EPA 600/R-14/262 I September 2014 I www.epa.gov/research
United States
Environmental Protection
Agency
Expedient Approaches for the
Management of Wastes Generated
from Biological Decontamination
Operations in an Indoor
Environment
EVALUATION OF WASTE SAMPLING AND
DECONTAMINATION PROCEDURES
Office of Research and Development
National Homeland Security Research Center
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EPA 600-R-14-262
September 2014
Expedient Approach for Decontamination of Biologicals
Indoor Environment -
Evaluation of Waste Decontamination Procedures
Assessment and Evaluation Report
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
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Disclaimer
The United States Environmental Protection Agency (EPA), through its Office of Research and
Development's National Homeland Security Research Center, funded and directed this investigation
through EP-C-09-027 with ARCADIS U.S., Inc. This report has been peer and administratively reviewed
and has been approved for publication as an Environmental Protection Agency document. It does not
necessarily reflect the views of the Environmental Protection Agency. No official endorsement should be
inferred. This report includes photographs of commercially available products. The photographs are
included for purposes of illustration only and are not intended to imply that EPA approves or endorses the
product or its manufacturer. Environmental Protection Agency does not endorse the purchase or sale of
any commercial products or services.
Questions concerning this document or its application should be addressed to:
M. Worth Calfee, Ph.D.
Decontamination and Consequence Management Division
National Homeland Security Research Center
U.S. Environmental Protection Agency (MD-E343-06)
Office of Research and Development
109 T.W. Alexander Drive
Research Triangle Park, NC 27711
Phone:919-541-7600
Fax:919-541-0496
E-mail: Calfee.Worth@epamail.epa.gov
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Acknowledgments
This effort was directed by the principal investigator from the Office of Research and Development's
(ORD) National Homeland Research Center (NHSRC), Decontamination and Consequence Management
Division (DCMD) utilizing the support from the US Environmental Protection Agency's (EPA's) Chemical,
Biological, Radiological, and Nuclear (CBRN) Consequence Management Advisory Division (CMAD)
within the Office of Emergency Management (OEM). The contributions of the entire team are
acknowledged.
Project Team:
Worth Calfee, Ph.D. (Principal Investigator)
US EPA, Office of Research and Development, NHSRC, DCMD
Research Triangle Park, NC 27711
Paul Lemieux, Ph.D.
US EPA, Office of Research and Development, NHSRC, DCMD
Research Triangle Park, NC 27711
Mario lerardi, Ph.D.
US EPA, Office of Solid Waste and Emergency Response, Office of Resource Conservation and
Recovery, Materials Recovery and Waste Management Division, Water Compliance Branch
Arlington, VA, 22202
Paul Kudarauskas
US EPA, Office of Solid Waste and Emergency Response, OEM, CBRN CMAD
Washington, DC, 20004
Jeanelle Martinez, Ph.D.
US EPA, Office of Solid Waste and Emergency Response, OEM, CBRN CMAD
Cincinnati, OH 45220
R. Leroy Mickelsen, M.S., P.E.
US EPA, Office of Solid Waste and Emergency Response, OEM, CBRN CMAD
Research Triangle Park, NC 27711
Randy Schademann
US EPA, Federal On-Scene Coordinator, Region 7
Lenexa, KS, 66219
This effort was completed under U.S. EPA contract #EP-C-09-027 with ARCADIS-US, Inc. The support
and efforts provided by ARCADIS-US, Inc. are acknowledged.
Ramona Sherman (Quality Assurance)
NHSRC, ORD, US EPA
Cincinnati, OH 45268
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The following peer reviewers of this report are also acknowledged for their input to this product:
Marshall Gray (EPA ORD), Cathy Young (EPA Region 1), John Martin (EPA Region 6).
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Table of Contents
Disclaimer iii
Acknowledgments iv
List of Tables x
List of Acronyms and Abbreviations xii
Executive Summary xiii
Summary of Results xiii
1 Introduction 1
1.1 Process 2
1.2 Project Objectives 3
1.3 Experimental Approach 3
1.3.1 Testing Sequence 3
1.3.2 Decontamination Strategy 4
1.3.3 Method Development for Neutralization 4
2 Materials and Methods 5
2.1 Facility Design 5
2.2 Test Coupon Preparation 5
2.2.1 Carpet and Upholstery 5
2.2.2 Paper 6
2.2.3 Nitrile Gloves 7
2.3 Spore Preparation 8
2.3.1 Coupon Inoculation and Test Preparation 8
2.4 Decontamination Procedure 8
2.5 Method Development for Neutralization 9
2.6 Test Matrix 9
2.6.1 Neutralization Method Development Test Matrix 9
2.6.2 Test Matrix 10
3 Sampling and Analytical Procedures 12
3.1 Sampling Strategy 12
3.1.1 Sponge-Stick™ Sampling 12
3.1.2 Extractive Sampling 12
3.1.3 Sample Preservation 13
3.1.4 Sampling Points 13
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3.1.5 Carpet and Upholstery 13
3.1.6 Paper Samples 14
3.1.7 PPE Samples 14
3.2 Sampling Frequency 15
3.2.1 Sample Quantities 15
3.3 Measurement Methods 16
3.3.1 Decontamination Solutions 16
3.3.2 Microbiological Samples 16
3.3.2.1 Sample Extraction 17
3.3.2.2 Sample Analysis 17
3.4 Data Analysis 17
3.4.1 Sampling Efficiency 17
3.4.2 Surface Decontamination Efficacy 17
3.4.3 Statistical Analysis 20
4 Results and Discussion 22
4.1 Sampling Methods Evaluation 22
4.1.1 Carpet Material 22
4.1.2 Upholstery Material 24
4.1.3 PPE Material 26
4.1.4 Paper Material 27
4.1.5 Sampling Methods Test Synopsis 29
4.2 Neutralization Methods Evaluation 30
4.2.1 Optimization of Neutralizer Concentration 30
4.2.2 Sample Hold Time Effects 31
4.2.3 Immersion Time Effects 31
4.2.4 Neutralization Tests Synopsis 32
4.3 Dunking/Immersion Decontamination Test Results 32
4.3.1 Carpet Decontamination Results 33
4.3.1.1 Sampling Methods Evaluation for Carpet 33
4.3.1.2 Carpet Decontamination Effectiveness 36
4.3.2 Upholstery Decontamination Results 37
4.3.2.1 Sampling Methods Evaluation for Upholstery 37
VII
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5.3 QA/QC Checks 50
5.4 Acceptance Criteria for Critical Measurements 52
5.5 Data Quality Audits 56
5.6 QA/QC Reporting 56
6 Summary and Recommendations 57
Appendix A: Miscellaneous Operating Procedures A1
MOP 3120 VHP Operation July 2013 signed A2
MOP 3128 A pH Adjusted Bleach Dec 2013 signed A15
MOP 3148 Chlorine Dioxide and Chlorite by HACH Nov 2012 signed A19
MOP 3165 Sponge Sample Collection July 2013 signed A23
MOP 3194 Procedure for Fabricating 18"x 18" Upholstery Coupons for Liquid Innoculation A31
MOP 3195 Immersion Decontamination Aug 2013 for Worth approval A36
MOP 6535a Bacterial Spore Plate Counting and Dilutions Jan 2013 signed A41
MOP 6562 Preparing Pre-Measured Tubes with Aliquoted Amounts of Phosphate Buffered
Saline with Tween 20 (PBST) A49
MOP 6565 Filter-Plate Method Feb 2013 signed A55
MOP 6580 Recovery of Bacillus spores from 3M Sponge Stick Samples Feb 2013 signed A58
MOP 6584 Replating Bacteria Spore Plates Nov 2012 A65
VIII
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List of Figures
Figure 2.1: Poly Hog Trough® 5
Carpet tile 6
Front of assembled upholstered coupon 6
Paper material 7
White disposable nitrile glove 7
Figure 2.2:
Figure 2.3:
Figure 2.4:
Figure 2.5:
Figure 3.1:
Figure 3.2:
Figure 4.1:
Figure 4.2:
Figure 4.3:
Figure 4.4:
Figure 4.5:
Figure 4.6:
Figure 4.7:
Figure 4.8:
Figure 4.9:
Material section shown with template during sampling with Sponge-Stick™ and
extraction 14
Sampling timeline 15
Effect of waste storage time on positive control recoveries (colony forming units
[CFU]) from carpet for the extractive and the Sponge-Stick™ methods 23
Effect of waste storage time on positive control recoveries (colony forming units
[CFU]) from upholstered material for the extractive and the Sponge-Stick™
methods 25
Effect of waste storage time on positive control recoveries (colony forming units
[CFU]) from personal protective equipment for the extractive method 26
Effect of waste storage time on positive control recoveries (colony forming units
[CFU]) from paper for the extractive method 28
The effects of sampling method and waste storage duration on recoveries (colony
forming units [CFU]) from carpet following decontamination with pH adjusted
bleach 33
The effects of immersion time in pH adjusted bleach on carpet decontamination
efficacy (colony forming units [CFU] log reduction in recovery) 37
The effects of sampling method and waste storage duration on recoveries (colony
forming units [CFU]) from upholstery following decontamination 38
The effects of immersion time in pH adjusted bleach on upholstery
decontamination efficacy (log reduction in colony forming units [CFU] in recovery) 40
The effects of waste storage duration on recoveries (colony forming units [CFU])
from personal protective equipment following decontamination
.42
Figure 4.10: Personal protective equipment decontamination efficacy by decontaminant type
(colony forming unit [CFU] log reduction) 44
Figure 4.11: Recoveries (colony forming units [CFU]) following a decontamination of paper with
pH adjusted bleach (immersion time: 15 minutes) 45
Figure 4.12: Recoveries (colony forming units [CFU]) following decontamination of paper with
diluted bleach (immersion time: 15 min) 46
Figure 4.13: Decontamination efficacy of pH adjusted bleach and diluted bleach on paper
(colony forming units [CFU] log reduction, immersion time: 15 min) 48
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List of Tables
Table 2.1: Neutralization Methods Test Matrix 10
Table 2.2: Measurement and Neutralization Methods 10
Table 2.3: Decontaminants and Accessibility 10
Table 2.4: Decontamination Procedures and Intensity 11
Table 2.5: Decontamination Test Sequence Event 11
Table 3.1: Coupon Types Used to Evaluate Waste Decontamination Procedures 13
Table 3.2: Number of Sample Types per Material Section per Sampling Sequence 16
Table 3.3: Number of Sample Types per Material Section per Sampling Sequence 21
Table 4.1: Effects of Waste Storage Time on Positive Control Recoveries from Carpet for the
Extractive and Sponge-Stick™ Sampling Methods 23
Table 4.2: Two-Sample Independent T-test Performance Parameters for Effects of Waste
Storage Time on Recoveries from Carpet by Sampling Method and
Decontamination Procedure 24
Table 4.3: Effects of Waste Storage Time on Positive Control Recoveries from Upholstered
Material forthe Extractive and Sponge-Stick™ Sampling Methods 25
Table 4.4: Two-Sample Independent T-Test Performance Parameters forthe Effects of Waste
Storage Time on Recoveries from Upholstery by Sampling Method 26
Table 4.5: Effects of Waste Storage Time on Positive Control Recoveries from Personal
Protective Equipment forthe Extractive Sampling Method 27
Table 4.6: Analysis of Variation Performance Parameters for Effects of Storage Time on
Recoveries from Personal Protective Equipment by Sampling Method and
Experiment 27
Table 4.7: Effects of Waste Storage Time on Positive Control Recoveries from Paper for the
Extractive Sampling Method 29
Table 4.8: Analysis of Variance Performance Parameters for Effects of Waste Storage Time
on Recoveries from Paper by Sampling Method and Experiment 29
Table 4.9: Preliminary Neutralization Optimization 30
Table 4.10: Effect of Sample Hold Time on Neutralizer Optimization 31
Table 4.11: Effect of Immersion Time on Spore Recovery (Colony Forming Units) from
Neutralized pH Adjusted Bleach-Exposed Carpet Samples, High and Low Spore
Concentrations 31
Table 4.12: Decontamination Test Sequence Event 32
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Table 4-13: Post-Decontamination Recoveries (Colony Forming Units [CPU]) from Carpet for
Extractive and Sponge-Stick™ Sampling Methods (Immersion Time: 15 min,
Decontaminant: pH adjusted Bleach) 34
Table 4-14: Post-Decontamination Recoveries (Colony Forming Units [CPU]) from Carpet for
Extractive and Sponge-Stick™ Sampling Methods (Immersion Time: 30 min,
Decontaminant: pH adjusted Bleach) 35
Table 4-15: Post-Decontamination Recoveries (Colony Forming Units [CPU]) from Carpet for
Extractive and Sponge-Stick™ Sampling Methods (Immersion Time: 60 min,
Decontaminant: pH adjusted Bleach) 35
Table 4.16: Analysis of Variance Performance Parameters for Effects of Post-Decontamination
Storage Time on Recoveries (Colony Forming Units [CPU]) from Carpet 36
Table 4.17: Decontamination Efficacy versus Immersion Time (Colony Forming Units Log
Reduction) for Carpet 36
Table 4.18: Post-Decontamination Recoveries (colony forming units [CPU]) from Upholstery for
Extractive and Sponge-Stick™ Sampling Methods (Immersion Time: 15 min) 39
Table 4.19: Analysis of Variance Performance Parameters for Effects of Post-Decontamination
Sample Storage Time on Recoveries (Colony Forming Units [CPU]) from
Upholstery (immersion Time 15 min) 39
Table 4.20: Decontamination Efficacy (Log Reduction in Recovery) for Upholstered Coupon
Decontamination Efficacy 40
Table 4.21: Recoveries (Colony Forming Units) Following Decontamination of Personal
Protective Equipment with pH Adjusted Bleach (Immersion Time: 15 min) 42
Table 4.22: Recoveries Colony Forming Units Following Decontamination of Personal
Protective Equipment with Diluted Bleach (Immersion Time: 15 min) 43
Table 4.24: Personal Protective Equipment Decontamination Efficacy (Log Reduction in
Recovery) 44
Table 4.25: Recoveries (Colony Forming Units [CFU]) Following Decontamination of Paper with
pH Adjusted Bleach (Immersion Time: 15 Min) 46
Table 4.26: Recoveries (Colony Forming Units [CFU]) Following Decontamination of Paper with
Diluted Bleach (Immersion Time: 15 Min) 47
Table 4.28: Paper Decontamination Efficacy (Log Reduction in Recovery, Immersion Time: 15
min) 47
Table 5.1: Instrument Calibration Requirements 49
Table 5.2: Quality Control Checks 51
Table 5.3: Critical Measurement Acceptance Criteria 53
Table 5.4: Data Quality Assessment 54
Table 6.1: Portion of Samples with No Viable Spores Detected After Decontamination 58
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List of Acronyms and Abbreviations
ANOVA Analysis of variance
ATCC American Type Culture Collection
CBRN Chemical, Biological, Radiological, and Nuclear
CPU Colony forming units
CMAD Consequence Management Advisory Division
COC Chain of custody
DCMD Decontamination and Consequence Management Division
EPA U.S. Environmental Protection Agency
FAC Free available chlorine
FIFRA Federal Insecticide, Fungicide, and Rodenticide Act
HSRP Homeland Security Research Program
HSPD Homeland Security Presidential Directives
ISO International Organization for Standardization
MOP Miscellaneous Operating Procedure
NHSRC National Homeland Security Research Center
NIST National Institute of Standards and Technology
OEM Office of Emergency Management
OPP Office of Pesticides Programs
ORD Office of Research and Development
OSWER Office of Solid Waste and Emergency Response
pAB pH-adjusted bleach
PARTNER Program to Align Research and Technology with the Needs of Environmental Response
PBST Phosphate buffered saline with Tween®20
PPE Personal protective equipment
QA Quality assurance
QAPP Quality Assurance Project Plan
QC Quality control
RH Relative humidity
RTP Research Triangle Park
SD Standard deviation
STS Sodium thiosulfate
VHP® Vaporized hydrogen peroxide
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Executive Summary
This project supports the mission of the U.S. Environmental Protection Agency's (EPA) Office of
Research and Development's (ORD) National Homeland Security Research Center (NHSRC) by
providing information relevant to the remediation of areas contaminated with biological agents.
The primary objective of this investigation was to determine the effectiveness of an expedient approach to
waste decontamination. Such approaches were utilized in previous bioterror remediation, although their
effectiveness has yet to be determined experimentally. To determine the effectiveness of
decontamination approaches, the current study evaluated an immersion-based approach to
decontaminate waste materials contaminated with Bacillus atrophaeus spores (surrogate for 8.
anthracis). The effectiveness of this decontamination approach was evaluated for high traffic commercial
carpet tile, nitrile gloves (personal protective equipment [PPE]), books, and upholstered seat pans that
are typical of porous material found in an indoor office or items expected to be generated during a
sampling and remediation (i.e., PPE). The decontamination and sampling strategies utilized herein were
selected by an EPA project team, which consisted of staff from EPA's Office of Research and
Development, EPA's Office of Solid Waste and Emergency Response, and EPA's Region 7. The
methods utilized were chosen based on their expected effectiveness and ease of use during remediation.
Test materials were inoculated with Bacillus spores at known locations and concentrations, and subjected
to prescribed decontamination procedures (i.e., immersion in decontaminant). After the decontamination
procedure, a sub-set of the test materials were sampled immediately (Day 0), then the items were bagged
and stored (to simulate waste handling/staging during a response). The simulated waste items were re-
sampled in a waste staging area after a drying time of 1 day (at least 18 hours), 7 days, and 30 days. A
subset of bagged, inoculated waste samples was left untreated and served as positive controls. The
efficacies of two decontamination solutions (dilute bleach and pH-adjusted bleach (pAB)) were
determined using immersion times varying from 15 minutes to 1 hour. Two sampling methods were used
for carpet and upholstery: extractive and surface sampling with 3M Sponge-Stick™. Only extraction-
based methods were utilized for PPE and books.
Summary of Results
Most waste materials were effectively decontaminated (greater than 6 log reduction) by a 15 minute
immersion in pAB, with the exception of carpet. Longer immersion times increased the efficacy of the
decontamination process on carpet, but a 60 minute immersion failed to provide more than a 4 log
reduction in viable spores. Decontamination of spores inside closed books was sometimes difficult, as
contact with the decontamination solution was not homogenous. Likewise, air pockets in gloves
prevented contact with the decontamination solution and could randomly provide complete protection to
spores on that surface. The pAB was found to achieve higher decontamination efficacies than diluted
bleach for all of the materials tested in this study.
In addition to decontamination efficacy, the collection efficiency of the two sampling methods (extractive
and Sponge-Stick™) used in this study were compared as a function of material and elapsed time from
inoculation and the time when the sample was collected. Analysis showed no significant effect of sample
storage time of up to 30 days on spore recovery, when using either sampling method. Results obtained
when using the Sponge-Stick™ approach showed that this sampling method results in an overestimation
xiii
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of the actual decontamination efficacy due to its lower recovery efficiencies compared to the extractive
sampling technique. These data suggest that the extractive sampling approach should be used whenever
wet porous materials are sampled.
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1 Introduction
This project supports the mission of the U.S. Environmental Protection Agency's (EPA) Office of
Research and Development's (ORD) Homeland Security Research Program (HSRP) by providing
information relevant to the decontamination of areas contaminated as a result of an act of terrorism.
Under Homeland Security Presidential Directives (HSPD)-5, 7, 8, and 10, the EPA, in a coordinated effort
with other federal agencies, is responsible for "developing strategies, guidelines, and plans for
decontamination of ...equipment, and facilities" to mitigate the risks of contamination following a biological
agent contamination incident.
EPA's National Homeland Security Research Center (NHSRC) aims to help EPA address the mission of
the HSRP by providing expertise and products that can be widely used to prevent, prepare for, and
recover from public health and environmental emergencies arising from terrorist threats and incidents.
One of NHSRC's missions is to provide expertise and guidance on the selection and implementation of
decontamination methods and provide the scientific basis for a significant reduction in the time, cost, and
to address the complexity of decontamination activities. The NHSRC's research supports the EPA's
Office of Solid Waste and Emergency Response (OSWER), Office of Pesticides Programs (OPP), and the
Regions. Close collaboration between the different program offices having homeland security
responsibilities is sought in order to rapidly increase EPA's capabilities to help the nation recover from a
terrorist event involving the intentional release of CBRN materials.
In 2001, the introduction of a few letters containing Bacillus anthracis (anthrax) spores into the U.S.
Postal Service system resulted in the contamination of several facilities and the deaths of two postal
employees. Although most of the facilities in which these letters were processed or received in 2001 were
heavily-contaminated, they were successfully decontaminated with approaches such as fumigation with
chlorine dioxide or vaporized hydrogen peroxide (VHP®). It is well agreed that additional quick, effective
and economical decontamination methods with the capacity to be employed over wide areas (outdoor
and indoor) are required to increase preparedness for such an incident. Fumigation was used in primarily
contaminated facilities that were heavily-contaminated. Other cleaning methods were used in less
heavily contaminated facilities such as those that were secondarily contaminated or those primarily
contaminated facilities that showed a minimal presence of anthrax spores. These other "expedient" or
"low-tech" methods included removal of contaminated items and/or on-site decontamination. For the
surface of a contaminated material, decontamination can be accomplished by physical removal of the
contaminant or by inactivation of the contaminant with antimicrobial chemicals. Physical removal could be
accomplished by removing spores from the material (i.e., physical cleaning) or via disposal of the
material. Inactivation of the contaminant can be done on-site (within the contaminated structure or on-
site) or after removal of the contaminated material prior to ultimate disposal (i.e., incinerated off-site). The
decision-makers' selection of the balance between the on-site and off-site destruction of spores was
facility-dependent and factored in many issues (e.g., physical state of the facility). One factor was that
such decontamination was unprecedented for the United States government and no sporicidal
technologies had been proven or registered for use against B. anthracis spores at the time. The cost of
waste management proved to be very significant and was complicated by the nature of the waste.
Since 2001, the emphasis for facility decontamination has been to identify and characterize efficacious
on-site decontamination methods and to optimize the decontamination/waste management paradigm; this
optimization could reduce decontamination time and cost. If proven effective, a lower-tech approach to
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decontaminating waste on-site could reduce overall decontamination costs by reducing the amount of
waste treatment required by off-site, specialized facilities (e.g., medical waste incinerators) and the risk
associated with transporting contaminated materials to such facilities. Developing and demonstrating
lower cost waste management solutions could increase EPA's readiness to respond to a wide area
release that would generate contaminated waste volumes much larger than those previously managed.
Response and remediation activities associated with this type of incident will require additional waste
handling, segregation, and staging. These additional requirements illustrate the need for efficacy data and
improved process knowledge to support assessments regarding decontamination operations and waste
management.
Waste items (ceiling tile and carpet) generated during a recent facility-scale test (BOTE study1), that were
decontaminated during the study by expedient methods (liquid bleach spray orspritz), bagged/managed
in a manner typical of a real remediation effort, stored (~6 months), and subsequently sampled showed
that significant quantities of the test organism (Bacillus atrophaeus) survived the treatment and
subsequent 6 month storage duration. This finding indicates that current waste management techniques
used during expedient decontamination efforts may generate waste items that have residual
contamination. Since the willingness of waste disposal facilities to accept waste items may depend partly
upon their contamination level, identification and demonstration of methodologies to effectively
decontaminate waste on-site during low-tech decontamination activities are of significant need. This
study evaluated several waste decontamination strategies that could be conducted on-site.
For waste generated during the decontamination of typical indoor office or indoor residential settings, no
sampling methods have been standardized. Waste items generated from the decontamination of building
interiors are expected to consist largely of porous materials1, which pose challenges to currently-available
sampling methods. To provide the data necessary to standardize a waste sampling method, extraction-
based and surface-wipe-based sampling methods were evaluated. Extraction-based sampling consisted
of excision and subsequent extraction of a portion of the material. Surface-wipe-based sampling
consisted of collection of the contaminant from the material surface with a Sponge-Stick™.
1.1 Process
This study investigated decontamination of selected materials by an immersion (dunking) approach of
waste materials contaminated with Bacillus atrophaeus spore inoculum (i.e., surrogates for 8. anthracis).
The effectiveness of this decontamination approach was evaluated for high traffic commercial carpet tile,
books, and upholstered seat pans that are typical of material found in indoor office, or items like nitrile
gloves (personal protective equipment [PPE]) that would be expected to be generated during sampling
and decontamination. Replicate sections of test materials were inoculated at known locations with a
targeted number of Bacillus spores. After decontamination, sections of the test material were sampled
immediately, and then bagged and stored (to simulate field waste handling procedures) in the waste
staging area. The decontaminated test materials were re-sampled after a drying time of 1 day (at least 18
hours), 7 days, and 30 days to determine if storage duration could increase the effectiveness of the
decontamination treatment (where decontamination treatment is defined, for the purpose of this study, as
a prescribed decontamination procedure). A subset of inoculated bagged waste samples was left
untreated and served as positive controls. The sampling strategies discussed herein have been selected
and optimized to determine the survival of B. atrophaeus spores following decontamination treatment.
The purpose of this study was to identify effective and efficient means to decontaminate waste on-site
(i.e., not requiring transport of an infectious agent and treatment at remote, specialized, off-site facilities),
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and to compare the efficiency of both detection methods used. The decontamination and sampling
strategies utilized were selected by an EPA project team, which consisted of staff from EPA's Office of
Research and Development, EPA's Office of Solid Waste and Emergency Response, and EPA's Region
7.
