EPA/600/R-20/217 | September 2020
www.epa.gov/homeland-security-research
United States
Environmental Protection
Agency
&EPA
Washing/Drying of Clothing
Contaminated with a Biological
Agent Such as Ba
Office of Research and Development
Homeland Security Research Program
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EPA 600/R-20/207
Washing/Drying of Biological Agent-Contaminated
Clothing
U.S. EPA Principal Investigator:
Shannon Serre
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
Prepared by:
Abderrahmane Touati
Jacobs
Contract EP-C-15-008
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DISCLAIMER
The research described in this article has been funded wholly or in part by the U.S.
Environmental Protection Agency Contract No. EP-C-15-008 to Jacobs Technology, Inc. It has
been subject to the Agency's review, and it has been approved for publication as an EPA
document. Mention of trade names or commercial products does not constitute endorsement or
recommendation for use.
Questions concerning this document, or its application should be addressed to the
following individual:
Shannon Serre, PhD
Office of Emergency Management (OEM)
Chemi cal/Bi ol ogi cal/Radi ol ogi cal/Nucl ear (CBRN)
Consequence Management Advisory Division (CMAD)
U.S. Environmental Protection Agency (MD-E343-06)
109 T.W. Alexander Drive
Research Triangle Park, NC 27711
E-mail Address: Serre. Shannon@epa.gov
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The U.S. Environmental Protection Agency (EPA) is charged by Congress with
protecting the Nation's land, air, and water resources. Under a mandate of national environmental
laws, the Agency strives to formulate and implement actions leading to a compatible balance
between human activities and the ability of natural systems to support and nurture life. To meet
this mandate, EPA's research program is providing data and technical support for solving
environmental problems today and building a science knowledge base necessary to manage our
ecological resources wisely, understand how pollutants affect our health, and prevent or reduce
environmental risks in the future.
The Center for Environmental Solutions and Emergency Response (CESER) within the
Office of Research and Development (ORD) conducts applied, stakeholder-driven research and
provides responsive technical support to help solve the Nation's environmental challenges. The
Center's research focuses on innovative approaches to address environmental challenges
associated with the built environment. We develop technologies and decision-support tools to
help safeguard public water systems and groundwater, guide sustainable materials management,
remediate sites from traditional contamination sources and emerging environmental stressors,
and address potential threats from terrorism and natural disasters. CESER collaborates with both
public and private sector partners to foster technologies that improve the effectiveness and
reduce the cost of compliance, while anticipating emerging problems. We provide technical
support to EPA regions and programs, states, tribal nations, and federal partners, and serve as the
interagency liaison for EPA in homeland security research and technology. The Center is a
leader in providing scientific solutions to protect human health and the environment.
Gregory Sayles, Director
Center for Environmental Solutions and Emergency Response
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ACKNOWLEDGMENTS
The principal investigator from the U.S. Environmental Protection Agency (EPA)
directed this effort with the support of a project team from across EPA. The
contributions of the individuals listed below have been a valued asset throughout this
effort.
EPA Project Team
Shannon Serre, OLEM/CMAD (Office of Land and Emergency
Management/Consequence Management Advisory Division)
Joseph Wood, CESER/HSMMD (Center for Environmental Solutions and Emergency
Response/ Homeland Security and Materials Management Division)
EPA Quality Assurance
Ramona Sherman, CESER/HSMMD
EPA Technical Reviewers
Elise Jakabhazy, OLEM/CMAD (Office of Land and Emergency
Management/Consequence Management Advisory Division)
Lukas Oudejans, CESER/HSMMD (Center for Environmental Solutions and Emergency
Response/ Homeland Security and Materials Management Division)
External Technical Reviewers
Barbara Alexander, Centers for Disease Control and Prevention, National Institute for
Occupational Safety and Health
Robert Miknis, United States Department of Agriculture, Animal and Plant Health
Inspection Service
Jacobs Technology. Inc.
Abderrahmane Touati
Francis Rob Delafield
Steve Terll
Denise Aslett
Ahmed Abdel-Hady
Kathleen May
Mariela Monge
Brian Ford
Wendy Coss
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EXECUTIVE SUMMARY
A large-area accidental or intentional release of Bacillus anthracis (Ba) spores may
subject emergency response teams as well as the general public to exposure and cross-
contamination through personal protective equipment (PPE) or personal clothing. The overall
purpose of this study was to assess the use of washers and dryers with a suitable decontamination
solution as a potential self-help practice to reduce and/or inactivate biological spores from
common clothing material. This approach may be used to lessen potential cross-contamination
among emergency first-responders, security personnel, or public citizens exposed to a wide area
B. anthracis spore release.
The overall decontamination approach was evaluated as a function of different
parameters such as wash temperature, wash cycle time, material clothing type, wash solution
type (water versus bleach-type solution), water rinse, and drying. Six material fabrics (cotton,
stretch denim, polyester fleece, spandex, cotton/polyester blend, and US Navy camouflage) were
evaluated to determine if the clothing material type had any effect on decontamination efficacy.
Spore viability (quantified by enumeration of colony forming units [CFU]) was evaluated at each
step in the experimental decontamination process to assess the potential for spores on clothing
materials to either be removed, inactivated, or re-distributed to the wash/rinse solutions. The
project included bench scale testing to simulate the washing and drying of contaminated clothes
with viable spores of Bacillus atrophaeus var. globigii (Bg) spores, a surrogate for Bacillus
anthracis (Ba) spores.
Scoping tests were conducted to identify potential solutions that could be used in a
household washing machine to inactivate Bacillus spores. These solutions were then tested using
cloth coupons, 14 in. x 14 in., and a compact washing machine with the optimal decontamination
parameters applied from the scoping tests. Results from the washing machine tests confirmed
that 1% diluted Clorox® chlorine-based bleach solution with the addition of a detergent using an
18-minute wash time was effective in decontaminating material fabric inoculated with ~107
spores per square foot. All materials had surface log reductions > 6. The increased surface
decontamination efficacies seen during this phase may have been due to the increased agitation
dynamic from the washing machines.
A drying sequence was tested following the wash sequence to see if drying added any
benefit to reduction of spores on the clothing. The addition of a drying sequence resulted in no
apparent increase in the decontamination efficacy of the diluted bleach solution. A similar
number of spores were detected on cotton material coupons following the wash cycles with and
without a drying step. The difference between the number of spores recovered between the two
procedures is made up almost entirely of spores recovered from HEPA filters placed on the air
outlet of the dryers. Due to the potential for spore re-aerosolization and lack of a significant
increase in decontamination efficacy, drying of washed contaminated clothing is not
recommended as a potential self-help practice to reduce cross-contamination on clothing.
The use of 1% diluted Clorox® chlorine-based bleach solution with the addition of a
detergent using an 18-minute wash time can be used to lessen potential cross-contamination on
clothing. Machine drying should be avoided as no additional benefit was observed from this
process and resulted in re-aerosolization of spores from the clothing.
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CONTENTS
DISCLAIMER ii
1.0 INTRODUCTION 1
Purpose of the Study 1
Project Description and Objectives 1
2.0 EXPERIMENTAL APPROACH 2
Phase I: Scoping Test (Liquid Decontamination Solution Testing) 2
Phase II: Bench-Scale Testing with Material Coupons 3
Phase III: Decontamination Testing using a Compact Washing Machine 4
Phase IV: Decontamination Testing using a Portable Washing Machine and Dryer 4
3.0 TESTING AND MEASUREMENT PROTOCOLS 5
Test Materials 5
Material Coupon Preparation 6
Material Coupon and Equipment Sterilization 6
Test Organism and Spore Inoculation 7
3.1.1 Liquid Spore Inoculation 7
3.1.2 Aerosol Deposition Apparatus (ADA) Assisted Inoculation of Surfaces 7
3.1.2.1 Phase II Spore Inoculation with MDI 7
3.1.2.2 Phases III and IV: Dry Spore Inoculation 8
Decontamination Solution Preparation 9
Neutralizing Agents for Test Samples 9
Washing and Drying Simulation Equipment 10
3.1.3 Orbital Shaker 10
3.1.4 Compact Washing Machine 11
3.1.5 Laundry Dryer 11
Washing and Drying Test Procedures 12
3.1.6 Phase I: Scoping Test 12
3.1.7 Phase II: (Bench-Scale Testing with Material Coupons) 12
3.1.8 Phase III: (Decontamination Testing using a Washer) 13
3.1.9 Phase IV: (Decontamination Testing using a Washer & Dryer) 14
Sample Neutralization Procedure 15
4.0 SAMPLING AND ANALYSIS APPROACH 18
Sampling Material and Equipment 18
Sampling Methods 20
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4.1.1 Cloth Material Coupon, and Liquid Samples 20
4.1.2 Wipe Sampling 20
4.1.3 Quality Assurance/Quality Control (QA/QC) Samples 21
4.1.3.1 Swab Samples 21
4.1.3.2 Material Samples 21
Sampling Containers 21
Sample Preservation 22
Sample Quantities 22
Preventing Cross-Contamination During Sampling 22
5.0 ANALYTICAL METHODS 24
Sample Extraction 24
5.1.1 Coupon Extraction 24
5.1.2 Wipe Sample 24
5.1.3 Swabs 24
Spiral- and Filter- Plating Procedures 24
Other Analytical Procedures 25
5.1.4 Determination of FAC by Titration 25
5.1.5 pH and Temperature Measurements 25
Decontamination Efficacy 25
6.0 RESULTS AND DISCUSSION 29
Phase I: Scoping Test 29
6.1.1 Extraction Efficacy 29
CFU, colony forming units; Stdev, standard deviation 29
6.1.2 Challenges with Filter Plating Residual Decontamination 30
6.1.3 Neutralization Efficacy 31
6.1.3.1 High Spore Inoculated Solutions 31
6.1.3.2 Low Spore Inoculated Solutions 32
6.1.4 Preliminary Washing Solutions Evaluation 33
6.1.5 Washing Parameters 34
6.1.6 Chlorine-Based Bleach with Surfactant 35
Phase II: Bench-Scale Testing with Material Coupons 37
6.1.7 Diluted Bleach Solution: Cotton Material 37
6.1.8 Diluted Bleach Solution with the addition of Detergent: Cotton Material 38
6.1.9 1% Diluted Bleach with the Addition of a Detergent: All Materials 39
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Phase III: Decontamination Testing using a Washing Machine 41
6.1.10 1% Diluted Bleach with Detergent: All Materials 41
6.1.11 1% Diluted Bleach Without the Addition of a Detergent: Cotton and Military
Materials 43
Phase IV: Decontamination Testing using a Washing Machine/Dryer 43
7.0 QUALITY ASSURANCE AND QUALITY CONTROL 45
Supplies and Consumables 45
Measurement Equipment Calibration 45
Criteria for Critical Measurements and Parameters 46
Control Checks 47
8.0 Summary 49
REFERENCES 51
Appendix A. Challenges with Filter Plating Residual Decontamination 52
Appendix B-1: Temporal Recoveries from Neutralized Test Solutions at High-Spore Loading
Concentrations 55
Appendix B-2: Temporal Recoveries from Neutralized Test Solutions at Low-Spore Loading
Concentrations 55
Appendix B-3: Phase I (Scoping). Decontamination Results with Wash Liquid Solutions 56
Appendix B-4: Phase I (Scoping): Decontamination Results with Diluted Bleach Alone 57
Appendix B-5: Phase I (Scoping): Decontamination Results with Diluted Bleach Plus Detergent 57
Appendix C-1: Phase II Spore Recovery and Redistribution for Diluted Bleach Solution on Cotton
Material 58
Appendix C-2: Phase II (Benchscale): 1% Diluted Bleach Solution, with the Addition of a
Detergent, Decontamination Results: All Materials 61
Appendix D-1: Phase III (Washer): 1% Diluted Bleach Solution, with the Addition of a Detergent,
Decontamination Results: All Materials 67
Appendix D-2: Phase III: 1% Diluted Bleach Solution without the Addition of a Detergent
Decontamination Results: Cotton and Military Materials 73
Appendix E-1: Phase IV (Washer and Dryer): 0.1% Diluted Bleach Solution, with the Addition of a
Detergent, Decontamination Results Using a Washer/Dryer: Cotton Material 75
FIGURES
Figure 3-1. Material Coupons Tested 5
Figure 3-2. Test Coupons (3- x 3-in stainless-steel and fabric material) 8
Figure 3-3. ADA with actuator and MDI, clamped onto test coupon, with stainless-steel sliding
panel closed (Panel A) and open ready for MDI actuation (Panel B) 8
Figure 3-4. ADA (a) with the assembled actuator (b) and MDI (c) 9
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Figure 3-5. Lab Line Incubator-Orbital Shaker fitted with a platform insert to secure Nalgene
bottles 10
Figure 3-6. Portable Mini Compact Twin Tub Washer Spin Dryer 11
Figure 3-7. Magic Chef Compact Laundry Dryer 11
Figure 6-1. 1 -mL aliquot filter plated from 10% bleach solution sample 30
Figure 6-2. 10-mL aliquots filter plated from 10% bleach solution sample 30
Figure 6-2. Temporal Recoveries from Neutralized Test Solutions Inoculated at High-Spore
Loading Concentration 32
Figure 6-3. Temporal Spore Recoveries from Neutralized Solutions Over Time 33
Figure 6-4. Wash Liquid Solutions Recovery Results 34
Figure 6-5. Chlorine-based bleach dilute solution decontamination efficacy 35
Figure 6-6. Diluted bleach solution with the addition of a detergent decontamination efficacy 36
Figure 6-7. Decontamination efficacy of a diluted chlorine-based bleach solution with and without
the addition of a detergent 37
Figure 6-8. Spore recovery and redistribution for different diluted bleach solutions 38
Figure 6-9. Spore recovery and redistribution of material coupons treated with 10% diluted
bleach without detergent and 1% diluted bleach with and without detergent 39
Figure 6-10. Bench-scale decontamination efficacy results for all material coupons using 1%
diluted 40
Figure 6-11. Spores recovered from material coupons using 1% diluted bleach solution with
detergent 40
Figure 6-12. Spores recovered from material coupons subjected to Dl water procedural testing
process 41
Figure 6-13. Pilot-scale washer decontamination efficacy results for all material coupons using
1% diluted bleach solution with detergent 42
Figure 6-10. Bench-scale versus pilot-scale spores recovered from material coupons subjected to
Dl water procedural testing process 42
Figure 6-15. Spores recovered from cotton and military material coupons using 1% diluted bleach
solution without the addition of detergent using washing machine 43
Figure 6-16. Spore distribution following a post-decontamination washing/drying cycle using 0.1%
diluted bleach solution with the addition of a detergent 44
Figure A-1. 1 -mL aliquot filter plated from 10% bleach solution sample 52
Figure A-2. 10-mL aliquots filter plated from 10% bleach solution sample 52
Figure A-3. Aliquots filter plated on GN-6 white filter from 10% bleach solution (A: 1-, B: 10-mL) 54
Figure A-4. Aliquots filter plated on PES black filter from 10% bleach solution (A: 1-, B: 10-mL) 54
Figure A-5. Aliquots filter plated on PES white filter from 10% bleach solution (A: 1-, B: 10-mL) 54
Tables
Table 3-1. Tested Material Coupons 6
Table 4-1. Sampling Materials and Equipment 19
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Table 4-2. Sample Quantities for Each Decontamination Test 22
Table 6-1. Material Coupon Sample Extraction Efficacy Evaluation 29
Table 7-1. Instrument Calibration Frequency 45
Table 7-2. Data Quality Acceptance Criteria for Critical Measurements 46
Table C1-1. Phase II (Benchscale): Decontamination Results for Cotton Material with a Bleach
Dilution Ratio of 1:1000 58
Table C1-2. Phase II (Benchscale): Decontamination Results for Cotton Material with a Bleach
Dilution Ratio of 1:100 59
Table C1-3. Phase II (Benchscale): Decontamination Results for Cotton Material with a Bleach
Dilution Ratio of 1:10 60
Table C2-1: Phase II (Benchscale): Decontamination Results for Cotton Material with 1% Diluted
Bleach Plus Detergent 61
Table C2-2. Phase II (Benchscale): Decontamination Results for Blend Material with 1% Diluted
Bleach Plus Detergent 62
Table C2-3. Phase II (Benchscale): Decontamination Results for Military Material with 1% Diluted
Bleach Plus Detergent 63
Table C2-4. Phase II (Benchscale): Decontamination Results for Polyester Material with 1%
Diluted Bleach Plus Detergent 64
Table C2-5. Phase II (Benchscale): Decontamination Results for Spandex Material with 1%
Diluted Bleach Plus Detergent 65
Table C2-6. Phase II (Benchscale): Decontamination Results for Denim Material with 1% Diluted
Bleach Plus Detergent 66
Table D1-1. Phase III (Washer): Decontamination Results for Cotton Material with 1% Diluted
Bleach Plus Detergent 67
Table D1-2. Phase III (Washer): Decontamination Results for Blend Material with 1% Diluted
Bleach Plus Detergent 68
Table D1-3. Phase III (Washer): Decontamination Results for Spandex Material with 1% Diluted
Bleach Plus Detergent 69
Table D1-4. Phase III (Washer): Decontamination Results for Polyester Material with 1% Diluted
Bleach Plus Detergent 70
Table D1-5. Phase III (Washer): Decontamination Results for Denim Material with 1% Diluted
Bleach Plus Detergent 71
Table D1-6. Phase III (Washer): Decontamination Results for Military Material with 1% Diluted
Bleach Plus Detergent 72
Table D2-1. Phase III (Washer): Decontamination Results for Cotton Material with 1% Diluted
Bleach and without Detergent 73
Table D2-2. Phase III (Washer): Decontamination Results for Military Material with 1% Diluted
Bleach and without Detergent 74
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Table E-1. Phase IV (Washer and Dryer): Decontamination Results for Cotton Material with 0.1%
Diluted Bleach Plus Detergent 75
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ACRONYMS AND ABBREVIATIONS
ADA
aerosol deposition apparatus
ASTM
ASTM International
Ba
Bacillus anthracis
Bg
Bacillus atrophaeus var. globigii
BioLab
HSMMD RTP Microbiology Laboratory
CESER
Center for Environmental Solutions and Emergency Response
CFU
colony-forming unit
CMAD
Consequence Management Advisory Division
DI
deionized
DTRL
Decontamination Technologies Research Laboratory
EPA
U.S. Environmental Protection Agency
EtO
ethylene oxide
FAC
free available chlorine
H2O2
hydrogen peroxide
HSMMD
Homeland Security and Materials Management Division
in.
inch
ISO
International Organization for Standardization
LR
log reduction
MDI
metered-dose inhaler
mg/L
milligram per liter
ml
milliliter
N
normality
NIST
National Institute of Standards and Technology
OLEM
Office of Land and Emergency Management
pAB
pH-adjusted bleach
PBST
phosphate-buffered saline with 0.05% Tween 20
PES
polyethersulfone
PPE
personal protective equipment
ppm
part per million
psi
pound per square inch
QA
quality assurance
QC
quality control
RTP
Research Triangle Park
Stdev
standard deviation
STS
sodium thiosulfate
TSA
tryptic soy agar
VHP
vaporous hydrogen peroxide
VpAB
volume of pH-adjusted bleach
VSTS
volume of sodium thiosulfate
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1.0 INTRODUCTION
The U.S. Environmental Protection Agency's (EPA's) Homeland Security Research
Program (HSRP) provides credible information to protect human health and the environment
from adverse impacts arising from terrorist threats and other contamination incidents. Within the
HSRP, the Center for Environmental Solutions and Emergency Response's (CESER) Homeland
Security and Materials Management Division (HSMMD) conducts research to provide expertise
and guidance on decontamination methods for wide area remediation efforts. This project
addresses HSRP strategic goals as described in detail in the Homeland Security Strategic
Research Action Plan (U.S. EPA, 2020).
