EPA 600/R-11/092 | September 2011 | www.epa.gov/ord
United States
Environmental Protection
Agency
Effectiveness of Physical
and Chemical Cleaning
and Disinfection Methods
for Removing, Reducing
or Inactivating Agricultural
Biological Threat Agents
Office of Research and Development
National Homeland Security Research Center
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EPA/600/R-11/092
SEPA
Effectiveness of Physical and Chemical Cleaning and Disinfection
Methods for Removing, Reducing or Inactivating Agricultural Biological
Threat Agents
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Disclaimer
The United States Environmental Protection Agency, through its Office of Research and Development's
National Homeland Security Research Center, funded and directed this investigation through EP-C-09-027
WA 1-35 with ARCADIS-US, 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. The
Environmental Protection Agency does not endorse the purchase or sale of any commercial products or
services. This report includes photographs of commercially available products. The photographs are
included for purposes of illustration only and are not intended to imply that the Environmental Protection
Agency approves or endorses the product or its manufacturer.
Questions concerning this document or its application should be addressed to the principal investigator on
this effort.
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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Foreword
Following the events of September 11, 2001, addressing the critical needs related to homeland security
became a clear requirement with respect to the U.S. Environmental Protection Agency's (EPA's) mission
to protect human health and the environment. Presidential Directives further emphasized EPA as the
primary federal agency responsible for the country's water supplies and for decontamination following a
chemical, biological, and/or radiological (CBR) attack. To support the EPA mission with respect to
response and recovery from incidents of national significance, the National Homeland Security Research
Center (NHSRC) was established to conduct research and deliver products that improve the capability of
the Agency to carry out its homeland security responsibilities.
One specific goal of NHSRC's research is to provide information on decontamination methods and
technologies that can be used in the response and recovery efforts resulting from a biological incident.
The complexity and heterogeneity of surface decontamination necessitates the understanding of the
effectiveness of a range of decontamination options. In addition to effective volumetric decontamination
approaches (e.g., facility fumigation), more rapidly deployable or readily available alternative surface
decontamination approaches have also been recognized as a tool to enhance the capabilities to respond
to and recover from such incidents.
Through working with EPA's Federal Partners (for example, Department of Homeland Security and
Department of Agriculture), NHSRC is attempting to understand and develop useful surface
decontamination procedures for agriculturally-relevant situations such as a foreign animal disease
incident. This report documents the results of a laboratory study to better understand the effectiveness
of surface cleaning and decontamination methods and to develop a readily-deployable treatment
procedure for surfaces contaminated with highly pathogenic biological agents. Studies such as this
advance Durability to respond and recover from incidents of national significance where biological agent
has contaminated commodities and facilities.
These results, coupled with additional information in separate NHSRC publications (available at
www.epa.gov/nhsrc) can be used to determine whether a particular decontamination technology can be
effective in a given scenario. NHSRC has made this publication available to assist the response
community to prepare for and recover from incidents involving biological contamination. This research is
intended to move EPA and its Federal Partners one step closer to achieving the nation's homeland
security goals and the agency's overall mission of protecting human health and the environment while
providing sustainable solutions to our environmental problems.
Jonathan Herrmann, Director
National Homeland Security Research Center
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Acknowledgments
This effort was initiated following the identification of knowledge gaps by the National Science and
Technology Council (NSTC) Subcommittee on Foreign Animal Disease Threats (FADT), Decon and
Disposal Working Group, which is co-chaired by the US EPA and the US Department of Agriculture (USDA).
Emergency response and remediation following a foreign animal disease (FAD) incident will involve
numerous federal agencies (particularly those listed), as well as state, local, and private entities. This project
addresses closing gaps in Durability to decontaminate and remediate facilities following an agro-terrorism
incident. Funding support by the US Department of Homeland Security (DHS) to complete this effort is
greatly appreciated.
This effort was directed by the principal investigator from ORD's National Homeland Research Center
(NHSRC), utilizing the support of a project team consisting of staff from across the US EPA, DHS, and
USDA. The contributions of the following individuals have been a valued asset throughout this effort:
• R. Leroy Mickelsen (US EPA/OSWER/OEM/National Decontamination Team)
• Carlton (Jeff) Kempter (US EPA/Office of Chemical Safety and Pollution Prevention)
• Joseph P. Wood (US EPA/ORD/NHSRC, Decontamination and Consequence Management Division)
• Michelle Colby (DHS)
• Lori Miller (USDA)
This effort was completed under U.S. EPA contract #EP-C-09-027 with ARCADIS-US, Inc. The support and
efforts provided by the ARCADIS-US, Inc. are gratefully acknowledged. The support provided by Tanya
Medley (U.S. EPA/ORD/NHSRC) in acquiring the vast quantities of supplies required for the completion of
this project is also appreciated.
Additionally, the authors would like to thank the peer reviewers for their significant contributions. Specifically,
the efforts of Doris Betancourt, Terry Stilman, and Alan Lindquist are recognized.
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Table of Contents
List of Tables vi
List of Figures vii
List of Acronyms viii
Executive Summary xi
1. Introduction 1
1.1 Objectives 1
1.2 Experimental Approach 2
1.2.1 Task I Approach 4
1.2.2 Task II Approach 5
1.3 Definition of Efficacy 6
1.3.1 Surface Efficacy 6
1.3.2 Ultimate Fate of Spores 9
2. Materials and Methods 10
2.1 Coupon Materials and Fabrication 10
2.1.1 Material Surfaces 10
2.1.2 Task I and Task II Coupons 11
2.2 Material Inoculation Procedure 12
2.2.1 Bacillus Spore Preparation 12
2.2.2 Coupon Inoculation Procedure 13
2.3 Experimental Approach 15
2.3.1 Task I-Small Chamber 15
2.3.2 Task II-Large Chamber (COMMANDER) 16
2.4 Decontamination Procedure 18
2.5 Test Matrix 19
2.6 Sampling and Analytical Procedures 21
2.6.1 Factors Affecting Sampling/Monitoring Procedures 22
2.6.2 Preparation for Sampling/Monitoring 22
2.6.3 Wipe Sampling 23
2.6.4 Rinsate Collection and Sampling 23
2.6.5 Bioaerosol Sample Collection 23
IV
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2.6.6 Sample Analyses 24
2.6.7 Coupon, Material, and Equipment Cleaning and Sterilization 24
3. Results and Discussion 26
3.1 Surface Sampling Results - Positive Controls 26
3.1.1 Task I 26
3.1.2 Task II 28
3.2 Task I: Evaluating Decontamination Procedures 29
3.2.1 Surface Sampling Results 29
3.2.2 Evaluation of the pH-Adjusted Bleach Application Procedure 31
3.2.3 Evaluation of the Spor-Klenz® Application Procedure 33
3.2.4 Ultimate Fate of Viable Spores 34
3.2.4.1 Aerosol Samples (Via-Cell®) - Task I 34
3.2.4.2 Rinsate-Taskl 36
3.3 Task II Results 36
3.3.1 Surface Sampling Results - Test Coupons 36
3.3.2 Ultimate Fate of Viable Spores 37
3.3.2.1 Aerosol Samples (Via-Cell®)-Task 11 37
3.3.2.2 Rinsate-Task II 38
3.4 Assessment of Operational Parameters 39
3.4.1 Time 39
3.4.2 Physical Impacts on Materials 39
3.4.3 Impact on Decontamination Workers 40
3.5 Summary of Results 40
4. Quality Assurance and Quality Control 43
4.1 Calibration of Sampling/Monitoring Equipment 43
4.2 Data Quality Indicator (DQI) Goals 44
4.2.1 Free Available Chlorine (FAC) Measurements 45
4.2.2 pH Measurements 45
4.2.3 Temperature Measurements 45
4.2.4 Pressure Measurements 46
4.2.5 Flow Measurements 46
4.2.6 CFU Counts 46
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4.3 Data Quality Audit 46
4.4 QA/QC Reporting 46
4.5 Amendments and Deviations from the Original QAPP 46
4.5.1 Formal Amendments 46
4.5.2 Deviations from the QAPP 48
5. References 49
Appendix A: Coupon Sterilization 51
Appendix B: Test Chamber and Equipment Cleaning Procedures 52
Appendix C: Miscellaneous Operating Procedures (MOPs) 54
Appendix D: Spore Deposition and Handling Procedures 78
Appendix E: Contamination Prevention and Quality Control Measures 80
Appendix F: Sampling Procedures 91
Appendix G: Sampling Analyses 100
Appendix H: Test Reports 102
List of Tables
Table 2-1. Test Matrix 20
Table 2-2. Cleaning Methods and Frequency for Common Test
Materials/Equipment 25
Table 3-1. Positive Control 27
Table 3-2. Task II Positive Controls 29
Table 3-3. Conditions for each Task 1 Test 30
Table 3-4. Bioaerosol Levels 35
Table 3-5. Rinsate Sample CFUs 36
Table 3-6. CFU Recovered from Task II Rinsate 39
Table 4-1. Laboratory Instrument Calibration Frequency 43
Table 4-2. Microbiology Laboratory Instrument Calibration Frequency 44
Table 4-3. Acceptance criteria and test values for critical measurements 45
Table 4-5. Coupon Sample Coding 47
VI
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List of Figures
Figure 1-1. Task I Test Approach Flow Chart 3
Figure 2-1. Pressure-treated Wood Coupon Front 10
Figure 2-2. Curing Concrete (left) and Final Concrete Coupons (right) 11
Figure 2-3. Sampling Template on Task II Pressure-treated Plywood Coupon 12
Figure 2-4. Task II Coupon Sampling Areas 14
Figure 2-5. Nine Dosing Chambers 14
Figure 2-6. Task I Decontamination Chamber 16
Figure 2-7. Spraying Through Center-aligned Port in the Small Chamber Door 16
Figure 2-8. Airlock in Foreground and Large Chamber (COMMANDER) 17
Figure 3-1. Positive Control and Material Coupon Loading for Task I 28
Figure 3-2. Material Surface Log Reduction for each Test Conducted 31
Figure 3-3. Efficacy of pH-Adjusted Bleach Tests. 32
Figure 3-4. Efficacy of Spor-Klenz® Tests 34
Figure 3-5. Efficacy of Task II Decontamination Procedures 37
Figure 3-6. Bioaerosol Levels during Task II 38
Figure 3-7. Corrosion on Pressure Washer Nozzle from Spor-Klenz® Contact 40
Figure E-4: Center of Spray during Task 1 Decontamination Procedures 86
Figure E-7: Dl Water Supply System 89
Figure F-1. Nalgene Analytical Filter Unit Connected to a Filter Unit. 97
Figure F-3 Via-Cell® BioAerosol Cassette 99
VII
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List of Acronyms
acm actual cubic meter
ADA Aerosol Deposition Apparatus
APPCD Air Pollution Prevention and Control Division
Ba Bacillus atropheus (formerly identified as Bacillus subtilis var.
niger, and B. globigii)
CBR Chemical, Biological, Radiological
CPU Colony Forming Unit(s)
COMMANDER Consequence Management and Decontamination Evaluation Room
DCMD Decontamination and Consequence Management Division
DGM dry gas meter
DHS US Department of Homeland Security
Dl Deionized
DPG U.S. Army Dugway Proving Grounds
DQI Data Quality Indicator
DQO Data Quality Objective
ECBC U.S. Army Edgewood Chemical Biological Center
EPA U.S. Environmental Protection Agency
FAC Free Available Chlorine
FAD Foreign Animal Disease
FADT Subcommittee on Foreign Animal Disease Threats
FIFRA Federal Insecticide, Fungicide, and Rodenticide Act
ft foot, feet
g gram(s)
g/L gram(s) per liter
H2O2 Hydrogen Peroxide
HVAC heating, ventilation and air conditioning
in inch(es)
INL Idaho National Laboratory
ISO International Organization For Standardization
VIM
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L liter
Lpm liters per minute
LR log(s) reduction
MDI Metered Dose Inhaler
min minute(s)
ml_ milliliter(s)
MOP Miscellaneous Operating Procedure
NHSRC National Homeland Security Research Center
NIST National Institute of Standards and Technology
NSTC National Science and Technology Council
ORD Office of Research And Development
OSWER Office of Solid Waste And Emergency Response
pH-AB pH-Adjusted Bleach
PBST Phosphate Buffered Saline with 0.05% TWEEN®-20
PPE Personal Protective Equipment
ppm parts per million
ppmv parts per million by volume
psi pounds per square inch
QA Quality Assurance
QAPP Quality Assurance Project Plan
QC Quality Control
RSD Relative Standard Deviation
RTU Ready-to-Use
SS stainless steel
sq square
STS sodium thiosulfate
TNTC too numerous to count
TSM Three-Step Method
USDA US Department of Agriculture
USG US Government
IX
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WAM Work Assignment Manager
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Executive Summary
This project supports the U.S. Environmental Protection Agency (EPA), through its National Homeland
Security Research Center (NHSRC) Decontamination and Consequence Management Division (DCMD), by
providing relevant information pertinent to the decontamination of contaminated animal facilities resulting
from an agro-terrorism incident or foreign animal disease (FAD) event. The primary focus of this project is to
evaluate and improve the effectiveness and practical application of in situ, cost-effective alternative
decontamination methods to remediate and restore areas contaminated by biological threat agents. These
decontamination techniques rely on equipment (garden hoses, portable chemical sprayers, power washers)
and application of liquid decontaminant solutions that are cost-effective and readily available.
The aim of this research was to assess the effectiveness of two decontamination application methods and
two decontaminants: the use of either a portable, battery-powered backpack sprayer or a motorized power
chemical sprayer to dispense antimicrobial solutions of either pH-adjusted bleach (pH-AB) or Ready-to-Use
(RTU) Spor-Klenz® onto contaminated surfaces. The performance of these two decontamination procedures
and two decontaminants was evaluated with respect to the physical removal, inactivation, and overall fate of
spores on "medium-sized" 35.6 cm by 35.6 cm (14 in by 14 in) and "large-sized" 101.6 cm by 101.6 cm (40
in by 40 in) pressure-treated wood and concrete pieces (coupons). These materials were chosen because
of their common occurrence in animal production facilities. Coupon materials were inoculated (loaded) with
1 x 106 - 5 x 106 8. atrophaeus spores using metered dose inhalers (MDIs) provided by the U.S. Army
Edgewood Chemical Biological Center (ECBC) according to a proprietary protocol. Bacillus spores were
used as surrogates for all FAD biological agents since they are highly resistant to chemical inactivation and
represent a conservative estimate of decontamination effectiveness. Each "medium-sized" coupon was
inoculated independently by being placed into a separate aerosol deposition apparatus (ADA) designed to fit
one 14 in by 14 in coupon of any thickness. For the "large-sized" coupons, inoculations with spores were
performed using nine ADAs aligned side-by-side (three rows of three) to cover the entirety of the surface. All
coupons were free of dirt or grime.
The effectiveness of each decontamination method was first evaluated using the "medium-sized" coupons in
a custom built test chamber, testing three coupons at a time in a vertical orientation, under varying
conditions (Task I). Ten different test runs were set up with variations in application methods and
antimicrobial solutions, as well as variations in spray time, rinse methods and time, and total contact time.
Results from the "medium-sized" coupon tests were then used to develop two decontamination procedures
applying antimicrobial solutions to "large-sized" coupons inside an enclosed, single-access-point chamber
designated as the "Consequence Management and Decontamination Evaluation Room (COMMANDER)"
(Task II). These tests were designed to evaluate the decontamination approach on a pilot scale. The pilot
scale offers not only more realistic assessment of the effectiveness of the decontamination procedures than
small scale testing (e.g., in a small chamber), but also more insights on the operational parameters such as
time, physical impacts on materials and equipment, impact on the remediation crew (e.g., physical exertion),
and spore cross-contaminations arising from the by-products of the decontamination processes (rinsate,
exhaust, and decontamination equipment).
The major findings from this study are as follows:
• pH-Adjusted bleach was highly effective (approximately 6 log reduction (LR)) on wood and
concrete when used with a thirty-minute contact time and two applications.
XI
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• Spor-Klenz was more effective on wood than on concrete.
®
• For concrete coupons, pH-adjusted bleach was more efficacious than Spor-Klenz .
• Reduction of the number of pH-adjusted bleach applications and contact time resulted in lower
decontamination efficacy for surfaces and greater amounts of spores detected in rinsate and
aerosol samples.
• Decontaination efficacy was similar between the two evaluated application devices (backpack
sprayer and pressurized sprayer) despite significant differences in volume of decontaminant
delivered to the coupon surface.
• Viable biological agent was detected in aerosol and rinsate (runoff) samples during all tests and
can therefore be a significant source of cross-contamination during a remediation
• Elimination of a rinse step from the decontamination procedure did not reduce surface
decontamination efficacy, and may be a viable option on materials not susceptible to corrosion.
• Worker fatigue may be of concern in an actual remediation as heat and exhaustion were
experienced by laboratory workers when conducting scale-up tests that required level C personal
protective equipment.
More specifically, most tests performed during Task I achieved the target efficacy from surfaces of greater
than 6 Log Reduction (LR), a widely accepted standard for demonstrating sporicidal efficacy (e.g., 1 LR
would be a reduction of 10, 2 LR would be a reduction of 100, 6 LR would be a reduction of 1 million, etc.).
The decontamination by means of pH-adjusted bleach was accomplished by a combination of removal and
inactivation of spores. Viable spores were found in both the rinsate and bioaerosol samples. Of the
procedures tested, those incorporating pH-adjusted bleach were more effective for decontamination on
concrete and wood than Spor-Klenz®. The lower log reduction (4 LR) seen in one test with wood may have
been the result of material demand (i.e., reduction in activity of the decontaminant though reaction with the
test material) in conjunction with a single application of the pH-adjusted bleach; one spray application does
not appear to provide enough pH-adjusted bleach to overcome the demand of wood. The surface LRs for
tests utilizing Spor-Klenz® were comparable to those with pH-adjusted bleach on treated wood, but
significantly lower on tests involving concrete (< 3 LR).
Based on the Task I results, the most effective decontamination procedures were developed for further
testing in Task II: the use of pH-adjusted bleach by backpack sprayer, sprayed on either concrete or wood,
and rinsed or not rinsed. These procedures all used two, 30-second spray times every 15 minutes, for a
total of 30 minutes of spray exposure per application. Procedure 1 included a rinse step, and Procedure 2
did not include this step. The results indicate that the two decontamination approaches were equivalent in
decontaminating the two types of materials. The results also suggest that rinsing is not needed for these
decontamination procedures to be effective on concrete and wood. However, if applications were to be
made to surface materials sensitive to bleach (e.g., stainless steel), rinsing might be desirable from that
standpoint as bleach and other aggressive oxidants are known to cause corrosion of numerous surfaces.
LRs were approximately 6 for concrete and just under 6 for wood.
The overall fate of the biological spores was assessed, not only for the viable spores recovered from the
surface of the materials, but also for fugitive viable agent escaping in the rinsate and aerosol fractions.
Aerosol samples collected using bioaerosol filter cassettes during testing with the "medium-sized" coupons
XII
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show that re-aerosolization of viable spores can be expected during the decontamination process. Although
one test with the "large-sized" coupons suggests that spores were dislodged during the first
decontamination step and were constantly removed from the chamber (due to air exchange) following that
release, further evaluation of the data indicates that there was likely cross-contamination and re-
aerosolization of ambient spores in the chamber. However, the data do indicate that spores can be
expected to be re-aerosolized in a field decontamination event and could be expected to travel through the
Heating, Ventilation and Air Conditioning (HVAC) system (if operating) during decontamination and
potentially spread contamination throughout a facility.
For most of the "medium-sized" coupon testing, the number of colony forming units (CPUs) recovered in the
rinsate was below the detection limit. However, in the tests where only one short application of pH-amended
bleach (pH-AB) was used, a large number of viable spores were physically removed from the surface during
the decontamination and rinse steps. Such rinsate would potentially cause contamination to spread if not
properly collected and treated.
The collection troughs for the "large-sized" coupon rinsates were immediately contaminated once brought
inside the test chamber during test set-up. However, the rinsate contamination was systematically higher for
the concrete coupons over the wood coupons and suggests that the contamination is coming from the
coupons themselves. The loose material from the concrete coupons might have dropped into the trough
while it was being placed under the coupon. Despite the occurrence of viable spores in the troughs prior to
testing, the data suggest that active spores were transferred to the rinsate, as viable spore abundance in
these samples increased by approximately 1 x 105 following the decontamination procedure that utilized a
rinse step.
XIII
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1. Introduction
Contamination of farm animal facility surfaces
and equipment during a Foreign Animal Disease
(FAD) outbreak could pose potential risks to
human and animal health following an incident.
Viable options for returning contaminated items to
pre-incident risk levels are of immediate need. In
response to data gaps/needs identified by the
National Science and Technology Council
(NSTC) Subcommittee on Foreign Animal
Disease Threats (FADT), Decon and Disposal
Working Group, which is co-chaired by the US
Department of Agriculture (USDA) and the US
Environmental Protection Agency (EPA), the
EPA's National Homeland Security Research
Center (NHSRC) conducted a study to measure
the effectiveness of selected physical and
chemical cleaning and disinfection methods for
removing, reducing or inactivating FAD threat
agents on different surface materials.
This project supports the missions of the USDA
and US Department of Homeland Security (DHS)
by increasing capabilities to respond and recover
from an agro-terrorism or Foreign Animal Disease
(FAD) incident. NHSRC's expertise in outdoor
decontamination testing and evaluation was
sought in order to advance the state of the
science and benefit all agencies involved. This
project also supports the mission of the NHSRC
by providing relevant information pertinent to the
decontamination of outdoor surfaces
contaminated during a biological incident and
supports the NHSRC's mission as delineated in
Homeland Security Presidential Directive 5, 7,
and 9.
During the decontamination activities following
the 2001 anthrax incidents, a combination of
removal and in situ decontamination was used.
The balance between the two was facility-
dependent and factored in many issues (e.g., the
nature of the contaminant, the physical state of
the facility, etc.). One factor was that such
remediation was unprecedented for the United
States Government (USG) and few technologies
had been proven for such a large-scale use at the
time. The cost of disposal proved to be very
significant and was complicated by the nature of
the waste (e.g., finding an ultimate disposal site).
Since 2001, a primary focus for facility
remediation has been improving the effectiveness
and practical application of in situ
decontamination methods and evaluating waste
treatment options to be able to provide
information necessary to make the
decontamination/disposal strategy more efficient
(i.e., less costly, less time-consuming, and more
efficacious).
1.1 Objectives
The primary objective of this study was to
address decontamination method gaps that
currently exist for response and recovery from an
FAD outbreak at an animal production facility.
Bacillus spores were used as surrogates for FAD
biological agents since they are highly resistant to
inactivation and represent a conservative
estimate of decontamination effectiveness.
A number of procedures using two active
decontamination solutions were evaluated, using
equipment expected to be available at such a
facility (i.e., garden hoses, pressure washers, and
portable chemical sprayers). The
decontamination agents tested were pH-adjusted
bleach (pH-AB) and Spor-Klenz® RTU, a broad
spectrum disinfectant and sporicide (details of
both decontaminants given in Appendix E -
Decontamination Process). The effectiveness of
combined steps of the procedures was tested on
"medium-sized" 35.6 cm by 35.6 cm (14 in by 14
in) pieces (coupons) of the selected materials
(Task I) and "large-sized" 101.6 cm by 101.6 cm
(40 in by 40 in) pieces (Task II). Both coupon
sizes are larger than those used commonly in
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other decontamination testing1"4, but smaller than
what will likely be encountered in the field (e.g.,
roadways, walkways, and walls). The medium-
sized coupons allow numerous materials and
decontaminants to be tested under varying
conditions with replication. In addition, 1 sq. ft.
size is the preferred surface area for wipe
sampling. The 35.6 cm by 35.6 cm (14 in by 14
in) coupons offer this surface area size for
decontamination and sampling. The large-sized
coupons were used to provide insight into and a
more realistic application of decontamination and
sampling methods. Operational parameters such
as time, physical impacts on materials, impact on
the remediation crew (e.g., physical exertion),
and fate of the viable spores (e.g., contamination
of equipment, wash water, filters) were also
determined.
1.2 Experimental Approach
The general approach used to meet the
objectives of this project was:
• Use of experimental chambers with controlled
environmental conditions, standardized
coupons and spore inocula;
• Contamination of medium- and large-sized
pieces of materials (coupons) via aerosol
deposition of bacterial spores;
• Quantitative assessment of spore
contamination by sampling positive control
coupons (coupons contaminated with the
bacterial spores in the same manner as test
coupons, but not subjected to the
decontamination treatment being tested prior
to sampling);
• Application of a prescribed decontamination
procedure to the test coupons and procedural
blanks;
• Quantitative assessment of residual
contamination by sampling test coupons and
procedural blanks;
• Quantitative and qualitative analysis of
decontamination procedure residues (e.g.,
waste water, aerosol samples);
• Determination of decontamination
effectiveness (comparison of results from
positive controls, negative controls and test
coupons); and
• Documentation of operational considerations
(e.g., cross-contamination, procedural time,
impacts on materials and personnel).
For the purposes of this project, effectiveness of
a procedure was evaluated by generating a
quantitative estimate of the reduction of viable
spores on a surface, measured as "log reduction".
In addition, determining the extent to which viable
spores were relocated to rinsate water (runoff) or
aerosol droplets is important for implications
regarding fugitive emissions and downstream
health risks.
Log Reduction (LR) can be defined as the
amount of reduction in viable spores required to
move the decimal one place, or reduce the
exponent in scientific notation by one. If starting
with one million spores, a log reduction of 2 would
result in a 99% reduction, or a change from 1 x
106 to 1 x 104. A 5 LR would be 99.999%
reduction, or a change from 1 x 106to 1 x101.
The general test approach for Task I is depicted
graphically in the flow chart shown in Figure 1-1.
Details of the types and numbers of materials
tested, as well as the procedures used for
contamination, decontamination, sampling and
testing, are described in Section 2 and in the
attached appendices.
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Typical Timeline
Day 1
Coupon
Fabrication
1
>
Coupon
Sterilization
Coupon
Contaminatior
Preparation
for
Test
Day 2
Day 3
Day 4
Load
Coupons
into their
respective
cabinets
I
Decontamination
Procedure
Application
\
t
N.