1.2 Project Objectives
The primary objective of this work was to estimate the efficacy of liquid-based decontamination
approaches for on-site treatment of bundled or bagged waste items (contaminated indoor office items that
would generally be placed in bags or bundled for transportation during the removal process) typically
generated during an anthrax clean-up response for an indoor office setting. While there are no
established decontamination procedures or performance criteria for 8. antfvac/s-laden waste items, it is
likely that waste disposal facilities will require post-decontamination sampling of waste items priorto
acceptance. The criterion for waste acceptance is also not known, and may differ between facilities and
states. Further, the amount of viable spore contamination within waste items is expected to vary widely.
To this end, it is impossible to evaluate the exact conditions (spore load, waste acceptance criteria, etc.)
expected to be encountered during an actual 8. anthracis remediation. This challenge is not unlike that
encountered in the evaluation of sporicidal decontaminant efficacies. It is impractical to evaluate all
potential sporicides under all conditions (spore load, material type, environmental conditions, etc.). To
address this challenge in the evaluation of sporicides and in the current waste decontamination
evaluation, and allow comparison across products or methods (respectively), a consistent challenge is
posed to evaluate effectiveness. For example, a 7 Log spore challenge (inoculation of simulated waste
items with ~ 5 x 107 spores) was used across all tests and materials. Consistent with sporicidal efficacy
tests used to register sporicides under FIFRA, the current study utilized the generally accepted criterion of
6 Log Reduction to consider an approach effective. Recovery of no viable spores following treatment was
considered highly effective.
An additional objective was to assess the collection efficiency of the two waste sampling methods utilized.
The collateral damage to materials during decontamination procedures was monitored. The ultimate
objective was to provide data to support development of a step-wise procedure(s) for on-scene
responders and decontamination teams to use for on-site waste treatment during responses involving the
indoor environment. Demonstrated waste decontamination procedures could reduce the cost and time of
a response by validation of decontamination method, in advance of an emergency incident, that could be
used to justify reducing the number of waste characterization samples required and/or reducing the
stringency of waste treatments required off-site.
1.3 Experimental Approach
1.3.1 Testing Sequence
The testing sequence used to meet the objectives of this project was:
1. Prepare material sections for each test material as described in Section 2.2.
2. Pre-punch material sections (carpet, upholstery, and book materials), and retain the excised sections
for use as 18 mm coupons for extraction-based sampling procedure. The nitrile gloves did not require
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the use of 18-mm punch coupons, but rather used the 1" tip of each finger for extraction-based
analysis.
3. Assemble the material sections by re-inserting the 18 mm excised coupons into their respective void
areas of the material section.
4. Sterilize the materials prior to inoculation using ethylene oxide or VHP® sterilization. Wait a minimum
of 7 days after sterilization before inoculating materials (See Section 2.3.1).
5. Inoculate 18 mm positive control and test material coupons (without removing from larger material
section), each fingertip of each nitrile glove PPE, and the target page samples.
6. Allow inoculum carrier to dry for at least 18 hours.
7. Apply the decontamination procedure to procedural blank material section batch then to the test
material section batch.
8. Collect the samples from material sections immediately upon completion of the decontamination
procedure.
9. After sample collection, bag the material batch such that the total weight does not exceed 35 Ibs per
bag.
10. Sample remaining bagged material sections in the waste staging area after a drying time of 1 day (at
least 18 hours), 7 days, and 30 days.
1.3.2 Decontamination Strategy
It is preferred that an on-site decontamination procedure be effective, yet generate liquid and solid waste
products that are easily disposable, and have minimal detrimental environmental impacts. Accordingly,
the current study evaluated decontaminants in order of accessibility (most to least): diluted bleach (0.5%
NaCIO) and pH adjusted bleach (pAB). Material decontamination was initiated with diluted bleach. In
those cases where diluted bleach proved ineffective (less than 6 log reduction in colony forming units of
B. atrophaeus) when used in conjunction with the dunking/immersion procedure, pAB was used and the
testing cycle repeated.
1.3.3 Method Development for Neutralization
The presence of decontamination solution components in the rinsate or extraction liquid (desorbed from
the coupon) could negatively bias colony forming unit (CPU) quantification results. Prior to the
decontamination testing sequence, neutralization tests were performed to determine the amount of
neutralizer liquid needed to quench each decontaminant/application combination. Decontaminant
neutralizers were added to liquid samples immediately after collection to quench their activity, resulting in
precise chemical exposure durations and lower recovery bias.
-------
2 Materials and Methods
2.1 Facility Design
All decontamination activities were conducted inside the spray booth area located in the EPA's Research
Triangle Park (RTP) facility in High Bay Room H122A, a single access point room containing ventilation
independent of the High Bay Building containing the room. The spray booth also served as the waste
staging area.
The immersion tank was a 10 ft3 (0.28 m3) Poly Hog Trough® (EZ Grout Corp., Waterford, OH) made of
virgin polyethylene with steel legs (http://www.ezgrout.com/products/masonry products/hog-trough.php)
(Figure 2.1). The overall dimensions of the tank were 26" x 54 1/2"x24" (66.04 cm x 138.43 cm x 60.96
cm).
Figure 2.1: Poly Hog Trough®.
2.2 Test Coupon Preparation
All the coupons were sterilized prior to use to prevent any background organisms from confounding the
tests; the books and the glove materials were fumigated with ethylene oxide using an Andersen (Haw
River, NC) EO-Gas® 333 sterilization system, while the carpet and the upholstered coupons were
fumigated via a Vaporous Hydrogen Peroxide (VHP®) sterilization cycle according to Miscellaneous
Operating Procedure (MOP) 3120 (Appendix A). To prevent cross contamination, on the day of testing,
procedural blank coupons were moved into the spray chamber, and procedural blank decontamination
occurred before decontamination of any inoculated coupons.
2.2.1 Carpet and Upholstery
The carpet coupons were ready-made 24" x 24" (0.61 m x 0.61 m) 100% nylon tiles, exuberant 00310
color type (Figure 2.2). Upholstered coupons (20" x 20" (0.51 m x 0.51 m)), were prepared according to
Miscellaneous Operating Procedure (MOP) 3194, with layers of foam and fabric layer adhered together
(Figure 2.3). MOP 3194 and other associated MOPs can be found in Appendix A.
-------
Figure 2.2: Carpet tile.
Figure 2.3: Front of assembled upholstered coupon.
For both the carpet and upholstered coupons, a 20" x 20" (2581 cm ) template was used. This template
(see Figure 3.1) was comprised of 4"x4" (103 cm2) grid size sections to create a 5 by 5 sampling grid. An
18 mm-diameter coupon was excised from the center of each grid section for sample inoculation. For
each sampling event, the 18 mm coupons were either removed for extraction, or left in place to be part of
the grid section that was sampled using the surface sampling method.
2.2.2 Paper
Paper samples consisted on the entire front cover (inside cover inoculated) along with the first page of the
Merck Manual of Medical Information (Second Home Edition, 2004) (Figure 2.4); and pages 955
(inoculated) plus one page before and two pages after (953-960), respectively. For sampling, the front
pages, and the middle pages were removed, and processed separately. Each page measured 9" by 6.5".
-------
THE
MEFCK
MANUAL
OF
MEDICAL INFORMATION
The woild's most widely used
t loi me twenty-fits) c
Figure 2.4: Paper material.
2.2.3 Nitrile Gloves
The material chosen to represent PPE waste consisted of powder free, 5.5 mils thick, 9" (23 cm) length,
white disposable nitrile gloves (McMaster Part#52555T15, www.mcmaster.com) illustrated in Figure 2.5.
The powder-free gloves are considered superior for applications where particulate contamination is a
concern. Whole gloves were utilized, however, the inside tips of each finger were inoculated and served
as replicates. Following decontamination, the terminal 1" (2.5 cm) of each finger was excised and
collected as an individual sample.
Figure 2.5: White disposable nitrile glove.
-------
2.3 Spore Preparation
The test organism for this investigation was a liquid spore suspension of 8. atrophaeus (strain: ATCC®
9372) in 29% ethanol solution. This bacterial species was formerly known as 8. subtilis var Niger and
subsequently 8. globigii. The spores were purchased from Yakibou, Inc. (Holly Springs, NC), at a
population of 1 x 109 colony forming units (CPU) per ml. The titer of the stock was confirmed at the start
of each testing event by the procedure detailed in MOP 6535a.
2.3.1 Coupon Inoculation and Test Preparation
Inoculation of the 18 mm coupons (carpet and upholstery), and the front cover and the middle pages of
the books were performed by aseptically applying 100 uL of a diluted spore solution to reach a target
concentration of 5 x 107 CPU recoverable from each sample. Fingertips of the nitrile gloves were
inoculated to reach the same target concentration. To simulate field conditions, where gloves are turned
inside out during doffing procedures, tested gloves were inoculated on the exterior surface, allowed to
dry for 18-24 hours, then turned inside out prior to use in testing.
2.4 Decontamination Procedure
The general decontamination procedure consisted of "dunking" a batch of coupons in the immersion tank
containing the decontamination solution for a prescribed immersion time. This decontamination
procedure was performed according to MOP 3195 "General Procedure for Immersion Decontamination",
included in Appendix A, and is described below:
1. Prepared decontaminant bath in chemical resistant container. Performed all required quality control
(QC) checks listed in Table 5-2.
2. Collected the material batch for immersion which consisted of 3 pre-punched sterile material sections
(contains the test samples) and enough non-punched sterile material sections (does not contain test
samples) to fill the waste storage bag (not to exceed 35 Ib (16 kg) when wet; amounts vary per
material). Of the material section batch, only three sections of the decontaminated material were
inoculated with Bacillus spores. For example, of a 35 Ib batch of carpet tiles, only 3 tiles contained
inoculated 18 mm coupons.
3. The material batch (not to exceed 35 Ib when wet) was submerged in the decontaminant bath and
subjected to the prescribed decontamination procedure.
4. Removed material sections and allowed them to drain briefly (15 minutes) over the decontaminant
bath. Immediately collected the post decontamination (T0) samples per material type.
5. Aseptically transfered decontaminated materials to a labeled material 55-60 Gallon Contractor's
storage bag (Uline Model S-19876) which remained closed in the waste staging area until the next
sampling event. Material types were bagged separately and arranged such that one inoculated
material section was located at the bottom of the batch, 1 was located in the middle, and one was
located on the top.
This procedure was repeated for each material using a single immersion container. Therefore, the
immersion container was sanitized between tests by removing all debris, wiping interior surfaces with
8
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Dispatch Hospital Cleaner Disinfectant Towels with Bleach wipes (Chlorox, Company, Oakland, CA),
rinsing interior surfaces with deionized water, then drying with 70% ethanol prior to the start of each test.
Testing was performed using a "clean team/dirty team" technique. The "dirty team" was responsible for
moving the material sections into and out of the immersion tank and performing the decontamination
procedure. The "clean team" was used for procedural blank, control and test sampling. Only dirty team
members handled contaminated items and only clean team members handled procedural blank coupons
and samples. New disposable lab coats were worn for each new material or contamination level. Fresh
gloves were donned prior to applying the decontamination procedure then changed before handling the
material section after completion of the decontamination procedure. Dirty team members could become
clean team members by donning a new set of protective garb (inner and outer gloves, lab coat, P95
mask, and hairnet).
2.5 Method Development for Neutralization
The presence of decontamination solution components in the rinsate or extraction liquid (desorbed from
the sample) could negatively bias spore recoveries. Prior to the decontamination testing sequence,
neutralization tests were performed to determine the amount of neutralizer liquid needed to quench
residual decontaminant produced from each decontaminant/application combination.
HACH® Method 10100 (http://www.hachco.ca: Hach Company, Loveland, CO) for high range bleach was
used to experimentally determine the amount of sodium thiosulfate (STS) required to neutralize in excess
the active ingredient (i.e., free available chlorine [FAC]) in pAB and diluted bleach. Due to the variation of
the amount of decontaminant solution on the coupons, excess of stoichiometric neutralization was
evaluated to ensure that it did not hinder the recovery of the spores. Analyses of the spores in the
optimized excess neutralizer solution was also evaluated at a 1 hour and a 24-hour hold time to see if the
lag time for processing the samples had an effect on the viable spore recoveries. Finally, the effect of
spore inoculation concentration and the decontamination time on the neutralization tests recoveries were
also evaluated.
2.6 Test Matrix
2.6.1 Neutralization Method Development Test Matrix
Samples collected from wet, decontaminated material sections may contain enough decontamination
liquid to confound enumeration analysis and therefore require neutralization. To determine the
appropriate amount of neutralizer needed to quench residual decontaminant, two sets of 5 un-inoculated
18 mm coupons from each test material (carpet, PPE, upholstered furniture, and paper) were immersed in
each fresh decontamination liquid (diluted bleach or pAB) fora prescribed soaking period corresponding
to each test condition. Each soaked coupon was then placed into a 50 ml conical tube containing
phosphate buffered saline with Tween®20 (PBST) (ICI Americas Inc., Bridgewater, NH) solution used for
extraction. One set of samples was spiked with 1 x 107 (High) and second set spiked with 1 x 102 (Low)
B. atrophaeus spores to observe the effects of wet sample collection on both high and low 8. atrophaeus
concentrations that could be present on actual test samples. Two additional sets of coupons were used
as controls; these samples were collected from materials not decontaminated, placed into PBST for
extraction, and spiked with 1 x 107 and 1 x 102 spores, respectively.
-------
The two populations (CPU recovered from decontaminated sample vs. CPU recovered from control
spiked sample) for each coupon type and test condition were then analyzed to determine if there was a
negative bias induced by the presence of the decontaminant solution in the extraction liquid. If such bias
was statistically significant, then neutralization tests were performed using STS as the neutralizing agent.
The amount of STS was determined based on the average STS required to neutralize the free available
chlorine (FAC) of the diluted bleach or pAB solutions following extraction of pre-spiked decontaminated
coupons, and the ability to obtain acceptable recoveries (within 0.5 log of the control coupon
concentration). The neutralization methods Test Matrix is shown in Table 2.1.
Table 2.1: Neutralization Methods Test Matrix
Test ID
STS1
STS2
STS3
STS4
Decontaminated Spike
5
5
5
5
Control Spike
5
5
5
5
Spike Amount
1x107CFU
1x107CFU
1x102CFU
1x102CFU
Extraction Liquid
PBST
PBST + STS
PBST
PBST+STS
CPU, colony forming units; PBST, phosphate buffered saline with Tween®20; STS, sodium thiosulfate
Aliquots of the bulk decontamination solution were collected and analyzed for the active ingredients using
the methods listed in Table 2.2 immediately (within 10 minutes) before use. Temperature readings of the
bulk decontamination solution were also taken immediately (within 10 minutes) before use.
Table 2.2: Measurement and Neutralization Methods
Decontamination Solution
Dilute Bleach (0.5% NaCIO)
pAB
Active Ingredient
Hypochlorite
Hypochlorite
Measurement Method
MOP3128-A
MOP3128-A
Proposed Neutralization Solution
STS
STS
MOP, Miscellaneous Operating Procedures; pAB, pH-adjusted bleach; STS, sodium thiosulfate
2.6.2 Test Matrix
The test matrix was initially devised to optimize the on-site decontamination procedures by maximizing
efficacy and minimizing the manual effort and hazard level of the procedure. The decontaminant
optimization process was designed to test decontaminants in order of accessibility. The most accessible
to the least, as shown in Table 2.3, were diluted bleach and pAB. The planned decontamination
procedures in order of increasing intensity were spray, immersion, rigorous immersion, and
immersion/spray, as shown in Table 2.4.
10
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Table 2.3: Decontaminants and Accessibility
Decontaminant
Diluted Bleach
pH- Adjusted Bleach
Decreasing Accessibility
{}
Table 2.4: Decontamination Procedures and Intensity
Decontamination Procedure
Spray
Immersion
Rigorous Immersion
Immersion/Spray
Increasing Intensity
\
/
Due to time constraints for each test (30 days to complete a full testing sequence), the testing approach
consisted of starting with the two most readily available decontaminants (diluted bleach and pAB) and the
immersion procedure. If the procedure were determined to be ineffective (<6 log reduction), a procedure
with a higher intensity (rigorous immersion) would be applied. If the procedure was determined to be
effective, then the procedure with a lower level of intensity would be applied (spraying). Tests were
identified by combining the decontaminant and the application procedures (Tables 2.3 and 2.4). Table
2.5 lists the actual test matrix that was performed, analyzed and described in this report. Please note that
the tests on carpet were performed at different dunking/immersion times of 15, 30, and 60 minutes,
respectively, from the top to the bottom of Table 2.5.
Table 2.5: Decontamination Test Sequence Event
Decontamination
Procedure
Immersion
Immersion
Immersion
Immersion
Rigorous Immersion
Immersion
Immersion
Rigorous Immersion
Decontaminant
Solution
pAB
pAB
Diluted Bleach
pAB
pAB
Diluted Bleach
pAB
pAB
Material
Type
Carpet
PPE
PPE
Upholstery
Carpet
Paper
Paper
Carpet
Exposure
Time
15 minutes
15 minutes
15 minutes
15 minutes
30 Minutes
15 minutes
15 minutes
60 minutes
Test Date
(DayO)
Sept 25,
Nov5,2013
Nov 19,
Dec 10,
Jan 23,
Feb4,
Feb18,
Mar 25,
End Date (Day
30)
Oct23, 2013
Dec 4, 2013
Dec 16, 2013
Jan 8, 2014
Feb20, 2014
Mar 4, 2014
Mar 20, 2014
Apr 23, 2014
pAB, pH adjusted bleach; PPE, personal protective equipment
11
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3 Sampling and Analytical Procedures
3.1 Sampling Strategy
Prior to each sampling event, all materials needed for sampling were prepared using aseptic techniques
and placed in a bin containing enough sampling kits, gloves and bleach wipes to accommodate all
required samples for the specific test. The materials specific to each protocol are included in the relevant
sections below.
In an effort to minimize the potential for cross-contamination during sampling, and in accordance with
aseptic technique, a sampling team was utilized, made up of a "sampler" (handling only the sampling
media, a "sample handler" (the only person to handle material coupons during the sampling event), and a
"support person," who in addition to being responsible for handing sterile templates to the sampler was
also responsible for handling sealed samples and disinfecting outer bags and containers for transport.
Within a single test, sampling of the material sections was completed for all procedural blank coupons
first before sampling of any test material or control sections. Sampling was done by collecting the
coupons for extraction first, then sponge sampling the remaining coupons according to the protocols
documented below. The surface area for all sponge samples was about 103 cm2 (16 in2) and the
diameter for all extraction coupons was 18 mm (0.71 in). Once sampling was complete, material sections
were returned to their original waste storage bag.
Sponge stick and stub sample integrity was maintained by storage of samples in triple containers (1 -
sample collection container, 2 - sterile bag, 3 - disinfected container holding all samples from a test). All
individual sample containers remained sealed while in the decontamination lab or in transport after the
introduction of the sample. The sampling person did not handle any samples after they were relinquished
to the support person during placement into the primary sample container.
Since the current sampling techniques are destructive, each coupon was sampled only once, however
each test was replicated 3-5 times (See Table 3.2.1). Test coupons and positive controls were sampled
in parallel for each sampling time sequence. Temperature, pH, and active ingredient measurements of
each decontaminant solution were performed prior to each decontamination procedure. The temperature
and relative humidity (RH) of the waste staging area were recorded by three strategically placed,
calibrated HOBO® Data Loggers (Onset Computer Corporation, Bourne, MA) temperature and RH
sensors. Additional measurements included quality control checks on the reagents and equipment used
in the decontamination procedure.
3.1.1 Sponge-Stick™ Sampling
3M Sponge-Stick™ with neutralizing buffer (part number SSL10 NB; 3M, St. Paul, MN) were used to
aseptically sample 103 cm2 (16 in2) areas on the carpet material sections and 79 cm2 (12.25 in2) areas on
the upholstery and paper material sections using sampling templates with 25 sampling areas as a guide
for Sponge-Stick™ sampling. Samples were collected according to MOP 3165.
3.1.2 Extractive Sampling
The 18 mm coupons for extractive sampling were removed from the sampling area and transferred in the
waste staging area into 50 ml sterile vials containing 10 ml PBST and the predetermined amount of
12
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neutralization liquid (STS). For PPE samples, excess pAB was captured in a separate vial for
subsequent analysis.
3.1.3 Sample Preservation
After sample collection for a single test was complete, all biological samples were transported to the
NHSRC Research Triangle Park (RTP) Biocontaminant Laboratory immediately, with appropriate chain of
custody form(s) and stored at 4 °C ± 2 °C until extraction. All samples were allowed to equilibrate at room
temperature for one hour prior to extraction and plating. Liquid samples were stored no longer than 24
hours prior to analysis. Samples of other matrices were stored no longer than 5 days before the primary
analysis. A typical holding time, prior to analyses, for most biological samples was 2 days.
3.1.4 Sampling Points
All the samples were collected from wet materials immediately after application of the decontamination
procedure, or after the required hold time (as bagged waste in the waste staging area), and neutralized
immediately after sample collection Table 3.1 lists the coupon types and the respective sampling
procedures.
Table 3.1: Coupon Types Used to Evaluate Waste Decontamination Procedures
Material
Carpet
Upholstered Furniture
Paper
PPE materials
Porous or
Non-porous
Porous
Porous
Porous
Non-porous
Material Description
Building material, high traffic,
commercial carpet tile, 24" x 24"
Upholstered seat pan, 20" x 20"
Book pages
Nitrile, powder free, disposable
exam gloves
Coupon/Sample
Size
18 mm punch / 101
mmx 101 mm
square
18 mm punch 789
mmx 89 mm square
Whole Front and
Middle pages/ 22.9
cmx 16.5 cm
2.5 cm tip of finger
Sampling
P raced ure(s)
Extraction / Sponge-
Stick™
Extraction / Sponge-
Stick™
Extraction
Extraction
PPE, personal protective equipment
3.1.5 Carpet and Upholstery
When sampling upholstery and carpet materials, a sterile template was placed on the coupon creating a
grid with an inoculated 18 mm coupon within the center of each 4"x4" area (Figure 3.1 shows a material
section with template during sampling). Designated areas on the coupon were sampled by either Sponge-
Stick™ or by removing a pre-punched 18 mm coupon for extraction sampling. When possible, Sponge-
Stick™ and extraction samples were taken from areas representing different parts of the coupon (center,
sides, and corners).
13
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Sponge Stick
18 mm coupon
(removed)
Sampling
template
18 mm coupon
(installed)
Material
section
Figure 3.1: Material section shown with template during sampling with Sponge-Stick™ and
extraction.
3.1.6 Paper Samples
Paper samples, designated paper front (PF) and paper middle (PM), included the front cover and first
page and middle pages, as described previously. The additional pages adjacent to those inoculated were
collected to account for any spores being relocated (via capillary action, etc.) to the adjacent pages during
inoculation and/or decontamination. Sterile razor cutters were used to excise the paper samples after
decontamination testing. Once excised, the paper samples were put inside a sterilized pre-labeled
stomacher bag along with 80 ml of PBST and a pre-determined volume of STS neutralizer, and mixed
altogether. Eight books were used for each sampling sequence (3 books: front and middle pages) and
inside cover page, 2 books for positive controls: front and middle pages, 2 books for field blank samples:
front and middle pages, and a 1 book for laboratory blank: front and middle pages for a total of 32 books
for the 4 test sequences for each decontamination method.
3.1.7 PPE Samples
Gloves were inoculated on the outside, and then aseptically turned inside-out to mimic removal and
placement into a decontamination line waste stream. Three gloves were inoculated for each test (3
samples from 3 inoculated fingers (thumb, middle, pinky) for each glove, resulting in 9 samples), one
glove for positive controls (5 samples from all 5 inoculated fingers), one glove for field blank sample (3
un-inoculated fingers), and one glove for laboratory blank sample (1 sample from 1 un-inoculated finger)
for a total of 6 gloves per test sampling sequence, or 24 gloves for the 4 test sequences for each
decontamination method.
14
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3.2 Sampling Frequency
After the waste decontamination procedure was executed, material sections were either sampled
immediately or bagged and stored in the waste staging area for subsequent sampling. Sampling after
storage occurred following several simulated waste storage durations, including 1 day (at least 18 hours),
7 days, and 30 days. Figure 3.2 outlines the sampling timeline for both sampling approaches (extraction
and Sponge-Stick™). The indicated number of test samples was collected from each of the 3 inoculated
material sections as well as from procedural blanks (un-inoculated coupons that were exposed to test
procedures) and positive control sections (inoculated coupons that were not exposed to test procedures).
This timeline was developed to model the hold times decontaminated materials may be subject to on-site
prior to being transported off site for final disposal. Three positive control and 3 procedural blank samples
were collected during each sampling event. Positive control coupons were inoculated concurrently with
test coupons so they would have the same bacterial aging times as the samples. The synchronization of
inoculation and sampling of positive control and test coupons was critical for accurate log reduction
analysis. Bagged untreated positive control materials were sampled at the same time (within 8 hours) as
their decontaminated counterparts.
• 3 test coupons
removed for
extraction
• 3 test coupons
Sponge- Stick™
sampled
• 3 positive
controls, 3
procedural blanks
• 3 test coupons
removed for
extraction
• 3 test coupons
Sponge- Stick™
sampled
• 3 positive
controls, 3
procedural blanks
• 3 test coupons
removed for
extraction
• 3 test coupons
Sponge- Stick™
sampled
• 3 positive
controls, 3
procedural blanks
• 3 test coupons
removed for
extraction
• 3 test coupons
Sponge- Stick™
sampled
• 3 positive
controls, 3
procedural blanks
Figure 3.2: Sampling timeline.