Purpose of the Study
The purpose of this study was to assess washing and drying clothes as a potential self-
help practice to reduce and/or inactivate a Bacillus anthracis (Ba) spore surrogate in common
clothing material.
Project Description and Objectives
The objective of this work was to determine the efficacy of washing and drying clothing
in the removal and/or inactivation of bacterial spores from contaminated clothing materials. The
overall process for data generation involved bench-scale testing to simulate the washing and
drying of contaminated clothes. Viable Bacillus atrophaeus var. globigii (Bg) spores, a surrogate
for Ba, were quantified throughout this process. Tests were designed to evaluate the removal or
inactivation efficacy of selected detergents and bleach. The effects of washing temperature and
drying time on various clothing materials were also evaluated.
The washing/drying approach was evaluated in terms of decontamination efficacy (spore
inactivation) as a function of different parameters such as wash temperature, wash cycle time,
material clothing type, wash solution type (water versus bleach-type solution), and water rinse.
To assess the potential for viable spores to be removed and/or inactivated from clothing
materials, clothing material coupons, and wash and rinse liquids used in the decontamination
process were collected and individually analyzed to determine the number of viable spores
(quantified as colony forming units [CFU]) in each type of sample. This analysis strategy
provided not only an assessment of spore inactivation on clothing materials, but also of spore re-
distribution from contaminated clothing materials to the wash/rinse liquids.
The decontamination effectiveness of the washing/drying process was determined by
calculating the difference in colony forming units (CFUs) observed for the post-treated material
coupon (test sample) and those observed for a similar material coupon that was inoculated but
not treated (control sample). Recoveries were expressed on a log-10 scale. The fate and the
overall redistribution of the spores were also determined by the CFUs recovered in the wash and
rinse solution.
Quality control (QC) samples such as procedural blanks (clothing material samples that
underwent the washing/drying treatment process but were not inoculated) and negative controls
(clothing material samples that were neither inoculated nor treated) were included to monitor for
cross-contamination.
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2.0 EXPERIMENTAL APPROACH
The experimental work was performed at the EPA's facilities in Research Triangle Park
(RTP), North Carolina. The HSMMD RTP Microbiology Lab (BioLab) quantified the number of
viable spores for all samples. This study was performed in four (4) consecutive phases:
• Phase I included scoping tests designed to identify the decontamination solution that
would be used in subsequent test phases. These scoping tests were conducted without
material coupons using an orbital shaker platform to simulate a washing sequence (see
Section 3.7.1). The wash solutions were inoculated with ~ 101 Bg spores using a liquid
inoculation approach.
• In Phase II the experimental design was based on an initial scoping test (Phase I) that
determined the testing decontamination parameters that were to be used in Phase II
(bench-Scale experiments). Phase II experiments used test coupons of 3 inch (in.) x 3 in.
clothing materials inoculated with ~ 101 Bg spores using an aerosol deposition apparatus
(ADA) fitted with an actuator and a metered-dose inhaler (MDI). See Section 3.1.2.1.
• Phase III experiments were conducted using a portable compact twin tub washer spin
dryer (see Section 3.7.2) to decontaminate 14-in. x 14-in. inoculated test fabric materials
using the ADA dry spore inoculation approach.
• Phase IV was conducted using the previous compact washer along with a standalone
front-load compact laundry dryer (see Section 3.7.3) to decontaminate 14-in. x 14-in.
inoculated test fabric materials that had been contaminated using the ADA dry spore
inoculation approach.
Phase I: Scoping Test (Liquid Decontamination Solution Testing)
This phase was designed to determine the type of decontaminant solution that was to be
used in Phase II. The target decontamination solutions were tested at a dilution ratio of 1:10 in
deionized (DI) water at two wash temperatures (72 °F and 120 °F) to determine if they are
sporicidal under these testing conditions. The solutions found to be sporicidal were tested
further.
Liquid spore inoculation was used during this scoping phase. The Bg liquid spore
suspension was purchased from Yakibou, Inc. (Apex, NC) at a population density of ~ 1 x 109
CFU per ml. The inoculation was performed by making a 1:10 dilution of the original stock, then
aseptically applying 100 |il to the 300-ml volume decontamination solution to reach a target
inoculation of 1.0 x io7 spores (see Section 3.4.1). The test parameters evaluated during this
Phase I test sequence were:
1. Type of Decontamination Solutions
1. Tide® Free & Gentle detergent (Free and Gentle, Proctor & Gamble, Cincinnati, OH;
"detergent" henceforth)
2. Clorox® concentrated germicidal bleach ("diluted bleach" henceforth)
3. Diluted Clorox® bleach (Clorox Corp., Oakland, CA), with the addition of detergent
(Tide®)
4. Clorox 2® Free & Clear stain remover and color booster, H2O2 bleach (Clorox® 2
henceforth).
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2. Bleach Solution Dilution Ratio
1. 15:100
2. 10:100
3. 5
100
4. 3
100
5. 1
100
6. 5
1000
7. 1
1000
8. 1
10000
3. Wash Cycle Time
1. 9-min
2. 18-min
3. 27-min
Phase II: Bench-Scale Testing with Material Coupons
A series of tests were conducted with chlorinated bleach solution with and without the
addition of a surfactant (Tide® liquid detergent) to determine the decontamination efficacy of
chlorinated bleach formulations on 3 in. x 3 in. fabric materials inoculated with ~ 101 Bg spores.
The fabric materials were inoculated using an aerosol deposition apparatus (ADA) fitted with an
actuator and a metered dose inhaler (MDI) (see Section 3.4.2). The experiments were conducted
using 300-ml volume decontamination solutions in 500-ml Nalgene® bottles (Nalge Nunc
International Corporation, Rochester, NY) at ambient temperatures. The test parameters
evaluated during this test sequence were:
1. Type of Decontamination Solutions
1. Clorox® diluted bleach
2. Clorox® diluted bleach with the addition of a liquid Tide® detergent
2. Material Coupons
1. 100% Cotton material
2. 100% Polyester fabric
3. Cotton/nylon military fabric
4. Cotton/polyester blend fabric
5. Spandex fabric
6. Stretch denim cotton
3. Bleach Solution Dilution Ratio
1. 1:100
2. 1:1000
4. Wash Cycle Time
1. 18-min
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Phase III: Decontamination Testing using a Compact Washing Machine
This series of tests was conducted with chlorinated bleach solution with and without the
addition of a surfactant (Tide® detergent) using a portable compact twin tub washer to confirm
the results obtained in Phase I and II testing sequences. This testing sequence included the
inoculation of larger 14-in. x 14-in. fabric material coupons (see Section 3.4.2.2) in triplicate
followed by typical wash procedures.
The concentration of treatment (ratio of bleach to water) was based on results from Phase
II. A limited number of tests without the addition of a detergent were performed at a high
dilution (1:1000) ratio to determine if the sustained agitation dynamic of the washing machines
has any positive impact on the decontamination efficacy when compared to similar chlorinated
bleach solutions with added detergents. The test parameters evaluated during this test sequence
were:
1. Type of Decontamination Solutions
A. Clorox® diluted bleach
B. Clorox® diluted bleach with the addition of liquid Tide® detergent
2. Bleach Solution Dilution Ratio
A. 1:100
B. 1:1000
3. Wash Cycle Time
A. 18-min
4. Material Coupons
A. 100% cotton material
B. Stretch denim cotton
C. 100% polyester fabric
D. Spandex fabric
E. Cotton/polyester blend fabric
F. Cotton/nylon military fabric
Phase IV: Decontamination Testing using a Portable Washing Machine and Dryer
This series of tests was designed to evaluate the effect of adding a drying step to the
decontamination efficacy following a wash cycle. The chlorinated diluted bleach solution
(1:1000 dilution ratio) with the addition of a liquid detergent was not found to be fully effective
during the previous testing phases. This diluted bleach solution, which left residual
contamination in the test material, was chosen as the target decontamination solution to evaluate
if adding a drying sequence would increase the overall decontamination efficacy. The test
parameters evaluated during this test sequence were:
A. Clorox® diluted bleach (1:1000) with the addition of Tide liquid detergent
B. 18-min wash cycle
C. 30-min drying cycle
D. 100% cotton material
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3.0 TESTING AND MEASUREMENT PROTOCOLS
This section describes the test materials, material coupon preparation, material coupon
and equipment sterilization, test organism and spore inoculation, decontamination solution
preparation, neutralizing agent for test material coupon, decontamination equipment,
decontamination procedures, and neutralization procedures.
Test Materials
The fabrics selected for coupon fabrication were purchased from JOANN stores
(Durham, NC) and from Amazon (Seattle, WA). The six (6) clothing materials evaluated during
this project are shown in Figure 3-1 and listed in Table 3-1.
Figure 3-1. Material coupons tested.
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Table 3-1. Tested Material Coupons
Material ID
Description
Item Number
A: Cotton
Keepsake Calico 100% Cotton Fabric Purple Tonal
JOANN# 1560390
B: Denim
Stretch Denim Cotton (70% Cotton, 28% Poly/ & 2% Spandex)
JOANN# 1617661
C: Polyester
Blizzard Fleece Fabric (100% Polyester)
JOANN # 3604352
D: Spandex
Performance Apparel Poly Spandex (92% Poly/8% Spandex)
JOANN# 12505251
E: Blend
Sew Classics Knits Interlock Solid Blend (40% Poly/60%
Cotton)
JOANN# 12505251
F: Military
US Navy NWU Nyco Camouflage (50% Nylon/50% Cotton)
Amazon (ASIN
#B00XUYNBKE)
Material Coupon Preparation
Test material (fabrics) and stainless-steel (for inoculation support) controls were used in
this study. The stainless-steel coupons (14-in by 14-in) were made from 16-gauge type 304 mill-
finished steel sheet (McMaster-Carr, Elmhurst, IL) using a hydraulic sheet metal shear, and
carbide de-burring tool for the edges. These stainless-steel coupons were used as a support for
the test material coupons (fabrics) during the inoculation process and as independent inoculation
controls.
Material Coupon and Equipment Sterilization
This project required that many activities be performed on material sections or coupons
intentionally contaminated (test samples and positive controls) and not contaminated (procedural
and control blanks). The treatment of test samples, positive controls, and procedural blanks
varied for each group. Hence, specific procedures were used to mitigate cross-contamination
among test samples, controls, and coupon inoculation equipment, such as the aerosol deposition
apparatus (ADA) for dry spore inoculation. Adequate cleaning of all common materials and
equipment was critical to prevent cross-contamination. The fabric coupon materials were used
as-is and were not sterilized.
The common materials (templates and actuators) were sterilized using an ethylene oxide
(EtO) sterilizer system (PN 333 EOGas®, Andersen Products, Haw River, NC). All the items
that were sterilized were packed in appropriate self-sealed EtO pouches, and placed in
appropriate large sterilization bags, along with a dosimeter, a Humidichip® relative humidity
stabilizer (Andersen Products), and an EtO cartridge. The sterilization bags were vacuum-sealed
and loaded into the EtO sterilizer for an 18-hour sterilization cycle.
Stainless-steel coupons and Nalgene bottles were sterilized using a 121 °C gravity cycle
autoclave (SV 120 scientific pre-vacuum sterilizer; STERIS Amsco, Mentor, OH). Sterility was
evaluated by swab sampling one coupon from each sterilization batch.
Prior to use, test equipment (washers, dryers, and AD As) were sterilized using Vaporized
Hydrogen Peroxide® (VHP). The hydrogen peroxide (H2O2) vapor was generated using a
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STERIS VHP 1000ED system loaded with a 35% H2O2 Vaprox® cartridge (American
Sterilization Company, Mentor, OH). Each sterilization cycle lasted 4 hours at 250 ppm VHP.
Test Organism and Spore Inoculation
Bg, a surrogate for the spore-forming bacterial agent Ba, was used for this project. Like
Ba, Bg is a soil dwelling, gram positive, spore forming, aerobic microorganism, but, unlike Ba, it
is non-pathogenic. Bg forms an orange pigment when grown on nutrient agar, a desirable
characteristic when there is a need to detect viable spores in environmental samples. Bg has a
long history of use in the biodefense community as a simulant for anthrax associated biowarfare
and bioterrorism events (Gibbons et al., 2011).
Bg spores were used at a concentration of about 107 spores per test. Liquid spore
inoculation was used during the scoping test (Phase I), while dry spore inoculation on test
coupons was used for Phase II (Bench-Scale Testing with Coupons), Phase III (Wash Cycle with
Coupons), and Phase IV (Wash Cycle and Drying with Coupons).
3.1.1 Liquid Spore Inoculation
Liquid spore inoculation was used during the scoping phase. The Bg liquid spore
suspension was purchased from Yakibou, Inc. (Apex, NC) at a population density of ~ 1 x 109
CFU per ml. The titer of the stock was confirmed at the start of each testing event by the BioLab.
The inoculation was performed during Phase 1 testing by making a 1:10 dilution of the original
stock, then aseptically applying 100 |il to the 300-ml volume of distilled water in a 500-mL
Nalgene bottle (see Section 3.8.1) to reach a target inoculation of 1.0 x 107 spores per sample.
3.1.2 A erosol Deposition Apparatus (ADA) Assisted Inoculation of Surfaces
The dry spore test organism for this study was a powdered spore preparation of Bg
(Gibbons et al.. 2011) mixed with silicon dioxide particles. This preparation was obtained from
the U.S. Army Dugway Proving Ground Life Sciences Division. The powdered stock preparation
procedure is described in Brown et al. (2007). After 80 to 90% sporulation, the suspension is
centrifuged to generate a preparation of approximately 20% 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 (Degussa, Frankfurt am
Main, Germany).
Prior to inoculation, an ADA of appropriate size was situated directly on top of a flat test
surface. The MDI + actuator assembly was placed in the actuator fitting on top of the ADA, then
the lid was opened, and the MDI was activated one time to deliver approximately 107 Bg spores
to the test surface under the ADA. Following inoculation, the lid was closed, and the MDI
assembly was removed. The ADA was left in place for at least 18 hours to allow time for the
spores to settle. This process was repeated for each inoculated surface (inoculation controls,
positive controls, and test materials).
3.1.2.1 Phase II Spore Inoculation with MDI
Test and positive control coupons (3 in. x 3 in.) were inoculated with ~ 101 Bg spores
using an aerosol deposition apparatus (ADA) fitted with an actuator and an MDI. Each material
coupon was aligned and placed on top of a sterile stainless-steel coupon (3 in. x 3 in.) as shown
in Figure 3-2. An ADA designed to inoculate a 1 V2 inch diameter area in the center of each
7
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coupon was situated on top of the coupon, clamped into place, then coupled with the actuator
holding the MDI, as shown in Figure 3-3 A. Once the actuator coupling and MDI were fitted into
place, the ADA sliding stainless steel cover was opened (as shown in Figure 3-3B) and the MDI
was actuated one time to make a single discharge onto the coupon. A 0.22 (am filter covered the
.ADA exhaust, as shown in Figure 3-2, to prevent spore escape into the test area. Spores were
allowed to settle on the test coupon for 18 hours before use.
Figure 3-2. Test Coupons (3- x 3-in. stainless-steel and fabric material).
Actuator
I — m —
Figure 3-3. ADA with actuator and MDI, clamped onto test coupon, with stainless-steel sliding
panel closed (Panel A) and open ready for MDI actuation (Panel B).
3.1.2.2 Phases ill and IV: Dry Spore Inoculation
The inoculation of the test material coupons was performed like the dry deposition
inoculation used for Phase 2, with the exception that the ADA was larger to accommodate a 14
in. x 14 in. coupon, as described by Calfee et al... 2013. Briefly, each coupon was contaminated
independently with a separate custom-made pyramid-shaped ADA. The ADA was designed to
overlay and cover a 12" x 12" surface area of each 14" x 14" coupon. The MDI and the actuator
were positioned in the ADA opening, as shown in Figure 3-4, located at the ADA's top center.
The MDI was discharged a single time to inoculate the 12" x 12" coupon area.
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Figure 3-4. ADA (a) with the assembled actuator (b) and MDI (c).
Decontamination Solution Preparation
The decontaminant solutions that were used in this project were Tide®, Clorox®
(chlorine bleach), and Clorox® Free and Clear (hydrogen peroxide bleach). The Tide® detergent
(Free and Gentle, Proctor & Gamble, Cincinnati, OH) dilution ratio was 1:10 which is more
concentrated than the manufacturer's recommendation of 1.56 ounces per load. Clorox®
concentrated germicidal bleach (The Clorox® Company, Oakland, CA) was used first at a 1:10
dilution ratio, far greater than the dilution ratio recommended in the manufacturer's instructions
(dilute ¥2 cup [120 mL] of concentrated product in a load of laundry [typically 26 gallons, or 98.4
L]). The optimal dilution ratio was determined during Phase 1 of the study.
Neutralizing Agents for Test Samples
The presence of a bleach-based decontamination solution on the sample surface, or in the
liquid effluent following a decontamination event, could negatively bias CFU quantification
results. Based on previous studies, sodium thiosulfate (STS) was proven to be an effective
neutralizer for bleach on porous and non-porous surfaces (Calfee et al., 2011), and was used
during the post-decontamination recovery of the sampling li qui ds and sample fabric. The volume
of STS to be added to the collection media was first determined by measuring free available
chlorine (FAC) in the sample using an iodometric method for the determination of chlorine
dioxide and chlorite using a FIACFI® Hypochlorite Test Kit (supplier: Model CN-HRDT, Fisher
Scientific, Waltham, MA).