Coupon
Drying
^m
Recovery of
aerosol and
rinsate
samples
Analysis
Figure 1-1. Task I Test Approach Flow Chart
The two materials investigated in this study were
concrete and pressure-treated wood. These
materials were chosen due to their common
occurrence in animal production facilities. Prior to
the start of testing, medium-sized 35.6 cm by
35.6 cm (14 in by 14 in) and large-sized 101.6 cm
by 101.6 cm (40 in by 40 in) coupons were
fabricated (see Section 2) for Task I and Task II,
respectively. The coupons were then sterilized
(see Appendix A). The 35.6 cm by 35.6 cm (14 in
by 14 in) coupons were sterilized in groups (by
autoclave for concrete and by STERIS VHP®
1000ED (STERIS Corporation, Mentor, OH) for
pressure-treated wood) identified by sterilization
batch number. The 101.6 cm by 101.6 cm (40 in
by 40 in) coupons were sterilized in place using
250 ppmv (parts per million volume) vaporized
hydrogen peroxide (H2O2) generated by a VHP®
1000EDfor4 hours.
Prior to use, all test equipment intended to come
in contact with coupons or samples was sterilized
via autoclave sterilization at 121 °C, 103 kPa (15
psi) or by a STERIS VHP® cycle at 250 ppmv
H2O2 for 4 hours. All laboratory work surfaces
were wiped with Dispatch® bleach wipes
(Caltech, Midland, Ml), rinsed with Dl water, and
dried with 70 percent ethanol (VWR, West
Chester, PA).
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In an actual incident, contaminated surfaces must
undergo an organic burden reduction step prior to
undergoing an effective decontamination with
chemicals. This study uses burden-free materials
and makes no attempt to determine the
effectiveness of decontamination of heavily soiled
materials since the removal of organic burden
and surface pre-cleaning are assumed. Burden
reduction steps would likely require significant
additional effort in an actual incident.However,
burden reduction may aid in surface
contamination removal. Further testing utilizing a
standardized burden on material surfaces is
currently underway to better understand the
effects of grime on decontamination efficacy.
1.2.1 Task I Approach
Day 1 of testing involved coupon inoculation and
preparation for testing on Day 2. The required
number of pre-sterilized test and positive control
coupons were loaded with the target spores. The
procedural blank coupons were also located with
the test and positive control coupons, but were
not intentionally loaded with the target organism.
The coupons remained isolated in independent
deposition devices throughout this time.
On Day 2, the inoculated (and procedural blank)
coupons were removed from the deposition
devices and loaded into their respective cabinets
(positive controls and test coupons into the Test
Coupon Cabinets and the procedural blanks into
the Procedural Blank Cabinet) until being
retrieved for use in the decontamination test.
Task I coupons were tested in the small chamber
(see Section 2.3.1) in a vertical orientation.
Procedural blank coupons were subjected to the
decontamination procedure first, followed by the
test coupons. The decontamination procedure
was completed on all test coupons of one
material type before moving on to the next
material. After the decontamination procedure
was applied to a coupon or set of coupons, the
coupons were moved to the appropriate cabinet
for drying (test coupons to the Decontaminated
Coupon Cabinet and procedural blanks to the
Procedural Blank Cabinet).
The temperature and pH of the pH-adjusted
bleach solution and Dl water, and the
temperature of the Spor-Klenz® were measured
at the initiation of a test and prior to the start of
each test set (i.e., material type). The flow rate
from the backpack sprayer (SRS-600 Propack,
SHURflo, Cypress, CA), the pressure washers
(John Deere 3300 psi, Model 020382 and Troy
Bilt 2550 psi, Model 020337), and the chemical
sprayer (Model# PP-UAG1003HU-K, UDOR,
USA) were measured at the start and end of
testing of each set of three coupons. The spray
pattern for the backpack sprayer was confirmed
(and adjusted as needed) prior to the start of a
test. The 25° nozzle was used with the pressure
washers. The chemical sprayer had an adjustable
nozzle similar to the garden hose. These
measurements were made to ensure that such
parameters were in accordance with the data
quality objectives (DQOs) defined for the project
(see Section 4). Adjustments were made as
necessary to achieve the desired set-points,
within the acceptable tolerances.
Although surface sampling of the coupons did not
occur until Day 3, several other samples were
collected to obtain additional information on the
fate of the spores. To assess the potential for
viable spores to be washed off the surfaces, all
liquid runoff (rinsate) generated in the
decontamination process was collected and
quantitatively analyzed. Rinsate samples were a
composite of all replicate coupons of a particular
material type per test. Quantitative analysis was
conducted on rinsate samples so that the
magnitude of spore relocation could be
determined. The volume of runoff liquid collected
for each coupon set was measured after
collection. To quench the decontaminant activity
in runoff samples during and after collection,
sufficient neutralizer was added to the sample
container prior to sample collection to prevent
sporicidal activity post sample collection and
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provide an accurate estimate of viable spores
leaving the contaminated surface in rinse water.
Soil or heavily soiled areas receiving biological
agent-laden runoff during remediation following
an actual FAD incident would be expected to
quench most decontaminants in a similar manner.
Bioaerosol samples, using Via-Cell® Bioaerosol
Sampling Cassettes (Part# VIA010, Zefon Int.,
Ocala, FL), were originally collected during
spraying operations (decontamination and rinse
steps) in the small chamber to assess the
potential for spores to be aerosolized during the
decontamination procedure (see Appendix F.4 for
details). Bioaerosol samples were collected from
the exhaust vent during some tests.
After the completion of each set of coupons, the
test chamber was cleaned in accordance with the
procedure described in Appendix B. A coupon set
for Task I includes all blank coupons or all
replicates of one material type. Cleaning
between sets reduced the potential for cross-
contamination of samples.
On Day 3, after at least 18 hours of drying,
sampling of the coupons was performed using
pre-wetted gauze wipes (Kendall, 8042) (see
section F.2.1). A sampled area of 1,175 cm2 (1.3
ft2) per coupon was used by sampling the interior
section of each coupon. A template was used to
cover the exterior 0.635 cm (0.25 in) of each 35.6
cm x 35.6 cm (14 in by 14 in) coupon leaving a
square, 34.29 cm by 34.29 cm (13.5 in by 13.5 in)
exposed for sampling. Surface sampling of each
test coupon was conducted only once using the
common method of wiping the surface with a
wipe in three directions (vertical, horizontal,
diagonal), completely covering the surface of the
coupon in each direction (Appendix F).
The primary analysis of the samples collected
(coupon, rinsate, and bioaerosol) occurred over a
three-day period for Task I (note: Day 1 of the
microbiological analysis was Day 3 of
experimentation). In general, the Microbiology
Laboratory extracted and plated the samples on
the day of receipt and then counted colonies the
next day. In instances when there was insufficient
time for wipe samples to be extracted and plated
on the day of receipt, they were refrigerated on
the day of receipt, with sample extraction and
plating on Day 2, and colony counting the
following day. Filter plating or additional dilution
plating was performed on an as-needed basis.
Appendix C contains Miscellaneous Operating
Procedures (MOPs), including the aerosol
deposition of spores. Appendices D through G
contain additional details of the contamination,
decontamination, and sampling and analysis
procedures, respectively.
1.2.2 Task 11 Approach
Task II followed a similar pattern, except that an
additional wipe sampling step to characterize
contamination levels was done before the
decontamination procedure, and the first step on
Day 2 in Figure 3-1 (loading coupons into their
respective cabinets) was not applicable. In
addition, the timeline was extended compared to
Task I, with the differences detailed below.
Day 1 of testing in the large chamber (referred to
as COMMANDER; see Section 2.3.2) involved
running a STERIS VHP® cycle in the
COMMANDER and airlock to sterilize both the
coupons and deposition devices.
On Day 2, the required number of test and
positive control coupons were loaded with the
target spores in COMMANDER in a horizontal
orientation (nine deposition devices per large
coupon, see Figure 2-5). Spores were allowed
to settle onto the coupon surface for at least 18
hours. The deposition devices were removed on
Day 3 and placed in the airlock. The 101.6 cm by
101.6 cm (40 in by 40 in) coupons were placed in
vertical positions inside COMMANDER, and the
deposition devices and the troughs underwent a
STERIS VHP® cycle in the airlock.
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Sterility checks (swab samples) were taken of the
troughs on Day 4, with the weekend being Days 5
and 6. On the morning of Day 7, provided the
troughs were not significantly contaminated (low
spore counts were not unexpected since the
coupons had been loaded with spores in the
airlock), the troughs were placed beneath their
assigned coupon inside COMMANDER and
another sterility check was taken. For the first
test, contamination of the organism of interest
was found in the troughs, so the troughs and
surfaces were wiped down with Dispatch® bleach
wipes and the airlock was subjected to another
STERIS VHP® cycle until no growth from sterility
samples was observed. Positive control samples
were taken immediately prior to the start of the
decontamination process.
Unlike Task I, all coupons were inside the test
chamber (COMMANDER) together. Completion
of the decontamination procedure as well as pre-
and post-decontamination sampling were done
sequentially, alternating between concrete and
pressure-treated wood coupons. Only pH-AB was
used for these Task II tests, and pH-AB was
applied with the backpack sprayer (SRS-600
Propack, SHURflo, Cypress, CA). During the first
test, a garden hose was used to rinse the
coupons with deionized (Dl) water following the
contact time with the decontaminant. Such rinse
steps have been included in low-tech remediation
of Bacillus anthracis contaminations, as rinsing is
thought to reduce the amount of corrosion due to
residual decontaminants and reduce the amount
of chlorine off-gassing in a facility post-
decontamination. Elimination of the final rinse
step during animal facility remediation is believed
to be a potential option; however, previously there
have been limited data to support making such
changes.
The troughs were used to collect the rinsate from
each coupon. Separate bioaerosol samples
were collected before, during, and after each
individual step of the decontamination process.
On Day 8, post-decontamination sampling was
conducted. A stainless steel template was used
to create the nine individual sample areas, each
30.5 cm by 30.5 cm (12 in by 12 in). Sampling
was conducted only once on any one of the nine
sampling locations per coupon.
1.3 Definition of Efficacy
The overall effectiveness of a decontamination
technique relies on the potential of the technology
to inactivate and/or remove the spores from
contaminated building material surfaces and the
ultimate disposition (or fate) of the spores that
would result in secondary contamination of by-
products (rinsate) and equipment that would
necessitate specific remediation strategies.
Surface decontamination efficacies are for the
complete procedure and for each specific
material. The ultimate fate of the spores is also
pertinent in assessing the overall remediation
strategy.
The efficacy of each decontamination method
(combination of steps) was determined based on
the number of viable spores collected from the
surface of the decontaminated coupon, as
compared to the number of viable spores
collected from the surface of control coupons (or
coupon areas) not subjected to decontamination
procedures. The number of viable spores was
measured as colony forming units, or CPU.
1.3.1 Surface Efficacy
CPU counts per coupon or coupon area were
calculated according to the equation shown in
MOP 6535a (Appendix C). The first step in the
calculation of overall efficacy of a treatment to
reduce contamination on the surface of the
coupons is a separate calculation of efficacy for
each individual coupon in a given set of
replicates. Efficacy is defined as the extent (by
log reduction, or LR) to which the agent extracted
from the coupons after the treatment with the
decontamination procedure is reduced below that
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extracted from positive control coupons (not
exposed to the decontamination procedure).
Efficacy was calculated for each test coupon
within each combination of decontamination
procedure (;) and test material (/) as:
where:
Cyc = the number of viable organisms recovered from c control coupons for the /
decontamination procedure and/ test material.
NJJC = the number of control coupons for the j test material, ; decontamination procedure
Njjk = number of viable organisms recovered on the K replicate test coupon for the /
decontamination procedure and/ test material.
The efficacy of the decontamination technique for
a specific surface material is evaluated by means
of the difference in the logarithm of the CPU
before decontamination and after
decontamination for that material. This value is
reported as a log reduction (LR) efficacy on the
specific material surface as defined in Equation 1-
2.
c=l
where:
the average log reduction of spores on a specific material
surface
(^ J?J J )/ /V
CJ
CJ
)/ A/" _
the average of the logarithm of the number of viable spores
(determined by CPU) recovered on the control coupons (C=
control, j = coupon number, and Nc is the number of coupons
(1,7))
the average of the logarithm of the number of viable spores
(determined by CPU) remaining on the surface of a
decontaminated coupon (S= decontaminated coupon, k =
coupon number, and Nt is the number of coupons tested (1 , k))
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When no viable spores were detected, the
detection limit of the sample was used, and the
efficacy reported as greater than or equal to the
value calculated by Eqn. 1-2. The detection limit
of a sample depends on the analysis method and
therefore may vary. The detection limit of a plate
was assigned a value of 0.5 CPU, but the fraction
of the sample plated varied. For instance, the
detection limit of a 0.1 ml plating of a 20 ml
sample suspension is 100 CPU (0.5 CPU / 0.1
ml * 20 ml), but if all 20 ml of the sample is
filter-plated, the detection limit is 0.5 CPU.
The standard deviation of LRj is calculated by
Eqn 1-3:
(1-3)
where:
so.
= standard deviation of T\\
LR,
the average log reduction of spores on a specific material
surface
the average of the log reduction of the k replicate test coupon
x'Jk = for the/ decontamination procedure and j test material.
I
_
x,Jk -
(1-4)
where:
the "mean of the logs", the average of the logarithm transformed
number of viable spores (determined by CPU) recovered on the
control coupons (C= control, j = coupon number, and Nc is the
number of coupons (1, j))
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CPU
ijk
number of CPU on the surface of the kl" decontaminated
coupon for the /
material.
; decontamination procedure and/ test
1.3.2 Ultimate Fate of Spores
The surface log reduction, as calculated in
accordance with Equation 1-4, depicts the
effectiveness of the decontamination in mitigating
the contamination on materials. The mitigation
could be due to inactivation of the spores on the
materials (i.e., due to the application of a
sporicide) or physical removal from the material
(e.g., washed/rinsed off or aerosolized). For
physical removal, viable spores may either
remain in the rinsate or be re-aerosolized due to
the decontamination activity itself. Understanding
the ultimate fate of the spores, not just the
surface log reduction, is critical to recognizing the
utility or appropriate implementation of the
decontamination process. Process parameters
(as well as the general nature of microbiological
sampling) prevented an exact accounting of the
fate of spores; however, qualitative
measurements were good indications of ultimate
fate. For the rinsate sample, the results are
reported as Total CFU and CFU per coupon. The
Via-Cell® air sample from the vacuum
containment cabinet or COMMANDER
atmosphere is reported as CFU per actual liter (L)
of air sampled.
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2. Materials and Methods
2.1 Coupon Materials and Fabrication
2.1.1 Material Surfaces
This section describes each material and how the
medium- and large-sized coupons were
fabricated. Both materials are considered porous.
1. Pressure-Treated Wood (Figure 2-1). The
material used for these coupons is 3/4 in
thick, 4 ft by 8 ft Georgia-Pacific ACQ-D
(alkaline copper quaternary type D) pressure-
treated plywood. Coupons were cut to size
(35.6 cm by 35.6 cm (14 in by 14 in) for Task
I, 101.6cm by 101.6cm (40 in by 40 in) for
Task II) with a table saw.
Figure 2-1. Pressure-treated Wood Coupon Front
2. Concrete (Figure 2-2). Quikrete
Sand/Topping mix was used to fabricate 1.5-
in thick coupons for Task I (35.6 cm by 35.6
cm (14 in by 14 in)) and 1.0-in thick coupons
for Task II (101.6 cm by 101.6 cm (40 in by
40 in)). The mix was prepared and poured
into forms. Surfaces were smoothed with a
hand trowel, then covered with plastic
sheeting and allowed to cure for 24 hours.
Once set, the coupons were removed from
the form and loose grit was sprayed from the
surface with a pressure washer. Task I
coupons were then stacked on a pallet where
they were further wetted and covered with
plastic to cure (more than 20 days). Task II
coupons were cured for five days in the shop
where they were fabricated.
10
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Figure 2-2. Curing Concrete (left) and Final Concrete Coupons (right)
2.1.2 Task I and Task II Coupons
The coupons made from each material for Task I
had dimensions of approximately 35.6 cm width
by 35.6 cm length (or approximately 14 in width
by 14 in length). The dimensions provided an
adequate edge for the spore deposition device to
seal to the coupon surface and allow for a
contaminated surface area of 1 ft by 1 ft. A
sample area of 1 sq ft is recommended for wipe
samples.11 Contamination procedures have been
developed, tested, and demonstrated by NHSRC
in other decontamination studies. The sampled
area of 1.3 sq ft per coupon was used for Task I
of this study by sampling the interior section of
each coupon. The thickness of the coupons
varied for each material based upon the
fabrication procedures determined to be the most
appropriate for each material type. However,
each material type had a uniform thickness for all
replicate coupons.
Task II coupons prepared from pressure-treated
wood and concrete were 101.6 cm by 101.6 cm
(40 in by 40 in), and, conceptually, equal to the 3
by 3 square of nine coupons used in Task I. Two
replicate coupons of each material were used for
each test in Task II. The template used to sample
individual coupon areas is shown in Figure-2-3.
All coupons were sterilized as described in
Appendix A. There were no visible or
documented changes to the structure of the
coupons as a result of sterilization.
For the purposes of this project, coupon sets
were defined as all blank coupons, groups of
replicate test coupons, and all positive control
coupons of the same material type.
11
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Figure 2-3. Sampling Template on Task II Pressure-treated Plywood Coupon
2.2 Material Inoculation Procedure
The investigation of the effectiveness of the
decontamination procedures required that a
target organism be applied to a "sterile" material
surface (i.e., material inoculation) at a precise
target loading (e.g., spores per piece of material
(or coupon)). This section provides detail on the
target organism and material inoculation
procedures used for this investigation.
2.2.1 Bacillus Spore Preparation
The test organism for this work consisted of a
Bacillus atrophaeus spore preparation infused
with silicon dioxide particles. This bacterial
species was formerly known as 8. subtilis var
niger and previous to that as 8. globigii. The
preparation was obtained from the U.S. Army
Dugway Proving Grounds (DPG) Life Science
Division. The preparation procedure is reported in
Brown et al.12 Briefly, after 80 - 90 percent
sporulation, the suspension was centrifuged to
generate a preparation of about 20 percent
solids. A preparation resulting in a powdered
matrix containing approximately 1 x 1011 viable
spores per gram was prepared by dry blending
and jet milling the dried spores with fumed silica
particles (Deguss, Frankfurt am Main, Germany).
The powdered preparation was loaded into
metered dose inhalers (MDIs) by the U.S. Army
Edgewood Chemical Biological Center (ECBC)
according to a proprietary protocol. The MDIs are
claimed to provide a consistent dose of 1E9
12
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spores per puff. Quality assurance documentation
is provided by ECBC with each batch of MDIs.
Control checks for each MDI were included in the
batches of coupons contaminated with a single
MDI as described in Section 2.2.2.
2.2.2 Coupon Inoculation Procedure
Coupons were inoculated (loaded) with spores of
8. atrophaeus from an MDI using the procedure
detailed in MOP 6561 (an EPA proprietary
method, patent pending). The large 101.6 cm by
101.6 cm (40 in by 40 in) coupons were placed
horizontally inside COMMANDER. Nine dosing
chambers were arranged on the large coupons,
overlapping the inside edges of the dosing
chambers. Clamps were placed along the outside
edge, and two bars spanning the width of the
coupon were clamped down to help stabilize the
internal edges for the second Task II test. Each
dosing chamber covered a coupon area, as
shown in Figure 2-4. Figure 2-5 shows the dosing
chambers in place.
Briefly, each coupon (or coupon area for Task II)
was contaminated independently by being placed
into a separate dosing chamber (aerosol
deposition apparatus or ADA) designed to fit one
35.6 cm by 35.6 cm (14 in by 14 in) coupon of
any thickness. In accordance with MOP 6561, the
MDI was discharged a single time into the dosing
chamber. The spores were allowed to settle onto
the coupon surfaces for a minimum period of 18
hours. After the minimum 18-hr period, the Task I
coupons were then removed from the dosing
chamber and moved to an isolated cabinet (Test
Coupon Cabinet) which contained all loaded
coupons for a single test. The Task II coupons
were moved to their test positions in the large
chamber following the deposition period. The
target recovery range was 1 x 107 CFU per
coupon.
The MDIs are claimed to provide 200 discharges
per MDI. The number of discharges per MDI was
tracked so that use did not exceed this value.
Additionally, in accordance with MOP 6561, the
mass of each MDI was determined after
completion of the contamination of each coupon.
To prevent inadequate inoculation of coupons
due to near-empty MDIs, if an MDI had a mass of
less than 10.5 g at the start of the contamination
procedure described in MOP 6561, it was retired
and a new MDI was used. For quality control of
the MDIs, an inoculation control coupon was run
as the first, middle, and last coupon inoculated
with a single MDI in a single test. The
contamination control coupon was a stainless
steel coupon (35.6 cm by 35.6 cm) inoculated in
accordance with MOP 6561, sampled in
accordance with Appendix F, and analyzed in
accordance with Appendix G.
A log was maintained for each set of coupons or
coupon areas that were dosed. Each record in
this log recorded a unique coupon identifier (see
Appendix D), the MDI unique identifier, the date,
the operator, the weight of the MDI before
dissemination into the coupon dosing device, the
weight of the MDI after dissemination, and the
difference between these two weights.
13
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Area 2
Area 4
Area 5
Area 6
Area 7
AreaS
Area 9
Figure 2-4. Task II Coupon Sampling Areas (BLUE indicates areas for positive controls)
Figure 2-5. Nine Dosing Chambers on a 101.6 cm by 101.6cm (40 in by 40 in) Coupon
14
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The handling of the inoculated coupons, including
movement to minimize or control spore dispersal,
is described in Appendix D.
2.3 Experimental Approach
2.3.1 Task I-Small Chamber
For Task I, application of the decontamination
procedures was done in a custom-built test
chamber shown in Figure 2-6. The chamber,
located in High-Bay Room 130 at EPA's
Research Triangle Park facility, has dimensions
of 1.2 m high by 1.2 m wide by 1.2 m deep (4 ft
high by 4 ft wide by 4 ft deep) and is designed to
accommodate three 35.6 cm by 35.6 cm (14 in by
14 in) coupons at a time in either orientation
(horizontal or vertical, see below). The chamber
is of solid stainless steel construction with the
exception of the front face and top which are
fabricated from clear acrylic plastic. The front face
acrylic section is a door allowing full access to the
inside of the chamber while standing outside. The
back stainless steel wall contains an assembly to
hold the vertically-oriented coupons (maximum
three 35.6 cm by 35.6 cm (14 in by 14 in)
coupons at one time).
A center-aligned hole in the chamber door is
outfitted with a swivel port (see Figure 2-7),
allowing spray nozzles to fit and align with the
middle of the coupons. The wand is inserted into
this center port and moved in and out as
necessary to maintain the correct distance from
the three coupons while accomplishing the spray
pattern described in Appendix E
(Decontamination application methods and
rinsing with water). Every effort was made to
perform this step consistently and maintain the
correct distance from all coupons. The port also
allows the chamber door to remain closed during
application of the decontamination solutions.
During the pressure-washing, rinsing steps with
the garden hose and the spraying of the
decontamination solutions with the backpack
sprayer, the front face door was closed and
sealed. The seal is designed to contain any
splashed liquid. Maintaining the door closed also
prevents exposure of the worker to the toxic
fumes from decontamination solution during
application.
The bottom of the chamber is pyramidal in shape
with a 7.6 cm (3 in) diameter drain in the center.
The drain can be closed or opened to either
collect or release the runoff from the coupons
during the decontamination procedure. The
bottom of the chamber has a 227 L (50 gal)
collection capacity.
The chamber is fitted with connections allowing
filtered air to enter and filtered exhaust to exit via
a readily accessible connection to the facility's air
handling system. Connection to facility point
exhaust results in a slight negative pressure
inside the spray chamber in relation to the room
within which it is contained. The chamber is also
designed to be easily decontaminated between
runs using either liquids orfumigants, as needed.
Decontamination of the chamber is discussed in
Appendix B.
15
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Figure 2-6. Task I Decontamination Chamber
Figure 2-7. Spraying through center-aligned port in the small chamber door
16
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Figure 2-8. Airlock in foreground and large chamber (COMMANDER) in background
2.3.2 Task II - Large Chamber (COMMANDER)
For Task II, application of the decontamination
procedures was done inside the Consequence
Management and Decontamination Evaluation
Room (COMMANDER) (Figure 2-8). This room is
an enclosed, single-access-point chamber that
meets the following criteria:
• Supports repeated fabrication of a
representative test environment (e.g.,
furnished office room, outdoor setting)
contained within the chamber
• Allows for release of biological organisms or
chemicals into the chamber (Biosafety Level
2, Chemical Safety Level 4)
• Under slight negative pressure in relation to
outside environment
• Allows for application of a decontamination
technology (including fumigation with toxic,
corrosive gases)
• Supports entry into the chamber during all of
the above-mentioned activities (in appropriate
personal protective equipment (PPE))
• External dimensions of 2.74 m by 3.66 m by
3.05 m high (9 ft by 12 ft by 10 ft high)
• Contains a 1.83 m by 1.83 m by 2.44 m high
(6 ft by 6 ft by 8 ft high) airlock with single air-
tight entry/exit port with a window
• Contains entry/exit ports in line with the
enclosure double door to allow for large
materials to be brought into or out of the
chamber
17
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• Complies with all relevant local and national
codes
2. After 15 minutes, reapply the liquid
decontaminant to material surface.
• For the current study, a trough was placed
under the coupons and curtains placed
around the coupons, in order to capture and
collect the runoff and spray during the
decontamination procedures. The curtains
were placed to act as a guide during the
decontamination steps to facilitate
maintaining the correct distances between
the nozzles and the surface of the coupon.
2.4 Decontamination Procedure
The two procedures tested for application of pH-
AB and Spor-Klenz® can be summarized with the
following sequential procedural steps.
Modifications made to the test matrix are detailed
in Section 2.5.