3.2.1 Sample Quantities
The number of samples collected for both the neutralization and on-site decontamination tests are
outlined in Table 3.2. This table includes not only the biological samples, but also samples collected to
describe the decontamination process for each test in the test matrix. Some tests required 18 mm
samples, sponge samples, or both. For tests that indicate both 18 mm and sponge samples were
collected, the sample quantities for sterility blanks, positive control samples, procedural blanks, and test
samples for both sample types were identical.
15
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Table 3.2: Number of Sample Ty
Test
On-Site
Decontamination
DaV (To Days' M Day
Ty Days' ' 30 Days)
Type of Sample
Material
STS
Carpet Coupons
Upholstered Coupons
PPE3
Paper
pes per Material Section per Sampling Sequence
Laboratory
Blank
E1
0
1
1
1
2
S2
0
1
1
0
0
Positive
Control
Samples
E
5
3
3
5
6
S
0
3
3
0
0
Field
Blanks
E
0
3
3
3
2
S
0
3
3
0
0
Test
Samples
E
5
3
3
15
6
S
0
3
3
0
0
Extractive
Samples
(Y/N)
Y
Y
Y
Y
Y
Sponge
Samples
(Y/N)
N
Y
Y
N
N
N, no; PPE, Personal Protective Equipment; Y, yes
1 Number of extractive samples; 2Number of Sponge-Stick™ samples; 3Each finger of a glove is considered one
sample; note that for the gloves, runoff samples were also collected.
3.3 Measurement Methods
In addition to the collection of material samples, temperature, pH, and active ingredient measurements of
each decontaminant solution were performed prior to each decontamination procedure. The temperature
and relative humidity (RH) of the waste staging area were recorded by three strategically placed,
calibrated HOBO® Data Loggers temperature and RH sensors. Additional measurements included quality
control checks on the reagents and equipment used in the decontamination procedure.
3.3.1 Decontamination Solutions
The pH-adjusted bleach was prepared as described in MOP 3128-A; in short, it consisted on diluting one
part Clorox® concentrated germicidal bleach (Clorox Corp., Oakland, CA) with eight parts of deionized
water and one part 5% (v/v) acetic acid (Fisher Scientific, Pittsburgh, PA; Part# 13025), or equivalent).
The pH was adjusted to 6.5-7.0 with 5% acetic acid, and the free available chlorine content was adjusted
to 6000-6700 ppm with deionized water after preparation. The pH-adjusted bleach was used within three
hours of preparation. The diluted bleach was prepared fresh prior to testing by mixing one part Clorox®
concentrated regular bleach with approximately 14 part of deionized water to reach a target FAC of about
6000 ppm. Safety precautions were taken to protect personnel from liberated chlorine gas produced as a
result of pH reduction of the bleach solution.
The free available chlorine (FAC) concentration of bleach formulations was measured according MOP
3148 based on ASTM Method D2022-89. In short, a 5 mL aliquot was mixed with a buffered potassium
iodide solution and iodometrically titrated with STS to a colorless end-point. The aliquot was taken and
analyzed immediately after formulation and mixing. The validity of the FAC measurement equipment
(Hach® High Range Bleach Test Kit, Method 10100 [Model CN-HRDT]) was confirmed through the
titration of a chlorite ion standard. The pH of each solution was measured with an Oakton Acorn® Series
pH 5 meter (Oakton Instruments, Vernon Hills, IL). This meter was calibrated daily.
3.3.2 Microbiological Samples
General aseptic laboratory technique to prevent cross-contamination was followed and was embedded in
MOPs used by the NHSRC RTP Biocontaminant Laboratory to recover and plate samples. Additionally,
16
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the order of analysis (consistent with the above) was as follows: (1) all blank coupons; (2) all
decontaminated coupons; then (3) all positive control coupons. Both coupon and Sponge-Stick™
extracts samples were diluted, plated and manually enumerated. Details of the extraction and analytical
procedures are provided below.
3.3.2.1 Sample Extraction
Extraction sample vials containing 18 mm coupons, phosphate-buffered saline with 0.05% TWEEN® 20
(PBST) (Sigma-Aldrich, Co, P/N P3563-10PAK [PBST]), and neutralizer were vortexed for 2 minutes to
further dislodge any viable spores. Each vial was briefly re-vortexed immediately before any solution was
withdrawn for dilution or filter plating. The Sponge-Stick™ samples were extracted according to the
validated CDC cellulose Sponge-Stick™ procedure as outlined in MOP 6580 "Recovery of Bacillus
Spores from 3M Sponge-Stick™ Samples".
3.3.2.2 Sample Analysis
Experimental samples were subjected to up to five-stage serial dilutions (10~1 to 10~5) in accordance with
MOP 6535a (a revision of MOP 6535 specifically for bacterial spores, attached in Appendix A), plated in
triplicate and incubated overnight at 35 °C ± 2 °C. Following incubation, CPU were manually enumerated
according to MOP 6535a. Samples that had fewer than the reportable limit of 30 CFU/plate of the
undiluted sample underwent further analysis according to MOP 6565 and/or MOP 6584. These MOPs
describe filter plating and re-plating, respectively.
3.4 Data Analysis
The total spore recovery for each method, material and time point was calculated by multiplying the mean
CPU counts from triplicate plates by the inverse of the volume plated (e.g., 1/0.1 ml or 10), by the dilution
factor, and finally by the volume of the sample extract (X ml for Sponge-Stick™ samples and Y ml for
extracted stubs).
3.4.1 Sampling Efficiency
To determine which of the two detection methods employed in the study was more efficient at detecting
viable spores on the waste materials tested, the sampling efficiency (SE) for each detection, all time
points and material types was calculated. SE is defined as the ratio of the measured mean sampled CPU
(CFUm) to that of the inoculums (CFU0):
(3-1)
3.4.2 Surface Decontamination Efficacy
The efficacy of each decontaminant was assessed by determining the number of viable organisms
remaining on each inoculated test coupon after decontamination and comparing this result to the number
of viable organisms extracted from the positive control coupons, which were inoculated but not
decontaminated. Excess decontamination solutions (in the form of rinsate) were also analyzed from PPE
samples to determine if the representative decontamination application washed the spores from the
surface of the PPE coupons or if the decontaminant inactivated the spores. These rinsate analysis results
were calculated and evaluated, but not used in the decontamination efficacy calculation.
17
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The surface decontamination efficacy is defined as the extent (as Iog10 reduction) by which viable spores
extracted from test coupons after decontamination were less numerous than the viable spores extracted
from positive control coupons. First, the logarithm of the CPU abundance from each coupon extract was
determined, and then the mean of those logarithm values was determined for each set of control and
associated test coupons, respectively. This value is reported as a log reduction on the specific material
surface as defined in Equation 3-2.
77 =^J -- ^ - (3-2)
''
N
where:
Surface decontamination effectiveness; the average log
77. = reduction of spores on a specific material surface (surface
material designated by;)
NC The average of the logarithm (or geometric mean) of the
2_, log(CFUcfk) ^ number of viable spores (determined by CPU) recovered on
— the control coupons (C indicates control and Nc is the number
c of control coupons)
The average of the logarithm (or geometric mean) of the
Nt
V \oe(CFU ) number of viable spores (determined by CPU) remaining on
k=\ = the surface of a decontaminated coupon (S indicates a
Ns decontaminated coupon and Ns is the number of coupons
tested).
When no viable spores are detected, a value of 0.5 CPU was assigned for CFUs,k, and the efficacy was
reported as greater than or equal to the value calculated by Equation 3-1.
The standard deviation of the average log reduction of spores on a specific material (r|j) is calculated by
Equation 3-3:
where:
or) _ Standard deviation of r|j, the average log reduction of spores
Vi on a specific material surface
18
-------
~j _ The average log reduction of spores on a specific material
'' surface (surface material designated byi)
The average of the log reduction from the surface of a
k decontaminated coupon (Equation 3-4)
Ns = Number of test coupons of a material surface type.
(3-4)
Where:
Represents the "mean of the logs" (geometric mean),
NC the average of the logarithm-transformed number of
2_l\og(CFUck~) ^ viable spores (determined by CPU) recovered on the
— control coupons (C = control coupons, Nc = number of
c control coupons, k = test coupon number and Ns is the
number of test coupons)
^ci i _ Number of CPU on the surface of the kth
CFLJc k —
decontaminated coupon
Total number (1 ,k) of decontaminated coupons of a
s material type.
The average surface decontamination effectiveness of the decontamination technique for spores
recovered on the material, independent of the type of material, was evaluated by comparing the
difference in the logarithm of the CPU before decontamination (from sampling of the positive control
coupons) and after decontamination (from sampling of the test coupons) for all the tested materials.
These data are calculated by determining the arithmetic mean of n for all material types according to
Equation 3-5 and reported as log reductions of spores for each decontamination technique.
(3-5)
Where /7 is the overall surface log reduction efficacy for the technique, and Nj is the total number of
coupon material types tested with that technique (;' indicates coupon material type).
19
-------
The standard deviation of f]7 is calculated by Equation 3-6:
where:
C*n _ Standard deviation of T\j, the overall surface log reduction
VT efficacy for the technique
TjT = Overall surface log reduction efficacy for the technique
_ The average log reduction of spores on a specific material
'' surface (surface material designated byi)
NJ = Number of coupon material types.
Decontamination procedures were considered effective if greater than 6 log reduction was achieved. A procedure
was considered highly effective if no viable spores were recovered following decontamination.
3.4.3 Statistical Analysis
To determine if either the extraction or sponge sampling method was better for collecting spores, a 2
independent t-test (or 2-factor analysis of variance [ANOVA]) analysis of the recovery from two sampling
methods was performed for each decontamination/material combination.
Single factor ANOVA was used to determine if time is a factor in the decontamination efficacy for each
material and each sampling method individually (see Table 3.3).
The Shapiro-Wilk test was used to check if the data sets used in the 1 or 2-factor ANOVA statistical
analysis came from normally distributed sample population. The Shapiro-Wilk test is designed to test for
normality of small data-size population (n < 50). The null hypothesis of this test is that the population is
normally distributed. In other word, if the p-value is less than 0.05 (95% confidence interval), the null
hypothesis is rejected and there is evidence that the data tested are not from a distributed population. In
contrary, if the p-value is greater than 0.05, then the population is normally distributed.
20
-------
Table 3.3: Number of Sample Types per Material Section per Sampling Sequence
Analysis
Paired t-test or 2 Factor ANOVA (To determine the
better detection method for this application, over
time). Performed for each material.
Single factor ANOVA (To determine if time is a factor
in decon for each material). This would be a group of
individual analyses whose results are compared.
Independent Variable
Detection method
(extract and sponge)
Time points
(for individual materials)
Dependent Variable(s)
Collection efficiency over time: calculated
from known inoculum and analysis of positive
controls at different time points
Decontamination efficacy over time
21
-------
4 Results and Discussion
This section presents the results of the overall effectiveness of the dunking or immersion of the waste to
reduce and/or inactivate spores of 8. anthracis from a contaminated surface of different types and
porosity. Effectiveness was determined by sampling the waste contents following decontamination and
comparing to sampling controls, which did not undergo the decontamination treatment. A 7 Log spore
challenge (inoculation of simulated waste items with ~ 5 x 107 spores) was used across all tests and
materials. Consistent with sporicidal efficacy tests used to register sporicides under FIFRA, the current
study utilized the generally accepted criterion of 6 Log Reduction to consider an approach effective.
Recovery of no viable spores following treatment was considered highly effective.
The results of the comparison of effectiveness of each decontaminant/application combination for each
material and the evaluation of the quantitative performance parameters are presented in Section 4-1.
Evaluation of the two sampling methods (extraction versus Sponge-Stick™) is discussed in Section 4-2.
The results for the neutralization tests performed prior to each decontamination sequence are presented
in Section 4-3. The results of the decontamination approach that utilized dunking or immersion of the
waste are reported in Section 4.4.The Shapiro-Wilk test was used to check if the data sets used in the 1
or 2-factor ANOVA statistical analysis came from normally distributed sample population. Only normally
distributed data sets are used for the various analyses. If a dataset is not normally distributed, it will be
discussed separately.
4.1 Sampling Methods Evaluation
Independent of the decontamination assessment, the two sampling methods (extractive and Sponge-
Stick™) were evaluated by comparing positive control recoveries at each sampling point (Day 0, Day 1,
Day 7, and Day 30) for both carpet and upholstery. Comparing recoveries of the two methods at any
particular time-point allowed a comparison of each method's performance, comparison of recoveries to
the starting inoculum shows the temporal effects on spore recovery through the duration of the
experiment (30 Days). Such temporal effects on the extractive sampling method were also evaluated for
both the PPE and the paper materials.
4.1.1 Carpet Material
Spore recoveries from carpet as a function of simulated waste storage time (sampling time delay) are
shown in Figure 4.1, and summarized in Table 4.1. The averaged recoveries were 1.37 x 107 (Standard
deviation, SD = 7.91 x 106, n= 33 samples) CFU using the extractive method (removal of a coupon from a
larger sample), and 5.02 x 105 (standard deviation (SD) = 4.45 x 105, n= 33 samples) CFU using the
Sponge-Stick™) sampling approach. The overall percent recovery of the extractive method when
compared to the starting inoculum (26.7% + 3.56%) was much higher than that of the Sponge-Stick™
sampling method (0.94% + 0.27%). A two-sample independent t-test for these data show a p-value less
than 0.001, confirming that the populations means between the 2 methods are significantly different. No
significant effects of storage time on recoveries were detected for either sampling method (Table 4.2).
22
-------
107,
10s-
105-
I I Extractive Method
Sponge Stick Method
Inoculum
Inoculum DayO Day1 Day 7
Waste Storage Time (Days)
Day 30
Figure 4.1: Effect of waste storage time on positive control recoveries (colony forming units
[CPU]) from carpet for the extractive and the Sponge-Stick™ methods.
Table 4.1: Effects of Waste Storage Time on Positive Control Recoveries from Carpet for the
Extractive and Sponge-Stick™ Sampling Methods
Waste Storage Time
Inoculum
DayO
Day1
Day/
Day 30
All Days (Combined)
Statistic
Average
SD
Average
SD
Average
SD
Average
SD
Average
SD
Average
SD
Recovery (CPU)
Extraction
Sponge-
Stick™
5.12 xio7
6.74 x106
1.62X107
9.66 x 106
1.42X107
7.46 x 106
1.29X107
9.98 x 106
1.13x107
5.32 x 106
1.37X107
7.91 x 106
6.51 x 105
4.49 x 105
4.81 x 105
4.61 x 105
2.67 x 105
1.85x105
5.32 x 105
5.38 x105
5.02 x 105
4.45 x105
Recovery (%)
Extraction
Sponge-
Stick™
Reference Measurement
31.7
27.8
25.2
22.0
26.7
3.56
1.3
0.9
0.5
1.0
0.94
0.27
CPU, colony forming units; SD, standard deviation
23
-------
Table 4.2: Two-Sample Independent T-test Performance Parameters for Effects of Waste Storage
Time on Recoveries from Carpet by Sampling Method and Decontamination Procedure
Sampling Method
(Waste Storage
Time)
Extractive Method
(Day 0,1 ,7, and 30)
Sponge-Stick™
Method (Day 0, 1,7,
and 30)
pH Adjusted Bleach Applied to Carpet
Immersion
15 min
Mean
P-
value
0.70
0.27
F-
value
0.38
1.66
Variance
P-
value
0.93
0.58
F-
value
0.07
0.61
Rigorous immersion
30 min
Mean
P-
value
0.17
0.24
F-
value
2.14
1.70
Variance
P-
value
0.54
0.74
F-
value
0.78
0.43
Rigorous immersion
60 min
Mean
P-
value
0.15
0.65
F-
value
2.29
0.58
Variance
P-
value
0.86
0.73
F-
value
0.25
0.45
pAB, pH adjusted bleach
4.1.2 Upholstery Material
Spore recoveries from upholstery material as a function of simulated waste storage time (sampling time
delay) are shown in Figure 4.2, and summarized in Table 4.3. The averaged recoveries were 1.73 x 107
(Standard deviation, SD = 1.16 x 107, n= 10 samples) CPU using the extractive method (removal of a
coupon from a larger sample), and 1.08 x 107 (standard deviation (SD) = 7.21 x 106 n= 10 samples) CPU
using the Sponge-Stick™ sampling approach. The overall percent recovery of the extractive method
when compared to the starting inoculum (34.2% + 12.3%) was higher than the Sponge-Stick™ sampling
method (18.9% + 10.37%) but within the same order of magnitude. A 2-sample independent t-Test
showed that at the 95% confidence interval, the difference of the population means is not significant (p =
0.116). Recoveries for both sampling methods declined significantly (p < 0.05, ANOVA) overtime (Table
4.3). Stain resistant coatings on the upholstery surface material may limit spore soaking into the fabric
during inoculation, resulting in a large fraction of the spores remaining on the material surface and
therefore explain the higher recoveries achieved from this material type.
24
-------
Positive Controls Recovery (CPU)
q. 4 4 Q,
T
1
T
1
T
1
1 Extractive Method
^Sponge Stick Method
1 Inoculum
T
1
1
1
Inoculum DayO Day1 Day 7 Day 30
Waste Storage Time (Days)
Figure 4.2: Effect of waste storage time on positive control recoveries (colony forming units
[CPU]) from upholstered material for the extractive and the Sponge-Stick™ methods.
Table 4.3: Effects of Waste Storage Time on Positive Control Recoveries from Upholstered
Material for the Extractive and Sponge-Stick™ Sampling Methods
Waste Storage
Time
Inoculum
DayO
Day1
Day?
Day 30
All Days
(Combined)
Statistic
Average
Average
SD
Average
SD
Average
SD
Average
SD
Average
SD
Recovery (CPU)
Extraction
Sponge-
Stick™
5.12 xio7
3.05 x107
6.95 x106
2.06 x 107
7.67 x 106
1.37x107
6.37 x 106
4.29 x 106
6.51 x 106
1.73x107
1.16x107
1.89X107
1.52X106
1.37X107
6.37 x 106
7.20 x 106
2.11 x106
7.91 x 106
5.20 x 106
1.08x107
7.21 x 106
Recovery (%)
Extraction
Sponge-
Stick™
Reference Measurement
53.2
35.9
23.9
23.9
34.2
12.0
33.0
23.9
12.6
6.04
18.9
10.4
CPU, colony forming units; SD, standard deviation
25
-------
Table 4.4: Two-Sample Independent T-Test Performance Parameters for the Effects of Waste
Storage Time on Recoveries from Upholstery by Sampling Method
Sampling Method
(Waste Storage Time)
Extractive Method (Day: 0, 1, 7, 30)
Sponge-Stick™ Method (Day: 0, 1, 7,
Mean
p-value
0.0094
0.0099
F-value
7.76
7.61
Variance
p-value
0.95
0.159
F-value
0.11
2.26
4.1.3 PPE Material
Spore recoveries from PPE material as a function of simulated waste storage time (sampling time delay)
are shown in Figure 4.3, and summarized in Table 4.5. Due to the irregular shape and small size of the
PPE samples only extractive sampling methods were used. The averaged CPU recoveries for two tests
which included 4 sampling events, with 5 samples (each finger of nitrile glove is considered a single
sample) are 2.88 x 107 (SD = 5.34 x 106, n= 40 samples). The overall percent recovery of the extractive
method for the PPE materials is 42.3% ± 4.60%. Recoveries for the extractive sampling method used for
the PPE samples were consistent overtime ((p >0.05, ANOVA); Table 4.6).
o
10'
-e io6
8
5
Extractive Method
Inoculum
Inoculum Day 0 Day 1 Day 7
Waste Storage Time (Days)
Day 30
Figure 4.3: Effect of waste storage time on positive control recoveries (colony forming units
[CPU]) from personal protective equipment for the extractive method.
26
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Table 4.5: Effects of Waste Storage Time on Positive Control Recoveries from Personal Protective
Equipment for the Extractive Sampling Method
Waste Storage Time
Inoculum
DayO
Day1
Day?
Day 30
All Days (Combined)
Statistic
Average
Average
SD
Average
SD
Average
SD
Average
SD
Average
SD
Recovery(CFU)
5.73 x107
2.70 x 107
5.99 x 106
2.68 x107
7.94 x106
2.18 x107
5.80 x 106
2.15 x107
4.59 x 106
2.88 x107
5.34 x106
Recovery (%)
Reference Measurement
47.1
46.7
38.0
37.4
42.3
4.60
CPU, colony forming units; SD, standard deviation
Table 4.6: Analysis of Variation Performance Parameters for Effects of Storage Time on
Recoveries from Personal Protective Equipment by Samplinc
Sampling Method
(Waste Storage Time)
Extractive Method (Day: 0, 1, 7, 30)
Mean
p-value
0.077
F-value
2.47
Variance
p-value
0.29
F-value
1.31
Method and Experiment
4.1.4 Paper Material
Spore recoveries from the front page paper "PF" and the middle page paper "PM" as a function of
simulated waste storage time (sampling time delay) are shown in Figure 4.4, and summarized in Table
4.7). Due to the highly porous and absorptive nature of paper, spores were expected to migrate from the
original location to subsequent pages in the book. As such, extraction-based methods were used on the
inoculated page, and the adjacent pages to optimize spore recovery. The averaged CFU recoveries for
the "PF" and "PM" are 7.68 x 106 (SD = 4.30 x 106, n= 23 samples) and 8.05 x 106 (SD = 3.95 x 106, n=
25 samples), respectively. The recoveries achieved by the extractive method for the "PF" and "PM"
sample materials were 17.3% + 7.5% and 18.3% + 5.5%, respectively. A two-sample independent t-Test
showed that at the 95% confidence interval, the difference of the spore recovery population means
between the "PF" Samples and the "PM" is not significantly (p = 0.75). Recoveries for the "PF" samples
seem to be less consistent (p= 0.032, ANOVA)) than the recoveries for the "PM" samples (p=0.267
ANOVA) over the 30 days sampling period (Table 4.8).
27
-------
•
LL
O
0)
If)
2 106-
o
O ;
1 '
D.
105-
T
1
T
1
1 | KM samples
1 1 PF Samples
1 1 Inoculum
j
Inoculum DayO Day1 Day 7
Waste Storage Time (Days)
Day 30
Figure 4.4: Effect of waste storage time on positive control recoveries (colony forming units
[CPU]) from paper for the extractive method.
28
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Table 4.7: Effects of Waste Storage Time on Positive Control Recoveries from Paper for the
Extractive Sampling Method
Waste Storage Time
Inoculum
DayO
Day1
Day?
Day 30
All Days (Combined)
Statistic
Average
Average
SD
Average
SD
Average
SD
Average
SD
Average
SD
Sample Location
Front Page
Recovery (CPU)
Recovery (%)
Middle Page
Recovery (CPU)
Recovery (%)
4.20 x 107
5.10 xio6
1.91 xio6
5.15 x106
2.36 x 106
6.99 x 106
4.77 x 106
1.18x107
4.58 x 106
7.68 x 106
4.30 x 106
12.1
12.3
16.6
28.1
17.3
7.5
5.98 x 106
8.00 x 105
7.71 x 106
2.04 x 106
6.09 x 106
4.51 x 106
1.09X107
5.85 x 106
8.05 x 106
3.95 x 106
14.2
18.3
14.5
26.0
18.3
5.5
CPU, colony forming units; SD, standard deviation
Table 4.8: Analysis of Variance Performance Parameters for Effects of Waste Storage Time on
Recoveries from Paper by Sampling Method and Experiment
Sample Location
Front Page
Middle Page
Mean
p-value
0.032
0.267
F-value
3.61
1.41
Variance
p-value
0.423
0.018
F-value
0.979
4.21
4.1.5 Sampling Methods Test Synopsis
The two sampling methods (extractive and Sponge-Stick™) were evaluated for overall and temporal
recoveries (Day 0, Day 1, Day 7, and Day 30) for both the carpet material and the upholstered coupons
using the results of the positive controls for each sampling event. The mean recoveries of the extractive
method, when compared to the reference inoculum plating, were much higher than that achieved by the
Sponge-Stick™ sampling method for both materials that were sampled by both methods. There were no
significant temporal effects on mean recoveries or variance for these two materials and sampling
methods. For the upholstered materials, the difference in the overall mean recoveries for both sampling
methods was found to be not significant; however, recoveries for both sampling methods declined
significantly overtime. The overall and temporal recoveries for the PPE and paper materials were found
to be consistent overtime.
29
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4.2 Neutralization Methods Evaluation
The presence of decontamination solution components in the rinsate or extraction liquid (desorbed from
the sample) could negatively bias spore recoveries via residual decontamination. Prior to each
decontamination testing sequence, neutralization tests were performed to determine the optimal
neutralization concentration (neutralizerto decontaminant), the effect of holding time between the time
the sample is collected and the time when the sample is analyzed, and the effect of the immersion time
on the amount of neutralizer required for full neutralization
4.2.1 Optimization of Neutralizer Concentration
To determine the optimal amount of neutralizer (STS) for each material/decontaminant, preliminary
neutralization tests were conducted. The samples were neutralized at different stoichiometric ratio of STS
to decontaminant (X); then the solutions (with the samples) were spiked with either ~2 x 102 spores (low
concentration) or~5 x 107 spores (high concentration) before analysis. The results are presented in
Table 4.9. The data collected showed that complete spore recovery can be obtained when the required
amount of STS, based on stoichiometric ratio, is applied for each material/decontaminant combination.
