Store DMA extract samples at 4°C until preparation for real-time PCR analysis (use photo-tray to
transport 2 ml tubes).
31. Follow laboratory cleanup protocol in Annex A.
Modified Heat Lysis Protocol for TO and T9 aliquots
1. For 1.5 ml Eppendorf tubes containing 100 (oL sample aliquot and 900 (oL 10 mM Tris buffer (pH 8),
keep tubes on ice until subsequent processing. If frozen, thaw tube contents prior to conducting steps
2-8 below.
2. Centrifuge at 13,000-14,000 rpm (maximum speed) at 4°C for 5 min.
3. Remove 900 (oL supernatant and resuspend pellet in remaining 100 (oL (keep on ice when not
aliquoting or vortexing). Take care not to disturb pellet and repeat centrifugation if material is not firmly
pelleted.
4. Heat for 10 min at 95°C.
5. Place tubes on ice to cool (1-2 min).
6. Centrifuge at 13,000-14,000 rpm (maximum speed) at 4°C for 5 min.
7. Transfer supernatant to new 1.5 ml tube taking care to not disturb pellet.
8. Store at -20°C until ready to perform PCR analysis.
Real-time PCR analysis of TO and T9 aliquots
1. In clean BSC or PCR-preparation hood, set up 96-well PCR plate with PCR mix (20 pL mix per well)
according to plate layout. For each sample, 3 replicate BG reactions were performed. Replicates
will follow format, A1, A2, A3, followed by the next set of replicates (A4-A6) rather than A1, B1, C1
(followed by D1-F1). Note: Columns are numbered 1-12 and rows are A-H. Leave columns 10 and
11 empty. Add PCR mix to the wells in column 12 for the DMA standard controls.
Note: One 96-well plate will accommodate samples and controls from one manifold (24 x 3 replicates
= 72 total, plus PCR standard controls).
TO and T9 extracts from the same sample should be analyzed on the same PCR plate in order to
make more consistent comparison between sample time-points.
2. Seal PCR plate and transfer to BSC.
3. Transfer 2 ml DMA extract tubes to the BSC.
4. Perform 1:10 dilution of TO and T9 samples:
Add 180 (oL of PCR-grade water to clean 2 ml Eppendorf tubes in a 96-well tube rack (8x12) and
label to match with the tube layout and labels of the 2 ml DMA extract tubes. Add "1/10 dil" to label.
Perform one dilution at a time. Mix DMA extract up and down 5 times and transfer 20 (oL to
appropriate 2 ml tube, maintaining the tube layout. Cap undiluted DMA extract tube and return to
tube rack. Cap diluted DMA extract tube, vortex briefly and place in appropriate tube rack. After all
dilutions are completed, store remaining undiluted extract tubes at -20°C. Store diluted DMA extract
tubes at 4°C until PCR is performed.
K-18
-------
5. Add diluted extract to PCR plate.
Open one diluted DMA extract tube at a time, and mix up and down 10 times and transfer 5 (oL to the
appropriate well on the PCR plate (containing 20 (oL of PCR mix).
6. Repeat 2 times for each sample or control diluted DMA extract following the PCR plate layout to yield
3 replicates per sample or control. When all transfers are complete, seal PCR plate with clear seal.
Change gloves. Store remaining diluted samples at -20°C.
7. Centrifuge sealed PCR plate for 1 min at 2,000 rpm.
8. Remove plate and place on photo-tray, change gloves, transfer PCR plate to ABI thermocycler.
9. Run PCR (see Annex B).
10. After thermal cycling completion, discard sealed PCR plate to waste. Autoclave. PCR plates with
amplified product are never to be opened in the laboratory.
11. Follow laboratory cleanup protocol in Annex A.
Data Analysis and Reporting
PCR standards:
DMA standards were generated for BG isolated from the CRP BG spore stock. BG DMA standards were
analyzed on each sample PCR plate. The DMA is prepared as follows: DMA is extracted from cultured
cells using a MasterPure™ Complete DMA and RNA Purification Kit (Epicentre® Biotechnologies Inc.),
followed by RNase treatment. The DMA concentration was measured with a Qubit™ fluorometer using
the PicoGreen™ assay (Invitrogen™, Quant-iT™ dsDNA HS assay kit for Qubit fluorometer, Cat. No.
Q32854). DMA quality was assessed by spectrophotometric analysis; the UV absorbance 260 to 280 ratio
should be > 1.8. Standard concentrations prepared in PCR-grade water will range from a high level, 1
ng/reaction (5 uL of a 200 pg/ uL stock solution) and a low level, 100 fg/reaction (5 uL of a 20 fg/uL stock
solution). Triplicate standards for each level was included on each sample plate, along with a negative
(no-template) control.
Results from genomic DMA standards should be within 2 Ct of expected values (reference Ct values)
based on previous testing (for at least 2 of 3 standards for each concentration), and the limit of detection
should be at least 100 fg. Each lab should establish their own reference Ct values on their ABI 7500 Fast
instrument. Automated analysis settings for baseline and threshold should be used. The absolute Ct
values are used qualitatively to determine whether high (1 ng) and low (100 fg) levels are consistently
detected and the 100 fg limit of detection is achieved. In addition, the no-template control should be non-
detect.
RV-PCR Results Interpretation
Results from negative control samples (laboratory controls and field blanks) should be non-detect.
Results from replicate PCR analyses from positive control samples should be within 3 Cts for ACt and
Ct(T9).
If the Ct(TO) and/or Ct(T9) is non-reactive (non-detect) for 45 cycles, the value(s) is arbitrarily set to 45 in
order to calculate ACt. For the BOTE, using B. atrophaeus (BG) spores (from CRP) on field samples
K-19
-------
(pre- and post-decon), the PCR performance criteria were set as ACt [Ct(TO)-Ct(T9)] > 6 (to represent at
least a two log difference in DMA concentration), and an T9 Ct of < 39 was used for BOTE data analysis.