Backpack Sprayer-Applied Decontaminant
1. Apply liquid decontaminant to material
surface using a pressurized backpack
sprayer.
2. After 15 minutes, reapply the liquid
decontaminant to material surface.
3. Once a total of 30 minutes have elapsed
since the first application, rinse the material
surface with distilled water using a garden
hose.
4. Allow material to dry overnight.
5. Sample material surface using sterile non-
cotton pre-moistened wipes.
Pressure Washer-Applied Decontaminant
1. Apply liquid decontaminant to material
surface using pressure washer and chemical
supply tank.
3. Once a total of 30 minutes have elapsed
since the first application, rinse the material
surface with distilled water using a pressure
washer.
4. Allow material to dry overnight.
5. Sample material surface using sterile non-
cotton pre-moistened wipes.
Determining the efficacy of the above-mentioned
procedures was the focus of this study, both with
respect to the physical removal and the
inactivation of spores.
This project employed the use of backpack
sprayers, pressure washers, nozzles, garden
hoses, pressure regulators, bleach, vinegar, and
Spor-Klenz®, as well as carboys, buckets for Dl
water, and containers for mixing the pH-adjusted
bleach solution. The specifications of the
materials and equipment used for the
decontamination procedural steps are detailed in
Table E-1 of Appendix E.
It was critical for this project that each step in the
decontamination procedure be implemented as
uniformly as possible for all coupons and tests.
Changes in technique during the study could lead
to highly variable and/or biased data and lead to
erroneous conclusions. Therefore, the methods
for each step were documented in detail to
provide as much standardization as possible.
Staff performing the decontamination procedures
practiced each step in advance and an attempt
was made to add measurable controls. Additional
details can be found in Appendix E.
The results of the testing provide information to
evaluate the effectiveness of a number of
procedures using two active decontamination
solutions for removing surface contamination.
Additionally, the testing provided information on
18
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viable spore disposition for consideration in the
development of remediation strategies (e.g.,
when/where the procedure might be considered
for application, need for water collection and
treatment, estimation of waste generation).
2.5 Test Matrix
Ten tests in Task I 35.6 cm by 35.6 cm (14 in by
14 in) coupons and two tests in Task I1101.6 cm
by 101.6 cm (40 in by 40 in) coupons were
performed. Table 2-1 identifies each procedural
step for each material type. The original test
matrix was amended as the tests progressed,
based on the results obtained. These changes
were adaptive (altering parameters based upon
results of previous tests) and in remediation of
unforeseen consequences of testing (replacing of
spray devices following failure of the initial device
due to incompatibility with the liquid
decontaminant).
• For Task II, cleaning of the chamber was
performed by running a STERIS VHP® cycle
as detailed in Section 1.2 after the completion
of each material type per test.
• For Task I, each test required six test
coupons, one procedural blank, and six
positive control coupons of each material
type. Hence, 13 coupons (total) were
required for each material type.
• For Task II, each test required two replicate
coupons, divided into five test coupon and
four positive control sample areas.
• Wipe sampling was used on both the
concrete and pressure-treated wood.
• Procedural blanks for Task I (coupons of
each material not intentionally loaded with the
target organism) were run first, followed by
the test coupons of each material type. The
procedural blank coupons were subjected to
the same procedural decontamination steps
as the test coupons. On the day of testing,
the coupons are moved to their respective
storage cabinets (positive controls and test
coupons into the Test Coupon Cabinets and
the procedural blanks to the Procedural Blank
Cabinet) to avoid potential cross-
contaminations between coupons. For Task I,
a maximum of three coupons were run at a
single time in the decontamination chamber.
Only one material type was run at a time.
• For Task I, cleaning of the chamber was
performed in accordance with Appendix B
after the completion of each material type per
test.
19
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Table 2-1. Test Matrix
Task
1
1
1
1
1
1
1
1
1
1
2
2
2
2
Test
1
2
3
4
5
6
7
8
9
10
C1
C1
C2
C2
Date of
Decon
10/12/2010
10/12/2010
12/14/2010
12/14/2010
10/27/2010
10/27/2010
11/17/2010
11/17/2010
1/18/2011
1/18/2011
2/8/2011
2/8/2011
2/24/201 1
2/24/201 1
Material
Concrete
Wood
Concrete
Wood
Concrete
Wood
Concrete
Wood
Concrete
Wood
Concrete
Wood
Concrete
Wood
Size
(in)
14"x14"
14"x14"
14"x14"
14"x14"
14"x14"
14"x14"
14"x14"
14"x14"
14"x14"
14"x14"
40"x40"
40"x40"
40"x40"
40"x40"
Replicates
(n)
6
6
6
6
6
6
6
6
6
6
2
2
2
2
Application
Sprayer
Sprayer
Chemical
Sprayer
Chemical
Sprayer
Sprayer
Sprayer
Pressure
Washer
Pressure
Washer
Sprayer
Sprayer
Sprayer
Sprayer
Sprayer
Sprayer
Decon
pH-AB
pH-AB
pH-AB
pH-AB
Spor-
Klenz®
Spor-
klenz®
Spor-
Klenz®
Spor-
Klenz®
pH-AB
pH-AB
pH-AB
pH-AB
pH-AB
pH-AB
Spray
Duration
(sec)
30
30
15
15
30
30
15
15
10
10
30
30
30
30
Reapplication
Time(min)
15
15
15
15
15
15
15
n/a
n/a
n/a
15
15
15
15
Rinse
Duration
(sec)
10
10
10
10
10
10
10
10**
10
10
30
30
NA
NA
No. of
Sprays
2
2
2
2
2
2
2
A ***
1
1
2
2
2
2
Total
Exposure
(min)
30
30
30
30
15*
15*
30
34
15
15
30
30
30
30
Coupons were inadvertently rinsed immediately after the second Spor-Klenz® spray, resulting in a total contact time of 15 minutes.
Rinse applied with garden hose due to power washer failure (34 minute contact time).
Power washer failed before second decontaminant application during first set of three replicate coupons. First set had one application; second set was not included in test results.
20
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In Tests 1 and 2, the backpack sprayer was used
to spray the coupons twice for 30 seconds with
pH-AB, followed by a 15-minute contact time after
each spray. This scheme resulted in a total
exposure (contact time) of 30 minutes before the
Dl rinse. Tests 5 and 6 were conducted
identically, except that Spor-Klenz® was used as
the decontaminant.
Due to concerns over compatibility between pH-
AB and the pressure washer, Tests 7 and 8 with
Spor-Klenz® were conducted first. Because of the
higher flow rate of the pressure washer versus
the backpack spray, the duration of the two
sprays was reduced to 15 seconds. The total
contact time for the concrete coupons remained
at 30 minutes. The concrete coupons were
subjected to the test procedure first, and the
procedure was completed as prescribed. Wood
coupons were tested second, and received the
first decontamination spray, but the pressure
washer could not be restarted to accomplish the
second application. Following only one
application of Spor- Klenz®, these coupons were
rinsed with Dl water using a garden hose after 34
minutes of exposure. Ultimately, the pressure
washer was rendered inoperable by the Spor-
Klenz®.
Tests 3 and 4 were conducted with pH-AB using
a chemical sprayer. Known incompatibility with
standard pressure washers prevented their use;
the UDOR chemical sprayer (Model# PP-
UAG1003HU-K, UDOR, USA) was chosen for
this test because it was made specifically for use
with chlorine (see Appendix E). These tests
involved two 15-second sprays of pH-AB with 15-
minute contact times after each spray (30 minute
total exposure), and a 10-second Dl water rinse
using the replacement pressure washer.
Based on the pH-AB results from Tests 1 through
4, Tests 9 and 10 reduced the pH-AB backpack
spray time to 10 seconds and involved just one
15-minute contact time prior to the Dl water rinse.
Tests C1 and C2 were conducted in
COMMANDER using two replicate coupons of
each material for each test. For both tests, the
backpack sprayer was used to spray the coupons
twice for 30 seconds with pH-AB, followed by a
15-minute contact time after each spray. The
difference between these tests was that the
coupons in C2 did not receive a Dl water rinse.
2.6 Sampling and Analytical Procedures
Three types of samples were included in this
project. Surface sampling procedures were used
to collect samples from the coupon materials. In
order to obtain the additional critical information
on the fate of the spores, several samples in
addition to the surface sampling of the coupons
were collected. To assess the potential for viable
spores to be washed off the surfaces, all liquids
used in the decontamination process were
collected and quantitatively analyzed. This
sample was a composite for all replicate coupons
of a particular material type per test. Quantitative
analysis was done on these rinsate samples to
provide for an order of magnitude determination
of the disposition of viable spores in this media.
To assess the potential for spores to be
aerosolized from coupon surfaces during
spraying or pressure washing, aerosol samples
were collected from the decontamination
chamber during any such activities. Quantitative
analyses were performed on these samples, so
that a concentration (viable spores per volume of
air) could be determined. These data are
important for understanding the potential for
contamination spread and worker risk during the
decontamination procedures. Any spores
released during this phase may also avoid
contact with the decontaminant and therefore
remain active. A second decontamination
procedure may be needed to decontaminate
aerosolized spores that redeposit elsewhere. The
materials and equipment used as well as the
sampling protocols for sampling are detailed in
Appendix F.
21
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2.6.1 Factors Affecting Sampling/Monitoring
Procedures
Sampling of coupon surfaces was done after
coupons that were wetted during the
decontamination procedure had become visibly
dry. Drying was allowed to occur in the
Decontaminated Coupon Cabinet or Procedural
Blank Cabinet, or inside COMMANDER (as
appropriate), facilitated by a slight air flow due to
a constant positive pressure. All coupons were
allowed to dry for at least 18 hours. The actual
time that each coupon was allowed to dry was
recorded.
2.6.2 Preparation for Sampling/Monitoring
Sampling kits for wipes were prepared as
specified MOP 6568 (see Appendix C). For Task
I, all laboratory surfaces intended for use during
sampling were wiped with Dispatch® bleach
wipes. Precut 50.8 cm by 50.8 cm (20 in by 20 in)
sheets of absorbent bench liner were used to
cover all work surfaces, replaced after each
phase of a test (e.g., coupon contamination is
considered one phase, decontamination another,
and surface sampling a third). Sampling was
conducted on only one coupon at a time. One
coupon was moved from the Decontaminated
Coupon Cabinet (test coupons), Test Coupon
Cabinet (positive controls), or Procedural Blank
Coupon Cabinet (procedural blanks) to the
sampling space located immediately outside (to
the front) of each cabinet. All coupons were
placed horizontally for sampling, regardless of
their orientation during the decontamination.
Within a single test, surface sampling of the
coupons was performed starting with coupons
from the lowest level of contamination and ending
with the highest level of contamination (i.e., all
procedural blank coupons first, followed by all test
coupons, and then all positive control coupons).
Surface sampling was performed by wipe
sampling in accordance with the protocols
included in Appendix F. The surface area for all
samples was 1175.8 cm2 (1.3 ft2).
A template was used to cover the exterior 0.635
cm (0.25 in) of each coupon leaving a square
(34.29 cm by 34.29 cm) exposed for sampling for
all coupons. The outer 0.635 cm of each coupon
was not sampled in order to avoid edge effects.
A sampling material bin was stocked with all
appropriate items (consistent with the protocols in
Appendix F) for each sampling event. The bin
contained enough wipe sampling kits to
accommodate all required samples for the
specific test. An additional kit was also included
for backup. Enough gloves and bleach wipes
needed to complete the test were available.
Templates (35.6 cm by 35.6 cm (14 in by 14 in))
with an interior opening of 34.3 cm by 34.3 cm
(13.5 in by 13.5 in) were wrapped in aluminum foil
and packaged in sterile autoclave-safe bags
(autoclave-sterilized by MOP 6570 using a one
hour gravity cycle, 10 templates per bag) and
transported with the original sterile coupons
(concrete and stainless steel procedural blanks).
These bags of templates were also included with
the sampling kits. A sample collection bin was
used to transport samples back to the
Microbiology Laboratory. The exterior of the
transport container was decontaminated by
wiping all surfaces with a Dispatch® bleach wipe
prior to transport from the sampling location to the
Microbiology Laboratory. To ensure the integrity
of samples and to maintain a timely and traceable
transfer of samples, an established and proven
chain of custody was strictly adhered to for each
test.
For Task II, a template (see Figure 2-3) was used
to create the nine individual sample areas, each
30.5 cm by 30.5 cm (12 in by 12 in). The
sampling templates were sterilized by VHP® or
Dispatch® wipes prior to sampling. Coupons were
sampled in the vertical position, one material at a
time.
22
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2.6.3 Wipe Sampling
To assess the effectiveness of the
decontamination procedure, wipe sampling was
performed for each coupon. Wipe sampling is the
method that is anticipated to be used following an
FAD incident. Wipe sampling is typically used for
small sample areas and is effective on
nonporous, smooth surfaces such as ceramics,
vinyl, metals, painted surfaces, and plastics.11
The general approach is that a moistened sterile
non-cotton pad is used to wipe a specified area to
recover bacteria, viruses, and biological toxins.11
The protocol that was used in this project is
described in Appendix F and has been adapted
from that provided by Busher et al.,11 Brown et
al.,12 and documented in the INL 2008 Evaluation
Protocols.13 Materials utilized in this study are
considered hard and porous. While wipe
sampling is not highly efficient on porous
materials, few other options exist. In addition,
preliminary data suggest that wipe sampling of
wood and concrete surfaces routinely allowed
recoveries of greater than 1 x 106 CFU when
surfaces were inoculated with 1 x 107 CFU per ft2.
Wipe sampling was therefore utilized for both
porous materials used in this study.
2.6.4 Rinsate Collection and Sampling
Decontamination procedures utilizing corrosive
liquids such as bleach will likely incorporate a
final rinse step following a prescribed contact time
with the decontaminant to reduce the potential for
damage to contacted surfaces. It is important to
determine if this "runoff' is a potential risk for
spread of contamination, so rinsate samples were
sampled and analyzed for viable spores following
decontamination.
For Task I, the runoff from the coupons
throughout the entire decontamination procedure
was collected for a given coupon set (material
type or all blanks). After all coupons from a single
set were moved to the Decontaminated Coupon
Cabinet or Procedural Blank Cabinet, the
chamber was rinsed with sterile Dl water. The
sterile runoff collection carboy was labeled and
the volume of liquid collected was recorded. The
decontamination liquid was neutralized by sodium
thiosulfate (STS) by placing the STS in the
collection vessel prior to commencement of the
decontamination steps. Neutralization was done
in order to standardize the results from all tests,
i.e., any sporicidal activity of the runoff was
eliminated once the runoff was captured in the
carboy preventing run-to-run variability due to
differences in the runoff composition.
Neutralization of the rinsate was used to simulate
a worst case field situation where the residual
killing power of the pH-AB or Spor-Klenz® would
be removed (i.e., due to material demand from
the collection surface (e.g., concrete or pressure-
treated wood)).
After collection, rinsate samples were
homogenized by shaking and 100 ml aliquots
were taken using aseptic technique according to
the protocol described in Appendix F. The
aliquots were submitted to the Microbiology
Laboratory for analysis at the conclusion of each
entire test.
For Task II, a trough blank was first collected by
adding 1 L of sterile Dl water to each trough and
taking three 100 ml aliquots for analysis. STS
was added to the trough prior to the start of the
decontamination procedure. The volume of
rinsate collected in each coupon's trough was
measured, and 100 ml aliquots were taken as for
Task I and submitted to the Microbiology
Laboratory for analysis.
2.6.5 Bioaerosol Sample Collection
To assess the potential for biological particles to
escape the surface of coupons during spraying
(decontamination and rinse steps) as aerosols,
bioaerosol samples were collected by actively
sampling (12 L/min) the air. ViaCell® bioaerosol
sampling cassettes were used to collect air from
the 1.2 m by 1.2 m (4 ft by 4 ft) spray chamber
23
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and from the COMMANDER chamber during the
decontamination procedures. Data obtained
from bioaerosol samples were used to indicate
whether reaerosolization is possible during
decontamination procedures.
2.6.6 Sample Analyses
Analyses of all samples were conducted in the
on-site Microbiology Laboratory. Phosphate
buffered saline with 0.05% TWEEN®-20 (PBST)
was used as the extraction buffer. After the
appropriate extraction procedure, as described in
Appendix F, the samples were plated, incubated,
and analyzed (CPU enumerated) in accordance
with MOP 6535a (see Appendix C). Appropriate
dilutions of the extracted sample (i.e., the initial
undiluted sample extraction dilution, and up to a
four-stage serial dilution (10~1 to 10~4)) were plated
depending on expected CPU concentration. For
example, the last two dilutions (10~3 and 10~4)
were not plated for a decontaminated sample if a
low CPU concentration was expected.
In addition to the analysis in MOP 6535a,
additional analysis procedures were used for
samples resulting in less than 30 CPU/sample in
the undiluted sample extract (e.g., wipe in the
extraction buffer). These analyses were
conducted in order to lower the current detection
limit associated with MOP 6535a. In accordance
with MOP 6565, Revision 2 (see Appendix C),
samples were filter plated.
The PBST was prepared according to the
manufacturer's directions and in accordance with
MOP 6562 (see Appendix C), dissolving one
packet in one liter of sterile water. The solution
was then vacuum-filtered through a sterile 0.22
urn filter unit to sterilize.
The extraction procedure used to recover spores
varied depending upon the different matrices
(wipes, Via-Cell ®cassette). The procedures are
described in Appendix F.
2.6.7 Coupon, Material, and Equipment
Cleaning and Sterilization
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 coupons that
were intentionally contaminated (test coupons
and positive controls) and not contaminated
(procedural blanks). The treatment of these three
groups of coupons (positive control, test, and
procedural blank) varied for each group. Hence,
specific procedures were put in place in an effort
to prevent cross-contamination among the
groups.
Due to the amount of waste and reusable items
(requiring decontamination after use) generated
during this testing (e.g., sterilization bags,
sampling templates, etc.), creation of a rigid plan
to segregate such items was imperative.
Reusable items were clearly distinguished and
separated from waste items after use and put in
distinct, segregated locations within the testing
area.
During the decontamination procedure for Task I,
one person (sample handler) was tasked with
moving the coupons to the decontamination
chamber. A different person was tasked with
moving the treated coupons to the appropriate
drying cabinet. Disposable laboratory coats were
used by the sample handler (tasked with moving
the coupons) to further minimize the potential of
cross-contamination. The sample handler donned
a new disposable laboratory coat after moving a
complete set of test samples (i.e., 6) from the
test coupon cabinet to the decontamination
chamber.
All bins, buckets, and containers remained closed
or covered unless in use (e.g., material being
placed into or extracted from the bin, bucket, or
container). Adequate cleaning of all common
materials and equipment was critical in
preventing cross-contamination.
24
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Each test in the experimental matrix included four
primary activities. These activities were
preparation of the coupons, execution of the
decontamination process (including sample
recovery), sampling, and analysis. Specific
management controls for each of these activities
are shown in Table 2-2. Appendix A details the
coupon sterilization procedures and Appendix B
describes the test chamber and equipment
cleaning procedures.
Table 2-2. Cleaning Methods and Frequency for Common Test Materials/Equipment
Material/Equipment
Decontamination Procedure
Chamber
Coupon Cabinets
Distilled water tanks (reservoir)
All work surfaces
Use
Contain coupons during
the application of the
decontamination
procedure being tested
Store coupons prior to
testing and/or sampling
Utilized during the garden
hose rinse and pressure
wash rinse procedures
Throughout each test
Cleaning Method
Washing with pH-adjusted
bleach solution, or wiping
with Dispatch® Bleach
Wipes, rinsing with Dl water
followed by ethyl alcohol
pH-Adjusted bleach solution
or wiping with Dispatch®
Bleach Wipes, rinsing with
Dl water followed by ethyl
alcohol
Bleach solution, soak
overnight
Maintaining the surface wet
with a pH-adjusted bleach
solution for 10 minutes
followed by wiping with 70%
ethyl alcohol before wiping
dry with a clean towelette.
Frequency
Before/after each test
and between test
materials
Before/after each test
Treated before each test
(within 48 hours of the
test start)
Before/after each use
(cleaning of surfaces
between handling of
replicate coupons during
sampling; cleaning
before/after moving all
contaminated coupons)
25
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3. Results and Discussion
The primary objective of this study was to
evaluate the efficacy of decontaminating building
material surfaces as a function of the
decontamination method parameters. The
parameters were chosen to improve application
efficiency while maintaining efficacy. In addition to
reduction of contamination from material
surfaces, the ultimate fate of the spores was also
a critical measurement objective. Combined, this
information can inform selection or further
development of appropriate, situation-specific
decontamination procedures. This section
discusses the results of individual
decontamination procedures and, when possible,
explores the ultimate fate of the spores and
decontamination worker exposure due to those
procedures.
3.1 Surface Sampling Results - Positive
Controls
3.1.1 Task I
Most standard or widely used laboratory methods
to test the efficacy of decontamination products
rely on the contamination of carriers (i.e., uniform
pieces of materials, also referred to as coupons)
with the target organism using a liquid
suspension.151617 Such methods offer the ability
to precisely contaminate the material in order to
maintain intra- and inter-test consistency. While
there are substantial benefits to using liquid
inoculation-based test methods in the laboratory
measure of efficacy, questions remain as to the
representativeness of the results with respect to
use in the field on materials contaminated with
aerosolized biological agent. Lee et al.18 describe
the development of a novel method to precisely
deposit aerosolized spores onto materials at a
target loading consistent with that used in liquid
inoculation-based methods, i.e., allowing the
determination of at least a six-log reduction due
to the decontamination process. The method
developed in that study was the predecessor of
the methods used in the current effort.
18
The method reported by Lee et al. was modified
to be used on the larger coupons required for the
current study. The target loading, based upon
recovery from the positive controls, was 1 x 106
spores per coupon with a relative standard
deviation (RSD) of 50 percent. The sampling
methods used for each material were based on
the results of the above-mentioned preliminary
comparison test, along with consultation with the
Project Team.
Surface sampling results from the positive control
coupons of each material demonstrate the ability
of the deposition and sampling methods to meet
the target criteria. Results shown in Table 3-1
confirm approximately a 6-log recovery (on
average) of viable spores from the material
surfaces of the positive controls.
26
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Table 3-1. CPU Abundance and RSD for Positive Controls
Decontamination
Procedure*
A= Test 1 & 2
B= Test 3 & 4
C= Test 5 & 6
D= Test 7 & 8
E=Test9&10
Stainless Steel Control
CFU/ft2
1.34E+07
2.02E+07
2.87E+07
1.64E+07
1.79E+07
RSD
27.03%
24.33%
20.95%
17.64%
3.20%
Concrete
CFU/ft2
1.71E+06
2.24E+06
1.93E+06
2.15E+06
1.07E+06
RSD
40.35%
28.09%
44.63%
53.90%
37.86%
Wood
CFU/ft2
3.28E+06
2.92E+06
3.87E+06
4.93E+06
1.07E+06
RSD
47.45%
55.45%
71.33%
44.66%
29.72%
Average
1.93E+07
18.63%
1.82E+06
40.96%
3.28E+06
49.72%
See Table 2-1 for additional details.
A = pH-AB applied with backpack sprayer (30 minute exposure).
B = pH-AB applied with chemical sprayer (30 minute exposure).
C = Spor-Klenz® applied with backpack sprayer (15 minute exposure).
D = Spor-Klenz® applied with pressure washer (30/34 minute exposure).
E = pH-AB applied with backpack sprayer (15 minute exposure).
Three stainless steel coupons were incorporated
into each test as control coupons indicative of the
deposition method. The smooth surface of
stainless steel allows for optimal recovery of
viable spores. Thus, the number of recovered
CPU is expected to be higher than from the
sample materials. During the inoculation
procedure of each Task I test, one stainless steel
coupon was loaded with spores before any other
coupons; one in the middle of the inoculation
series; and one at the end. Thus, these
inoculation control coupons could be used to
verify the consistency of the spore dispersion
apparatus.
The average spore recovery from the positive
controls of each material typically fell within 1 log
of the stainless steel controls (Figure 3-1). As
mentioned above, sampling from the rough,
heterogeneous surfaces of concrete and wood
was expected to yield lower, more variable CFU
than sampling from stainless steel. Recovery
from concrete was lower than recovery from
wood. During the wipe sampling of concrete, fine
particles were present on the sampling surfaces
prior to sampling, despite power-washing the
coupons prior to sterilization. During sampling,
fine particles on the surface of the coupon would
cluster together, forming larger masses that
would stay behind on the surface of the coupon,
presumably with an unknown quantity of the
target organism.
27
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Positive Control and Material Loading
l.OOE+08
LOQE+07
l.OOE+06
l.OOE+05
l.OOE+04
l.OOE+03
l.OOE+02
1.00E+Q1
l.OOE+00
I I I I I I I I I
II I I I 1 I I I
I I I I I 1 I I I
I I I I I I I I I
123456783 10
Test
iAvgCFU/Sample(SS)
i Avg CPU/Sample
(Material)
Figure 3-1. Positive Control and Material Coupon Loading for Task I
The variation in positive control CPU for each
material (Table 3-1) was higher than the
anticipated 50 percent for some tests. For the
wood coupons in Procedure B and C (Tests 4
and 6), the outliers were higher than the
averagepossibly because of natural variations in
the coupon surface: a few coupons were
smoother than normal and offered superior
recovery from wipe samples. For the concrete
coupons in Procedure D (Test 7), the outlier was
lower than anticipated.
3.1.2 Task II
For Task II, Areas 1,3,7, and 9 for positive
control determination of the inoculated coupons
were sampled immediately prior to the
decontamination procedure. The CFU recovered
from these sampled areas would be compared to
the CFU enumeration recovered from different
areas of the same inoculated coupon after the
decontamination procedure was complete. This
procedure is very similar to the efficacy method
that would be used in a field event. There were
duplicate coupons for each material. The positive
control results are shown in Table 3-2, below.
The positive controls for concrete vertical coupon,
replicate A (CVA) in Test C1 were lower than
anticipated, but still high enough to provide a
potential 6-log reduction. CVA may have had a
lower inoculation concentration based on
irregularities during inoculation.