Table 4.9: Preliminary Neutralization Optimization
Decontaminant
pH Adjusted
Bleach
Diluted Bleach
Spore Inoculum
[CPU]
~5x107
~2x102
~5x107
~2x102
Stoichiometric
Ratio
1.3-1.5X
2.5X
1.3-1.5X
2.5X
1.3-1.5X
1.3-1.5X
Material
Type
Carpet
Upholstery
Paper
PPE
Carpet
PPE
Carpet
Upholstery
Paper
PPE
Carpet
PPE
Carpet
Upholstery
Paper
PPE
Carpet
Upholstery
Paper
PPE
Spore Recovery [CPU]
Mean CPU
2.57 x107
4.46 x107
4.79 x107
5.16x107
3.89 x107
4.88 x107
2.78 x102
3.24 x102
1.45x102
9.97 x101
2.40 x102
3.91 x101
3.39 x107
4.56 x107
1.11x107
4.83 x107
2.78 x102
3.12x102
1.77x102
1.62x102
SD
8.84 x106
2.94 x106
3.13x106
6.60 x106
7.79 x106
2.13x106
4.91 x101
4.42 x101
6.76 x101
1.66 x101
1.75x101
3.58 x10'1
2.95 x106
4.11 x106
7.87 x106
2.15x106
4.91 x101
7.71 x101
5.78 x101
4.43 x101
30
-------
4.2.2 Sample Hold Time Effects
The effect of sample hold time on the neutralizer efficacy was evaluated for both the pAB and diluted
bleach at two different spore concentrations: ~2 x 102 CPU (low concentration) or ~5 x 107 CPU (high
concentration). The processing lag time at 24 hours after sampling compared to within 1 hour of a
sampling event did not show any bias in the spores' recoveries for both types of decontaminants and
spore concentrations (Table 4.10).
Table 4.10: Effect of Sample Hold Time on Neutralizer Optimization
Decontaminant
pH Adjusted
Bleach
Diluted Bleach
Spore Inoculum [CPU]
~5x107
~2x102
~5x107
~2x102
Hold Time
[Hours]
1h
24 h
1h
24 h
1h
24 h
1h
24 h
Spore Recovery [CPU]
Mean CPU
3.89 x107
3.87 x107
2.64 x102
SD
7.79 x106
8.12 x106
2.87 x101
TNC
3.39 x107
5.25 x107
2.78 x102
1.69x102
2.95 x106
8.62x 106
4.91 x101
1.17x102
CPU, colony forming units; pAB, pH adjusted bleach; SD, standard deviation; TNC, Tests Not Conducted
4.2.3 Immersion Time Effects
Two neutralization tests, at two spore concentrations (~ 2 x 102 and ~ 5 x 107 CPU), were completed to
determine the effect of an extended immersion time (60 min instead of 15 min) on the neutralization
effectiveness. The amount of neutralizer liquid needed for each decontaminant/application combination
was found to be dependent on the immersion time for porous materials, meaning that the carpet material
was not saturated at the 15 min immersion time; thus requiring more neutralizer volume. The results
presented in Table 4.11 show that when the required amount of STS is added, based on stoichiometric
ratio, complete neutralization is achieved independently of immersion time.
Table 4.11: Effect of Immersion Time on Spore Recovery (Colony Forming Units) from Neutralized
pH Adjusted Bleach-Exposed Carpet Samples, High and Low Spore Concentrations
Decontaminant
pH Adjusted
Bleach
Spore Inoculum [CPU]
~5x107
~2x102
Immersion Time
[Minutes]
15 min
60 min
15 min
60 min
Spore Recovery [CPU]
Mean CPU
3.89 x107
1.46x107
2.40 x102
1.57x102
SD
7.79 x106
1.35 x106
1.75x101
1.85x101
CPU, colony forming units; SD, standard deviation
31
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4.2.4 Neutralization Tests Synopsis
The neutralization tests determined that the excess STS stoichiometric neutralization is dependent on the
type of material used; full recoveries of the spores inoculated on material spores were obtained with 2.5 X
(stoichiometric ratio) for the carpet/pAB combination, and 1.3 X was sufficient for the carpet/diluted bleach
combination. The ratio of 1.5 X was found adequate for upholstered material with either pAB or diluted
bleach as decontaminant, and the same ratio was found for PPE/diluted bleach and paper/pAB
decontaminants. Time lag between the time the sample was neutralized and the time it was processed
over a 24 hour period, immersion time, and spore inoculum concentration did not appear to bias the spore
recoveries if adequately neutralized.
4.3 Dunking/Immersion Decontamination Test Results
The results presented in this section report the overall effectiveness of the decontamination treatment for
each material/decontaminant/procedure combination. Material sections of the test materials were
sampled immediately after the decontamination treatment, and were bagged and re-sampled and
analyzed after a drying time of 1 day (at least 18 hours), 7 days, and 30 days. A subset of bagged waste
samples was left untreated and served as positive controls. The test matrix for the dunking/immersion
evaluation is presented in Table 4.12.
Table 4.12: Decontamination Test Sequence Event
Decontamination
Procedure
Immersion
Immersion
Immersion
Immersion
Rigorous Immersion
Immersion
Immersion
Rigorous Immersion
Decontaminant
Solution
pH Adjusted Bleach
pH Adjusted Bleach
Diluted Bleach
pH Adjusted Bleach
pH Adjusted Bleach
Diluted Bleach
pH Adjusted Bleach
pH Adjusted Bleach
Material Type
Carpet
PPE
PPE
Upholstery
Carpet
Paper
Paper
Carpet
Exposure / Hold
Time
15 minutes
15 minutes
15 minutes
15 minutes
30 Minutes
15 minutes
15 minutes
60 minutes
Test Date
(DayO)
Sept 25, 2013
Nov5,2013
Nov19,2013
Dec 10, 2014
Jan 23, 2014
Feb4,2014
Feb 18, 2014
Mar 25, 2014
End Date (Day
30)
Oct23,2013
Dec 4, 20 13
Dec 16, 2013
Jan 8, 2014
Feb 20, 2014
Mar 4, 2014
Mar 20, 2014
Apr 23, 2014
pAB, pH adjusted bleach; PPE, personal protective equipment
32
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4.3.1 Carpet Decontamination Results
Material sections (coupons) simulating carpet wastes were subjected to various immersion-based
decontamination procedures. The results presented in this section report the effectiveness of these
procedures as a function of waste storage time, decontamination method, and sampling method.
4.3.1.1 Sampling Methods Evaluation for Carpet
The two sampling methods (extractive and Sponge-Stick™) were evaluated for overall recoveries and for
recoveries following various simulated waste storage durations (Day 0, Day 1, Day 7, and Day 30). The
evaluation was performed for each decontaminant type following each decontamination event (15, 30,
and 60 minutes) using 3 carpet samples for each sampling method at each time point. A multiple-
population ANOVA statistical analysis was used to determine if recoveries differed significantly as a
function of storage time, the amount of time between decontamination and sample collection.
Spore survival on carpet following each decontamination approach, and after various waste storage
duration are shown in Figure 4.5, and summarized in Table 4.13, 4.14, and 4.15 for immersion times of
15, 30, and 60 minutes, respectively (note that samples were not collected for Day 7 [15 minutes
immersion time] for logistical reasons).
scovery (CFU)
4 4 q, c
i — f — i
1
1
Immersion
ime: 60 mm 1 1 E
-r
T
Extractive Sampling
sponge-Sticks™
I
Q. -,
o 1Q2-
c m°
1 Immersion Time: 30 min 1
±
T
T
I
T
Ł m6
d
T
T
• i
Immersion Time: 15 min
T
I
— IT-
•
1
i — T — i
-L
T
DayO Day1 Day 7 Day 30
Waste Storage Time (Days)
Figure 4.5: The effects of sampling method and waste storage duration on recoveries (colony
forming units [CFU]) from carpet following decontamination with pH adjusted bleach.
The mean combined spore recoveries (CFU recovered) calculated for the 15, 30, and 60 minutes
immersion times are respectively 3.36 x 104 + 2.39 x 104 (n= 27 samples), 1.27 x 104 + 9.55 x 103 (n= 36
samples), and 1.44 x 103 + 1.67 x 103 (n= 36 samples) using the extractive method (removal of a coupon
33
-------
from a larger sample). For the Sponge-Stick™ sampling approach, the corresponding results are
respectively 5.78 x 102 + 9.78 x 102 (n= 27 samples), 2.50 x 102 + 6.30 x 102 (n= 36 samples), and 1.03 x
101 + 2.78 x 102 (n= 36 samples). The recoveries obtained with the extraction-based sampling was up to
2 orders of magnitude higher than the Sponge-Stick™ sampling method, which may have biased the
calculation of the decontamination efficiency with the latter sampling technique. No viable spores were
detected in some replicate samples using the Sponge-Stick™ method, while spores were detected in all
the samples collected using the extraction-based method.
Table 4-13: Post-Decontamination Recoveries (Colony Forming Units [CFU]) from Carpet for
Extractive and Sponge-Stick™ Sampling Methods (Immersion Time: 15 min,
Decontaminant: pH adjusted Bleach)
Recoveries (Immersion Time: 15 min, Decontaminant: pH adjusted Bleach)
Coupon
ID
C01
C02
COS
All
Coupons
Statistic
Average
SD
Average
SD
Average
SD
Average
SD
Extraction Sampling Method
Sponge-Stick™ Sampling Method
Waste Storage Time
ODay
1.62x104
8.62 x 103
5.13x104
5.15 x104
1.85x104
9.20 x 103
2.87 x 104
3.15 x104
1 Day
3.80 x 104
1.38x104
2.91 x 104
1.12x104
1.62x104
2.37 x 103
2.31 x 10"
2.46 x 10"
30 Day
3.46 x 10"
1.50x10"
4.97 x 10"
1.47x10"
4.89 x 10"
3.23 x 10"
4.89 x 10"
3.23 x 10"
ODay
6.04 x10'1
2.75 x10'2
3.53 x 102
1.59x102
4.44 x 101
7.28 x 101
1.33x102
1.88x102
1 Day
2.72 x 103
1.69x103
6.80 x 102
5.40 x 102
8.38 x102
5.75 x102
1.41 xio3
1.35x103
30 Day
1.19x102
4.89 x 101
3.86 x 102
3.99 x 102
5.98 x101
5.70 x 101
1.88x102
2.53 x102
SD, standard deviation
34
-------
Table 4-14: Post-Decontamination Recoveries (Colony Forming Units [CPU]) from Carpet for
Extractive and Sponge-Stick™ Sampling Methods (Immersion Time: 30 min,
Decontaminant: pH adjusted Bleach)
Recoveries (Immersion Time: 30 min, Decontaminant: pH adjusted Bleach)
Coupon
ID
C01
C02
COS
All
Coupons
Statistic
Average
SD
Average
SD
Average
SD
Average
SD
Extraction Sampling Method
Sponge-Stick™ Sampling Method
Waste Storage Time
ODay
2.59x104
1.28x101
1.29x10i
7.63x103
1.82x104
9.02x103
1.90x104
1.04x104
1 Day
1.46x104
8.14x103
5.82x103
3.18x103
1.56x104
8.97x103
9.81 x 1Q3
2.68x103
7 Day
3.55 x 1Q3
7.43 x 1Q2
2.20 x 1Q4
1.40x104
7.07 x 1Q3
3.26 x 1Q3
1.45x104
1.11 x1Q4
30 Day
1.50x104
5.47x103
1.79x100
7.47x10-1
2.76x103
1.36x103
8.91 x 1Q3
6.48x103
ODay
4.38x102
6.91 x 1Q2
1.11x103
1.92x103
6.64x101
9.15x101
5.38x102
1.12x103
1 Day
2.23x102
2.47x102
2.13x102
1.78x102
6.10x102
8.87x102
3.49x102
5.08x102
7 Day
1.03x102
6.11x101
1.97x101
1.46x101
2.05x102
3.02x102
1.09x102
1.74x102
30 Day
8.74x100
4.91 x 1QO
1.79x100
7.47x10-1
3.25x100
3.94x100
4.59x100
4.49x100
SD, standard deviation
Table 4-15: Post-Decontamination Recoveries (Colony Forming Units [CPU]) from Carpet for
Extractive and Sponge-Stick™ Sampling Methods (Immersion Time: 60 min,
Decontaminant: pH adjusted Bleach)
Recoveries (Immersion Time: 60 min, Decontaminant: pH adjusted Bleach)
Coupon ID
C01
C02
COS
All
Coupons
Statistic
Average
SD
Average
SD
Average
SD
Average
SD
Extraction Sampling Method
Sponge-Stick™ Sampling Method
Waste Storage Time
ODay
1.69x103
1.53x103
3.90x103
2.28x103
1.01 x1Q3
2.76x102
2.0x103
1.90x103
1Day
3.19x103
4.14x103
2.07x103
4.08x102
1.43x103
4.76x102
1.36x103
1.01 x1Q3
7 Day
8.68x101
9.08x101
8.93x102
8.75x102
4.51 x 102
3.70x102
6.72x102
5.92x102
30 Day
9.75x102
8.75x102
3.95x100
1.78x100
8.99x102
7.53x102
8.61 x 102
6.66x102
ODay
6.47x10-1
2.00x10-2
6.77x10-1
1.51 xlO-2
8.99x10-1
3.83x10-1
7.41 x 10-1
2.26x10-1
1Day
1.58x101
1.72x101
5.18x100
7.21 xlQo
5.05x100
6.09x100
8.67x100
1.12x101
7 Day
6.47x101
8.59x101
1.67x101
2.01 x 101
3.86x100
2.10x100
2.84x101
5.21 x 101
30 Day
3.02x100
1.04x10-
1
3.95x100
1.78x100
3.07x100
1.15x10-
1
3.34x100
1.00x100
SD, standard deviation
35
-------
At the 95% confidence level, no significant effects of sample waste storage time on recoveries were
detected for the extraction-based sampling method. At least one significant interaction (15 minute) was
noted for the Sponge-Stick™ sampling approach (Table 4.16, significant is shown in bold).
Table 4.16: Analysis of Variance Performance Parameters for Effects of Post-Decontamination
Storage Time on Recoveries (Colony Forming Units [CPU]) from Carpet
Sampling Method
(Waste Storage Time)
Extractive Method
(Day: 0,1,7,30)
Sponge-Stick™
Method (Day: 0,1,7,
Immersion Time
15min
Mean
P-
value
0.25
0.014
F-
value
1.48
5.17
Variance
P-
value
0.76
0.026
F-
value
0.28
4.25
30 min
Mean
P-
value
0.122
0.278
F-
value
2.083
1.342
Variance
P-
value
0.897
0.279
F-
value
0.198
1.339
60 min
Mean
P-
value
0.13
0.14
F-
value
2.029
1.982
Variance
P-
value
0.224
0.171
F-
value
1.534
1.778
4.3.1.2 Carpet Decontamination Effectiveness
The results of the carpet decontamination tests are presented in Table 4.17 and in Figure 4.6. The
decontamination effectiveness is presented as the mean Log10 reduction in CPU recovered, from all
samples within a particular material and treatment. For example, recoveries following sampling at all
storage times were averaged to yield one estimate of recovery for that particular treatment. This
aggregate approach was utilized since the ANOVA indicated no significant interaction between sample
storage time and recovery.
Table 4.17: Decontamination Efficacy versus Immersion Time (Colony Forming Units Log
Reduction) for Carpet
Sampling Method
(Waste Storage Time)
Extractive Method (Day:
0,1,7,30)
Sponge-Stick™
Method (Day: 0, 1,7, 30)
Immersion Time
15 min
Average
2.72
3.99
SD
0.32
1.15
30 min
Average
3.37
4.34
SD
0.34
1.05
60 min
Average
3.91
4.72
SD
0.75
0.61
CPU, colony forming units; SD, standard deviation
36
-------
5-
'E:
o
'•& 4-
Efficacy (Log10 CPU Reduc
D -^ NJ CO
I I Extractive Sampling
IZZl Sponge-Sticks™
T
1
15rrin
SOmin
Immersion Time
60min
Figure 4.6:
The effects of immersion time in pH adjusted bleach on carpet decontamination
efficacy (colony forming units [CPU] log reduction in recovery).
The mean combined Log10 reduction in spore recoveries for the 15 , 30, and 60 minutes immersion times
are respectively 2.72 + 0.32 (n= 27 samples), 3.37 + 0.34 (n= 36 samples), and 3.92 + 0.76 (n= 36
samples) using the extractive method (removal of a coupon from a larger sample). For the Sponge-
Stick™ sampling approach, the corresponding results are respectively 3.99 + 1.15 (n= 27 samples), 4.34
+ 1.05 (n= 36 samples), and 4.72 + 0.61 (n= 36 samples). These results suggest that increasing the
immersion time increases the efficacy of the decontamination technique. As mentioned previously,
results obtained using the Sponge-Stick™ approach may result in overestimation of the actual
decontamination efficacy due to the relatively lower recovery of this sampling technique. Interestingly,
complete decontamination of carpet was not achieved with pAB, even when the 60 minute immersion
procedure was rendered. Viable spores were recovered by both sampling approaches, for at least one
replicate sample from all immersion times.
4.3.2 Upholstery Decontamination Results
Samples simulating upholstered waste were subjected to a 15 minutes pAB immersion-based
decontamination procedure. The results presented in this section report the effectiveness of these
procedures, as a function of waste storage time, decontamination method, and sampling method.
4.3.2.1 Sampling Methods Evaluation for Upholstery
The two sampling methods (extractive and Sponge-Stick™) were evaluated for overall recoveries and
recoveries following various simulated waste storage durations (Day 0, Day 1, Day 7, and Day 30). The
evaluation was performed for pAB decontaminant following each a 15 minutes decontamination time
using 3 carpet samples for each sampling method at each time point. A multiple-population ANOVA
statistical analysis was used to determine if recoveries differed significantly as a function of storage time,
the amount of time between decontamination and sample collection.
37
-------
Spore survival on upholstery material the 15 minutes decontamination approach, and after various waste
storage duration are shown in Figure 4-7, and summarized in Table 4.18. The mean combined spore
recoveries (CPU recovered) for the 15 minutes immersion times in pAB are respectively 8.58 x 101 + 1.78
x 102 (n= 36 samples), and 4.22 x 101 + 1.46 x 1032 (n= 36 samples) for the extraction-based method and
the Sponge-Stick™ method. A two sample Paired t-Test showed that at the 95% confidence interval, the
difference in the spore recovery populations means between the 2 sampling techniques is not significant
(p = 0.75). The average values were based partially upon detection limit, as no viable spores were
detected in some replicate samples.
Recovery (CPU)
1
_
-------
Table 4.18: Post-Decontamination Recoveries (colony forming units [CPU]) from Upholstery for
Extractive and Sponge-Stick™ Sampling Methods (Immersion Time: 15 min)
Recoveries (Immersion Time: 15 min, Decontaminant: pH Adjusted Bleach*)
Coupon
ID
C01
C02
COS
All
Coupons
Statistic
Average
SD
Average
SD
Average
SD
Average
SD
Extraction Sampling Method
Sponge-Stick™ Sampling Method
Sampling Interval
ODay
1.08x100
3.63x10-1
5.57x100
8.41 x 100
2.11 x1Q2
3.38x102
7.24x101
1.98x102
1 Day
5.40 x 1Qo
7.70x100
1.17x101
1.91 x1Q1
3.30 x 1Q2
5.63 x 1Q2
1.15x102
1.92x102
7 Day
4.56x101
7.74x101
6.25x10-1
7.81 x 10-3
5.92x100
7.60x100
3.27x100
4.43x101
30 Day
7.12x10-1
1.39x10-2
2.92x102
5.05x102
6.62 x 1Qo
9.00 x 1Qo
2.68x100
5.38 x 1Qo
ODay
8.48x10-1
5.04x10-2
9.09x10-1
1.14x10-1
1.05x100
5.19x10-1
9.37x10-1
2.82x10-1
1 Day
9.02x10-1
6.06x10-2
8.56x10-1
2.35x10-2
8.20x10-1
1.56x10-2
8.59x10-1
1.12x101
7 Day
6.81 x 10-1
2.21 x 10-2
1.20x100
1.03x100
6.17x10-1
2.10x100
2.84x101
5.21 x 1Q1
30 Day
6.50x10-1
9.14x10-3
2.92 x 1Q2
5.05 x 1Q2
6.37x10-1
1.15x10-1
3.34 x 1Qo
1.00x100
SD, standard deviation
* Note: values based partially upon detection limit, as no viable spores were detected in some replicate samples.
At the 95% confidence level, no significant effects of sample hold times on recoveries were detected for
either sampling method (Table 4.19).
Table 4.19: Analysis of Variance Performance Parameters for Effects of Post-Decontamination
Sample Storage Time on Recoveries (Colony Forming Units [CPU]) from Upholstery
(immersion Time 15 min)
Immersion Time: 15 min, Decontaminant: pH Adjusted bleach *
Sampling Method
(Waste Storage Time)
Extractive Method (Day: 0, 1, 7, 30)
Sponge-Stick™ Method (Day: 0, 1, 7,
30)
Mean
p-value
0.58
0.41
F-value
0.67
0.995
Variance
p-value
0.58
0.41
F-value
0.67
0.997
Note: values based partially upon detection limit, as no viable spores were detected in some replicate samples.
4.3.2.2 Upholstered Coupon Decontamination Effectiveness
The results of the upholstery decontamination tests are presented in Table 4.20 and in Figure 4.8. The
decontamination effectiveness is presented as the mean Log10 reduction in CPU recovered, from all
samples within a particular material and treatment. The decontamination effectiveness is presented as
the mean Log10 reduction in CPU recovered, from all samples within a particular material and treatment.
For example, recoveries following sampling at all storage times were averaged to yield one estimate of
39
-------
recovery for that particular treatment. This aggregate approach was utilized since the ANOVA indicated
no significant interaction between sample storage time and recovery.
The averaged combined Log10 CPU decontamination efficacy for the 15 minutes immersion time is 6.4 +
0.6 (n= 36 samples) using the extractive method (removal of a coupon from a larger sample). For the
Sponge-Stick™ sampling approach, the corresponding result is 6.8 + 0.7 (n= 36 samples). Both
sampling techniques seem to lead to the same decontamination efficiency suggesting that the 15 minutes
immersion pAB procedure on upholstered coupons is very effective (>6 log reduction). The higher
efficacy values observed for upholstery, as compared to carpet, might be explained by the lower
absorption of the inoculum by the upholstery material during inoculation. This phenomenon likely resulted
in more spores remaining on the upholstery coupon surface, and might have been more easily accessed,
and therefore killed, by the decontaminant.
Table 4.20: Decontamination Efficacy (Log Reduction in Recovery) for Upholstered Coupon
Decontamination Efficacy
Immersion Time: 15 min, Decontaminant: pH Adjusted Bleach
Sampling Method (Waste Storage Time)
Extractive Method (Day: 0, 1, 7, 30)
Sponge-Stick™ Method (Day: 0, 1, 7, 30)
Mean LR
6.4
6.8
SD
0.6
0.7
LR, log reduction; SD, standard deviation
7-
6-
?
••8
-g 5-
O
f 3-
§ 2-
LU
1-
0-
I I Extractive Sampling
I I Sponge-Sticks™
15 min
mmersion time
Figure 4.8: The effects of immersion time in pH adjusted bleach on upholstery decontamination
efficacy (log reduction in colony forming units [CFU] in recovery).
40
-------
4.3.3 PPE Decontamination Results
Material sections (coupons) simulating PPE waste were subjected to various immersion-based
decontamination procedures, and stored for up to 30 days to simulate waste staging during an actual
anthrax incident. Samples (glove sections) were collected at each time point, and spore survival was
determined. The results presented in this section report the effectiveness of these procedures, as a
function of waste storage time, and decontamination method. PPE material coupons were immersed in
either pAB or diluted bleach for an overall immersion time of 15 minutes.
4.3.3.1 The Effects of Waste Storage Duration on Recoveries from PPE following
Decontamination
For each experimental trial, the PPE material sections were bagged following the decontamination
treatment, and samples (glove tips) were collected at various days (Day 0, Day 1, Day 7, and Day 30)
during simulated waste storage. Spore survival was determined at each time point. The PPE sampling
technique consisted of collecting 3 inoculated fingers (1st, 3rd and 5th fingers starting with the thumb) from
each glove separately. Spores were recovered by extraction of individual glove fingers in PBST. Five
gloves were used to produce 15 test samples (3 samples from 3 inoculated fingers for each glove) one
glove for positive controls (5 samples from 5 inoculated fingers), one glove for field blank sample (3 un-
inoculated fingers), and one glove for laboratory blank sample (1 sample from 1 un-inoculated finger) for
a total of 8 glove per test sampling sequence, or 32 gloves for the 4 test sequences for each
decontamination procedure. Field blanks were not inoculated, but were subjected to the decontamination
treatment. Lab blanks were not inoculated, and were not subjected to a decontamination treatment.
ANOVA was used to determine if recoveries differed significantly as a function of storage time.