The ACt > 9 criterion represents an increase in DMA concentration at T9 relative to detectable DMA at TO,
if any, as a result of the presence of viable spores in the sample that germinated and propagated during
the 9 hr of incubation in growth medium. If no PCR data is obtained for one or more replicates, these data
should not be averaged to obtain an average Ct value. For example, if 2 of 3 replicates yield PCR data,
these two data alone are used to generate an average Ct value. However, attempts should be made to
repeat the PCR analysis until consistent results are obtained for all three replicates.
RV-PCR and Culture Results Reporting
Qualitative results or positive/negative (presence/absence) data for BG spores were reported for both RV-
PCR and culture analyses for each sample and control. In addition, the data reports by event included
the following for each sample and control: 1) Average ACt value (with standard deviation, SD); 2) Average
CPU/sample value corrected for dilution factor; 3) Source of culture result; and 4) Data from PCR
confirmation of culture result. A report template is shown in Annex F. In addition, the results for all three
events were summarized using the summary form shown in Annex F. Sample barcodes from field
samples and blanks were linked with sample data using a barcode scanner/software system, and final
reports included sample barcode information. Excel spreadsheets with results were provided in order to
link laboratory data with field identification data. While quantitative data were provided, the comparison
of RV-PCR and culture methods was based on qualitative results (positive/negative or
presence/absence).
K-20
-------
Annex A: Lab Cleanup Procedure
Lab Cleanup Protocol
• Dispose of all biological materials in autoclave bags (double bagged)
• Autoclave all waste materials at end of work for the day
• Decontaminate counters and all equipment with fresh bleach (1 volume water and 9 volumes
commercial bleach), followed by 70% isopropanol, and finally rinse with Dl water.
K-21
-------
Annex B: Bacillus atrophaeus (BG) Real-time PCR assay conditions
Real-time PCR analysis of Bacillus atrophaeus subsp. globigii (BG) on the
AB 7500 Fast Real-Time PCR Thermocycler (in fast mode)
Reagents:
Primers (Biosearch Technologies, Cat. No. P1R-1) and probes (Biosearch Technologies, Cat. No.
DLO-FB1-1)
TaqMan 2X Universal Master Mix with UNG and AmpliTaq Gold (Applied Biosystems, Cat. No.
4305719)
Molecular Biology grade distilled water, RNase- and DNase-free (Teknova, Cat. No. W3350)
PCR Reagents Mix:
Reagent
TaqMan 2X Universal PCR Master Mix
Forward primer, Bg42F, 10 |j,M
Reverse primer, Bg104R, 10 |j,M
Probe, Bg60F/BHQ1, 1 |aM
Molecular Biology Grade Water
Template DNA
TOTAL
Volume (|J_)
12.5
1.25
1.25
2.5
2.5
5
25
Final
Concentration
1X
0.5|aM
0.5 |aM
0.1 |aM
N/A
Variable
Equipment:
ABI 7500 Fast Real-Time PCR Thermocycler
Optical Fast 96-well plates (Applied Biosystems, Cat. No. 4366932)
Optical adhesive plate covers (Applied Biosystems, Cat. No. 4311971)
Thermocycling Conditions:
STEPS
UNG incubation
AmpliTaq Gold
PCR, 45 cycles
K-22
-------
Temperature
Time
HOLD
50°C
2 min
activation
HOLD
95°C
10 min
Denaturation
95°C
5s
Annealing/extension
60°C
20s
*Fast Ramp: 3.5°C/s up and 3.5°C/s down.
The 8. atrophaeus (BG) assay (forward primer, Bg42F: CGCGCCCGAGGACTTAA; reverse primer,
Bg104R: ATGTCAAGAAACCGCCGTC; and fluorogenic probe, Bg60F/BHQ1: FAM-
TCTCGTAAAGGGCAGCCCGCAAG-BHQ1; 63-bp amplicon) was developed at Lawrence Livermore
National Laboratory (LLNL) to specifically target the recF gene of 8. atrophaeus.
K-23
-------
Annex C: Consumables
Description
Catalog #
Units
Supplier
Item part
Number
PCR Materials
PCR plates
PCR plates seals
TaqMan Universal PCR
master mix
PCR probes
PCR primers (pair)
96 Well Hard Plates Costar-
black 1 00/cs
2.0 ml Screw cap tubes
(500 bag; Eppendorf)
PCR water- ultra pure,
Molecular Biology Grade)
4346906 or
4366932
4311971
4305719
DLO-FB1-1
P1R-1
29442-922
20170-237
W3350-06
20/box or
cs of 1 0
boxes
1 00/box
(10)5mL
bottles
1 |j,mol
1 |j,mol
cs
bag
1 L
Applied
Biosystems, Inc.
Applied
Biosystems, Inc.
Applied
Biosystems Inc.