The FAC in the test coupon phosphate-buffered saline with 0.05% Tween 20 (PBST)
solution (Tween®, Sigma-Aldrich, St. Louis, MO), along with the volume of diluted bleach
collected in the wash and rinse, was used to calculate the volume of STS (Vsts) needed for
neutralization using Equation 3-1 adapted from the standard procedure for sample titration using
the FIACFI test kit.
9
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T7 _ FAC (mg/L) • VDB {ml)
VSTS NSrs • 35453 ' (3_1)
Where:
FAC = Free available chlorine (in milligrams per liter [mg/L])
Vdb = Volume of diluted bleach (rnL)
Nsts = Normality (N) of STS (2.26 N)
35453 = Equivalent weight (milligrams) per electron
Washing and Drying Simulation Equipment
Various equipment was used to simulate a washing and/or drying decontamination
scheme. Phases I and II used an orbital shaker to simulate the liquid/liquid agitation dynamic of
a washer, while Phase III and IV used an off-the-shelf compact washer and dryer.
3.1.3 Orbital Shaker
A Lab Line Incubator-Orbital Shaker (Figure 3-5) fitted with an orbital shaking
mechanism on a bench-scale platform, designed to hold 500-mL Nalgene bottles, was used to
simulate a washing machine.
Figure 3-5. Lab Line Incubator-Orbital Shaker fitted with a platform insert to secure Nalgene
bottles.
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3.1.4 Compact Washing Machine
A portable compact twin tub washer spin dryer (Model No. KP1040600A, Kuppet, Oslo,
Norway), shown in Figure 3-6, was chosen for this study to scale up the tests conducted under
Phase 2. The Twin Tub design function allows washing and spinning dry a load of laundry at the
same time. Washing is conducted in one tub while the other tub can be used for spin drying. The
total capacity for this machine is 8 lbs. for the washer and 5 lbs. for the spin cycle with 5 preset
3.1.5 Laundry Dryer
A standalone front load compact 1.5 cubic-feet capacity laundry dryer (Model No.
MCSDRY15W, Magic Chef, Wood Dale, IL)), shown in Figure 3-7, was used to evaluate the
effect of adding a drying step on the decontamination efficacy following a wash cycle. The dryer
was operated under a "FTigh Fleat" position for optimum drying with a timer set at 30 minutes.
Figure 3-7. Magic Chef compact laundry dryer.
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Washing and Drying Test Procedures
Decontamination procedures with sampling protocols were devised for each testing phase
as described in the next sections. All procedures described in these methods should be conducted
under sterile conditions to prevent contamination of the sample with background contaminants
and to prevent cross-contamination.
3.1.6 Phase I: Scoping Test
The detailed testing sequences for Phase I (scoping test without test coupons) is described
in the following section:
1. Aseptically pour 270 ml of sterile DI water into each sterile 500-ml Nalgene bottle.
2. Add 30 mL of treatment liquid (detergent or bleach).
3. Use a second Nalgene bottle containing 300 ml of DI water, to be treated in the same
water bath, so that the water temperature inside the bottles can be measured.
4. Heat the Nalgene bottles in a water bath until the desired water temperature (either 72
°F or 120 °F) is achieved.
5. Add liquid inoculum to reach a target concentration of 1.0 x 107 spores per bottle
(100 |iL as per Section 3.4.1).
6. Place bottles in an orbital shaker fitted with a platform designed to hold 500-ml
Nalgene bottles.
7. Shake at ambient temperature and 250 rpm for a 9-minute cycle.
8. Add 25 mL of 2N STS, and mix.
9. Analyze the wash liquid for the presence of spores.
3.1.7 Phase II: (Bench-Scale Testing with Material Coupons)
The detailed decontamination testing sequence for Phase II is described in the following
section:
1. Place coupon (inoculated or control) in a 500-ml Nalgene bottle.
2. Aseptically pour 300 mL of sterile DI water, 10% bleach in water (treatment), or
diluted bleach with detergent into each sterile 500-ml Nalgene bottle.
3. Place bottles in an orbital shaker fitted with a platform designed to hold 500-ml
Nalgene bottles.
4. Shake at 250 rpm at ambient temperature for a 9-minute cycle.
5. Transfer coupon to labeled 500-mL Nalgene bottle and add 300 mL of DI water at
ambient temperature.
6. Add 40 mL 2N STS to the wash liquid solution, and mix.
7. Take sample of the neutralized wash solution (Wash Sample).
8. Place bottles with the coupons in an orbital shaker fitted with a platform designed to
hold 500-ml Nalgene bottles.
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9. Shake at 250 rpm and ambient temperature for a 9-minute cycle.
10. Add 40 mL 2N STS to the rinse solution and mix.
11. Take sample of the rinse (Rinse sample).
12. Transfer coupon to labeled Stomacher® bag(P/N BA6141/CLR Seward Ltd.,
Worthing, West Sussex, United Kingdom) (Coupon sample).
13. Analyze the wash liquid, rinse liquid and coupons for the presence of spores.
3.1.8 Phase III: (Decontamination Testing using a Washer)
The detailed decontamination testing sequence for Phase III (washer) with test coupons is
as summarized below:
1. Day 1: Spore Inoculation:
1. Dose 3 fabric coupons of each material (3 per test), and 2 steel coupons
(inoculation controls) with 107 CFU Bg spores using AD As (section 3.4.2.2).
2. Leave coupons, procedural blank and inoculation controls covered with AD As for
18 hrs.
2. Day 2: Decontamination Testing
1. Wipe sample the stainless-steel inoculation controls and negative controls.
2. Place fabric material negative and fabric material positive controls into
individually labeled Stomacher bags.
3. Collect 100 mL of tap water for sterility check.
4. Prepare 14.0 L of the 1% dilute bleach solution in tap water.
5. Titrate the 1% dilute bleach solution to verify concentration.
6. Add 4.2 mL Tide® detergent to the 14 L of 1% dilute bleach solution to be used
as wash water.
7. Titrate the solution to determine the amount of 2N STS needed for
decontamination solution neutralization.
8. Collect 100 mL of this solution for sterility check.
9. Pour 3.25 L of this solution into each of 4 compact washers (3 for testing, one for
procedural blank).
10. Place inoculated fabric material coupons into 3 test washers and the procedural
blank into the washer designated for it.
11. Set the wash cycle to 9 minutes for each washer. At the end of 9 minutes reset the
timer to another 9 minutes for a total wash time of 18 minutes.
12. At the end of 18 minutes, move the coupons to the spin side of the washer and
spin for 5 minutes.
13. As the spin cycle proceeds, drain the wash water from each washer into an
adequately sized sterile vessel.
14. From each vessel pipette 300 mL of the wash solution to a sterile Nalgene bottle
that contains 5.0 mL of 2N STS.
15. After the spin cycle, place the coupon back in the wash side and add 3.25 L of tap
water only from the remaining sterile pitchers. Set the rinse cycle for 9 minutes.
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16. At the end of the rinse cycle, add 50 mL 2N STS to the rinse water containing the
cloth coupons. Turn on the washer briefly to ensure good mixing.
17. Drain the rinse water into the same pitcher the tap water was in and transfer the
fabric material coupons to the spin basket and spin for 5 minutes.
18. Pour 300 mL of the rinse water into sterile Nalgene bottles.
19. At the end of the second spin cycle, transfer the test fabric material coupons into a
Stomacher bag for analysis.
20. Analyze all wash solution, rinse water, and material fabric coupons for the
presence of spores.
3.1.9 Phase IV: (Decontamination Testing using a Washer & Dryer)
The detailed decontamination testing sequence for Phase IV is similar to the Phase III
testing sequence with the inclusion of a drying sequence. The changes in the testing and
sampling approaches are shown in italics in the following test sequence.
1. Day 1: Spore Inoculation:
1. Dose 6 cotton material coupons (2 duplicate sample coupons for each test), and 2
steel coupons (inoculation controls) with 107 CFU Bg spores using AD As.
2. Leave coupons, procedural blank and inoculation controls covered with AD As for
18 hrs.
2. Day 2: Decontamination Testing
1. Wipe sample the stainless inoculation controls and stainless-steel negative
controls.
2. Place fabric material negative and fabric material positive control coupons into
individual Stomacher bags.
3. Collect 100 mL of tap water for sterility check.
4. Prepare 14.0 L of 1% dilute bleach solution in tap water.
5. Add 4.2 mL Tide® detergent to the 14 L of 1% dilute bleach solution.
6. Titrate a sample of the wash solution to determine the amount of 2N STS needed
for wash solution neutralization.
7. Collect 100 mL of the wash solution for sterility check.
8. Pour 3.25 L of this solution into each of 4 compact washers (3 for testing, one for
procedural blank).
9. Place a set of duplicate inoculated fabric material coupons into each of 3 washers,
and a set of duplicate non-inoculated cloth coupons in the fourth washer
(procedural blank).
10. Set the wash cycle for 9 minutes for each. At the end of 9 minutes reset the timer
to another 9 minutes for a total wash time of 18 minutes.
11. At the end of 18 minutes, move the duplicate coupons to the spin side of the
washer and spin for 5 minutes.
12. As the spin cycle proceeds, drain the wash solution from each washer into an
adequately sized sterile vessel.
13. From each vessel pipette 300 mL of the wash solution to a sterile Nalgene bottle
that contains 5.0 mL of 2N STS.
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14. After the spin cycle, place the duplicate coupons back in the wash side and add
3.25 L of tap water only from the remaining sterile pitchers. Set the rinse cycle for
9 minutes.
15. At the end of the rinse cycle, add 50 mL 2N STS to the rinse water with cloth
coupons. Turn on the washer briefly to ensure good mixing.
16. Drain the rinse water into the same pitcher the tap water was in and transfer the
duplicate cloth coupons to the spin basket and spin for 5 minutes.
17. Pour 300 mL of the rinse water into sterile Nalgene bottles.
18. At the end of the second spin cycle transfer one test coupon (Coupon 1) per set of
duplicates into a labeled Stomacher bag for analysis and transfer the second
(Coupon 2) to a separate dryer for a 30-min drying sequence.
19. At the end of 30 minutes drying time, transfer Coupon 2 into a Stomacher bag for
analysis.
20. Remove the discharge filter from each dryer and transfer to Stomacher bags for
analysis.
21. Analyze all wash water, rinse water, coupons, and dryer HEP A filter for the
presence of spores.
Sample Neutralization Procedure
Neutralization tests were designed to determine the neutralizer's effectiveness in stopping
the activity of the residual decontamination solution in the collected samples (wipe, runoff, and
rinse) before microbiological analysis. To test for the efficacy of neutralization at low
concentrations of spores, 10% and 1% dilute bleach solutions were neutralized with appropriate
amounts of 2N sodium thiosulfate (STS) for the chlorine-based solution and potassium
permanganate (KMnC>4) and sulfuric acid (H2S) for the bleach-based solutions, and then
inoculated with a known concentration of spores (~5 x 102 CFU for low concentration and 107
CFU for high concentration). These samples were plated immediately and again after 4 days in
order to test for any residual decontamination effects.
To test for the possible toxicity of the STS solution, the amount of 2N STS used to
neutralize the respective dilute bleach solutions, was added to PBST, and to a separate set of
samples with the same amount of STS added to DI water. These samples were plated on the
same days as the neutralized bleach solutions. All samples were compared to positive controls
consisting of inoculated PBST and inoculated DI water. The neutralization procedure for each
test sample is summarized below:
Chlorine-based Solution Neutralization
1. Day 1 Activities
1. Perform concentration check of inoculum to be used.
2. Ensure that enough filter sterilized STS is on hand to be used for the test.
3. Filter sterilize 3 liters of DI water to be used in the test.
4. Sterilize 6 Erlenmeyer flasks and 6 stir bars.
2. Day 2 Activities
1. Prepare 2 Erlenmeyer flasks with 200-mL of PBST with sterile stir bar.
2. Prepare 2 Erlenmeyer flasks with 200-mL of DI water with sterile stir bar.
15
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3. Titrate initial bleach stock solution to be used. Ensure FAC is within the QC
criteria.
4. Make 200 mL of a 1:10 dilution of stock bleach (10% Dilute Bleach solution used
for the 3 test samples and 1 procedural blank sample).
5. Calculate amount of STS needed to neutralize the total 200-mL stock solution of
10% Dilute Bleach.
6. Add 2x the amount of STS needed to the stock dilute bleach solution and place on
stir plate for 5 minutes to ensure appropriate mixing of STS and bleach.
7. Add this same amount of STS to one of the flasks containing DI water and one of
the flasks containing PBST (solutions used to check for and STS toxicity).
8. Re-titrate the now neutralized 10% bleach stock solution and ensure that FAC
measurement is 0.
9. Dispense 40 mL aliquots of each of these solutions into four 50-mL conical tubes.
(There should be 4 tubes with 40-mL of the neutralized dilute bleach solution, 4
with the PBST and STS, and 4 with the DI water and STS)
10. Repeat steps 4-9 except using a 1:100 dilute bleach stock and appropriate amount
of STS for that solution.
11. Inoculate all test samples at the target Bg spores concentration from a liquid stock
solution prepared in PBST.
12. Plate all samples immediately.
3. Day 6 Activities
1. Plate all test samples with same volumes used on initial plating to ensure no residual
decontamination occurred.
Hydrogen Peroxide-based Solution Neutralization
1. Day 1 Activities
1. Perform concentration check of inoculum to be used.
2. Sterilize 6 Erlenmeyer flasks and 6 stir bars.
2. Day 2 Activities
1. Transfer approximately 5 mL of the 1:10 diluted hydrogen peroxide-based
solution to a tared 250 mL flask. Record weight of H202 sample.
2. Add 40 mL of 5% H2SO4, then dilute to about 150 mL with DI water.
3. Place the flask containing the sample on a stir plate, then titrate to the first
permanent pink color with IN KMnC>4. Record required volume of KMnC>4.
4. To determine % H2O2 (wt/wt %), use the following equation.
% H2O2 (by weight) = (mL KMnCU) / 1000 x N / 2 x (34.0146 g/mol) / (g of sample) x 100,
Where: N = Normality of the standard KMnC>4
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5. Inoculate all test samples at the target Bg spore concentration from a liquid stock
solution prepared in PBST.
6. Plate all samples immediately
Day 6 Activities
1. Plate all test samples with same volumes used on initial plating to ensure no
residual decontamination occurred.
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4.0 SAMPLING AND ANALYSIS APPROACH
The inoculation of the test solutions and fabric coupons, decontamination testing, and
sampling took place in the EPA/RTP Decontamination Testing Research Facility (High Bay
Room H130A). Care was taken so that the inoculation area and the sampling area were separated
to avoid cross-contamination.
The following sections discuss sampling material and equipment, sampling methods,
quality assurance/quality control (QA/QC) samples, sample containers, sample preservation,
sample quantities, and prevention of cross-contamination of sampling and monitoring equipment.
Sampling Material and Equipment
Before sampling, all materials needed for sampling were prepared using aseptic
techniques. In addition, general sampling supplies were sterilized and disinfected for each
sampling event. A sampling material bin was stocked for each sampling event. The bin contained
enough sampling kits to accommodate all required samples for the specific test. Table 4-1 lists
the materials that were used for sampling. In addition to the items listed in the table some other
items that are needed include: sterile forceps, conical tubes, Stomacher bags, sampling data
sheets, permanent marker or pre-printed sample labels, and PPE.
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Table 4-1. Sampling Materials and Equipment
Material or Equipment
Description
Disposable laboratory coats
Kimberly-Clark Kleenguard™ A10 light duty apparel (P/N 40105)
PBST
PBS with 0.05% Tween 20 (Sigma Aldrich USA, P/N: P3563-
10PAK)
Sterile sampling bags
TWIRL'EM® sterile sampling bags (Labplas Inc. Corporation
Canada, Quebec, Canada) Overpack Size 10 by 14 inches
Inner bag size: 5.5 by 9 inches (wipe)
Sample bag size: 5.5 by 9 inches
1 L or 500 ml Nalgene bottles
Nalgene bottle (Part No. 2187-0032, Thermo Fisher Scientific,
Waltham, Massachusetts [supplier])
Bleach wipes
Dispatch® bleach wipes (Clorox Co., Oakland, CA) or
Hype-Wipe®) towelettes (Current Technologies, Indianapolis, IN)
Polyester-rayon blend wipes for
sampling
Kendall Curity™ Versalon all-purpose, absorbent gauze sponges, 2
by 2 inches, sterile packed, #8042, four-ply (Covidien PLC, Dublin,
Ireland)
Swabs
BactiSwab® swab (Remel Inc., San Diego, CA)
(http: //www .reme line .com/Industrial/C ollectionT ransport/Bacti Swab
.aspx; last accessed luly 2, 2020)
1.5 cubic ft. electric dryer
Magic Chef® - Compact 1.5 cu. ft. Electric Dryer in White. Model #
MCSDRY15W (MC Appliance Corp., Wood Dale, IL)
Washing machine, portable with
spin dryer
Kuppet washing machine, portable compact twin tub washer spin
dryer; 13 lb capacity; Model # KP1040600A (Kuppet, Oslo, Norway)
Clorox® concentrated germicidal
bleach
Clorox® 121 oz. Concentrated Germicidal Bleach, UPC:
4460030798
Clorox 2®
Clorox 2® - Free & Clear Stain Remover & Color Booster, UPC:
44600300481
Tide® detergent
Tide® Original Liquid Laundry Detergent, UPC: 037000138822
(Proctor & Gamble, Cincinnati, OH)
Additional measurements prior to or during the decontamination procedure application
were required to ensure quality control during testing. These measurements include quality
control checks on the reagents and equipment being used in the decontamination procedure. A
sampling data log sheet was maintained for each sampling event (or test) that included each
sampling event, the date, test name, sample IDs, and other test details such as test temperature,
sample volumes, and sample extraction time. The following sections discuss the sampling
protocol, sample types, and frequency of sampling and monitoring events.
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Sampling Methods
The major classes of operational samples for this study are summarized below:
1. Cloth material coupon
2. Liquid (wash solution and rinse)
3. Surface wipe samples
4.1.1 Cloth Material Coupon, and Liquid Samples
The cloth material coupons, and liquid samples (wash solution and rinse) were collected
as described in the testing sequence (see Section 3.8.). Within each sampling event, each
sampling coupon was placed in a Stomacher bag with 100 ml of sterile PBST, the wash and rinse
solutions were pipetted into sterile 500-ml Nalgene bottles.
4.1.2 Wipe Sampling
Wipe sampling was performed on stainless-steel coupons: three (3) inoculation controls
to verify the consistency of spore application associated with each MDI, and one (1) negative
control to monitor for cross-contamination. The general approach was that a moistened sterile
polyester-rayon blend wipe (Curity™ all-purpose sponges #8042, 2-in x 2-in, 4-ply, Covidien
PLC, Dublin, Ireland) was used to wipe a specified area to recover the target organism. Sampling
was conducted on one coupon at a time. All coupons were placed horizontally for sampling.