28
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Table 3-2. Task II Positive Controls
Material
Stainless Steel
Concrete (CVA)
Concrete (CVB)
Wood (TWA)
Wood (TWB)
Testd*
Avg. CFU/ft2
1.49E+07
5.52E+05
2.98+06
2.51+06
2.34+06
RSD (%)
27.3%
48.1%
23.1%
35.7%
16.8%
Test C2*
Avg. CFU/ft2
4.51+06
3.26+06
5.29+06
1.96+06
2.39+06
RSD (%)
19.2%
27.6%
31.3%
21.1%
20.6%
Test C1 = pH-AB applied with backpack sprayer (30 minute exposure) with Dl water rinse.
Test C2 = pH-AB applied with backpack sprayer (30 minute exposure); no Dl water rinse.
CVA and CVB, and TWA and TWB, are designations for the replicate coupons.
3.2 Task I: Evaluating Decontamination
Procedures
3.2.1 Surface Sampling Results
To determine the most effective decontamination
procedure and to determine which parameters
were necessary to achieve desired results,
several individual procedures were tested within
the test matrix to determine their effect on overall
efficacy. Several parameters were modified:
application method, spray time, contact time, and
overall exposure. Several novel approaches were
used in the current study to provide a more
directly visible tie of laboratory efficacy testing to
field application of decontamination methods
(e.g., use of aerosol deposition of biological agent
instead of a liquid inoculation, use of field
sampling methods instead of coupon extraction
methods, and use of large coupons). This section
details the results with conclusions that can be
drawn from tests completed in this study.
The conditions for each Task I test are shown in
Table 3-3. Most tests performed during this task
achieved the target log reduction of greater than
6 LR. Figure 3-2 shows the efficacy in terms of
log reduction (LR) of the decontamination
technique averaged for all material surfaces for
each test.
29
-------
Table 3-3. Conditions for each Task 1 Test
Test
1
2
3
4
5
6
7
8
9
10
Material
Concrete
Wood
Concrete
Wood
Concrete
Wood
Concrete
Wood
Concrete
Wood
Application
Backpack
Sprayer
Backpack
Sprayer
Chemical
Sprayer
Chemical
Sprayer
Backpack
Sprayer
Backpack
Sprayer
Power
Washer
Power
Washer
Backpack
Sprayer
Backpack
Sprayer
Decon
pH-AB
pH-AB
pH-AB
pH-AB
Spor-
Klenz®
Spor-
klenz®
Spor-
Klenz®
Spor-
Klenz®
pH-AB
pH-
AB
Spray
Time
(sec)
30
30
15
15
30
30
15
15
10
10
Reapplication
Time (min)
15
15
15
15
15
15
15
n/a
n/a
n/a
Number
of
Sprays
2
2
2
2
2
2
2
1
1
1
Contact
Time
(min)
30
30
30
30
15
15
30
34
15
23
Rinse
Method
Garden
hose
Garden
hose
Power
washer
Power
washer
Garden
hose
Garden
hose
Power
Washer
Garden
Hose
Garden
Hose
Garden
Hose
U#
6.54
6.77
6.60
6.74
1.63
6.80
2.80
6.99
6.30
4.04
*LR values represent surface log reduction only.
The decontamination by means of pH-adjusted
bleach was accomplished by a combination of
removal and inactivation of spores. Viable spores
were found in both rinsate and Via-Cell® air
samples (discussed below). Of the procedures
tested, those incorporating pH-adjusted bleach
(Tests 1-4, 9-10) were typically most effective (>
6 log reduction) for decontamination. The lower
log reduction in Test 10 may be a result of
material demand in conjunction with a single
application; one spray application may not
provide enough pH-adjusted bleach to overcome
the demand of wood. The surface log reductions
for tests utilizing Spor-Klenz® (Tests 5-8) were
comparable to those with pH-adjusted bleach on
treated wood (Tests 6,8), but significantly lower
on tests involving concrete (Tests 5,7). Reduced
efficacy of peroxide-based decontaminants on
concrete is consistent with results from previous
studies,19 and suggests that this material may
catalyze the destruction of peroxide. Interestingly,
efficacy of Spor-Klenz® on wood (Test 6) was not
negatively affected by the inadvertent rinse (and
therefore reduced contact time) following the
second spray application. These results suggest
Spor-Klenz® is highly efficacious on wood and is
consistent with previous studies.19
30
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Log Reduction
I Log Reduction
3456
TestH
9 10
Figure 3-2. Material Surface Log Reduction for each Test Conducted
The stability of all concrete coupon samples was
called into question when a sample that yielded
'too numerous to count' (TNTC) originally failed to
produce any viable CPU six weeks later. The
PBST extraction buffer was not strong enough to
neutralize the alkalinity of the concrete wipe
sample; the extracted sample had a pH of 12
(following the 6-week storage at 4 C). The pH of
the sample matrix may have inactivated the
spores over this time period. This result has
minimal effect upon study results as most
samples were processed within hours of
collection. Further, decontamination efficacy was
calculated based upon control samples, so
antimicrobial activities post-sample collection
would have equally impacted recovery from
positive controls and thus have little impact on
overall efficacy. Another complication arising from
samples collected from concrete was that debris
from these wipe samples clogged the 200 uL
pipette tips used for dilution plating. These tips
were graduated to allow for a visual check that
the micropipette dispensed the correct volume.
While tips with a larger orifice did allow passage
of concrete debris, they did not possess
graduations and therefore did not provide the
same quality assurance during plating.
3.2.2 Evaluation of the pH-Adjusted Bleach
Application Procedure
For Tests 1-4 and 9-10, pH-adjusted bleach was
the sporicidal agent of choice, due in part not only
to the previously demonstrated efficacy of this
commonly-available solution, but also to the
evident incompatibility of Spor-Klenz® with the
selected equipment. To optimize the efficacy of
low-tech decontamination procedures, several
parameters were varied during the course of
testing.
• Tests 1 & 2: Apply one 30-sec pH-AB
spray with backpack sprayer, repeat 30-sec
spray after 15 minutes, and then rinse with Dl
water using a garden hose after 15 minutes
(30 minute contact time).
• Tests 3 & 4: Apply one 15-sec pH-AB
spray with chemical sprayer, repeat 15-sec
spray after 15 minutes, and then rinse with Dl
water using a pressure washer after 15
minutes (30 minute contact time).
• Tests 9 & 10: Apply one 10-sec pH-AB
spray with backpack sprayer and then rinse
31
-------
with Dl water using a garden hose after 15
minutes (15 minute contact time).
Figure 3-3 shows the LR from the material
coupon surface during decontamination in
comparison to the number of spores collected in
the rinsate (note: bars with values preceded by
"<" are detection limit values). The single
application (in addition to a shorter spray
duration) resulted in a lower total efficacy rate due
to the presence of active spores in the rinsate.
For tests with 30 minute contact time, the overall
surface spore removal was very consistent
across both spray durations and material types.
The shorter spray duration yielded a higher
number of viable spores in the rinsate. This
higher number of spores in the rinsate along with
the lower total efficacy for a single application of
pH-adjusted bleach suggests that a single
application would not be as effective or useful for
decontamination as two short applications. The
effectiveness of the chemical sprayer is
consistent with the backpack sprayer used in the
remainder of the pH-adjusted bleach tests. Direct
comparison of these two methods is complicated
by a shorter spray duration used in the chemical
sprayer tests. The flow rate for the backpack
sprayer is approximately 0.017 L/sec. A 30-
second spray dispenses 0.51 L of liquid onto the
coupon surface. The flow rate for the chemical
sprayer is approximately 0.185 L/sec. A 15-
second spray dispenses 2.75 L of liquid onto the
coupon surface. These results suggest that
smaller amounts of pH-adjusted bleach solution
can be just as effective as much larger amounts.
Application of decontaminant with both the
backpack sprayer and the chemical sprayer
resulted in compete wetting of the coupon
surface. The increased volume of
decontaminant applied with the chemical sprayer
likely only increased runoff from the coupon
surface and not exposure of spores to
decontaminant.
I Concrete LR
I Concrete Rinsate
Wood LR
I Wood Rinsate
2 Long 2 Short 1 short
Backpack Sprays with spray, 15
Sprays, 30 Power minute
minute washer, 30 contact,
contact, minute Rinse
Rinse contact,
Rinse
2 Long 2 Short 1 short
Backpack Sprays with spray, 15
Sprays, 30 Power minute
minute washer, 30 contact,
contact, minute Rinse
Rinse contact,
Rinse
Figure 3-3. Efficacy of pH-Adjusted Bleach Tests.
32
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3.2.3 Evaluation of the Spor-Klenz Application
Procedure
Tests 5-8 utilized Spor-Klenz® as the sporicidal
agent. To optimize the efficacy of this
decontamination method, several parameters
were modified during the course of testing.
• Tests 5 & 6 : Using a backpack sprayer,
apply one 30-sec Spor-Klenz® spray, repeat
30-sec spray after 15 minutes. Coupons were
inadvertently rinsed with Dl water
immediately after the second spray, resulting
in a contact time of 15 minutes.
• Tests 7 & 8: Using a pressure washer,
apply one 15-sec Spor-Klenz® spray, repeat
15-sec spray after 15 minutes, and then rinse
with Dl water after 15 minutes. (Actual
parameters varied; see discussion below)
Figure 3-4 shows that Spor-Klenz® was much
more effective as a sporicidal agent on wood
coupons than on concrete coupons (note: bars
with values preceded by "<" are detection limit
values). The lack of viable spores in the Spor-
Klenz concrete test rinsate (values in Figure 3-4
are detection limit values) could indicate that the
amount of sodium thiosulfate (STS) used to
neutralize the Spor-Klenz® was not adequate; the
neutralization equivalents used were those of
other researchers (USEPA Evaluation Report19)
and the Spor-Klenz® activity in the rinsate was not
independently verified. If the Spor-Klenz® was not
sufficiently neutralized, the spores may have
continued to be inactivated after the Dl rinse until
the samples were analyzed by the Microbiology
Laboratory. Another plausible explanation is that
despite the low decontamination efficacy on
concrete coupon surfaces, few viable spores
were relocated to the rinsate fraction.
The parameters for Test 8 (pressure washer on
wood coupons) were not met as there were
unforeseen malfunctions with the pressure
washer (apparent vapor lock). Test 8 received
only one contact time with Spor-Klenz® and was
rinsed with Dl water after a 34-minute total
exposure time. Overall, Spor-Klenz® seems to be
as effective as pH-AB on treated wood but less
effective on concrete. Further testing would be
necessary to determine its relative effectiveness
on other commonly tested materials.
33
-------
8.00
0.00
I Concrete LR
I Concrete Rinsate
Wood LR
I Wood Rinsate
2 Long Backpack 2 Short Sprays 2 Long Backpack 2 Short Sprays
Sprays, 30 minute with Power Sprays, 30 minute with Power
contact, Rinse washer, 30 minute contact, Rinse washer, 30 minute
contact, Rinse contact, Rinse
Figure 3-4. Efficacy of Spor-Klenz Tests
3.2.4 Ultimate Fate of Viable Spores
An overall assessment of the decontamination
procedural steps considers not only the viable
spores recovered from the surface of the
materials, but also those dislodged from the
coupon either through re-aerosolization (as
sampled by the Via-Cell ®) or into the rinsate.
3.2.4.1 Aerosol Samples (Via-Cef) - Task I
The chamber used for Task 1 decontamination
was designed for maximum air flow in order to
protect laboratory workers from the hazardous
fumes emitted by the decontamination procedure.
The aerosol sampling strategy initiallycalled for
sampling at a height and distance away from the
coupons typical of the breathing zone of a
decontamination worker. These bioaerosol
sample data are reported as CPU per liter of air
sampled, or roughly CPU per two breaths of air.
The data are shown in Table 3-4.
34
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Table 3-4. Bioaerosol Levels
Test ID
1
2
3
4
5
6
7
8
9
10
CFU/L in Aerosol
11.8
5.41
70.5
48.3
46.0
6.37
2.01
6.71
391
386
Sprayer Type
Backpack Sprayer
Backpack Sprayer
Chemical Sprayer
Chemical Sprayer
Backpack Sprayer
Backpack Sprayer
Power Washer
Power Washer
Backpack Sprayer
Backpack Sprayer
These samples were collected only during the
active spraying and are the maximum expected
concentrations in the test chamber. These
concentrations should not be viewed as a
maximum possible exposure, because it is
anticipated that the high rate of air exchange in
the chamber removed many of the spores upon
resuspension. The time interval over which these
concentrations might have been sustained is also
unknown. In a real-world area with less air
exchange, the concentrations experienced could
be much higher.
There was concern during testing that the
bioaerosol sample towards the front of the
chamber may not be representative of
concentrations throughout the chamber due to
high flow rates from the exhaust duct at the rear.
For Tests 7 and 8, a Via-Cell® cartridge was
placed in the duct to monitor the spores exiting
the chamber. These, combined with the total flow
rate of the duct, could provide a total number of
CPU re-aerosolized. These samples were not
part of the original sampling strategy, and there
were numerous difficulties due to the design of
both the Via-Cell® cartridges and the sampling
location. The volume of air sampled is not directly
known due to the failure of an engineering
control, but can be estimated by the sample flow
rate and the sample time. These estimations
suggest a concentration of nearly 7 x 104 CFU/L
could be re-aerosolized. When extrapolated from
the short duration of the sample collection, this
concentration represents approximately 1 x 108
total CPU re-aerosolized from the coupon
surfaces. Again, because real-time data were not
collected, the duration of time over which these
concentrations might be sustained is unknown, or
what the total number (or fraction) of spores re-
aerosolized might be. The data do show that re-
aerosolization of viable spores can be expected
during the decontamination process. This re-
aerosolization has a broad impact on the efficacy
of in-situ decontamination. Table 3-4 shows that
in Tests 9 andl 0, at least one order of magnitude
higher total number CPU re-aerosolized were
present as compared to the rest of the tests. This
result parallels the higher number of spores left in
the rinsate following a single application of pH-
adjusted bleach (see Fig. 3-3). These tests
employed only one application with pH-adjusted
bleach coupled with a shorter contact time. Re-
35
-------
aerosolized spores could settle on previously
decontaminated surfaces and thus complicate
remediation efforts. The purpose of the collection
of the bioaerosol samples was to determine if
the potential exists for re-aerosolization during
decontamination application procedures and not
to assess exposure quantitatively.
Table 3-5. Rinsate Sample CPUs
Rinsate
Test#
1
2
3
4
5
6
7
8
9
10
Total
CPUs
<24
<26
<44
100
<25
<27
<29
<27
13000
83000
3.2.4.2 Rinsate - Task I
For most Task 1 testing, the number of CPU
recovered in the rinsate was below the detection
limit and is shown in Table 3-5, below. However,
for Tests 9 and 10, a large number of viable
spores were physically removed from the surface
during the decontamination and rinse steps;
these spores could potentially re-contaminate
treated surfaces if not properly collected and
inactivated. When no viable spores were
detected, a value of 0.5 CPU was assigned as the
detection limit of the plated amount, and the
CPUs were reported as less than the detection
limit.
Table 3-5 shows that approximately 8x10 CPU
were present in the rinsate from Tests 9-10.
These tests employed only one application with
pH-adjusted bleach. These coupons had less
contact time with pH-adjusted bleach, which
resulted in less chemical inactivation. More viable
spores on the coupon at the time of rinsing led to
a higher number of viable spores in the rinsate.
3.3 Task II Results
3.3.1 Surface Sampling Results-Test Coupons
Based on the Task I results, the decontamination
procedure that was most effective was developed
for further testing in Task II: the use of pH-
adjusted bleach by backpack sprayer, sprayed on
either concrete or wood and rinsed with a garden
hose. In addition, in an effort to shorten the
required time for facility decontamination, a
36
-------
second test in Task II was conducted, similar to
the first test except the rinse step was omitted.
Both procedures used two 30-second spray times
every 15 minutes, for a total of 30 minutes
exposure per application. Again, Procedure 1
included a rinse step, and Procedure 2 did not
include this step. The results are shown in Figure
3-5. The results indicate that the two
decontamination approaches were equivalent in
decontaminating the two types of materials. The
results also suggest that a rinse step is not
needed for these decontamination procedures to
be effective on concrete and wood. However, if
applications were to be made to surface materials
sensitive to bleach (e.g., stainless steel), rinsing
might be desirable. On surfaces and materials
where corrosion is not a concern, elimination of
the rinse step could streamline the
decontamination process and significantly reduce
the amount of contaminated wastewater
generated. At facilities with minimal ventilation,
a rinse step may be necessary to reduce chlorine
off-gassing after decontamination.
7.00
6.00
5.00
4.00
3.00
2.00
1.00
0.00
Task II Efficacy (Log Reduction)
I Concrete
I Wood
2 Backpack Sprays, 30 min,
Rinse
2 Backpack Sprays, 30 min
Figure 3-5. Efficacy of Task II Decontamination Procedures
3.3.2 Ultimate Fate of Viable Spores
3.3.2.1 Aerosol Samples (Via-Cell®) - Task II
The Task II Bioaerosol sample results (Figure 3-
6) show some ambiguity. Test C2 suggests that
spores were dislodged during the first
decontamination step and were constantly
removed (due to air exchange) following that
release. During Test C1, however, a single
aerosol sample was two orders of magnitude
above samples taken two minutes before and two
minutes after. Given that no intentional activity
was done that could have initiated the spike, this
high spike in CFU in such a short amount of time
may be the result of cross contamination.
37
-------
u_
U
,0*
Viacells
•Test Cl
•Test C2
Figure 3-6. Bioaerosol Levels during Task II
Discarding this outlier, the aerosol data trend
downward as the decontamination progresses,
beginning before the decontamination steps were
started. Thus, not only is cross-contamination
likely, but the original presence of the spores is
due to either control sampling or re-aerosolization
of ambient spores in the COMMANDER
chamber. This result prevented decoupling of
airborne spore concentration from environmental
conditions such as air exchange rate and the
decontamination procedure itself. Hence, unlike
in Task I where ambient airborne spore
concentration can be shown to increase due to
decontamination steps, the data from Task II
neither supports nor refutes this proposition due
to the complex activity inside COMMANDER
before and during the decontamination.
The Task II aerosol sample results shown in
Figure 3-6 represent around 9 x 104 re-
aerosolized spores during Test C2,
demonstrating that airborne spores can be
expected in a field decontamination event. The air
exchange rate in COMMANDER is higher than
could be anticipated in a typical indoor
environment, and so could be seen as a best
case scenario (i.e., expect higher airborne spore
concentrations in atypical indoor environment
with a lower air exchange rate). During
decontamination, re-aerosolized spores could be
expected to move into and through the HVAC
system (if operating) during decontamination,
thereby spreading contamination to other areas of
the facility. In an outdoor environment, or in an
indoor facility typical of FAD operations with
higher airflow, these airborne contaminants could
be removed during decontamination and perhaps
contaminate areas adjacent to the initial
contamination zone or primary contaminated
facility.
3.3.2.2 Rinsate-Taskll
For Task II, the rinsate collection troughs were
immediately contaminated once brought inside
the COMMANDER chamber. The fact that the
contamination rate seems systematically higher
for concrete coupons than wood coupons
suggests that the contamination is coming from
the coupons themselves and not from the
common environment. The loose material from
the concrete coupons might have dropped into
the trough while it was being placed under the
coupon. While this complicates interpretation of
the data, the CFU counts after the
decontamination procedure were higher for Test
38
-------
C1, suggesting that active spores were
transferred to the rinsate (Table 3-6). No CPU
were detected following the decontamination
procedure in Test C2. Failure to detect any CPU
seemed unlikely given the presence of spores
before the decontamination began. Perhaps
excess STS may have caused inhibition of spore
outgrowth during cultivation of sample extracts.
For Test C1, the STS was quite dilute (due to the
presence of the rinse water), so less than 1
percent of the total amount used was present on
the filter. For Test C2, STS represented 40
percent of the total rinsate.
Table 3-6. CPU recovered from Task II Rinsate
Coupon
CVA
CVB
TWA
TWB
Testd
Rinsate before Decon
2.30E+04
2.00E+04
3.30E+03
3.33E+03
Rinsate after Decon
1.30E+05
2.84E+05
1.51E+05
1.41E+05
TestC2
Rinsate before Decon
4.93E+03
1.87E+03
1.00E+03
3.73E+02
Rinsate after Decon
<105
<398
<75
<205
3.4 Assessment of Operational Parameters
3.4.1 Time
The time required to decontaminate a batch of
coupons depended on the decontamination
procedure being applied. Experience using the
backpack sprayer decontamination procedure in
Task II suggested that 350 sq ft can be
decontaminated by one person before a second
application would be needed, which works out to
700 sq ft/hour. The rinse step could be performed
quickly at the end of a 4-hour shift, suggesting
that 2000-2500 sq ft could be decontaminated per
worker per 4-hour shift. Such an application
would require approximately 18 gallons/hour of
sporicide. This volume would require that the
backpack sprayer (5-gallon capacity) be refilled
every 15 minutes. Due to safety concerns with
fatigue while wearing a NFSA Class C suit,
cooling vests may be necessary to sustain a 4-
hour shift, especially in hot weather.
3.4.2 Physical Impacts on Materials
Treated wood and concrete showed no signs of
physical changes after being decontaminated.
Spor-Klenz® was incompatible with the
commercial off-the shelf pressure washer due
probably to its low pH. The apparent vapor lock
on the day of decontamination was probably an
effect of corrosion, as seen on the nozzle the
following morning (see Figure 3-7).
39
-------
Figure 3-7. Corrosion on Pressure Washer Nozzle from Contact withSpor-Klenz
3.4.3 Impact on Decontamination Workers
For the Task I study, the actual decontamination
procedure was moderately intensive with minor
discomfort at certain points in the procedure. The
procedure included standing in an upright position
while decontaminating materials.. Most individual
tasks were ergonomic in nature.
Task II tests were performed inside
COMMANDER in a completely different
environment than Task I tests. Since
COMMANDER is an enclosed space in which
chlorine levels are a safety hazard to any
member of the remediation crew, level B
HAZMAT suits were required for any
decontamination event in COMMANDER.
Supplied air respirators were used and 5 min
escape bottles were carried by personnel inside
the chamber. The supplied air was fed to the
respirators using air lines mounted inside
COMMANDER. The backpack sprayers were left
on the floor and the remediation crew simply
sprayed the coupons from a stationary position.
Space is limited inside COMMANDER, causing
otherwise simple tasks to require coordination
between team members. Although temperatures
approximated normal room temperature inside
COMMANDER, heat stress was a potential factor
while doing work wearing level C suits, so cooling
vests were worn inside the suits. At the end of a 2
hr decontamination cycle (including wipe
sampling upon entry), the crew was very fatigued
and the ice packs in the cooling vests had often
melted. For a member of an actual field crew,
there would be the added weight of a supplied air
cylinder and the need to carry the backpack
sprayer from position to position. In addition, if a
supplied air cylinder is being worn on the back, it
would probably be difficult to wear the backpack
sprayer correctly. Wearing a supplied air cylinder
would necessitate constant lifting of the sprayer
and all of the health and safety risks that are
inherent in such actions.
3.5 Summary of Results
Most tests performed during Task I achieved the
target efficacy from surfaces of greater than 6 LR,
a widely accepted standard for demonstrating
40
-------
sporicidal efficacy (e.g., 1 LR would be a
reduction of 10, 2 LR would be a reduction of
100, 6 LR would be a reduction of 1 million, etc.).
The decontamination by means of pH-adjusted
bleach was accomplished by a combination of
removal and inactivation of spores. Viable spores
were found in both the rinsate and bioaerosol
samples. Of the procedures tested, those
incorporating pH-adjusted bleach were more
effective for decontamination on concrete and
wood than Spor-Klenz®. The lower LR (4 LR)
seen in one test with wood may have been the
result of material demand (i.e., reduction in
activity of the decontaminant though reaction with
the test material) in conjunction with a single
application of the pH-adjusted bleach. One spray
application does not appear to provide enough
pH-adjusted bleach to overcome the demand of
wood. The surface LRs for tests utilizing Spor-
Klenz® were comparable to those with pH-
adjusted bleach on treated wood, but significantly
lower on tests involving concrete (< 3 LR).
Based on the Task I results, the most effective
decontamination procedures were developed for
further testing in Task II: the use of pH-adjusted
bleach by backpack sprayer, sprayed on either
concrete or wood, and rinsed or not rinsed. These
procedures all used two 30-second spray times
every 15 minutes, for a total of 30 minutes spray
exposure per application. Procedure 1 included a
rinse step, and Procedure 2 did not include this
step. The results indicate that the 2
decontamination approaches were equivalent in
decontaminating the two types of materials. The
results also suggest that rinsing is not needed for
these decontamination procedures to be effective
on concrete and wood. However, if applications
were to be made to surface materials sensitive to
bleach (e.g., stainless steel), rinsing might be
desirable from that standpoint as bleach and
other aggressive oxidants are known to cause
corrosion of numerous surfaces. LRs were
approximately 6 for concrete and just under 6 for
wood.
The overall fate of the biological spores was
assessed, not only for the viable spores
recovered from the surface of the materials, but
also fugitive viable agent escaping in the rinsate
and aerosol fractions. Aerosol samples
collected using bioaerosol filter cassettes during
testing with the "medium-sized" coupons show
that re-aerosolization of viable spores can be
expected during the decontamination process.
Although one test with the "large-sized" coupons
suggests that spores were dislodged during the
first decontamination step and were constantly
removed from the chamber (due to air exchange)
following that release, further evaluation of the
data indicates that there was probably cross-
contamination and re-aerosolization of ambient
spores in the chamber. However, the data do
indicate that spores can be expected to be re-
aerosolized in a field decontamination event and
could be expected to travel through the HVAC
system (if operating) during decontamination and
potentially spread contamination throughout a
facility.
For most of the "medium-sized" coupon testing,
the number of CPU recovered in the rinsate was
below the detection limit. However, in the tests
where only one short application of pH-AB was
used, a large number of viable spores were
physically removed from the surface during the
decontamination and rinse steps. Such rinsate
would potentially cause contamination to spread if
not properly collected and treated.