Biosearch
Technologies
Biosearch
Technologies
GSS
GSS
Teknova
4346906 or
4366932
4311971
4305719
DLO-FB1-1
P1R-1
29442-922
20170-237
W3350-06
Culture Materials
ISA (BD Bacto)
TSB Media (BD Difco)
Petri dishes 60x1 5
LAZY-L-SPREADERS
STERILE case of 500
10 uL Inoculating Loop
Yellow, Sterile
MicroFunnel Filter Funnels
(0.45 urn MCE membrane;
236950
211825
25373-085
101100-886
12000-810
4800
500 g
500 g
cs
500/cs
1 000/cs
25/bg
50/cs
VWR
VWR
GSS
GSS
VWR
VWR
90002-706
90000-378
25373-085
101100-886
12000-810
28143-544
K-24
-------
Pall Sciences)
Vacuum Filtration Manifold
(3-place manifold, 47 mm,
stainless steel)
xx2504735
ea
Millipore
xx2504735
Buffers/Reagents
Potassium phosphate,
KH2PO4
Tween® 80 9005-65-6
EtOH 200 proof absolute,
anhydrous
PBST Buffer, Sterile
Butter-field's Buffer, Sterile
NaOH
Filter system (PES, 0.2|am)
P0662-500G
103170
111ACS200
P0201
U190
S8045-500G
87006-066
500 g / bottle
100mL
1 L
1 L
0.5L(10/cs)
500 g
1 L(pkof 12)
Sigma-Aldrich
MP Biomedicals
Trans Meridian
UCI/QUANTIUM
CHEM
Teknova
Hardy
Diagnostics
Sigma
VWR
P0662-500G
103170
111ACS200
P0201
U190
S8045-500G
87006-066
K-25
-------
DMA extraction and purification reagents
Promega Magnesil Kit
Promega Salt Wash
Promega Paramagnetic
Particles
Promega Lysis Buffer
Promega Anti-foam Reagent
Promega Elution Buffer
Promega Alcohol Wash,
Blood
Promega
Promega
Promega
Promega
Promega
Promega
Promega
MD1360
MD1401
MD1441
MD1392
MD1431
MD1421
MD1411
VWR
VWR
VWR
VWR
VWR
VWR
VWR
PAMD1360
PAMD1401
PAMD1441
PAMD1392
PAMD1431
PAMD1421
PAMD1411
Vacuum filtration materials
Tygon Tubing 1/4" ID 1/2"
OD
Nalgene 2 Liter Bottle
Nalgene Venting Cap
Vacuum pump, Model DOA-
P704, or similar
3870E DOOR BELLOWS
ASSEMBLY KIT - Part for
vacuum pump
3870E AIR JET VALVE
(BLACK TOP) - Part for
vacuum pump
3870E DOOR GASKET
(Door Seal) - Part for
vacuum pump
3870E FILL/VENT MESH
CHAMBER FILTER
(Stainless Steel) - Part for
vacuum pump
3870E PLUNGER VALVE
KIT (3mm) - Part for vacuum
BH-95636-00
BH06257-20
BH06258-10
EW-07061-40
TUK030-2150
TUJ034-2149
TUG074-2146
MIF062-2126
TUK082-2155
bx
ea
ea
ea
ea
ea
ea
ea
ea
Cole-Parmer
Cole-Parmer
Cole-Parmer
Cole-Parmer
A & A Dental &
Medical Services
A & A Dental &
Medical Services
A & A Dental &
Medical Services
A & A Dental &
Medical Services
A & A Dental &
BH-95636-00
BH06257-20
BH06258-10
EW-07061-40
TUK030-2150
TUJ034-2149
TUG074-2146
MIF062-2126
TUK082-2155
K-26
-------
pump
3870E PLUNGER VALVE
KIT (6mm) - for vacuum
pump
3870E SAFETY VALVE (40
PSI) - for vacuum pump
TUK086-2156
TUV065-2166
ea
ea
Medical Services
A & A Dental &
Medical Services
A & A Dental &
Medical Services
TUK086-2156
TUV065-2166
K-27
-------
Sample processing materials
30 ml Screw blue-cap tubes
Polyethylene caps, blue with
pull-rings
Polyethylene caps, red,
tapered
Monofilament polyester
mesh disc
Monofilament polyester
mesh sheet, cut into 2" x 2"
squares
Quick turn tube fitting
polypropylene, female cap
Whatman Autocups
[available from VWR under
misc-supplies]
Disposable Nylon Forceps
100 ml Reagent Reservoirs
(1 00/case)
Bioblocks for dilutions ( 96
wells/ 2ml_ per well)
T3242S
57935K16
T-14, red
93185T17
9218T13
AP17FLPOOP
1602-0465
12576-933
8086
662000
100/cs
100/pk
100/pk
ea
12'x1'sheet
pk
250/cs
1 00/pack
cs
20/case
E & K Scientific
McMaster Carr
Caplugs
McMaster Carr
McMaster Carr
Ark-Plas
Products, Inc.
VWR
GSS
Thermo Fisher
E & K Scientific
T3242S
57935K16
T-14, red
93185T17
9218T13
AP17FLPOOP
1502-
0465(Whatman)
12576-933
8086
662000
Pipettors and tips for PCR and DMA extraction and purification
1 000 uL Filter ITS Tips
200 uL Filter ITS Tips
20 uL Filter ITS Tips
L-1000 ITS Pi petto r
L-100LTSPipettor
L-200 ITS Pipettor
L-20 ITS Pipettor
L- 10 ITS Pipettor
RT-L10F
RT-L1000F
RT-L200F
L1 000 ITS
L100LTS
L200 ITS
L20 ITS
L10LTS
cs
cs
cs
ea
ea
ea
ea
ea
Rainin
Rainin
Rainin
Rainin
Rainin
Rainin
Rainin
Rainin
RT-L20F
RT-L1000F
RT-L200F
L1 000 ITS
L 100 ITS
L200 ITS
L20 ITS
L1 0 ITS
K-28
-------
Carousel Stand
CR-7
ea
Rainin
CR-7
General laboratory supplies
Diamond Grip Latex Gloves
X-Small
Diamond Grip Latex Gloves
Small
Diamond Grip Latex Gloves
Medium
Diamond Grip Latex Gloves
Large
Diamond Grip Latex Gloves
X-Large
VWR Autoclave Bags 25x35
VWR 5.0 mL Freezer Vials
Corning 50 mL conical tubes
Corning 15 mL conical tubes
BD Sharps Containers
32916-498
32916-506
32916-500
32916-502
32916-503
14220-042
66008-400
21008-714
21008-678
BD305551
cs
cs
cs
cs
cs
cs
bag
cs
cs
cs
GSS
GSS
GSS
GSS
GSS
GSS
GSS
GSS
GSS
GSS
32916-498
32916-506
32916-500
32916-502
32916-503
14220-032
66008-400
89004-367
89004-370
BD305551
General laboratory supplies
Kaydry EX-L Wipers
Bleach gallon bottles case
Disposable lab coats w/cuffs
VWR, Bleach wipes 10
pkg/cs
Autoclaved ampoules
21903-021
37001-060
CV9841N
47735-634
101101-788
cs
cs
cs
1 0pkg/cs
20 ampoules/
box
GSS
GSS
GSS
GSS
GSS
21903-021
37001-060
CV9841N
37001-060
14220-030
Serological pipettor and tips for manual RV-PCR
K-29
-------
Portable pipet aid
50 ml serological pipettes,
VWR
10 ml serological pipettes,
VWR
25 ml serological pipettes,
VWR
5 ml serological pipettes,
VWR
4-000-100
29442-440
29442-430
29442-436
29442-422
ea
bag
bag
bag
bag
VWR
GSS
GSS
GSS
GSS
4-000-100
53283-712
53283-708
53283-710
53283-706
Sample materials
Wipes (Kendall Versalon)
8042
3000/case
GSS
89004-507
Acronyms: bx, box; cs, case; ea, each; pk, package
Government Scientific source (GSS)
K-30
-------
Annex D: Buffers and Media Preparation
1 M Sodium Hydroxide Solution (NaOH)
Note: Label all bottles and flasks with reagent name, date and initials.