Recoveries (CFU) following a decontamination treatment as a function of waste storage time are shown
in Figure 4.9 and summarized in Table 4.21 and 4.23 for pAB and diluted bleach, respectively. Note that
for the pAB decontamination procedure, the decontamination consistently achieved greater than 6 Log
reductions, but for one sample (1 sample from one finger among the 15 samples (COS) showed little or
no decontamination. As mentioned previously, this low efficacy was presumably due to incomplete
contact of the decontaminant within the glove. The pAB was found to be more effective than the diluted
bleach using the immersion decontamination procedure. One data set, which included the finger that was
not decontaminated, did not pass the Shapiro-Wilk test for normality since the null hypothesis is rejected
and there is evidence that the data tested are not from a normally distributed population. Therefore, the
difference in the spore recovery populations means between the two sampling techniques was not
assessed.
41
-------
6x10
5x10*-
LL
U
O
UL
Ł
0.
E 3x10
ra
in
c
g
'ro
• 2x10'
Q
— 1x10
O
Q_
0 -
pAB
Diluted Bleach
Day 0
Day 30
Day 1 Day 7
Waste Storage Time(Days)
Figure 4.9: The effects of waste storage duration on recoveries (colony forming units [CPU])
from personal protective equipment following decontamination.
Table 4.21: Recoveries (Colony Forming Units) Following Decontamination of Personal Protective
Equipment with pH Adjusted Bleach (Immersion Time: 15 min)
Recoveries (Immersion Time: 15 min, Decontaminant: pAB)
Coupon ID
C01
C02
COS
C04
COS
All coupons
Statistic
Average
SD
Average
SD
Average
SD
Average
SD
Average
SD
Average
SD
Sampling Interval
ODay
5.70 x 1Q-1
2.43 x 1C'2
5.57 x 1Q-1
1.31 xlO'2
5.42 x 1Q-1
5.62 x 1Q-3
5.54 x 1Q-1
7.78 x 1C'3
5.49 x 1Q-1
2.89 x 1Q-3
5.55 x 1Q-1
1.48X10'2
1 Day
5.76 x 1Q-1
2.25 x10'2
5.94 x1Q-1
5.88 x10'3
5.92 x 1Q-1
8.89 x 1Q-3
5.81 x 1Q-1
1.97X10'2
5.90 x 1C'1
1.20X1Q-2
5.87 x 1C'1
1.48X10'2
7 Day
1.32x10°
1.11x10°
6.33 x 1Q-1
4.59 x 1Q-3
6.62 x 1Q-1
5.78 x 1Q-2
6.45 x 1C'1
2.94 x 1C'2
6.53 x 1C'1
2.28 x 1C'2
7.83 x 1C'1
5.06 x 1C'1
30 Day
5.53 x 1C'1
3.48 x 1C'3
5.57 x 1C'1
1.27X10'2
7.66 x 1C'1
3.66 x 1Q-1
7.66 x 1Q-1
3.66 x 1Q-1
7.43 x105
1.29x106
1.49x105
5.75 x105
SD, standard deviation
Note: values based partially upon detection limit,
as no viable spores were detected in some replicate samples.
42
-------
Table 4.22: Recoveries Colony Forming Units Following Decontamination of Personal Protective
Equipment with Diluted Bleach (Immersion Time: 15 min)
Recoveries (Immersion Time: 15 min, Decontaminant: Diluted Bleach)
Coupon ID
C01
C02
COS
C04
COS
All coupons
Statistic
Average
SD
Average
SD
Average
SD
Average
SD
Average
SD
Average
SD
Sampling Interval
ODay
6.16 xio3
1.03x104
1.12x104
1.94x104
1.94x106
1.80x106
1.47x103
2.53 x 103
2.61 x 104
1.52x104
3.97 x 105
1.05x106
1 Day
6.15 x10'1
8.67 x 1C'2
4.09 x 106
7.09 x 106
2.35 x 104
4.06 x 104
4.39 x 106
3.66 x 106
1.28x104
2.22 x 104
1.70X106
3.70 x 106
7 Day
6.17 x10'1
4.33X10-3
1.24x10°
1.08x10°
1.50x102
2.59 x 102
6.38 x10'1
1.59X1Q-2
5.19x10"
8.99 x 104
1.04x104
4.02 x 104
30 Day
1.45x10°
9.27 x 10-1
1.24x10°
1.06x10°
3.15 x105
5.46 x 105
3.15 x105
5.46 x 105
1.30X106
2.26 x 106
3.25 x 105
1.02X106
SD, standard deviation
4.3.3.2 PPE Decontamination Effectiveness
The results of the PPE decontamination tests are presented in Table 4.24 and in Figure 4.10. The
decontamination effectiveness is presented as the mean Log10 reduction in recoveries (CFU), from all
samples within a particular material and treatment. The decontamination effectiveness is presented as
the mean Log10 reduction in CFU recovered, from all samples within a particular material and treatment.
For example, recoveries following sampling at all storage times were averaged to yield one estimate of
recovery for that particular treatment. Only extraction-based sampling methods were utilized for PPE.
The mean combined Log10 CFU decontamination efficacy for the 15 minutes immersion time in pAB was
7.57 + 0.86 (n= 60 samples), and 5.45 + 2.51 (n= 60 samples) for the 15 minutes immersion time in
diluted bleach. The pAB/15 minutes immersion procedure did achieve greater than 6 log reduction for all
samples, with the exception of one sample (1 finger of the glove). The decontamination efficacy with
diluted bleach ranged from 0.22 to 7.58 log reduction. These data suggest that achieving complete
coverage of the decontaminant with all PPE surfaces is challenging. This is not unexpected, as PPE
such as gloves may have trapped air inside that may prevent decontaminant from contacting all interior
surfaces. Since gloves are inverted when doffed and therefore contaminants are likely concentrated on
the inside of the glove, decontamination of PPE waste with this method may have difficulty in achieving
complete kill throughout the entire contents of the waste. Full decontamination with either decontaminant
is more probable if the immersion time is greater than the permeation time for these decontaminant/PPE
material decontamination procedures. The permeation time of bleach through nitrile gloves is greater
than 480 minutes.
43
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Table 4.24: Personal Protective Equipment Decontamination Efficacy (Log Reduction in Recovery)
Decontaminant
pH adjusted bleach
Diluted Bleach
Immersion Time
15 min
Mean Log
Reduction
7.57
5.45
Standard
Deviation
0.86
2.51
7-
S
••8
.
-J
O 4-
15 min
Immersion time
Figure 4.10: Personal protective equipment decontamination efficacy by decontaminant type
(colony forming unit [CPU] log reduction).
4.3.4 Paper Decontamination Results
Material sections (coupons) simulating Paper waste were subjected to various immersion-based
decontamination procedures, and stored for up to 30 days to simulate waste staging during an actual
anthrax incident. Samples were collected at each time point, and spore survival was determined. The
results presented in this section report the effectiveness of these procedures, as a function of waste
storage time, decontamination method, and sampling method. Paper material coupons were immersed in
either pAB or diluted bleach for an overall immersion time of 15 minutes.
4.3.4.1 The Effects of Waste Storage Time on Paper Decontamination Efficacy
For each experimental trial, batches of contaminated books and non-contaminated books were subjected
to a 15-minutes decontamination treatment, and samples were collected using the extractive method at
various days (Day 0, Day 1, Day 7, and Day 30) during simulated waste storage. Spore survival was
determined at each time point. The paper samples consist of paper front "PF"" that included the
44
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inoculated front cover of the Merck Manual of Medical along with the first page, and paper middle "PM"
samples that included the inoculated page 955, and one page before and two pages after (pages 953-
960).
Spore survival data for the Paper are shown in Figures 4.11 and 4.12, and summarized in Tables 4.25
and 4.26 for pAB and diluted bleach, respectively. The middle pages showed lower decontamination
efficacies than the front pages for the pAB decontamination procedures. This may have been due to the
protection of the spores inside the books when they were closed. Diluted bleach decontamination was
almost negligible for either front or middle pages as shown for Day 0 and Day 1 sampling events. The
residual bleach in the papers seems to continue its decontamination overtime suggesting that the off-
gassing of the paper continued to be efficacious.
The pAB and the diluted bleach spore survival data did not pass the Shapiro-Wilktest for normality since
the null hypothesis is rejected and there is evidence that the data tested are not from a normally
distributed population. Therefore, the effect of storage time on spore recoveries, and the difference in
decontamination efficiency between the two decontaminants, could not be assessed.
6x103
JD
Q_
I
O
5x1(f-
4x1(f-
3x1(f-
2x1(f-
0-
DayO
Day 1 Day 7
Waste Storage Time
Day 30
Figure 4.11: Recoveries (colony forming units [CFU]) following a decontamination of paper with
pH adjusted bleach (immersion time: 15 minutes).
45
-------
Table 4.25: Recoveries (Colony Forming Units [CPU]) Following Decontamination of Paper with
pH Adjusted Bleach (Immersion Time: 15 Min).
Coupon ID
C01
C02
COS
Average
SD
Front Page Sample Type
Middle Page Sample Type
Waste Storage Time
ODay
7.09X10'1
7.09X10'1
6.74x10'1
6.97x10'1
2.05X10'2
1 Day
7.22x10'1
7.35x10'1
7.63x10'1
7.40 x10'1
2.09X10'2
7 Day
7.09 x10'1
7.22 x10'1
7.77 x10'1
7.36 x10'1
3.63x10-2
30 Day
4.04 x101
7.70 x10'1
7.70 x10'1
1.40x101
2.29 x101
ODay
5.96 x103
1.03 x102
7.09X10'1
2.02 x103
3.41 x 103
1 Day
6.74x10'1
7.22x10'1
1.37 x101
5.05x10°
7.53x10°
7 Day
1.46x102
7.63 x10'1
7.93 x10'1
4.90 x101
8.36 x101
30 Day
4.04 x102
4.04 x101
8.08 x101
1.75x102
1.99x102
SD, standard deviation
Note: values based partially upon detection limit, as no viable spores were detected in some replicate samples
5x106
4x106-
5
^ 3x106-
CD
Q.
I
g 2x106
CO
I
Q
to
S.
1x106-
0-
DayO
Day 1 Day 7
Waste Storage Time
Day 30
Figure 4.12: Recoveries (colony forming units [CFU]) following decontamination of paper with
diluted bleach (immersion time: 15 min).
46
-------
Table 4.26: Recoveries (Colony Forming Units [CPU]) Following Decontamination of Paper with
Diluted Bleach (Immersion Time: 15 Min).
Coupon ID
C01
C02
COS
Average
SD
Front Page Sample
ODay
7.09x100
8.08x101
2.91 x 1Q6
9.70x105
1.68x106
1Day
1.65x106
7.63x10-1
7.35x10-1
5.52x105
9.55x105
7 Day
2.02x100
8.60x10-1
9.19x10-1
1.27x100
6.54x10-1
30 Day
4.04 x 1Qo
2.02x100
2.69x100
2.92 x 1Qo
1.03x100
Middle Page Sample
ODay
1.84x106
6.74x10-1
6.74x10-1
6.12x105
1.06x106
1Day
1.92x104
4.99x106
1.39x102
1.67x106
2.87 x 1Q6
7 Day
8.08x101
7.52x101
7.09x10-1
5.23x101
4.47x101
30 Day
3.31 x 1Q3
4.04x100
1.12x103
1.48x103
1.68x103
SD, standard deviation
Note: values based partially upon detection limit, as no viable spores were detected in some replicate samples
4.3.4.2 Paper Decontamination Effectiveness
The results of the Paper decontamination tests are presented in Table 4.28, and illustrated in Figure 4.13.
The decontamination effectiveness is presented as the mean Log10 reduction in recoveries (CFU), from
all samples within a particular material and treatment. The decontamination effectiveness is presented as
the mean Log10 reduction in CFU recovered, from all samples within a particular material and treatment.
For example, recoveries following sampling at all storage times were averaged to yield one estimate of
recovery for that particular treatment.
The mean combined decontamination efficacies (Log10 CFU Reductions) for the front and middle pages
after a 15 minutes immersion time in pAB was 6.6 + 0.5 (n= 24 samples) and 5.4 + 1.3 (n= 24),
respectively. Following a 15 minute immersion in diluted bleach, the respective decontamination
efficacies were 6.0 + 1.8 (n= 24 samples) and 4.5 + 2.4 (n= 24). The data suggest that a 6 Log reduction
in recoverable spores is more easily obtained for front pages than those in the middle of the books. Full
decontamination may have been achieved by using a longer immersion time and/or by opening the books
during the immersion process.
Table 4.28: Paper Decontamination Efficacy (Log Reduction in Recovery, Immersion Time: 15
min)
Decontaminant
pH Adjusted
Bleach
Diluted Bleach
Sample Location
Front Page
Average
6.6
6.0
SD
0.5
1.8
Middle Page
Average
5.4
4.5
SD
1.3
2.4
SD, standard deviation
47
-------
7-
1 '
"° 5-
(2
I?*-
0
I3'
§ 2-
g •
1-
n-
CZlpAB
I 1 Diluted Bleach
Front Page Middle Page
Paper Sample Type
Figure 4.13: Decontamination efficacy of pH adjusted bleach and diluted bleach on paper (colony
forming units [CPU] log reduction, immersion time: 15 min).
48
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5 Quality Assurance
This project was performed according to an approved Category III Quality Assurance Project Plan
(QAPP). Sufficient detail of the methods outlined in the QAPP are provided within this report.
5.1 Sampling, Monitoring, and Analysis Equipment Calibration
Operating procedures for the maintenance and calibration of all laboratory and NHSRC RTP
Biocontaminant Laboratory equipment were prepared. All equipment was verified as being certified
calibrated or having the calibration verified by EPA's Air Pollution Prevention and Control Division on-site
(Research Triangle Park, NC) Metrology Laboratory at the time of use. Standard laboratory equipment
such as balances, pH meters, biological safety cabinets and incubators were routinely monitored for
proper performance. Data gathered with the HOBO thermistors were processed using the factory
calibration. Calibration of instruments was done at the frequency shown in Table 5.1. Any deficiencies
were noted. The instrument was adjusted to meet calibration tolerances and recalibrated within 24 hours.
If tolerances were not met after recalibration, additional corrective action was taken, possibly including,
recalibration or/and replacement of the equipment.
Table 5.1: Instrument Calibration Requirements
Equipment
Thermometer
pH Meter
HOBO® RH Sensor
Stopwatch
Micropipettes
Clock
Scale
Calibration/Certification
Compare to independent NIST thermometer (this
is a thermometer that is recertified annually by
either NIST or an International Organization for
Standardization (ISO)-1 7025 facility) value once
per quarter.
Perform a 2 point calibration with standard buffers
that bracket the target pH before each use.
Compare to calibrated RH sensor prior to use.
Compare against NIST Official U.S. time at
http://nist.time.aov/timezone.cai?Eastern/d/-5/iava
once every 30 days.
All micropipettes will be certified as calibrated at
least once per year. Rainin™ Pipette liquid
handling devices are recalibrated by gravimetric
evaluation of pipette performance to
manufacturer's specifications every six months by
the supplier (Rainin Instruments, Mettler Toledo,
Greifensee, Switzerland).
Compare to office U.S. Time
every 30 days.
Check calibration with Class 2 weights
Expected Tolerance
±1°C
±0.1 pH units
±5%
±1 min/30 days
±5%
±1 min/30 days
+ 0.1% weight
NIST, National Institute of Standards and Technology; RH, relative humidity
5.2 Data Quality
The primary objective of this project (Task 1: Evaluation of Waste Decontamination Procedures) was to
estimate the efficacy of liquid-based decontamination approaches for on-site treatment of bundled or
bagged waste items typical of an indoor office setting that had been contaminated with B. anthracis
49
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spores. The QAPP in place for this project was followed with deviations that have been documented in
the laboratory notebook. These deviations did not affect data quality.
5.3 QA/QC Checks
Uniformity of the material sections was a critical attribute to assure reliable test results. Uniformity was
maintained by obtaining a large enough quantity of material that multiple material sections and coupons
could be constructed with presumably uniform characteristics. Samples and test chemicals were
maintained to ensure their integrity. Samples were stored away from standards or other samples which
could cross-contaminate them.
Supplies and consumables were acquired from reputable sources and were NIST traceable when
possible. Supplies and consumables were examined for evidence of tampering or damage upon receipt
and prior to use, as appropriate. Supplies and consumables showing evidence of tampering or damage
were not used. All examinations were documented and supplies were appropriately labeled. Project
personnel checked supplies and consumables prior to use to verify that they met specified task quality
objectives and did not exceed expiration dates.
Quantitative standards do not exist for biological agents. Quantitative determinations of organisms in this
investigation did not involve the use of analytical measurement devices. Rather, the CPU were
enumerated manually and recorded. QC checks for critical measurements/parameters are shown in Table
5.2. These checks also served as data quality indicator goals. The acceptance criteria were set at the
most stringent level that can be routinely achieved. Positive controls and procedural blanks were included
along with the test samples in the experiments so that well-controlled quantitative values could be
obtained. Background checks were also included as part of the standard protocol. Replicate coupons
were included for each set of test conditions. Standard operating procedures using qualified, trained and
experienced personnel were used to ensure data collection consistency. The confirmation procedure,
controls, blanks, and method validation efforts were the basis of support for biological investigation
results. If necessary, training sessions were conducted by knowledgeable parties, and in-house practice
runs were used to gain expertise and proficiency prior to initiating the research.
Tests with conditions falling outside of these criteria were rejected and repeated upon approval by the
EPA project team.
Background contamination was controlled by sterilization of test materials and use of aseptic technique,
procedural blank controls, and a pure initial culture. Aseptic technique was used to ensure that the culture
remains pure. Procedural blank controls were run in parallel with the contaminated materials. Assuming
that the procedural blank controls showed no CPU, the observed colonies from inoculated coupons
indicated surviving spores from the inoculated organisms provided they were consistent with the expected
colony morphology (i.e., orange color, round form, flat elevation, rough texture, and undulate margin).
50
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Table 5.2: Quality
QC Sample
Procedural Blank
(coupon without
biological agent)
Positive Control
(Sample from
material coupon
contaminated with
biological agent but
not subjected to
the test conditions)
Blank plating of
microbiological
supplies
BlankTrypticSoy
agar Sterility
Control
(plate incubated,
but not inoculated)
Ohlorinp
ronrpntration
PH
Field blank
samples
f Control Checks
Information
Provided
Controls for sterility of
materials and methods
used in the procedure.
Initial contamination
level on the coupons;
allows for
determination of log
reduction; controls for
confounds arising from
history impacting
bioactivity; controls for
special causes.
Shows plate's ability to
support growth.
Controls for sterility of
supplies used in
dilution plating
Controls for sterility of
plates.
Concentration of free
available chlorine
(FAC) in the fresh pH
adjusted bleach or
diluted bleach solution
Effective concentration
of hydrogen ions in
solution
The level of
contamination present
during sampling
Frequency
1 per test
3 or more
replicates
per test
3 of each
supply per
plating event
Each plate
1 peruse
1 peruse
1 per sampling
event
Acceptance
Criteria
No observed colony
forming units (CFU)
For high inoculation,
target loading of 1 x 107
CFU per sample with a
standard deviation of <
0.5 log. (5x106-5x107
CFU/sample); For low
inoculation, target loading
of1 x102CFUper
sample with a standard
deviation of < 0.25 log.
(56 -177 CFU/sample);
Grubbs outlier test (or
equivalent).
No observed growth
following incubation
No observed growth
following incubation.
6000-6700 ppm for fresh
pH adjusted bleach or
diluted bleach
>6.5 and <7.0 for fresh
pH adjusted bleach
Non-detect
Corrective
Action
Reject results of test
coupons on the same order
of magnitude, Identify and
remove source of
contamination.
Outside target range:
correct loading procedure
for next test and repeat
depending on decided
impact.
Outlier: evaluate/exclude
value.
Sterilize or dispose of
source of contamination.
Re-plate samples.
All plates are incubated prior
to use, so any contaminated
ones will be discarded.
Reject solution, replace
reagents and prepare a new
solution
Reject solution, replace
reagents and prepare a new
solution
Clean up environment.
Sterilize sampling materials
before use.
Several management controls were put in place in order to prevent cross-contamination. This project was
labor intensive and required that many activities be performed on material sections or coupons that were
intentionally contaminated (test samples and positive controls) or intentionally not-contaminated
(procedural blanks). The treatment of these three groups of test areas (positive control, test, and
procedural blank) varied for each group. Hence, specific procedures were put in place to prevent cross-
contamination among the groups. Adequate cleaning of all common materials and equipment was critical
51
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in preventing cross-contamination; therefore, all common materials were fumigated using a VHP® or
ethylene oxide sterilant, then swab sampled for sterility prior to each use.
There are four primary activities for each test in the experimental matrix. These activities are preparation
of the coupons, execution of the decontamination process (including sample recovery), sampling, and
analysis. Specific management controls for each of these activities are described below.
5.4 Acceptance Criteria for Critical Measurements
The data quality objectives define the critical measurements needed to address the stated objectives and
specify tolerable levels of potential errors associated with simulating the prescribed decontamination
environments. The following measurements were deemed to be critical to accomplish part or all of the
project objectives:
• Chlorine concentration, determined by measuring FAC in decontaminant solutions.
• pH
• Temperature
• RH
• Time
• Decontamination time
• Plated volume
• Spore log reduction
Data quality indicators for the critical measurements were used to determine if the collected data met the
data quality objectives. The critical measurement acceptance criteria are shown in Table 5.3. The target
values and actual test parameters for each run are shown in Table 5.4.
The tests were conducted so that all the critical parameters are within the measurements accepted
criteria listed in Table 5.4. When one of the test parameters did not meet the test target value, the test
method was repeated or modified to reach test target values and therefore achieve 100% completeness
for the task. For example, if the target FAC concentration in the bleach (decontaminant) solution was not
met, the solution was either re-prepared or adjusted according to the procedure in MOP 3128-A. Test RH
values were adjusted with data from calibrated RH sensors. Similarly, if the CFU count for bacterial
growth didn't fall under the target range, the sample was either filtered or re-plated.
52
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Table 5.3: Critical Measurement Acce
Critical Measurement
Plated volume
CFU/plate
FAC
Exposure time
PH
RH/temp of chamber
Measurement
Device
Pipette
Hand counting
HACH® Method
10100 -Digital
Titrator
Timer
Oakton® pH Meter
HOBO® U1 2 Sensor
Dtance Criteria
Accuracy
±2%
±10 % (between 2 counters)
±1%
±1 second
±0.01pH
±2.5% from 10% to 90%
Detection Limit
NA
1CFU
1 Digit (0.5 g/L
Chlorine)
1 second
NA
NA
Completeness
100%
100 %
100%
100%
100%
60%
CPU, colony forming units; FAC, free available chlorine; NA, Not applicable
Plates were quantitatively analyzed (CFU/plate) using a manual counting method. For each set of results
(per test), a second count was performed on 25 percent of the plates within the quantification range
(plates with 30 - 300 CFU). All second counts were found to be within 10 percent of the original count.
There are many QA/QC checks used to validate microbiological measurements. These checks include
samples that demonstrate the ability of the NHSRC RTP Biocontaminant Laboratory to culture the test
organism, as well as to demonstrate that materials used in this effort do not themselves contain spores.
The checks include:
• Negative control coupons: sterile coupons that underwent the decontamination process
• Field blank coupons: sterile coupons carried to the decontamination location but not decontaminated
• Laboratory blank coupons: sterile coupons not removed from NHSRC RTP Biocontaminant
Laboratory
• Laboratory material coupons: includes all materials, individually, used by the NHSRC RTP
Biocontaminant Laboratory in sample analysis
• Positive control coupons: coupons inoculated but not fumigated
53
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Table 5.4: Data Quality Assessment
Decontaminant
pH Adjusted
Bleach
pH Adjusted
Bleach
Diluted Bleach
pH Adjusted
Bleach
pH Adjusted
Bleach
Decontamination
Procedure
Immersion
Immersion
Immersion
Immersion
Rigorous
Immersion
Material
Type
Carpet
Personal
Protective
Equipment
Personal
Protective
Equipment
Upholstery
Carpet
Chlorine Concentration (FAC) - per 5
mL Bleach titrated
Target
Value
(ppm)
6000 - 6700
6000 - 6700
5700- 6300
6000 - 6700
6000 - 6700
Test
Value
(ppm)
6650
6610
6049
6390
6470
Frequency
Once before
testing
Once before
testing
Once before
testing
Once before
testing
Once before
testing
pH
Target
Value
6.5-
7.0
6.5-
7.0
-11
6.5-
7.0
6.5-
7.0
Test
Value
6.61
6.64
11.09
6.6
6.79
Frequency
Once
before
testing
Once
before
testing
Once
before
testing
Once
before
testing
Once
before
testing
Chamber Parameters (HOBO®)
RH
46
42
31
25
20
Temp
71
64
61
61
62
Frequency
Data recorded at
5 min intervals
for the duration
of the test
Data recorded at
1 min intervals
for the duration
of the test
Data recorded at
5 min intervals
for the duration
of the test
Data recorded at
5 min intervals
for the duration
of the test
Data recorded at
3 min intervals
for the duration
of the test
54
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Diluted Bleach
pH Adjusted
Bleach
pH Adjusted
Bleach
Immersion
Immersion
Rigorous
Immersion
Paper
Paper
Carpet
5700- 6300
6000 - 6700
6000-6700
5989
6209
6550
Once before
testing
Once before
testing
Once before
testing
-11
6.5-
7.0
6.5-
7.0
11.39
6.75
6.87
Once
before
testing
Once
before
testing
Once
before
testing
21
38
63
62
Data recorded at
3 min intervals
for the duration
of the test
Data recorded at
3 min intervals
for the duration
of the test
HOBO® Data Not Available this test
FAC, free available chlorine
55
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5.5 Data Quality Audits
This project was assigned QA Category III and did not require technical systems or performance
evaluation audits.