The wipe sampling kits were processed in the BioLab by aseptically removing the wipes
from their packing and placing them into an unlabeled sterile 50-mL conical tube (Cat. No. 14-
959-49A, Fisher Scientific, Waltham, MA) using sterile forceps (Part No. 7190 Busse Hospital
Disposables, Hauppauge, NY). Each transferred wipe was then moistened by adding 2.5 mL of
sterile PBST and capped. The bags and conical tubes need to be labelled with the correct sample
information. The general sampling approach for the MDI inoculation controls is summarized
below:
1. Don a fresh pair of sterile boot covers and personal protective equipment (PPE)
(laboratory coat, nitrile gloves, P-95 respirator, and safety glasses).
2. Remove the clamps from the Blank MDI control coupon, and then lift the ADA off the
coupon.
3. Wipe the surface of the coupon horizontally, using a consistent amount of pressure and
using S-strokes to cover the designated sample area of the coupon.
4. Fold the wipe in half, concealing the exposed side and then wipe the same surface
vertically using the same S-stroke technique.
5. Fold the wipe again and roll it so that it fits into a conical tube.
6. Place the folded wipe into the conical tube.
7. Place the conical tube in a sterile Twirl'Em® bag (Labplas Inc. Corporation Canada,
Quebec, Canada).
8. Sterilize the outside of the Twirl'Em bags using bleach wipes.
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9. Place the sample in the collection bin.
Repeat this process for the 3 MDI control coupons and the 1 MDI negative control.
4.1.3 Quality Assurance/Quality Control (QA/QC) Samples
QA/QC samples were collected as indicators of sterility of sample materials and
equipment before and after a sampling event. The QA/QC samples including swab and material
samples are discussed next.
4.1.3.1 Swab Samples
Swab samples for biological data quality indicators were used for sterility checks on
coupons, materials, and equipment before use in the testing. A single, pre-moistened swab
sample was collected from each item and coupon.
The general approach for swab sampling was to use a moistened swab (BactiSwab®
collection and transport system, R12100, Remel, Thermo Fisher Scientific, Waltham, MA) to
wipe a specified area to recover bacterial spores. The swab sampling procedure is described
below:
1. Through the sleeve, crush the moistening ampule of the BactiSwab™ swab at midpoint.
2. Hold BactiSwab™ tip end up for at least five seconds to allow the medium to wet the swab.
3. Open the package and remove the BactiSwab™ swab.
4. Label the plastic tube.
5. Remove the cap-swab from the plastic tube.
6. Swab the surface while spinning the cap-swab between the thumb and index fingers.
7. Return cap-swab to tube.
8. Date and initial each sample tube.
4.1.3.2 Material Samples
QA/QC material samples were collected to provide information on the level of
contamination possibly present during sampling due to contaminated materials. These samples
were the unexposed field blank (sterility) samples. Also, a blank plating of microbiological
supplies served as the control for sterility checks of supplies used in dilution plating.
Sampling Containers
For each wipe sample, the primary sample container used was an individual, sterile, 50-
ml conical tube. Secondary and tertiary containment consisted of sterile sampling bags.
Cloth and filter coupons were placed into Stomacher closure bags (P/N BA6141/CLR
closure bags, Seward Laboratory System Inc., Islandia, NY), with 100 ml of PBST. Secondary
and tertiary containment consisted of a second and third Stomacher bag.
Liquid effluent samples were collected into individual sterile specimen cups or Nalgene
bottles and placed inside pre-labeled, sterile bags for secondary containment.
Samples were transferred to the BioLab for microbiological analysis in sterile, primary,
independent packaging within sterile, secondary containment containing the collected samples
for analysis.
21
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Sample Preservation
After sample collection, sample integrity was maintained by storing samples in quadruple
containers: first, a sample container; second, a sterile bag; third, a sterile bag with its exterior
sterilized during sample packaging; and fourth, a sterile container holding all samples from a
testing event. All individual sample containers remained sealed while in the decontamination
laboratory and during transport.
Sample Quantities
Each decontamination event was evaluated in triplicate. Table 4-2 lists the total number
of each type of sample.
Table 4-2. Sample Quantities for Each Decontamination Test
Sample Type
Number of Replicates
Test Coupon
3 (Phases II, and III); 6 (Phase
IV)
Test Coupon Wash Cycle Solution
3
Test Coupon Water Rinse
3
Procedural blank wash cycle liquid
1
Procedural blank rinse cycle liquid
1
Negative control coupon
1
Positive control coupon
3
Inoculation control
3-4
Swab sample
1 per item
Preventing Cross-Contamination During Sampling
Several administrative controls were put into place to prevent cross-contamination. This
project is labor intensive and requires that many activities be performed on coupons that are
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 the effort to
prevent cross-contamination among the groups.
Before each test, all materials and equipment needed for sampling were sterilized as
discussed in Section 3-8. and handled using aseptic techniques. Non-powdered, surgical gloves
were used during sampling. Individually wrapped, pre-moistened bleach wipes were placed in
sterile sampling bags. Dispatch® bleach wipes were also used for sterilization of work surfaces,
sample containers, and outside sampling bags. A sampling material bin was stocked for each
sampling event based on the sample quantity. The bin contained enough wipe sampling kits to
accommodate all required samples for each specific test. The following test protocol was
implemented for each test.
Table 4-3 lists the cleaning methods and their frequency for common test materials and
equipment.
22
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Table 4-3. Cleaning Methods and Frequency for Common Test Materials and Equipment
Material or
Equipment
Use
Cleaning Method
Frequency
Glassware, Nalgene
bottles, and DI tanks
Testing and sample
collection
Autoclaving
Before each test
All Work Surfaces
Throughout each test
Wiping with isopropyl
alcohol
Before and after each
use
ADAs, washer, and
dryer
Inoculation and
testing
VHP
Before each test
23
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5.0 ANALYTICAL METHODS
The BioLab analyzed samples either qualitatively for spore presence (quality control,
swab samples) or quantitatively for the number of viable spores recovered per sample. Results
were reported in colony forming units (CFUs) per unit volume. Details of the extraction and
analysis procedures are provided in the next sections.
Sample Extraction
Spores were extracted using the appropriate method for each sample type, as described
below. After extraction, aliquots were removed for either dilution plating or filter plating, as
appropriate for expected spore recoveries.
5.1.1 Coupon Extraction
Each material sample coupon was aseptically placed in Stomacher bags (P/N
BA6141/CLR Seward Ltd., Worthing, West Sussex, United Kingdom) along with 100-ml of
sterile PBST. The coupon was extracted in a Stomacher 400 Circulator machine (Seward Ltd.,
Worthing, West Sussex, United Kingdom) for 1 minute at 260 rpm. The excess liquid was
recovered from the material coupon while in the bag, and then the coupon was aseptically
retrieved and discarded. The eluent was transferred to a sterile specimen cup (P/N B120210,
Starplex Scientific Corp., Cleveland, TN, USA) and sonicated for 10 minutes in a Branson
Ultrasonics™ cleaner (Model 8510R-MT, Emerson Electric Co., St Louis, MO, USA) and was
then vortexed (Multi-Tube Vortexer, P/N 58816-115, VWR, Radnor, PA) continuously for
2 minutes prior to plating.
5.1.2 Wipe Sample
Wipe samples were extracted in 20 mL of sterile PBST as extraction buffer by agitating
the tubes with a vortex mixer set to the maximum rotation for 2 min in 10-s intervals. After
extraction, the sample buffer was subjected to serial dilution and analyzed using procedures
described in Section 5.2. All extracts were stored at 4 ± 2 °C.
5.1.3 Swabs
Each swab was removed from the packaging in a Biological Safety Cabinet (BSC), then
rotated across the tryptic soy agar (TSA) media surface in a zig-zag pattern until the entire
circumference of the swab contacted the media surface. Plates were incubated at the appropriate
temperature and time based on the Bacillus strain used in the test. Swab plates were manually
observed to determine "growth" or "no growth" of the target organism.
Spiral- and Filter- Plating Procedures
Sample extracts requiring dilution were plated in triplicate using an Autoplate® 5000
spiral plater (Autoplate® 5000, Advanced Instruments Inc., Norwood, MA). The automated
spiral plater deposits the sample in exponentially decreasing amounts across a rotating agar plate
in concentric lines to achieve three 10-fold serial dilutions on each plate. Plates with Bg samples
were incubated at 35 ± 2 °C for 18 to 20 hours. Colonies on each plate were enumerated using a
QCount® colony counter (Advanced Instruments Inc., Norwood, MA).
Positive control samples were diluted 100-fold in PBST before spiral plating, while
samples of unknown concentration were plated with no dilution and with a 100-fold dilution.
24
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Samples with known low concentrations were plated with no dilution. The QCount® colony
counter automatically calculates the CFU/mL in a sample based on the dilution plated and the
number of colonies that developed on the plate. The QCount® records the data in an MS Excel
spreadsheet.
Only spiral plates meeting the threshold of at least 30 CFUs were used for spore recovery
estimates. After quantitation with the QCount® colony counter, samples with plate results
below the 30-CFU threshold were either re-spiral plated with a more concentrated sample aliquot
or filter-plated to achieve a lower detection limit. The filter plate volume was based on the CFU
data from the QCount® result. Filter plating was performed using 100-ml capacity micro funnel
units with 0.45 |im Pall® filters (Item # 4852, Pall Corporation, Laboratory, Port Washington,
NY) and a vacuum manifold (Pall Corporation, Laboratory, Port Washington, NY). The filters
were placed onto TSA plates and incubated at 35 ± 2 °C for 20 to 24 hours before manual
enumeration.
The test samples, procedural blanks, and sterility controls were filter plated with 1/10/100
ml aliquots, 1/10 ml/remainder aliquots or various aliquots until 2 plates were obtained within
the countable range for filters (15-150 CFU/filter).
Other Analytical Procedures
5.1.4 Determination ofFACby Titration
Measurements ofFAC were performed using an iodometric method that uses a HACH®
digital titrator and a HACH® reagent titration kit (Model #16900, HACH Company, Loveland,
CO). The titration procedure can be found in the HACH® digital titrator manual (https://pim-
resources.coleparmer.com/instruction-manual/24908-0Q.pdf).
5.1.5 pH and Temperature Measurements
Measurements of pH and temperature of the bleach solutions were performed daily using
a calibrated pH meter (Oakton® Acorn™ pH 5, OAKTON Instruments, USA). The temperature
sensor included with the pH meter was factory-calibrated and checked monthly by comparison of
the displayed value to a NIST-certified thermometer.
Decontamination Efficacy
Data reduction was performed on measurements of the total spores (CFU) recovered from
each sampled surface coupon material, liquid samples and rinse samples. The following groups
of samples were collected for each decontamination testing sequence and used to determine the
effectiveness of the remediation technique.
• MDI control coupons: Stainless steel coupons inoculated at the same time as the fabric
material coupons and sampled by sponge wipe. These coupons are inoculated at the
beginning, middle, and end of each inoculation campaign to assess the stability of the
MDI during the inoculation operation.
• Positive control: Sample from fabric material coupon contaminated with biological
agent but not subjected to experimental conditions.
• Procedural positive control: Representative materials that were inoculated and
subjected to a water wash cycle.
25
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• Test coupons: Inoculated fabric coupons that went through a decontamination
treatment.
• Procedural blank: Sterile non-inoculated material that was subjected to the same
decontamination process as the inoculated test samples.
• Negative control material: Sterile non-inoculated material that was not subjected to a
decontamination process.
• Post-decontamination (Post-decon) liquid wash sample: Aliquot sample of the
decontamination solution following a decontamination event.
• Post-decon liquid rinse sample.: Aliquot sample of the water rinse following a
decontamination event.
• Procedural liquid wash sample: Aliquot sample of the water solution following a
water-only wash event.
• Procedural liquid rinse sample: Aliquot sample of the water rinse following a water-
only wash event.
Decontamination/removal efficacy was expressed as a log reduction (LR) of the viable
Bg colony forming units (CFU) recovered following treatment of coupon material from the
average of the inoculated positive control samples recovered CFU. Typically, for laboratory
assessments of decontamination efficacy, for a 1 x 106 colony forming units (CFU) challenge or
greater, a LR > 6 is considered effective (U.S. EPA. 2018). and when no viable spores are
recovered after decontamination treatment, the method is considered highly effective.
Decontamination/Removal efficacy for each testing procedure is defined as log reduction
(LR) of the average viable Bg spores recovered from the treated test material sample from the
average of the inoculated positive control samples for that material. This value will be reported
as a log reduction on the specific material cloth as defined in Equation 5-1.
£log (CFUct) X^g(CFUst)
1
k=\
k=1
N
(5-1)
where:
Surface decontamination effectiveness; the average log
= reduction of spores on a specific material coupon (designated
by /).
Nc
2>g(CFt/Cii)
The average of the logarithm (or geometric mean) of the
number of viable spores (determined by CFUs) recovered on
the positive control coupons (C indicates control and Nc is the
number of control coupons).
k=1
26
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Nt
5>g (CFUsa)
k=1
N.
The average of the logarithm (or geometric mean) of the
_ number of viable spores (determined by CFUs) remaining on
the coupon material (S indicates a decontaminated coupon and
Ns is the number of coupons tested).
When no viable spores are detected on a decontaminated coupon, a value of 1 CFU will
be assigned for extracted sample, k, and the efficacy will be reported as greater than or equal to
the value calculated by Equation 5-1.
The standard deviation of the average log reduction of spores on a specific cloth material
) is calculated using Equation 5-2:
SDnr
where:
Ik-*,)3
k=l
Ns-1
SD.
7, =
Standard deviation of ^7, the average log reduction of spores
on a specific material cloth.
The average log reduction of spores on a specific material
cloth (specific material designated by /').
The average of the log reduction from a decontaminated
coupon (Equation 5-3).
JCk =
Ns = Number of test coupons of a material coupon type.
(5-2)
N „
X((log(CFC/c)-log(CFf/a))
=
where:
k=l
Ns
(5-3)
Nr
log (CFUc) =
Xlog (CFUCJe)
k=1
N,
c
The "mean of the logs" (geometric mean), the
average of the logarithm-transformed number
= of viable spores (determined by CFU)
recovered on the control coupons (C =
control coupons, Nc = number of control
coupons, k = coupon number, and Ns is the
27
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number of test coupons).
CFUsm =
Number of CFUs recovered from the klh
decontaminated coupon.
N.
s
_ Total number (l,k) of decontaminated
coupons of a material type.
28
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6.0 RESULTS AND DISCUSSION
This section discusses the impact of washing and drying as a potential method to reduce
and/or inactivate biological spores from common clothing material. The overall process for data
generation involved bench scale testing to simulate the washing and drying of contaminated
clothes. Viable Bg spores, a surrogate for B. cmthrcicis (Ba), were quantified throughout this
process.
Section 6-1 describes the scoping phase (Phase I) during which a bench-scale washing
machine simulation (without material coupons) was used to evaluate potential decontamination
solutions and test parameters (e.g., temperature, decontamination solution dilution ratio, and
wash cycle times) for use in subsequent test phases. Section 6-2 discusses Phase II in which
bench-scale experiments were conducted with inoculated cloth coupons (3 in. x 3 in.) and the
optimal decontamination parameters observed during Phase I. Section 6-3 discusses the Phase III
pilot-scale experiments performed with off-the-shelf washers, decontamination solutions
identified in Phase I, and larger inoculated cloth coupons (14 in. x 14 in.). Section 6-4 discusses
the results of Phase IV using the combination of a washer/dryer during the decontamination
process. Finally, Section 6-5 summarizes the results of the overall study.
Phase I: Scoping Test
Experiments began with testing the extraction efficiency of the procedure to be used
when processing the cloth material coupons and then evaluating the decontamination efficacy of
proposed decontamination solutions at different temperatures. Subsequent tests determined
neutralizer effectiveness to prevent residual decontamination past set washing time points.
Finally, effective decontamination solutions were challenged by adjusting washing parameters,
including adjusting decontamination solution dilution ratios and adjusting wash cycle times. All
tests in this phase, with the exception of the first extraction efficiency test, were performed using
a liquid spore inoculation and without cloth coupons, which were used in the later phases of this
project. The test progression results are described in the next sections.
6.1.1 Extraction Efficacy
The recovery of spores from five (5) cotton positive control coupons (3 in. x 3 in.) was
tested. Coupons were placed in a Stomacher bag filled with 100-mL of sterile PBST, and
extracted using the Stomacher 400 Circulator for 1 min at 260 rpm extraction method. Results
(Section 5.1.1) were compared to the recovery of spores from three (3) 14 in. x 14 in. stainless-
steel control coupons using the wipe sampling method and extracted in 20-mL of sterile PBST
extraction buffer (Section 5.1.2). Table 6-1 displays average recoveries for the cloth extraction
procedure compared to the stainless-steel controls.
Table 6-1. Material Coupon Sample Extraction Efficacy Evaluation
Coupon Type
Number of
Samples
Average
(CFU)
Stdev (CFU)
Stainless Steel
3
3.Ox107
7.7 xlO6
Cotton Material
5
2.7x 107
2.4 xlO6
CFU, colony forming units; Stdev, standard deviation
29
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6.1.2 Challenges with Filter Plating Residua1 Decontamination
The filtration and subsequent plating of 1-mL and 10-mL aliquots through white Pall®
filters (Pall Life Sciences, Ann Arbor, MI, P/N 4804 MicroFunnel™ with GN-6 Metricel®
membrane with a 0.45 jim pore size made from a mixed cellulose ester) resulted in unexpected
recoveries for Clorox bleach post-decon samples. The 1 ml filters had too many colonies to count
while the 10 ml filters were either non-detect or had very few colonies. Filter plates from the
same selected sample, plated at the same time, are shown in Figures 6-1 and 6-2, respectively.
Figure 6-1. 1-mL aliquot filter plated from 10% bleach solution sample.
Figure 6-2. 10-mL aliquots filter plated from 10% bleach solution sample.
These unexpected results raised questions about all results obtained with the filter plating
of the chlorinated bleach solutions. A scoping test (Appendix A) was performed to evaluate
whether the different 0.45 jam Pall filter membrane materials (GN-6 white P/N 4804 mixed
30
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cellulose ester, black P/N 4805 and white P/N 4802 made of polyethersulfone [PES]) were
influencing the recovery results. The black Pall filters are typically used to analyze lighter
colored colonies (e.g., Btk colonies are white).
The results of the scoping test demonstrated that the GN-6 white Pall filters are not
suitable for plating chlorine-containing samples. All subsequent filter plating for this type of
sample were performed with the PES type (black or white filters).
6.1.3 Neutralization Efficacy
This series of tests was performed to determine the neutralizer's ability to quench the
sporicidal activity of a bleach-based decontamination solution before microbiological analysis.