1 Add 10 g NaOH to 200 ml Milli-Q H2O in a 500 ml glass bottle
2 Mix with magnetic stirrer
3 After NaOH pellets are dissolved, bring final volume to 250 ml with Milli-Q H2O
10X Wash Buffer (250 mM KH2PO4, pH 7.4)
Note: Label all bottles and flasks with reagent name, date and initials.
Addition of NaOH is required if Milli-Q H20 has low pH value (pH ~5).
1 Dissolve 34 g KH2PO4 in 500 ml Milli-Q H2O
2 Add enough 1 M NaOH to bring to pH 7.2 (> 200 ml of 1 M NaOH)
3 Bring volume to 1 L with Milli-Q H2O
4 Filter sterilize using 1 L, 0.22 micron PES filtering system with disposable bottle
1X Wash Buffer (25 mM KH2PO4, pH 7.4)
Note: Label all bottles and flasks with reagent name, pH level, date and initials.
1 Add 100 ml 10X Wash Buffer to 900 ml Milli-Q H2O
2 Mix with magnetic stirrer, when mixed, measure pH
3 Filter sterilize using 1 L, 0.22 micron PES filtering system with disposable bottle
Butterfield's Buffer (0.31 mM KH2PO4, pH 7.2; Hardy Diagnostics Cat. No. U190)
Note: This equates to 42.5 mg KH2PO4/L.
Label all bottles and flasks with reagent name, pH level, date and initials.
1 Add 12.5 ml 1X Wash Buffer (pH 7.4) and bring volume to 1 L with Milli-Q H2O
2 Mix with magnetic stirrer, when mixed, measure pH; adjust to 7.2 if necessary
3 Autoclave, or filter sterilize using 1 L, 0.22 micron PES filtering system with disposable bottle
K-31
-------
Phosphate-Buffered Saline Tween-20 (PBST) Buffer (Teknova, Cat. No. P0201,1 L, sterile)
137mMNaCI
2.7 mM KCI
4.3 mM Na2HP04
1.4 mM KH2PO4
0.05% Tween-20
High Salt Wash Buffer (207 mM KH2PO4, pH 6.0)
Note: Label all bottles and flasks with reagent name, date and initials. Addition of NaOH is required if
Milli-Q H20 has low pH value (pH ~5).
1 Dissolve 28.2 g KH2PO4 in 500 ml Milli-Q H2O
2 Add enough 1 M NaOH to bring to pH 6.0 (> 100 ml of 1 M NaOH)
3 Bring volume to 1 L with Milli-Q H2O; Mix well.
4 Filter sterilize using 1 L, 0.22 micron PES filtering system with disposable bottle
K-32
-------
Extraction Buffer w/Tween (0.25 mM KH2PO4, 30% EtOH, 0.05% Tween 80)
Note: Label all bottles and flasks with reagent name, date and initials.
1 Add 500 ml Milli-Q H2O to 1 L bottle or flask
2 Add 1 ml 10X Wash Buffer
3 Add 300 ml 200 proof ethanol
4 Add 0.5 ml Tween® 80
5 Bring volume to 1 L with Milli-Q H2O; Mix well.
6 Filter sterilize using 1 L, 0.22 micron PES system with disposable bottle
Extraction Buffer w/o Tween (0.25 mM KH2PO4, 30% EtOH)
Note: Label all bottles and flasks with reagent name, date and initials.
1 Add 500 ml Milli-Q H2O to 1 L bottle or flask
2 Add 1 ml 10X Wash Buffer
3 Add 300 ml 200 proof ethanol
4 Bring volume to 1 L with Milli-Q H2O; Mix well.
5 Filter sterilize using 1 L, 0.22 micron PES system with disposable bottle
Tryptic Soy Broth (TSB) medium
Note: Label all bottles and flasks with reagent name, date and initials.
1 Add 20 g Bacto Tryptic Soy Broth powder in 1 L glass bottle with screw cap
2 Add 500 ml Milli-Q H2O
3 Place on hotplate and gently mix with spin bar.
4 Autoclave (121°C at >15 psi for 15 minutes)
Tryptic Soy Agar (TSA)
