EPA/600/R-15/228 I September 2015
www.epa.gov/homeland-security-research
United States
Environmental Protection
Agency
Assessment of Bacill spore
inactivation on indoor surfaces using
commercially-available cleaning
products
Office of Research and Development
Homeland Security Research Program
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Disclaimer
Disclaimer
The U.S. Environmental Protection Agency through its Office of Research and Development funded the
research described here under Contract Number EP-C-09-027 with Arcadis U.S., Inc. It has been subjected
to the Agency's review and has been approved for publication. Note that approval does not signify that the
contents necessarily reflect the views of the Agency. Mention of trade names, products, or services does not
convey official EPA approval, endorsement, or recommendation.
Questions concerning this document or its application should be addressed to:
Shannon Serre, Ph.D.
Decontamination and Consequence Management Division
Homeland Security Research Program
U.S. Environmental Protection Agency (MD-E343-06)
Office of Research and Development
109 T.W. Alexander Drive
Research Triangle Park, NC 27711
Phone:919-541-3817
Fax:919-541-0496
E-mail: Shannon.Serre@epa.gov
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Disclaimer
Acknowledgments
Contributions of the following individuals and organization to this report are gratefully
acknowledged:
U.S. Environmental Protection Agency (EPA)
Francisco J. Cruz
Marshall Gray
Paul Kudarauskas
Eletha Brady-Roberts
Ramona Sherman
Joseph P. Wood
Arcadis, Inc.
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List of Acronyms and Abbreviations
Contents
Disclaimer i
Acknowledgments ii
List of Acronyms and Abbreviations vi
Executive Summary viii
1. Introduction 1
1.1. Objectives 3
2. Experimental Approach 4
2.1. Materials 6
2.2. Inoculation of Coupons 8
2.3. Preparation of Decontamination Solution 9
2.3.1 Clorox® Concentrated Germicidal Bleach 9
2.3.2 Lysol® Mold & Mildew Blaster 11
2.3.3 Tilex® Mold & Mildew Remover 12
2.3.4 Clorox® Clean-Up Cleaner + Bleach 13
2.3.5 Clorox® Disinfecting Bleach Foamer 14
2.4. Characterization of Decontamination Solutions 15
2.4.1 Concentration of Sodium Hypochlorite (NaOCI) versus Free Available Chlorine and pH 16
2.4.2 Temperature 17
2.5. Decontamination Procedure 18
2.5.1. Test Matrix 22
3. Sampling and Analytical Procedures 25
3.1. Types of Samples 25
3.2. Sampling Procedures 26
3.2.1 Wipe Sampling 26
3.2.2 Liquid Effluent Sampling 27
3.2.3 Solid Waste Sampling 27
3.2.4 Aerosol Sampling 27
3.3. QA/QC Samples 28
3.3.1 Swab Samples 28
3.3.2 Material Samples 28
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List of Acronyms and Abbreviations
3,3,3 Decontamination Solution Samples
4. Determination of Sporicidal Effectiveness
5. Results and Discussion
5.1. Characterization of Decontamination Solutions
5 1.1 FAC Concentration
5.1.2 pH and Temperature
5.2. Application Characterization
5.3. Decontamination Results
5.3.1. Inoculation Results
5.3.2. Log Reduction Results
5.4. Fate of Spores
5.4.1. Exhaust air concentrations
5.4.2. Liquid waste concentrations
5.4.3. Solid waste concentrations
6. Summary
7. References
Tables
Table 2-1. Description of Building Materials for Decontamination Testing
Table 2-2. Grime Recipe
Table 2-3. Concentration of Sodium Hypochlorite (%) and pH of Decontamination Solutions
Table 2-4. Rate Constants (k2) of NaOCI Decompositions with Respect to Strength and Temperature*
Table 2-5. Phase I Test Matrix
Table 2-6. Phase II Test Matrix
Table 5-1. Air Sample Results
Table 5-2. Run-off Samples Results
Table 5-3. Rinsate Sample Results
Table 5-4. Solid-waste samples results
28
29
32
32
32
33
35
36
36
36
42
42
42
43
44
46
6
8
16
18
23
24
42
43
43
44
iv
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List of Acronyms and Abbreviations
Figures
Figure 2-1. Test Coupons 7
Figure 2-2. Packaging of Clorox® Concentrated Germicidal Bleach (a) and Clorox® Germicidal Bleach (b) 9
Figure 2-3. Packaging of Splash-less Clorox® Concentrated Germicidal Bleach 11
Figure 2-4. Packaging of Lysol® Mold & Mildew Blaster 12
Figure 2-5. Packaging of Tilex® Mold & Mildew Remover 13
Figure 2-6. Packaging of Clorox® Clean-Up Cleaner + Bleach 14
Figure 2-7. Packaging of Clorox® Disinfecting Bleach Foamer 15
Figure 2-8. Distribution of Free Chlorine Species in Aqueous Solutions (from reference 11) 17
Figure 2-9. Decontamination Chamber (Left) and Application of Diluted Bleach Using Rag 19
Figure 3-1. Spray Chamber Exhaust Duct (white arrow shows the sampling point location) 27
Figure 5-1. FAC of Diluted Bleach Solutions Prepared In-House (Phase I) 33
Figure 5-2. pH of Diluted Bleach Solutions Prepared In-House (Phase I) 34
Figure 5-3. Temperature of Diluted Bleach Solutions Prepared In-House (Phase I) 35
Figure 5-4. Decontaminant Specific LR values for Phase I (values in green - average target
decontamination efficacy of LR>6) 38
Figure 5-5. Decontaminant Specific LR values for Phase II (values in green - average target
decontamination efficacy of LR>6) 39
Figure 5-6. Material Specific LR Values for Phase I (values in green - average target decontamination
efficacy of LR>6) 40
Figure 5-7. Material Specific LR Values for Phase II (values in green - average target decontamination
efficacy of LR>6) 41
Appendices
Appendix A. Characterization of decontaminants and decontamination procedures
Appendix B. Miscellaneous Operating Procedures
Appendix C. Decontamination Efficiency Results
Appendix D: Quality Assurance
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List of Acronyms and Abbreviations
List of Acronyms and Abbreviations
ADA
Aerosol Deposition Apparatus
APIC
Association for Professionals in Infection Control and Epidemiology
ATCC
American Type Culture Collection
B.
Bacillus
CDC
Centers for Disease Control and Prevention
CFU
Colony Forming Unit(s)
COC
Chain of Custody
COTS
Commercial Off the Shelf
DCMD
Decontamination and Consequence Management Division
DHS
Department of Homeland Security
Dl
Deionized
DNA
Deoxyribonucleic Acid
DPG
Dugway Proving Ground
DQI
Data Quality Indicator
DQO
Data Quality Objective(s)
DTRL
Decontamination Technologies Research Laboratory
EPA
U. S. Environmental Protection Agency
fl. oz
Fluid Ounce
ft
Feet or Foot
ft2
Square Feet or Foot
FAC
Free Available Chlorine
HSRP
Homeland Security Research Program
HVAC
Heating Ventilation and Air Conditioning
HVLP
High Volume Low Pressure
In
Inch(es)
L
Liter
LD50
Lethal Dose, 50%
LR
Log Reduction
MDI
Metered Dose Inhaler
Ml
Milliliter
Min
Minute
MOP
Miscellaneous Operating Procedure
NIST
National Institute of Standards and Technology
OPP
Office of Pesticide Programs
OSHA
Occupational Safety and Health Administration
OSWER
Office of Solid Waste and Emergency Response
oz
Ounce
pAB
pH-Adjusted Bleach
PEG
Polyethylene Glycol
PPE
Personal Protective Equipment
PPm
Part(s) Per Million
RH
Relative Humidity
RSD
Relative Standard Deviation
RTU
Ready to Use
QAPP
Quality Assurance Project Plan
QUATS
Quaternary Ammonium Compounds
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List of Acronyms and Abbreviations
RH
Relative Humidity
RTP
Research Triangle Park
RTU
Ready To Use
RNA
Ribonucleic Acid
SD
Standard Deviation
SNL
Sandia National Laboratories
STEL
Short-Term Exposure Limit
STS
Sodium Thiosulfate
TSA
Tryptic Soy Agar
UV
Ultraviolet (light)
VHP
Vaporous Hydrogen Peroxide
WA
Work Assignment
WACOR
Work Assignment Contract Officer's Representative
vii
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Executive Summary
Executive Summary
This project supports the mission of the U.S. Environmental Protection Agency's Office of Research and
Development's Homeland Security Research Program (HSRP) by providing information relevant to the
decontamination of areas contaminated as a result of an act of terrorism or other incident.
This project evaluated "low tech"/"self-help" expedient bioagent decontamination options of porous and
nonporous building materials using bleach solutions. In Phase I, the sporicidal effectiveness of various
dilutions of germicidal bleach and diluted bleach with surfactant was investigated. In Phase II, bleach-based
ready-to-use (RTU)-commercial-off-the-shelf (COTS) cleaning and disinfecting products were used.
The efficacy of diluted germicide and bleach with surfactants was evaluated on common building materials
contaminated with aerosolized Bacillus atrophaeus spores (surrogate for Bacillus anthracis). Evaluation
exercised two types of operational procedures: Phase I procedures- application of in-house prepared
decontaminant (diluted bleach) via rag or sponge -10 minutes processing - water rinse using clean rag or
sponge, and Phase II procedure - application of ready-to-use/commercial-off-the-shelf (RTU-COTS)
decontaminant via spray -10 minutes processing - water rinse using clean rag. Estimates of the number of
spores re-aerosolized and rinsed off from the decontaminated material surface were also performed.
For the Phase I procedure, the average log reduction (LR) values ranged from 3.6 to 7.8. The
decontamination effectiveness was dependent on bleach dilution, with dilution at 1:5 determined to provide a
satisfactory sporicidal action. The superior mechanical removal of spores associated with the more abrasive
rag application provided better sporicidal efficacy for nonporous materials, and the vertical penetration
offered by use of a sponge (into the sponge) seemed to improve the decontamination efficacy for porous
materials.
Addition of surfactants improved spore removal efficacy - the target decontamination rate (average LR > 6)
was achieved for 100% of the materials decontaminated with the rag procedure using bleach with surfactant-
based liquid sporicide. Similarly, all tests in Phase II performed with RTU-COTS products containing bleach,
various surfactants, and additives resulted in the full decontamination of all materials tested (average LR
values from 6.7 to 8.0).
These data suggest that "low-tech"/"self-help" decontamination technology can provide greater than 6 log
reductions of viable spores on the common building materials tested. The use of RTU-COTS materials
removes additive-bleach compatibility issues and a priori eliminates any potential errors in preparation of
diluted bleach and surfactant-amended bleach formulation.
The estimates of the fate of spores suggest that the low-tech liquid sporicide-based decontamination process
leads to physical removal of spores from decontaminated surfaces, with a consequent transport of viable
spores to the post-decontamination liquid and solid waste. Spores can also 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. The final "self-help" method tutorial/ instructional document resulting
from this project should therefore emphasize the importance of using the required personal protective
equipment (PPE). Users of this guidance should follow PPE recommendations on the product label when
using these products. Mixing of these products with other chemicals is also not recommended.
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Assessment of Bacillus spore inactivation on
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1. Introduction
The Department of Homeland Security (DHS) and other appropriate Federal departments and agencies
have been tasked to develop comprehensive plans which "provide for seamless, coordinated Federal, state,
local, and international responses to a biological attack." As part of these plans, the U.S. Environmental
Protection Agency (EPA), in a coordinated effort with DHS, is responsible for "developing strategies,
guidelines, and plans for decontamination of persons, equipment, and facilities" to mitigate the risks of
contamination following a biological weapons attack. EPA's Homeland Security Research Program (HSRP)
provides expertise and products that can be widely used to prevent, prepare for, and recover from public
health and environmental emergencies arising from terrorist threats and incidents.
Decontamination can be defined as the process of inactivating or reducing a contaminant in or on humans,
animals, plants, food, water, soil, air, areas, or items through physical, chemical, or other methods to meet a
cleanup goal. In terms of the surface of a material, decontamination can be accomplished by physical
removal of the contaminant or via inactivation of the contaminant with antimicrobial chemicals, heat,
ultraviolet (UV) light, etc. Physical removal could be accomplished via in situ removal of the contaminant
from the material or by physical removal of the material itself (i.e., disposal). Similarly, inactivation of the
contaminant can be conducted in situ or after removal of the material for ultimate disposal. Following the
2001 anthrax incidents, a combination of removal and in situ decontamination was used [1-5].The balance
between the two procedures were facility-dependent and factored in many issues (e.g., physical state of the
facility) [1-5], One factor was that such remediation was unprecedented for the United States Government,
and no technologies had been proven for such use at the time. The cost of disposal proved to be significant
and was complicated by the nature of the waste (e.g., finding an ultimate disposal site). Since 2001, a
primary focus for facility remediation has been improving the effectiveness and practical application of in
situ decontamination methods and evaluating waste treatment options to be able to provide information
necessary to optimize the decontamination/disposal paradigm. This optimization has a significant impact on
reducing the cost of and time for the remediation effort.
Quick, effective and economical decontamination methods that have the capacity to be employed over wide
areas (outdoor and indoor) required to increase emergency preparedness are the focus of the HSRP's
research, which supports the Office of Solid Waste and Emergency Response (OSWER) and the Office of
Pesticide Programs (OPP). Decontamination methods being tested by the HSRP include various "high-tech"
technologies like fumigations and "low-tech" approaches like combined mechanical and chemical
procedures (vacuum, scrub/wash and bleach). If proven effective, "lower-tech" approaches involving
washing and cleaning with readily available equipment, washes and off-the-shelf sporicides would
significantly increase the Nation's readiness to respond to a wide-area contaminant release.
Low tech decontamination approaches employing liquid and physical cleaning methods have been used
following the intentional or accidental release of Bacillus (B.) anthracis spores in secondarily contaminated
areas, (i.e., areas contaminated with a biological agent tracked from primary contaminated sites, often via
materials contaminated/cross-contaminated with anthrax; e.g. letters), or in primary contaminated facilities
(i.e., directly exposed to intentional or accidental release of anthrax spores) showing a minimal presence of
the B. anthracis spores [1-5], These methods included combinations of disposal of contaminated items,
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Assessment of Bacillus spore inactivation on
indoor surfaces using commercially-available
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vacuuming, and the use of liquid sporicides (e.g., pH-amended bleach solution) or a combination of
mechanical and chemical procedures (vacuum, scrub/wash, and bleach) [1-5],
Bleach, (5.3%-8.3% NaOCI by weight for regular and concentrated off-the-shelf products) is the most
popular chlorinating agent in use today. When added to water, bleach hydrolyzes as hypochlorous acid,
sodium ion and hydroxide:
NaOCI + H2O -» HOCI + Na+ + OH"
Additionally, some of the HOCI dissociates into hydrogen ion and hypochlorite ion:
HOCI -» H+ + OCI-
Both HOCI and OCI- are oxidants and effective germicides. HOCI is the stronger and more effective of the
two species [6-7],
Bleach is effective against a broad range of microorganisms at low contact times (5-10 minutes). The
Centers for Disease Control and Prevention (CDC), Occupational Safety and Health Administration (OSHA),
and Association for Professionals in Infection Control and Epidemiology (APIC) guidelines recommend
bleach as a broad spectrum germicide to disinfect hard surfaces contaminated by blood spills and tough-to-
kill pathogens such as Clostridium difficile spores and norovirus, both of which are resistant to disinfection
by quaternary ammonium compounds (QACs) [6], For resistant organisms and surfaces that are highly
soiled, the CDC recommends a 1:10 dilution of 5.25% -6.15% bleach (5250 parts per million (ppm) -6150
ppm sodium hypochlorite solution). As a strong oxidizer, bleach reacts with nucleic acids (DNA/RNA), lipids
and fatty acids associated with the cell membrane and destroys the cellular activity of structural and
functional proteins [6], Chlorine may affect a variety of metabolic processes in bacteria including inhibition
of metabolic enzymes and inhibition of membrane-mediated active transport processes and respiratory
activity [7], There is no evidence of bacteria or viruses developing resistance to the powerful oxidizing
action of bleach when used at recommended dilutions [6],
Addition of simple surfactants to liquid sporicides may improve decontamination efficacy because some
surfactants can prevent the formation of biofilms due to surface tension that pulls apart microorganisms
upon contact. A new generation of antibacterial/antiviral products known as "nanoemulsion chemicals" are
formulations made from detergents and oil in water [8-9], These antimicrobial nanoemulsions fuse with lipid-
containing organisms. This fusion is enhanced by the electrostatic attraction between the cationic charge of
the emulsion and the anionic charge on the pathogen, which ultimately destabilizes the pathogen lipid
membrane, resulting in cell lysis and death [9],
Optimized low tech decontamination approaches can be especially useful for self-help decontamination
purposes of secondary contaminated areas, like residential dwellings. However, additional information is
needed to predict the decontamination efficacy of off-the-shelf sporicidal agents deployed over complex and
challenging material types and developing safe and efficient concepts of operation that are both easily
accessible and understandable to the general public.