5.6 QA/QC Reporting
Quality Assurance (QA)/QC procedures were performed in accordance with the QAPP for this
investigation.
56
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6 Summary and Recommendations
The pAB immersion-based waste decontamination procedure, when performed on carpet material
coupons, showed that decontamination efficacy increases with increasing immersion time. The mean
combined Log10 CPU reductions for the 15, 30, and 60 minutes immersion times were respectively 2.72 +
0.32, 3.37 + 0.34, and 3.91 + 0.51 using the extraction-based sampling method (removal of a coupon
from a larger sample). As mentioned, for this study, a log reduction of 6.0 was considered effective. The
same pAB immersion-based procedure, when applied to upholstered coupons, resulted in a much higher
decontamination efficacy (6.4 + 0.6 Log Reduction) for an equal immersion time of 15 minutes, and using
the same sampling method.
The mean combined Log10 CPU reductions for PPE materials immersed for 15 minutes in pAB and diluted
bleach are respectively 7.57 + 0.86 and 5.45 + 2.51. The pAB/15 minutes immersion procedure did
achieve greater than 6 log reduction for all but one sample. When PPE waste decontamination was
attempted with immersion in diluted bleach for 15 minutes, the efficacy ranged from 0.22 to 7.58 log
reduction. Difficulties in wetting all interior surfaces of PPE materials may explain the wide range of
decontamination efficacies observed for this material. One potential explanation for why this effect was
less evident during the attempted decontamination with pAB, is that the higher volatility of pAB (compared
to diluted bleach) may have resulted in higher exposures of spores to chlorine gas regardless of whether
these spores were located on wetted areas.
The mean combined Log10 CPU reductions for the front and middle pages of a book material, when a 15
minutes immersion time in pAB was utilized are 6.6 + 0.5 and 5.4 + 1.3, respectively. When the paper
material decontamination was attempted with a 15 minute immersion in diluted bleach 15, the respective
efficacies were 6.0 + 1.8 and 4.5 + 2.4. These data suggest that decontamination of paper materials fully
exposed to the decontaminant (i.e., cover and front pages of a book) is more efficient than of those
materials shielded from the liquid decontaminant (i.e., middle of the book pages). Agitation methods
designed to expose all pages of books or similar waste items may increase the efficacy of treatment for
this type of waste.
For the material/decontamination procedures that achieved more than 6 Log reductions, there were still
instances in which full decontamination was not achieved. The residual spores detected in the samples
were due to the inability of the decontamination solution to have full contact with all the surface areas of
the materials tested as shown in Table 6.1. Carpet data is not included in this table since it never
achieved 6 Log reductions.
Overall, the data from these tests demonstrate that immersion time (contact time) is a critical parameter in
waste decontamination, as demonstrated in this study for carpet material. The pAB decontaminant
achieved greater spore reductions than diluted bleach for all of the materials tested in this study.
Materials such as carpet may prove difficult to achieve complete spore inactivation with these techniques,
especially in a field setting where large quantities of the materials are requiring treatment. Such large-
scale application of this method may prove logistically challenging.
The two sampling methods (extractive and Sponge-Stick™) were evaluated for both carpet and
upholstered materials. For these materials, the extraction-based method consistently achieved higher
recoveries. As a consequence of its lower recoveries, the Sponge-Stick™ method resulted in over-
57
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estimation of the decontamination efficacies. If possible, utilization of extraction-based methods for waste
sampling provides improved sensitivity of detection. However, these methods might not be easily
deployed in the field, and could generate samples that are not as easily analyzed in the laboratory.
Representative extraction samples can be difficult to collect for certain materials. For example, it was
found, during preliminary exercises, that carpet (specifically, the backing) and upholstery materials were
difficult to cut with simple hand tools. For aseptic sampling purposes specific to this study, these
materials were pre-cut for extractive sampling during coupon material preparation however; this would not
be the case during a field sampling event. Damaged material samples and poor sample collection
technique are a few of the issues to be encountered with extractive sampling. Furthermore, preliminary
testing showed that the required laboratory analysis method was specific to the material. Variations in
neutralizer volumes, extraction solution volumes, extraction vessels, etc. were necessary for each
material during analysis. An effective surface sampling technique that could be utilized for the majority of
materials would result in an efficient and economical sample analysis process. More work is needed to
develop and characterize effective waste sampling methods.
Table 6.1: Portion of Samples with No Viable Spores Detected After Decontamination
Sampling Method
Extractive method
Sponge-Stick™
Material Type
Upholstery
Paper Front Page
Paper middle Page
PPE
Carpet (15-min Immersion Time)
Carpet (30-min Immersion Time)
Carpet (60-min Immersion Time)
Upholstery
Carpet (15-min Immersion Time)
Carpet (30-min Immersion Time)
Carpet (60-min Immersion Time)
Post-Decon Samples with No Viable Spores
Detected / Total Number of Samples Collected
pAB
19/36
12/13
5/11
58/60
0/27
0/36
1/36
34/36
9/27
4/36
13/36
Diluted Bleach
^^^^^^_
9/12
4/12
28/60
\
Overall, numerous knowledge gaps and capability gaps were identified during the current study. Some of
these gaps include:
• What are the logistical challenges in scaling up waste decontamination and sampling
methods for a wide area release?
• What are the most efficient waste sampling methodologies for various waste streams?
• Do current waste management approaches affect contaminant resuspension or aerosol
generation?
• What are the likely waste acceptance criteria at waste management facilities that may be
able to handle these wastes? How will that impact sampling and decontamination methods
(e.g., liquids, residual chemicals, spore loading, etc.)?
58
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• What are the likely volumes of waste liquids being generated and how those will need to be
managed as a result of these methods?
• Will sample processing labs accept these sample types? Are the sample types optimized
for preferred analytical methods?
Within this list are many research gaps for which future studies are needed.
References
1. U.S. EPA. Technical Brief- Bio-response Operational Testing and Evaluation (BOTE) Project.
U.S. Environmental Protection Agency, Washington, DC, EPA/600/S-12/001, 2012.
59
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Appendix A: Miscellaneous Operating Procedures
MOP 3120 VHP® Operation
MOP 3128-A Procedure for preparing pH-Adjusted Bleach Solution
MOP 3148 lodometric Method for the Determination of Chlorine Dioxide and Chlorite using the
HACH Test Kit
MOP 3165 Sponge Sample Collection Protocol
MOP 3194 Procedure for Fabricating 18" x 18" Upholstery Coupons for Liquid Inoculation
MOP 3195 General Procedure for Immersion Decontamination
MOP 6535a Serial Dilution: Spread Plate Procedure to Quantify Viable Bacterial Spores
MOP 6562 Preparing Pre-Measured Tubes with Aliquoted Amounts of Phosphate Buffered Saline
with Tween 20 (PBST)
MOP 6565 Filtration and Plating of Bacteria from Liquid Extracts
MOP 6580 Recovery of Bacillus Spores from 3M Sponge-Stick™ Samples
MOP 6584 Procedure for Replating Bacteria Spore Extract Samples
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MOP-3120
Revision 5
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Miscellaneous Operating Procedure (MOP) 3120:
VHP Operation
Prepared by:
Rob Delafield, ARCADIA Technical Lead Author
Reviewed by:
Dahman Tgaati, ARC ADIS'Project Manager
Date: 6/26/2013
Date: 6/26/2013
Approved by:
. A
Worth Calfee, EPA W0fk Assignment Manager
I ./
Date: 6/26/2013
Prepared for
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
Prepared by
ARCADIS U.S., Inc.
4915 Prospectus Drive, Suite F
Durham, NC 27713
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MOP 3120
TITLE: VHP Operation
SCOPE: Outlines setup and operation of the VHP 1000-ED.
PURPOSE: Ensure the decontamination and/or sterilization of the COMMANDER chamber
and/or airlock contents.
1.0 OVERVIEW
The VHP has no method to control the concentration generated in the chamber. Therefore the
target concentration must be achieved and maintained through the set up of the operating
parameters. These values may need to be adjusted as material type and volume vary.
2.0 SAFETY
A Ensure all personnel in the room are aware that VHP is going to be dispensed in
COMMANDER or the airlock.
A Activate the warning lights outside the door from the control room and outside the
exterior door.
A Verify that the enclosure air monitor is calibrated and functioning properly.
3.0 SETUP
Starting parameters may be as follows:
PHASE
(DH)de-humidification
(CD) conditioning
(DC) de-contamination
(AR) aeration
(A A) aux aeration
TIME (hr:min:sec)
00:00:00
00:30:00
06:00:00
04:00:00
00:00:00
INJECTION RATE (g/min)
12.0 (Maximum rate)
12.0
The times and injection rates can be adjusted after start-up if needed. Airflow is set and
maintained at 17scfm for all cycles.
4.0 PRE-START UP
a. Use the chamber SCADA system to verify the supply and exhaust blowers for the
chamber are off. On the 485 Com screen, the buttons for SC 101 and SC 102 blowers
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should be red (Figure 1). Click them with the mouse to change them from green to red
if necessary.
File View Alarm Security Window Help
SC-101 Cbr Supply Blower
EPA DCMD Chamber - Drives and Stulz AHU Communication
Stulz Coils
Air Proving Failure Alarm
Temperature Display F/C
Pump Failure Alarm
Drain Failure Alarm
Water Detection Alarm
SC-102 Cbr Exhaust Blower
SC-211 Airlock Exhaust Blower
Stulz Registers
DC-BUS Voltage
&EPA
J Start | | ^ PAC Display Runtime ...
ote Humidity Display
oyiiern On/Off Status
Room Temperature Setpoint
Analog Input 6 Minimum C
Dg Input 6 Maximum [
Room Humidity Setpoint
Analog Input 7 Minimum C
Analog Input 7 Maximum [
Password 1
Figure 1. 485 Com screen showing interface for the SC 101 and SC 102 blowers
b. Check that the filtered exhaust valves on top of the chamber are closed (the two valves
labeled Chamber Exhaust (Filtered) in Figure 2), and that the center valve is closed
(the valve labeled Chamber Exhaust Bypass). This Bypass circuit is not longer
functional and has been blanked off.
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Figure 2. Top of the chamber showing the valve locations
c. Check that the chamber supply valve is closed (labeled Chamber Supply Air in Figure 3
and shown open).
d. Ensure that the ATI sensor is in the chamber and not covered.
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Figure 3. Top of the chamber showing the Chamber Supply Air valve location
e. Securely close the chamber door.
f. Ensure the supply and return lines of the VHP are connected to the chamber (Figure 4).
Keep in mind that the supply line gets hot during operation which softens the tubing. This
makes it susceptible to kinking if stressed.
g. Ensure the chamber P-trap is filled with water (Figure 4).
h. Connect the Emergency Stop cable to the back of the Steris 1000ED (Figure 5).
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Figure 4. Locations of VHP return and supply ports and the COMMANDER P-trap
The Enclosure ATI is wired through the E-Stop switch to shut down the VHP operation
in the event the concentration in the enclosure reaches 5 ppm. This is a latching action
and will require pressing the alarm reset (A\R) on the ATI monitor to unlatch. Of course,
check the concentration on the SCADA to ensure it is safe to enter the Enclosure.
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Figure 5. Emergency Stop connection at the DC INPUT
5.0 VHP OPERATION
a. Fill out a VHP safety checklist and insert in the Steris Log book.
b. Log in to the Steris 1000ED. The VHP has a touch screen. A pen works well for keying
in selections. Select Run.
c. From OPERATOR menu, enter Username= 3, Password= 3. Select HiOi Fill. Fill
reservoir to 1850 grams.
d. It may be necessary to fill or refill the supply bottle. If so, then:
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1) Turn the knob above the bottle door to Replace (This knob only turns clockwise).
Wearing a lab coat, nitrile gloves and safety glasses, remove the bottle by sliding
forward.
2) Fill the bottle from the 5 gal container, replace the bottle and slide all the way back.
3) Turn the knob to Engage. Unless you manually stopped the fill process, it will
automatically resume. If manually stopped, press Start to resume.
e. Press X to return to the operator menu and select run cycle.
f. Choose the cycle named chamber (or airlock).
g. Start the cycle by pressing the green icon.
h. Answer YES to run regeneration after completion of cycle. Record dryer and scale values
in the "Steris log" notebook.
i. Monitor the conditioning phase for desired concentration, (typically 250 or 400 ppm).
j. If necessary, press Cycle Setup and increase the time and/or the injection rate to achieve
target (use the right arrow key to advance to the second screen). Press X to exit and save
changes.
NOTE: Frequent changes to injection rates can cause injection rate deviation trips.
k. Monitor decontamination phase and adjust injection rate and/or time if necessary to
achieve set point conditions. Typical targets are 250 ppm for 4 hours and 400 ppm for 6
hours.
6.0 CYCLE COMPLETION
Open chamber supply and filtered exhaust valves, then turn on the chamber supply and
exhaust blowers to aerate the chamber. Chamber is safe to enter when the concentration falls
below 1.0 ppm. (See Section 9.0 on chamber entry).
7.0 DECON of the AIRLOCK
a. Place a fan inside the airlock and make sure it is turned on.
b. Ensure the VHP hoses are connected to the airlock ports. The VHP supply hose should
always be as short as possible.
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c. A short extension tube is used on the supply port inside the airlock to separate the
supply and return.
d. Check the FfeCh sensor location to be sure it is attached to the hanger on the left-hand
side wall.
e. Check with one other person that all items are in airlock, especially Bis and bin lids.
Also review that the VHP connection is correct (Figure 6).
Figure 6. VHP connections
f. At the SCADA 485 Com screen (Figure 7), ensure the blower is off (SC-211). Mouse
click to turn the blower off (red) if necessary.
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File View Alarm Security Window Help
SC-101 Cbr Supply Blower
EPA DCMD Chamber - Drives and Stulz AHU Communication
Stulz Coils
Air Proving Failure Alarm
Temperature Display F/C
Pump Failure Alarm
Drain Failure Alarm
Water Detection Alarm
Blower Stauts
Humidifier Status
SC-102 Cbr Exhaust Blower
SC-211 Airlock Exhaust Blower
Stulz Registers
&EPA
Remote Humidity Displc,,
System On/Off Status
Room Temperature Setpc
Room Temperature Setpc
Room Temperature Setpc
Analog Input 6 Minimum C
Analog Input 6 Maximum L
Room Humidity Setpoint
Analog Input 7 Minimum C
Analog Input 7 Maximum [
Password 1
ARCADIS
Start] | ^ PAC Display Runtin
Figure 7. 485 Com Screen showing SC-211 Blower interface
g. Turn supply damper off (shown off in Figure 8).
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Figure 8. Airlock Supply Damper shown in the off position
h. Close exhaust dampers/valves (Airlock Exhaust (Filtered) shown open in Figure 9).
i. Put up the WARNING sign on the airlock door.
j. Verify the airlock drain is closed.
k. Seal bottom of the airlock door with duct tape.
1. Run the COMMANDER program per Section 5.0.
m. Close Enclosure doors.
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Figure 9. Filtered Airlock Exhaust shown in the open position
8.0 AERATION OF AIRLOCK
a. Open filtered exhaust valve near the floor (Figure 9).
b. Open supply air damper at waist level (Figure 8).
c. Turn on airlock blower from the SCADA "485 Com" screen (Figure 7). SC-211 will be
red - make it green.
9.0 CHAMBER ENTRY
Check the ATI enclosure sensor reading on the SCADA. The reading should be less than 1 ppm
for entry into the enclosure.
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Use 0.1 to 3 ppm ffcCh Drager tubes (P/N 81010414) per manufacturer's directions to monitor
concentration for safe entry (MOP 3187). If the measured concentration is above 1 ppm, more
aeration is required before opening the door.
10.0 RH PROBE FAILURE TRIP
A common problem with the VHP is a PvH probe failure trip. If under certain conditions moisture
gets on the probe, the alarm cannot be cleared by usual methods. Should this happen these steps
can be taken to clear the alarm (this procedure is not outlined in the manual).
a. Enter the service mode (use the same ID and password).
b Select "Calibration"
c. Select "Cycle Airflow". You should here the fan start and the flow rate ramp-up to set
point. This dries the sensor. Allow it to run for 30 seconds.
d. Selecting the red X in the bottom right corner will terminate the fan.
e. Continue selecting the red X to return to the operation screen. You should now be able to
reset the alarm.
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Revision 2
Dec 2013
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Miscellaneous Operating Procedure (MOP) 3128-A:
Procedure for Preparing pH-Adjusted Bleach Solution
Prepared by:
Reviewed by: _
Dahrnan Tojiaii, ARCADIS-iroject Manager
Approved by:
Lukas QudejanS; EP|\ WACOR
Date: 12/11/2013
Stella McDonald, ARCADIS MOP Author
Date: 12/11/2013
Date: 12/11/2013
Prepared for
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
Prepared by
ARCADIS U.S., Inc.
4915 Prospectus Drive, Suite F
Durham, NC 27713
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MOP 3128-A
TITLE: PROCEDURE FOR PREPARING pH-ADJUSTED BLEACH SOLUTION
SCOPE: This MOP describes a procedure for reproducibly preparing the pH-adjusted
bleach solution.
PURPOSE: The purpose of this MOP is to ensure the solution meets QA specifications for
each test.
Equipment/Reagents:
Draeger or personal chlorine (C12) monitor [Oakton Acorn Series pH 5 meter or
equivalent
Plastic or glass funnel
. Triple rinsed container suitable for transporting hazardous solutions
Oakton pH 7 (pH = 7.00 +/- 0.01 @ 25°C) buffer or equivalent
. Concentrated Clorox Commercial Solutions Germicidal Bleach (LOWE'S p/n 174273 ),
less than 1 year old
. 5% v/v Acetic Acid (Ricca Chemical, p/n 7732-18-5 or equivalent)
. Deionized water
1.0 PROCEDURE
1.1 Calibrate pH Meter
1. Turn meter on (Figure 1). Meter will automatically enter pH mode.
2. Rinse electrode thoroughly with DI water. DO NOT wipe the electrode.
3. Dip both the electrode and temperature sensor into pH 7.00 buffer solution. The glass
bulb must be completely immersed into the sample. Stir gently, and wait for the reading
to stabilize (about 40 seconds).
4. Press CAL key to enter the calibration mode. The display will momentarily flash "CA" to
indicate Calibration. The display will show the current uncalibrated reading, blinking
while in calibration mode.
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Figure 1. Oakton pH meter
5. Allow the reading to stabilize. The meter will automatically recognize 7.00, 4.01, or
10.00 buffers.
6. Record the uncalibrated value in the laboratory notebook. Press Enter key once to
confirm calibration. The LCD displays "CO" to indicate the calibration point has been
confirmed. The meter exits calibration mode and returns to measurement mode.
7. Record the pH buffer measurement and temperature (Press MODE key to select
parameter) in the appropriate lab notebook.
1.2 Bleach Preparation
1. Dilute concentrated germicidal bleach to regular germicidal bleach by making a 2:1 dilution
with deionized water. For example, to prepare 750 ml of regular germicidal bleach, add 250
ml (1 part) of deionized water to 500 ml (2 parts) of concentrated germicidal bleach.
2. The pH-adjusted bleach should consist of 80% deionized water, 10% germicidal bleach
(prepared in Step 1) and, 10% acetic acid. For example, to prepare 10 L of solution, combine
1 L of prepared regular germicidal bleach, 2 L of deionized water, 1 L of acetic acid, and 6 L
of deionized water in that order. Prepare the solution in a container that accommodates the
total volume of solution and a funnel if necessary. Record the total volume as Vstartin the lab
notebook.
3. Seal the mixing container and gently agitate for mixing. Place the pH probe into the solution
and measure the pH (target pH = 6.8). If pH is above 7.0, add small increments of acetic acid.
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If below 6.5, add germicidal bleach. Refer to the QAPP to determine if adjustments are
permitted. Record the volume required for adjustment as Vadd. Calculate Vtotai as Vstart + Vadd
in the lab notebook.
4. Measure the free available chlorine (FAC) per MOP 3148. The target FAC is 6350 mg/L.
The acceptable range is 6000 mg/L< FAC < 6700 mg/L.
a) If FAC exceeds the acceptable range, dilute the total volume with deionized water by the
percent difference between the target FAC and the actual FAC.
Dilution volume = [(actual - target) + target] x (Vtotai)
b) If the FAC is less than the acceptable range, add bleach according to the following
equations:
Additional volume of bleach = (target - actual)/ target x Vtotai
Recalculate Vtotai according to the all additions and repeat steps 3 and 4 until both
parameters are met. Record the final FAC, pH, temperature, and time in the lab
notebook.
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Miscellaneous Operating Procedure (MOP) 3148:
lodometric Method for the Determination of Chlorine Dioxide and
Chlorite using the HACH Test Kit
Prepared by: /U^A^uc^w ,. jv^.^v ^~ ^ Date: 11/15/2oi2
Stella McDonald, ARCADIS Work Assignment Leader
Reviewed by: ^ fe^^^~ J> Date: 11/15/2012
Dahman Tduati, ARCADIS Project Manager
/
Approved by: / /_ Date: 11/15/2012
Worth Car&erEPA Work Assignment Manager
Prepared for
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
Prepared by
ARCADIS U.S., Inc.
4915 Prospectus Drive, Suite F
Durham, NC 27713
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MOP-3148
TITLE: IODOMETRIC METHOD FOR THE DETERMINATION OF CHLORINE
DIOXIDE AND CHLORITE USING THE HACK TEST KIT
SCOPE: This MOP is intended for measurement of bleach (FAC) or chlorine dioxide (C1O2)
in the DTRL.
PURPOSE: This document provides the standard procedure for sample titration using the
HACK Test Kit.
Equipment
HACK digital titrator
. Magnetic stir bar
. Buret
Reagents
HACK digital titrator cartridge (2.26N stabilized sodium thiosulfate (STS), cat. No.
26869-01)
HACK starch indicator solution (cat. No. 349-32)
. 6N Hydrochloric Acid (HC1)
. Phosphate buffer concentrate
Potassium Iodide
. Deionized water
1. PROCEDURE
1.1 Preparation of Potassium Iodide Phosphate Buffer (KIPB) Solution
Add 5 mL of phosphate buffer concentrate and 50 g KI to a 1.0 L volumetric flask. Bring up to
1 .OL with deionized water.
1.2 Preparation of Sample
1. Insert a clean delivery tube into the 2.26N STS titrant solution cartridge. Attach the cartridge
to the titrator body (see Figure 1).
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J' Delivery Tube
Reagent Cartridge
Figure 1. Digital Titrator body, delivery tube, and reagent cartridge
2. Flush the delivery tube by turning the delivery knob to eject a few drops of titrant. Reset the
counter to zero and wipe off the tip.
3. In a 250 ml beaker, add 20 mL of KIPB and 5 mL of sample
4. Fill beaker to about the 200 mL mark with deionized water.
5. Add a stir bar and place beaker on a stir plate
1.3 Titration: C1O2 and Chlorite
For F AC measurements of bleach, proceed directly to Step 3
1. Place the delivery tube tip into the solution and titrate with 2.26N STS until the solution is pale
yellow. From the Digital Titrator, record the number of digits required (A).
2. Calculate the volume of titrant delivered (VA):
VA (ml) = A/800
3. Reset the counter to zero and add ~5 mL of 6N HCL to beaker.
4. Titrate with 2.26 N STS until the solution is pale yellow, add 1 dropper of starch indicator
and continue titration until the solution becomes colorless. Record the number of digits
required (B).
5. Calculate the volume of titrant delivered (Vs):
VB(ml) = B/800
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Calculations
In the following equations, 5 represents the sample size in mL, 2.26 represents the normality of the
STS, the other constants are the equivalent weights (mg/eq) per electron, and VA and VB are as
defined previously.
Bleach (ppm FAC) = VB * 2.26 *35453 / 5
Chlorine dioxide (ppm) = VA * 2.26 * 67452 / 5
Chlorite (ppm) = (VB - 4 * VA) *2.26 * 16863 / 5
If VB is not greater than 4 * VA, then the solution contains chlorine and must be reformulated.
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Miscellaneous Operating Procedure (MOP) 3165:
Sponge Sample Collection Protocol
Prepared by:
Date: 7/17/2013
Stella McDonald, ARCADIS Work Assignment Leader
Reviewed by:
Date: 7/17/2013
Dahman^fouati, ARCADIS Project Manager
Approved by:
/
Date: 7/17/2013
Worth Calfee, EPA Work Assignment Manager
Prepared for
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
Prepared by
ARCADIS U.S., Inc.
4915 Prospectus Drive, Suite F
Durham, NC 27713
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MOP 3165
TITLE:
SCOPE:
PURPOSE:
SPONGE SAMPLE COLLECTION PROTOCOL
This MOP outlines the procedure for collecting spores using a 3M Sponge-Stick™.
To provide a procedure for the collection of spore samples using a Sponge-Stick™
in a consistent and repeatable manner.