1 Add 20 g Bacto Tryptic Soy Broth powder in 1 L bottle or flask
2 Add 8 g Bacto Agar powder
3 Add 500 ml Milli-Q H2O
4 Place on hotplate and gently mix with spin bar.
5 Autoclave (121°C at >15 psi for 15 minutes)
6 Place on hotplate and gently mix with spin bar.
7 Allow agarto cool down to 45°C before pouring.
8 In BSC, pour 20 ml of solution in each Petri dish using a serological pipette.
K-33
-------
1 Annex E: Sample processing protocol for the BOTE
Vvlpewith support in 30-mL sample tube
Add 20-mL Extraction Buffer with Tween
Vortex 20 min
2
Mix briefly; Transfer 14-mL to 50-mL tube
Add 14-mL Extraction Bufferw/o Tween
Vortex 10 min
Mix briefly; Transfer 14-mL to same 50-mL tube
Mix by pipetting; Transfer 13-mL to filter cup
forRV-PCR analysis
Manual RV-PCR Protocol
Transfer 13-rnL from remaining extract solution
to another 50-mL tube
Centrifuge 30 min, 4000 rpm, 4°C
Remove 9.5 mLto waste (or archive)
Suspend pellet in remaining 3.5 mL
(10° suspension)
Use0.5 mL of 10° suspension fordilution series
Plate3 reps of 100 uL onTSA plates (10'1 final)
Perform dilution series
(0.5 mL in 4.5 mL)
Butterfield Buffer (10-1 and 10'2)
Incubate at 35°C, 18-24 hr
Count BG colonies
~2.7 rnLforfurther processing (10° suspension)
_L
FilterO.5 mL through
Microfunnel filter
Place filter on TSA plate
(10° final)
Transfer remaining 10° suspension
to 15-mL tube;
Centrifuge 30 min, 4000 rpm, 4°C;
Transfer supernatant to waste
Plate 3 reps of 100 uL on TSA
plates (10-2 and 10~3 final)
BG= Bacillus atrophaeus subsp. globigii
lncubateat35°C,18-24 hr
Count BG colonies
Add5-mLTSB medium; Suspend
pellet; Incubate at 35°C, 18-24 hr
Incubate at35°C, 18-24 hr
Count BG colonies
Two presumptive BG colonies for each sample were confirmed by real-time PCR.
If no colonies were available and the enrichment culture was turbid, the
enrichment culture was tested by real-time PCR.
If turbid, streak onto TSA for isolation
Check for BG colonies
K-34
-------
Collect and wash spores in filter cup
Add 3.5 ml TSB medium to filter cup
Vortex filter cups
Take TO aliquot for
DMA concentration and purification
Incubate filter cups
9 hr, 35°C, 230 rpm
Vortex filter cups
Take T9 aliquot for
DMA concentration and purification
Real-time PCR analysis of
TO and T9 aliquots:
Data reporting
K-35
-------
Annex F: Data Reporting Forms
2
3
BOTE Report Form
Laboratory:
Samples Event:
Sample
Barcode/
ID
NC
PC
Culture (24-48 hr)
Average
CFU/
Sample
Source of
Culture
Result*
Culture-
PCR (Ct)**
Culture
Result*
positive/
negative
Positive
Negative
RV-PCR (9 hr)
Average
ACt
Std Dev
ACt
Ct(T9) < 39;
ACt>6
RV-PCR Result
(positive/
negative)
Notes
* Positive or negative culture determination based on the serial dilution (Plates), filter funnel plate (FF), and/or enrichment culture (EC),
restreak from enrichment culture (RS), or Promega-extracted concentrated EC (EC-PE). Positive culture result obtained if >0 BG colonies
are present on serial dilution or filter membrane plates, and/or BG-positive PCR results are obtained (Ct < 35) from colonies and/or the EC
or RS. N/A = Not applicable; No CFUs for analysis and EC not turbid and/or no growth from RS.
** Cycle threshold (Ct) values obtained from real-time PCR analysis of selected colonies and/or enrichment culture (EC); Undetermined =
Not detected
Positive RV-PCR result based on Ave. ACt > 6 and Ave. T9 Ct < 39
PC = Positive Control; NC = Negative Control; TB = True Blank
4
5
K-36
-------
1 BOTE Sample Analyses Summary
BOTE
Event1
MFP
Pre-
VHP
Post-
VHP
Pre-
Bleach
Post-
Bleach
Pre-CD
Post-CD
Total
Total
Sample
No.2
Samples
RV-PCR
and
Culture
Pos
RV-PCR
and
Culture
Neg
RV-PCR
Pos
and
Culture
Neg
RV-PCR
Neg
and
Culture
Pos
Total
Agreement3
(%)
False
Positive3
(%)
False
Negative3
(%)
True
Blanks
RV-PCR
and
Culture
Neg
Negative
Controls
RV-PCR
and
Culture
Neg
Positive
Controls
RV-PCR
and
Culture
Pos
2 nMFP = Mandatory Full Participation (background samples); VHP = Vaporous Hydrogen Peroxide; CD = Chlorine Dioxide
3 2Total sample number includes surface and QC samples, and does not include True Blanks or laboratory negative and positive controls.
4 3Note that each sample was divided into two equal parts for parallel RV-PCR and culture analyses; as a result, variability could be observed for
5 the samples with low spore levels. Percentages are based on surface and QC samples and do not include True Blanks or laboratory negative
6 and positive controls.
K-37
-------
Appendix L
Spatial Analysis Methodology
-------
Spatial Analysis Methodology
One of the noticeable characteristics of the data was that within a test event, some combinations of
method and object appeared to be dramatically different in their magnitude of CFUs/cm2. This appears
to create a large amount of variability when using geostatistical methods on the raw values.
Furthermore, a few extremely high values tend to have a large influence on geostatistical maps. To
produce maps with clearer gradients, a process was performed where similar method and object
combinations were put into strata, and the raw values within a stratum were assigned their distribution
percentiles.
Inspection of method, object, texture, and orientation combinations led to the following nine strata.
Samples within a strata tend to have distribution of CFUs/cm2 within an individual test event. For
different Object and Method combinations within the same strata, this implies the mean CFUs/cm2 and
standard deviations were somewhat similar, and also made some logical sense.
1. Floor Sponge Stick - Sponge stick samples taken on the floor.
2. Floor Vacuum - Vacuum samples taken on the floor.
3. Ceiling Sponge Stick - Sponge stick samples taken on the ceiling.
4. Ceiling Vacuum - Vacuum samples taken on the ceiling.
5. Non-Metal Desk/Workbench Sponge Stick - Sponge stick samples taken on a desk or
workbench, non-metal surface, facing up.