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Assessment of Bacillus spore inactivation on
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1.1. Objectives
The primary objective of this study was to develop a simple "self-help" decontamination approach for
building surfaces contaminated with Bacillus atrophaeus spores (i.e., surrogates of Bacillus anthracis). "Self-
help" decontamination was defined as in situ surface-disinfection method not requiring specialized materials
or equipment (i.e., cleaning supplies/products available at a local hardware store would be used). The
optimized method must be easily understandable to the general public/responders without specialized
training.
Several procedures using bleach-based decontamination solutions were evaluated, using basic tools
expected to be easily accessible at residential dwellings (i.e., cleaning rags and sponges). The
decontamination agents tested were diluted germicidal concentrated bleach and bleach with surfactants
solutions and four commercial RTU-COTS bleach-based disinfecting/cleaning products (details of
decontaminants are given in Section 2.4). The effectiveness of decontaminants/proposed self-help
procedures was tested on 14 x14 inch coupons of the porous and nonporous common building materials
(details given in Section 2.2). The operational parameters of decontamination (material compatibility of
decontaminants with test materials, effectiveness of decontamination solution delivery as a function of the
type of cleaning medium and medium-specific loading volume) were considered important to understand the
sporicidal activity of a bleach-based decontamination process and were characterized in addition to
sporicidal effectiveness. The fate of the viable spores during decontamination (transfer from decontaminated
surface to air) and transfer to post-decontamination solid and liquid waste were also determined. The fate or
partitioning of the spores was studied to determine whether the loss of spores from the surface were a result
of inactivation or physical removal from the surface.
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Assessment of Bacillus spore inactivation on
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2. Experimental Approach
This section describes the test materials, test facilities and equipment, general decontamination approach
and test conditions, and the methods that were used to evaluate the data related to the project objectives.
Testing was conducted at EPA's Research Triangle Park facility (RTP, NC, USA). Sampling and analytical
procedures are described in Section 3.
The general experimental approach was:
Preparation of representative samples of nonporous and porous building materials; the 35.6 cm by
35.6 cm (14x14 in) coupon area was selected as a suitable surface for decontamination and
sampling.
Selection of bleach-based formulations and commercial COTS products for decontamination; all
products used in this study are obtainable from national retailers. The products used in these
studies were titrated to verify the hypochlorite concentration as this concentration will change as the
product sits on the shelf.
Contamination of model building surfaces (coupon) with standardized inocula of B. atrophaeus
spores using an aerosol deposition method, followed by quantitative assessment of pre-
decontamination spore loading by sampling positive control (non-decontaminated) coupons (n = 3
per test)
Application of the decontamination procedure on test coupons (n = 3 to 5 coupons per each
decontamination procedure tested); test coupons were decontaminated with various solutions of
bleach and bleach with additives using different decontamination procedures, followed by
quantitative determination of viable spores. Quantitative assessment of residual (background)
contamination was performed by sampling of procedural blanks. The transfer of viable spores to
post-decontamination waste and air was done by qualitative analysis of decontamination procedure
residues, i.e., decontamination solution run-off, rinse water waste, expended decontamination tools
(rags and sponges) and in aerosol samples.
Determination of decontamination effectiveness as a function of the procedure/decontaminant and
material type. Decontamination effectiveness was measured as log reduction (LR), defined as the
amount of reduction in viable spores required to move the decimal one place, or reduce the
exponent in scientific notation by one. The target LR for decontamination studies is usually LR>6
(>99.9999% reduction). A 6 LR target is consistent with sporicidal efficacy tests used to register
sporicides under the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA). Recovery of no
viable spores following treatment was considered highly effective.
This project was conducted in two phases:
1) Phase I - determining the effectiveness and operational parameters for a "low-tech" or "self-help"
approach for decontamination of B. anthracis surrogate from selected building surfaces using a
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Assessment of Bacillus spore inactivation on
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simple liquid sporicide (diluted germicidal concentrated bleach) deployed via an uncomplicated
wipe-wait-rinse technique. The experimental design assumed bleach dilution at ratios starting at
10:1 and working down to a 1:1 ratio if necessary. The candidate processing times were 10 and 30
minutes. Addition of surfactant to the bleach solution was also tested for the combination of bleach
dilution and processing time that was determined to provide > 6 log spore inactivation/removal for
bleach without additives. Lastly, effect of grime/organic matter presence on the selected building
surface (laminate flooring) surface was also tested for the procedure that provided the highest
sporicidal activity for all materials (1:5 bleach with surfactant decontamination solution, applied via
rag with processing time of 10 minutes).
2) Phase II - determining the effectiveness of a "low-tech" or "self-help" approach for decontamination
of B. anthracis surrogate from selected building surfaces using bleach-based RTU-COTS products
deployed via spray-wait-rinse technique, using wiping media and processing time optimized in
Phase I.
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Assessment of Bacillus spore inactivation on
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2.1. Materials
The representativeness and uniformity of test materials are essential in achieving defensible evaluation
results. Material representativeness means that these materials are typical of those currently used in
buildings in terms of quality, surface characteristics, and structural integrity. In this effort, representativeness
will be assured by selecting test materials that are typical of those found in residential dwellings, and that
meet industry standards or specifications for indoor use, and by obtaining those materials from appropriate
suppliers. Material uniformity means that all these material coupons are equivalent for purposes related to
testing. Uniformity will be maintained by obtaining and preparing a quantity of material sufficient to allow
multiple test samples to be prepared with presumably uniform characteristics (i.e., test coupons will be cut
from the interior rather than the edge of a large piece of material). Documented procedures were
established for coupon preparation to ensure material uniformity.
Coupons (14 x14 inch) were prepared from selected nonporous and porous building materials (glass,
finished wood flooring, grouted ceramic tile, and painted wallboard (Table 2-1, Figure 2-1) that are typical of
the materials found in residential dwellings that meet industry standards or specifications for indoor use.
Table 2-1. Description of Building Materials for Decontamination Testing
Material
Description
Manufacturer/
Supplier Name
Coupon Surface
Size, L x W (inches)
Material Preparation
Glass
Borosilicate glass, thickness
1/4 inch
McMaster-Carr
14x14
- Clean with water and detergent
- Rinse with water and wipe dry
- Sterilize (autoclave)
Finished wood
flooring
7-5/8 x 50-3/4 inch laminated
wood locking flooring; smooth
surface
Project Source; color-
/Lowe's/Winchester
Oak
14x14
- Remove particles by wiping clean with
water and wipe dry
- Sterilize (VHP)*
Grouted ceramic
tile
12" x 12" Ceramic mosaic tile
grouted with cementitious
sanded premixed grout
Lowe's/American
Olean and TEC Invision
14x14
- Remove particles by wiping clean with
water and wipe dry
- Sterilize (VHP)
Painted
wallboard
1/2-in x 4-ftx 8-ft drywall
panel painted with premium
latex interior flat paint in white
Lowe's/National
Gypsum Company and
Behr
14x14
- Remove particles by wiping clean with
water and wipe dry
- Sterilize (VHP)
"Vaporous hydrogen peroxide.
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a. Finished wood flooring
c. Painted wallboard
Figure 2-1. Test Coupons
Assessment of Bacillus spore inactivation on
indoor surfaces using commercially-available
cleaning products
b. Glass
d. Grouted mosaic tile
The glass coupons were sterilized prior to use by steam autoclave utilizing a gravity cycle program. The
remaining materials (painted wallboard, finished wood flooring and grouted tile) were sterilized using
Vaporized Hydrogen Peroxide® (VHP). The hydrogen peroxide vapor in this study was generated using a
STERIS VHP 1000ED generator (referred to as Vaporized Hydrogen Peroxide®, or VHP) loaded with a 35%
H2O2 Vaprox® cartridge. The sterility of the coupons was verified through the use of negative control
samples.
For tests with grime (test 25, Table C-1, Appendix C), coupons were loaded with grime using a high volume,
low pressure (HVLP) sprayer per Miscellaneous Operating Procedure (MOP) 3163 (Appendix B). Grime
consisting of 94% fine dust, 3% soot and 3% biological materials was prepared in-house using the
procedure developed by Sandia National Laboratories (SNL) (recipe was adopted from "Evaluation of
Surface Sampling Method Performance for Bacillus spores on Clean and Dirty Outdoor Surfaces"
developed by Sandia National Laboratories [10]). The detailed recipe forgrime is shown in Table 2-2.
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Table 2-2. Grime Recipe
Vendor
Part#
Component Name
Relative composition of
individual component [%]
Relative composition of
classes component [%]
Powder Technology Inc., PTI
PP2G4 A2 fine
Arizona fine dust
94.00%
94% Fine dust
Powder Technology Inc., PTI
Raven 410
Carbon black
2.50%
3% Soot
National Institute of Standards and
Technology, NIST*
SRM 1650b
Diesel particulate
0.25%
Auto Parts
Off the shelf
Motor oil
0.13%
Fisher Scientific
AC 16436-0050
a-Pinene,97%
0.13%
Fisher Scientific
S755301
Lycopodium
1.00%
3% Biological materials
Polysciences Inc.
7673
Ragweed pollen
1.00%
Polysciences Inc.
7670
Paper Mulberry pollen
1.00%
*National Institute of Standards and Technology
2.2. Inoculation of Coupons
Inoculation of test and positive control coupons with spores of B. atrophaeus was performed via aerosol
deposition using a metered dose inhaler (MDI).
The test organism for this work was a powdered spore preparation of B. atrophaeus (American Type Culture
Collection (ATCC) 9372) and silicon dioxide particles. This bacterial species was formerly known as B.
subtilis var. niger and subsequently B. globigii. The preparation was obtained from the U.S. Army Dugway
Proving Ground (DPG) Life Science Division. Briefly, after 80 - 90 percent sporulation, the suspension was
centrifuged to generate a preparation of approximately 20 percent solids. A preparation resulting in a
powdered matrix containing approximately 1x1011 viable spores per gram was prepared by dry blending and
jet milling the dried spores with fumed silica particles (Deguss, Frankfurt am Main, Germany). The powdered
preparation was loaded into metered dose inhalers (MDIs). Control checks for each MDI were included in
the batches of coupons contaminated with a single MDI.
Coupons (test and positive controls) were inoculated with spores of B. atrophaeus from an MDI confirmed to
deliver E+07 spores per discharge. Results of inoculation of positive control coupons are given in Section
5.3.1). Each coupon was contaminated independently by being placed into a separate dosing chamber into
a separate dosing chamber (aerosol deposition apparatus or ADA) designed to fit one 35.6 cm by 35.6 cm
(14 inch (in) by 14 in) coupon of any thickness. The MDI was discharged a single time into the dosing
chamber. The number of discharges per MDI was tracked so that use did not exceed 75% of the calculated
capacity. Additionally, the weight of each MDI was determined after completion of the contamination of each
coupon. The inoculation control coupons (14 in by 14 in sterile stainless steel coupons) were inoculated as
the first, middle, and last coupons within a single group of coupons inoculated by any one MDI within a
single test. Prior to decontamination testing, spores were allowed to settle onto the coupon surfaces for a
minimum period of 18 hours (overnight).
8
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Assessment of Bacillus spore inactivation on
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cleaning products
2.3. Preparation of Decontamination Solution
Two types of sporicides were tested: solutions prepared in-house using Clorox® Concentrated Germicidal
Bleach (The Clorox® Company, USA) and Clorox® Splash-less Concentrated Bleach (The Clorox®
Company, USA). RTU-COTS recipes used for preparation of in-house diluted bleach solutions are given in
Table A-1 (Appendix A). RTU-COTS products were used as is. More detailed information on each sporicide
can be found in Sections 2.4.1 through 2.4.5.
2.3.1 Clorox® Concentrated Germicidal Bleach
This Clorox® Concentrated Germicidal Bleach is a newer product of the Clorox® Company that replaces
Regular Germicidal Bleach (Figure 2-2). Per the Clorox® Concentrated Germicidal Bleach label sodium
hypochlorite concentration in this product is 8.25% (compared to 6.15% in Regular Germicidal Bleach). Per
manufacturer's instructions, 1/2 cup of concentrated product shall be diluted in 1 gallon of water (1:32
dilution ratio). For disinfection, the recommended surface contact time should be at least five minutes. The
dilution ratios used in this study were lower; from 1:10 to 1:5, with a contact time of 10 minutes.
NOTE: The concentrated product that is being sold in a smaller and more ergonomic bottle
with a yellow band (Figure 2-2 a) is easily distinguishable from its non-concentrated
predecessor (Figure 2-2b). However, a clear recommendation should be made in the
instructional document resulting from this project to always use the new generation
(concentrated) product for preparation of liquid decontaminant solution.
CONCENTRATED
Blanqueador Germicida
Ku-ausi «u xiiuua k«<
PELIGR0:i • •
a. b.
Figure 2-2. Packaging of Clorox® Concentrated Germicidal Bleach (a) and Clorox® Germicidal Bleach (b)
9
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Assessment of Bacillus spore inactivation on
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cleaning products
Clorox® Splash-less Concentrated Bleach (The Clorox® Company, USA; Figure 2-3) was used to test the
effect of surfactant addition on sporicidal effectiveness of diluted bleach. The concentration of sodium
hypochlorite in this product is 3.985% (compared to 8.25% in Regular Concentrated Germicidal Bleach).
The diluted solution of bleach with surfactant to be used in decontamination testing had the same working
concentration of sodium hypochlorite as in diluted bleach without surfactant liquid decontaminant (see
characterization of bleach and bleach with surfactant solutions in Section 2.5). Note that COTS bleach
should be used before the expiration date as sodium hypochlorite concentrations will decrease with age.
Clorox® Splash-Less formulation has multiple surfactants in addition to sodium hypochlorite:
Cetyl betaine: surfactant - zwitterionic (amphoteric; cationic/anionic) surfactant - cleaning agent,
used as thickener and foam stabilizer.
Cocamidopropyl betaine (coco betaine): zwitterionic (amphoteric; cationic/anionic) surfactant - high-
foaming agent/foam booster, also used as thickener and foam stabilizer and viscosity enhancer;
has anti-static properties; moderate emulsifier.
Sodium xylene sulfonate: anionic surfactant - hydrotrope; solubilizes hydrophobic compounds in
aqueous solutions; is generally used to stabilize other ingredients in a cleaning product to maximize
effectiveness of the formula. It is also useful as a co-thickener (in combination with other
ingredients) in cleaning products.
10
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Assessment of Bacillus spore inactivation on
indoor surfaces using commercially-available
cleaning products
CONCENTRATED
SPLASH LESS L1/
Figure 2-3. Packaging of Splash-less Clorox® Concentrated Germicidal Bleach
Utilization of RTU-COTS bleach with surfactant product was preferred over addition of other commercially
available cleaning product/surfactant into bleach-based liquid sporicide formulations. Using a commercial
product is a "mistake-proof approach" and removes compatibility issues from preparation of a surfactant-
amended bleach formulation. Use of a COTS product is especially important if the surfactant-amended
bleach is eventually to be prepared by a person without specialized training for self-help decontamination
purposes.