The neutralizer sodium thiosulfate (STS) was used in a 2x stoichiometry with the chlorinated
bleach solutions. Both high (~ 7 x io7) and low (~5 x 102) Bg spore loading levels were tested to
determine the effect of spore concentration on the neutralization effectiveness. The potential
sporicidal activity of STS was also evaluated using the lower spore loading levels. The results of
these tests are described in the next section.
6.1.3.1 High Spore Inoculated Solutions
Samples were prepared using chlorine-based Clorox® diluted bleach, hydrogen peroxide-
based bleach solutions (Clorox 2®), or Tide® detergent diluted in a 1:10 ratio with DI water.
Chlorine-based solution was neutralized using 2N-STS neutralizer that was added to each sample
(using a 2x stoichiometric ratio) to evaluate its effectiveness in neutralizing the decontamination
effects of the hypochlorite. The hydrogen peroxide-based bleach solutions and detergent (Tide®)
were neutralized using 5% sulfuric acid (H2S) and lN-KMn04 (potassium permanganate).
Samples containing the 10% dilute bleach-based solution and Tide® detergent with the
neutralization agent were then inoculated with ~ 7 x 101 Bg spores. Spore recoveries from these
samples were compared to recoveries from non-neutralized samples containing only phosphate
buffered saline with 0.05% Tween 20 (PBST). All samples were analyzed for the presence of
viable spores on the same day they were inoculated and again four 4 days later to evaluate the
potential for residual decontamination.
The results illustrated in Figure 6-2, and detailed in Appendix B-l. show that same-day
(Day 0) sample analysis yielded a mean spore recovery (6.8 x 107 spores) from the neutralized
bleach samples that was surprisingly about 70% of the average recovery (9.8 x 107 spores) from
the positive control PBST reference samples. These recoveries were expected to be more similar.
However, the average recovery from these same chlorine-based bleach solution samples (3.9 x
107 spores), analyzed four days later, was only 48% of the average recovery from the PBST
positive controls (8.1 x 107 spores), thereby suggesting some residual decontamination. Both
neutralized hydrogen peroxide-based and the detergent (Tide®) solutions' recoveries were found
to be stable over time.
31
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Q
W
+}
T3
O
O
<1)
(Z
O
o
d)
o
c
05
<1)
1E+08
1E+08 -
8E+07 -
6E+07 -
4E+07 -
2E+07 -
0E+00
PBST Germicidal Bleach Clorox 2®
Diluted Test Solution
Tide
Figure 6-2. Temporal recoveries from neutralized test solutions inoculated at high-spore loading
concentration.
6.1.3.2 Low Spore Inoculated Solutions
To test for the efficacy of neutralization of Clorox® chlorine-based diluted bleach
solution at low spore concentration, 10% and 1% dilute bleach solutions were neutralized with
appropriate amounts of 2N STS and then inoculated with Bg liquid inoculum at a tested
concentration of 3.77x 103. Each sample was inoculated with 133 [xL for a target inoculation of 5
x 102 spores.
These samples were then plated immediately and again after 4 days to test for any
residual decontamination effects. To test for the possible toxicity of the STS solution the amount
of 2N STS used to neutralize the respective dilute bleach solutions was added to PBST. All
samples were compared to positive controls consisting of inoculated PBST and inoculated DI
water.
The results for this series of tests are illustrated in Figure 6-3 and detailed in Appendix B-
2. The results confirm the high concentration results with a decrease in spore recovery over time.
This decrease in recovery is also shown in DI water solution containing STS which suggests
potential toxicity of the STS solution on viability of the spores. All the post-decontamination
samples henceforth were analyzed the same day to minimize biasing the results.
32
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Day 0 Day 4
Day of Sample Analysis
H20 ~ PBSTl I PBST + STS (10) ~ PBST + STS (1) ~ H20 +STS 10
H20O + STS (1)1 110% Neutralized Diluted Bleach + STS (10)
1% Neutralized Diluted Bleach + STS (1)
Figure 6-3. Temporal spore recoveries from neutralized solutions over time.
6.1.4 Preliminary Washing Solutions Evaluation
The target decontamination solutions were tested at two wash temperatures (72 °F and
120 °F) and at a dilution ratio of 1:10 in DI water to determine potential sporicidal efficacy. The
test solutions were mixed in sterile 2-L Erlenmeyer flasks. Test solution aliquots (300 mL) were
transferred to sterile 500-mL Nalgene bottles, heated to the desired test temperature in a water
bath (the 120 °F samples only), and then inoculated with ~1 x 101 Bg spores from a liquid spore
stock. Using an orbital shaker set at 250 rpm, each sample was agitated for a total duration of 9
min. After the 9-min cycle, samples were immediately processed and analyzed for potential
viable spores. The tested solutions were as follows:
1. Tide® detergent
2. Clorox® diluted bleach
3. Clorox® 2
The spore recoveries for the two non-chlorinated solutions (Clorox® 2, and Tide®
laundry detergent) were about the same levels as the spore recoveries for the DI water solution,
as shown Figure 6-4 and detailed in Appendix B-3. Full decontamination with non-recoverable
spores was observed for the chlorine-based Clorox® diluted bleach solution. A two-way
ANOVA analysis showed that at the 0.05 level of confidence of the mean spore recoveries from
the tested decontamination solutions were significantly different (P-Value =0.00) but the
33
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interaction between the decontamination solutions and solution temperatures was not significant
(P-Value = 0.98). The non-chlorinated bleach solutions, found not effective, were not tested
further, and the wash solutions were maintained at ambient laboratory conditions henceforth.
o
CO
107-|
CD
o
to
D
LL
C)
105i
o
o
103-
(1)
CN
O
101 i
O
o
] 72 °F
120 °F
-------
The results, shown in Figure 6-5, and summarized in Appendix B-4, demonstrated that
none of the solutions resulted in full decontamination of spores. The largest log reduction was
obtained using a 10% bleach solution with a 27-minute simulated wash time. 1% bleach
solutions had negligible decontamination efficacy at all wash times.
Q
W
T
3
LL
O
cr
LU
c
o
-4—'
03
c
E
05
H—'
£=
O
O
Q
£=
TO
0 2 4 6 8 10 12 14 16
Diluted Bleach Concentration (%)
Figure 6-5. Chlorine-based bleach dilute solution decontamination efficacy.
6.1.6 Chlorine-Based Bleach with Surfactant
Following observations from the previous tests that none of the diluted bleach
concentrations had full decontamination, it was postulated that the hydrophobic nature of the
spores may have contributed to these results. The hydrophobic properties of the spores may have
led to clumping and prevented full interactions between the spores and the decontamination
solutions. A new series of tests was designed to include the addition of a commercial off-the-
shelf surfactant (Tide®) detergent to the bleach decontamination solutions. Manufacturer's
directions were used to add the appropriate amount of detergent to each of the bleach
decontamination solutions, 300 |iL of Tide® detergent per liter of decontamination solution.
Samples were inoculated with ~2 x io7 spores and subjected to a series of tests evaluating
simulated wash times and dilution ratios. The following wash times and dilution ratios were
evaluated.
1. Bleach Solution Dilution Ratio
1. 15:100 (15% diluted bleach)
2. 10:100 (10% diluted bleach)
3. 5:100(5% diluted bleach)
4. 3:100 (3% diluted bleach)
5. 1:100 (1% diluted Bleach)
6. 5:1,000 (0.5% diluted bleach)
35
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7. 1:1,000 (0.1% diluted bleach)
8. 1:10,000 (0.01% diluted bleach)
2. Wash Cycle Time
1. 9-min
2. 18-min
3. 27-min
The results, shown in Figure 6-6 and summarized in Appendix B-5, indicate that full
decontamination was obtained using a 1% diluted bleach solution with the addition of a detergent
with simulated wash times of 18 and 27 minutes. Diluted bleach solutions containing less than
1% bleach had minimal decontamination efficacies with the exception of the 0.5% germicidal
bleach solution with a 27-minute wash time which resulted in only a 3-log reduction of spores.
Higher concentrated bleach solutions resulted in similar inactivation as with the 1% diluted
bleach. The 18-minute wash times provided similar results to the 27-minute wash times and were
used for all subsequent tests.
Figure 6-6. Diluted bleach solution with the addition of a detergent decontamination efficacy.
As shown in Figure 6-7, the addition of a detergent to the diluted bleach solutions, with
an 18-minute wash time, increased the decontamination effectiveness by orders of magnitude.
This effect is most pronounced with the 1% diluted bleach solutions in which there is ~7 log
reduction difference (6.7 log reduction and 1.0 log reduction) between the solutions with and
without detergent respectively. The hydrophobic nature of the spores may have led to spore
clumping in the liquid bleach solutions and offered some protection to the entrapped spores from
the decontaminant agent. The addition of the detergent may have decreased the clumping of the
spores and made them more susceptible to the bleach solution. Surfactants such as Tween 80
have been reported to improve spore extraction efficiency from various solutions (Da Silva et al..
8
n
0 2 4 6 8 10 12 14 16
Diluted Bleach Concentration (%)
36
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2011). The surfactant-containing detergent may have reduced spore-spore interactions or spore-
container interactions such that spores were more evenly dispersed in solution and thereby more
readily in contact with the decontamination agent.
Figure 6-7. Decontamination efficacy of a diluted chlorine-based bleach solution with and without
the addition of a detergent.
Phase II: Bench-Scale Testing with Material Coupons
A series of tests was conducted with the germicidal bleach solution with and without the
addition of a surfactant (Tide® liquid detergent) to determine the decontamination efficacy of the
different bleach formulations. The coupons used for this series of tests and the remainder of tests
were all inoculated with ~ 101 Bg spores using the method described in Section 3.4.2.1. The
following sections discuss the results of these tests.
6.1.7 Diluted Bleach Solution: Cotton Material
The method development tests showed that chlorinated germicidal bleach was the most
effective off-the-shelf decontaminant of Bacillus spores. A series of tests was designed to
determine the conditions that would provide optimal decontamination conditions while using the
most diluted bleach. These tests were run using an 18-min wash cycle on inoculated cotton
materials with no detergent added. The tests were run at three bleach dilution ratios (1:10 (10%
bleach), 1:100 (1% bleach), and 1:1000 (0.1% bleach).
The results for these tests are shown in Figure 6-8 and summarized in Appendix C-l. An
increase in the concentration of the germicidal bleach solution resulted in a decrease of spores
recovered on the cotton material coupons. The 10% bleach resulted in no-detectable spores on
the cotton material as well as the post decontamination rinse samples. Close to a 6 LR (5.76 LR),
and just over 4 LR (4.45 LR) were observed on the coupon material for the 1% and 0.1% bleach
solutions with detectable spores found in both the post decontamination wash and rinse samples.
8
0 With Detergent
—0— Without Detergent
-| 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
0 2 4 6 8 10 12 14 16
Diluted Bleach Concentration (%)
37
-------
1
CD 10 -J
Material Coupon
Wash Solution
Sample Type
Rinse Solution
Procedural (Dl Water)
1% Diluted Bleach
0.1% Diluted Bleach
10% Diluted Bleach
Figure 6-8. Spore recovery and redistribution for different diluted bleach solutions.
Procedural positive experiments were also conducted following the same parameters as
the decontamination tests without any bleach added to the decontamination solutions. These tests
were used to evaluate the redistribution of spores during the wash and rinse procedure. In both
tests a < 2 LR was observed for the cloth surfaces and a large redistribution of spores to the wash
and rinse samples were noted. These results suggest that the inactivation of the spores occurs
both on the surface of the material coupons and in the wash solution.
6.1.8 Diluted Bleach Solution with the addition of Detergent: Cotton Material
After the previous tests showed a greater decontamination efficacy, using an aerosol
deposition and cloth coupons, and the results from the Phase I tests demonstrated the increased
decontamination efficacy by adding a surfactant to the wash solutions, an additional test was
conducted to evaluate the effectiveness of lowering the concentration of the germicidal bleach
decontamination solutions in conjunction with the addition of detergent. This test was conducted
using the same parameters as the previous tests apart from the addition of a detergent to the 1%
bleach solution.
Results shown in Figure 6-9 confirmed previous results that the addition of a detergent to
the bleach solutions increased the decontamination efficacy. The 1% diluted bleach with the
addition of a detergent achieved a similar log reduction compared to the 10% diluted bleach
without the addition of a detergent (6.8 LR and 7.2 LR, respectively) with non-detectable spores
on the material coupons. Both of these solutions were at least 1 log greater in their
decontamination efficiencies compared to the 1% bleach without the addition of a detergent
(<5.8 LR). Recoverable spores were found in both the 1% and 10% diluted bleach without
detergent wash solutions.
38
-------
10'-
Q
co io6
ii
O)
>
o
o
-------
u 7.
C£
o
03
o
y=
LU
E
03
-1—¦
£=
O
O
0
Q
(D
O
03
t
=3
CO
03
q3
-1—¦
03
7.2
6.9
4.7
Cotton Blend Spandex Polyester Military
Material Coupon Type
Denim
Figure 6-10. Bench-scale decontamination efficacy results for all material coupons using 1% diluted
bleach.
104
Q
CO
103
0)
>
o
0
0)
01
S
u_
O
102
O)
o
101
CO
0)
10°
Coupon Material
Wash Solution
Rinse Solution
Cotton iBIend Spandex Polyester Military
Material Coupon Type
Denim
Figure 6-11. Spores recovered from material coupons using 1% diluted bleach solution with
detergent.
40
-------
Each material also underwent a procedural positive wash during which they were
subjected to the same wash and rinse times without the addition of bleach. The procedural
positive test results are shown in Figure 6-12, < 2 LR was obtained on the procedural positive
material samples regardless of the type of material used. These results suggest that the spores
were completely inactivated by the bleach solution rather than just redistributed during the
procedure.
107 -
u
10
T—
+
1t)G i
s '
>*
>
105 -i
O
O
0)
E£
104-
D
Ll
O
10-1' 1
o
m
102 1
O
_l
c
ro
a)
101 i
10° -
T
Cotton
rf~
~i —^^^^~
Blend Spandex Polyester Military Denim
Material Coupon Type
j Coupon Material |
Wash Solution
Rinse Solution
Figure 6-12. Spores recovered from material coupons subjected to DI water procedural testing
process.
Phase III: Decontamination Testing using a Washing Machine
Results from Phase II tests showed that a 1:100 (1% Clorox® diluted bleach) dilution
ratio with the addition of a detergent provided acceptable decontamination results (> 6 LR) for
all materials with the exception of the military and denim cloth. The series of tests in Phase III
used this optimized decontamination solution on larger material coupons, 14 in. x 14 in., using
off-the-shelf washing machines (see Section 3.7.2).
6.1.10 1% Diluted Bleach with Detergent: All Materials
The decontamination results for all six materials tested with the washers are shown in
Figure 6-13 and summarized in Appendix D-l. The diluted bleach solution (dilution ratio: 1:100)
with the addition of a detergent resulted in non-detectable spores on the surface of the material
and in both the liquid washing and rinse solutions. These results confirm the results obtained in
Phase II. Interestingly, both the military and denim materials had no-detectable spores on their
surfaces. This effect may have been due to the increased agitation from the washing machines,
which may have liberated spores embedded within the materials, making them more susceptible
to decontamination.
41
-------
8
§
li-
CS 7
5
1 6
e
UJ
1
2
E 4
5-
2 -
3
CO
6 9
8.7
T
.4 ! 3 4
8.9
Cotton Blend Spendex Polyester Military
Material Coupon Type
Denim
Figure 6-13. Pilot-scale washer decontamination efficacy results for all material coupons using 1%
diluted bleach solution with detergent.
Confirming the observation that the increased agitation of the washing machines liberated
more spores from the clothing materials, procedural positive coupon samples all had a greater
log reduction during this phase of testing compared to the bench scale tests in Phase II. These
results are shown in Figure 6-14.
Q
10'-i
10
©
>
8
o
—I
c
-------
6.1.11 1 % Diluted Blench Without the Addition of a Detergent: Cotton and Military
Materials
A limited number of tests were performed to determine whether the use of detergent
increased the decontamination efficacy of the bleach solutions given the increased agitation from
the washing machines. Cotton and military cloth coupons were tested in the washing machines
using a 1:100 dilution ratio (1% diluted bleach solution) with and without the addition of a
detergent. Cotton was chosen because it was the material with the most data having been used
for all scoping tests. The military material was chosen for this series of tests because it had the
lowest surface log reduction during the Phase II tests and was used to challenge these test
conditions.
The results for these tests are shown in Figure 6-15, and detailed in Appendix D-2. The
germicidal bleach solution (dilution ratio: 1:100), with and without the addition of detergent,
resulted in similar decontamination efficacies. These results confirm that the agitation dynamic
of the washers increased physical removal of the spores from the surface of the coupons to the
wash solution, thus resulting in a more efficient decontamination process.
Q 107
W
A- 106
> 105
o
Ll_
o
o
g* 102
_l
0) 101
10°
o<
,0^
Cotton Material
Military Material
G°
*?
,0^
,0^
Material Coupon Type
Figure 6-15. Spores recovered from cotton and military material coupons using 1% diluted bleach
solution without the addition of detergent using washing machine.
Phase IV: Decontamination Testing using a Washing Machine/Dryer
A series of tests was designed in this phase to evaluate the effect of adding a drying step
to the decontamination process. Re-aerosolization of spores which were not decontaminated
during the wash cycle were also of interest. HEPA filters were placed on the air inlet and outlet
of the dryer to eliminate any cross contamination from outside sources and to capture any spores
43
-------
potentially released during the drying process. 14 in. x 14 in. cotton coupons were inoculated
with 107 spores and were first subjected to an 18-minute wash cycle followed by a 5-minute spin
cycle, 9-minute rinse, additional 5-minute spin and finally a drying cycle using "High Heat" for
30 minutes.
Diluted bleach solution with a high dilution ratio (1:1000) with the addition of a liquid
detergent that was used previously in Phase II did not result in full decontamination of the
inoculated fabric material; hence, this condition was tested here to determine if the addition of
heating improved the inactivation of spores, when compared to a washing step alone.
The results for this test are illustrated in Figure 6-16 and summarized in Appendix E-l.
The addition of a drying sequence resulted in no apparent increase in the decontamination
efficacy of the diluted bleach solution. A similar number of spores were detected on the cotton
coupons following both procedures. The difference between the number of spores recovered
between the two procedures is made up almost entirely of spores recovered from the HEPA
filters placed on the air outlet of the dryers. Due to the potential for spore re-aerosolization and
lack of an increase in decontamination efficacy, drying of washed contaminated clothing is not
recommended as a potential self-help practice to reduce cross-contamination among an affected
population. It is suggested to air dry the clothing following washing.
e6°
^>CP
Post-Treatment Sample Type
Figure 6-16. Spore distribution following a post-decontamination washing/drying cycle using 0.1%
diluted bleach solution with the addition of a detergent.