6. Non-Metal Table/Shelves/Countertop/Nightstand Sponge Stick - Sponge stick samples taken
on a table, shelves, countertop, or nightstand, non-metal surface, facing up.
7. Metal Sponge Stick - Sponge stick samples taken on a metal surface facing up.
8. Furniture Vacuum - Vacuum samples taken on furniture facing up.
9. Wall Vacuum - Vacuum samples taken on a wall, inclined surface.
Some samples were removed from this analysis because of small sample sizes that didn't allow them to
be properly compared to other method and object combinations. For instance, there were very few
swab samples taken on an individual test event on an individual floor. Samples taken on vents and
monitors were other samples not included in this analysis.
Within an individual stratum, the raw CFUs/cm2 values are ranked, and the ranks are converted to
percentiles. For example, if there were 10 samples ranked highest to lowest, they would be assigned
the percentiles 0.95, 0.85, 0.75, 0.65, 0.55, 0.45, 0.35, 0.25, 0.15, 0.05, which are the centers of 10
intervals of 10 percent each. All samples were then put into VSP with their stratum percentile as the
response variable.
This nonparametric approach produced geostatistical maps that appear to have some degree of
gradients.
L-2
-------
l.nged Estimat
Q
c
Figure L.I. Kriged Estimates of the Contamination on the First Floor Prior to the pH-Adjusted Bleach
Decontamination (square = vacuum, diamond = swab, circle = sponge stick).
t.rfged Esttmat
• Room 205ft.
11
Figure L.2. Kriged Estimates of the Contamination on the Second Floor Prior to the pH-Adjusted Bleach
Decontamination (square = vacuum, diamond = swab, circle = sponge stick).
L-3
-------
Figure L.3. Kriged Estimates of the Contamination on the First Floor Prior to the CIO2 Decontamination
(square = vacuum, diamond = swab, circle = sponge stick).
Figure L.4. Kriged Estimates of the Contamination on the Second Floor Prior to the CIO2
Decontamination (square = vacuum, diamond = swab, circle = sponge stick).
L-4
-------
l.nged Estimat
D
Q
c
i - 1
|B..m m|
Q
Figure L.5. Kriged Estimates of the Contamination on the First Floor Prior to the VHP Decontamination
(square = vacuum, diamond = swab, circle = sponge stick).
Figure L.6. Kriged Estimates of the Contamination on the Second Floor Prior to the VHP
Decontamination (square = vacuum, diamond = swab, circle = sponge stick).
L-5
-------
170K
150K-
140K-
90K-
70K-
60K
30K-
OK
i' i' i' i' |' i' i' i' i' |' i' i' i' i' |' i' i' i' i' |' i' i' i' i' |' i' i' i' i' |' i' i' i' i' |' i' i' i' i' |' i' i' i' i' |' i' i' i' i' |' i' i' i' i'
0 100 200 300 400 500 600 700 800 900 1000 1100
Distance
Figure L.7. Variogram for the First Floor Contamination Prior to the pH-Adjusted Bleach
Decontamination.
2200
2000
1800
1600
1200-
1000
800
600
400
200 H
0
0 100 200 300 400 500 600 700 800 900 1000 1100
Distance
Figure L.8. Variogram for the Second Floor Contamination Prior to the pH-Adjusted Bleach
Decontamination.
L-6
-------
1.1-
1-
0.9-
0.8-
Efl.6-
rtO.5-
0.3-
0.2-
0.1-
g
.
,/^'
/
T
\
•
0 100 200 300 400 500 600 700 800 900 1000 1100
Distance
Figure L.9. Variogram for the First Floor Contamination Prior to the CIO2 Decontamination.
On
OH
Ł n G
I05-
01
0.2-
0.1-
0
• m
* s*~^~ • * *
f
I
•
'
100 200 300 400 500 600 700 800 900 1000 11
10
Distance
Figure L.10. Variogram for the Second Floor Contamination Prior to the CIO2 Decontamination.
i.j
1.2-
1.1-
0.9-
vt 0.0-
I"
rt 0.6
O 0.5-
0.4-
0.3-
0.2-
0.1-
0
100
200
300
400
500 600
Distance
700
800
900
1000
1100
Figure L.ll. Variogram for the First Floor Contamination Prior to the VHP Decontamination.
1-7
-------
100
200
300
400
700
800
900 1000 1100
500 600
Distance
Figure L.12. Variogram for the Second Floor Contamination Prior to the VHP Decontamination.
n
D
d
Figure L.13. Kriged Estimates of the Contamination on the First Floor after the VHP Decontamination
(square = vacuum, diamond = swab, circle = sponge stick).
L-8
-------
Figure L.14. Kriged Estimates of the Contamination on the Second Floor after the VHP Decontamination
(square = vacuum, diamond = swab, circle = sponge stick).
L-9
-------
0.65
0.6-
0.55-
0.5-
0.45-
rt 0.4-
^0.35-
5 0.3-
U0.25-
0.2-
0.15-
0.1-
0.05-
50 100 150 200 250 300
Distance
350
400
450
500
Figure L.15. Variogram for the First Floor Contamination after the VHP Decontamination.
ro
E
PS
O
1.4
1.3
1.2
1.1
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
50
100 150 200 250 300 350 400 450
500
Distance
Figure L.16. Variogram for the Second Floor Contamination after the VHP Decontamination.