Surfactants used in Clorox® Splash-less Concentrated Bleach (sodium xylenesulfonate, cocoamidopropyi
betaine, cetyl betaine) are commonly used in a variety of other cleaning products designed for efficient
removal of tough dirt and grease, e.g., Spic and Span®, which would be possible candidates for sporicidal
additions to bleach.
Recipes for preparation of bleach and bleach with surfactant solutions used in this study are given in Table
A-1 (Appendix A).
2.3.2 Lysol® Mold & Mildew Blaster
Lysol® Mold and Mildew Blaster with bleach is recommended for cleaning and disinfection purposes. The
ingredients are water, sodium hypochlorite (2%), sodium hydroxide, sodium chloride, laurylamine oxide, and
fragrance.
11
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Assessment of Bacillus spore inactivation on
indoor surfaces using commercially-available
cleaning products
Lysol® Mold and Mildew Blaster is sold in 28 ounce (oz.) and 32 oz. bottles and is available from major
national retailers at a price of approximately $0.10/fluid (fl.) oz. The current packaging of this product is
shown in Figure 2-4,
Figure 2-4. Packaging of Lysol® Mold & Mildew Blaster
Per manufacturer's information, this product is bactericidal, fungicidal, and virucidal.
2.3.3 Tilex® Mold & Mildew Remover
Tilex® Mold & Mildew Remover is intended to disinfect and kill mold and mildew on hard nonporous
surfaces. The ingredients are water, sodium hypochlorite (2.4%), sodium hydroxide, dimethicone/silica/
polyethylene glycol distearate antifoam, fragrance, lauramine oxide, and sodium silicate.
Tilex® Mold and Mildew Remover is sold in 16 oz. and 32 oz. bottles and is available from major national
retailers at a price of approximately $0.12/fl. oz. The current packaging of this product is shown in Figure 2-
5.
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Assessment of Bacillus spore inactivation on
indoor surfaces using commercially-available
cleaning products
Figure 2-5. Packaging of Tilex® Mold & Mildew Remover
2.3.4 Clorox® Clean-Up Cleaner + Bleach
Clorox® Clean-Up Cleaner + Bleach is a multipurpose cleaner for cleaning, disinfection, and deodorization of
most hard nonporous household surfaces. The ingredients are water, sodium hypochlorite (1.84%), sodium
hydroxide, dimethicone/silica/polyethylene glycol (PEG) distearate antifoam, fragrance, lauramine oxide,
and sodium silicate.
Clorox® Clean-up Cleaner + Bleach is sold in 32 oz. bottles and is available from major national retailers at a
price of approximately $0,09/fl. oz. It is also available in 64 oz. and 180 oz. refill bottles ($0.07-0.10/fl. oz.).
The current packaging of this product is shown in Figure 2-6.
13
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Assessment of Bacillus spore inactivation on
indoor surfaces using commercially-available
cleaning products
Clean-Up
Cleaner* Bleach
Umpiadot * CltKO
Figure 2-6. Packaging of Clorox® Clean-Up Cleaner + Bleach
2.3.5 Clorox® Disinfecting Bleach Foamer
Clorox® Disinfecting Bleach Foamer is used as a cleaning agent as well as an antimicrobial agent designed
specifically for bathroom surfaces. The ingredients are water, sodium hypochlorite (2.4%), sodium
hydroxide, dimethicone/silica/PEG distearate antifoam, fragrance, lauramine oxide, sodium silicate, and
alkyl dimethyl benzyl ammonium chloride (quaternary ammonium compounds or QUATS). Clorox®
Disinfecting Bleach Foamer is sold in 30 oz. bottles and is available from major national retailers at a price
of approximately $0.11/fl. oz. The current packaging of this product is shown in Figure 2-7.
14
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Assessment of Bacillus spore inactivation on
indoor surfaces using commercially-available
cleaning products
SPR**
EVE#*
y Spopr
, - Disinfecting
if Bleach Foamer
^ vj for the Bathroom
Faamleg Blescb Action, St rob Less'
Figure 2-7. Packaging of Clorox® Disinfecting Bleach Foamer
2.4. Characterization of Decontamination Solutions
The baseline values for pH, chlorine concentration (% sodium hypochlorite and free available chlorine
(FAC)) and temperature were established experimentally prior to testing via tripiicate measurements for
each liquid decontaminantto be tested, including RTIJ products. The importance of these parameters for
characterization of liquid sporicides is explained in Section 2.5.1 through 2.5.3.
The averages from these measurements were then used as the target threshold of FAC and pH for each
decontamination solution prepared in-house and used in ongoing assessments of quality assurance (QA)
objectives in Phase I (Section 6.2). Time of preparation of each batch of diluted bleach was also noted. For
Phase II, RTU-COTS products were used as is, after the initial testing of FAC, pH and temperature
parameters (Table 2-3).
Baseline characteristics (% sodium hypochlorite and pH as per Safety Data Sheet (SDS), product label and
values delivered experimentally) of decontaminants used in Phase I and Phase II are listed in Table 2-3.
15
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Assessment of Bacillus spore inactivation on
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cleaning products
Table 2-3. Concentration of Sodium Hypochlorite (%) and pH of Decontamination Solutions
Sporicide
% Sodium Hypochlorite
PH
SDS
Label
Tested
Value***
SDS
Tested
Value
Prepared in-house (Phase I testing)
Clorox® Concentrated Germicidal Bleach
1:10 dilution
0.50-1.5*
0.83*
0.67
>11*
11.4
Clorox® Concentrated Germicidal Bleach
1:5 dilution
1.0-3.0*
1.65*
1.37
>11*
11.6
Clorox® Splashless Concentrated Bleach
1:5 dilution equivalent
0.40-2.1**
1.65**
1.55
>11**
11.8
COTS (Phase II testing)
Lysol® Mold and Mildew Blaster
1.0-2.5
2.00
2.52
12.3-12.7
12.2
Tilex® Mold and Mildew Remover
O
I
cn
0
2.40
2.49
12.4-12.8
12.6
Clorox® Clean-Up Cleaner + Bleach
0
1
cn
0
1.84
2.11
12.4-12.8
12.6
Clorox® Disinfecting Bleach Foamer
0
1
cn
0
2.40
2.15
-12.2
12.6
* Full strength ClOPOX® Concentrated Germicidal Bleach concentration perSDS: 5-15%, per label: 8.3%; pH per SDS ~11.9
** Full strength C lOPOX® Concentrated Splashless Bleach concentration per SDS: 1-5%, per label: 3.985%; pH per SDS ~12.5
*** As determined byiodometric titration (n = 3)
Detailed results from characterization of decontamination solutions are given in Section 5-1 and Tables A-2
and A-3 (Appendix A).
2.4.1 Concentration of Sodium Hypochlorite (NaOCI) versus Free Available Chlorine and
PH
Sodium hypochlorite (NaOCI) is the active ingredient (oxidizing agent) in bleach. For consumer products,
the sodium hypochlorite strength is most commonly expressed as weight percent [wt%] of sodium
hypochlorite (the weight of the sodium hypochlorite per 100 parts of solution).
The definition most commonly used to describe the oxidizing power of bleach-based products used in the
decontamination research is grams per liter [g/L] of free available chlorine (FAC). FAC is any residual
chlorine that is available to react with sources of bacteria or other contaminants. FAC is the sum of all of the
chemical species that contain a chlorine atom in the 0 or +1 oxidation state and are not combined with
ammonia or other organic nitrogen. Some species of FAC that might be present in aqueous solutions are:
Molecular chlorine: CI2
Hypochlorous acid: HOCI
Hypochlorite: OCI"
Trichoride: Cb- a complex formed by molecular chlorine and the chloride ions (CI ) [11],
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Assessment of Bacillus spore inactivation on
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cleaning products
In addition to free available chlorine concentration, pH significantly changes relative effectiveness of bleach
as a disinfectant, since different species of chlorine ions are more prevalent at different pH levels. In the pH
range 6-9, HOCI and OCI- are the main chlorine species [11], Depending on pH level, the ratio of these two
free chlorine species changes (Figure 2-8 adapted from reference 11).
100
OCI
Cl2
HOCI
u
<
¦—
c
0 1 23456789 10 11 12
PH
Figure 2-8. Distribution of Free Chlorine Species in Aqueous Solutions (from reference 11)
Figure 2-8 shows that chlorine hydrolysis into HOCI is almost complete at pH < 4. Dissociation of HOCI into
OCI- begins at approximately 5.5 pH and increases dramatically thereafter. The formation of HOCL is
important because HOCI and OCI- do not have the same effectiveness as disinfectants. HOCI can be 80-
100% more effective as a disinfectant than OCh Optimum disinfection occurs at pH 5 to 6.5 where HOCI is
the prevailing species of free chlorine present. As pH rises above that level, the ratio shifts towards being
primarily OCh At pH 7.5, the ratio is about even. When the pH value rises to 8 or higher, OCI- is the
dominant species. Therefore, assuming the concentration of the CI2 species is constant, the higher the pH
of the solution rises above 5.5, the lower the oxidation capability and disinfecting power of the FAC [11], In
conclusion, knowing the FAC and pH of bleach solution is important to have a more complete picture of its
disinfecting power.
Results for test-specific pH and FAC measurements of decontamination solutions are given in Section 5.1
and in Table A-2 and A-3 (Appendix A).
2.4.2 Temperature
Sodium hypochlorite solutions have limited storage stability. Decomposition of sodium hypochlorite solutions
will occur due to the following two reactions [7]:
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Assessment of Bacillus spore inactivation on
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Transformation into chlorate: 3 NaOCI —> 2 NaCI + NaCICb
Release of oxygen: 2 NaOCI —> 2 NaCI + O2
In a good quality sodium hypochlorite solution, the chlorate decomposition pathway accounts for
approximately 90% of the total decomposition. Increasing temperature, hypochlorite concentration and ionic
strength (salt content) will increase the reaction rate of both reactions to about the same extent. UV light
(sunlight) also catalyzes both decomposition reactions. The decomposition of sodium hypochlorite is
minimized in the range of pH 12 to 13 (between 0.025 and 0.35% excess sodium hydroxide). Below pH 11,
the rate of formation of chlorate will increase dramatically. In the pH range of 13 to 13.5, there is only a
minor increase in the decomposition rate [7],
Bleach decomposition is dependent on temperature. For any given temperature, the higher the solution
strengths, the faster the bleach decomposes. Briefly, for every 10 °C increase in storage temperature, the
sodium hypochlorite will decompose at an increased rate factor of approximately 3.5 [7], The rate constants
(k2) of sodium hypochlorite decompositions with respect to strength and temperature are given in Table 2-4,
below [7],
Table 2-4. Rate Constants (k2) of NaOCI Decompositions with Respect to Strength and Temperature*
Temperature,
°C
Sodium Hypochlorite
15.89%
13.46%
10.82%
7.93%
4.74%
55
250
189
138
98.2
65.5
45
80.7
58.7
43.9
30.2
19.3
35
23.1
17.0
12.2
8.43
5.45
25
6.33.
4.68
3.22
2.19
1.58
15
1.65
1.15
0.80
0.53
0.30
*from reference [7]
In this study, the temperature of the decontamination solutions was monitored carefully to determine pot-life
of decontamination solution batches. A temperature increase greater than 5 °C was a threshold designating
that preparation of a new batch of the decontamination solution is needed (see Section 6.2 for details).
Results of temperature monitoring of diluted bleach solutions are given in Section 5.1.
2.5. Decontamination Procedure
For each decontamination test, sets of building material coupons were inserted into the test coupon holders
of the small spray test chamber (Figure 2-9) in a vertical position. Chamber dimensions are 4 ft high by 4 ft
wide by 4 ft deep, and it is designed to accommodate three 14 in x 14 in coupons at a time in horizontal or
vertical orientation. In this study, only the vertical orientation assembly was utilized. The chamber was
constructed of stainless steel with the exception of the front face and top that were clear acrylic plastic. The
acrylic door was fitted with three ports, one per coupon, to allow the insertion of the backpack delivery
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Assessment of Bacillus spore inactivation on
indoor surfaces using commercially-available
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system in the direct middle of each vertical coupon. In this study, the decontamination solution was applied
by hand, and the door was open during application to imitate real-life iow-tech decontamination (Figure 2-9).
After application of decontaminant, the chamber door was closed. The project personnel were wearing
personal chlorine gas monitors at all times. Chlorine emissions during applications of decontamination
agents were below the OSHA short-term exposure limit (STEL).
The reverse-pyramid design of the chamber bottom allowed for collection of runoff from the coupons during
the decontamination procedure through a central (3 inch in diameter) drain. The bottom of the chamber has
a 189 liter (L) (50 gallon) collection capacity.
Figure 2-9. Decontamination Chamber (Left) and Application of Diluted Bleach Using Rag
The chamber exhaust exits via a readily accessible connection to the facility's air handling system. Aerosol
samples were collected from the chamber exhaust duct using Via-Cell® BioAerosol Cassettes (Zefon
International, Inc., Ocala, FL, USA). The sampling point was eight diameters downstream and two
diameters upstream of any flow disruptions.
Decontamination procedural steps were as follows:
1. Inoculation of coupons
- Three to five (3-5) test coupons in Phase I and Phase II work, respectively, and three (3) positive
control coupons were inoculated with spores of B, atrophaeus via aerosol deposition using a
metered dose inhaler (MDI) and Aerosol Deposition Apparatus (ADA); details of the inoculation
procedure are described in Section 2,3.
- After aerosol deposition, spores were allowed to settle overnight.
2. Preparation of test chamber
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Assessment of Bacillus spore inactivation on
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cleaning products
- The test chamber was sterilized using pH-amended bleach solution; the pH-adjusted bleach (pAB)
solution used for cleaning surfaces in equipment in both the decontamination and microbiology
laboratories is prepared as a 1:10 dilution of bleach in deionized (Dl) water, pH-adjusted to ~6.8
using glacial acetic acid.
- Using the back sprayer, the interior surfaces of the test chamber were sprayed with pAB, and kept
wet with solution for 10 minutes, and then rinsed with Dl water.
- After rinsate drained from the chamber, the chamber valve was closed and the interior of the
chamber was wiped down with isopropyl alcohol or ethanol.
3. Verification of critical operational parameters of decontamination solutions
- Measurements of pH, FAC and temperature of bleach-based decontamination solution, or
- Measurements of pH and temperature for RTU products
o Results were recorded in a laboratory notebook.
4. Installation of aerosol sampling device in exhaust duct
- A Zefon Via-Cell® Bioaerosol Sampling Cassette (p/n VIA010) coupled with EPA Method 5 Dry
Gas meter box was used to collect air samples using the procedure described in Section 3.6
5. Installation of coupons in test chamber
- Coupons were aseptically installed in the test chamber in the vertical orientation.
- The position of each coupon was recorded.
6. Application of a prescribed decontamination sequence
In Phase I, bleach-based liquid decontaminant was applied using a sterilized rag or sponge wetted
in the bucket of diluted bleach solution. The rag used was a 12 in by 12 in cotton cloth (Walmart,
Durham, NC). The excess decontaminant was removed by gentle squeezing (wiping medium
remained well wetted; this step was only to avoid dripping-wet wiping media), then
decontamination solution was applied onto the coupon surface using a zigzag stroke technique.
The coupon cleaning (wiping) action started at the upper right corner of the coupon. Using a zigzag
stroke, the rag or sponge was firmly moved down and up the entire coupon surface. After
application of decontaminant, the wiping medium was double-bagged in sterile bags for
microbiological analysis.
In Phase II, RTU-COTS decontaminants were sprayed using the product-specific original spray
bottle as provided by manufacturer (i.e., RTU products were not transferred to any secondary
spray bottles or sprayers); each RTU-COTS solution will be applied onto the coupon surface until
the surface is visibly wet, starting at the upper right corner of the coupon. Spraying then continued
20
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Assessment of Bacillus spore inactivation on
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cleaning products
from right to left, then moved slightly downward and continued from left-to-right, then right-to-left,
until the entire coupon surface was covered with decontamination product.