The collection troughs for the "large-sized"
coupon rinsate were immediately contaminated
once brought inside the test chamber during test
set-up. However, the rinsate contamination was
systematically higher for the concrete coupons
over the wood coupons. The contamination may
be coming from the coupons themselves. The
loose material from the concrete coupons might
have dropped into the trough while it was being
placed under the coupon. Despite the occurrence
of viable spores in the troughs prior to testing, the
data suggest that active spores were transferred
41
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to the rinsate as viable spore abundance in these
samples increased by approximately 1 x 105
following the decontamination procedure that
utilized a rinse step.
The major findings from this study are as follows:
• pH-Adjusted bleach was highly effective
(approximately 6 LR) on wood and
concrete when used with a thirty-minute
contact time and two applications.
• Spor-Klenz® was more effective on
wood than on concrete.
• For concrete coupons, pH-adjusted
bleach was more efficacious than Spor-
®
Klenz .
• Reduction of the number of pH-adjusted
bleach applications and contact time
resulted in lower decontamination
efficacy for surfaces and greater
amounts of spores detected in rinsate
and aerosol samples.
• Decontamination efficacy was similar
between the two evaluated application
devices (backpack sprayer and
pressurized sprayer) despite significant
differences in volume of decontaminant
delivered to the coupon surface.
• Viable biological agent was detected in
aerosol and rinsate (runoff) samples
during all tests, and can therefore be a
significant source of cross-
contamination during a remediation.
• Elimination of a rinse step from the
decontamination procedure did not
reduce surface decontamination efficacy
and may be a viable option on non-
corrosive materials.
• Worker fatigue may be of concern in an
actual remediation as heat and
exhaustion were experienced by
laboratory workers when conducting
scale-up tests that required level C
personal protective equipment.
42
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5. Quality Assurance and Quality Control
This project was performed under an approved
Category III Quality Assurance Project Plan titled
Effectiveness of Physical and Chemical Cleaning
and Disinfection Methods for Removing,
Reducing or Inactivating Agricultural Biological
Threat Agents (DCMD 3.41 B) (August 2010)5
5.1 Calibration of Sampling/Monitoring
Equipment
There were standard operating procedures for the
maintenance and calibration of all laboratory and
Microbiology Laboratory equipment. All
equipment was verified as being certified
calibrated or having the calibration validated by
EPA's Air Pollution Prevention and Control
Division (APPCD) on-site (RTP, 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.
Calibration of instruments was done at the
frequency shown in Tables 4-1 and 4-2. 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 4-1. Laboratory Instrument Calibration Frequency
Equipment
Thermometer
pH meter
Stopwatch
Clock
Analytical balance
Pressure Gauge
Sampling Pump Flow Rate
Calibration/Certification
Compare to independent NIST thermometer ( a
thermometer that is recertified annually by either
NIST or an International Organization for
Standardization (ISO)-17025 facility) value once per
quarter
Perform a single point calibration with standard
buffers daily.
Compare against NIST Official U.S. time at
(http://nist.time.goV/timezone.cgi7Eastern/d/-5/java)
monthly.
Compare to office U.S. Time (5), www.NIST.time.qov
at the start of each test (before coupon loading).
All analytical balances will be certified as calibrated
at time of use. Balances are recalibrated by the
Metrology Laboratory using standards. Evaluation
of balance performance to manufacturer's
specifications conducted yearly.
Compare to independent NIST Pressure gauge
annually.
Compare to a NIST certified and calibrated soap
bubble meter monthly
Expected
Tolerance
±1°C
± 0.1 pH units
±1second/min
±1 min/30
days
±5%
+2 psi
+ 1 Lpm
43
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Table 4-2. Microbiology Laboratory Instrument Calibration Frequency
Equipment
Thermometer
Pipettes
Analytical balance
pH Meters
Clock
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)-17025 facility) value once per quarter.
All micropipettes will be certified as calibrated at time of use. Pipettes are
recalibrated by gravimetric evaluation of pipette performance to
manufacturer's specifications every twelve months by supplier (Rainin
Instruments/Ovation) or credible calibration service.
All analytical balances will be certified as calibrated at time of use. Balances
are recalibrated by the Metrology Laboratory using standards. Evaluation of
balance performance to manufacturer's specifications conducted yearly.
Perform a 2-point calibration with standard buffers that bracket the target pH
daily.
Compare to office U.S. Time @ www.NIST.time.gov at the start of each test
(before coupon loading).
Expected
Tolerance
±1°C
±5%
±5%
± 0.1 pH units
±1 min/30 days
5.2 Data Quality Indicator (DQI) Goals
Target acceptance criteria for the critical
measurements are shown in Table 4-3 along with
precision goals.
44
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Table 4-3. Acceptance criteria and test values for critical measurements
Measurement Parameter
Free Available Chlorine (FAC) in pH-
adjusted bleach solution
pH of pH-adjusted bleach solution
Temperature of liquids
Head pressure of rinse water
Pressure of backpack sprayer
Flow rate of backpack sprayer
Flow rate of pressure washer
Positive control CFUs
CFU abundance on dilution plate**
CFU abundance on filter plate
Target Value
6000 - 6700 ppm
>6.5 pH <7.0
18-28°C
55-65 psi
30-40 psi
850-950 mL/min
10-11 kg/min
5x106 -5x107
CFU per ft2
30 - 300 CFU per
plate
<100
Test Value
6200-6800*
6.5-6.8
10.7*-25.1
60
30-36
990M104*
8.3*-8.7*
2.0 x 10s* - 4.9 x 10s
Taskl
6.8x104*-6.5x105*
Task 2
19* -296 CFU per plate
0-89 CFU
Outside the target range
This requirement only for plates used for quantification; plates outside this range were not used for quantification.
5.2.1 Free Available Chlorine (FAC)
Measurements
The Hach High Range Bleach Test Kit was used
to titrate a standard solution of 1000 ppm
NaCIO2. The Hach test kit returned a value
within 10 percent of the standard. The pH-
adjusted bleach FAC measurement was higher
than the target value for Test 2 during
decontamination of wood coupons due to a
personnel oversight. The LR for Test 2 may
have been slightly elevated in regards to the
other tests. As there were spores detected for
these samples, the overall effect of the slight
elevation of FAC is not expected to be
significant.
5.2.2 pH Measurements
The Oakton pH probe was calibrated with
certified pH 7.0 buffer solution per manufacturer's
instructions at the start of each test day. All the
results were within the specified target range.
5.2.3 Temperature Measurements
The contamination prevention protocol required
the deionized water reservoir to be filled the day
of testing to minimize cross-contamination.
Protocol for the daily filling of the deionized
reservoir consisted of the following steps: the
morning of testing, the reservoir was filled with a
diluted bleach solution, let sit for one hour,
emptied, triple-rinsed with Dl water, then refilled
with Dl water for testing. Therefore, the water
temperature was dependent on the room
temperature, and measurements outside the
target range were recorded. The temperature of
the Dl rinse water is expected to have minimal
effect on project results, therefore was allowed to
remain outside specification without corrective
action.
45
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5.2.4 Pressure Measurements
All pressure measurements were consistently
within specification.
5.2.5 Flow Measurements
The target flow rates listed in the QAPP for the
backpack sprayer were based on water; this
study used a non-water solution. The sprayer was
set to its lowest setting to provide a spray pattern
of 16-in diameter from a distance of 3 ft. The
same can be said for the pressure washer. In
general, the flow rates were consistent between
tests and did not affect the intra-test
comparisons.
5.2.6 CPU Counts
Twenty-five percent of all plates containing
significant growth (30-300 CPUs) were counted
by a second person, and fell within 10 percent of
the initial count. The positive control spore counts
were in a few instances below the target counts;
however, all were within an order of magnitude of
the target count and were deemed acceptable by
the EPA Work Assignment Manager (WAM).
Further, in some instances, the CPU abundances
on the dilution plates were taken as they were
when their values were near the lower CPU
acceptance criterion of 30.
5.3 Data Quality Audit
The ARCADIS QA Manager reviewed the final
report and randomly selected portions of reported
data to trace from the initial acquisition through
reduction to final reporting to ensure the integrity
of the reported results. Data from two tests from
Task I were selected (Test 2 and Test 7) and one
test from Task II (Test C2). For each of these
tests the following documentation was
reviewed: laboratory notebook entries,
laboratory test reports, and data tables within the
final report. Any discrepancies between reported
results and raw data files were brought to the
attention of the ARCADIS WAL and revised
and/or corrected as appropriate.
5.4 QA/QC Reporting
QA/QC procedures were performed in
accordance with the QAPP for this investigation.
5.5 Amendments and Deviations from the Original
QAPP
5.5.1 Formal Amendments
During the course of the projects, some
amendments were added to the QAPP by the
EPA WAM in response to data results or
equipment failures. These amendments, listed
below, were submitted by e-mail to the EPA QA
officer for formal approval.
Amendment 1 (10/12/2010)
Wipe Sampling Protocol (page 31, step 2b)
amended to read, "The wipe will be moistened by
adding 2.5 ml of sterile phosphate buffered
saline with 0.005% TWEEN-20" (instead of 5
ml).
Amendment 2 (10/22/2010)
Table 4-5, Coupon Sample Coding, was
replaced. The original table outlined a sample
nomenclature that did not permit easy
identification of control samples.
46
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Table 4-5: Coupon Sample Coding
Coupon Identification: T-S-M-NN
T/C
(Test Number)
S
(Sample Type)
M
(Material)
NN
(Sample number)
Code
1 -10
P
T
PX
FX
LX
R
VS
VD
S
CV
TW
SS
Dl
XX
##
Test Number preceded with T for Task l(Table 3-2)
and preceded with C for tests in Task II (Table 3-4)
Positive control wipe sample
Test wipe sample
Procedural blank
Field Blank
Lab Blank
Rinsate
Aerosol sample (Viacell Suspended in chamber)
Aerosol sample (Viacell in Duct)
Swab sample
Concrete (vertical orientation)
Pressure Treated Wood (vertical orientation)
Stainless Steel
Dl Water
Blank
Replicate number or sample area number
APPCD Microbiology Laboratory Plate Identification: T-S-M-NN-R-D
T-S-M-NN
Replicate
Dilution
As above
R
D
A-C
1 x10°-1 x104
Amendment 3 (11/08/2010)
Based on data from the first four tests, the Spor-
Klenz® power washer testing (Tests 7 and 8) was
changed to utilize an application rate of 15
seconds per 3 coupons (rather than 30 seconds)
(section 3.1.5.3.2).
Amendment 4 (11/22/2010)
During tests 7 and 8, the John Deere pressure
washer failed due to incompatibility with Spor-
Klenz®. Section 3.1.5.3.2 of the QAPP was
amended to state that sporicides would be
applied to the coupons via a Chemical Sprayer
47
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(Model# PP-UAG1003HU-K) made by UDOR
USA. The pressure washer would still be used to
rinse the decontaminant from the coupons once
the contact time had been achieved. In addition,
the remainder of tests would be conducted using
the following replacement pressure washer:
Troy Bilt
M# 020337
S# 1017273115
Max psi=2550
Max GPM=2.3
5.5.2 Deviations from the QAPP
Most of the data quality indicators for the critical
measurements were within their specified target
ranges as indicated in Table 4-3. However, in
some instances, some small deviations were
noted such as deionized water temperatures,
sprayer flow rates, or CPU counts. These small
deviations in measurements, although critical,
were consistent throughout the tests and did not
affect the intra-test comparisons
Amendment 5 (02/01/2011)
Based on test results, Task II decontaminant
spray to the large 101.6 cm by 101.6 cm (40 in by
40 in) coupons would be for 30 seconds per
application (rather than 90 seconds).
Amendment 6 (02/23/2011)
The last two Task II tests would be conducted
exactly as the first two tests, except that a rinse
step would not be conducted following the contact
time.
48
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6. References
1. Test Method for Quantitative Sporicidal
Three-Step Method (TSM) to Determine
Sporicidal Efficacy of Liquids and Vapor or
Gases on Contaminated Carrier Surfaces
(ASTME2414).
2. Determination of Efficacy of Liquid Sporicides
Against Spores of Bacillus subtilis on a Hard
Nonporous Surface Using the Quantitative
Three-Step Method (TSM) (AOAC 2008.05)
3. Rogers, J.V.; Richter, W.R.; Choi, Y.W.;
Flemming, E.J.; Shesky, A.M.; Cui, J.; Taylor,
M.L.; Riggs, K.B.; Willenberg, Z.J.; Stone,
H.J. Evaluation of Spray-Applied Sporicidal
Decontamination Technologies; Technology
Evaluation Report for Joe P. Wood of U.S.
Environmental Protection Agency: RTP, NC,
September 2006.
http://www.epa.qov/nhsrc/pubs/600r06146.pd
f, last assessed August 8, 2011.
4. Rastogi, V.K.; Wallace, L.; Smith, L.S.; Ryan,
S.P.; Martin, G.B. Quantitative Method to
Determine Sporicidal Decontamination of
Building Surfaces by Gaseous Fumigants,
and Related to Laboratory-scale Studies.
Applied and Environmental Microbiology
2009, 75(11), 3688-3694.
5. ARCADIS U.S., Inc. Quality Assurance
Project Plan for the Effectiveness of Physical
and Chemical Cleaning and Disinfection
Methods for Removing, Reducing or
Inactivating Agricultural Biological Threat
Agents (DCMD 3.41B).. Prepared under
Contract No. EP-C-09-027, Work Assignment
No. 1-35. U.S. Environmental Protection
Agency, National Homeland Security
Research Center, Research Triangle Park,
NC. July 2010. (Available Upon Request)
6. USEPA. Anthrax Spore Decontamination
using Bleach (sodium hypochlorite).
Pesticides: Topical & Chemical Fact Sheets.
Available at.
http://www.epa.gov/pesticides/factsheets/che
micals/
bleachfactsheet.htm.
7. Cousins, C.M.; Allan, C.D. Sporicidal
Properties of Some Halogens. Journal of
Applied Bacteriology 1966, 30 (1), 168 - 174.
8. Brazis, A.R.; Leslie, J.E.; Kabler, P.W.;
Woodward, R.L. The Inactivation of Spores of
Bacillus globigii and Bacillus anthracis by
Free Available Chlorine. Applied Microbiology
1958, 6(5), 338-342.
9. Babb, J.R.; Bradley, C.R.; Ayliffe, G.A.J.
Sporicidal Activity of Glutaraldehydes and
Hypochlorites and Other Factors Influencing
their Selection for the Treatment of Medical
Equipment. Journal of Hospital Infection
1980, 1,63-75.
10. ARCADIS U.S., Inc. Determination of the
Efficacy of Spore Removal from Carpets
using Commercially-available WetA/acuum
Carpet Cleaning Systems, Assessment and
Evaluation Report. Prepared under Contract
No. EP-C-04-023, Work Assignment No. 4-50
for U.S. Environmental Protection Agency,
National Homeland Security Research
Center, Office of Research and
Development, Research Triangle Park, NC.
Submitted March 2011. (In Preparation)
11. Busher, A.;Noble-Wang; J.; Rose, L. Surface
Sampling. In Sampling for Biological Agents
in the Environment; Emanual, P., Roos, J.W.,
Niyogi, K., Eds.; ASM Press: Washington,
DC, 2008; pp 95-131.
49
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12. Brown, G.S.; Betty, R.G.; Brockmann, J.E.;
Lucero, D.A.; Souza, C.A.; Walsh, K.S.;
Boucher, R.M.; Tezak, M.; Wilson, M.C.;
Rudolph, T. Evaluation of a Wipe Surface
Sample Method for Collection of Bacillus
Spores from Nonporous Surfaces. Applied
and Environmental Microbiology 2007, 73 (3),
706-710.
13. Sanderson,W.T.; Hein, M.J.; Taylor, L;
Curwin, B.D.; Kinnes, G.M.; Seitz, T.A.;
Popovic, T.; Holmes, H.T.; Kellum, M.E.;
McAllister, S.K.; Whaley, D.N.; Tupin, E.A.;
Walker, T.; Freed, J.A.; Small, D.S.; Klusaritz,
B.; Bridges, J.H. Surface Sampling Methods
for Bacillus anthracis Spore Contamination.
Emerging Infectious Diseases 2002, 8
14. After Action Report- Danbury Anthrax
Incident, U.S. EPA Region 1, September 19,
2008.
15. Rastogi, V. K., Wallace, L.; Smith, L.S.; Ryan,
S.P; Martin, B.. Quantitative method to
determine sporicidal decontamination of
building surfaces by gaseous fumigants, and
issues related to laboratory-scale studies.
Applied and Environmental Microbiology
2009, 75:3688-3694.
16. Rogers, J. V.; Sabourin, C.L.; Choi, Y.W.;
Rudnicki, D.C.; Riggs, K.B.; Taylor,M.L;
Chang, J. Decontamination assessment of
Bacillus anthracis, Bacillus subtilis, and
Gee-bacillus stearothermophilus spores on
indoor surfaces using a hydrogen peroxide
gas generator. Journal of Applied
Microbiology 99 (4):739- 748.
17. Tomasino, S. F., et al. 2010. Use of
alternative carrier materials in AOAC official
method 2008.05, efficacy of liquid sporicides
against spores of Bacillus subtilis on a hard,
nonporous surface, quantitative three-step
method. J. AOAC Int. 93:259-276.
18. Lee, SD; Ryan, S.P.; Snyder, E.G. 2011.
Development of an Aerosol Surface
Inoculation Method for Bacillus Spores.
Applied and Environmental Microbiology.
77(5): 1638-1645.
19. Calfee, MW2010. USEPA Technology
Evaluation Report- Biological Agent
Decontamination Technology Testing,
EPA/600/R-10/087, September 2010.
http://oaspub.epa.qov/eims/eimscomm.qetfile
?p download id=498369 (Assessed August
8,2011)
50
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Appendix A: Coupon Sterilization
Task I: Pressure-Treated Wood Coupons 35.6 cm by 35.6 cm (14 in by 14 in)
®
The pressure-treated wood coupons underwent sterilization using a STERIS VHP sterilization cycle. This
cycle entails the use of a STERIS VHP®ARD hydrogen peroxide (H2O2) generator. The coupons were
individually enclosed in H2O2 vapor-permeable sterilization bags (General Econopak, Inc.; Steam
Component Autoclave Bag, White, 20" by 20"; Item # 62020TW) and exposed to H2C>2at250 ppmvfor240
minutes by maintaining this minimum concentration in the airlock of COMMANDER. The coupons were
sterilized in batches. The number of coupons per batch was determined so that all coupons in the chamber
would be exposed to the vapor without shielding (e.g., no coupons were physically on top of others) and
appropriate mixing of the H2O2 occurs in the chamber. After sterilization, coupons of the same type were
placed in a sterile container for storage prior to use and transport to the testing location. The container was
marked with the contents, including the date of sterilization. One coupon from each material type and
sterilization cycle was sampled according to the sterilization sampling procedure described in Appendix F.
The samples from each material were analyzed qualitatively for the presence of any potentially confounding
contamination. Batches found to have the presence of contamination were re-sterilized. If after a second
sterilization cycle the batch was determined to still be contaminated, all coupons from the batch were
discarded.
Test parameters such as temperature, relative humidity and concentration were monitored and recorded to
ensure STERIS's defined quality standards were met. The quality of the cycle was considered in compliance
with STERIS's label as long as all parameters were within the manufacturer's specifications.
Task I: Concrete Coupons 35.6 cm by 35.6 cm (14 in by 14 in)
The STERIS VHP® sterilization cycle described above was determined to be inadequate for the sterilization
of the concrete coupons. These coupons were therefore sterilized by steam autoclave utilizing a one-hour
gravity cycle program consistent with an APPCD Microbiology Laboratory internal MOP 6570 (included in
Appendix C). Confirmation of sterilization was conducted as described above with respect to the coupons
sterilized using the STERIS VHP sterilization cycle. Prior to sterilization, concrete coupons were cleaned by
pressure-washing each with water to remove excess grit and loose agglomerations of concrete.
Task II: Pressure-Treated Wood and Concrete Coupons 101.6 cm by 101.6 cm (40 in by 40 in)
®
The large coupons used for Task II underwent sterilization using the STERIS VHP sterilization cycle
described above for the Task I pressure-treated wood coupons, but the large coupons were not enclosed in
sterilization bags. These coupons were tested in COMMANDER following sterilization.
MDI Control Check Stainless Steel Coupons
In addition to the test materials, metered dose inhaler (MDI) control coupons made of stainless steel 35.6
cm by 35.6 cm (14 in by 14 in) were also used as coupon inoculation controls. These coupons were
sterilized prior to use by steam autoclave utilizing a one-hour gravity cycle program consistent with an
APPCD Microbiology Laboratory internal MOP 6570. Confirmation of sterilization was done by sampling.
51
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Appendix B: Test Chamber and Equipment Cleaning Procedures
The pH-adjusted bleach solution to be used for cleaning surfaces of equipment in both the decontamination
and microbiology laboratories will be prepared as a 1:10 dilution of bleach in Dl water, pH-adjusted to ~6.8
using glacial acetic acid.
The following steps will be followed for cleaning the decontamination chamber between each material type
and before/after each test.
a. Using the backpack sprayer, the interior surfaces will be kept wet with pH-adjusted bleach solution for
10 minutes.
b. With the drain open, the surfaces will then be rinsed with Dl water. The rinsate will be collected in a
carboy and ultimately discarded.
c. After ensuring all rinsate is removed from the chamber, the valve will be closed in preparation for the
next test.
d. A mop assembly with a disposable pad will be used to wipe down the interior of the chamber with
isopropyl alcohol or ethanol.
e. The pad will be then removed and placed in a bucket of amended bleach solution for decontamination
prior to disposal.
The following steps will be followed for cleaning the work surfaces before and after use.
(R)
a. Wet all surfaces with pH-adjusted bleach solution or using Dispatch bleach wipes.
b. Rinse with Dl water.
c. Wet and wipe surfaces with isopropyl alcohol or ethanol.
d. Air dry prior to re-use.
e. Alternatively, cover paper can be used and replaced before/after each use.
The sampling templates will be autoclaved before/after each use.
The following steps will be followed for cleaning the coupon cabinets before and after use.
a. Wet and wipe all surfaces with pH-adjusted bleach solution or using Dispatch bleach wipes.
b. Rinse with Dl water.
c. Wet and wipe surfaces with isopropyl alcohol or ethanol.
d. Air-dry prior to re-use.
The gaskets used during the contamination procedure were cleaned via fumigation with the STERIS VHP®
sterilization cycle. This cycle entails the use of a STERIS VHP® ARD hydrogen peroxide (H2O2) generator
and exposure of all components of the wet/dry vacuum to \-\2®2 at 1000 ppmvfor60 minutes by maintaining
this constant concentration in a decontamination chamber.
Bins used in the study will either be filled with pH-adjusted bleach and left covered for at least 60 minutes,
rinsed with Dl water, and air-dried or cleaned by the following procedure:
®
a. Wet and wipe all surfaces with pH-adjusted bleach solution or using Dispatch bleach wipes.
52
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b. Rinse with Dl water.
c. Air-dry prior to re-use.
Alternatively to the use of pH-adjusted bleach for the sterilization of the materials and equipment used in
each decontamination test, the STERIS VHP sterilization cycle may be used. The equipment/materials will
be placed in either the ~900 cu. ft. stainless steel chamber, or the COMMANDER main chamber or airlock.
The sterilization cycle shall be a minimum of 250 ppmv H2O2 for at least 4 hours. Dehumidification to less
than 40% RH shall be done prior to the injection of H2O2 vapor. A minimum of 1000 ppmv-hours (dose or
CT = concentration by time) shall be achieved with the concentration above the minimum target of 250
ppmv (i.e., the CT clock shall be stopped if the concentration falls below this value.). VHP will be used for
all sterilization events in COMMANDER.
53
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Appendix C: Miscellaneous Operating Procedures (MOPs)
MOP 3135 Procedure for Sample Collection using BactiSwab™ Collection and Transport Systems
MOP 6535a: Serial Dilution: Spread Plate Procedure to Quantify Viable Bacterial Spores
MOP 6561: Aerosol Deposition of Spores onto Material Coupon Surfaces Using the Aerosol Deposition
Apparatus (An EPA proprietary method, unable to be disclosed at the time of writing this
report, patent pending)
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 6567: Recovery of Bacillus Spores from Wipe Samples
MOP 6568: Aseptic Assembly of Wipe Kits
MOP 6570: Use of STERIS Amsco Century SV 120 Scientific Prevacuum Sterilizer
MOP 6571: Recovery of Bacillus Spores from Via-cell Aerosol Sampling Cassettes
54
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MOP 3135
TITLE:
Procedure for WA 1-25: Procedure for Sample Collection using BactiSwab Collection
and Transport Systems
SCOPE: This MOP describes the procedure for collecting swab samples for Low Tech
Decontamination Technique Testing
PURPOSE: The purpose of this MOP is to ensure that all swab sampling is performed in a
consistent manner.
Equipment/Reagents
• Disposable laboratory coat
• Nitrile examination gloves
• P95 Respirator
• Shoe covers
• Bouffant cap
• Safety glasses
• BactiSwab™ Collection and Transport System
1.0 PROCEDURE
1. Enter the COMMANDER airlock wearing appropriate, project-specific PPE (at a minimum gloves, laboratory
coat, and safety glasses), making sure the airlock door is closed.
,TM
2. Through the sleeve, crush the BactiSwab ampule at midpoint.
3. Hold BactiSwab™ tip end up for at least five seconds to allow the medium to wet the swab.
4. Open the package and remove the BactiSwab
TM
55
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5. Label the plastic tube appropriately using the following scheme:
X-Y-N where,
X is the test number,
Y is the material abbreviation, and N is the material number.
6. Remove the cap-swab from the plastic tube.
7. Swab the surface following the recommend guidelines for each material while spinning the cap-swab
between the thumb and index fingers.
a. Brushes (B).
Pull the cap-swab through the brush bristles using one continuous stroke moving top to bottom and left to
right.
b. Nozzles (N).
Swab around the squeegee, inside the divisions, and inside the opening for the hose attachment.
c. Buckets (P).
Swab the sides and the bottom surfaces in an " S " pattern.
d. Brush Handles (BH).
Swab the top quarter of the handle top to bottom then bottom to top, turning the handle as you go.
e. Hoses (VH).