44
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7.0 QUALITY ASSURANCE AND QUALITY CONTROL
All test activities were documented in laboratory notebooks and with digital photographs.
The documentation included, but was not limited to, a record for each decontamination
procedure, any deviations from the quality assurance project plan, and physical impacts on
materials. All tests were conducted in accordance with established EPA Decontamination
Technologies Research Laboratory (DTRL) and CESER/HSMMD RTP Microbiology
Laboratory (BioLab) procedures to ensure repeatability and adherence to the data quality
validation criteria set for this project.
The following sections discuss the measurement equipment calibration, the criteria for
the critical measurements and parameters, data quality indicators, and the quality assurance (QA)
and quality control (QC) checks for the project.
Supplies and Consumables
Test materials must be uniform to ensure reliable test results. Uniformity was maintained
by obtaining a large enough quantity of material so that multiple coupons could be constructed
with presumably uniform characteristics.
To determine the sampling efficiency, there must be assurances that the enumeration of
viable spores is representative of the actual number of spores present and not the result of
contamination or faulty culture. The QC checks are discussed in Section 7.4 to provide this
assurance.
Measurement Equipment Calibration
The DTRL and Biolab have standard operating procedures for the maintenance and
calibration of all laboratory equipment. All equipment is verified as being certified calibrated or
by having the calibration validated by the Air Methods and Characterization Division's
Metrology Laboratory at the time of use. Table 7-1 summarizes the instrument calibration
frequency.
Table 7-1. Instrument Calibration Frequency
Equipment
Calibration/Certification
Expected Tolerance
Thermometer
Compare to independent NIST thermometer (a thermometer
recertified annually by either NIST or an ISO-17025
facility) value once per quarter
+1 °C
Stopwatch
Compare to official U.S. time at time.gov every 30 days
+1 minute every 30 days
Micropipettes
Certified as calibrated at time of use; recalibrated by
gravimetric evaluation of performance to manufacturer's
specifications every year
±5%
Scale
Compare reading to Class S weights
±1%
Graduated cylinder
Collect effluent at specified time.
± 1 mL
45
-------
Criteria for Critical Measurements and Parameters
The data quality objects define the critical measurements needed to address the stated
objectives and specify tolerable levels of potential error associated with the decontamination
event. The following measurements have been deemed critical to accomplishing the project
objectives:
• pH and temperature of the diluted bleach solution and any other liquid solution being
used
• Sodium hypochlorite concentration as free available chlorine (FAC) of the diluted
bleach decontamination solution (quantitative)
• Washing solution volume
• Water rinse volume
• Washing time
• Drying time
• Washing solution temperature
Data quality indicator goals for the critical measurements are used to determine if the
collected data meet the objectives established for this project. All the acceptance criteria for the
critical parameters listed in Table 7-2 were met for this project.
Table 7-2. Data Quality Acceptance Criteria for Critical Measurements
Critical Measurement
Analysis Method
Accuracy/Precision
Acceptance Criteria
CFU per plate
Spiral plater/QCount
50% RSD among
the triplicate plating
50% RSD
Temperature of incubation
chamber
NIST-traceable
thermometer (daily)
± 2 °C
38 ±2 °C
Decontamination time
NIST-calibrated
stopwatch
± 1 minute/hour
± 2 minutes (2 x ± 1
min)
FAC
Na2S2C>3/KI titration
± 0.06 gram per liter
± 10% of target value
pH
pH meter /NIST-traceable
buffer solutions
± 0.01 pH unit
10% RSD
Titration/extraction/sample/neu
tralizer volumes volume and
extraction volumes
Burette
± 1 mL
± 10% of target value
Water bath Temperature
Thermometer
± 1 °C
± 10% of target value
RSD = Relative standard deviation
46
-------
Control Checks
Quantitative standards do not exist for biological agents. Viable spores were counted
using a QCount® colony counter. Counts generated that were either greater than 300 or less than
30 were considered outside of the targeted range. If the count of colony-forming units for
bacterial growth did not fall within the target range, the sample was either filtered or replated.
Replates and filter plates were enumerated manually.
Before each batch of plates were enumerated on the QCount®, a QC plate was analyzed,
and the result was verified to be within the range indicated on the back of the QC plate. As the
plates were being counted, a visual inspection of colony counts made by the QCount® software
was performed. Obvious count errors made by the software were corrected by adjusting the
settings (e.g., colony size, light, and field of view) and recounting or by manually removing or
adding colonies as needed.
The acceptance criteria for the critical CFU measurements were set at the most stringent
level that could be achieved routinely. Positive controls were included along with the test
samples in the experiments so that spore recovery from the different surface types could be
assessed. Background checks were also included as part of the standard protocol to check for
unanticipated contamination. Replicate coupons were included for each set of test conditions to
characterize the variability of the test procedures.
Additional QC samples were collected and analyzed to check the ability of the BioLab to
culture the test organism, as well as to demonstrate that materials used in this effort did not
contain spores. The checks included the following:
• Procedural blank coupons: Material coupons sampled in the same fashion as test
coupons but not contaminated with surrogate organism prior to sampling.
• Stainless-steel positive control coupons: Coupons inoculated in tandem with the test
coupons and meant to demonstrate the highest level of contamination recoverable
from an inoculation event.
Additional QC checks for BioLab procedures are listed in Table 7-3. These provide
assurances against cross-contamination and other biases in microbiological samples.
Table 7.3. QA/QC Sample Acceptance Criteria
QC Sample
Information Provided
Frequency
Acceptance Criteria
Positive control (sample from
material coupon contaminated
with biological agent but not
subjected to decontamination
Initial contamination
level on the coupons;
allows determination
of log reduction *(LR)
3 or more
replicates per
test
Target loading of 1 x 107 CFU
per sample, with standard
deviation of < 0.5 log (5 x 106
to 5 x io7 CFU per sample)
Procedural blank (test
material coupon without
biological agent)
Controls for sterility of
materials and methods
used in the procedure
1 per test
No observed CFU
47
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QC Sample
Information Provided
Frequency
Acceptance Criteria
Laboratory blank (TSA
sterility control; plate
incubated but not inoculated)
Controls for sterility of
plates
Each plate
incubated for
at least 18 but
less than 24
hours
No observed growth after
incubation
Inoculation control (replicate
of microbiological dilution
plates)
Controls for
repeatability of results
3 per dilution
Counts greater than 30, and
less than 300 are reportable;
standard deviation must be
< 100%; Grubbs outlier test or
equivalent
Blank plating of
microbiological supplies
Controls for sterility of
supplies used in
dilution plating
3 of each
supply per
plating event
No observed growth after
incubation
Procedural blank
Contamination level
present during
sampling
1 per
sampling
event
Non-detect
Field blank coupon
Verify the sterility of
materials used to
analyze viable spore
count
3 per material
per test
No detectable spores
MDI control (wipe sample
from stainless-steel coupon
contaminated with biological
agent)
Initial contamination
level on coupons;
shows plate's ability to
support growth
3 replicates
per MDI use
Target loading CFU per
sample, with a standard
deviation of < 0.5 log
The QA/QC control test results for the whole sampling campaign are listed for each test
result tabulated in Appendices B, C, D, and E for Phases I, II, III, and IV respectively. Sterility
checks were conducted for all the equipment and materials and listed for each test in the result
section. Most of the control blanks (negative controls), sampling procedure blanks (procedural
blanks), and the inoculum control blanks (stainless-steel control blanks) were non-detectable.
Some control blanks were found to be contaminated, but they had little or no effect on the final
results. The detection limit for each sample was set at 1 CFU per sample volume extracted.
48
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8.0 Summary
The main purpose of this study was to evaluate potential decontamination solutions that
can be efficacious in removal and/or inactivation of bacterial spores from contaminated clothing.
The overall process of data generation included bench scale testing to simulate the washing and
drying of contaminated clothes with viable spores of Bg. The washing/drying decontamination
approach was evaluated as a function of different parameters such as wash temperature, wash
cycle, material clothing type, wash solution type (water versus bleach-type solution), water rinse,
and drying. Six materials (cotton, stretch denim, polyester fleece, spandex, cotton/polyester
blend, and US Navy camouflage)) were evaluated to determine if the clothing material type had
any effect on the overall decontamination approach.
Results from the first phase indicated that oxygen-based diluted (10%) bleach solutions
(Clorox®2, and Tide® Free & Gentle laundry detergent) were not sporicidal. Full
decontamination with non-recoverable spores was observed for the Clorox® chlorine-based
diluted bleach solution. The effect of temperature on the decontamination efficacy of the wash
solutions was found to be negligible at the two temperatures that were evaluated.
The non-chlorinated bleach solutions were not tested further, and the wash solutions were
maintained at ambient laboratory conditions henceforth. Additionally, Phase I experiments
demonstrated that the addition of a detergent to the chlorine-based diluted solution allowed a
significant decrease in concentration (down to 1%) while still maintaining adequate
decontamination efficacy (> 6 LR). The addition of Tide® detergent may have caused the
reduction of spore clumping in liquid-contaminated solution; hence increasing the effectiveness
of the bleach solution.
Phase II testing confirmed results from the Phase I tests that the 1% Clorox® chlorine-
based bleach solution was a highly effective decontamination approach. This solution provided a
> 6 LR for all cloth materials (cotton, blend, spandex, and polyester) except for denim and
military fabrics (5.7 and 4.7 LR, respectively). The lower decontamination efficiencies of these
two fabrics may have been the result of a denser more complex weave and a greater number of
spores being entrapped within these cloths compared to the other 4 materials. It should also be
noted that greater decontamination efficacies were observed during this phase of testing
compared to the Phase I tests. This effect may have been caused by the aerosol inoculation of
material coupons during Phase II tests compared to the liquid inoculation used during Phase I
tests.
Results from Phase III confirmed results obtained in Phase II, that 1% diluted Clorox®
chlorine-based bleach solution with the addition of a detergent using an 18-minute wash time
was effective in decontaminating fabric inoculated with ~107 spores per square foot. All
materials had surface log reductions > 6. The increased surface decontamination efficacies seen
during this phase may have been due to the increased agitation dynamic from the washing
machines.
In Phase IV a drying sequence was added to the wash sequence. The addition of a drying
sequence resulted in no apparent increase in the decontamination efficacy of the diluted bleach
solution. A similar number of spores were detected on cotton material coupons following the
wash cycles with and without a drying step. The difference between the number of spores
recovered from the coupons between washing and drying is made up almost entirely of spores
49
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recovered from HEPA filters placed on the air outlet of the dryers. Due to the potential for spore
re-aerosolization and lack of an increase in decontamination efficacy, drying of washed
contaminated clothing is not recommended as a self-help practice to reduce cross-contamination.
The use of 1% diluted Clorox® chlorine-based bleach solution with the addition of a
detergent using an 18-minute wash time can be used to lessen potential cross-contamination on
clothing. As with any process there are many variables which can have an impact on the efficacy
of this approach. Some of these variables include how the material is agitated in the washing
machine (e.g., how the material folds or crumples in the wash/dryer cycle), volume of the
washing machine and impact of the agitation mechanism, clothing load, and organic load (e.g.,
soiling of clothing).
One of the primary goals of this project was to demonstrate the effectiveness of washing
and drying cycles for inactivating Bacillus spores for clothing. The process parameters identified
in this project could be used as a risk reduction option in the event of a wide area release of
Bacillus anthracis.
Safety Considerations
• Spores can be released to the air and can therefore be a significant source of cross-
contamination during a remediation and can pose health risks to persons performing
decontamination tasks.
• When using bleach or other disinfectants and cleaners follow the instructions on the labels for
precautions, use, and personal protective equipment (PPE) recommendations.
• Never mix bleach products together with vinegar or ammonia.
• Residual spores may be contained in the washing machine following a washing cycle with
contaminated clothing. An empty cycle should be completed in the washing machine using
bleach to inactivate any remaining spores.
50
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REFERENCES
Baron, P. A., et al. 2008. "Development of an aerosol system for uniformly depositing bacillus
anthracis spore particles on surfaces." Aerosol Science and Technology, 2008, 42(3), 159-
172.
Brown, G.S., et al. 2007. "Evaluation of a wipe surface sample method for collection of Bacillus
spores from nonporous surfaces " Appl Environ Microbiol, 2007, 73(3), 706-710.
Busher, A.; Noble-Wang, J.; and Rose, L. 2008. P. Emanuel, J. Roos, and K. Niyogi (Editors).
Surface Sampling for Biological Agents in the Environment, pp. 95-131. Washington DC:
ASM Press.
Calfee, M.W.; Lee, S.D.; and Ryan, S.P. 2013. "A rapid and repeatable method to deposit
bioaerosols on material surfaces." J Microbiol Methods, 2013 Mar, 92(3), 375-380.
Da Silva, S.M.; Filliben, J. J.; and Morrow, J.B. 2011. "Parameters affective spore recovery from
wipes used in biological surface sampling." Appl Environ Microbiol, 77(7), 2374-2380.
Kabashima, J.; Giles, D. K.; and Parrella, M. P. 1995. "Electrostatic sprayers improve pesticide
efficacy in greenhouses." California Agriculture, July-August, 1995. Volume 49 (4).
Lee, S. D.; Ryan, S. P.; and Snyder, E. G. 2011, "Development of an aerosol surface inoculation
method for Bacillus spores.'" Appl Environ Microbiol, 2011, 77(5), 1638-1645.
U.S. Environmental Protection Agency. 2018. Product Performance Test Guidelines OCSPP
810.2100: Sterilants, Sporicides, and Decontaminants, Guidance for Efficacy Testing, EPA
712-C-17-003; Washington, DC.
U.S. Environmental Protection Agency. 2020. Homeland Security Strategic Research Action
Plan-2019-2022. EPA 601K20002. March 2020. https://www.epa.gov/research/homeland-
securitv-strategic-research-action-plan-2019-2022
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Appendix A. Challenges with Filter Plating Residual Decontamination
While processing the Clorox® bleach post-decontamination samples, the filtration and
subsequent plating of 1-mL and 10-mL aliquots through white Pall® filters (P/N 4804
MicroFunnel™ with GN-6 Metricel® membrane with a 0.45 jam pore size made from a mixed
cellulose ester) resulted in unexpected recoveries. The 1 ml filters had too many colonies to
count (too numerous to count, TNTC) while the 10 ml filters were either non-detect or had very
few colonies. Photos of filter plates from the same selected sample, plated at the same time, are
shown in Figures A-l and A-2, respectively.
Figure A-l. 1-mL aliquot filter plated from 10% bleach solution sample.
Figure A-2. 10-mL aliquots filter plated from 10% bleach solution sample.
These unexpected results raised questions about all the results obtained via filter plating
of chlorinated bleach solutions. A scoping test was performed to evaluate whether the different
0.45 jam Pall filter membrane materials (GN-6 white P/N 4804 mixed cellulose ester, black P/N
4805 and white P/N 4802 made of polyethersulfone [PES]) were influencing the recovery results.
The same samples with 10% bleach and W and 2* STS were filter plated and plated on
all three types of filters. Aliquots of 1, 10, and 100 ml were filter plated with no extra rinse. All
the PES (black and white filters) filters had colony growth at the expected levels, while the GN-6
52
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white filters were found to have no growth at the 10-ml and 100-ml aliquots. The data results for
the scoping tests are presented in Table A-l and pictures of the 10% bleach filtered samples are
shown in Figures A-3 through A-5 for all three filters for the 1-ml and 10-mL aliquots.
These results demonstrate that the GN-6 white Pall filters are not suitable for plating
chlorine-containing samples. All subsequent filter plating for this type of sample was performed
with the PES type (black or white filters).
Table A-l. Filter Plating Results for Different Types of Filters
Sample ID
CFU GN-6 White
Filters
CFU PES White
Filters
CFU PES Black
Filters
Volume Plated
(mL)
152-10%-2 x STS-01
150
91
79
1.0
4
TNTC
TNTC
10.0
6
TNTC
TNTC
100.0
152-10%-2 x STS-02
141
88
49
1.0
1
TNTC
TNTC
10.0
2
TNTC
TNTC
100.0
152-10%-2 x STS-03
87
123
64
1.0
2
TNTC
TNTC
10.0
3
TNTC
TNTC
100.0
152-Water-2x STS-01
117
109
104
1.0
TNTC
TNTC
TNTC
10.0
TNTC
TNTC
TNTC
100.0
-------
A B
Figure A-3. Aliquots filter plated on GN-6 white filter from 10% bleach solution (A: 1-, B: 10-mL)
A B
Figure A-5. Aliquots filter plated on PES white filter from 10% bleach solution (A: 1-, B: 10-mL)
54
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Appendix B-l: Temporal Recoveries from Neutralized Test Solutions at High-Spore
Loading Concentrations
Wash Solution
PBST
Diluted Bleach
Clorox 2® Free and
Clear, H2O2 Bleach
Tide®
Analysis
(Day)
Test #
CFU
CFU
%
CFU
%
CFU
%
Day 0
1
1.0 xio8
6.1 xlO7
62
9.2 xlO7
95
1.0 xio8
106
2
1.1 xlO8
7.0 xlO7
71
8.4 xlO7
86
1.1 xlO8
110
3
8.3 xlO7
7.5 xlO7
77
8.8 xlO7
90
1.1 xlO8
115
Average
9.8 xlO7
6.8 xlO7
70
8.8 xlO7
90
1.1 xlO8
110
Stdev
1.3 xlO7
7.1 xlO6
7.2
4.2 xlO6
4.3
4.2 xlO6
4.3
Day 4
1
8.3 xlO7
3.5 xlO7
43
7.2 xlO7
89
9.0 xlO7
111
2
8.2 xlO7
3.7 xlO7
46
7.1 xlO7
87
9.0 xlO7
112
3
7.7 xlO7
4.6 xlO7
57
7.2 xlO7
89
8.8 xlO7
109
Average
8.1 xlO7
3.9 xlO7
48
7.2 xlO7
89
8.9 xlO7
111
Stdev
3.3 xlO6
6.0 xlO6
7.5
9.2 xlO5
1
1.0 xlO6
1.2
Appendix B-2: Temporal Recoveries from Neutralized Test Solutions at Low-Spore
Loading Concentrations
Processing Day
Day 0
Day 4
Recoveries (CFU/sample)
Average
Stdev
%
Average
Stdev
%
H20
3.7 xlO2
1.5 xio1
NA
3.6 xio2
4.4 xio1
NA
PBST
3.6 xlO2
3.9 xio1
99
4.1 xio2
4.0 xio1
116
PBST + STS 10
3.9 xlO2
1.5 xio1
106
3.9 xio2
2.3 xio1
109
PBST + STS 1
3.8 xlO2
6.0 xlO1
104
3.9 xlO2
2.9 xio1
109
H20 + STS 10
4.3 xlO2
5.3 xlO1
118
3.6 xlO2
4.6 xlO1
100
H20+ STS1
4.7 xlO2
3.4 xlO1
128
3.6 xlO2
4.6 xlO1
100
10% Neutralized Diluted Bleach
3.9 xlO2
1.5 xlO1
105
3.1 xlO2
2.1 xlO1
87
1% Neutralized Diluted Bleach
4.2 xlO2
3.9 xlO1
115
2.9 xlO2
5.1 xlO1
82
Note: STS 1 or 10 corresponds to the same amount of STS added to 1 or 10% neutralized diluted bleach
solution
-------
Appendix B-3: Phase I (Scoping). Decontamination Results with Wash Liquid Solutions
Wash Solution
Wash Solution
Temperature
Spores Recovered
(CFU)
Decontamination Efficacy
(LR [CFU])
°F
Per test
Average
Stdev
Per test
Average
Stdev
Procedural
Positive
Control (DI
Water)
72
3.54 xlO7
2.9 xlO7
8.7 xlO6
Not Applicable
2.30 xlO7
120
4.26 xlO7
2.3 xlO7
2.7 xlO7
3.87 xlO6
Tide®
Detergent
72
6.81 xlO7
7.0 xlO7
2.5 xlO6
-0.4
-0.4
0.02
7.17 xlO7
-0.4
120
6.72 xlO7
6.6 xlO7
1.5 xlO6
-0.5
-0.5
0.01
6.51 xlO7
-0.4
Clorox® 2
Hydrogen
Peroxide-based
Bleach
72
5.88 xlO7
5.5 xlO7
5.1 xlO6
-0.2
-0.2
0.04
5.16 xlO7
-0.2
120
4.47 xlO7
5.4 xlO7
1.3 xlO7
-0.3
-0.4
0.11
6.33xl07
-0.4
Clorox®
Concentrated
Germicidal
Bleach
72
3
3
0.0
7.0
7.0
0.00
3
7.0
120
3
3
0.0
6.9
6.9
0.00
3
6.9
Green shading denotes the sample detection limit.