- The time required for application of decontamination solution on each coupon was recorded.
Loading volumes of decontaminant are given in Section 5.2.
- The runoff of decontaminant was collected in a clean sterile carboy loaded with neutralizer (sodium
thidosulfate) that was placed under the drain of the chamber. The volume of neutralizer was
determined in a series of preliminary experiments for single set (three coupons) of porous and
nonporous material/wiping medium and/or decontamination solution combination.
- After a 10-minute processing time, the coupon surface was wiped using a new sterilized rag or
sponge wetted in sterile water; the coupon surface was wiped with a wetted wiping medium using
a zigzag stroke technique. The coupon water rinse (wiping) action always started at the upper right
corner of the coupon. Using a zigzag stroke, the cloth or sponge was firmly moved down and up
the entire coupon surface.
- The runoff of rinse water was collected into a separate clean sterile carboy loaded with neutralizer
that was placed under drain of the chamber. The volume of neutralizer was determined in series of
preliminary experiments using a single set (three coupons) of porous and nonporous
material/wiping medium used for water rinse of decontaminant combination.
7. Post-decontamination procedures
Immediately after the rinse step was completed, the collected runoff and rinsates were
characterized (volume, FAC, pH, temperature) and were taken for microbiological analyses.
Coupons were moved to designated storage cabinets and allowed to dry overnight.
- Visual inspection of the decontaminated building surfaces was then performed. Changes in color,
reflectivity, and roughness were assessed qualitatively and observations were made and
documented in the laboratory notebook and by digital photographs when surface change was
observable.
- After completion of the entire decontamination procedure, coupons were then moved to designated
Coupon Storage Cabinets (non-contaminated control coupons were transferred to the Blank
Coupon Cabinet, non-decontaminated coupons were transferred to the Positive Coupon Cabinet,
decontaminated coupons were moved to Test Coupon Cabinet) and allowed to dry overnight.
Coupons positions were recorded in the Coupon Tracker (Appendix).
8. Sampling and transfer of samples to the HSRP RTP Biocontaminant Laboratory for microbiological
analysis
- On the following day, coupons were sampled using sampling techniques described in Section 3.2.1
and transferred to the laboratory for microbiological analysis. Samples were transferred in sterile
primary independent packaging within sterile secondary containment containing logical groups of
samples for analysis. All samples were accompanied by a completed chain of custody (COC) form.
9. Determination of decontamination efficacy
- After the HSRP RTP Biocontaminant Laboratory has performed a quantitative assessment of
viable spores for each type of sample, the determination of surface decontamination efficacy will
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Assessment of Bacillus spore inactivation on
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be performed (comparison of viable spore concentrations from positive controls and test coupons;
see Section 4 for details).
2.5.1. Test Matrix
Table 2-5 and Table 2-6 show the test matrix for Phase I and Phase II, respectively. The Phase I test matrix
was developed as the tests progressed, based on the results (decontamination efficacy, LR obtained; see
Figure 5-4 and 5-6 for details). The Phase II test matrix used processing time and rinse wiping medium
selected after obtaining Phase I results (i.e., 10 minutes and cotton rag for rinse water application).
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Assessment of Bacillus spore inactivation on
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Table 2-5. Phase I Test Matrix
Test ID
Material Type
Decontamination Agent
Application Mode
of Decontaminant
Application Mode
of Rinse Water
Processing time
1
Finished wood flooring
Bleach 1:10
Rag
Rag
10 minutes
2
Glass
Bleach 1:10
Rag
Rag
10 minutes
3
Painted wallboard
Bleach 1:10
Rag
Rag
10 minutes
4
Grouted ceramic tile
Bleach 1:10
Rag
Rag
10 minutes
5
Finished wood flooring
Bleach 1:5
Rag
Rag
10 minutes
6
Glass
Bleach 1:5
Rag
Rag
10 minutes
7
Painted wallboard
Bleach 1:5
Rag
Rag
10 minutes
8
Grouted ceramic tile
Bleach 1:5
Rag
Rag
10 minutes
17
Finished wood flooring
Bleach 1:5
Sponge
Sponge
10 minutes
18
Glass
Bleach 1:5
Sponge
Sponge
10 minutes
19
Painted wallboard
Bleach 1:5
Sponge
Sponge
10 minutes
20
Grouted ceramic tile
Bleach 1:5
Sponge
Sponge
10 minutes
21
Finished wood flooring
Bleach with surfactant 1:5
Rag
Rag
10 minutes
22
Glass
Bleach with surfactant 1:5
Rag
Rag
10 minutes
23
Painted wallboard
Bleach with surfactant 1:5
Rag
Rag
10 minutes
24
Grouted ceramic tile
Bleach with surfactant 1:5
Rag
Rag
10 minutes
25
Finished wood flooring + grime
Bleach with surfactant 1:5
Rag
Rag
10 minutes
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Assessment of Bacillus spore inactivation on
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Table 2-6. Phase II Test Matrix
Test ID
Material Type
Decontamination Agent
Application Mode
of Decontaminant
Application Mode
of Rinse Water
Processing time
1
Finished wood flooring
Lysol® Mold and Mildew Blaster
Spray
Rag
10 minutes
2
Glass
Lysol® Mold and Mildew Blaster
Spray
Rag
10 minutes
3
Painted wallboard
Lysol® Mold and Mildew Blaster
Spray
Rag
10 minutes
4
Grouted ceramic tile
Lysol® Mold and Mildew Blaster
Spray
Rag
10 minutes
5
Finished wood flooring
Tilex® Mold and Mildew Remover
Spray
Rag
10 minutes
6
Glass
Tilex® Mold and Mildew Remover
Spray
Rag
10 minutes
7
Painted wallboard
Tilex® Mold and Mildew Remover
Spray
Rag
10 minutes
8
Grouted ceramic tile
Tilex® Mold and Mildew Remover
Spray
Rag
10 minutes
9
Finished wood flooring
Clorox® Clean-Up Cleaner + Bleach
Spray
Rag
10 minutes
10
Glass
Clorox® Clean-Up Cleaner + Bleach
Spray
Rag
10 minutes
11
Painted wallboard
Clorox® Clean-Up Cleaner + Bleach
Spray
Rag
10 minutes
12
Grouted ceramic tile
Clorox® Clean-Up Cleaner + Bleach
Spray
Rag
10 minutes
13
Finished wood flooring
Clorox® Disinfecting Bleach Foamer
Spray
Rag
10 minutes
14
Glass
Clorox® Disinfecting Bleach Foamer
Spray
Rag
10 minutes
15
Painted wallboard
Clorox® Disinfecting Bleach Foamer
Spray
Rag
10 minutes
16
Grouted ceramic tile
Clorox® Disinfecting Bleach Foamer
Spray
Rag
10 minutes
24
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Assessment of Bacillus spore inactivation on
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3. Sampling and Analytical Procedures
3.1. Types of Samples
Four major classes of operational samples were collected:
Surface samples (see Section 3.2.1 for sampling procedures). Each test listed in Tables 2-5 and 2-6
included surface samples from each material collected in triplicate (Phase I) orquintuplicate (Phase
II), positive control surface sampling in triplicate (Phase I and Phase II), and procedural blank
surface samples (one per each Phase I and Phase II test).
Liquid effluent samples (see Section 3.2.2 for sampling procedures). These samples were collected
to assess the potential for viable spores to be washed off the surfaces. Composite samples of liquid
waste (runoff of bleach solution and water rinsate) were collected and analyzed quantitatively for
each test.
Solid waste samples (see Section 3.2.3 for sampling procedures). Post-decontamination solid
waste (rags and sponges) were important for understanding the fate of the spores and the transfer
process as well as for determination of safe disposal of post-decontamination waste.
Aerosol samples (see Section 3.2.4). Via-Cell® BioAerosol Cassette samples were collected during
each decontamination and procedural blank test. These samples were used to estimate the
occurrence and magnitude of fugitive emissions of viable B. atrophaeus spores during the
decontamination process.
Additional QA/QC samples (see Section 3.3) included:
Swab (sterility check) samples
Material samples/field samples
Decontamination solution samples.
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Assessment of Bacillus spore inactivation on
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3.2. Sampling Procedures
Within a single test, surface sampling of the materials was completed for all procedural blank coupons first
before sampling of any test material was performed. Surface sampling was done by wipe sampling in
accordance with the protocols documented below. Prior to the sampling event, all materials needed for
sampling were prepared using aseptic techniques. The materials specific to each protocol are included in
the relevant sections below. The general sampling supplies were sterile or sterilized/disinfected for each
sampling event. To ensure the integrity of samples and to maintain a timely and traceable transfer of
samples, an established and proven chain of custody was strictly adhered to for each test.
3.2.1 Wipe Sampling
Wipe sampling is typically used for small sample areas and is effective on nonporous smooth surfaces such
as ceramics, vinyl, metals, painted surfaces, and plastics. The general approach is that a moistened sterile
non-cotton pad is used to wipe a specified area to recover bacteria, viruses, and biological toxins.
Sampling kits for wipes were prepared as specified in MOP 6568 (see Appendix B). All laboratory surfaces
intended for use during sampling were wiped with Dispatch® bleach wipes (Clorox, Oakland, CA). Precut
50.8 cm by 50.8 cm (20 in by 20 in) sheets of absorbent bench liner were used to cover all work surfaces,
replaced after each phase of a test (e.g., coupon contamination is considered one phase, decontamination
another, and surface sampling a third). Sampling was conducted on only one coupon at a time. One coupon
was moved from the Decontaminated Coupon Cabinet (test coupons), Test Coupon Cabinet (positive
controls), or Procedural Blank Coupon Cabinet (procedural blanks) to the sampling space located
immediately outside (to the front) of each cabinet. All coupons were placed horizontally for sampling,
regardless of their orientation during the decontamination.
Within a single test, surface sampling of the coupons was performed starting with coupons from the lowest
level of contamination and ending with the highest level of contamination (i.e., all procedural blank coupons
first, followed by all test coupons, and then all positive control coupons).
Surface sampling was performed by wipe sampling in accordance with the protocols described in MOP
3144 (Appendix B). The surface area for all samples was 1175.8 cm2 (1.3 ft2). A template was used to cover
the exterior 0.635 cm (0.25 in) of each coupon, leaving a square (34.29 cm by 34.29 cm) exposed for
sampling for all coupons. The outer 0.635 cm of each coupon was not sampled to avoid edge effects.
A sampling material bin was stocked with all appropriate items for each sampling event. The bin contained
enough wipe sampling kits to accommodate all required samples for the specific test. An additional kit was
also included for backup. Enough gloves and bleach wipes needed to complete the test were available.
Templates (35.6 cm by 35.6 cm (14 in by 14 in)) with an interior opening of 34.3 cm by 34.3 cm (13.5 in by
13.5 in) were wrapped in aluminum foil and packaged in sterile autoclave-safe bags (autoclave-sterilized
using a one hour gravity cycle, 10 templates per bag) and transported with the original sterile coupons
(concrete and stainless steel procedural blanks). These bags of templates were also included with the
sampling kits. A sample collection bin was used to transport samples back to the Microbiology Laboratory.
The exterior of the transport container was decontaminated by wiping all surfaces with a Dispatch® bleach
wipe prior to transport from the sampling location to the HSRP RTP Biocontaminant Laboratory for analysis.
The samples were stored at 4 ± 2 °C until processed.
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Assessment of Bacillus spore inactivation on
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3.2.2 Liquid Effluent Sampling
Liquid effluents from the decontamination process were collected using the central drain of the test
chamber. Liquid effluents were collected into pre-weighted sterilized containers (amber glass bottles)
preloaded with neutralizer- 2N STS (sodium thiosulfate) solution. Since neutralizer was added to the
collection vessel before collection of liquid samples, the active (sporicidal) ingredient was neutralized as
sample was being collected (not after collection). The runoffs and rinsates were collected as one composite
sample for each test set. For each test, the total mass of liquid effluents collected was recorded for
comparison of the collection vessel final weight versus the initial weight value. After collection, triplicate 100
mL aliquots were taken via aseptic technique using a new 100 mL sterile serological pipette and sterile 4 oz.
container. The liquid effluent aliquots were then double-contained in sterile bags and transported to the
HSRP RTP Biocontaminant Laboratory for analysis. The samples were stored at 4 ± 2 °C until processed.
3.2.3 Solid Waste Sampling
Once the application of bleach solution in Phase I was finished, the wiping medium (rag or sponge) was
aseptically collected to 10" x 15" Twirl Em bag (Fisher cat no. 01 -002-53) preloaded with neutralizer. This
primary contained sample was then weighed, placed in a secondary sterile 10" x 15" bag and transported to
the HSRP RTP Biocontaminant Laboratory for microbiological analysis. Samples were refrigerated at 4 ± 2
°C until processed.
3.2.4 Aerosol Sampling
Aerosol samples were collected from the chamber exhaust duct (Figure 3-1) using Via-Cell® BioAerosol
Cassettes®. The sampling point was eight diameters downstream of and two diameters upstream of any
flow disruptions.
Figure 3-1. Spray Chamber Exhaust Duct (white arrow shows the sampling point location)
A 4-in diameter galvanized duct that is 44 in long was attached to the chamber to allow for precise flow
measurements and sampling. The duct was attached to the chamber using a coupling and a 90-degree
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Assessment of Bacillus spore inactivation on
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elbow. The sampling port was located 32 in (8 diameters) downstream from the 90-degree elbow that is
connected to the chamber and 12 in (3 diameters) from the bend in the flexible duct that connects to the
main exhaust (the sampling point location is indicated with a white arrow symbol on Figure 3-1). The
exhaust air was sampled using a Via-Cell® and dry meter box operated according to MOP 3155. After
sampling, the Via-Cell® cassette was placed into the special foil bag (provided by the manufacturer) and zip-
closed. The red safety seal label was applied over the top of the foil bag opening to ensure sample integrity
until analysis. The primary bag containing the cassette was then placed inside a pre-labeled 5.5 in x 15 in
sterile bag for secondary containment. For each test day, there was a field blank (plain, unused Via-Cell®
cassette). The samples were stored at 4 ± 2 °C until processed.
3.3. QA/QC Samples
3.3.1 Swab Samples
MOP 3135 (Appendix B) was used for collecting swab samples. The general approach was to use a
moistened swab to wipe a specified area to recover bacterial spores. Swab samples were collected from all
decontamination procedure equipment before and after use. Swab samples were also collected from
materials before use as sterility checks.
3.3.2 Material Samples
Material samples provided information on the level of contamination possibly present during sampling due to
contaminated materials, e.g., unused wipe kits and unused Via-Cell® cassettes were transferred from/to or
to the HSRP RTP Biocontaminant Laboratory for microbiological analysis. The samples were referred to as
unexposed field blank samples. There were also blank plating of microbiological supplies to check for
sterility of supplies used in dilution plating.
3.3.3 Decontamination Solution Samples
Samples of decontamination solutions prepared in-house (Phase I) were evaluated for critical
parameters (pH, FAC and temperature) prior to each test. The RTU-COTS products were
experimentally evaluated for pH, FAC and temperature prior to start of Phase II testing, then pH and
temperature were recorded prior to each decontamination test.
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Assessment of Bacillus spore inactivation on
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4. Determination of Sporicidal Effectiveness
The sporicidal effectiveness (efficacy) of a decontamination technique is a measure of the ability of the
method to inactivate and/or remove the spores from a contaminated material surface (i.e., represented by
coupons in this study). It is evaluated by measuring the difference in the logarithm of the measured colony
forming units (CFU) before decontamination (determined from sampling the positive control coupons) and
after decontamination (determined from sampling the test coupons) for the same type of material. The
number of viable spores was measured as CFU and reported as a log reduction on the specific material
surface as defined in Equation 3-1.