Swab inside and outside the hose opening that attaches to the nozzle.
f. Vacuums (V).
56
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Randomly swab the folds of the HEP A filter, swab the bottom of the vacuum lid, then swab the walls and
bottom of the canister. Swab the inside of the exhaust port.
8. Return cap-swab to tube.
9. Date and initial each sample tube. Enter this information into the laboratory notebook.
10. Complete the chain of custody form and relinquish the samples to the BioLab.
57
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BL MOP NO. 6535a 4-8-2009, rev. 2.0
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 (sterile deionized water, buffered peptone water or phosphate buffered saline)
Trypticase Soy Agar plates
Microliter pipettes with sterile tips
Sterile beads placed inside a test tube (will be used for spreading samples on the agar surface)
Vortex mixer
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 Trypticase Soy agar 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
58
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by vortexing for 10 seconds, and immediately pipette 100 |jl_ 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 ISA 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.
5- 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.
6- Incubate the plates overnight at 32 °C - 37 °C (incubation conditions will vary depending on the
organism's optimum growth temperature and generation time.)
7- Enumerate the colony forming units (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).
Since each dilution was tested in triplicate, determine the average of the triplicate plate
abundances. Plates suitable for counting must contain between 30 - 300 colonies.
Calculations
Total abundance of spores (CPU) within extract:
(Avg CPU / volume (ml_) plated) X (1 / tube dilution factor) X extract volume
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For example:
Tube Dilution Volume plated Replicate CPU
10~3 100|jL (0.1 ml) 1 150
10~3 100|jL (0.1 ml) 2 250
10~3 100|jL (0.1 ml) 3 200
Extract total volume = 20 ml
(200 CPU/0.1 ml) X (1/10"3) X 20 ml =
(2000) X (1000) X 20 = 4.0 X107
Note: The volume plated (ml) and tube dilution can be multiplied to yield a 'decimal factor' (DP). DP can
be used in the following manner to simplify the abundance calculation.
Spore Abundance per ml_ = (Avg CPU) X (1 / DP) X extract volume
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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
(Dl) 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
urn cellulose acetate filter) for sterilization. Complete this procedure 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.
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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.
1. Prepare the hood by wiping down with ethanol, followed by bleach, followed by Dl water and a
clean Kimwipe or Texwipe. 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 1/4 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.
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
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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 Dl water and a clean
Kimwipe or TexWipe.
4.0 PREPARING 900 jiL PBST TUBES FOR USE DURING EXPERIMENTATION
1. Prepare the hood by wiping down with ethanol, followed by bleach, followed by Dl water and a
clean Kimwipe or Texwipe. Then stock the hood with the following items if they are not already there:
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 uL micropipette. - 1000 uL 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.
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3. Add 900 |jl_ 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 uL 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 uL 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 uL 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 900 |jL 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.
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 Dl water and a clean
Kimwipe or TexWipe.
BL MOP NO. 6565 03-14-2011, rev. 3
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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 (CPU) 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 decontamination rinsates.
Materials: Petri dishes with appropriate agar
0.2 urn pore-size disposable analytical filter units (2-3 per sample)
P1000 pipette and sterile tips
Sterile forceps
Pipetman and sterile serological pipettes
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/QC'd media.
(Note: for analysis of 5 to 30 ml extracts, 1 ml and remainder should be filtered: for 31 to 200 ml
samples, 1 ml, 10 ml, and remainder should be filtered: for samples over 200 ml, more filter
samples may be needed)
(Note #2: For previously plated samples where 10-19 CFU were observed, replatinq using a 400
uL inoculum, and plates where 20 - 29 CFU were observed, replatinq using a 200 ul inoculum can
be executed rather than filter plating. For inocula greater than 200 ul, a sterile spreader should be
used rather than the bead method).
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)
4- Using a P1000, sterile tip, and aseptic techniques, immediately following vortexing, pipette 1 ml of
the extract 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.
6- Using a sterile serological pipette, rinse the filter unit by pipetting 10 ml of sterile deionized water
along the inner sides of the unit while it is under vacuum.
7- Aseptically remove the filter from the filter apparatus using sterile forceps and lay the filter onto the
agar surface within the Petri dish (spore side up).
8- Vortex the liquid extract vigorously for 10 seconds.
9- Use the appropriate volume serological pipette to transfer the remaining aliquots into their
respective filtration units (one at a time).
10- Repeat steps 5 through 7 taking time to vortex or mix the sample 10 seconds immediately before
removing an aliquot.
Important: Be sure to note and record the volume of the "remainder" sample.
11 - Incubate all plates at the optimal growth temperature for the organism used for 16 - 28 hours.
12- Enumerate and record the number of CPU on each plate.
Data Calculations
Utilize the following equation to determine the total abundance of recovered spores:
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= CFUx- Extract
'filtered
where N is the total number of spores recovered in the extract, CPU is the abundance of colonies
on the agar plate, VExtmct is the total volume of the extract (before any aliquots were removed),
the volume of the extract filtered.
MOP 6567
Title: RECOVERY OF BACILLUS SPORES FROM WIPE SAMPLES
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Scope: This MOP outlines the procedure for recovering Bacillus spores from wipe samples
Purpose: To aseptically extract and quantify Bacillus spores from wipe samples in order to determine
viability and obtain quantifiable data.
1.0 MATERIALS
• PPE (gloves, laboratory coat, safety goggles)
• Biological Safety Cabinet (Class II)
• pH-Amended bleach
• Deionized water
• 70% Solution of denatured ethanol
• Kimwipes
• Dispatch® bleach wipes
• Non-regulated waste container
• 50 ml sterile conical tubes containing 20 ml of sterile phosphate buffered saline with Tween® 20
solution (PBST) (MOP 6562)
• Vortex mixer
• Cart
• Wire or foam rack for 50 ml conical tubes
• Tryptic soy agar plates
• 900 uL tubes of sterile PBST
• Pipettor and pipette tips for dilutions
• Incubator set to appropriate growth temperature for target organism (35 °C or 55 °C)
• Light box for counting colonies
• Laboratory notebook
• QAPP for project that is utilizing the wipe samples
2.0 PROCEDURE
1 . Begin by donning PPE (gloves, laboratory coat, and protective eyewear).
2. Obtain wipe samples that may contain Bacillus spores. Wipe samples should be received as one
wipe/sponge in a sterile 50 mL conical tube delivered in secondary containment. Make certain that all
of the samples are labeled. Review any chain of custody forms that may accompany the samples to
ensure that all of the labels are consistent and that there is no notable variation in the samples. If
variation has occurred, make a note of it in the notebook.
3. Clean the workspace (biological safety cabinet) by wiping surfaces with pH-amended bleach, next with
deionized water, and lastly with a 70-90 % solution of denatured ethanol. Wipe with a Kimwipe to
remove any excess liquid. Make sure the workspace is clean and free of debris. Gather all
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necessary items to perform the task, place these items on a clean cart beside the biological safety
cabinet, within arm's reach so that once the procedure has begun, the task may be performed without
interruptions.
4. Discard gloves and replace with fresh pair.
5. One at a time, under the biological safety cabinet, remove the sample tube containing the wipe sample
from the secondary containment bag in which it arrived. Using the Dispatch® bleach wipes, wipe each
sample tube with one wipe, and then wipe it with a clean Kimwipe. Discard the used bleach wipe and
the used Kimwipe in the secondary containment bag and place them in the non-regulated waste
container. Remove gloves and don a fresh pair of gloves. Repeat this procedure for every sample.
After each sample has been cleaned, place the tubes containing the wipe samples in an appropriate-
sized wire or foam rack to hold the tubes in an upright vertical position.
6. Leaving the tubes in the rack underneath the biological safety cabinet, aseptically add 20 ml of PBST
solution (this should be in a pre-measured, sterile conical tube, per MOP 6562) to each sample tube
containing a wipe, one a time. Remove the rack containing wipe samples from the hood when all
samples have had the PBST added. Place the rack with the samples on the cart.
7. Using the procedure to clean the biological safety cabinet, as found in Step 3, clean the biological safety
cabinet again. Afterwards don a fresh pair of gloves.
8. Using a vortex mixer, agitate the wipe samples, four at a time, in a biological safety cabinet, for ten
second bursts for two minutes total. Make certain to clean the biological safety cabinet after each set
of four samples and change gloves between each set of samples.
Note: The reason that four samples are done at one time is to limit the time between agitation
and plating. The samples need to be processed immediately after agitation, and agitation of
more than four samples at a time leaves too much time between agitation and spread plating.
9. Using tryptic soy agar media plates that are appropriately labeled with the sample number, dilution set
and date, complete dilution plating for the wipe samples immediately after the two-minute agitation step
is completed. The samples should also be agitated again for ten seconds directly prior to removing an
aliquot from the sample tube. Each dilution tube should also be agitated for ten seconds prior to removal
of aliquots. Dilutions should be completed using the techniques and methodology as described in
MOP 6535a, and the 900 uL tubes should be made with sterile PBST to stay consistent with
materials/solutions. Plating in this manner should be repeated for all samples, with any changes in
protocol noted in the laboratory notebook.
10. Once the dilution plating has been completed, the plates are to be placed in an incubator. For non-
thermophilic Bacillus species, the plates should be placed at 35 °C ± 2 °C for 12-24 hours. For
thermophilc Bacillus species, such as Gee-bacillus stearothermophilis, the plates should be incubated at
55 °C ± 2°C for 12-24 hours. The target Bacillus organism that will be used for the wipe samples will be
specific to the project and noted in the QAPP.
11. After the plates have incubated for a sufficient amount of time (12-24 hours) and the growth from any
Bacillus colonies is quantifiable, the colonies should be manually counted using the light box and the
data should be properly recorded as dictated per project by the QAPP. All results will be checked for
quality assurance and all data will be reported to the proper personnel as listed in the QAPP.
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MOP 6568
Title: ASEPTIC ASSEMBLY OF WIPE KITS
Scope: This MOP outlines the procedure for the aseptic assembly of wipe kits.
Purpose: To aseptically assemble kits that will be used to collect wipe samples from which
quantifiable data will be derived.
1.0 MATERIALS
PPE (gloves, laboratory coat, safety goggles)
Biological Safety Cabinet (Class II)
pH-Adjusted bleach
Deionized water
70% Solution of denatured ethanol
Kimwipes
Sterile, sealed Twirl-em® bags in two sizes, 10"x15" and 5.5"x9"
Sterile Kendall 4-ply all-purpose sponges
Sterile, disposable thumb forceps
50 ml conical tubes containing 5 ml PBST tubes (MOP 6562)
Sharpie
Wire or foam rack for 50 ml conical tubes
Secondary containment such as a large Tupperware bin
Laboratory notebook
QAPP for project that is utilizing the wipe samples
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2.0 PROCEDURE
2.1 Preparation for Wipe Kit Assembly
Prior to wipe kit assembly, 50 ml sterile conical tubes containing 5 ml of sterile PBST and a sterile 2-ply
sponge must be put together. They are assembled in the following manner:
1. Begin by donning PPE (gloves, laboratory coat, and protective eyewear).
2. Clean the workspace and biological safety cabinet by wiping surfaces with pH-adjusted bleach, followed
by deionized water, and then with a 70% solution of denatured ethanol. Wipe the surfaces with a
Kimwipe to remove any excess liquid. Make sure the workspace is clean and free of debris. Gather
all necessary items to perform the task, place these items on a clean cart beside the biological safety
cabinet, within arm's reach so that once the procedure has begun, the task may be performed without
interruptions.
3. Discard gloves and replace with fresh pair.
4. Place the sterile 50 ml conical tubes containing 5 ml PBST tubes under the biological safety cabinet in
a foam or wire rack designed to hold 50 ml conical tubes. Using two sterile, disposable thumb forceps,
aseptically transfer one half of a 4-ply sterile all-purpose sponge to each of the tubes. Complete the
transfer by using the two forceps together to first separate the 4-ply sponge in half to create two 2-ply
sponges. Then remove a cap from one of the tubes, carefully fold one of the 2-ply sponges using the
forceps together and aseptically place it in the opening of the tube so that it sits at the top portion of the
tube, while the 5 ml of PBST remains at the bottom of the tube. Replace the cap on the tube. Repeat
this process until all of the tubes have sponges in them. Once all of the tubes contain sterile sponges,
then label the tube rack appropriately with the action completed, the date and your initials and place the
tubes on the shelf. These tubes are shelf-stable for up to three months.
2.2 Assembly of Wipe Kits
Wipe kits are assembled in the following manner:
1 . No more than 48 hours prior to testing or collecting samples, assemble the wipe kits. Wipe kits can be
assembled outside the biological safety cabinet, in a dry, clean area. Make certain to use proper PPE,
including gloves, while handling all wipe kit materials. Gather all materials to assemble the kits before
assembly. These materials include:
50 ml conical tubes containing both a sterile wipe sponge and 5 ml PBST
Twirl-em
Sharpie
Twirl-em® bags in two sizes, 1 0"x1 5" and 5.5"x 9"
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Vortex mixer
2. Obtain a copy of the labeling scheme for the samples. This scheme may be detailed in the QAPP.
For each wipe kit, use a Sharpie
containing the sponge and PBST.
For each wipe kit, use a Sharpie and label a large 10" x 15" Twirl-em® bag and a 50 ml conical tube
3. Once all of the tubes are labeled, use the vortex mixer on the highest setting to agitate the tube. This
will mix the sponge, which was placed at the top of the tube, with the 5 ml of PBST.
4. Open the labeled, 10" x 15" Twirl-em® bags one at a time. Place the labeled, agitated tubes in the 10"
x 1 5" Twirl-em® bags that have the corresponding label (that matches the tube). Add a non-labeled,
sealed 5.5" x 9" Twirl-em® bag into the 10"x 15" Twirl-em® bag, along with the tube containing the wipe
sponge to complete the wipe kit assembly. Record the time and date on which the wipe kits were
assembled in the laboratory notebook; include the labeling schematic for the wipe kits.
5. Place the assembled wipe kits into a secondary containment, such as a large Tupperware bin. Use
within 48 hours. When moving the kits to a sampling location, always have them in secondary
containment.
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BL MOP NO. 6570 July 2010
Title: Use of STERIS Amsco Century SV 120 Scientific Prevacuum Sterilizer
Scope: Basic instructions for use of the autoclave
Purpose: To outline proper use of the autoclave, using preprogrammed cycles, to effectively sterilize
media, supplies, or waste.
Materials
Amsco Century SV 120 Scientific Prevacuum Sterilizer
Items to be sterilized (liquids, solids, waste, etc)
Pouches to contain materials to be sterilized and maintain that state until later use
Aluminum foil Autoclave Indicator Tape
Sterilization Verification Ampoules
Thermally resistant gloves or tongs
Deionized (Dl) water
Procedure:
Basic start up:
1. Turn on the autoclave. The power switch is located behind the door in the top right corner. The
digital touch screen on the front of the unit will power up and indicate that a memory test is in
progress.
2. After the memory test is complete, the device will request that it be flushed. This flushing should be
conducted daily to minimize scaling inside the boiler. The flush valve is located on the bottom left of
the device (yellow handle). Actuate the valve to the open position and then press the "Start Timer"
button on the touch screen. The flush will run for 5 minutes and will alert at completion with a single
chime.
3. Once the flush is complete, close the flush valve and press the "Continue" button on the touch
screen. The screen should then return to its default menu which has two choices "Cycle Menu" and
"Options"
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Basic Operation:
1. Prepare any items that need to be sterilized. The items must be carefully wrapped or sealed in
sterilization pouches in order to maintain sterility when removed from the autoclave. Examples of
this wrap or seal include: wrapping any orifices with aluminum foil, placing whole items in
autoclave pouches, loosely applying a cap on a bottle (to allow for the pressure changes inside).
2. Once prepared, each item should be outfitted with a sterility indicator. This indicator can be
completed by applying a small piece of autoclave indicator tape to an item or by utilizing an
autoclave pouch with a built-in sterility indicator strip. These indicators provide a visual verification
that the sterilizing temperature (121 °C) was reached.
3. To add items to the autoclave, open the autoclave door by pressing down on the foot pedal on the
bottom right corner on the front of the device.
4. Place items that need to be sterilized into the autoclave, adding or moving racks to accommodate
the load. If liquids are being autoclaved, then they must have secondary containment (usually a
large plastic autoclave-safe tray) to contain any fluids in the event of a leak, spill or boil-over.
5. Once the autoclave is loaded, press the foot pedal to close the autoclave door.
6. Once the door is sealed, a menu of the cycles can be seen by pressing the button on the touch
screen labeled "Cycle Menu". Then choose the appropriate cycle by touching the corresponding
button. If the cycle chosen is the one desired for the sterilization process, press the "Start Cycle"
button. Otherwise, press "Back" to return to the prior menu screen.
7. After the cycle has started, the type of cycle, the number of the cycle, the items placed in the
autoclave during the cycle, the time, whether or not a QC ampoule was included in the load, and
the initials of the person starting the cycle must be recorded in the autoclave log book, located in
the drawer across form the unit labeled "Autoclave Supplies."
8. QC ampoules, usually Raven Prospore Ampoules with Geobacillus stearothermophilus, are added
to one cycle each day to ensure that the autoclave is functioning properly. These ampoules are
used according to manufacturer's instructions.
9. Upon completion of a cycle, the autoclave will alarm with a repeating beep for approximately one
minute. Any time after this alarm starts, it is safe to open the main door (take caution because the
steam escaping the chamber will be very hot when the door is opened). The contents of the
autoclave will be very hot; thermal protection for the hands is therefore required to remove the items
(thermally resistant cloth gloves or tongs).
10. Place the contents of the autoclave in an appropriate place to cool and close the autoclave door
using the foot pedal.
Cycles:
Gravity Cycles:
Gravity cycles are used to sterilize glassware and other utensils which are not submerged in nor contain any
volume of liquid. These cycles are typically used for "dry" materials.
Currently there are two different gravity cycles programmed for daily operations: a 1 -hour cycle and a 30-
minute cycle. The time that the chamber is held at the sterilization temperature (121 °C) is the only
difference between these two cycles. The different sterilization times allow for the compensation of the
various sizes of materials and more resilient organisms. The 30-minute cycle is primarily used fora small
quantity of material. The 1-hour cycle is used for large loads or items containing a large amount of
contamination. The 1-hour cycle is recommended for inactivation of gram positive spore-forming bacteria.
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Liquid Cycles:
Liquid cycles are used to sterilize a variety of liquids and solutions. The solutions are typically mixed prior to
sterilization. It is important to have secondary containment to contain any fluids in the event of a leak, spill or
boil-over. The 30-minute liquid cycle is used to sterilize small volumes of liquid (usually less than 2 L total).
When attempting to sterilize any volume larger than 2 L, the 1-hour liquid cycle should be used to ensure
complete sterilization. The 1-hour liquid cycle is the preferential cycle used as the destruction cycle for
waste. In the event of materials (liquid or otherwise) being contaminated/exposed to microorganisms, the 1-
hour liquid cycle will be used as the initial means of decontamination. When completing a decontamination
cycle, if there is no liquid inside a container, then deionized water must be added to the container or the item
must be submerged prior to the start of the cycle. Only items that are being decontaminated can go in
destruction cycles. Decontamination cycles cannot be mixed with sterilization cycles.
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MOP 6571
Title: RECOVERY OF BACILLUS SPORES FROM VIA-CELL® AEROSOL SAMPLING
CASSETTES
Scope: This MOP outlines the procedure for recovering Bacillus spores from Via-Cell® aerosol
sampling cassettes
Purpose: To aseptically extract and quantify Bacillus spores from Via-Cell® samples in order to
determine viability and obtain quantifiable data.
MATERIALS
• Via-Cell® Bioaerosol Sampling Cassettes (Zefon International, Ocala, FL, Part# VIA010)
• PPE (gloves, laboratory coat, safety goggles)
• Biological Safety Cabinet (Class II)
• pH-Amended bleach
• Deionized water
• 70% Solution of denatured ethanol
• Kimwipes
• Dispatch® bleach wipes
• Non-regulated waste container
• 50 ml sterile conical tubes containing appropriate volume of buffer
• Vortex mixer
• Cart
• Wire or foam rack for 50 ml conical tubes
• Sterile blade
• Sterile, disposable forceps
• Tryptic soy agar plates
• 900 uL tubes of sterile PBST
• Pipettor and pipette tips for dilutions
• Incubator set to appropriate growth temperature for target organism (35 °C or 55 °C)
• Light box for counting colonies
• Laboratory notebook
• QAPP for project that is utilizing the wipe samples
PROCEDURE
1. Begin by donning fresh PPE (gloves, laboratory coat, and protective eyewear).
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2. Obtain Via-Cell® samples that may contain Bacillus spores. Via-Cell® samples should be received as
one Via-Cell® cassette delivered in secondary containment. Make certain that all of the samples are
labeled. Review any chain of custody forms that may accompany the samples to ensure that all of the
labels are consistent and that there is no notable variation in the samples. If variation has occurred,
make a note of it in the notebook.
3. Clean the workspace (biological safety cabinet) by wiping surfaces with pH-amended bleach, next with
deionized water, and lastly with a 70-90 % solution of denatured ethanol. Wipe with a Kimwipe to
remove any excess liquid. Make sure the workspace is clean and free of debris. Gather all necessary
items to perform the task, place these items on a clean cart beside the biological safety cabinet, within
arm's reach so that once the procedure has begun, the task may be performed without interruptions.
4. Discard gloves and replace with fresh pair.
5. One at a time, under the biological safety cabinet, remove the sample cassette. Using a sterile blade,
cut through the tape around the outside of the cassette. Twist apart the cassette and discard the top
portion not containing the sample slide (portion of the cassette where the sample is collected). Using
sterile, disposable forceps, remove the slide and place into the appropriate amount of buffer solution.
Repeat this procedure for every sample.
6. Using the procedure to clean the biological safety cabinet, as found in Step 3, clean the biological safety
cabinet again. Afterwards don a fresh pair of gloves.
7. Using a vortex mixer, agitate the Via-Cell® samples, four at a time, in a biological safety cabinet, for ten
second bursts for two minutes total. Make certain to clean the biological safety cabinet after each set
of four samples and change gloves between each set of samples.
Note: The reason that four samples are done at one time is to limit the time between agitation
and plating. The samples need to be processed immediately after agitation, and agitation of
more than four samples at a time results in excessive lag-time between agitation and plating.
8. Using tryptic soy agar (or other appropriate growth media) media plates that are appropriately labeled
with the sample number, dilution set and date; conduct dilution plating for the Via-Cell® samples
immediately after the two-minute agitation step is completed.-The samples should also be agitated
again for ten seconds directly prior to removing an aliquot from the sample tube. Each dilution tube
should also be agitated for ten seconds prior to removal of aliquots. Dilutions should be completed using
the techniques and methodology as described in MOP 6535a, and the 900 uL tubes should be made
with the appropriate buffer to stay consistent with materials/solutions. Plating in this manner should be
repeated for all samples, with any changes in protocol noted in the lab notebook.
9. Once the dilution plating has been completed, the plates are to be placed in an incubator. For non-
thermophilic Bacillus species, the plates should be placed at 35 °C ± 2 °C for 18-24 hours. For
thermophilc Bacillus species, such as Gee-bacillus stearothermophilis, the plates should be incubated at
55 °C ± 2 °C for 18-24 hours. The target Bacillus organism that will be used for the wipe samples will be
specific to the project and noted in the QAPP.
10. After the plates have incubated for a sufficient amount of time (18-24 hours) and the growth from any
Bacillus colonies is quantifiable, the colonies should be manually counted using the light box and the
data should be properly recorded as dictated per project by the QAPP. All results will be checked for
quality assurance and all data will be reported to the proper personnel as outlined in the QAPP.
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Appendix D: Spore Deposition and Handling Procedures
The handling of the contaminated coupons for Task I, including movement to minimize or control spore
dispersal, was done in accordance with the MOP 6561 (a proprietary method unable to be disclosed at the
time of writing this report). One person was tasked with removing the clamps holding the dosing chamber to
the coupon and the removal of the dosing chamber and gasket from the coupon. A second person was then
tasked with moving the coupon to the proper location (e.g., test and positive control coupons to the Test
Coupon Cabinet and blank coupons to the Blank Coupon Cabinet).
For Task II, two personnel were used to move the 101.6 cm by 101.6 cm (40 in by 40 in) coupons into their
vertical positions in COMMANDER following removal of the deposition devices. This was the only time the
coupons were handled, and this handling occurred a minimum of two days prior to sampling.
For Task I, the Test Coupon Cabinet was a steel cabinet (48 in wide by 24 in deep by 78 in high) with twelve
shelves each 6 in apart. Each cabinet held a total of 36 coupons, so two Test Coupon Cabinets were
needed for a test. These cabinets were labeled as Test Coupon Cabinet 1 and Test Coupon Cabinet 2. Test
and positive control coupons were arranged in each cabinet according to material types. A single material
type was not split among cabinets. Procedural blank coupons of each material/orientation to be used in a
single test were contained in a separate isolated cabinet (Blank Coupon Cabinet) of similar construction,
however, with dimensions of 48 in wide by 24 in deep by 36 in high with 3 shelves.
Each MDI was claimed to provide 200 discharges. The number of discharges per MDI was tracked so that
use did not exceed this value. Additionally, in accordance with MOP 6561 (a proprietary method), the weight
of each MDI was recorded after completion of the contamination of each coupon. If an MDI weighed less
than 10.5 g at the start of the contamination procedure, the MDI was retired and a new MDI used. For
quality control of the MDIs, a contamination control coupon was run as the first, middle, and last coupon
contaminated with a single MDI in a single test. The contamination control coupon was a stainless steel
coupon (1.2 ft by 1.2 ft) that was contaminated, sampled, and analyzed.
A log was maintained for each set of coupons that were dosed via the method of MOP 6561 (a proprietary
method). Each record in this log recorded a unique coupon identifier (see Table D-1), the MDI unique
identifier, the date, the operator, the weight of the MDI before dissemination into the coupon-dosing device,
the weight of the MDI after dissemination, and the difference between these two weights. The coupon codes
were pre-printed on the log sheet prior to the start of coupon contamination (dosing).