MATERIALS
• 3M Sponge-Sticks™ (P/N SSL10NB), hereafter referred to as 'sponge'
• One Seward stomacher bag (P/N BL6041/CLR) per kit
• Disposable gloves
• Sterilized sampling templates
• One Fisher Sterile sampling bag with flat wire enclosure (7" x 12", P/N 14-955-194) per kit
• One Fisher Sterile sampling bag with flat wire enclosure (10" x 14", P/N 01-002-53) per kit
for overpack
• Dispatch wipes
1.0 PREPARATION
All materials needed for collection of each sample will be prepared in advance using aseptic
technique. A sample kit for a single sponge sample will be prepared as follows:
1.1 One stomacher bag will be uniquely labeled as specified in the project QAPP.
1.2 A 10" x 14" bag will be labeled with the same ID as the stomacher bag.
1.3 One stomacher bag, and one 9.5" x 12" unlabeled bag will be placed in the overpack bag.
1.4 A sterile Sponge-Stick will be added to the overpack bag.
1.5 Each prepared bag is one sampling kit.
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2.0 PROCEDURE FOR 12" X 12" SAMPLING AREAS
NOTE: For sampling surface dimensions not outlined directly in this MOP, follow the
12" x 12" sampling procedure for areas larger than 3" x 3" and the 2" x 2"
procedure for areas smaller than 3" x 3". The area must be at least 1.5" x 1.5" to
accommodate the dimensions of the sponge stick itself. Number of passes will
vary with the dimensions of the surface being sampled. It is important that each
pass overlaps the previous pass, and that each direction (horizontal/vertical/
diagonal) is sampled as described in this MOP.
A two person team will be used, employing aseptic technique throughout. The team will consist of
a sampler and a sample handler. In some cases, a third person may be needed to move samples.
Throughout the procedure, the support person will log anything they deem to be significant into the
laboratory notebook.
In general, the team works from the least contaminated sample set (i.e., control blanks) towards the
most contaminated sample set (i.e., positive controls).
All members shall wear dust masks to minimize potential contamination of the samples.
Depending on the situation, respiratory protection beyond a dust mask may be required to protect
the sampling team (e.g., SAR; this will be specified in the project QAPP). New disposable lab
coats are required for the sample handler when changing between different types of materials or
when direct contact between the coupon and lab coat occurs.
2.1 The sampler will don sterile gloves and place the disposable template over the area to be
sampled.
2.2 The support person will remove a sample kit from the sampling bin and record the sample
tube number on the sampling log sheet next to the corresponding coupon code just
recorded.
2.3 The sampler and support person will verify the sample code and ensure that the correct
coupon and location are being sampled.
2.4 The support person will:
a) Open the outer sampling bag touching the outside of the bag.
b) Touching only the outside of the (10" x 14") bag, remove the sponge, and hand it to
the sampler.
c) Remove the stomacher bag, being careful to not touch the inside of the outer sampling
bag, and open it touching only the outside.
2.5 The sampler will remove the sterile sponge from its package. Grasp the sponge near the top
of the handle. Do not handle below the thumb stop.
2.6 The sampler will wipe the surface to be sampled using the moistened sterile sponge by
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laying the widest part of the sponge on the surface, leaving the leading edge slightly lifted.
Apply gentle but firm pressure and use an overlapping 'S' pattern to cover the entire
surface with horizontal strokes (Figure 1). Use the other hand to hold the template during
sampling, being careful not to touch the surface.
Figure 1. First pass with sponge - horizontal strokes using one side of the sponge
2.7 The sampler will turn the sponge over and wipe the same area again using vertical ' S'-
strokes (Figure 2).
Figure 2. Second pass with sponge - vertical strokes using the other side of the sponge
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2.8 The sampler will the use the edges of the sponge (narrow sides) to wipe the same area using
diagonal 'S'-strokes (Figure 3). The sponge will be flipped to use the opposite side
immediately after the longest stroke at opposite corners.
Figure 3. Third pass with sponge - diagonal strokes using the edges of the sponge
2.9 The sampler will use the tip of the sponge to wipe the perimeter of the sampling area
(Figure 4).
Figure 4. Final (fourth) pass with sponge - perimeter wipe using the tip of the sponge
2.10 The sample handler will open the stomacher bag, careful not to touch the inside of the bag.
2.11 The sampler will place the end of the sponge in the bag, holding the handle outside the
opening of the bag.
2.12 The sample handler will grasp the sponge from outside of the bag, and help the sample break
off the handle of the sponge. The handle below the thumbstop should not touch the inside of
the stomacher bag.
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2.13 The sample handler will securely seal the stomacher bag and wipe the outside with a
disinfecting wipe.
2.14 The sample handler will then place the stomacher bag inside the unlabeled sterile bag.
2.15 The sample handler will place this in the overpack bag and wipe the overpack bag with
disinfecting wipes.
2.16 The sample handler will place the overpack bag in the sample bin.
NOTE: Remove excessive air from the re-sealable plastic bags to increase the number of
samples that can be shipped in one container.
2.17 The sampler will dispose of the template if present. The coupon handler will remove the
coupon, if present.
2.18 Both members will remove outer gloves and discard. Clean gloves should be worn for each
new sample.
3.0 PROCEDURE FOR 2" X 2" COUPONS
A two person team will be used, employing aseptic technique throughout. The team will consist of
a sampler and a sample handler. In cases where coupons are mobile, a third person will be needed
to move coupons. Only the coupon handler will handle coupons.
Throughout the procedure, the support person will log anything they deem to be significant into the
laboratory notebook.
In general, the team works from the least contaminated sample set (i.e., control blanks) towards the
most contaminated sample set (i.e., positive controls).
All members shall wear dust masks to minimize potential contamination of the samples.
Depending on the situation, respiratory protection beyond a dust mask may be required to protect
the sampling team (e.g., SAR; this will be specified in the project QAPP). New disposable lab
coats are required for the sample handler when changing between different types of materials or
when direct contact between the coupon and lab coat occurs.
3.1 The support person will remove a sample kit from the sampling bin and record the sample
tube number on the sampling log sheet next to the corresponding coupon code just
recorded.
3.2 The sampler and support person will verify the sample code and ensure that the correct
coupon and location are being sampled.
3.3 The support person will:
d) Open the outer sampling bag touching the outside of the bag.
e) Touching only the outside of the (10" x 14") bag, remove the sponge, and hand it to
the sampler.
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f) Remove the stomacher bag, being careful to not touch the inside of the outer sampling
bag, and open it touching only the outside.
3.4 The sampler will remove the sterile sponge from its package. Grasp the sponge near the top
of the handle. Do not handle below the thumb stop.
3.5 Align the sponge with the widest part in contact with the surface to be sampled in the upper
left corner, as seen in Figure 5. Sample by moving the sponge down to the bottom edge of
the sampled area along the left edge applying even pressure to the sponge tip.
3.6 Align the sponge with the widest part in contact with the surface on the same side as in Step
3.5 in the upper right corner. Sample by moving the sponge down to the bottom edge of the
sampled area along the right edge applying even pressure to the sponge tip.
1
Figure 5. Sponge on 2" x 2" coupon (Side A)
3.7 Rotate the coupon 90 degrees.
3.8 Flip the sponge over and repeat Steps 3.5 and 3.6.
3.9 Flip the sponge on one side (the flat part of the handle will be in the vertical orientation) and
sample horizontally across the coupon from upper left corner to lower right, overlapping
30% each stroke.
3.10 Rotate the coupon 90 degrees.
3.11 Flip the sponge to the other side and repeat Step 3.9.
3.12 The sample handler will open the stomacher bag, careful not to touch the inside of the bag.
3.13 The sampler will place the end of the sponge in the bag, holding the handle outside the
opening of the bag.
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MOP-3165
Revision 3
July 2013
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3.14 The sample handler will grasp the sponge from outside of the bag, and help the sample break
off the handle of the sponge. The handle below the thumbstop should not touch the inside of
the stomacher bag.
3.15 The sample handler will securely seal the stomacher bag and wipe the outside with a
disinfecting wipe.
3.16 The sample handler will then place the stomacher bag inside the unlabeled sterile bag.
3.17 The sample handler will place this in the overpack bag and wipe the overpack bag with
disinfecting wipes.
3.18 The sample handler will place the overpack bag in the sample bin.
NOTE: Remove excessive air from the re-sealable plastic bags to increase the number of
samples that can be shipped in one container.
3.19 Both members will remove outer gloves and discard. Clean gloves should be worn for each
new sample.
4.0 REFERENCES
Sponge sample collection protocol adapted from:
National Validation Study of a Cellulose Sponge Wipe-Processing Method for Use after Sampling
Bacillus anthracis Spores from Surfaces. Rose, Laura J.; Hodges, Lisa; O'Connell, Heather;
Noble-Wang, Judith. Appl. Environ. Microbiol. 2011, 77(23):8355.
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MOP 3194
Revision 0
August 2013
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Miscellaneous Operating Procedure (MOP) 3194:
Procedure for Fabricating IS" X IS" Upholstery Coupons
for Liquid Inoculation
Prepared by:
Stelfa McDonald, ARCADIS Work Assignment Leader
Date: 8/9/2013
Reviewed by:
^
Dahman Touatf^ARCADIS ProjeCfMaTiager
Date: 8/9/2013
Approved by:
Worth Carree*, EPA Work Assignment Manager
Date: 8/9/2013
Prepared for
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
Prepared by
ARCADIS U.S., Inc.
4915 Prospectus Drive, Suite F
Durham, NC 27713
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MOP 3194
Revision 0
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MOP 3194
TITLE:
SCOPE:
PROCEDURE FOR FABRICATING 18" X 18" UPHOLSTERY COUPONS FOR
LIQUID INOCULATION
This MOP describes the procedure for constructing 18" x 18" upholstery coupons
with the foam and fabric layers adhered together.
PURPOSE: The purpose of this MOP is to ensure consistent manufacturing (materials and
procedure) of these coupons.
1.0 INTRODUCTION
Section 2.0 details the fabrication procedure for the material coupons. Section 3.0 describes how
the 18 mm coupon punches (for liquid inoculation) are created.
2.0 FABRICATION OF 18" X 18" UPHOLSTERY COUPONS
The materials to be used for the fabrication of the upholstery coupons are detailed in the table
below.
Material
Upholstery Fabric
Upholstery Foam
Upholstery Adhesive
Plywood
Description
Bryant
Indoor/Outdoor Pine
I"x24"x 108" High
Density Upholstery
Foam
3M™ Foam Fast 74
Spray Adhesive Clear
3/4" Pine plywood
Vendor
www.fabric.com
OnlineFabricStore
3M™
Lowe's
Part Number
0298925
124108-2645
62495049504
35677
Prepare the upholstery coupons as follows:
1. Cut an 18 in x 18 in piece of foam padding, an 18 in x 18 in x 3/4 in piece of plywood (not
pressure treated), and a 24 in x 24 in piece of upholstery fabric.
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MOP 3194
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2. Place the 18 in x 18 in piece of foam padding in the center of an 18 in x 18 in x 3/4 in piece
of plywood.
3. Spray two layers of 3M™ FoamFast 74 to the surface of the foam then, quickly cover with a
24 in x 24 in piece of upholstery fabric to cover the foam.
4. Fold excess fabric underneath and staple to the back side of the plywood backing as shown in
Figures la and b.
Figure 1. Front (a) and back (b) of assembled upholstery cushion
3.0 FABRICATION OF 18 MM PUNCHES
1. Place a 17.5" x 17.5" grid with 3.5" x 3.5" sections on the surface of the coupon (Figure 2).
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MOP 3194
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17.5"
3.5"
3.5"
Figure 2. 17.5" x 17.5" Punch Grid
2. Place an 18 mm punch at the center of a 3.5" x 3.5" section of the grid and punch through the
upholstery and foam, stopping at the plywood (DO NOT PUNCH THROUGH THE
PLYWOOD)
3. Retain the 18 mm punch
4. Continue until 18mm punches have been removed from each section of the grid (Figure 3).
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18 mm
coupon
(removed)
Punch grid
o o o o
V/o o o
0000
18 mm
coupon
(installed)
Material
section
Figure 3. Punch Grid on Upholstery Coupon with 18mm Punches
MOP 3194
Revision 0
August 2013
Page 5 of 5
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MOP 3195
Revision 0
September 2013
Page 1 of 5
Miscellaneous Operating Procedure (MOP) 3195:
General Procedure for Immersion Decontamination
Prepared by:
Stelfa McDonald, ARCADIS Work Assignment Leader
Reviewed by:
Dahman TouatffARCADIS Proj-etfMaTiager
Approved by:
Worth Calfee, EPA WopJ< Assignment Manager
Date: 9/6/2013
Date: 9/6/2013
Date: 9/6/2013
Prepared for
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
Prepared by
ARCADIS U.S., Inc.
4915 Prospectus Drive, Suite F
Durham, NC 27713
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MOP 3195
Revision 0
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MOP 3195
TITLE: GENERAL PROCEDURE FOR IMMERSION DECONTAMINATION
SCOPE: This MOP details the procedure for immersion decontamination of carpet,
upholstery, personal protective equipment (PPE; nitrile gloves), and books.
PURPOSE: The purpose of this procedure is to ensure all immersion decontaminations are
performed in a consistent manor.
Equipment and Supplies:
• 75-Gallon immersion tank (10 cu ft Poly Trough, EZ Grout Corporation, p/n HTP10)
• Polypropylene mesh (McMaster Carr, p/n 30145T51)
• Stir rod
• Oakton pH 5 meter, Acorn Series
• Ventilating polypropylene mesh bag, white, 21" wide x 31 Va" high (McMaster Carr, p/n
9883T63)
• Decontaminant solution (see Table 1)
1.0 INTRODUCTION
Prior to beginning any decontamination procedure, all test materials, equipment, and supplies
should be prepared as described in the work assignment's QAPP. While containing
decontaminant solution, the immersion tank should be placed on a spill deck.
Table 1 lists several decontaminants, their active ingredient(s), and the appropriate preparation
and analysis method.
Table 1: Active Ingredients and Titration Methods for Select Decontaminants
Decontaminant
Diluted Bleach
pH-Adjusted Bleach
(pAB)
Spore-Klenz®
Active Ingredient
Hypochlorite
Hypochlorite
Hydrogen peroxide (H2O2)
and peracetic acid (PAA)
Preparation
Method
MOP 3181
MOP3128-A
Per product label
Analysis Method
lodometric Method
(MOP 3 128- A)
lodometric Method
(MOP 3 128- A)
Ceric Sulfate Titration
(MOP 3 196)
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2.0 CARPET AND UPHOLSTERY
1. Prepare approximately 35 gallons of decontaminant solution (see Table 1) in the immersion
tank. Perform the necessary data quality indicator (DQI) checks to verify the solution is
within the quality specifications stated in the QAPP.
2. Lower the sterilized mesh (ethylene oxide or vapor ffcCh sterilization) into the immersion
tank containing the decontaminant solution so that it covers the bottom and walls of the tank.
3. Analyze the solution for active ingredient(s) (see Table 1), pH, and temperature then discard
the sample.
4. Place the entire batch of material sections into the immersion tank (see QAPP for batch
information).
5. Allow to soak for the predetermined immersion time (nominally 15 minutes).
6. After soaking for the required amount of time, remove the material sections from the
immersion tank by lifting the mesh and allow to drain over the immersion tank for 5 minutes.
7. Place the inoculated coupons in the established sampling area for sample collection. Place the
uninoculated material sections (do not contain 18 mm inoculated coupons) directly into the
waste storage bag oriented horizontally.
8. Collect samples from inoculated material sections per the QAPP. Then place horizontally in
the same waste storage bag used in Step 7 in the following order:
• 1 inoculated coupon
• 2 uninoculated coupons
• 1 inoculated coupon
• 2 uninoculated coupons
• 1 inoculated coupon
9. Collect a sample of the residual decontaminant solution.
10. Analyze the residual decontaminant solution for the active ingredient(s) (see Table 1), pH,
and temperature.
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3.0 NITRILE GLOVES (PPE)
1. Prepare approximately 35 gallons of decontaminant solution (see Table 1) in the immersion
tank. Perform the necessary DQI checks to verify the solution is within the quality
specifications stated in the QAPP.
2. Place the entire batch of gloves (see QAPP for batch information) into a sterilized
polypropylene mesh bag (ethylene oxide or vapor IHbCh sterilization).
3. Lower the mesh bag containing the gloves into the immersion tank containing the
decontaminant solution so that it covers the gloves. If necessary, add weights to the bag to
keep it submersed.
4. Allow to soak for the predetermined immersion time (nominally 15 minutes).
5. After soaking for the required amount of time, remove the gloves from the immersion tank
by lifting the mesh bag and allow to drain over the immersion tank for 5 minutes.
6. Place the inoculated (white) gloves in the established sampling area for sample collection.
Place the uninoculated (blue) gloves directly into the waste storage bag.
7. Collect samples from the inoculated gloves per the QAPP. Then place in the same waste
storage bag used in Step 6.
8. Collect a sample of the residual decontaminant solution.
9. Analyze the residual decontaminant solution for the active ingredient(s), pH, and
temperature.
4.0 BOOKS
1. Prepare 35 gallons of decontaminant solution in the immersion tank (see Table 1). Perform
the necessary DQI checks to verify the solution is within the quality specifications stated in
the QAPP.
2. Place the entire batch of books (see the QAPP for batch information) into a sterilized
polypropylene mesh bag (ethylene oxide or vapor IHbCh sterilization).
3. Lower the mesh bag containing the books into the immersion tank containing the
decontaminant solution so that it covers the books.
4. Allow to soak for the predetermined immersion time (nominally 15 minutes).
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5. After soaking for the required amount of time, remove the books from the tank by lifting the
mesh bag and allow to drain over the immersion tank for 5 minutes.
6. Place the inoculated books in the established sampling area for sample collection. Place the
uninoculated books directly into the waste storage bag.
7. Collect samples from the inoculated books per the QAPP. Then place into the same waste
storage bag as the uninoculated books (Step 6).
8. Collect a sample of the residual decontaminant solution.
9. Analyze the residual decontaminant solution for the active ingredient(s) (see Table 1), pH,
and temperature.
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MOP 6535a
Revision 4
January 2013
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Miscellaneous Operating Procedure (MOP) 6535a:
Serial Dilution: Spread Plate Procedure to Quantify Viable
Bacterial Spores
Prepared by:
IS Work Assignment Leader
Reviewed by:
Nicole Griffin GaichaMan,
Date: 2/11/2013
Date: 2/11/2013
......
Dahman TtiARCADlS^rojecfManager
Approved by:
Date: 2/11/2013
Worth Calfee, EPA Work Assignment Manager
Prepared for
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
Prepared by
^/YHK
* .j\CrtL?i^
ARCADIS U.S., Inc.
4915 Prospectus Drive, Suite F
Durham, NC 27713
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MOP 6535a
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MOP 6535a
TITLE: SERIAL DILUTION: SPREAD PLATE PROCEDURE TO QUANTIFY
VIABLE BACTERIAL SPORES
SCOPE: Determine the abundance of bacterial spores in a liquid extract
PURPOSE: Determine quantitatively the number of viable bacterial spores in a liquid
suspension using the spread plate procedure to count colony-forming units (CPU)
Materials:
• Liquid suspension of bacterial spores
• Sterile centrifuge tubes
• Diluent as specified in QAPP or Test Plan (e.g., sterile water, Phosphate Buffered Saline with
Tween 20 (PBST))
• Media plates as specified in QAPP or Test Plan (e.g., Trypticase Soy Agar (TSA) plates)
• Microliter pipettes with sterile tips
• Sterile beads placed inside a test tube (used for spreading samples on the media surface
according to MOP 6555 (Petri Dish Media Inoculation Using Beads) or cell spreaders
• Vortex mixer
1.0 PROCEDURE (This protocol is designed for 10-fold dilutions.)
1. For each bacterial spore suspension to be tested label microcentrifuge tubes as follows: 10"1,
10"2, 10"3, 10"4, 10"5, 10"6... (The number of dilution tubes will vary depending on the
concentration of spores in the suspension). Aseptically, add 900 uL of sterile diluent to each
of the tubes.
2. Label three media plates for each dilution that will be plated. These dilutions will be plated in
triplicate.
3. Mix original spore suspension by vortexing thoroughly for 30 seconds. Immediately after the
cessation of vortexing, transfer 100 uL of the stock suspension to the 10"1 tube. Mix the 10"1
tube by vortexing for 10 seconds, and immediately pipette 100 uL to the 10"2 tube. Repeat
this process until the final dilution is made. It is imperative that used pipette tips be
exchanged for a sterile tip each time a new dilution is started.
4. To plate the dilutions, vortex the dilution to be plated 10 seconds, immediately pipette 100
uL of the dilution onto the surface of a media plate, taking care to dispense all of the liquid
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from the pipette tip. If less than 10 seconds elapses between inoculation of all replicate
plates, then the initial vortex mixing before the first replicate is sufficient for all replicates of
the sample. Use a new pipette tip for each set of replicate dilutions.
5. Carefully and aseptically spread the aliquotted dilution on the surface of the media either by
use of glass beads (MOP 6555) or cell spreader (the method used may be directed in the
QAPP or Test Plan) until the entire sample is distributed on the surface of the agar plate.
Repeat for all plates.
6. Incubate the plates for the optimum time period at the optimum growth temperature for the
target organism (incubation conditions will vary depending on the organism's optimum
growth temperature and generation time. This information can be found in Sergey's Manual
of Determinative Bacteriology or it will be provided with the ATCC certification.
7. Manually enumerate the colony forming units (CPU) on the media plates by manually
counting with the aid of a plate counting lamp and a marker (place a mark on the surface of
the Petri dish over each CPU when counting, so that no CPU is counted twice). A hand held
tally counter or an electronic counting pen may be used to assist the person counting, but
may not be used as the primary source for the count.
Quality control (QC) requirements for bacterial enumeration will be addressed per QAPP or
test plan. However, in general, the following QC practices should always be adhered to:
a. The arrangement of plates and tubes, and the procedure for preparing dilutions and
enumerating CPU should be done the exact same way each time. This helps prevent
systematic errors and often helps determine the cause of problems when a discrepancy is
found.
b. A visual check of the graduated pipette tip should be made during each use to ensure the
pipette is pulling properly.
c. Samples should acclimate to room temperature for 1 hour prior to plating.
d. Samples should be processed (extracted and plated) from the least contaminated to the
most contaminated.
e. When a target range of CPU is known, three dilution factors are plated to bracket the
expected results (0, -1, and -2, if the -1 dilution factor was the target).
f. Enumerated colonies and results should be verified that the results are the target
organism, and that second counts have been performed. Second counts must be
completed on 25% of significant data, and must be within 10% of the first count. If CPUs
are found to have more than a 10% difference between first and second counts, then a
third count is to be completed.
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g. Pictures should be taken of any plates that are contaminated or have results out of the
normal
Record all quantitative data in the "Serial Dilution/Plating Results Sheet". Target range for
statistically significant counts is 30-300 CPU. Data that fall out of the 30-300 CPU range are
addressed in MOP 6584 (Procedure for Replating Bacteria Spore Extract Samples) and MOP
6565 (Filtration and Plating of Bacteria from Liquid Extracts).
2.0 CALCULATIONS
Total abundance of spores (CFU) within extract:
(Avg CFU / volume (mL) plated) x (1 / tube dilution factor) x extract volume
For example:
Tube Dilution Volume plated Replicate CFU
10'3 lOOjiL(O.lmL) 1 150
10'3 lOOjiL(O.lmL) 2 250
10'3 lOOjiL(O.lmL) 3 200
Extract total volume = 20 mL
(200 CFU / 0.1 mL) x (1/10'3) x 20 mL =
(2000) x (1000) x 20 = 4.0 x 107 CFU
Note: The volume plated (mL) and tube dilution can be multiplied to yield a 'decimal factor'
(DF). DF can be used in the following manner to simplify the abundance calculation.
Spore Abundance per mL = (Avg CFU) x (1 / DF) x extract volume
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MOP 6535a
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Serial Dilution/Plating Results Sheet
Page 1 of
TEST INFORMATION
EPA Project No.
Technician Name
Technician Signature
PI
Test Date
Test No.
RESULTS
Date:
Sample ID
Volume Plated:
Plate
Repl.
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
Tube Dilution
10°
KT1
102
KT5
10^
10s
Iff*
NOTES:
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MOP 6535a
Revision 4
January 2013
Page 6 of 8
Page 2 of
Sample ID
Plate
Repl.
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
10°
101
102
KT5
10^
10s
Iff*
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MOP 6535a
Revision 4
January 2013
Page 7 of 8
Page.
of
Sample ID
Plate
Repl.
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
10°
101
102
KT5
10^
10s
Iff*
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NOTES:
MOP 6535a
Revision 4
January 2013
Page 8 of 8
Page.
of
Sample ID
Plate
Repl.
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
10°
101
102
KT5
10^
10s
Iff*
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MOP 6562
Revision 1
February 2013
Page 1 of 6
Miscellaneous Operating Procedure (MOP) 6562:
Preparing Pre-Measured Tubes with Aliquoted Amounts of
Phosphate Buffered Saline with Tween 20 (PBST)
Prepared by:
Date: 2/12/2013
Nicole Griffin GaJch^Iian,
IS Work Assignment Leader
Reviewed by:
Date: 2/12/2013
Dahman louati, ARCADKTProject Manager
Approved by:
Date: 2/12/2013
Worth Calfee, EPA Work Assignment Manager
Prepared for
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
Prepared by
ARCADIS U.S., Inc.
4915 Prospectus Drive, Suite F
Durham, NC 27713
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MOP 6562
TITLE: PREPARING PRE-MEASURED TUBES WITH ALIQUOTED AMOUNTS
OF PHOSPHATE BUFFERED SALINE WITH TWEEN 20 (PBST)
SCOPE: This MOP provides the procedure for preparing PBST.
PURPOSE: This procedure will ensure that that the PBST is prepared correctly and that all
measured tubes are filled aseptically.