Nc Nt
2 log (CFUCJt) ^ log (CFUSJt)
77=— — (3-1)
'' Nc Ns
where:
Surface decontamination effectiveness; the average log
Tj = reduction of spores on a specific material surface (surface
material designated by /)
Nc The average of the logarithm (or geometric mean) of the
X! \og(CFUc k) _ number of viable spores (determined by CFU) recovered on the
— control coupons (C indicates control and Nc is the number of
Nc control coupons)
The average of the logarithm (or geometric mean) of the
number of viable spores (determined by CFU) remaining on the
surface of a decontaminated coupon (S indicates a
decontaminated coupon and Ns is the number of coupons
tested).
2 log (CFUst)
k=1
N.
When no viable spores were detected, a value of 0.5 CFU was assigned to the maximum plated volume to
determine the detection limit for CFUs.k, and the efficacy was reported as greater than or equal to the value
calculated by Eqn.3 -1.
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Assessment of Bacillus spore inactivation on
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The standard deviation of the average log reduction of spores on a specific material (r|i) is calculated by
Eqn. 3-2:
SD.=
N,
I(*
k= 1
Ns-1
(3-2)
where:
SD,
Standard deviation of r|i, the average log reduction of spores on
a specific material surface
71,
The average log reduction of spores on a specific material
surface (surface material designated by /)
Xk =
The average of the log reduction from the surface of a
decontaminated coupon
and,
Ns = Number of test coupons of a material surface type.
N,
X((log(OT',)-log(CFŁ/,.,))
xt =
k=l
Ns
(3-3)
where:
Xlog(CfI/Cll)
log(CFt/c) =
k=l
Represents 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 = test coupon number and Ns is the
number of test coupons)
CFUsk =
Number of CFU on the surface of the kth decontaminated
coupon
Ns
Total number (1 ,k) of decontaminated coupons of a
material type.
The surface log reduction, as calculated in accordance with Equation 3-1, represents the effectiveness of
the decontamination in mitigating the contamination on selected building materials. The overall
decontamination is a cumulative effect of inactivation of the spores resulting from oxidative stress after
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Assessment of Bacillus spore inactivation on
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application of a bleach-based sporicide or due to physical removal of spores from the material via wiping
action. For physical removal, viable spores may either remain in the liquid waste or be re-aerosolized.
Understanding the ultimate fate of the spores, not just the surface log reduction, is critical to recognizing the
utility or appropriate implementation of the decontamination process. Process parameters (as well as the
general nature of microbiological sampling) will not allow exact accounting of the fate of spores, or closing of
the microbiological loading mass balance. However, the quantitative measurements of spores in post-
decontamination waste and exhaust air are good indications of ultimate fate of viable spores. For the liquid
and solid samples, the results are reported as total CFU per sample. The test-specific re-aerosolization
rates are reported as CFU per volume of exhaust air sampled.
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Assessment of Bacillus spore inactivation on
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5. Results and Discussion
The primary objective of this study was to evaluate the efficacy of simple "self-help" methods for
decontamination of building material surfaces using bleach-based decontamination solutions (prepared in
house and commercially available products) basic tools expected to be easily accessible at residential
dwellings (i.e., cleaning rags and sponges).
In addition to reduction of contamination from material surfaces, the fate of the spores was also considered
important information for selection of the most effective decontamination procedure. The method that offers
a satisfactory decontamination efficacy (> 6 LR), can be then translated to an optimized method (method
manual, method tutorial) that is easily understandable to the general public/responders without specialized
training. Any users of the products described in this report shall follow PPE recommendations listed on the
label of the respective products.
This section discusses the results of characterization of decontamination solutions (Section 5.1), operational
differences between individual decontamination procedures tested (Section 5.2) and the result of the
determination of decontamination efficacy for each method/material combination (Section 5.3). The
procedure-specific ultimate fate of the spores is discussed in Section 5.4.
5.1. Characterization of Decontamination Solutions
5.1.1 FAC Concentration
Concentration of FAC in the working decontamination solution was measured via by sodium thiosulfate/
potassium iodide titration using a commercial digital titrator (Hypochlorite (Bleach) HACH Test Kit, Model
CN-HRDT; Cat No. 26871-00, Hach Company, Loveland, CO, USA). In Phase I, samples were taken from
the solution container In Phase II samples were taken from the RTU-COTS product spray-bottles.
The concentration of free available chlorine in freshly prepared batches of diluted bleach solution in Phase I
experiments was 6,600 ± 180 ppm for 1:10 bleach solution (n = 7), 14,000 ± 280 ppm for 1:5 bleach solution
(n = 11), and 15,600 ± 670 ppm for 1:5 bleach with surfactant solution (Figure 5-1). Test-specific data for in-
house prepared decontamination solutions are given in Table A-2 (Appendix A). The FAC, pH, and
temperature were measured prior to testing and these are referred to as the initial assessments in Figures
5-1 through 5-3. These parameters were also measured prior to the decontamination tests and these are
shown as ongoing assessments in Figures 5-1 through 5-3.
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Assessment of Bacillus spore inactivation on
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FAC [ppm]
18,000
15,000
12,000
9,000
6,000
3,000
1
1
1
1
1
1
1
1
*
1
¦
1
1
1
1
1
1
1
1
m m
¦ ¦ ~ ~
¦—
¦ ¦
¦ ¦
u m ¦ ¦
1-1
¦
lj u
1:5 bleach with surfactants
1:5 bleach
¦
¦
II
II
~
~
m
~LHJ initial
assessments
" ongoing assessments
i:iu bleach
maxiumum threshold
throchnlrl
minimum threshold
Figure 5-1. FAC of Diluted Bleach Solutions Prepared In-House (Phase I)
The FAC concentration in RTU-COTS products was 22,100 ± 1950 ppm. Product-specific FAC
concentrations fordecontaminants used in Phase II experiments are given in Table A-3 (Appendix A).
5.1.2 pH and Temperature
The pH and temperature measurements of decontamination solutions were performed using an Oakton pH
meter equipped with a pH probe and a thermocouple probe (Oakton pH 5+, OAKTON Instruments, Vernon
Hills, IL, USA).
The pH of freshly prepared batches of diluted bleach solution, as measured with the Oakton pH meter, in
Phase I experiments was 11.4 ± 0.01 for 1:10 bleach solution (n = 7), 11.4 ± 0.17 for 1:5 bleach solution (n
= 11), and 12.1 ±0.01 for 1:5 bleach with surfactant solution (Figure 5-2). Test-specific data for in-house
prepared decontamination solutions are given in Table A-2 (Appendix A).
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Assessment of Bacillus spore inactivation on
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14
13
12
11
10
9
8
7
Figure 5-2. pH of Diluted Bleach Solutions Prepared In-House (Phase I)
Average temperature of freshly prepared batches of diluted bleach in Phase I of this study was 22.7 °C ±
1.2 °C. Test-specific data are shown in Figure 5-3 and in Table A-2 (Appendix A). Even though all
experiments in this study were performed in the environment-controlled laboratory, the temperature
variations of bleach solutions shown some minimal variation between batches prepared during colder and
warmer weather conditions - batched prepared during the February-May timeframe (initial assessment
tests, 1:10 dilution tests and majority of 1:5 dilution tests) had a post-mixing temperature of < 23 °C, batches
prepared in June-August (latest four tests for 1:5 dilution bleach and all tests for 1:5 bleach with surfactants),
had post-mixing temperature of > 23 °C (Figure 5-3). For RTU-COTS solutions used in this study
temperature of decontaminants was 24.8 °C ± 1.2 °C (Table A-3, Appendix A).
PH
A A . . A
\ / A /
t A A A A
¦
i
A A A A
AAA
S\
/
1:10 bleact
1:5 blea
ch
AAA initial assessments
ongoing assessments
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Assessment of Bacillus spore inactivation on
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cleaning products
27.0
26.0
25.0
24.0
23.0
22.0
21.0
20.0
19.0
18.0
17.0
16.0
15.0
Figure 5-3. Temperature of Diluted Bleach Solutions Prepared In-House (Phase I)
5.2. Application Characterization
The critical procedural steps of decontamination - average surface loading volume of decontaminant (in
milliliters (mL) per coupon, [ml_/coupon]), average application time per coupon (seconds per coupon
[sec/coupon]) and average processing time (in minutes per test [min/set]) were controlled and documented
for each test.
5.2.1. Average application time and surface loading volumes and
Loading volumes were determined gravimetrically by weighing the wetted and expended (post-
decontamination) wiping medium. The determined decontaminant loading rates varied significantly (average
34 mL per coupon, standard deviation (SD) = 17.3 mL, relative standard deviation (RSD) = 51%) between
Phase I tests when solution was applied using a rag or a sponge (Table A-2). The reason for this variability
is probably the variation in strength when the wiping medium was pressed against the material surface.
There was no obvious correlation between the surface loading (volume of decontaminant applied per
coupon) and decontamination efficacy within the variation of the loading used in this study - samples with
the highest average log reduction values (LR>7, e.g. test 21 and 22) were decontaminated on days with low
to average surface loadings (27.1 ± 9.4 mL and 42 ± 4.1 mL per coupon, respectively; see Table A-2 and C-
1 in Appendix A and C). Tests that had the lowest average log reduction values (LR<6) for generally
Temperature [°C]
• •
• • » » •
/
—-
/
\
O o O
}
|
V O o o
\
•
•
•
•
til
o
c
o
•
•
•
t
1:5 bleach w
rith surfactants
1:10 bleach
1:5 bleach
ooo
initial assessments
•••
ongoing assessments
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Assessment of Bacillus spore inactivation on
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efficacious 1:5 bleach concentration (test 18 and 19) had above-average or high surface loadings of
decontaminant (85.3 ± 42.2 mL and 34.7 ± 23.5 mL per coupon, respectively; Table A-2 and C-1 in
Appendix A and C). A more systematic study is needed to investigate how the manual application pressure
(and consequent mechanical removal of spores) versus amount of decontamination solution applied to the
surface can affect the decontamination efficacy.
The Phase II replicate applications were very consistent between each type of RTU-COTS decontamination
products: 20 ± 3.8 mL for Lysol® Mold & Mildew Blaster, 17.4 ± 0.86 mL for Tilex® Mold & Mildew Remover,
19.7 ± 4.4 mL for Clorox® Clean-up Cleaner + Bleach, 41.6 ± 1.83 mL Clorox® Disinfecting Bleach Foamer)
(Table A-3, Appendix A). The higher loading observed for Clorox® Disinfecting Bleach Foamer is probably
due to the foaming characteristics of this product (more solution had to be sprayed for the surface to appear
to be "visibly wet" - surface looks wet after the foam collapses). There was no correlation between amount
of RTU-COTS sprayed onto surface and decontamination efficacy (Table A-3 and C-2 in Appendix A and C)
- Phase II tests all had decontamination efficacies greater than 6 logs.
The average time need for application of the decontamination solution either via wiping medium (Phase I) or
via spraying (Phase II) was approximately 8-20 seconds per 14x14 inch coupon (Table A-2 and A-3,
Appendix A), and varied very slightly (RSD<10%) between decontamination product and material type
combinations, suggesting that the proposed decontamination methods are quickly and reproducibly
deployable, and that obtaining a visible coverage with decontaminant (wet surface) is a good indicator of the
successful application of the decontaminant.
5.3. Decontamination Results
5.3.1. Inoculation Results (Positive Controls)
In this study, test and positive control coupons of porous and nonporous building materials have been
inoculated with aerosol-deposited Bacillus spores at 1 E7. Test specific results for positive controls
(CFU/sample) are given in Table C-1 and C-2 (Appendix C). The average CFU recovered from positive
control coupons in Phase I was 2.3 E7, with an average coefficient of variance of 25% (Table C-1, Appendix
C). In Phase II, the average surface loading was 4.2 E7, with an average coefficient of variance of 29%
(Table C-2, Appendix C). The average CFU recovery values for the positive controls indicate that the initial
spore loading allowed decontamination experiments with a target sporicidal effectiveness > 6 log reduction
(LR). The average variability suggests a reproducible surface-sampling of spores. Only three tests had a
variation in positive control CFU higher than 50% percent for some tests, probably because of natural
variations in the coupon surface roughness. Presumably, test coupon sampling was similarly affected,
hence the variability in the initial loading/CFU recovery of positive controls was not considered a source of
methodical bias in calculations of the log reduction values for tests with higher (>50%) variability in CFU
recovery.
5.3.2. Log Reduction Results
Tables C-1 and C-2 in Appendix C and Figure 5-4 and 5-5 summarize decontamination product-specific log
reduction values. These data suggest that bleach-based liquid sporicide provides greater than 6 log
reduction of spore loading on most of the common building materials tested, at concentrations of
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Assessment of Bacillus spore inactivation on
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hypochlorite of approximately 1.5% or greater, using either in-house bleach solutions applied via cotton rag
processed for 10 minutes and wiped with water using clean cotton rag, or RTU-COTS applied via spraying,
processed for 10 minutes and wiped with water using a clean cotton rag. The sponge application tested in
Phase I seemed to be less efficacious than a cotton rag application, perhaps due to less scrubbing action
offered by the soft sponge.
Material-specific results (Figures 5-6 and 5-7) suggest that efficient decontaminants (bleach dilution 1:5,
bleach with surfactant dilution 1:5, all bleach-based RTU-COTS tested) performed similarly, independent of
the type of material treated. The average lower LR observed for glass and bleach dilution 1:10 applied by
sponge is due to the presence of an outlier that has surface decontamination efficacy almost 2 logs below
an average LR for this test (LR=4.1 compared to LR of 7.7 and 5.3 for the remaining two samples in a
subset).
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Assessment of Bacillus spore inactivation on
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Surface decontamination efficacy [LR +/- SD]
Finished wood flooring Glass ¦ Painted wallboard Grouted ceramic tile ¦ Finished wood flooring + grime
5f8
¦¦
Bleach 1:10 via rag, 10 min
Bleach 1:5 via rag, 10 min
Bleach 1:5 via sponge, 10 min
Bleach 1:5 with surfactants via rag, 10 min
Figure 5-4. Decontaminant Specific LR values for Phase I (values in green - average target decontamination
efficacy of LR>6)
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Assessment of Bacillus spore inactivation on
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Surface decontamination efficacy [LR +/- SD]
9.0
I Finished wood flooring Glass ¦ Painted wallboard Grouted ceramic tile
Lysol Tilex Clorox Cleanup + Bleach Clorox Bleach Foamer
Figure 5-5. Decontaminant Specific LR values for Phase II (values in green - average target decontamination
efficacy of LR>6)
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Assessment of Bacillus spore inactivation on
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Surface decontamination efficacy [LR +/- SD]
10.0
Finished wood flooring ¦ Finished wood flooring + grime Glass Grouted ceramic tile Painted wallboard
9.0
?-8 7,7
Figure 5-6. Material Specific LR Values for Phase I (values in green - average target decontamination efficacy of
LR>6)
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Assessment of Bacillus spore inactivation on
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Surface decontamination efficacy [LR +/- SD]
Finished wood flooring Glass Grouted ceramic tile Painted wallboard
7.8 7.8 7.8 7-9
7.5 , 7,5 1
8.0
7.8 7.8 7^8
Figure 5-7. Material Specific LR Values for Phase II (values in green - average target decontamination efficacy of
LR>6)
A comparison between Phase I and Phase II results suggest that RTU-COTS offer superior
decontamination efficacy (» 6 LR) for all building materials tested. The efficiency of RTU-COTS is not
statistically significant among tested products (one-way ANOVA, p-value=0.58, F=0.666 LR). This step is
also important in terms of method compatibility with bleach-sensitive materials (e.g., stainless steel). Results
show a 1-2 Log level in rinsates for Phase 1 and Phase II.
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Assessment of Bacillus spore inactivation on
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5.4. Fate of Spores
The exposure to B. anthracis re-aerosolized during the decontamination operations is a potential source of
exposure to personnel performing cleaning. The lethal dose where 50% of the population would be killed
(LD50) for B. anthracis in humans is not definitively known, but based on animal data is estimated to be in
the range of 4,100-10,000 inhaled spores. The survival of spores in the post-decontamination waste brings
into consideration other possible routes of accidental exposure of the person performing decontamination
through cuts and scrapes, i.e., via a cutaneous pathway.