Additionally, after a coupon was dosed via the above procedure, the coupon was labeled with the unique
identifier using the coding outlined in Table D-1. The label was printed on the side of the coupon using a
permanent marker (e.g., black or silver Sharpie®). The sampling team maintained an explicit laboratory log
which included records of each unique sample number and its associated test number, contamination
application, any preconditioning and treatment specifics, and the date treated. Each coupon was marked
with only the material descriptor and unique code number. Once the coupons were transferred to the
APPCD Microbiology Laboratory for plate counts, each sample was additionally identified by replicate
number and dilution.
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Table D-1. Coupon Sample Coding
Coupon Identification: T-S-M-NN
T/C
(Test Number)
S
(Sample Type)
M
(Material)
NN
(Sample number)
Code
1 -10
P
T
PX
FX
LX
R
V
S
CV
TW
SS
Dl
XX
##
Test Number preceded by T for Task 1 (Table 2-1)
and preceded by C for tests in Task 2 (Table 2-1)
Positive control wipe sample
Test wipe sample
Procedural blank
Field Blank
Laboratory Blank
Rinsate
Aerosol sample
Swab sample
Concrete (vertical orientation)
Pressure Treated Wood (vertical orientation)
Stainless Steel
Dl Water
Blank
Replicate number or sample area number
APPCD Microbiology Laboratory Plate Identification: T-S-M-NN-R-D
T-S-M-NN
Replicate
Dilution
As above
R
D
A-C
1 x10°-1 x104
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Appendix E: Contamination Prevention and Quality Control Measures
Coupon Storage Cabinets
On the decontamination procedure test day, the procedural blank, test, and positive control coupons have
been placed into the appropriate cabinets. A total of three cabinets were used to contain the coupons prior
to decontamination (one for the procedural blanks and two containing the contaminated (positive controls
and test) coupons). One additional cabinet was used to store test coupons for drying after the
decontamination procedure had been applied. The cabinets with their intended purpose are listed in Table
E-1.
Table E-1. Coupon Storage Cabinets
Cabinet Name
Description
Test Coupon Cabinet #1
Test Coupon Cabinet #2
For storage of contaminated coupons (both positive control and
test coupons); each cabinet can hold 36 coupons, so two
cabinets will be needed for all tests
Procedural Blank Cabinet
For storage of procedural blank coupons; the cabinet will be
under slight positive pressure in order to prevent contamination
from the test environment (i.e., laboratory) and allow passive air
flow to promote drying.
Decontaminated Coupon Cabinet
For storage of all test coupons after application of the
decontamination procedure; the cabinet will be under slight
positive pressure in order to prevent contamination from the
test environment (i.e., laboratory) and allow passive air flow to
promote drying.
Material and Equipment
The material and equipment used for the decontamination procedure were standardized as much as
possible and are listed in Table E-2. Decontamination steps are described in the subsequent sections of this
Appendix.
Table E-2. Material and Equipment Used in the Decontamination Procedural Steps
Material/Equipment
Pressure Washer
Pressure Washer
Chemical Sprayer
Description
John Deere 3300 psi, 3.2 gallon per minute,
Model 020382
Troy Bilt 2550 psi, 2.3 gallon per minute Model 020337
UDOR USA, Model* PP-UAG1003HU-K
300PSI@Maxof10.5GPM
AG Spray Gun
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Material/Equipment
Backpack Sprayer (Total of 2
units)
Bleach
Vinegar
Container for mixing pH-adjusted
bleach solution
Spor-Klenz®
Nozzle
Garden hose
Pressure regulator
Bucket of Dl water
Carboy container (Total of 9)
Pump
Description
SRS-600 Propack Rechargeable Electric Backpack Sprayer
(SHURflo, Cypress, CA), 4 Gallon, 12 Volt
http://leqacv.shurflo.com/pdf/industrv/qeneral/91 1/91 1-624.pdf
Ultra Clorox® Regular Bleach (EPA Reg. No. 67619-8)
(http://vwwv.clorox.com/products/overview.php?prod_id=clb)
6.15% sodium hypochlorite; <1% sodium hydroxide
(http://vwwv.thecloroxcompany.com/products/msds/
bleach/cloroxregularbleach0505_.pdf)
5% v/v technical grade acetic acid
5 gallon plastic carboy
STERIS Spor-Klenz® Ready-To-Use (EPA Reg. No. 1043-119)
Peracetic acid /Hydrogen Peroxide liquid decontaminant
http://vwwv.st.eris. com/prod ucts/view.cfm?id=253
Standard Adjustable-Flow Garden Hose Nozzle, Standard Brass, 4"
Length, McMaster-Carr, P/N 7484T1
75 ft.; 5/8 in diameter
Bronze Pressure Regulator-Plumbing-Code Rated Standard, 3/4"
NPT Female, 25-75 PS
3 gallons in a 5-gallon plastic pail
Carboys; Nalgene; Heavy Duty; polypropylene; Autoclavable; Leak
proof. For full vacuum applications up to 8 Hours; USP class VI,
vacuum rated for intermittent vacuum use only; 83B Closure size;
capacity: 5.25 gal. (20 L)
NSF-Certified Rotary Vane Pump for Water with Motor, Brass, 4.3
Max GPM, 3/4 Horsepower
pH-Adjusted Bleach Solution
The pH-adjusted bleach (pH-AB) solution was prepared by mixing 8 parts sterile distilled water with 1 part
5% acetic acid, and 1 part Clorox® bleach. The pH was then adjusted to 6.5 - 7.0 by adding more vinegar,
and FAC was adjusted to 6000 - 6700 ppm by diluting with water. The pH-AB solution was prepared just
prior to the initiation of testing on a particular day and was used within a window of 3 hours from the time of
preparation. After 3 hours, the bleach solution was discarded and a fresh pH-AB solution was prepared.
However, a single preparation was used within a single coupon set. Additionally, technical grade acetic acid
(5% v/v) was used instead of off-the-shelf white vinegar. This change was expected to reduce the variability
in the pH-AB solution for the purpose of this study.
The pH-AB solution was applied to each coupon using a backpack sprayer (Method 1) and with a chemical
sprayer (Method 2) (see Table 2-1).
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Spor-Klenz® Ready to Use (RTU) Solution
Spor-Klenz® RTU is a broad spectrum disinfectant and sporicide that is registered with EPA under FIFRA
(registration #1043-119). Spor-Klenz® is a mixture of 1.0% hydrogen peroxide, 0.08% peroxyacetic acid,
and 98.92% inert proprietary ingredients. The Ready-to-Use (RTU) variety of Spor-Klenz® was used for this
study, as opposed to the concentrate (registration #1043-120), to reduce the variation between experiments.
Preparation of diluted Spor-Klenz® from the concentrate for each day of testing would introduce unwanted
variation. Spor-Klenz® RTU requires no dilution prior to use. A new container of Spor-Klenz® RTU was used
for each day of testing. Unused Spor-Klenz® RTU was discarded appropriately. Since Spor-Klenz® RTU is
produced under manufacturer quality assurance criteria, only temperature was imposed as a critical
measurement for this liquid (see Section 4).
Spor-Klenz® has sterilant/sporicidal claims for nonporous surfaces when a 5.5 hour (20 °C) contact time is
used. While this contact time far exceeds the planned contact times for this study, our test aims to evaluate
technologies at conditions that are realistic of their use in homeland security-related remediation events.
Maintaining a 5.5 hour contact time in an animal facility would likely be unrealistic for the amount of surface
area needing to be decontaminated. Prior EPA research10 on post-anthrax incident carpet cleaning has
suggested that Spor-Klenz® RTU can be effective at much shorter contact times, so a contact time of 30
minutes was utilized.
Backpack Sprayer Application of Decontaminant
Prior to the start of testing, the spray pattern from the backpack sprayer was tested by spraying at the
appropriate distance (1 ft) onto a piece of 1.2 ft by 1.2 ft blue construction paper mounted in the position of
the test coupon. The spray was discharged into the center of the paper and the pattern was visually
assessed for consistency with that shown in Figure E-1. The diameter of the spray was checked to ensure
that the spray was within the acceptable limits (set at 16 in).
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Figure E-1. Backpack Sprayer - Spray Pattern (diameter of spray = 16 in) from Tests 9 and 10
For Task I, the spray wand from the backpack sprayer (SRS-600 Propack, SHURflo, Cypress, CA; see
Figure E-2) was inserted into the center port on the chamber. For Task II, the spray wand was inserted
between the curtains. From a distance of 1 ft, the coupons were sprayed to completely wet (or flood) the
surface of the materials. Each set of three Task I coupons was sprayed twice for 30 seconds with pH-AB
and Spor-Klenz®, with a third set sprayed once for 10 seconds with pH-AB. Task II coupons were sprayed
twice for 30 seconds with pH-AB.
The spray wand was moved back and forth to cover the surface of all three coupons evenly and completely
(Task I) or moved back and forth while moving downward to cover the surface of Task II coupons
completely. The Task I coupons were sprayed with three side-to-side strokes moving downward, starting
first from the top of the left-most coupon, across all three coupons, and finishing at the bottom of the right-
most coupon in the decontamination chamber. This step was repeated as often as necessary to satisfy the
required spray duration. The decontamination staff practiced the movement before the tests until the sprayer
could be operated in a repeatable manner. Data recorded included both the duration of the step and the
time of day when the step was started for each coupon.
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Figure E-2. Backpack sprayer
The constant spray pressure of 35 psi was maintained by the backpack sprayer. At this constant pressure,
the flow rate was maintained at 1046 mL/min (average over all tests) with a cone spray pattern of 16 in
diameter when held at a distance of 1 ft from the surface. For Task I, the spray wand was inserted at the
same distance in the port. The spray pattern is shown in Appendix E.
The flow rate was checked at the start and end of each test and before and after use on each coupon set to
ensure proper operation of the sprayer. The flow rate was measured by spraying into a graduated cylinder
for 10 seconds and measuring the final volume. A 30 minute contact time, with two applications, one at 0
minutes and one at 15 minutes, would be optimal and most realistic of effort expended during an actual FAD
remediation. For example, the decontamination solution would be reapplied once (once 15 minutes has
elapsed) during the 30 minute contact time. Section 2.5 presents the Test Matrix and describes how it was
modified based on these initial test results.
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Pressure Washer Application of Decontaminant
Commercial pressure washers are not recommended for use with bleach. Being concerned about the
effects of the pH-AB on this equipment, the first pressure washing application test (John Deere 3300 psi,
Model 020382; see Figure E-3) was conducted with Spor-Klenz®. A fixed volume of Spor-Klenz® was to be
dispensed onto the coupon surface by pressure washing for a fixed amount of time (planned as two sprays
of 15 seconds per set of three coupons). The supply line of the pressure washer was connected by the
garden hose to a reservoir containing the decontamination solution (at the final concentration). This
reservoir was the sole supply of liquid to the pressure washer during the application procedure. The
contents of the reservoir were therefore not diluted with water during use of the sprayer. The Task I coupons
were sprayed with side-to-side strokes starting first from the top of the left-most coupon, working downward
and the spray was moved across all three coupons in the decontamination chamber (Figure E-4). The start
time and duration for this action were recorded and spray of the coupons was performed as consistently as
possible across all coupons. A 25° angle nozzle was used with the pressure washer at full throttle. At a
distance of 3 ft from the coupon surface, this nozzle produces a fan of approximately 12 in. The nozzle was
oriented so that the fan was vertical.
Figure E-3. John Deere pressure washer
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Figure E-4: Center of spray during Task 1 decontamination procedures
Large volumes of rinsate were expected. For example, spraying the surface of a set of coupons with a 3300
psi/3.2 gpm pressure washer for a total of 30 seconds would generate 1.6 gallons of rinsate.
After the decontamination solution had been applied to the first set of coupons, the pressure washer was
rinsed with Dl water by connecting a second supply hose (a hose dedicated for distilled water only) to the
pressure washer and actuating the pressure washer for 30 seconds into a waste container. The coupons
were then rinsed with Dl water using the pressure washer.
During the second set of three coupons, the pressure washer was noticeably being negatively affected by
the Spor-Klenz® (i.e., running irregularly). Further, upon attempting to accomplish the second application of
sporicide during Test 8, the pressure washer failed to start initially and then ran roughly. The initial
application of the Spor-Klenz® was made to the third set of three coupons (second material), but the
pressure washer was unable to be restarted to make the second planned application for wood. This
procedural anomaly is noted in the Table 2-1 footnote.
Chemical Sprayer Application of Decontaminant
Due to the detrimental effects of Spor-Klenz® on the pressure washer, a chemical sprayer (Model# PP-
UAG1003HU-K, UDOR, USA; see Figure E-5) was obtained to conduct the Task I pH-AB decontamination
procedure. This procedure was conducted in the same manner as the procedure for the pressure washer in
Section 2.4.1.2 above (two sprays of 15 seconds per set of three coupons). The only other variation was
that a new pressure washer (Troy Bilt 2550 psi, Model 020337; see Figure E-6) was used for the Dl rinse
step.
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Figure E-5. Chemical sprayer
Figure E-6. Troy Bilt pressure washer
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Rinsing with Water
Rinsing of the coupons was accomplished using a standard garden hose (with nozzle) for Method 1
(backpack sprayer decon application) and for all Task II tests. For the pressure washer application of Spor-
Klenz® (Method 2), the pressure washer was also used to rinse the first two sets of coupons (first material).
However, when the pressure washer could not be restarted after the initial application on the first set of
coupons for the second material, the garden hose (with nozzle) was used to rinse the coupons. For the
chemical sprayer application of pH-adjusted bleach, an alternative (Troy Bilt) pressure washer was used for
the rinse step.
The water used in this study was Dl water produced by a Dracor water purification system (Model 34RC3).
An Oakton pH probe was maintained in the water reservoir to continually monitor the pH and temperature.
For Task I, the three coupons were sprayed with side-to-side strokes starting first from the top of the left-
most coupon, working downward and the spray was moved across all three coupons in the decontamination
chamber. Subsequent passes overlapped the previous by 50 percent. This was done from the central port
on the chamber. For Task II, the coupon was sprayed starting at the top left in an alternating left to right,
right to left motion, moving downward such that strokes overlapped by 50 percent, and finishing at the
bottom right corner. For both Tasks, the start time and duration for this action was recorded and was
performed as consistently as possible across all coupons.
Rinsing with a Garden Hose
For the garden hose, the water was supplied to the nozzle through a 75 ft garden hose of 5/8 in diameter.
The head pressure was maintained constant at approximately 60 psi using a pressure regulator listed in
Table E-1 of Appendix E. The water was supplied via a closed loop system having a 60-gallon tank as the
reservoir and a pump to provide pressurized stream and continual recirculation (Figure E-7). Via adjustment
of the nozzle, the spray pattern was controlled to be 1 ft in diameter measured at 3 ft from the nozzle.
Application was for 10 seconds during Task I and for 30 seconds during Task II Test C1.
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Figure E-7: Dl water supply system
Rinsing with a Pressure Washer
For the pressure washer, the water was supplied via a 75 ft garden hose of 5/8 in diameter. A circulation
pump was used to supply water from the tank to the pressure washer. The original pressure washer used
for the Spor-Klenz® test reports a pressure of 3300 psi and a flow rate of 3.2 gallons per minute. The
replacement pressure washer used for the pH-AB test rinse reports a pressure of 2550 psi and a flow rate of
2.3 gallons per minute. The 25° angle nozzle attachment was used during this study. Application was for 10
seconds during Task I.
Quality Control Measures
Additional measurements prior to or during the decontamination procedure application are also required in
order to ensure quality control in the testing. These measurements include quality control checks on the
reagents and equipment being used in the decontamination procedure. The pH and chlorine concentration
of the pH-adjusted bleach solution have been shown to have a significant impact on the inactivation of
Bacillus species spores. After preparation of the pH-adjusted bleach solution, the pH was measured using
an Oakton pH probe. Additionally, the pH was measured during the decontamination testing after each set
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of coupons was run within a test. The CI2 concentration was measured after preparation of the pH-adjusted
bleach solution by Hach High Range Bleach Test Kit, Method 10100 (Model CN-HRDT). The temperature
was also measured after the mixture was prepared and prior to the start of a new set of coupons within a
test using a NIST-traceable thermometer.
The water pressure at the head of the garden hose (i.e., faucet) was controlled with a pressure regulator.
The pressure was confirmed prior to each use of the hose. The flow rate and spray pattern from the hose
were checked prior to the start of the decontamination test. The flow rate was measured using an inline flow
meter. The spray pattern was visually verified to be nominally a 1 ft diameter (10 - 14 in) at the coupon
surface from a distance of 3 ft between the nozzle and coupon face.
The pressure wash rinse used the 25° attachment, to minimize the amount of overspray and maximize the
surface area covered by the spray pattern. This nozzle was also deemed the most appropriate for field use.
The chemical sprayer had an adjustable nozzle similar to the garden hose, and the pattern was set at 1 ft
diameter from a distance of 3 ft.
The time for application of each procedural step and time between procedural steps on each coupon was
measured using a stopwatch and recorded in the laboratory notebook.
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Appendix F: Sampling Procedures
F.1 Sampling Material and Equipment
The materials and equipment used for sampling are listed in Table F-1.
Table F-1. Material and Equipment Used in Sampling
Material/Equipment
Nonpowdered sterile surgical gloves
Nonpowdered nonsterile surgical gloves
Dust Masks
Disposable laboratory coats
Disposable Bench Liner
Phosphate Buffered Saline
50 ml_ conical tubes
Sterile sampling bags
Bleach wipes
Wipes
Swabs
Carboys (2)
Analytical Filter Units
Vacuum pump
Tubing
Description
KIMTECH PURE* G3 Sterile Nitrile Gloves, Kimberly-
Clark (VWR P/N HC61 1 10 for extra-large; VWR P/N
HC61 1 90 for large; VWR P/N HC61 1 80 for medium)
Exam gloves (Fisherbrand Powder-Free Nitrile Exam
Gloves, Fisher P/N 19-1 30-1 597D (for large); 19-1 30-
1597C (for medium))
3M Particulate Respirator 8271 , P95
Kimberly-Clark Kleenguard A10 Light Duty Apparel, P/N
40105
®
Phosphate Buffered Saline with TWEEN 20 (Sigma
Aldrich, P/N: P3563-10PAK)
(R)
BD Falcon BlueMax Graduated Tubes, 15 mL (Fisher
Scientific P/N 14-959-70C)
Fisherbrand Sterile Sampling Bags (TWIRL'EM )
Overpack Size : 10" by 14", P/N 01-002-53
Inner bag size: 5.5" by 9" (wipe);
Sample Bag Size: 5.5" by 9 "
Dispatch® Bleach Wipes, P/N 69260
Kendall Curity Versalon absorbent gauze sponge 2" by 2"
sterile packed (rayon/polyester blend)
(http://www.mfasco.com/)
(R)
Bacti Swab
(http://www.remelinc.com/lndustrial/
CollectionTransport/BactiSwab.aspx)
Nalgene autoclavable carboys with tabulation
(20 L) (Fisher Cat# 02-690-23)
1 50 mL Nalgene Analytical Filter Units (0.2 urn Cellulose
Acetate) (Fisher Cat# 130^020)
Gas oil-free vacuum Pump with adjustable suction (Fisher
Cat#0 1-092-25)
Fisher PVC clear tubing (1/2" i.d., 1/16" thickness) (Fisher
Cat#14-169-7J)
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Material/Equipment
Filter cassettes
Sampling pump
Description
Fisher PVC clear tubing (3/8" i.d., 1/16" thickness) (Fisher
Cat#14-169-7G)
Fisher PVC clear tubing (vacuum tubing)
(3/8" i.d., 1/8" thickness)
(Fisher Cat* 1 4-1 69-7H)
®
Via-Cell Bioaerosol Sampling Cassette P/N VIA010
http://www.zefon.com/store/via-cell-bioaerosol-sampling-
cassette.html
Isokinetic Method 5 Source Sampling Console
Model 51 1E
http://www.apexinst.com/products/consoles.htm
F.2 Surface Sampling Procedures
Within a single Task I test, surface sampling of the coupons was completed for all procedural blank coupons
first, followed by all test coupons, and then followed by all positive control coupons. Task II coupon areas
were tested on different days, in the following order: blanks (day X), positive control (day X), test (day X).
Surface sampling was done by wipe sampling in accordance with the protocol documented below. The
surface area for all samples was 1.3 sq ft. A template was used to cover the exterior 0.25 in of each Task I
coupon leaving a 13.5 in by 13.5 in square exposed for sampling. The outer 0.25 in around each coupon
was not sampled in order to avoid unrepresentative edge effects. A large stainless steel template covering
the entire coupon was used for Task II sampling. This template also prevented the outer edges from being
sampled, and provided a 0.5 in border between samples
Prior to the sampling event, all materials needed for sampling were prepared using aseptic technique. The
materials specific to each protocol are included in the relevant sections below. In addition, general sampling
supplies were needed. A sampling material bin was stocked for each sampling event, using the information
included in these sampling protocols. The bin contained enough wipe sampling kits to accommodate all
required samples for the specific test. Additional kits were also included for backup. Enough prepared
packages of gloves and bleach wipes were also included in the bin. Extra gloves and wipes were also
included. Task I templates (1.2 ft by 1.2 ft with an interior opening of 13.5 in by 13.5 in) were prepared,
sterilized, and packaged in sterile bags (7 templates per bag). These bags of templates were included with
the sampling kits. A sample collection bin was used to transport samples back to the APPCD Microbiology
Laboratory. The exterior of the transport container was decontaminated by wiping all surfaces with a bleach
wipe or towelette moistened with a solution of pH-adjusted bleach prior to transport from the sampling
location to the APPCD Microbiology Laboratory.
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F.2.1 Coupon Wipe Sampling
Wipe sampling is typically used for small sample areas and is effective on nonporous, smooth surfaces such
as ceramics, vinyl, metals, painted surfaces, and plastics. The general approach is that a moistened sterile
non-cotton pad is used to wipe a specified area to recover bacteria, viruses, and biological toxins. The
protocol that was used in this project is described below and has been adopted from that provided by
Busher et al.11 Brown et al.12, and documented in the INL 2008 Evaluation Protocols.13 None of these
references provides a validated wipe procedure for Bacillus spores, as a validated sampling procedure does
not currently exist.
The following procedure was used in this study for Task I wipe sampling of each coupon surface:
1. A three-person team was used, employing aseptic technique throughout. The team consisted of a
sampler, coupon handler, and support person.
2. All materials needed for collection of each sample were prepared in advance using aseptic technique. A
sample kit for a single wipe sample was prepared as follows:
a. Two sterile sampling bags (10" by 14", 5.5" by 9 ") and a 50 ml conical tube, capped, were labeled
in accordance with Appendix D. These bags and conical tube had the same label. The 5.5" by 9"
labeled sterile sampling bag was referred to as the sample collection sterile sampling bag.
b. A dry sterile wipe was placed in an unlabeled sterile 50 ml conical tube using sterile forceps and
aseptic technique. The wipe was moistened by adding 5 ml of sterile phosphate buffered saline
with 0.005% TWEEN®-20. The tube was then sealed.
c. The labeled 50 ml conical tube, capped, the unlabeled conical tube containing the pre-moistened
wipe, and the 5.5" by 9" labeled sampling bag were placed into the 10" by 14" labeled sterile
sampling bag. Each labeled sterile sampling bag contained a labeled 50 ml conical tube (capped),
an unlabeled capped conical tube containing a pre-moistened wipe, and an empty labeled sterile
sampling bag.
d. Each prepared bag was one sampling kit.
3. All members of the sampling team donned a pair of sampling gloves (a new pair per sample); the
sampler's gloves were sterile sampling gloves. All members wore dust masks to further minimize
potential contamination of the samples.
4. The coupon handler removed the coupon from the appropriate cabinet and placed the coupon on the
sampling area. The sampling area was covered with a new piece of laboratory bench cover for each
coupon.
5. The support person recorded the coupon code on the sampling log sheet.
6. The support person removed a template from the bag and handed it to the sampler.
7. The sampler placed the template onto the coupon surface.
8. The support person removed a sample kit from the sampling bin and recorded the sample tube number
on the sampling log sheet next to the corresponding coupon code just recorded.
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9. The support person:
a. Opened the outer sterile sampling bag touching the outside of the bag.
b. Touching only the outside of the overpack bag, removed and opened the unlabelled conical tube
and poured the pre-moistened wipe onto the sample.
c. Discarded the unlabeled conical tube.
d. Maneuvered the labeled 50 ml conical tube to the end of the outer sterile sampling bag and
loosened the cap.
e. Removed the cap from 50 ml conical tube immediately preceding the introduction of the sample
into the tube.
10. The sampler:
a. Wiped the surface of the sample horizontally using S-strokes to cover the entire sample area of the
coupon using a consistent amount of pressure.
b. Folded the wipe concealing the exposed side and then wiped the same surface vertically using the
same technique.
c. Folded the wipe over again and rolled up the folded wipe to fit into the conical tube.
d. Carefully placed the wipe into the 50 ml conical tube that the support person was holding, being
careful not to touch the surface of the 50 ml conical tube or plastic sterile sampling bag.
11. The support person then immediately closed and tightened the cap to the 50 ml conical tube and slid
the tube back into the sample collection sterile sampling bag.
12. The support person then put the 50 ml conical tube into the empty labeled 5.5" by 9" sampling bag and
sealed the bag.
13. The support person then sealed the outer sample collection bag now containing the capped 50 ml
conical tube (containing the sample wipe) inside a sealed 5.5" by 9" sample collection bag.
14. The support person then decontaminated the outer sample bag by wiping it with a Dispatch® bleach
wipe.
15. The support person then placed the triple-contained sample into the sample collection bin.
16. If sampling from the coupon was completed, the coupon handler moved the coupon and template to the
appropriate location for archival or discarding.
17. All members of the sampling team removed and discarded their gloves.
18. Steps 3 - 17 were repeated for each sample to be collected.
A very similar method was used for collecting the samples for Task II coupons. Changes were necessitated
by the orientation of the coupon and the use of areas as samples.
1. A two-person team was used, employing aseptic technique throughout. The team consisted of a
sampler and a coupon handler.