-------
Appendix B-4: Phase I (Scoping): Decontamination Results with Diluted Bleach Alone
Wash
Time
9 min
18 min
27 min
Recovery
Recovered (CFU)
LR (CFU)
Recovered (CFU)
LR (CFU)
Recovered (CFU)
LR (CFU)
Dilution
Ratio
Average
Stdev
Average
Stdev
Average
Stdev
Average
Stdev
Average
Stdev
Average
Stdev
1%
5.3 106
4.4 105
0.41
0.04
1.4 106
5.6 105
1.02
0.17
3.0 105
2.7 105
1.95
0.78
3%
3.1 106
9.1 105
0.65
0.13
1.5 105
1.5 105
2.12
0.44
2.5 104
2.1 104
2.87
0.46
5%
4.5 105
2.9 104
1.48
0.03
5.0 102
3.8 102
4.52
0.33
6.4 102
3.2 102
4.38
0.28
10%
1.1 103
3.1 102
4.09
0.11
1.0 103
9.8 102
4.46
0.83
2.6 101
2.5 101
5.88
0.48
15%
9.6 101
1.0 102
5.35
0.54
7.6 102
8.9 102
4.78
1.13
5.1 102
6.0 102
4.75
0.77
PCs1
6.4 106
7.3 106
NA1
PCs1; Positive Controls; NA2: Not Applicable
Appendix B-5: Phase I (Scoping): Decontamination Results with Diluted Bleach Plus
Detergent
Wash
Time
9 min
18 min
27 min
Recovery
Recovered (CFU)
LR (CFU)
Recovered (CFU)
LR (CFU)
Recovered (CFU)
LR (CFU)
Dilution
Ratio
Average
Stdev
Average
Stdev
Average
Stdev
Average
Stdev
Average
Stdev
Average
Stdev
0.01%
1.8 xio7
3.1 xio5
0.03
0.01
1.9 xlO7
1.4 xlO6
0.02
0.03
1.8 xlO7
2.2 xlO5
0.00
0.05
0.10%
1.8 xio7
4.4 xlO5
0.04
0.01
8.9 xlO6
7.7 xlO6
0.44
0.36
1.3 xlO7
2.2 xl06
0.16
0.05
0.50%
7.4 xio6
1.7 xlO5
0.45
0.01
1.9 xlO6
1.5 xlO5
1.05
0.03
2.0 xlO4
1.5 xlO4
3.16
0.51
1%
4.3 xio4
3.0 xlO4
2.64
0.32
3
0.01
6.72
0.00
3
0.01
6.72
0.00
3%
4.4 xio3
3.6 xlO3
3.75
0.45
3
0.004
6.75
0.00
4
1.9
6.66
0.17
5%
3.3 xio2
9.5 xlO"1
4.96
0.00
4.8 xlO2
8.2 xlO2
6.08
1.52
3
0.001
6.96
0.00
10%
2.3 xio3
3.9 xlO3
5.34
1.64
8.1 xlO2
1.4 xlO3
5.88
1.55
2.0 xlO2
3.4 xlO2
6.18
1.28
15%
3.0 x 10~
5.0 xlO2
5.90
1.22
9
4.5
6.57
0.28
5.0 xlO1
7.9 xlO1
6.36
0.90
0%
2.1 xlO7
6.1 xlO6
Green shading denotes the sample detection limit.
57
-------
Appendix C-l: Phase II Spore Recovery and Redistribution for Diluted Bleach Solution
on Cotton Material
Table Cl-1. Phase II (Benchscale): Decontamination Results for Cotton Material with a Bleach
Dilution Ratio of 1:1000
Sample Media
Type of Samples
Recoveries
Average
(CFUs)
Stdev
Coupon
Samples
Inoculation Controls
CFU
1.1 xlO7
1.9 xlO6
Log(CFU)
7.04
0.07
Positive Controls
CFU
5.9 xlO6
1.5 xlO6
Log(CFU)
6.76
0.11
Procedural Positive Controls
CFU
4.6 xlO5
3.3 xlO5
Log(CFU)
5.57
0.35
Log Reduction
1.47
0.35
Post Decon Fabric Sample
CFU
242
179
Log(CFU)
2.31
0.30
Log Reduction
4.45
0.30
Liquid wash
water Samples
Procedural Wash Liquid Sample
CFU
8.2 xlO6
1.1 xlO6
Log(CFU)
6.91
0.06
Post Decon Wash Liquid Sample
CFU
34
23
Log(CFU)
1.46
0.96
Procedural Rinse Liquid Sample
CFU
5.4 xlO5
9.4 xlO4
Log(CFU)
5.73
0.08
Post Decon Rinse Sample
CFU
77
7
Log(CFU)
1.89
0.04
Procedural
Blanks
Test Coupon
CFU/coupon
2
Wash Liquid Solution
CFU/mL
0.01
Rinse Liquid Solution
CFU/mL
0.01
Sterility Checks
Sterility Check for Decon Solution
CFU/ml
0.01
Sterility Check for DI-H20
CFU/mL
0.01
Green shading denotes the sample detection limit and blue shading denotes log CFU.
-------
Table Cl-2. Phase II (Benchscale): Decontamination Results for Cotton Material with a Bleach
Dilution Ratio of 1:100
Sample Media
Type of Samples
Recoveries
Average
(CFUs)
Stdev
Inoculation Controls
CFU
1.1 xlO7
9.2 xlO5
Log(CFU)
7.04
0.04
Positive Controls
CFU
7.3 xlO6
1.0 xlO6
Log(CFU)
6.86
0.07
Coupon
CFU
3.4 xlO5
1.4 xlO5
Samples
Procedural Positive Controls
Log(CFU)
5.50
0.20
Log Reduction
1.54
0.20
CFU
31
32
Post Decon Fabric Sample
Log(CFU)
1.10
0.92
Log Reduction
5.76
0.92
Procedural Wash Liquid Sample
CFU
9.9 xlO6
1.0 xlO6
Log(CFU)
6.99
0.04
Post Decon Wash Liquid Sample
CFU
47.8
77.53
Liquid wash
Log(CFU)
1.03
0.96
water Samples
Procedural Rinse Liquid Sample
CFU
5.7 xlO5
1.2 xlO5
Log(CFU)
5.75
0.09
Post Decon Rinse Sample
CFU
215
231
Log(CFU)
2.12
0.56
Procedural
Blanks
Test Coupon
CFU/coupon
1
Wash Liquid Solution
CFU/mL
0.01
Rinse Liquid Solution
CFU/mL
0.01
Sterility Check for Decon Solution
CFU/ml
0.01
Sterility Checks
Sterility Check for DI-H20
CFU/mL
0.01
Negative Control Coupon
CFU/coupon
7
Inoculation Control Blank
CFU/coupon
5
Green shading denotes the sample detection limit and blue shading denotes log CFU.
-------
Table Cl-3. Phase II (Benchscale): Decontamination Results for Cotton Material with a Bleach
Dilution Ratio of 1:10
Sample Media
Type of Samples
Recoveries
Average
(CFUs)
Stdev
Coupon
Samples
Inoculation Controls
CFU
1.8 xlO7
5.2 xlO6
Log (CFU)
7.25
0.13
Positive Controls
CFU
2.2 xlO7
6.8 xlO6
Log (CFU)
7.32
0.15
Procedural Positive Controls
CFU
2.5 xlO6
2.7 xlO5
Log (CFU)
6.39
0.05
Log Reduction
0.86
0.05
Post Decon Fabric Sample
CFU
1.3
0.02
Log(CFU)
0.1
0.01
Log Reduction
7.2
0.01
Liquid Samples
Procedural Wash Liquid Sample
CFU
1.5 xlO7
3.28 xlO6
Log (CFU)
7.18
0.10
Post Decon Wash Liquid Sample
CFU
4.4
2.16
Log(CFU)
0.61
0.20
Procedural Rinse Liquid Sample
CFU
1.8 xlO6
6.2 xlO5
Log (CFU)
6.23
0.16
Post Decon Rinse Sample
CFU
3.3
0.02
Log (CFU)
0.52
0.00
Procedural
Blanks
Test Coupon
CFU/coupon
1
Wash Liquid Solution
CFU/mL
0.01
Rinse Liquid Solution
CFU/mL
0.01
Sterility Checks
Sterility Check for Decon Solution
CFU/ml
0.01
Sterility Check for DI-H20
CFU/mL
0.01
Negative Control Coupon
CFU/coupon
1
Inoculation Control Blank
CFU/coupon
3
Green shading denotes the sample detection limit and blue shading denotes log CFU.
-------
Appendix C-2: Phase II (Benchscale): 1% Diluted Bleach Solution, with the Addition of
a Detergent, Decontamination Results: All Materials
Table C2-1: Phase II (Benchscale): Decontamination Results for Cotton Material with 1% Diluted
Bleach Plus Detergent
Sample Media
Type of Samples
Recoveries
Average
Stdev
Inoculation Controls
CFU
1.1 xlO7
9.2 xlO5
Log(CFU)
7.04
0.04
Positive Controls
CFU
7.3 xlO6
1.1 xlO6
Log(CFU)
6.86
0.07
Coupon
Samples
CFU
3.5 xlO5
1.4 xlO5
Procedural Positive Controls
Log(CFU)
5.51
0.20
Log
Reduction
1.35
0.20
CFU
1.2
0.01
Post Decon Fabric Sample
Log(CFU)
0.08
0.003
Log
Reduction
6.78
0.28
Procedural Wash Liquid
CFU
9.9 xlO6
9.9xl05
Sample
Log(CFU)
6.99
0.04
Post Decon Wash Liquid
CFU
3.0
0.01
Liquid Samples
Sample
Log(CFU)
0.48
0.001
Procedural Rinse Liquid
CFU
5.8 xlO5
1.2 xlO5
Sample
Log(CFU)
5.75
0.09
Post Decon Rinse Sample
CFU
3.0
0.02
Log(CFU)
0.48
0.002
Procedural
Blanks
Test Coupon
CFU/coupon
1
Wash Liquid Solution
CFU/mL
0.01
Rinse Liquid Solution
CFU/mL
0.01
Sterility Check for Decon
Solution
CFU/ml
0.01
Sterility Checks
Sterility Check for DI-H20
CFU/mL
0.10
Negative Control Coupon
CFU/coupon
7
Inoculation Control Blank
CFU/coupon
1
Green shading denotes the sample detection limit and blue shading denotes log CFU.
-------
Table C2-2. Phase II (Benchscale): Decontamination Results for Blend Material with 1% Diluted
Bleach Plus Detergent
Sample Media
Type of Samples
Recoveries
Average
Stdev
Coupon
Samples
Inoculation Controls
CFU
2.2 xlO7
6.7 xlO6
Log (CFU)
7.33
0.14
Positive Controls
CFU
3.0 xlO7
2.8 xlO6
Log (CFU)
7.30
0.06
Procedural Positive Controls
CFU
5.8 xlO5
3.7 xlO5
Log (CFU)
5.69
0.32
Log
Reduction
1.64
0.32
Post Decon Fabric Sample
CFU
2.1
1.53
Log (CFU)
0.26
0.29
Log
Reduction
7.04
0.29
Liquid Samples
Procedural Wash Liquid
Sample
CFU
1.8 xlO7
4.8 xlO6
Log (CFU)
7.25
0.12
Post Decon Wash Liquid
Sample
CFU
285
489
Log (CFU)
1.30
1.41
Procedural Rinse Liquid
Sample
CFU
4.7 xlO5
2.5 xlO5
Log (CFU)
5.63
0.22
Post Decon Rinse Sample
CFU
9.2
8.01
Log (CFU)
0.85
0.39
Procedural
Blanks
Test Coupon
CFU/coupon
3
Wash Liquid Solution
CFU/mL
0.01
Rinse Liquid Solution
CFU/mL
0.01
Sterility Checks
Sterility Check for Decon
Solution
CFU/ml
0.01
Sterility Check for DI-H20
CFU/mL
0.01
Negative Control Coupon
CFU/coupon
0.03
Inoculation Control Blank
CFU/coupon
1
Green shading denotes the sample detection limit and blue shading denotes log CFU.
-------
Table C2-3. Phase II (Benchscale): Decontamination Results for Military Material with 1% Diluted
Bleach Plus Detergent
Sample Media
Type of Samples
Recoveries
Average
Stdev
Coupon Samples
Inoculation Controls
CFU
1.4 xlO7
2.3 xlO6
Log(CFU)
7.14
0.07
Positive Controls
CFU
1.3 xlO7
1.9 xlO6
Log(CFU)
7.10
0.07
Procedural Positive Controls
CFU
1.3 xlO6
3.7 xlO5
Log(CFU)
6.09
0.13
Log
Reduction
1.05
0.13
Post Decon Fabric Sample
CFU
291
75
Log(CFU)
2.44
0.11
Log
Reduction
4.65
0.11
Liquid Samples
Procedural Wash Liquid
Sample
CFU
1.4 xlO7
5.8 xlO5
Log(CFU)
7.13
0.02
Post Decon Wash Liquid
Sample
CFU
348
130
Log (CFU)
2.52
0.19
Procedural Rinse Liquid
Sample
CFU
1.3 xlO6
6.5 xlO5
Log(CFU)
6.08
0.22
Post Decon Rinse Sample
CFU
940
527
Log(CFU)
2.93
0.25
Procedural
Blanks
Test Coupon
CFU/coupon
1
Wash Liquid Solution
CFU/mL
0.01
Rinse Liquid Solution
CFU/mL
0.01
Sterility Checks
Sterility Check for Decon
Solution
CFU/ml
0.01
Sterility Check for DI-H20
CFU/mL
0.01
Negative Control Coupon
CFU/coupon
1
Inoculation Control Blank
CFU/coupon
1
Green shading denotes the sample detection limit and blue shading denotes log CFU.
-------
Table C2-4. Phase II (Benchscale): Decontamination Results for Polyester Material with 1%
Diluted Bleach Plus Detergent
Sample Media
Type of Samples
Recoveries
Average
Stdev
Coupon Samples
Inoculation Controls
CFU
1.0 xlO7
3.0 xlO6
Log(CFU)
6.99
0.13
Positive Controls
CFU
1.2 xlO7
8.5 xlO5
Log(CFU)
7.09
0.03
Procedural Positive Controls
CFU
1.7 xlO6
2.5 xlO5
Log(CFU)
6.21
0.06
Log Reduction
0.77
0.06
Post Decon Fabric Sample
CFU
1
0.01
Log(CFU)
0.10
0.00
Log Reduction
6.99
0.00
Liquid Samples
Procedural Wash Liquid
Sample
CFU
1.0 xlO7
2.2 xlO6
Log(CFU)
6.99
0.10
Post Decon Wash Liquid
Sample
CFU
3
0.01
Log (CFU)
0.46
0.00
Procedural Rinse Liquid
Sample
CFU
2.1 xlO6
1.6 xlO5
Log(CFU)
6.32
0.03
Post Decon Rinse Sample
CFU
5
3.5
Log (CFU)
0.64
0.27
Procedural Blanks
Test Coupon
CFU/coupon
1.0
Wash Liquid Solution
CFU/mL
0.01
Rinse Liquid Solution
CFU/mL
0.01
Sterility Checks
Sterility Check for Decon
Solution
CFU/ml
0.01
Sterility Check for DI-H20
CFU/mL
0.01
Negative Control Coupon
CFU/coupon
1
Inoculation Control Blank
CFU/coupon
1
Green shading denotes the sample detection limit and blue shading denotes log CFU.
-------
Table C2-5. Phase II (Benchscale): Decontamination Results for Spandex Material with 1% Diluted
Bleach Plus Detergent
Sample Media
Type of Samples
Recoveries
Average
Stdev
Coupon Samples
Inoculation Controls
CFU
1.5 xlO7
1.3 xlO6
Log(CFU)
7.18
0.04
Positive Controls
CFU
1.9 xlO7
2.8 xlO6
Log(CFU)
7.27
0.06
Procedural Positive Controls
CFU
9.5 xlO5
3.5 xlO5
Log(CFU)
5.96
0.15
Log Reduction
1.22
0.15
Post Decon Fabric Sample
CFU
1.2
0.01
Log(CFU)
0.08
0.00
Log Reduction
7.19
0.00
Liquid Samples
Procedural Wash Liquid
Sample
CFU
1.5 xlO7
2.6 xlO6
Log(CFU)
7.18
0.07
Post Decon Wash Liquid
Sample
CFU
37
51
Log(CFU)
1.24
0.65
Procedural Rinse Liquid
Sample
CFU
6.6 xlO5
9.8 xlO4
Log(CFU)
5.82
0.06
Post Decon Rinse Sample
CFU
3
0.01
Log(CFU)
0.49
0.00
Procedural Blanks
Test Coupon
CFU/coupon
1
Wash Liquid Solution
CFU/mL
0.01
Rinse Liquid Solution
CFU/mL
0.02
Sterility Checks
Sterility Check for Decon
Solution
CFU/ml
0.01
Sterility Check for DI-H20
CFU/mL
0.01
Negative Control Coupon
CFU/coupon
1
Inoculation Control Blank
CFU/coupon
1
Green shading denotes the sample detection limit and blue shading denotes log CFU.