5.4.1. Exhaust air concentrations
The composite air samples were taken using ViaCell® BioAerosol cassettes during application and
exposure (processing) and rinsing phases of each decontamination test (i.e., results in Table 5.5.are for
spores re-aerosolized from three or five coupons, respectively). Table 5-1 shows CFU detected for each
type of material decontaminated in Phase I and Phase II (test specific results are given in Table C-1 and C-2
in Appendix C). These results suggest that decontamination procedures in Phase I and II have similar spore
re-aerosolization rates. The highest number of viable spores detected in all air samples was approximately
10 CFU per cubic foot (Table C-1 and C-2, Appendix C). Although this value appears to be low, it should be
emphasized that it represents the re-aerosolized spores from five 14x14 inch coupons (total area of
approximately 6.0 ft2). The decontamination cycle phase-specific aerosolization rates were not
systematically investigated in this study, but the initial scrubbing or spraying stage is presumably the phase
when most of the spores are aerosolized.
Table 5-1. Air Sample Results
Material type (n=number of tests)
Phase I
Phase II
CFUffi3<±SD)
Finished wood flooring (n = 4)
7.9E-01 (±1 4E+00)
3.5E+00 (±3.2E+00)
Grimed finished wood flooring (n = 1)
3.84+00
NA*
Glass (n = 4)
1,3E+00(±1.7E+00)
4.0E+00 (±4.4E+00)
Painted wallboard (n = 4)
2.3E+00 (±3.4E+00)
1.2E+00 (±1.6E+00)
Grouted ceramic tile (n = 4)
1.0E+00 (±1.0E+00)
2.0E+00 (±3.2E+00)
* not applicable - grimed surfaces were not tested in Phase II
5.4.2. Liquid waste concentrations
Tables 5-2 and 5-3 show CFU recovered from the liquid waste (run-off and rinsate) samples; test specific
results are given in Table C-1 and C-2 (Appendix C).
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Assessment of Bacillus spore inactivation on
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cleaning products
Table 5-2. Run-off Samples Results
Material type (n = number of tests)
Phase I
Phase II
CFU/sample<± SD)
Finished wood flooring (n = 4)
1.3E+03 (±1 1E+03)
2.3E+02 (±4.5E+02)
Grimed finished wood flooring (n = 1)
6.6E+01
NA*
Glass (n = 4)
1.1E+03 (±1.4E+03)
6.5E+00 (±6.7E+00)
Painted wallboard (n = 4)
2.6E+03 (±2.8E+03)
5.3E+00 (±5.3E+00)
Grouted ceramic tile (n = 4)
1.8E+03 (±3.2E+03)
4.0E+00 (±6.3E+00)
* not applicable - grimed surfaces were not tested in Phase II
Table 5-3. Rinsate Sample Results
Material type (n = number of tests)
Phase I
Phase II
CFU/sample(± SD)
Finished wood flooring (n=4)
1.2E+01(±1.9E+01)
3.2E+01 (±4.9E+01)
Grimed finished wood flooring (n=1)
3.6E+01
NA*
Glass (n=4)
5.6E+01(±1.4E+01)
9.7E+00 (±6.9E+00)
Painted wallboard (n=4)
1,2E+02(±2.2E+02)
2.4E+00 (±2.9E+00)
Grouted ceramic tile (n=4)
1.4E+01(±2.1E+01)
3.4E+00 (±3.4E+00)
* not applicable -grimed surfaces were not tested in Phase II
The average number of spores detected in the Phase I liquid waste was at about an average 3 log level in
runoff samples and an average 1-2 log level in rinsate samples. In Phase II, the liquid waste was less
contaminated, at an average 1-2 log level for runoff and average 0.5-1 log level in rinsates. These results
were anticipated, since the Phase I procedures - with one extra step that involves mechanical
wiping/scrubbing - were expected to result in a greater rate of physical removal of spores/higher spore
transfer to the rinsate. In real-life decontamination scenarios, runoff and rinsate would not be neutralized,
hence the sporicidal action of the decontaminant would have continued, resulting in reduction or complete
deactivation of biological burden in the waste.
5.4.3. Solid waste concentrations
Table 5-4 shows CFU recovered from the post-decontamination solid waste samples (expended rags and
sponges); test specific results are given in Table C-1 and C-2 (Appendix C).
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Assessment of Bacillus spore inactivation on
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cleaning products
Table 5-4. Solid-waste samples results
Material type (n = number of tests)
Phase I
Phase II
CFU/sample<± SD)
Wiping medium# 1 (application of decontaminant step)
Finished wood flooring (n = 4)
34E+03(±6.9E+03)
NA*
Grimed finished wood flooring (n = 1)
44E+01
NA***
Glass (n = 4)
14E+04(±24E+04)
NA*
Painted wallboard (n = 4)
7.7E+04(±1.0E+05)
NA*
Grouted ceramic tile (n = 4)
3.5E+04(±2.9E+04)
NA*
Wiping medium # 2 (rinse step)
Finished wood flooring (n = 2-4)
9.0E+01 (±1.2E+02)
4.0E+02 (±5.1E+02)
Grimed finished wood flooring (n = 1)
34E+00
NA**
Glass (n = 24)
8.3E+01 (±1 1E+02)
8.5E+01 (±1.5E+02)
Painted wallboard (n = 24)
4.8E+03 (±6.8E+03)
5.2E+02 (±1.0E+03)
Grouted ceramic tile (n = 24)
1.1E+03 (±1.5E+03)
4.6E+01 (±7.9E+01)
* not applicable - decontaminants were sprayed on In Phase II; ** not applicable - grimed surfaces were not tested in Phase II
The contamination of expended wiping media used in Phase I and II is similar to or higher than the levels
observed in the liquid waste (typically around 1-4 log level). In Phase I, rags and sponges used for
application of bleach were more contaminated than rinse media. Similarly to contaminated liquid waste,
the residual biocontamination of solid waste generated in real-life scenarios would most likely decrease
overtime (rags and sponges in this study were neutralized to allow qualitative analysis of spores). Still, it
is extremely important to emphasize the importance of use of appropriate personal protective equipment
and proper disposal of these waste (e.g., triple bagging) after the self-help decontamination procedure is
concluded.
6. Summary
Most tests performed during Phase I and II achieved the target efficacy from surfaces of > 6 LR. The
decontamination was achieved by a synergistic action of inactivation of spores by oxidizing agent (bleach)
and mechanical removal. Of the procedures tested, those incorporating RTU-COTS containing bleach with
surfactant/surfactants were more effective (» 6LR). The results indicate that the Phase II decontamination
approaches were comparable in decontaminating all types of tested materials (both porous and nonporous)
The fate of the biological spores showed secondary fugitive emissions of spores into aerosol fractions,
which suggests that spores may be expected in indoor environments during self-help decontamination and
potentially spread contamination throughout a residential dwelling and/or heating, ventilation and air
44
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Assessment of Bacillus spore inactivation on
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cleaning products
conditioning (HVAC) system. Viable spores were also found in liquid and solid post-decontamination waste
that can therefore be a source of cross-contamination during decontamination and a potential human
exposure hazard to persons performing self-help cleaning. It is noted that the reaerosolization of spores was
measured for spore surface concentrations where these methods would not be recommended.
Impact of Study
One of the primary goals of this project was to demonstrate the effectiveness of RTU-COTS products for
inactivating Bacillus spores on a variety of surfaces. The products identified in this project could be used by
a homeowner as an option that could be used as a risk reduction measure in the event of a wide area
release of Bacillus anthracis. The RTU-COTS products could also be used in conjunction with high tech
methods (e.g., fumigation) to treat hot spots that remain after treatment.
Safety Guidelines
• Spores can also 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. The final "self-help"
method tutorial/ instructional document resulting from this project should therefore emphasize the
importance of using the required personal protective equipment (PPE).
• Users of this guidance should follow PPE recommendations on the product label when using these
products. When using bleach or other disinfectants and cleaners follow the instructions on the labels for
precautions, use, and personal protective equipment (PPE) recommendations.
• Personal Protective Equipment (PPE) should be used when using cleaning and disinfectant products. PPE
may include protection for skin and eyes such as gloves and goggles. Other PPE should be employed only
per product labels.
• Never mix cleaning products together, unless specifically allowed by the product label.
45
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Assessment of Bacillus spore inactivation on
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cleaning products
References
Canter, D.A. Remediating Sites with Anthrax Contamination: Building on Experience. Proceedings
of the 2003 AWMA/EPA Indoor Air Quality Problems and Engineering Solutions Specialty
Conference and Exhibition, July 21-23, 2003, RTP, NC.
U.S. EPA. After Action Report - Danbury Anthrax Incident, U.S. EPA Region 1, September 19,
2008.
U.S. EPA. Effectiveness of Cleaning and Disinfection Methods for Removing, Reducing or
Inactivating Agricultural Biological Threat Agents. U.S. Environmental Protection Agency,
Washington, DC, EPA/600/R-11/092, 2011.
Calfee, M.W; Ryan, S.P; Wood, J.P.; Mickelsen, L.; Kempter, C.; Miller, L.; Colby, M.; Touati, A.;
Clayton, M.; Griffin-Gatchalian, N.; McDonald, S.; Delafield, R. Laboratory Evaluation of Large-
Scale Decontamination Approaches. J. Environ. Microbiol. 2012,112, 874-882.
Canter, D.A.; Gunning, D.; Rodgers, P.; O'Connor, L.; Traunero, C.; Kempter, C.J. Remediation of
Bacillus anthracis Contamination in the U.S. Department of Justice Mail Facility. Biosecur. Bioterror.
2005, 3(2), 119-27.
The Clorox Company. Bleach Facts, http://www.cloroxprofessional.com/industrv/health/knowledge-
expertise/facts-about-bleach, last accessed August 10. 2015.
Powell Fabrication and Manufacturing. Sodium Hypochlorite General Information
Handbook.httpV/www.powellfab.com/technical information/files/810.pdf, last accessed August 10.
2015.
Hamouda, T.; Hayes, M.M.; Cao, Z.; Tonda, R.; Johnson, K.; Wright, D.C.; Brisker, J.; Baker, J.R. A
Novel Surfactant Nanoemulsion with Broad-Spectrum Sporicidal Activity against Bacillus Species.
J. Infectious Diseases 1999,180, 1939-1949.
The Michigan Nanotechnology Institute for Medicine and Biological Sciences (MNIMBS).
Antimicrobial Nanoemulsions.http://nano.med.umich.edu/platforms/Antimicrobial-
Nanoemulsion.html, last accessed August 10. 2015.
Einfeld, W.; Boucher, R.M.; Tezak, M.S., Wilson, M.C; Brown, G.S. Evaluation of Surface Sampling
Method Performance for Bacillus Spores on Clean and Dirty Outdoor Surfaces. Sandia National
46
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Assessment of Bacillus spore inactivation on
indoor surfaces using commercially-available
cleaning products
Laboratories. SAND2011-4085. http://prod.sandia.gOv/techlib/access-control.cqi/2011/114085.pdf,
last accessed August 10. 2015.
Spahl R.J., 2012. The Myron L Company. FCE™: Groundbreaking Measurement of Free Chlorine
Disinfecting Power in a Handheld Instrument. http://www.mvronl.com/PDF/fcetr.pdf. last accessed
August 10. 2015.
47
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Assessment of Bacillus spore inactivation on
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cleaning products
Draft Report
Appendix A
Appendix A. Characterization of decontaminants and decontamination procedures
Table A-1. Phase I Decontamination Solution Recipes
Clorox® Concentrated Germicidal Bleach 1:10 dilution
Dl water [mL]
9000 mL
Clorox® Concentrated Germicidal Bleach [mL]
1000 mL
Clorox® Concentrated Germicidal Bleach 1:5 dilution
Dl water [mL]
8000 mL
Clorox® Concentrated Germicidal Bleach [mL]
2000 mL
Clorox® Splashless Bleach 1:5 dilution equivalent
Dl water [mL]
5860 mL
Clorox® Splashless Bleach [mL]
4140 mL
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Assessment of Bacillus spore inactivation on
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Draft Report
Appendix A
Table A-2. Characterization of decontamination solutions and process parameters in Phase I
Test ID
Material Type
Decontamination
Aqent
Application
Mode
Decontamination Solution Characterization
Run-off Character!