L-10
-------
Appendix M
The iPad as an Electronic Laboratory Notebook
-------
Introduction
EPA/ORD/NHSRC participated in a multi-agency test program and exercise called the Bio-Response
Operational Testing and Evaluation (BOTE) program. The BOTE project is an interagency project to
conduct and evaluate field-level facility biological remediation activities of various decontamination
technologies directed at Bacillus anthracis (anthrax) over 2 distinct phases: Phase 1, a field study to
evaluate the effectiveness of several different decontamination technologies; and Phase 2, a multi-
agency exercise to address interagency roles and responsibilities for an anthrax response in a field
setting. The BOTE testing occurred at a facility located on-site at the Idaho National Laboratory (INL) (see
Figure 1). The BOTE project used the surrogate biological warfare agent (BWA) organism Bacillus
atrophaeus instead of Bacillus anthracis, although most safety protocols, measures to prevent cross-
contamination, and personal protective equipment (PPE) requirements were performed as if the real
organism were present.
Figure 1. BOTE Test Facility (with and without tent)
The BOTE project included a detailed cost analysis. In order to perform this cost analysis, many
parameters associated with the testing activities (e.g., sampling, decontamination, waste management)
needed to be observed and logged as they were happening. In particular, there was an observer inside
the test facility during sampling and some of the decontamination operations to track the level of effort
to perform some of the sampling and decontamination activities. This observer needed to be suited up
in "Level C" PPE consisting of a Tyvek suit, gloves, booties, and a full-face respirator. Since this observer
would be making observations about complex activities it would be necessary for this person to have a
laboratory notebook with which to record their observations. However, due to the fact that the
observer would be wearing PPE and passing through a personnel decontamination line that included a
wash with soapy water and bleach as they left the facility after their tasks were complete, a
conventional paper laboratory notebook would not be feasible. A data logging solution was needed that
had the following requirements:
• Ability to create custom forms for data entry
• Ability to withstand passing through the personnel decontamination line with soapy water
and/or bleach
• Ability to operate inside Ziploc bags for extended periods of time
M-2
-------
• Ability to wirelessly transmit data to a server so that in the event the Ziploc bags fail during
decontamination, and the device was destroyed, no data would be lost
• Ability to operate for a minimum of 8 Hours on battery charge
The following hardware options were explored:
• Tablet PC
• PDA
• Apple iPad (1st generation)
The Tablet PC was an attractive option because: 1) it could operate Windows XP and be able to follow at
least to a certain extent the ORD standard configurations for computer hardware; 2) custom forms and
applications could be created using various application frameworks such as LabVIEW; 3) Tablet PCs
could transmit data wirelessly to a remote server; and 4) Tablet PCs have large screens which would
make visibility good for the user in PPE.
Unfortunately all the Tablet PCs that were evaluated had cooling fans inside the housing. This would
make them not operable for extended periods of time inside a Ziploc bag. For this reason the Tablet PC
was rejected for our application.
PDAs that use Windows CE, PalmOS, or other handheld operating systems were promising in that: 1)
they could operate inside a Ziploc bag for extended periods of time; and 2) they could easily transmit
data over a wireless network.
Unfortunately, PDAs have small LCD screens on the order of 3 inches square. This is less than optimal
for the user in PPE both from a visibility standpoint, and from an ability to easily enter free-form text.
PDAs are not approved configurations for use inside the EPA network, which could cause problems from
an approval standpoint. For this reason, PDAs were selected as a second choice for our application.
The Apple iPad was evaluated as an option as well. Advantages the iPad has include: 1) a relatively large
backlit screen; 2) it can easily transmit data over a wireless network, including over the newer 5G
protocol; 3) a native spreadsheet app called "Numbers" that allows the creation of custom forms and
can import and export Excel files; 4) a touch pad that can operate inside Ziploc bags with a stylus; 5) long
battery life - up to 10 hours; and 6) the absence of an internal fan - the iPad can maintain
manufacturer's recommended operating temperatures even while bagged up for long hours.
The main disadvantages of the iPad involve the unsupported nature of the hardware in that iPads are
not approved for use on EPA networks (the same problem as the PDAs). For this reason, the wireless
network exchange capabilities were not tested until we arrived at the test site.
The iPad was our first choice for our application.
Configuration
M-3
-------
The configuration that was settled on for the device was the 32 GB Wi-Fi only model since it was not
necessary to maintain wireless network capability away from the test site, plus there are inherent
bureaucratic difficulties associated with signing up for a fee-based subscription service. Figure 2 depicts
the unit (iPad 1st generation) that was purchased. The "Numbers" spreadsheet app was installed on the
iPad.
Figure 2. Apple iPad
Purchasing the iPad
The iPad retailed at $599 in the desired configuration. It took several months of wrangling with OSIM to
get the approval to purchase the device, even with a strong justification. It was purchased with the
Government Bankcard. Additional accessories that were purchased included: 1) a case/cover; 2) an
additional AC adapter/charger; 3) an SD card reader; and 4) an external keyboard.
Custom Forms for Data Entry
The Numbers spreadsheet app can read and write its own proprietary file format as well as being able to
output a PDF file or import/export MS Excel 2003 (xls) worksheets. Tabbed worksheets on both
Numbers and Excel are interchangeable. Numbers has some additional functionality that is particularly
useful in the iPad environment. First, there are some additional data types including a "stars" data type
that can be used to semi-quantitatively characterize numbers using a 1 through 5 stars scale. Second, by
applying a title to a table of spreadsheet cells imported from an Excel worksheet or created from scratch
in Numbers, a limited ability to easily develop custom forms is activated, where each row in the
spreadsheet is given a custom form tab within Numbers for entering data in that row, based on the
column headings in the titled table of cells on the first page. As additional instances of the form are
created, additional rows are created in the spreadsheet and populated with data from the forms as the
user inputs it. Figure 3 shows a Numbers spreadsheet and associated custom forms that are created
from that spreadsheet.
M-4
-------
Swab Wipe Vac
Upstairs Residence"!
Carpet
Wall
Desk
Room Air
IH
a
LS
a
LJ
L
L-
0
U
u
* \
u
a
LI
U
120/1
12/9/1
1?*W1
930 | 1020
dpo s?n
. . . . .1^^!"" —
Swab Wipe Vac
Upstairs Office!