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All materials needed for collection of each sample were prepared in advance using aseptic technique. A
sample kit for a single wipe sample was prepared as follows:
a. Two sterile sampling bags (10" by 14", 5.5" by 9 ") and a 50 ml conical tube, capped, were labeled
in accordance with Appendix D. These bags and conical tube had the same label. The 5.5" by 9"
labeled sterile sampling bag was referred to as the sample collection sterile sampling bag.
b. A dry sterile wipe was placed in an unlabeled sterile 50 ml conical tube using sterile forceps and
aseptic technique. The wipe was moistened by adding 5 ml of sterile phosphate buffered saline
with 0.005% TWEEN®-20. The tube was then sealed.
c. The labeled 50 ml conical tube, capped, the unlabeled conical tube containing the pre-moistened
wipe, and the 5.5" by 9" labeled sampling bag were placed into the 10" by 14" labeled sterile
sampling bag. Hence, each labeled sterile sampling bag contained a labeled 50 ml conical tube
(capped), an unlabeled capped conical tube containing a pre-moistened wipe, and an empty
labeled sterile sampling bag.
d. Each prepared bag was one sampling kit.
2. While wearing gloves, the sampling team affixed a sterile sampling template to the sample. No
personnel touched the coupon surface itself. Gloves were removed and discarded following template
placement.
3. All members of the sampling team each donned a pair of sampling gloves (a new pair per sample); the
sampler's gloves were sterile sampling gloves. All members wore dust masks to further minimize
potential contamination of the samples.
4. The support person recorded the coupon code and area on the sampling log sheet.
5. The support person removed a sample kit from the sampling bin and recorded the sample tube number
on the sampling log sheet next to the corresponding coupon code just recorded.
6. The support person:
e. Opened the outer sterile sampling bag touching the outside of the bag.
f. Touching only the outside of the overpack bag, removed and opened the unlabeled conical tube
and poured the pre-moistened wipe into the hands of the sampler.
g. Discarded the unlabeled conical tube.
h. Maneuvered the labeled 50 ml conical tube to the end of the outer sterile sampling bag and
loosened the cap.
i. Removed the cap from 50 ml conical tube immediately preceding the introduction of the sample
into the tube.
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7. The sampler:
a. Squeezed out the excess liquid from the wipe.
b. Wiped the surface of the sample horizontally using S-strokes to cover the entire sample area of the
coupon using a consistent amount of pressure.
c. Folded the wipe concealing the exposed side and then wiped the same surface vertically using the
same technique.
d. Folded the wipe over again and roll up the folded wipe to fit into the conical tube.
e. Carefully placed the wipe into the 50 ml conical tube that the support person was holding, being
careful not to touch the surface of the 50 ml conical tube or plastic sterile sampling bag.
8. The support person then immediately closed and tightened the cap to the 50 ml conical tube and slid
the tube back into the sample collection sterile sampling bag.
9. The support person put the 50 ml conical tube into the empty labeled 5.5" by 9" sampling bag and
sealed the bag.
10. The support person then sealed the outer sample collection bag now containing the capped 50 ml
conical tube (containing the sample wipe) inside a sealed 5.5" by 9" sample collection bag.
11. The support person then decontaminated the outer sample bag by wiping it with a Dispatch® bleach
wipe.
12. The support person then placed the triple-contained sample into the sample collection bin.
13. All members of the sampling team removed and discarded their gloves.
14. Steps 4-17 were repeated for each sample to be collected.
F.2.2 Swab Sampling
Swab sampling was used for sterility checks on coupons and equipment prior to use in the testing. A single
swab sample was collected from each item and coupon. MOP 3135 was followed (see Appendix C), which
employs a pre-moistened swab.
F.3 Rinsate Collection and Sampling Procedures
During application of the decontamination procedure for each set of Task I coupons, the drain in the
decontamination test chamber remained open. The runoff from the coupons throughout the entire
decontamination procedure being tested was collected for a given coupon set (material type or all blanks)
into a vessel which was pre-dosed with sodium thiosulfate (STS). The volume of STS needed to neutralize
the total volume of decontamination liquid to be applied was determined by titration, and was set to 150%
excess. After all coupons from a single set had been moved to the Decontaminated Coupon Cabinet or
Procedural Blank Cabinet, the chamber was rinsed with Dl water. For Task II, a rinsate collection vessel
(trough) was placed under the coupon, and curtains arranged so that splashing rinsate drained into the
trough. The trough was also dosed with enough STS to neutralize the decontamination liquid.
Analysis of the liquid was accomplished by filter-plating triplicate 100 ml aliquots of each rinsate sample.
The collection procedure for the 100 ml aliquots was performed as follows:
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1. Sampler donned a face mask, pair of examination gloves, disposable laboratory coat, and bouffant cap.
2. The contents of the carboy were agitated to ensure homogeneous mixing.
3. The carboy cap was removed.
4. Using a new 50 ml sterile pipette tip, 100 ml of sample was aseptically pipetted into a sterile 100 ml
container.
Step 4 was repeated until triplicate samples were obtained.
The rinsate aliquots are triple-contained and transported to the Microbiology Laboratory for submission and
analysis at the conclusion of the entire test according to MOP 6565 (see Appendix C). Briefly, spores in the
rinsate sample were collected onto 0.2 urn pore-size analytical filters by vacuum filtration (Figure F-1). The
filter was then placed (particulate side up) onto bacterial growth media and incubated 18 ± 2 hours at the
optimal growth temperature. After incubation, colonies were enumerated on the filter surface by visual
inspection as shown in Figure F-2 for Ba agent.
Figure F-1. Nalgene Analytical Filter Unit connected to a Filter Unit.
97
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Figure F-2. Ba CPU on a Filter.
F.4 Aerosol Sampling Procedures
The use of high-pressure hoses and pressure washers is expected to generate aerosols. There is potential
for generated aerosols to contain viable spores removed from the coupon surfaces. Bioaerosol samples
were collected from the decontamination chamber during all spraying activities. Zefon Via-Cell® Bioaerosol
Sampling Cassettes (Figure F-3) were used to collect aerosol samples. During aerosol sample collection,
the air concentration of chlorine gas (during pH-amended bleach application) or hydrogen peroxide vapor
(during Spor-Klenz® application) was also monitored.
The Via-Cell® sampler was operated and analyzed according to the manufacturer's recommendations.
(http://www.zefon.com/analytical/downloadA/ia-Cell Lab Manual Booklet.pdf). During Task I, separate
aerosol samples were collected during the liquid decontamination application and the Dl water rinse
application. During Task II, separate aerosol samples were collected before each decontamination step, two
samples during the decontamination step, and after the decontamination procedure to provide some
baseline data similar to the procedural blank during Task I. The aerosol samples were analyzed according
to MOP 6571 (see Appendix C).
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Figure F-3 Via-Cell BioAerosol Cassette
Filters are analyzed to determine viable CPU collected per volume of air sampled.
The following sampling procedure was used to collect the Via-Cell® samples:
1. With a clean pair of gloves, the Via-Cell® was removed from the foil pouch. The cassette and the pouch
were labeled with the sample ID.
2. The small blue plug was removed and the cassette connected to the dry gas meter pump.
3. A leak-check was performed by turning on the pump with the inlet to the Via-Cell® closed capped off.
The flow of air should have stopped. If not, all connections were checked.
4. The cap of the Via-Cell® was removed and affixed in the ambient air around the coupon to be
decontaminated.
5. The starting volume on the dry gas meter (DGM) was recorded and the timer reset.
6. When time to collect a sample, the two switches on the meter box for the pump and the timer were
simultaneously turned on. The sample ID, the time of day and the meter temperature were recorded.
7. The valve settings on the meter box were adjusted so that the delta H pressure reading was 1.1" water.
8. At the end of sample collection, the two switches on the meter box for the pump and the timer were
simultaneously turned off. The final reading on the DGM, the meter temperature, and the elapsed time
were recorded.
9. The cap of the Via-Cell® was replaced and the pump disconnected. The outlet plug was reinserted.
10. The Via-Cell® was placed in the foil pouch. The exterior of the pouch was wiped with a Dispatch® wipe,
and placed in secondary containment.
F.5 Sample Preservation
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After sample collection, sample integrity was maintained by storage of samples in quadruple containers
(1 - sample collection container, 2 - sterile bag, 3 - sterile bag with exterior sterilized during sample
packaging process, 4 - sterile container holding all samples from a test). All individual sample containers
remained sealed while in the decontamination laboratory or in transport after the introduction of the sample.
The locking lid on the container holding all samples remained closed except for the brief period it was
opened for sample introduction by the support person of the sampling team. The sampling person did not
handle any samples after they were relinquished to the support person during placement into the primary
sample container.
After sample collection for a single test was complete, all samples were transported to the APPCD
Microbiology Laboratory immediately, with appropriate chain of custody form(s).
In the APPCD Microbiology Laboratory, all samples were stored in the refrigerator at approximately 4 °C
until they were analyzed. All samples were allowed to stabilize at room temperature prior to analysis.
F.6 Sample Holding Times
All samples were stored in accordance with Section F.5 and no longer than 10 days before being analyzed.
A typical holding time for most samples was less than or equal to 2 days.
During the analysis procedure, samples could be stored in the refrigerator overnight after extraction and
prior to the dilution plating. All samples were allowed to equilibrate to room temperature and were vortexed
for 10 seconds prior to plating.
Appendix G: Sampling Analyses
G.1 Sample Analyses
The APPCD Microbiology Laboratory located in E-288 of the RTP, NC, campus facility analyzed all samples
to quantify the number of viable spores per sample. For all sample types, phosphate buffered saline with
0.05% TWEEN®-20 (PBST) was used as the extraction buffer. After the appropriate extraction procedure, as
described in the sections to follow, the buffer was subjected to a four-stage serial dilution (10~1 to 10~4),
plated, incubated, and analyzed (CFU abundance) in accordance with MOP 6535a (see Appendix C).
In addition to the analysis in MOP 6535a, two additional analysis procedures were used for samples
resulting in less than 30 CFU/sample in the zero tube (undiluted sample). These analyses were conducted
in order to lower the current detection limit associated with MOP 6535a. First, 1 mL of the extract was filter
plated in accordance with MOP 6565 (see Appendix C). The remainder of the sample was then filter plated
in accordance with the MOP 6565.
The PBST was prepared according to the manufacturer's directions and in accordance with MOP 6562 (see
Appendix C), dissolving one packet in one liter of sterile water. The solution was then vacuum-filtered
through a sterile 0.22 urn filter unit to sterilize.
The extraction procedure used to recover spores was varied depending upon the different matrices (wipes,
liquids, filter cassettes). These procedures are described in the following subsections.
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G.1.1 Recovery from Wipe Samples
The recovery of the spores from the wipe samples was done as follows (MOP 6567, Appendix C):
1. The analyst donned a fresh pair of gloves. Gloves were changed periodically (at least between batches)
or after direct contact with a sample to reduce contamination.
2. The 50 ml conical tube containing the sample wipe was removed from the double sterile bag and wiped
with a bleach wipe. The analysts changed gloves after the wipe step.
3. A volume of 20 ml of PBST was added to each 50 ml conical tube by aseptically pouring a pre-
measured volume.
4. The sample was then vortexed for 2 minutes in 10 second bursts, leaving the wipe in the same tube.
5. If the sample sat for more than one minute after Step 4, the sample was re-vortexed individually to
homogenize prior to dilution plating. To complete dilution plating, the conical tube was uncapped and
the cap placed underside up on the Bio Safety Cabinet surface while the aliquot was removed from the
tube. Immediately after the aliquot was removed, the cap was aseptically replaced.
6. Each sample was processed individually. Steps 1-5 were repeated for each sample in the batch.
Dilution plating occurred as described in Section G.1.
G.1.2 Recovery from Liquid
Abundance of viable spores in the rinsate samples was determined by filtration of rinsate aliquots (MOP
6565). Filter samples were cultured on bacterial growth media, and recovery was determined by
enumerating colony forming units (CPU). The abundance of spores in the original runoff water was
determined by multiplying the calculated abundance of spores per milliliter of aliquot by the total runoff
volume.
G.1.3 Recovery from Air Sample
The extraction of the spores from the filters was done in accordance with MOP 6571 (see Appendix C). In
short, the filter housing allows for in-situ extraction using 2 ml Dl water. This suspension was then dilution
plated in triplicate in accordance with Section G.1. The concentration of spores in the air was determined by
dividing the total abundance of spores by the total sampled air volume.
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Appendix H: Test Reports
DCMD 3.41 B: Effectiveness of Physical and Chemical Cleaning and Disinfection Methods for Removing, Reducing
or Inactivating Agricultural Biological Threat Agents
»> Test Report «<
Test Date:
10/12/2010
Test Number: 1 & 2
Sampling Date:
Sampling Team:
Test Team: R. Delafield. S Terll
K. Egler. S. Payne
10/13/2010
R. Delafield.
S.Terll,
S. Payne
Analysis Date:
10/13/2010
Analyst: C. Slone. N. Griffin
Surface Samples
Material
Stainless Stee
Concrete
Wood
Blank
Sample Type
wipe
wipe
wipe
wipe
Positiv
Avg. CPU/sample
1 83E+Q7
2.33E+06
4.46E+Q6
NA
5 Controls
Mean of
Logs
7.25
6.34
6,62
NA
RSD (%)
27 03%
40.35%
47.45%
NA
Blank Coupons
CPU/ sample
1.4E+01
6.3E-01
NA
Test
Avg.
CPU/sample
6.32E-01
7.24E-01
6.25E-01
Coupons
Mean of
Logs
-0.20
-0.16
-0.20
RSD (%}
2.7%
35.8%
0.0%
LR
6.54
6.77
NA
SD
0.01
0.13
NA
Detection
limit
value?
TRUE
FALSE
TRUE
Decon Sets
Blanks
Concrete
Wood
Rinsate
(Total CPUs)
2.6SE+01
<23.73
<26.05
Ambient Air
CFU./L
<0.0292
1.18E+01
5.41E+00
Detection limit values are in
Yellow
Observations/Comments'
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Project No. RN990271.0035
Revision 0.0
March 2011
DCMD 3.41 B: Effectiveness of Physical and Chemical Cleaning and Disinfection Methods for Removing, Reducing
or Inactivating Agricultural Biological Threat Agents
>» Test Report <«
Test Date: 12/14/2010 Sampling Date: 12/15/2010 Analysis Date: 12/17/2010
Test Number: 3 and 4
Sampling Team:
Rob Delafield
Test Team: Stella Payne
Material
Stainless Stee
Concrete
Wood
Blank
Steve Terll
Sample Type
wipe
wipe
wipe
wipe
Rob Delafield, Analyst: Griffin Gatachalian, Slone
Stella Payne,
Steve Terll
Surface Samples
Positiv
Avg. CPU/sample
2.75E+07
3.05E+06
3.97E+06
NA
s Controls
Mean of
Logs
7.43
6.46
6.55
NA
Decon Sets
Blanks
Concrete
Wood
Rinsate
(Total CPUs)
<28.18
<43.75
100.92
Ambient Air during
decon
CFU/L
< 0.0584
70.50
48.33
Ambient
Air during
CFU/L
<0.19
0.12
<0.12
RSD (%)
24.33%
28.09%
55.45%
NA
Blank Coupons Test Coupons
Avg. Mean of
CPU/ sample CPU/sample Logs |RS
6.7E-01 7.64E-01 -0.14 36
5.9E-01 6.53E-01 -0.19 3
NA 5.65E+00 0.41 12!
Detection limit values are in Yellow
Detection
limit
D(%) LR SD value?
.6% 6. 60 1 0.13 FALSE
7% 6.74 0.02 TRUE
5.8% NA NA FALSE
Observations/Comments: The only deviation from the test parameters was that one set (3) of each coupon type was rinsed for 15 seconds instead of
10 seconds. The blanks (2 coupons) were rinsed for 10 seconds instead of 7 seconds. The viacells during decon (VD) and for rinse (VR) are for all 6 test
coupons of each type.
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March 2011
DCMD 3.41 B: Effectiveness of Physical and Chemical Cleaning and Disinfection Methods for Removing,
Reducing or Inactivating Agricultural Biological Threat Agents
>» Test Report <«
Test Date: 10/27/2010 Sampling Date: 10/28/2010 Analysis Date:
Test Number: 5 and 6
Sampling Team: Rob Delafield, Analyst: Griffin Gatachalian, Slone
Rob Del afield
Test Team: Matt Clayton
Material
Stainless Stee
Concrete
Wood
Blank
Steve Terll
Sample Type
wipe
wipe
wipe
wipe
Stella Payne,
Steve Terll
Surface Samples
Positiv
Avg. CPU/sample
3.90E+07
2.62E+06
5.27E+06
NA
Decon Sets
Blanks
Concrete
Wood
Rinsate
(Total CPUs)
<11.74
<24.93
<27.35
Ambient Air
CFU/L
0.06
45.95
6.37
Observations/Comments:
s Controls
Mean of
Logs RSD
7.58 20.9
6.38 44.6
6.65 71.3
NA N/
Blank Coupons Test Coupons
Avg. Mean of
(%) CPU/ sample CPU/sample Logs RJ
5%
3% 7.7E-01 6.06E+04 4.75 4
3% 8.0E-01 7.03E-01 -0.15
\ NA 6.60E-01 -0.18 1
Detection limit values are in Yellow
Detection
limit
3D(%) LR SD value?
0.2% 1.63 0.19 FALSE
18% 6.80 0.02 TRUE
1.6% NA , NA TRUE
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Project No. RN990271.0035
Revision 0.0
March 2011
DCMD 3.41 B: Effectiveness of Physical and Chemical Cleaning and Disinfection Methods for Removing, Reducing
or Inactivating Agricultural Biological Threat Agents
>» Test Report <«
Test Date:
11/17/2010
Test Number: 7 and 8
Sampling Date:
Sampling Team:
Rob Delafield
Test Team: Kim Egler
Steve Terll
11/18/2010
Rob Delafield,
Stella Payne,
Steve Terll
Analysis Date:
11/19/2010
Analyst: Griffin Gatachalian, Slone
Surface Samples
Material
Sample Type
Positive Controls
Mean of
Avg. CPU/sample | Logs
RSD (%)
Blank Coupons
CPU/ sample
Test Coupons
Avg. Mean of
CPU/sample | Logs |RSD(%)
LR
SD
Detection
limit
value?
Stainless Steel
Concrete
Wood
Blank
wipe
wipe
wipe
wipe
2.23E+07
2.93E+06
6.71 E+06
NA
7.34
6.40
6.79
NA
17.64%
53.90%
44.66%
NA
6.5E+00
6.3E-01
NA
7.30E+03
6.32E-01
1.00E+00
3.60
-0.20
-0.03
117.4%
1.8%
47.1%
2.801
6.99.
0.55
0.01
NA
NA
FALSE
TRUE
FALSE
Decon Sets
Blanks
Concrete
Wood
Rinsate
(Total CFUs)
Ambient Air in Duct
CFU/L
<28.88
<27.12
<0.0457
0.23
0.28
Ambient
Air in
Chamber
CFU/L
6.19
2.01
6.71
Detection limit values are in
69204.15225
Yellow
Observations/Comments:
Equipment failure prevented the second decon application to the T8-T-TW (3-5) test coupons.
The T8-T-TW (3-5) test coupons were rinsed with the garden hose after a total contact time of 34 minutes.
The T8-T-TW (6-8) test coupons were not done as a result of the equipment failure.
For these tests the application time of the decontaminate was reduced to 15 seconds from 30 seconds. The rinse time remained 10 seconds.
A second application was applied after 15 minutes for a total contact time of 30 minutes.
The duct viacell fell apart and was pulled inside the duct during the concrete decon and rinse.
The ambient air in duct data is not available because the meter box ran backwards due to the vacuum created by the exhaust.
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March 2011
DCMD 3.41 B: Effectiveness of Physical and Chemical Cleaning and Disinfection Methods for Removing, Reducing or
Inactivating Agricultural Biological Threat Agents
>» Test Report <«
Test Date: 1/18/2011 Sampling Date: 1/19/2011 Analysis Date: 1/19/2011
Test Number: 9 and 10
Sampling Team:
Rob Delafield
Test Team: Stella Payne
Material
Stainless Stee
Concrete
Wood
Blank
Steve Terll
Sample Type
wipe
wipe
wipe
wipe
Posit!
Avg. CPU/sample
2.43E+07
1 .46E+06
1 .88E+06
NA
Decon Sets
Blanks
Concrete
Wood
Rinsate
(Total CPUs)
<5.57
1.31E+04
8.32E+04
Ambient Air during
decon
CFU/L
#DIV/0!
390.66
385.54
re Controls
Mean of
Logs
7.39
6.14
6.25
NA
Ambient Air
during rinse
CFU/L
#DIV/0!
0.36
4.78
Surface
RSD (%)
3.20%
37.86%
29.72%
NA
Rob Delafield, Analyst: Griffin Gatachalian, Slone
John Nash,
Steve Terll
Samples
Blank Coupons Test Coupons
Avg. Mean of
CPU/ sample CPU/sample Logs RS
1.3E+01 7.02E-01 -0.17 33
9.4E+00 1.50E+03 2.21 17J
NA 4.14E+00 0.33 121
Detection limit values are in Yellow
Detection
limit
D(%) LR SD value?
3% 6.30 0.12 FALSE
j.1% 4.04 1.19 FALSE
.3% NA , NA FALSE
Observations/Comments: Due to the power washer running out of gas, the contact time on the second set of treated wood (T1 0-T-TW-(6-8)) was
22 min 55sec instead of 15 minutes. There did not
seem to be any significant change in efficacy based on this variation.
The concrete results are based on a single spore on one coupon, so, while not a detection limit value, it is just above the detection limit.
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Revision 0.0
March 2011
DCMD 3.41 B: Effectiveness of Physical and Chemical Cleaning and Disinfection Methods for Removing, Reducing or Inactivating Agricultural
Biological Threat Agents
>» Test Report «<
Test Date: 2/8/2011 Sampling Date: 2/9/2011 Analysis Date: 2/10/2011
Test Number:
Test Team:
Material
Stainless Steel "
Concrete (CVA)
Concrete (CVB)
Wood (TWA)
Wood (TWB)
Decon Sets
Decon 1
Decon 2
Rinse
C1
Rob Delafield
Stella Payne
Tim McArthur
Sample Type
wipe
wipe
wipe
wipe
wipe
Sampling Team: Rob Delafield,
Matt Clayton,
Tim McArthur
Analyst: Griffin Gatachalian, Slone
Surface Samples
Pos
Avg. CPU/sample
2.02E+07
7.51E+05
4.06E+06
3.42E+06
3.18E+06
Ambient Air
Before
CFU/L
N'7.47
25.27
1.50
Ambient Air During
CFU/L
12.75
42.15
0.15
Itlve Controls
Mean of
Logs
7.29
5.84
6.60
6.51
6.50
Ambient Air
After
CFU/L
13.55
0.62
0.23
Observations/Comments :
RSD (%)
27.27%
48.1%
23. 1 %
35.7%
16.8%
Detection limit
Blank Coupons
CPU/ sample
Avg. CPU/
sample
6.06E-01
7.46E-01
1.28E+01
8.00E+00
Coupon
CVA
CVB
TWA
TWB
Rinsate before
Decon
Total CPU
2.30E+04
2.00E+04
3.30E+03
3.33E+03
values are in Yellow
Test Coupons
Mean of Logs RSD (%)
-0.22 2.1%
-0.15 37.8%
0.94 86.6%
0.75 89.1%
Rinsate after Decon
Total CPU
1.30E+05
2.84E+05
1.51E+05
1.41E+05
Detection
limit
LR SD value?
6.1 0.01 TRUE
6.7 0.14 FALSE
5.6 0.45 FALSE
5.7 0.44 FALSE
There was high contamination of air in COM M ANDER prior to decon steps - this reduces usefulness of ViaCell data during and after Decon steps
Rinsates were also contaminated before decon, but levels after decon were higher.
Only 3 positive control samples for CWA
107
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Draft Report
Project No. RN990271.0035
Revision 0.0
March 2011
DCMD 3.41 B: Effectiveness of Physical and Chemical Cleaning and Disinfection Methods for Removing, Reducing or Inactivating Agricultural
Biological Threat Agents
»> Test Report «<
Test Date: 2/24/2011 Sampling Date: 2/25/2011 Analysis Date: 2/28/2011
Test Number: C2
Sampling Team:
Rob Delafield
Test Team: Matt Clayton
Material
Stainless Steel
Concrete (CVA)
Concrete (CVB)
Wood (TWA)
Wood (TWB)
Tim McArthur
Sample Type
wipe
wipe
wipe
wipe
wipe
Rob Delafield,
Stella Payne,
Tim McArthur
Analyst: Griffin Gatachalian, Slone, Levine
Surface Samples
Positive Controls
Mean of
Avg. CPU/sample Logs
8.33E+06 6.99
4.43E+06 6.63
7.20E+06 6.84
2.67E+06 6.42
3.25E+06 6.50
Decon Sets
Decon 1
Decon 2
Ambient Air
Before
CFU/L
4.07
1.15
Ambient Air
Ambient Air During After
CFU/L CFU/L
27.93 2.97
0.91 0.07
Observations/Comments:
RSD (°/cJ
1.92E-01
27.6%
31.3%
21 . 1 %
20.6%
Detection limit
Blank Coupons
CPU/ sample
Avg. CPU/
sample
7.13E+01
4.49E+00
1.25E+01
1.67E+01
Coupon
CVA
CVB
TWA
TWB
Rinsate before
Decon
Total CPU
4.93E+03
1.87E+03
1.00E+03
3.73E+02
values are in Yellow
Test Coupons
Mean of Logs RSD (%)
1.27 \^9&5^^^
0.09 193.0%
0.81 82.7%
0.92 95.4%
Rinsate after Decon
Total CPU
<105.11
< 398. 33
<74.65
< 204. 67
Some rinsate samples have returned lower CPUs in subsequent plating (see C2-R-CVA-2) - Possibly due to a decontaminating agent in the rinsate itself. (Bleach
Detection
limit
LR SD value?
5.4 1.10 FALSE
6.8 0.68 FALSE
5.6 0.67 FALSE
5.6 0.67 FALSE
STS)?
No rinse during this test.
108
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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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