(neutralized
zation
Rinsate Characterization
Process Characterization
FAC
[ppm]
CI2
[g/L]
NaCIO
[%]
pH
T
[°C]
NaCIO
[%]
pH
T
[°C]
NaCIO
[%]
pH
T
[°C]
Average Surface
Loading Volume
[mL+/-SD]
Average Application Time
[h:min:sec +/¦ SD]
Average Processing Time
[h:min:sec +/¦ SD]
Initial
Characterization
Bleach 1:10
6400
6.40
0.67
11.4
22.4
\
1
Finished wood
floorinq
Bleach 1:10
Rag
6570
6.57
0.69
11.4
21.5
ND
4.27
21.90
ND
10.33
22.10
25.9
0.95
0:00:14
0:00:03
0:23:00
0:04:00
2
Glass
Bleach 1:10
Rag
6570
6.57
0.69
11.4
21.5
ND
9.34
22.00
ND
10.69
21.90
26.8
10
0:00:19
0:00:01
0:11:00
0:02:00
3
Painted
wallboard
Bleach 1:10
Rag
6770
6.77
0.71
11.4
21.6
ND
3.86
21.70
ND
9.43
21.90
37.4
5.3
0:00:17
0:00:00
0:10:00
0:00:00
4
Grouted
ceramic tile
Bleach 1:10
Rag
6770
6.77
0.71
11.4
21.6
ND
5.21
21.70
ND
9.91
21.80
21.9
9.6
0:00:17
0:00:01
0:10:00
0:00:00
Initial
Characterization
Bleach 1:5
13000
13.0
1.36
11.6
21.7
\
5
Finished wood
floorinq
Bleach 1:5
Rag
13962
13.96
1.47
11.6
21.7
ND
3.89
22.00
ND
7.26
22.10
16.7
6.2
0:00:15
0:00:02
0:10:00
0:00:00
6
Glass
Bleach 1:5
Rag
13962
13.96
1.47
11.6
21.7
ND
3.18
22.60
ND
9.49
22.30
28.2
4.1
0:00:16
0:00:02
0:10:00
0:00:00
7
Painted
wallboard
Bleach 1:5
Rag
13581
13.58
1.43
11.6
21.7
ND
5.64
22.30
ND
8.47
21.80
27.03
13.7
0:00:14
0:00:02
0:10:00
0:00:00
8
Grouted
ceramic tile
Bleach 1:5
Rag
13581
13.58
1.43
11.6
21.7
ND
3.57
21.90
ND
5.58
22.30
16.3
9.7
0:00:15
0:00:02
0:10:00
0:00:00
17
Finished wood
floorinq
Bleach 1:5
Sponge
14102
14.10
1.48
11.3
24.4
ND
4.15
24.30
ND
6.87
24.40
31.6
14.8
0:00:07
0:00:01
0:10:00
0:00:00
18
Glass
Bleach 1:5
Sponge
14102
14.10
1.48
11.3
24.4
ND
4.01
24.10
ND
9.51
24.20
85.3
42.2
0:00:07
0:00:01
0:10:00
0:00:00
19
Painted
wallboard
Bleach 1:5
Sponge
14302
14.30
1.50
11.3
23.5
ND
6.12
24.10
ND
9.69
23.90
34.7
23.5
0:00:07
0:00:02
0:10:00
0:00:00
20
Grouted
ceramic tile
Bleach 1:5
Sponge
14302
14.30
1.50
11.3
23.5
ND
4.11
23.90
ND
10.21
23.80
61.2
8
0:00:06
0:00:00
0:10:00
0:00:00
Initial
Characterization
Bleach with
surfactant 1:5
14800
14.8
1.55
11.8
22.8
\
21
Finished wood
floorinq
Bleach with
surfactant 1:5
Rag
16265
16.27
1.71
12.1
24.6
ND
9.72
25.60
ND
8.93
25.50
27.1
9.4
0:00:12
0:00:02
0:10:00
0:01:00
22
Glass
Bleach with
surfactant 1:5
Rag
16265
16.27
1.71
12.1
24.6
ND
9.14
25.7
ND
5.15
25.7
42.5
4.1
0:00:13
0:00:01
0:10:00
0:01:00
23
Painted
wallboard
Bleach with
surfactant 1:5
Rag
15364
15.36
1.61
12.0
24.7
ND
5.64
22.3
ND
8.47
21.8
29.7
15
0:00:14
0:00:01
0:10:00
0:00:00
24
Grouted
ceramic tile
Bleach with
surfactant 1:5
Rag
15365
15.37
1.61
12.0
24.7
ND
3.57
21.9
ND
5.58
22.3
31.4
4.5
0:00:13
0:00:01
0:10:00
0:00:00
25
Finished wood
flooring + grime
Bleach with
surfactant 1:5
Rag
14702
14.70
1.54
12.2
24.6
ND
8.86
25.1
ND
6.77
25.2
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Assessment of Bacillus spore inactivation on
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Draft Report
Appendix A
Table A-3. Characterization of Decontamination Solutions and Process Parameters in Phase II
Test ID
Material type
Decon Agent
Application
Mode
Decontamination Solution Characterization
Run-off Characterization
(neutralized)
Rinsate Characterization
Process Characterization
FAC
[PPm]
CI2
[g'L]
NaCIO
[%]
PH
T
[°C]
NaCIO
[%]
PH
T
[°C]
NaCIO
[%]
PH
T
[°C]
Average
Surface
Loading
Average
Application Time
[h:min:sec +/¦ SD]
Average
Processing Time
[h:min:sec +/¦ SD]
Initial
Characterization
24024
24.02
2.52
12.2
24.3
\
\
\
\
1
Finished wood
flooring
Lysol® Mold & Mildew
Blaster
Spray/Rag
\
\
\
ND
7.24
22.30
ND
9.96
21.10
16.80
2.60
0:00:09
0:00:01
0:10:30
0:00:36
2
Glass
Lysol® Mold & Mildew
Blaster
Spray/Rag
\
ND
6.77
22.40
ND
9.59
22.30
16.80
2.60
0:00:09
0:00:01
0:11:00
0:00:48
3
Painted
wallboard
Lysol® Mold & Mildew
Blaster
Spray/Rag
\
ND
8.24
21.90
ND
9.16
22.60
21.10
0.80
0:00:08
0:00:01
0:10:00
0:00:00
4
Grouted ceramic
tile
Lysol® Mold & Mildew
Blaster
Spray/Rag
\
ND
3.68
23.10
ND
8.52
22.60
24.60
1.80
0:00:10
0:00:01
0:10:00
0:00:00
Initial
Characterization
23743
23.74
2.49
12.6
24.6
\
\
\
X
5
Finished wood
flooring
Tilex® Mold & Mildew
Remover
Spray/Rag
ND
9.50
22.60
ND
9.64
22.20
17.90
0.50
0:00:10
0:00:01
0:10:18
0:00:30
6
Glass
Tilex® Mold & Mildew
Remover
Spray/Rag
\
\
\
ND
10.04
23.70
ND
9.91
23.80
17.20
0.80
0:10:00
0:00:00
7
Painted
wallboard
Tilex® Mold & Mildew
Remover
Spray/Rag
\
\
\
ND
9.66
22.50
ND
9.12
22.20
16.20
1.40
0:00:11
0:00:02
0:10:00
0:00:00
8
Grouted ceramic
tile
Tilex® Mold & Mildew
Remover
Spray/Rag
ND
9.50
22.60
ND
9.64
22.20
18.10
2.10
0:00:10
0:00:01
0:10:00
0:00:00
Initial
Characterization
20117
20.12
2.11
12.6
24.8
\
\
X
9
Finished wood
flooring
Clorox® Clean-up Cleaner +
Bleach
Spray/Rag
ND
10.08
21.40
ND
9.98
21.40
16.10
0.90
0:00:08
0:00:01
0:10:00
0:00:00
10
Glass
Clorox® Clean-up Cleaner +
Bleach
Spray/Rag
ND
10.32
21.20
ND
10.00
21.50
16.10
0.90
0:00:07
0:00:01
0:10:00
0:00:00
11
Painted
wallboard
Clorox® Clean-up Cleaner +
Bleach
Spray/Rag
\
ND
NA
NA
ND
NA
NA
21.40
2.20
0:00:10
0:00:01
0:10:00
0:00:00
12
Grouted ceramic
tile
Clorox® Clean-up Cleaner +
Bleach
Spray/Rag
\
ND
NA
NA
ND
NA
NA
25.00
2.00
0:00:11
0:00:00
0:10:32
0:00:54
Initial
Characterization
20518
20.52
2.15
12.6
25.4
\
\
\
\
\
13
Finished wood
flooring
Clorox® Disinfecting Bleach
Foamer
Spray/Rag
\
\
\
ND
11.27
22.10
ND
9.48
22.30
42.60
3.40
0:00:20
0:00:01
0:10:00
0:00:00
14
Glass
Clorox® Disinfecting Bleach
Foamer
Spray/Rag
\
ND
10.95
26.4
ND
8.82
22.8
39.30
2.20
0:00:18
0:00:01
0:10:00
0:00:00
15
Painted
wallboard
Clorox® Disinfecting Bleach
Foamer
Spray/Rag
ND
11.95
21.8
ND
9.93
17.5
40.90
3.50
0:00:16
0:00:01
0:10:00
0:00:00
16
Grouted ceramic
tile
Clorox® Disinfecting Bleach
Foamer
Spray/Rag
ND
11.07
21.6
ND
8.69
22
43.40
3.90
0:00:17
0:00:01
0:10:00
0:00:00
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Assessment of Bacillus spore inactivation on
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Draft Report
Appendix B
Appendix B. Miscellaneous Operating Procedures
MOP 3135
TITLE: Procedure for Sample Collection using BactiSwab™ Collection and Transport
Systems
SCOPE: This MOP describes the procedure for collecting swab samples for Low Tech
Decontamination Technique Testing
PURPOSE: The purpose if this MOP is to ensure all swab sampling is performed in a
consistent manner.
Equipment/Reagents
• Disposable lab coat
• Nitrile examination gloves
• P95 Respirator
• Shoe covers
• Bouffant cap
• Safety glasses
• BactiSwab™ Collection and Transport System
1.0 PROCEDURE
1. Before starting the swabbing procedure, make sure you are wearing the appropriate, project-
specific PPE (at a minimum gloves, lab coat, and safety glasses).
2. Through the sleeve, crush the BactiSwab™ ampule at midpoint.
3. Hold BactiSwab™ tip end up for at least five seconds to allow the medium to wet the swab.
4. Open the package and remove the BactiSwab™.
5. Label the plastic tube appropriately using the following scheme:
X-Y-N where,
X is the test number,
Y is the material abbreviation, and
N is the material number
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Draft Report
Appendix B
6. Remove the cap-swab from the plastic tube.
7. Swab the surface while spinning the cap-swab between the thumb and index fingers.
Swabbing should be conducted by following the recommend guidelines for each material as
detailed in the project documentation (usually the QAPP).
8. Return cap-swab to tube.
9. Date and initial each sample tube. Enter this information into the lab notebook.
10. Complete the chain of custody form and relinquish the samples to the BioLab.
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Assessment of Bacillus spore inactivation on
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Draft Report
Appendix B
MOP-3144
TITLE:
SCOPE:
PURPOSE:
PROCEDURE FOR WIPE SAMPLING OF COUPONS
This MOP describes the procedure for wipe sampling both small and large
coupons.
The purpose of this MOP is to ensure consistent and representative sampling of
such coupons.
EQUIPMENT (quantities are per sampling kit)
• Sterile sampling bag (10" x 14") - outer bag
• Sterile sampling bag (5.5" x 9") - inner "sample collection sterile sampling bag"
• Two sterile 50 mL Falcon Blue-Max™ Polypropylene Conical Tubes
• Sterile Kendall (ref. # 8402) 4-ply all-purpose sponge
• Sterile phosphate buffered saline with 0.005% TWEEN®-20, prepared according to
MOP -6562
• Pipette or other method for aseptic dispensing of 5 mL liquid
• Sterile Posi-grip© forceps
• P-95 Particulate Respirators - to prevent contamination and for respiratory protection.
(Specific projects may require additional respiratory protection and will be addressed in the
project Quality Assurance Project Plan (QAPP), e.g., SAR)
• Powder-free Nitrile gloves (support person) and Kimtech Pure G3 Sterile Nitrile gloves
(sampler)
• Dispatch® bleach wipes
1.0 PREPARATION
1. All materials needed for collection of each sample will be prepared in advance
using aseptic technique. A sample kit for a single wipe sample will be prepared
as follows:
a. Two sterile sampling bags (10" x 14", 5.5" x 9") and a 50 mL conical
tube, capped, will be uniquely labeled as specified in the project QAPP.
These bags and conical tube will have the same label. The 5.5" x 9"
labeled sterile sampling bag will be referred to as the sample collection
sampling bag.
b. A sterile all-purpose sponge will be placed in an unlabeled sterile 50 mL
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Appendix B
conical tube using sterile forceps and aseptic technique. The all-
purpose sponge will be moistened by adding 2.5 mL of sterile
phosphate buffered saline with 0.005% TWEEN®-20. The tube will then
be capped.
c. The labeled 50 mL conical tube (capped), the unlabeled conical tube
containing the pre-moistened all-purpose sponge, and the 5.5" x 9"
labeled sampling bag will be placed into the 10" x 14" labeled sampling
bag. Hence, each labeled sampling bag will contain a labeled 50 mL
conical tube (capped), an unlabeled capped conical tube containing a
pre-moistened all-purpose sponge, and an empty labeled sampling
bag.
d. Each prepared bag is one sampling kit.
2.0 SAMPLING PROCEDURE FOR SMALL (2"x2" or 14"x14") COUPONS
1. A three person team will be used, employing aseptic technique throughout. The team will
consist of a sampler, sample handler, and support person.
2. Throughout the procedure, the support person will log anything they deem to be significant
into the laboratory notebook.
3. In general, the team works from the least contaminated sample set (i.e., control blanks)
towards the most contaminated sample set (i.e., positive controls).
4. The sampling team will each don a pair of sampling gloves (a new pair per sample, non-
sterile, as they will only be handling non-sterile items); the sampler's gloves shall be sterile
sampling gloves as they are the only member of the team in contact with the sample. All
members shall wear dust masks to further minimize potential contamination of the samples.
Depending on the situation, respiratory protection beyond a dust mask may be required to
protect the sampling team (e.g., SAR; this will be specified in the project QAPP). New
disposable lab coats are required for the sample handler when changing between different
types of materials or when direct contact between the coupon and lab coat occurs.
5. The sample handler will remove the coupon from the appropriate cabinet (if necessary) and
place it on the sampling area, being careful to handle the coupon only around the edges.
6. The support person will record the coupon code on the sampling log sheet.
7. For some coupons the sampling area is evident from pre-drawn outlines. For these coupons,
proceed to step 10, as steps 8-9 are not necessary.
8. The support person will remove a template from the bag and aseptically unwrap it such that
the sampler may grab it wearing sterile gloves.
9. The sampler will place the template onto the coupon surface and align it such that the edges
of the coupon are visible through the holes on the template.
10. The support person will remove a sample kit from the sampling bin and record the sample
tube number on the sampling log sheet next to the corresponding coupon code just recorded.
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11. The sampler and support person will verify the sample code and ensure that the correct
coupon and location are being sampled.
12. The support person will:
a. Open the outer sampling bag touching the outside of the bag.
b. Touching only the outside of the (10" x 14") bag, remove and open the unlabeled
conical tube and pour the pre-moistened all-purpose sponge onto the sample or into
the sampler's hands.
c. Discard the unlabeled conical tube.
d. Remove the sample collection sample bag (5.5" x 9"), being careful to not touch the
inside of the outer sampling bag, and open it touching only the outside.
e. Maneuver the labeled 50 ml_ conical tube to the end of the outer sterile sampling bag
and loosen the cap.
f. Remove the cap from 50 ml_ conical tube immediately preceding the introduction of the
sample into the tube.
13. The sampler will:
a. Wipe the surface of the sample horizontally using S-strokes to cover the entire sample
area of the coupon using a consistent amount of pressure.
b. Fold the all-purpose sponge concealing the exposed side and then wipe the same
surface vertically using the same technique.
c. Fold the all-purpose sponge over again and roll up the folded sponge to fit into the
conical tube.
d. Carefully place the all-purpose sponge into the 50 ml_ conical tube that the support
person is holding, being careful not to touch the surface of the 50 ml_ conical tube or
plastic sterile sampling bag.
14. The support person will then immediately close and tighten the cap to the 50 ml_ conical tube
and slide the tube back into the sample collection sampling bag and seal it.
15. The support person will then wipe the sample collection sampling bag with a Dispatch® bleach
wipe and place it into the outer sampling bag.
16. The support person will then seal the outer sample collection bag now containing the capped
50 ml_ conical tube (containing the all-purpose sponge) inside a sealed 5.5" x 9" sample
collection bag.
17. The support person will then decontaminate the outer sample bag by wiping it with a
Dispatch® bleach wipe.
18. The support person will then place the triply contained sample into the sample collection bin.
19. All members of the sampling team will remove and discard their gloves.
20. Steps 2 - 19 will be repeated for each sample to be collected.
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3.0 SAMPLING METHOD FOR LARGE (4'x4' or larger) COUPONS
3.1 Sample Layout
The sampling of large coupons is carried out using a sample grid to divide the large coupons into
representative sections. These sections are then numbered and selected to be sampled at
different times during the course of the experiment as a blank, a control, or an experimental group
sample. This selection grid is pre-determined and the Project Quality Assurance Project Plan
(QAPP) may overrule the template shown in Figure 1 if otherwise specified.
As in the example below, the first cell is sampled as a Blank before contamination. Starting in cell
3, every third cell is sampled as a positive Control. This sample is to be taken post-contamination
and before decontamination. Every cell directly following a Control cell is sampled as
Experimental and is taken following decontamination. The sample kit labeling will be based on this
grid and the sampling team must ensure to correctly sample the coupons based on this template.
1
Blank
2
3
Control
4
Experimental
5
6
Control
7
Experimental
8
9
Control
10
Experimental
11
12
Control
13
Experimental
14
15
Control
16
Experimental
Figure 1. 4' x 4' Material Section Template and Sample Grid
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3.2 Sampling Procedure
1. A two-person team will be used, employing aseptic technique throughout. The team will
consist of a sampler and a support person.
2. Throughout the procedure, the support person will log anything they deem to be significant
into the laboratory notebook.
3. The sampling team will each don a pair of sampling gloves (a new pair per sample, non-
sterile, as they will only be handling non-sterile items); the sampler's gloves shall be sterile
sampling gloves as they are the only member of the team in contact with the sample. All
members shall wear dust masks to further minimize potential contamination of the samples.
Depending on the situation, respiratory protection beyond a dust mask may be required to
protect the sampling team (e.g., SAR; this will be specified in the project QAPP).
4. The support person will record the coupon code on the sampling log sheet.
5. The sampler will place the template onto the coupon surface (using clamps as necessary).
6. The support person will remove a sample kit from the sampling bin and record the sample
tube number on the sampling log sheet next to the corresponding coupon code just recorded.
7. The sampler and support person will verify the sample code and ensure that the correct
coupon and location (cell) is being sampled.