Start End Time Samplerl Sampled Samplers Notes
Carpet
Wall
Desk
File cabinet
Room Air
u
U
ti
u
u
u
L
c
u
a
u
u
ISJ
u
LI
11/8/10 8<15
11/8/10 900
11/8/10 1002
11/8/10 1200
850
910
1006
1300
**** •
** • • •
***• •
*** ••**•••
* ** ••**•••
*....*....
Broom sample ID
Light swi'ch
In drswer
Carpet
Air
SP etiok
8w«b
W,pe
Dale 18/9/10
Start Time 5««
End Time 145
Samplorl * * * • •
* *
* * ** •
HOLM I X«
Figure 3. Custom Forms in Numbers Spreadsheet
Ability to Operate Inside Ziploc Bags.
The iPad fits inside a large capacity Ziploc bag. The unit was then double bagged and sealed. A handle
was rigged up using duct tape. A stylus was used to enter data on the iPad through the Ziploc bags. The
unit operated acceptably by a PPE-clad user.
The double bagging process enabled the iPad to successfully endure an indefinite number of
decontamination operations, using a combination of bleach/vinegar and soapy water without getting
wet and/or failing.
Transferring Files
The iPad has several ways to transfer files back and forth from a user's office computer. Files can be
transferred from the iPad using a direct cable (a USB variant) hookup to a PC or Mac running iTunes or
wirelessly over a network via email, Apple's iCIoud service, or a "WebDAV" server running on a Mac or
PC. On a PC running Windows, WebDAV is a variation of Windows File Sharing that allows the HTTP
protocol to be used to access remote servers. It is built into Windows 7.
A WebDAV server was set up on an onsite PC running Windows 7 to receive data from various sources,
including the iPad. Once the data were entered into the iPad, the user clicks on the "My Spreadsheets"
button in the upper left corner of the screen (see Figure 4). This will yield the list of available
spreadsheet data files on the iPad. Using gestures the user brings the spreadsheet that is desired to be
transferred (in this case, the file "Sampling copy") to the center of the display. Clicking on the icon at the
bottom-left of the screen brings up a selection list for export options (see Figure 5). Selecting "Copy to
WebDAV" yields the screen in Figure 6. Selecting the appropriate file format and using standard file
management dialog boxes (see Figure 7) allows the user to select the desired destination directory on
M-5
-------
the WebDAV server. The ability for the iPad to access 5G wireless networks was particularly useful for
the BOTE tests because there was an unusually large amount of Wi-Fi activity at the site, which resulted
in the normal Wi-Fi connectivity intermittently dropping out, whereas the 5G connectivity was much less
prone to interference.
Figure 4. iPad File Selection Screen
My SpnxlilmU (4 ol 51
Figure 5. iPad File Transfer Selection Menu
M-6
-------
Figure 6. iPad Export File Format Option Screen
Figure 7. iPad File Management Dialog Box
Use as Lab Notebook Outdoors
The original plan was to use a conventional laboratory notebook to log data from outdoor sources, such
as the waste disposal processing, sampling and decontamination team preparation, radio traffic
reporting team locations and activities, and the personnel decontamination line. However, high winds
on the site made use of the conventional laboratory notebook very difficult outside. Therefore the data
entry Excel worksheets that were initially used to transcribe the lab notebook results into electronic
form were imported to the iPad and data entry was done directly on the iPad using the Numbers
application.
Adaptability to Situational Needs
M-7
-------
As the testing was going on there was a need to overlay some notes over building floor plans, to denote
the locations of certain items inside the rooms. These notes needed to be made while inside the
contaminated facility and brought outside. The necessity for PPE made it impossible to use paper to
record these notes. The building floor plans were loaded onto the iPad as jpg files and a freeware
application called "DrawFree" was downloaded from the iPad App Store. DrawFree allows the user to
make free form doodles on an imported background image. The building floor plans were emailed to
me and EPA WebMail was accessed through the Safari web browser on the iPad and the floor plans
were saved to the iPad's photo storage memory. DrawFree imported the floor plans as background
images from the iPad's photo storage. We color coded the notes for different types of items that were
placed in different rooms, and Figure 8 is an example of the output. In spite of limitations in the
fineness of the input due to the size of the stylus tip, the locations for fans (F), biological indicators (B),
sensors for H2O2 (S), and aerators (A) can be clearly identified.
Figures. Example Output from DrawFree
Problems
The iPad problems that occurred were minor and workarounds were developed. The iPads were not
immune to the ongoing Wi-Fi problems that were occurring at the site. These problems were corrected
usually by either rebooting the iPads or rebooting the routers. Data entry into Numbers sometimes
resulted in spreadsheet cells with the wrong format (e.g., a text format instead of a number format or a
number format instead of a date or time format). These data type issues were easily correctable once
the data were imported into Excel. Occasionally linefeed characters were found after the last character
or number in some cells when Numbers spreadsheets were imported into Excel. These were easily
deleted.
Conclusions
Special requirements from a high visibility field test that the EPA participated in necessitated the use of
innovative data logging techniques. After evaluating several potential options, Apple iPads were used as
electronic laboratory notebooks to log data both inside a building while the observer was wearing PPE,
M-8
-------
and outside the building during periods of high winds. The battery life was very good, with the (Pads'
batteries lasting through long work days. The lack of a convection fan enabled the iPad to operate
inside Ziploc bags and thus endure decontamination operations without damage. The "Numbers"
spreadsheet easily exchanged data with MS Excel and also provided functionality to create custom data
forms. Wireless networking capability and flexible file exchange capabilities simplified subsequent data
archival and analysis. The iPads were very successful hardware solution for these tasks and may have
good potential for routine use by ORD for certain situations.
M-9
-------
United States
Environmental Protection
Agency
PRESORTED STANDARD
POSTAGE & FEES PAID
EPA
PERMIT NO. G-35
Office of Research and Development (8101R)
Washington, DC 20460
Official Business
Penalty for Private Use
$300
------- |