-------
Table C2-6. Phase II (Benchscale): Decontamination Results for Denim Material with 1% Diluted
Bleach Plus Detergent
Sample Media
Type of Samples
Recoveries
Average
Stdev
Coupon Samples
Inoculation Controls
CFU
1.5 xlO7
8.6 xlO6
Log (CFU)
7.14
0.22
Positive Controls
CFU
2.3 xlO7
1.4 xlO6
Log (CFU)
7.36
0.03
Procedural Positive
Controls
CFU
2.3 xlO7
2.1 xlO6
Log (CFU)
6.71
0.18
Log Reduction
0.44
0.18
Post Decon Fabric Sample
CFU
113
122
Log (CFU)
1.64
0.95
Log Reduction
5.73
0.95
Liquid Samples
Procedural Wash Liquid
Sample
CFU
2.1 xlO7
2.1 xlO6
Log (CFU)
7.32
0.04
Post Decon Wash Liquid
Sample
CFU
100
76
Log (CFU)
1.90
0.39
Procedural Rinse Liquid
Sample
CFU
1.5 xlO6
2.1 xlO5
Log (CFU)
6.18
0.06
Post Decon Rinse Sample
CFU
5.0
1.73
Log(CFU)
0.68
0.17
Procedural Blanks
Test Coupon
CFU/coupon
1
Wash Liquid Solution
CFU/mL
0.01
Rinse Liquid Solution
CFU/mL
0.01
Sterility Checks
Sterility Check for Decon
Solution
CFU/ml
0.01
Sterility Check for DI-H20
CFU/mL
0.01
Inoculation Control Blank
CFU/coupon
1
Green shading denotes the sample detection limit and blue shading denotes log CFU.
-------
Appendix D-l: Phase III (Washer): 1% Diluted Bleach Solution, with the Addition of a
Detergent, Decontamination Results: All Materials.
Table Dl-1. Phase III (Washer): Decontamination Results for Cotton Material with 1% Diluted
Bleach Plus Detergent
Sample Media
Type of Samples
Recoveries
Average
Stdev
Coupon
Samples
Inoculation Controls
CFU
2.6 xlO7
6.2 xlO6
Log(CFU)
7.40
0.10
Positive Controls
CFU
1.4 xlO7
3.0 xlO6
Log(CFU)
7.12
0.10
Procedural Positive Controls
CFU
8.4 xlO4
1.8 xlO4
Log (CFU)
4.92
0.09
Log Reduction
2.49
0.09
Post Decon Fabric Sample
CFU
2
0.9
Log (CFU)
0.26
0.19
Log Reduction
6.87
0.19
Liquid Samples
Procedural Wash Liquid Sample
CFU
3.0 xlO7
5.7 xlO6
Log(CFU)
7.48
0.09
Post Decon Wash Liquid Sample
CFU
32.5
0.00
Log(CFU)
1.51
0.00
Procedural Rinse Liquid Sample
CFU
3.2 xlO6
6.2 xlO5
Log(CFU)
6.50
0.09
Post Decon Rinse Sample
CFU
32.5
0.00
Log(CFU)
1.51
0.00
Procedural
Blanks
Test Coupon
CFU/coupon
1
Wash Liquid Solution
CFU/mL
0.01
Rinse Liquid Solution
CFU/mL
0.01
Sterility Checks
Sterility Check for Decon Solution
CFU/ml
0.01
Sterility Check for DI-H20
CFU/mL
0.01
Negative Control Coupon
CFU/coupon
2
Inoculation Control Blank
CFU/coupon
1
Green shading denotes the sample detection limit and blue shading denotes log CFU.
-------
Table Dl-2. Phase III (Washer): Decontamination Results for Blend Material with 1% Diluted
Bleach Plus Detergent
Sample Media
Type of Samples
Recoveries
Average
Stdev
Coupon
Samples
Inoculation Controls
CFU
3.2 xlO7
6.9 xlO6
Log(CFU)
7.49
0.10
Positive Controls
CFU
2.4 xlO6
3.4 xlO5
Log (CFU)
6.37
0.06
Procedural Positive Controls
CFU
9.8 xlO4
1.7 xlO4
Log (CFU)
4.99
0.07
Log Reduction
2.50
0.07
Post Decon Fabric Sample
CFU
1
0.12
Log(CFU)
0.23
0.03
Log Reduction
5.91
0.03
Liquid Samples
Procedural Wash Liquid Sample
CFU
2.9 xlO7
4.4 xlO6
Log (CFU)
7.46
0.07
Post Decon Wash Liquid Sample
CFU
32.5
0.00
Log(CFU)
1.51
1.41
Procedural Rinse Liquid Sample
CFU
3.5 xlO6
7.9 xlO6
Log (CFU)
6.53
0.10
Post Decon Rinse Sample
CFU
32.5
0.00
Log (CFU)
1.51
0.00
Procedural
Blanks
Test Coupon
CFU/coupon
2
Wash Liquid Solution
CFU/mL
0.01
Rinse Liquid Solution
CFU/mL
0.01
Sterility Checks
Sterility Check for Decon Solution
CFU/ml
0.01
Sterility Check for DI-H20
CFU/mL
0.01
Negative Control Coupon
CFU/coupon
2
Inoculation Control Blank
CFU/coupon
1
Green shading denotes the sample detection limit and blue shading denotes log CFU.
-------
Table Dl-3. Phase III (Washer): Decontamination Results for Spandex Material with 1% Diluted
Bleach Plus Detergent
Sample Media
Type of Samples
Recoveries
Average
Stdev
Coupon
Samples
Inoculation Controls
CFU
2.4 xlO7
2.3 xlO6
Log(CFU)
7.37
0.04
Positive Controls
CFU
2.2 xlO7
2.1 xlO6
Log(CFU)
7.34
0.04
Procedural Positive Controls
CFU
1.9 xlO5
7.3 xlO4
Log(CFU)
5.25
0.16
Log Reduction
2.08
0.16
Post Decon Fabric Sample
CFU
5
3.2
Log(CFU)
0.64
0.30
Log Reduction
6.69
0.30
Liquid Samples
Procedural Wash Liquid Sample
CFU
2.6 xlO7
3.3 xlO6
Log(CFU)
7.41
0.06
Post Decon Wash Liquid Sample
CFU
32.5
0.00
Log(CFU)
1.51
0.00
Procedural Rinse Liquid Sample
CFU
3.6 xlO6
9.1 xlO5
Log(CFU)
6.55
0.11
Post Decon Rinse Sample
CFU
32.5
0.00
Log (CFU)
1.51
0.00
Procedural
Blanks
Test Coupon
CFU/coupon
2
Wash Liquid Solution
CFU/mL
0.01
Rinse Liquid Solution
CFU/mL
0.01
Sterility Checks
Sterility Check for Decon Solution
CFU/ml
0.01
Sterility Check for DI-H20
CFU/mL
0.01
Negative Control Coupon
CFU/coupon
2
Inoculation Control Blank
CFU/coupon
1
Green shading denotes the sample detection limit and blue shading denotes log CFU.
-------
Table Dl-4. Phase III (Washer): Decontamination Results for Polyester Material with 1% Diluted
Bleach Plus Detergent
Sample Media
Type of Samples
Recoveries
Average
Stdev
Coupon
Samples
Inoculation Controls
CFU
1.7 xlO7
6.9 xlO6
Log(CFU)
7.18
0.23
Positive Controls
CFU
1.0 xlO7
2.1 xlO6
Log(CFU)
6.89
0.40
Procedural Positive Controls
CFU
3.6 xlO5
9.0 xlO4
Log(CFU)
5.55
0.11
Log Reduction
1.53
0.11
Post Decon Fabric Sample
CFU
3.2
0.06
Log(CFU)
0.50
0.01
Log Reduction
6.39
0.01
Liquid Samples
Procedural Wash Liquid Sample
CFU
1.7 xlO7
3.5 xlO6
Log(CFU)
7.21
0.10
Post Decon Wash Liquid Sample
CFU
32.5
0.00
Log(CFU)
1.51
0.00
Procedural Rinse Liquid Sample
CFU
2.8 xlO6
2.9 xlO5
Log(CFU)
6.45
0.04
Post Decon Rinse Sample
CFU
32.5
0.00
Log (CFU)
1.51
0.00
Procedural
Blanks
Test Coupon
CFU/coupon
3
Wash Liquid Solution
CFU/mL
0.01
Rinse Liquid Solution
CFU/mL
0.01
Sterility Checks
Sterility Check for Decon Solution
CFU/ml
0.03
Sterility Check for DI-H20
CFU/mL
0.01
Negative Control Coupon
CFU/coupon
3.7 x 104
Inoculation Control Blank
CFU/coupon
3
Green shading denotes the sample detection limit and blue shading denotes log CFU.
-------
Table Dl-5. Phase III (Washer): Decontamination Results for Denim Material with 1% Diluted
Bleach Plus Detergent
Sample Media
Type of Samples
Recoveries
Average
Stdev
Inoculation Controls
CFU
2.6 xlO7
1.9 xlO7
Log (CFU)
7.26
0.48
Positive Controls
CFU
2.4 xlO7
6.5 xlO5
Log (CFU)
7.39
0.01
Coupon
CFU
2.0 xlO5
6.8 xlO4
Samples
Procedural Positive Controls
Log (CFU)
5.28
0.14
Log Reduction
2.10
0.14
CFU
3
0.16
Post Decon Fabric Sample
Log (CFU)
0.44
0.03
Log Reduction
6.95
0.03
Procedural Wash Liquid Sample
CFU
4.8 xlO7
6.3 xlO6
Log(CFU)
7.67
0.06
Post Decon Wash Liquid Sample
CFU
32.5
0.00
Liquid Samples
Log(CFU)
1.51
0.00
Procedural Rinse Liquid Sample
CFU
5.4 xlO6
4.2 xlO5
Log (CFU)
6.73
0.03
Post Decon Rinse Sample
CFU
43
19
Log (CFU)
1.61
0.17
Test Coupon
CFU/coupon
2
Procedural
Blanks
Wash Liquid Solution
CFU/mL
0.0
Rinse Liquid Solution
CFU/mL
0.0
Sterility Check for Decon Solution
CFU/ml
0.0
Sterility Checks
Sterility Check for DI-H20
CFU/mL
0.0
Negative Control Coupon
CFU/coupon
2
Inoculation Control Blank
CFU/coupon
5
Green shading denotes the sample detection limit and blue shading denotes log CFU.
-------
Table Dl-6. Phase III (Washer): Decontamination Results for Military Material with 1% Diluted
Bleach Plus Detergent
Sample Media
Type of Samples
Recoveries
Average
Stdev
Inoculation Controls
CFU
2.1 xio7
6.2 xlO6
Log(CFU)
7.30
0.14
Positive Controls
CFU
1.2 xlO7
1.7 xlO6
Log(CFU)
7.09
0.06
Coupon
CFU
8.1 xlO4
2.0 xlO4
Samples
Procedural Positive Controls
Log(CFU)
4.90
0.10
Log Reduction
2.19
0.10
CFU
2
0.03
Post Decon Fabric Sample
Log(CFU)
0.20
0.01
Log Reduction
6.89
0.01
Procedural Wash Liquid Sample
CFU
1.7 xlO7
3.6 xlO6
Log(CFU)
7.22
0.09
Post Decon Wash Liquid Sample
CFU
32.5
0.00
Liquid Samples
Log (CFU)
1.51
0.00
Procedural Rinse Liquid Sample
CFU
2.7 xlO6
4.3 xlO5
Log(CFU)
6.43
0.07
Post Decon Rinse Sample
CFU
32.5
0.00
Log(CFU)
1.51
0.00
Procedural
Blanks
Test Coupon
CFU/coupon
2
Wash Liquid Solution
CFU/mL
0.01
Rinse Liquid Solution
CFU/mL
0.01
Sterility Check for Decon Solution
CFU/ml
0.01
Sterility Checks
Sterility Check for DI-H20
CFU/mL
0.01
Negative Control Coupon
CFU/coupon
2
Inoculation Control Blank
CFU/coupon
1
Green shading denotes the sample detection limit and blue shading denotes log CFU.
72
-------
Appendix D-2: Phase III: 1% Diluted Bleach Solution without the Addition of a
Detergent Decontamination Results: Cotton and Military Materials.
Table D2-1. Phase III (Washer): Decontamination Results for Cotton Material with 1% Diluted
Bleach and without Detergent
Sample Media
Type of Samples
Recoveries
Average
Stdev
Coupon
Samples
Inoculation Controls
CFU
2.6 xlO7
6.2 xlO6
Log (CFU)
7.40
0.10
Positive Controls
CFU
1.4 xlO7
3.0 xlO6
Log (CFU)
7.12
0.10
Procedural Positive Controls
CFU
8.4 xlO4
1.8 xlO4
Log (CFU)
4.92
0.09
Log Reduction
2.49
0.09
Post Decon Fabric Sample
CFU
3
1.6
Log (CFU)
0.47
0.27
Log Reduction
6.66
0.27
Liquid Samples
Procedural Wash Liquid Sample
CFU
3.0 xlO7
5.7 xlO6
Log (CFU)
7.48
0.09
Post Decon Wash Liquid Sample
CFU
32.5
0.00
Log(CFU)
1.51
0.000
Procedural Rinse Liquid Sample
CFU
3.2 xlO6
6.2 xlO5
Log (CFU)
6.50
0.09
Post Decon Rinse Sample
CFU
32.5
0.00
Log (CFU)
1.51
0.000
Procedural
Blanks
Test Coupon
CFU/coupon
37
Wash Liquid Solution
CFU/mL
0.01
Rinse Liquid Solution
CFU/mL
0.01
Sterility Checks
Sterility Check for Decon Solution
CFU/ml
0.01
Sterility Check for DI-H20
CFU/mL
0.01
Negative Control Coupon
CFU/coupon
2
Inoculation Control Blank
CFU/coupon
1
Green shading denotes the sample detection limit and blue shading denotes log CFU.
73
-------
Table D2-2. Phase III (Washer): Decontamination Results for Military Material with 1% Diluted
Bleach and without Detergent
Sample Media
Type of Samples
Recoveries
Average
Stdev
Coupon
Samples
Inoculation Controls
CFU
3.7 xlO7
1.6xl07
Log(CFU)
7.54
0.18
Positive Controls
CFU
1.2 xlO7
1.7 xlO6
Log (CFU)
7.09
0.06
Procedural Positive Controls
CFU
8.1 xlO4
2.0 xlO4
Log(CFU)
4.90
0.10
Log Reduction
2.19
0.10
Post Decon Fabric Sample
CFU
2
0.89
Log (CFU)
0.30
0.17
Log Reduction
6.79
0.17
Liquid Samples
Procedural Wash Liquid Sample
CFU
1.7 xlO7
3.6 xlO6
Log (CFU)
7.22
0.09
Post Decon Wash Liquid Sample
CFU
32.5
0.00
Log(CFU)
1.51
0.00
Procedural Rinse Liquid Sample
CFU
2.7 xlO6
4.3 xlO5
Log (CFU)
6.43
0.07
Post Decon Rinse Sample
CFU
32.5
0.00
Log (CFU)
1.51
0.00
Procedural
Blanks
Test Coupon
CFU/coupon
2
Wash Liquid Solution
CFU/mL
0.01
Rinse Liquid Solution
CFU/mL
0.01
Sterility Checks
Sterility Check for Decon Solution
CFU/ml
0.01
Sterility Check for DI-H20
CFU/mL
0.01
Negative Control Coupon
CFU/coupon
2
Inoculation Control Blank
CFU/coupon
1
Green shading denotes the sample detection limit and blue shading denotes log CFU.
-------
Appendix E-l: Phase IV (Washer and Dryer): 0.1% Diluted Bleach Solution, with the
Addition of a Detergent, Decontamination Results Using a Washer/Dryer: Cotton
Material.
Table E-l. Phase IV (Washer and Dryer): Decontamination Results for Cotton Material with 0.1%
Diluted Bleach Plus Detergent
Sample
Media
Type of Samples
Recoveries
Average
Stdev
Inoculation Controls
CFU
2.4 xlO7
7.1 xlO6
Log (CFU)
7.37
0.11
Positive Controls
CFU
1.4 xlO7
3.0 xlO6
Log (CFU)
7.12
0.10
CFU
8.4 xlO4
1.8 xlO4
Procedural Positive Controls
Log (CFU)
4.92
0.09
Log Reduction
2.49
0.09
Coupon
Samples
CFU
2.2 xlO3
2.6 xlO3
Post Wash Fabric Sample
Log (CFU)
3.14
0.52
Log Reduction
3.98
0.52
CFU
1.1 xlO3
1.1 xlO3
Post Drying Fabric Sample
Log (CFU)
2.87
0.44
Log Reduction
4.25
0.44
Aerosol Samples
CFU
3.7 xlO2
2.8 xlO2
Log(CFU)
2.48
0.34
Procedural Wash Liquid Sample
CFU
3.0 xlO7
5.7 xlO6
Log (CFU)
7.48
0.09
Post Decon Wash Liquid Sample
CFU
1.3 xlO3
4.7 xlO2
Liquid
Log (CFU)
3.08
0.15
Samples
Procedural Rinse Liquid Sample
CFU
3.2 xlO6
6.2 xlO5
Log (CFU)
6.50
0.09
Post Decon Rinse Sample
CFU
1.3 xlO4
3.8 xlO3
Log (CFU)
4.11
0.12
Procedural
Blanks
Washer Test Coupon
CFU/coupon
1
Dryer Test Coupon
CFU/coupon
1
Dryer Filter
CFU/coupon
1
75
-------
Sample
Media
Type of Samples
Recoveries
Average
Stdev
Wash Liquid Solution
CFU/ml
0.01
Rinse Liquid Solution
CFU/mL
0.01
Sterility Check for Decon Solution
CFU/ml
0.01
Sterility
Sterility Check for DI-ILO
CFU/mL
0.01
Checks
Negative Control Coupon
CFU/coupon
2
Inoculation Control Blank
CFU/coupon
1
Green shading denotes the sample detection limit and blue shading denotes log CFU.
76
-------
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Environmental Protection
Agency
PRESORTED STANDARD
POSTAGE & FEES PAID
EPA
PERMIT NO. G-35
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