1.0 PREPARING STERILE PHOSPHATE BUFFERED SALINE WITH TWEEN 20
(PBST)
Phosphate Buffered Saline with Tween 20 (PBST) is prepared 1 L at a time in a 1 L flask.
1. Add 1 packet of SIGMA Phosphate Buffered Saline with Tween 20 (P-3563) to 1 L of
deionized (DI) water.
2. Shake vigorously to mix until dissolved.
3. Label bottle as "non-sterile PBST" and include date and initials of person who made
PBST.
4. Filter sterilize into two 500 mL reagent bottles using 150 ml bottle top filter (w/ 33mm
neck and .22 jam cellulose acetate filter) for sterilization. Complete this by pouring the
liquid into the non-sterile PBST into the top portion of the filtration unit 150 ml at a time,
while using the vacuum to suck the liquid through the filter. Continue to do this until 500
ml have been sterilized into a 500 ml bottle. Change bottle top filter units between each
and every 500 ml bottle.
5. Change label to reflect that the PBST is now sterile. Include initials and date of
sterilization. The label should now include information on when the PBST was initially
made and when it was sterilized and by whom.
6. Each batch of PBST should be used within 90 days.
2.0 PREPARING 20 ML/5 ML PBST TUBES FOR USE DURING
EXPERIMENTATION
Twenty (20) ml or five (5) ml of the prepared PBST will be added to each sterile 50-ml
conical tube as detailed below. Each flat of conical tubes contains 25 tubes, so one 500 ml
sterile bottle of PBST should fill approximately one flat when 20 ml tubes are needed and
four flats when 5 ml tubes are needed.
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1. Prepare the hood by wiping down with ethanol, followed by bleach, followed by DI water
and a clean Kimwipe or Techwipe. Then stock the hood with the following items if they
are not already there:
The flats of sterile conical tubes you need to fill with PBST.
Sufficient bottles of sterile PBST to fill these tubes.
- Ample 25 ml serological pipettes (at least 3 per flat) for 20 ml transfers and 10
ml serological pipettes for the 5 ml transfers.
Serological pipetter (automatic, hand-held pipette).
Burner and striker.
2. Light the burner and adjust the flame for a width adequate to flame the lips of the PBST
bottles.
3. Take one flat of sterile conical tubes and loosen each cap on the outside edges (about 1A
turn).
4. Open a serological pipette and insert into the serological pipetter, taking care to not touch
the tip to any surface.
5. Hold the pipetter with the first three fingers of your right (or dominant) hand. With your
left hand (or non-dominant hand), pick up a bottle of the PBST and use the bottom of
your right hand to unscrew the lid. Place the lid upside down on the benchtop and quickly
flame the lip of the bottle. Turn the bottle and repeat, taking care to thoroughly flame the
lip without getting the glass so hot that it shatters.
6. Inset the tip of the pipette into the bottle and fill to the 20 ml line. Flame the bottle lip and
place the bottle on the benchtop.
NOTE: If the tip of the pipette touches the outside of the bottle or any other
surface in the hood, consider it contaminated. Discard the pipette
and reload a new one.
7. Quickly pick up one of the tubes that you have loosened the cap on, and use the bottom
of your right hand to remove the cap. Completely discharge the entire pipette into the
tube, taking care to not touch anything with the tip of the pipette. Recap the tube and
place back into the flat (the lid does not have to be tight - you will tighten the lids after
you have completed filling the 10 outside tubes).
NOTE: If the tip touches the outside or rim of the tube (or any other surface
in the hood), consider the tube and pipette contaminated. Discard
both the tube and the pipette.
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8. Pick up the PBST bottle and flame the lip. Repeat Steps 6 and 7 until all 10 of the tubes
on the outside of the flat have been filled. Flame the lip of the PBST bottle and replace
the cap. Slide the used pipette back into the plastic sleeve and put to the side of the hood
for disposal. Then tighten the lid of each tube you just filled. But rather than placing it
back into its original spot in the flat, switch it for the empty tube from the next row.
When this has been completed, go around the outside of the flat again and loosen the lids
of these 10 tubes. Repeat steps 4 through 7 to fill and cap these tubes.
9. This same procedure is used to fill the middle row of tubes from the flat, and if more
than one flat of tubes is being filled, can be done at the same time as the outside rows of a
second flat.
10. When all tubes have been filled, label each flat as follows, and place on the shelf in room
E390B:
"PBST Tubes (20 ml or 5 ml)"
Date prepared
Your initials
11. These tubes should be made at least 14 days before they need to be used so that they can
be verified as sterile. Any tubes that are cloudy or that have any floating matter/turbidity
should be discarded. The tubes are stable for and should be used within 90 days.
3.0 CLEANUP FOR 20 ML/5 ML PBST TUBES
1. Dispose of the used pipettes in the nonregulated waste.
2. Plug in the serological pipetter so that it can recharge.
3. Replace any unused PBST in the liquid containment on the shelf. Make sure that the
bottle is labeled as having been opened (date opened and initials of whomever used it).
4. Turn off the burner.
5. Wipe down the hood benchtop with ethanol, followed by bleach, followed by DI water
and a clean Kimwipe or Tech Wipe.
4.0 PREPARING 900jiL PBST TUBES FOR USE DURING EXPERIMENTATION
1. Prepare the hood by wiping down with ethanol, followed by bleach, followed by DI water
and a clean Kimwipe or Techwipe. Then stock the hood with the following items if they
are not already there:
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A sterile beaker of microcentrifuge tubes.
Sufficient tubes of sterile PBST to fill these tubes (PBST may be aseptically
transferred to 50 ml conical tubes for an easier aseptic transfer to the
microcentrifuge tubes- it is easier than working from a 500 ml reagent bottle.
Make certain that these 50 ml conical tubes are labeled to when the PBST was
made, sterilized, etc.).
1000 jiL micropipette.
1000 jiL sterile pipette tips
Microcentrifuge tube racks.
Labeled beaker or waste container used to hold non-regulated waste, such as
tips, under the hood.
2. Carefully remove the microcentrifuge tubes one at a time from the beaker and close the
top on each one before placing it in the tube rack. Place the tubes in the rack skipping
every other row. Fill up two racks doing this.
3. Add 900 jiL of PBST to the microcentrifuge tubes by aseptically transferring the PBST
from the sterile 50 ml conical tube containing the PBST. Do this by using the 1000 jiL
micropitte and tips. Change tips whenever after two rows of tubes are completed or
whenever a contamination event (such as touching the outside of the 50 ml tube or the
microcentrifuge tube) occurs. Put the dirty tips in the beaker or container used to contain
waste (tips, tubes) in the hood. If any 900 jiL tubes are contaminated during the transfer,
dispose of them in the waste container used to hold tips under the hood. If a new box of
tips has to be opened, make certain the date it was opened and initials of the person who
opened it are clearly labeled on the box.
4. After both racks are full, carefully move all the tubes from one rack to fill in the empty
rows on the other rack. In this manner, one rack should be completely filled with tubes at
this point.
5. Label the rack of tubes as "Sterile 900 jiL PBST Tubes", along with the name of the
person who completed the transfer, along with the date. Also, include the date that the
original stock of PBST was made and the date it was sterilized, along with the initials of
the person who completed those steps.
5.0 CLEANUP FOR 900jiL PBST TUBES
1. Dispose of the waste that was put in the labeled beaker or waste container (micropipette
tips and tubes) in the nonregulated waste. Then, place this beaker in the "To be
decontaminated via sterilization- contaminated glassware" bin or if it is a disposable
container, then it can be put in the non-regulated waste container.
2. Put the unused sterile tips and the micropipetter back in its original location.
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3. Replace any unused 50 ml conicals of PBST in the liquid containment on the shelf. Make
sure that the tube is labeled as having been opened (date opened and initials of whomever
used it). If the tube could possibly be contaminated in any way, dispose of it in non-
regulated waste.
4. Wipe down the hood benchtop with ethanol, followed by bleach, followed by DI water
and a clean Kimwipe or Tech Wipe.
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MOP 6565
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Miscellaneous Operating Procedure (MOP) 6565:
Filtration and Plating of Bacteria from Liquid Extracts
Prepared by:
Date: 2/15/2013
Nicole Griffin G;
Work Assignment Leader
Reviewed by:
Date: 2/15/2013
Dahman Totrati, ARCADIS-Project Manager
Approved by:
Date: 1/8/2014
Worth Calfee, EPA Work Assignment Manager
Prepared for
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
Prepared by
ARCADIS
ARCADIS U.S., Inc.
4915 Prospectus Drive, Suite F
Durham, NC 27713
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MOP 6565
TITLE: FILTRATION AND PLATING OF BACTERIA FROM LIQUID EXTRACTS
SCOPE: This MOP outlines the procedure for filtration and subsequent cultivation of
bacterial spores from a liquid extract.
PURPOSE: This method is deployed when results from spread-plate methods yield less than
30 colony-forming units (CFU) per plate. This method allows a lower limit of
detection for bacterial recovery/survivorship assays. This method can also be
used to analyze liquid samples such as decon rinsates.
Materials:
• Petri dishes with appropriate agar
• 0.2 um to 0.45 um Pore-size disposable analytical filter units
• PI000 pipette and sterile 1000 uL tips
• Serological pipette
• Serological pipette tips
• Sterile forceps
• Sterile deionized water (in -10 mL aliquots)
1.0 PROCEDURE
1. For each liquid sample to be analyzed, gather the required number of disposable analytical
filter units and Petri dishes containing the desired sterilized/quality control checked (QC'd)
media.
NOTE #1: For analysis of 5 to 30 mL extracts, 1 mL and remainder should be filtered;
for 31 to 200 mL samples, 1 mL, lOmL, and remainder should be filtered;
for samples over 200 mL, additional filter samples may be needed and will
be determined on an individual basis.
NOTE #2: For previously plated samples where 10-19 CFU were observed, replate
using a 400 uL inoculums: for plates where 20 - 29 CFU were observed,
replating using a 200 uL inoculum can be executed rather than filter plating
(see MOP 6584).
2. Label plates.
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3. Vortex liquid extract vigorously for 2 minutes, using 10 second bursts (for larger volume
samples, a vigorous mixing by shaking of the sample container can be substituted for vortex
mixing). Directly prior to removing an aliquot, again vortex or agitate for 10 seconds.
4. For 1 mL to 10 mL filters (using a pipettor of appropriate size, sterile tip, and aseptic
techniques), immediately following vortexing, pipette the pre-determined amount of extract
into a 50 mL conical tube containing -10 mL of sterile deionized water. Vortex the aliquotted
solution and the water together and then aseptically pour into one of the filter units.
5. Apply vacuum to the filter unit to pull the liquid through the filter and collect the spores on
the surface of the filter. Immediately following, rinse the filter unit by aseptically adding -10
mL of sterile deionized water along the inner sides of the unit while it is under vacuum.
For aliquots greater than 10 mL that need filtering, pipette the extract directly into the filter
unit using a sterile serological pipette. Immediately following, rinse the filter unit by
aseptically adding -10 mL of sterile deionized water along the inner sides of the unit while it
is under vacuum.
NOTE #3: Be sure to note and record the volume of the sample.
6. Aseptically remove the filter from the filter apparatus using sterile forceps, and roll the filter
onto the agar surface within the Petri dish (spore side up).
7. Incubate all plates at the optimal growth temperature and time period for the specific
organism.
8. Following incubation, enumerate and record the number of CFU on each plate. Be certain to
record the volumes of the amount filtered on the data sheets.
2.0 DATA CALCULATIONS
Utilize the following equation to determine the total abundance of recovered spores:
V
N = CFU:
Extract
~tr
' filtered
Where, N is the total number of spores recovered in the extract, CFU is the abundance of
colonies on the agar plate, Vsxtract is the total volume of the extract (before any aliquots were
removed), and Vpnteredis the volume of the extract filtered.
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MOP 6580
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Miscellaneous Operating Procedure (MOP) 6580:
Recovery of Bacillus Spores from 3M Sponge-Stick™ Samples
Prepared by:
Work Assignment Leader
Reviewed by:
Date: 2/12/2013
Date: 2/12/2013
Dahman IXmti, ARCADEvProject Manager
Approved by:
Date: 2/12/2013
Worth Calfee, EPA Work Assignment Manager
N*. „.,*""'•""'
Prepared for
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
Prepared by
/" A "
ARCADIS U.S., Inc.
4915 Prospectus Drive, Suite F
Durham, NC 27713
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MOP 6580
TITLE: RECOVERY OF BACILLUS SPORES FROM 3M SPONGE-STICK™
SAMPLES
SCOPE: This MOP provides the procedure for recovering spores from 3M Sponge-
Stick™ samples.
PURPOSE: To extract and quantify bacterial spores from 3M Sponge-Stick™ samples
using a highly repeatable procedure.
MATERIALS
• pH-amended bleach
• 70-90 % Solution of denatured ethanol
• Deionized (DI) water
• Kimwipes
• 3M Sponge-Stick™ samples (P/N SSL10NB), hereafter referred to as 'sponge'
• Seward Stomacher® bags (P/N BA6041/CLR)
• Phosphate buffered saline with 0.05% TWEEN®20 (PBST) (SIGMA-ALDRICH, Co,
P/NP3563-10PAK)
• MicroFunnel Disposable Filter Funnels, Pall Life Sciences (VWR P/N 55095-060) or
Nalgene Sterile Analytical Filter Unit (Fisher P/N 130-4020)
• Disposable polystyrene serological pipettes (5mL and lOmL)
• Tryptic Soy Agar (TSA) plates
• Vortex mixer
• Disposable sterile lOjil loops
• Disposable sterile forceps
• Disposable gloves
• Cell spreaders or glass beads for spreading
• Racks for 15 mL and 50 mL centrifuge tubes
• Sterile, plastic, screw-cap 50 mL centrifuge tubes (e.g. Fisher Cat# 14-959-49A)
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• Sterile, plastic, screw-cap 15 mL centrifuge tubes (e.g. Fisher Cat# 14-959-49D)
• Pipette tips with aerosol filter for 1 mL and 200 uL
1.0 PREPARATION
Personnel must be familiar with all procedures prior to start.
1.1 Equipment Preparation
a) Begin by donning personal protective equipment (PPE) such as gloves, lab coat,
and protective eyewear.
b) Clean the workspace (Biological Safety Cabinet; BSC) by wiping surfaces with pH-
amended bleach, next with DI water, and lastly with a 70-90% solution of denatured
ethanol. Allow any excess liquid to dry prior to beginning procedure. Make sure the
workspace is clean and free of debris.
c) Assemble equipment in the BSC as needed: vortex mixer, filtration manifold,
automatic pipettors, tips, racks, etc.
d) Assemble extra supplies, such as stomacher and reagents, near BSC.
1.2 Supply Preparation
a) Unpack shipping containers directly into a BSC.
b) If sponges are not in Stomacher® bags, label one Stomacher® bag for each sponge
and place in a bag rack.
c) Label two sterile 50 mL centrifuge tubes for each sponge sample and place in tube
rack.
d) For each sample, label TSA plates on the agar side of the plate with the sample
number and the appropriate dilution factors, as per MOP 6535a (SerialDilution:
Spread Plate Procedure to Quantify Viable Bacterial Spores).
e) Label two additional plates for filter-plate analysis.
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2.0 PERFORM SPORE EXTRACTION, ELUTION, AND CULTURE PROCEDURE
2.1 Dislodge Spores from the Sample Sponges
a) Begin by donning a new pair of gloves. All subsequent procedures involving
manipulation of sponges or spore suspensions must be carried out in a BSC.
(Stomaching may occur outside the BSC when samples are double-contained inside
the indicated bags.)
b) If the sponges are not in Stomacher® bags, aseptically transfer each sponge to a
Stomacher® bag (labeled during step 1.2b) using sterile disposable forceps. Change
forceps between samples.
c) Aseptically add 90 mL of PBST to each bag that contains a sponge.
d) Stomach sponges in the PBST by completing the following:
• Make certain the Stomacher® is set to MANUAL. Program the Stomacher®
speed to 260 RPM and the timer to 1 minute.
• Open the Stomacher® door by raising the lid fully upward and back. The
DOOR OPEN icon will be displayed.
• Place the stomacher bag containing the sponge sample into a second stomacher
bag to contain any leakage in the event the primary containment is
compromised. Place the combined bags such that 50 to 60 mm of the top
portions protrude above the bag clamp, while making certain that the sponge
sample rests evenly between the homogenizer paddles.
• Close the door to the Stomacher®. The DOOR OPEN icon will no longer be
illuminated.
• Stomach each sponge for 1 min by pressing the START button.
• When the cycle ends, the Stomacher® will stop. If there is an emergent reason
to stop the stomacher during the 1 minute stomaching period, press the red
button or the power button to do so prior to opening the Stomacher®. Stopping
the Stomacher® by opening the door can damage the equipment.
• Open the door of the Stomacher® and remove the bags containing the sponge.
Grab the sponge from the outside of the bag with your hands. Move the sponge
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to the top of the bag while using your hands to squeeze excess liquid from the
sponge.
• Remove and discard the sponge 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 before
beginning the concentration step.
2.2 Remove Sponge 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.
2.3 Concentrate Sponge Elution Suspension (Optional)
a) Centrifuge 50 mL centrifuge tubes
• Prior to daily use and before placing tubes into centrifuge, follow MOP 6558
(Centrifuge Cleaning Procedure] for cleaning this equipment.
• Add centrifuge tubes to rotor, evenly distributing weight.
• Centrifuge tubes at 3500 x g for 15 min. Do not use the brake option on the
centrifuge to slow the rotor, as re-suspension of pellet may occur.
b) Carefully remove about 42mL of 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 or side.
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c) Vortex and sonicate tubes as follows:
• Set vortex mixer to level 10 and touch activation.
• Turn on sonicator water bath.
• Vortex tubes for 30 sec.
• Transfer tubes to sonicator bath and sonicate for 30 sec.
• Repeat vortex and sonication cycles two additional times.
d) Remove suspension from one tube with a sterile 5 mL pipette and place it in the
other tube of the same sample. The combined result is the final sponge elution
suspension.
e) Measure final volume of the final sponge elution suspension with 5 mL pipette and
record on tube and data sheet.
f) Repeat steps (e) through (i) for all samples.
2.4 Serially Dilute and Plate the Final Spore Elution Suspension
a) Use MOP 6535a to serially dilute and plate samples.
NOTE: If the samples are turbid, wide-orifice pipette tips may be used to prevent
clogging of pipette tips.
b) 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. Manually
enumerate CPU of target organism and record data.
• If the CPU is <300/plate, record actual number.
• If the CPU is >300/plate, record as "too numerous to count" (TNTC)
2.5 Capture Spores on Filter Membranes and Culture on TSA
Choose one of the following to methods to filter the final spore elution suspension:
a) Complete filter plating using MOP 6565 (Filtration and Plating of Bacteria from
Liquid Extracts).
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b) Complete filter plating using the following method:
1) Place two 0.45 um (pore-size) Microfunnels on a Pall vacuum manifold (Pall Cat#
15403).
2) 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.
3) Make certain that the manifold vacuum valve is closed. Turn on the vacuum.
4) With the vacuum valve closed, place 10 mL of PBST into each filter cup.
5) Add 1.0 mL of the final sponge elution suspension to each filter cup.
6) Open valves and allow the suspension to flow through the filter, close the valve.
7) Rinse the walls of each Microfunnel cup with 10 mL of PBST. Reopen the valve to
allow the suspension to flow through the filter.
8) Close the valve, turn off the vacuum pump. Slowly reopen the valve to equalize the
pressures.
9) Squeeze the walls of the Microfunnel cup gently and separate the walls from the
base holding the filter. Remove each filter membrane with sterile disposable 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.
10) Record exact volume of the sponge elution suspension filtered on each plate. It
should be 1 mL. (Greater sample volumes may be used to lower detection limits)
11) Repeat steps (1) through (8) for all each sample.
12) 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. Manually
enumerate CPU of target organism and record data.
• If the CPU is <300/plate, record actual number.
• If the CPU is >300/plate, record as "too numerous to count" (TNTC)
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MOP 6584
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Miscellaneous Operating Procedure (MOP) 6584:
Procedure for Replating Bacteria Spore Extract Samples
Prepared by:
Date: 11/15/2012
Nicole Griffin Ga
Work Assignment Leader
Re viewed by:
Date: 11/15/2012
Dahman Towati, ARCADIS-Project Manager
Approved by:
Date: 11/15/2012
Worth Caflfee, EPA Work Assignment Manager
Prepared for
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
Prepared by
ARCADIS
ARCADIS U.S., Inc.
4915 Prospectus Drive, Suite F
Durham, NC 27713
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MOP 6584
TITLE:
SCOPE:
PRUPOSE:
Materials:
PROCEDURE FOR REFLATING BACTERIA SPORE EXTRACT
SAMPLES
Determine the abundance of bacterial spores in a liquid extract that has
previously been plated.
This method is deployed when results from spread-plate methods yield a
relative standard deviation (RSD) value greater than 50 or colonies outside
of the acceptable range (30-300 CPU).
Liquid suspension of bacterial spores
5.0 mL sterile centrifuge tubes (e.g., USA Scientific 3882-7600) containing 2700 jiL
diluent (sterile deionized water, buffered peptone water or phosphate buffered saline)
Trypticase Soy Agar plates
Pipettes with sterile tips
Sterile beads placed inside a test tube (will be used for spreading samples on the agar
surface), or sterile disposable cell spreaders
Vortex mixer
1.0 DETERMING THE TARGET DILUTION
1. Using the original data (most likely obtained initially using MOP 6535a), locate the
dilution set where the mean number of colony forming units (CPU) was nearest the
30 to 300 range. This is the target dilution.
2. The target dilution will be noted on the tracking sheet, along with the sample ID and
test number. Each work assignment will have its own tracking sheet.
3. For each sample, 100 jiL, 200 jiL and 400 jiL aliquots of the dilutions indicated in
Table 1 shall be plated in triplicate.
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Table 1.
Average CPU in
Target Dilution
1-50
51-150
151-300
Volumes to Replate per Dilution
lOx less dilute than
Target (lO^1)
lOOjil
Target Diluti on (10X)
200|il, 400|il
100|il, 200|il, 400|il
lOOul
lOx more dilute
than Target (10X+1)
200nl, 400nl
Upon collection of results, at least one set of triplicate plates must contain all three
data points within the acceptable (30-300 CPU) range to consider the replate
successful. If none of the three sets of triplicate plates contain all data within the
rage, repeat replate procedure.
2.0 PROCEDURE
1. For each bacterial spore suspension to be tested label 2700 jiL diluent
2.
3.
4.
-2
•v4
V6
microcentrifuge tubes as follows: 10 , 10 , 10 , 10 , 10 , 10 ... (The number of
dilution tubes will vary depending on the concentration of spores in the
suspension.
For each liquid sample to be replated, gather the required number of 2700 jiL
diluent tubes and Petri dishes containing the desired sterilized/QC'd media.
Label three Trypticase Soy agar plates for each dilution that will be plated with
the sample ID and volume to be plated. These dilutions will be plated in
triplicate.
Mix original spore suspension by vortexing thoroughly for 30 seconds.
Immediately after the cessation of vortexing, transfer 300 jiL of the stock
suspension to the 10"1 tube. Mix the 10"1 tube by vortexing for 10 seconds, and
immediately pipette 300 jiL to the 10"2 tube. Repeat this process until the final
dilution is made. It is imperative that used pipette tips be exchanged for a sterile
tip each time a new dilution is started.
5. To plate the dilutions, vortex the dilution to be plated for 10 seconds, then
immediately pipette the desired volume (100, 200, or 400 jiL) of the dilution onto
the surface of a TSA plate, taking care to dispense all of the liquid from the
pipette tip. If less than 10 seconds elapses between inoculation of all replicate
plates, then the initial vortex mixing before the first replicate is sufficient for all
replicates of the sample. Use a new pipette tip for each set of replicate dilutions.
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6. Carefully pour the sterile glass beads onto the surface of the ISA plate with the
sample and shake until the entire sample is distributed on the surface of the agar
plate. Aseptically remove the glass beads. Repeat for all plates.
7. Incubate the plates overnight at 32°C - 37°C (incubation conditions will vary
depending on the organism's optimum growth temperature and generation time.)
8. Enumerate the CPU on the agar plates by manually counting with the aid of a
plate counting lamp, and a marker (place a mark on the surface of the Petri dish
over each CPU when counting, so that no CPU is counted twice).
9. Since each dilution was tested in triplicate, determine the average of the triplicate
plate abundances. Only those data between 30-300 colonies are suitable for use
in data calculation formulas below. High variability below 30 CPU, and high
probability of co-located CPU above 300 are the reasons that only data within this
range are acceptable for further reduction.
3.0 DATA CALCULATIONS
Total abundance of spores (CFU) within extract:
(Avg CFU / volume (mL) plated) X (1 / tube dilution factor) X extract volume
For example:
Tube Dilution Volume plated Replicate CFU
10'3 lOOjiL(O.lmL) 1 150
10'3 lOOjiL(O.lmL) 2 250
10'3 lOOjiL(O.lmL) 3 200
Extract total volume = 20 mL
(200 CFU / 0.1 mL) X (1/10'3) X 20 mL =
(2000) X(1000) X 20 = 4.0X107CFU
Note: The volume plated (mL) and tube dilution can be multiplied to yield a 'decimal
factor' (DF). DF can be used in the following manner to simplify the abundance
calculation.
Spore Abundance per mL = (Avg CFU) X (1 / DF) X extract volume
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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
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