8. The support person will:
a. Open the outer sampling bag touching the outside of the bag.
b. Touching only the outside of the (10" x 14") bag, remove and open the unlabeled
conical tube and pour the pre-moistened all-purpose sponge onto the sample or into
the sampler's hands.
c. The unlabeled conical tube is retained for Step 9.
d. Remove the sample collection sample bag (5.5" x 9") being careful to not touch the
inside of the outer sampling bag and open it touching only the outside.
e. Maneuver the labeled 50 ml_ conical tube to the end of the outer sterile sampling bag
and loosen the cap.
f. Remove the cap from 50 ml_ conical tube immediately preceding the introduction of the
sample into the tube.
9. The sampler will:
a. For a vertical coupon, the sampler will squeeze excess moisture from the sampling
sponge to prevent dripping down the sampling surface. The excess moisture is caught
in the unlabeled conical tube from Step 8c, and is then discarded.
b. Wipe the surface of the sample using S-strokes to cover the entire sample area of the
coupon (inside the grid) using a consistent amount of pressure.
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c. Fold the all-purpose sponge concealing the exposed side and then wipe the same
surface vertically using the same technique.
d. Fold the all-purpose sponge over again and roll up the folded sponge to fit into the
conical tube.
e. Carefully place the all-purpose sponge into the 50 ml_ conical tube that the support
person is holding being careful not to touch the surface of the 50 ml_ conical tube or
plastic sterile sampling bag.
10. The support person will then immediately close and tighten the cap to the 50 ml_ conical tube
and slide the tube into the sample collection sampling bag and seal it.
11. The support person will then wipe the sample collection sampling bag with a Dispatch® bleach
wipe and place it into the outer sampling bag.
12. The support person will then seal the outer sample collection bag now containing the capped
50 ml_ conical tube (containing the all-purpose sponge) inside a sealed 5.5" x 9" sample
collection bag.
13. The support person will then decontaminate the outer sample bag by wiping it with a
Dispatch® bleach wipe.
14. The support person will then place the triply contained sample into the sample collection bin.
15. All members of the sampling team will remove and discard their gloves.
16. Steps 2 - 15 will be repeated for each sample to be collected.
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MOP-3155
TITLE PROCEDURE FOR VIA-CELL AIR SAMPLING
SCOPE: This MOP describes the procedure for air sampling using the Via-Cell Bioaerosol
Sampling Cassette.
PURPOSE: The purpose of this MOP is to ensure consistent and representative air sampling.
EQUIPMENT
• Zefon Via-Cell® Bioaerosol Sampling Cassette (p/n VIA010)
• EPA Method 5 Dry Gas meter box within annual calibration
• 1/4" I.D. vacuum tubing
• Labeled 5.5" x 15" sterile bag for tertiary containment
• Writing pen
• Project notebook
1.0 PREPARATION
1. Verify from the package that the Via-Cell cassette is within its expiration date. If not, discard.
2. Label the Via-Cell cassette with the appropriate sample I.D according to the test plan or
Quality Assurance Project Plan (QAPP).
3. In the project laboratory notebook, record the cassette sample ID, lot number, and expiration
date.
2.0 AIR SAMPLING
There are two areas of operation for Via-Cell air sampling: 1) at the cassette and 2) at the meter
box. These two areas of operation may be performed by the same person or different people
depending on the situation. When air sampling inside COMMANDER while occupied, for instance,
a two-person team will be necessary; one person inside to connect the cassette and the other
outside to start and stop the dry gas meter.
1. Wearing nitrile gloves, tear open the foil package using the tear strip on top. Use care
when opening, as this package is re-sealable and is required to be used after sampling for
transport to the laboratory for analysis.
2. Remove Via-Cell from the package (see Figure 1).
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*«•! brak*™
Figure 1. Disassembled Via-Cell, showing cap (far left), package, and cassette
with outlet plug.
3. Remove the blue outlet plug from the cassette and place it into the foil for safe keeping
(seen at the bottom of the green Via-Cell cassette in Figure 1).
4. Connect the Via-Cell sampler outlet to the dry gas meter using vacuum tubing and position
the cassette in the desired location. The Via-Cell sampler is capable of operating in any
vertical or horizontal orientation and in confined spaces.
5. Perform a leak-check on the cassette by pulling a vacuum on the inlet cap. The meter box
flow rate should be zero.
6. Remove the large blue inlet cap for the cassette and place into the foil package for
safekeeping.
7. Record the dry gas meter's initial volume.
8. At the meter box, turn on the pump and set the sampling pump to a flow rate of 15 Ipm.
Over the pressure drop of the Via-Cell cassette, this is at a delta H of 1.1" water as read
on the front of the meter box. Pull a sample for the desired amount of time, monitoring the
delta H every ten minutes. When sampling is completed, and with new gloves, replace the
blue plug in the outlet and the blue cap over the inlet.
9. Record the sampling time and final volume. Check to ensure flow rate was 15 Ipm.
10. Place the Via-Cell cassette into the special foil bag and zip it closed. Apply the red safety
seal label over the top of the foil bag opening to ensure sample integrity until analysis.
11. Place foil bag containing cassette inside a pre-labeled 5.5" x 15" sterile bag for tertiary
containment.
12. Submit the cassette to the Biocontaminant Laboratory along with a Chain-of-Custody
(COC); the cassette should be analyzed according to MOP 6571 within 24 hours of
collection. The COC form should include the collection time and the analysis by time and
date in the comments section.
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13. Each sample should be associated with a laboratory blank (plain, unused Via-Cell
cassette) and a field blank of at least 150 liters of clean air in the same area as samples
are collected.
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MOP-3163
TITLE: AEROSOL APPLICATION OF GRIME ON MATERIAL COUPONS
SCOPE: This MOP describes the procedure for applying grime in a reproducible manner.
PURPOSE: The purpose of this MOP is to standardize grime application to material coupons
using a HVLP sprayer.
EQUIPMENT:
• HVLP Sprayer with can
• Croix CH-10 Turbine with air line
• Grime (2.5g + 4.5g per coupon set)
• 95% Ethanol (50mL + 100mL / coupon set)
• Safety glasses
• Lab coat
• Nitrile Gloves
• Non-sterile cups
• Measuring tape
• Calibrated scale
• Spatula
• Coupons to be sprayed
• Timer
• Dispatch® wipes
• Kim wipes
Note: You will need a buddy during spraying.
1.0 GRIME PREPARATION
1.1 Solution Preparation
Prepare the grime/ethanol solution using the balance and a non-sterile cup.
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a. Tare an empty cup and add 7.0 g of grime.
b. Pour the grime into the can (seen in Figure 1) inside a fume hood.
c. Add 150mL 95% ethanol to the can.
Figure 1. HVLP Sprayer can
d. Weigh the can and contents and record in the lab notebook. Empty can weight is 146.5g.
e. Connect the can to HVLP sprayer and seal using the lever on the top of the can.
1.2 Coupon Setup
a. Check the QAPP, or other controlling document, to determine which coupons will be
sprayed.
b. Remove the coupons from their sterilization pouch and place the coupons against the
back baffle in the walk-in hood in H122 as seen in Figure 2.
c. Note their position in the hood during spraying on the coupon tracker using coupon IDs.
d. Mark a line on the table in the hood 9" away from the bottom of the coupons with tape to
use as a guide during spraying. The spray nozzle should be 9" from the bottom of the
coupons during spraying.
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Figure 2. Coupon Setup
Sprayer Settings
Figure 3. HVLP Sprayer
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Dial 1 - The default position is seen in the Figure 4 below. This orients the fan in a diagonal
spray. Line up all sharpie marks on the nozzle.
Dial 2 - Turn the dial clockwise to closed. The sharpie marks should line up when fully closed.
From the closed position, turn the dial two complete turns counter-clockwise. (The
sharpie mark will pass and then line up with the mark on the body of the sprayer). This is
the default position. If this is not set up properly no liquid will be sprayed.
Dial 3 - Turn the dial clockwise to closed. The sharpie marks should line up when fully closed.
From the closed position, turn the dial two complete turns counter-clockwise. (The
sharpie mark will pass and then line up with the mark on the body of the sprayer). This is
the default position.
Figure 4. Dial 1
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Dial 4 - Turn the dial clockwise to closed. The sharpie marks should line up when fully closed.
From the closed position, turn the dial six complete turns counter-clockwise. (The
sharpie mark will pass five times and then line up with the mark on the body of the
sprayer). This is the default position.
Figure 5, Dial 3 and 4
Set all dials to their default position to prepare for grime application.
2.0 SPRAY PROCEDURE
a. Ensure all sprayer settings are correct (as per Section 1.3).
b. Don all appropriate PPE (i.e., labcoat, gloves, safety glasses).
c. Connect the turbine hose to the bottom of the HVLP sprayer using the quick-connect
fitting.
d. Ensure the valve on the quick connect fitting is turned on and have a buddy power on the
turbine.
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Note: The HVLP sprayer will constantly spray air whenever the turbine is on. Make
sure you have the sprayer pointed into the hood at all times when the turbine
is powered on.
e. Shake can for 15 seconds prior to beginning spray.
f. Start spray and timer simultaneously. Spray two 1,5-min applications with a 1 min break in
between sprays. Record time in lab notebook. The spray pattern is shown below in Figure
6.
g. Spray top to bottom in 8 passes in the upright position (as seen in Figure 6, Steps 1 and
2).
h. Once at the bottom of the coupon, spray 4 passes bottom to top turning the sprayer
sideways (as seen in Figure 6, Steps 3 and 4). This is increases the flowrate.
i. Repeat Steps g and h until 90 sees have elapsed. Stop spraying and stop timer.
j. After a 1 min break, shake the can for 15 sees, then resume spraying and start timer.
k. Repeat Steps g and h for an additional 90 sees. Stop spraying and stop timer. Record stop
time.
I. Once spraying is complete, weigh can and contents and record the value in the lab
notebook.
m. Determine the volume sprayed based on an average solution density of 0.85 g/ml_.
n. Mark the appropriate column on the coupon tracker to indicate the specified coupons have
been coated with grime.
o. Allow 15 minutes for the coupons to dry before removing them from the hood.
p. Unless stated otherwise by a QAPP or other controlling document, the coupons should be
handled without touching the front surface, placed horizontally, and covered with a
cleaned (decontaminated) puffing pyramid with gasket (see MOP 3161).
q. Before Section 3.0 or in between each sample set, ensure the sharpie marks on the HVLP
sprayer are clear and bold. If unclear, redraw them.
r. For each additional coupon set:
1) Add 95% ethanol to the can equal to the volume sprayed in the previous test.
2) Add 1g of grime for every 21 ml_ of ethanol added.
3) Weigh the can and contents and record the value in the lab notebook.
4) Attach the can to the HVLP sprayer.
5) Repeat Section 2.0 of this MOP.
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• v* t;
\
TJpri^Lt
Sideways
PliiSSiS
Wm
Sideways
Figure 6. Spray pattern
3.0 CLEAN UP
a. Record final weight of spray can.
b. Rinse spray can with ethanol, then Dl water. Wash the can with soapy water, rinse,
and allow to dry.
c. Disconnect sprayer from hose.
d. Turn off turbine blower, coil hose, and move to a convenient location.
e. Wipe down the empty hood with Dispatch and Kimwipes, until no black residue
remains.
f. Rinse with Dl water.
g. Allow to dry.
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MOP 3198
TITLE: PROCEDURE FOR PREPARING DILUTED BLEACH SOLUTION FOR WA 4-59
PARAMETRIC BLEACH TESTING
SCOPE: This MOP describes a procedure for reproducibly preparing diluted bleach
solutions for use in WA 4-59 testing.
PURPOSE: The purpose of this MOP is to ensure the solution meets quality assurance (QA)
specifications for each test.
1.0 EQUIPMENT/REAGENTS
• Clorox® Concentrated Germicidal Bleach; - sodium hypochlorite concentration 8.25%
NOTE: This is a new product of the Clorox Company that will replace Regular
Germicidal Bleach
• Clorox® Splash-less Concentrated Bleach - sodium hypochlorite concentration 3.95 %
• HACH Test Kit - Hypochlorite (Bleach) D.T (5-15% as Cl2 Model CN-HRDT; Cat No.
26871-00)
• 15 L Carboy (sterilized)
• 5000 mL Graduated cylinder
• 1000 mL Graduated cylinder
• 250 mL Erlenmeyer flask
• Deionized (Dl) water
• Oakton Acorn Series pH 5 meter or equivalent
• Triple-rinsed container suitable for transporting hazardous solutions
• Oakton pH 4, 7 and 10 (pH = 4.00 ± 0.01, 7.00 ± 0.01 and 10.00 ± 0.01 @ 25 °C) buffer
or equivalent
• Dispatch® wipes
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NOTE: Diluted Bleach solution should be prepared fresh each day.
NOTE: Sterile equipment needs to be sterilized prior to testing either by autoclaving (in
Biocontaminant Laboratory or H-122A) or by ethylene oxide (EtO) fumigation using
the EtO sterilization system located in H-222.
A Safety Requirements:
o PRE Required: Safety glasses, lab coat, nitrile gloves
o All work performed in a chemical fume hood
2.0 PROCEDURE
2.1 Calibrate pH Meter
1. Turn meter on (Figure 1).
2. Rinse electrode thoroughly with D! water. DO NOT wipe the electrode.
Figure 1. Acorn pH5
Meter
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3. Dip both the electrode and temperature sensor into pH 4.00 buffer solution. The glass bulb
must be completely immersed into the sample. Stir gently and wait for the reading to stabilize
(about 40 seconds).
4. Press the CAL key to enter the calibration mode. The display will momentarily flash "CA" to
indicate Calibration. The display will show the current uncalibrated reading, blinking while in
calibration mode.
5. Allow the reading to stabilize. The meter will automatically recognize 4.00, 7.00 or 10.00
buffers.
6. Record the uncalibrated value in the laboratory notebook. Press the Enter key once to
confirm calibration for the 4.00 buffer. The LCD displays "CO" to indicate the calibration point
has been confirmed. The meter exits calibration mode and returns to measurement mode.
7. Record the pH buffer measurement and temperature (press MODE key to select parameter) in
the appropriate lab notebook.
8. Repeat step 4 and 7 for the 7.00 and 10.0 buffers.
2.2 Diluted Bleach Preparation
2.2.1 1:10 Dilution
(theoretical concentration of hypochlorite 0.83%)
1. Add 9000 ml_ of Dl water to sterilized (autoclaved) carboy of at least 15 L capacity.
2. Prepare 10 L of diluted bleach by adding 1000 ml_ of Clorox Concentrated Germicidal
Bleach® to a carboy. Cap the container. Mix solution (by hand) for 1 min. This will result in a
1:10 ratio solution (target concentration = 0.83% sodium hypochlorite). Record the time and
date on the carboy and in the laboratory notebook.
3. Test the pH and temperature of bleach solution in a carboy. Record in the laboratory
notebook. Compare pH to the pH-threshold value established for the 1:10 dilution in
preliminary testing. If the % difference >10%, re-prepare decontamination solution.
4. Measure the Free Available Chlorine (FAC) concentration of the bleach solution using the
HACH kit (Section 2.4). Record in the laboratory notebook. Compare the FAC to the FAC
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threshold value established for the 1:10 dilution in preliminary testing. If the % difference
>10%, re-prepare decontamination solution.
5. Compare to pH with the threshold value established for the 1:1 dilution in preliminary testing.
If the % difference >10%, re-prepare decontamination solution.
2.2.2 1:5 Dilution
(theoretical concentration of hypochlorite 1.65%)
1. Add 8000 ml_ of Dl water to sterilized (autoclaved) carboy of at least 15 L capacity.
2. Prepare 10 L of diluted bleach by adding 2000 ml_ of Clorox Concentrated Germicidal
Bleach® to a carboy. Cap the container. Mix solution (by hand) for 1 min. This will result in a
1:5 ratio solution (target concentration = 1.65 % sodium hypochlorite). Record the time and
date on the carboy and in the laboratory notebook.
3. Test the pH and temperature of bleach solution in a carboy. Record in the laboratory
notebook. Compare the pH to the pH-threshold value established for the 1:5 dilution in
preliminary testing. If the % difference >10%, re-prepare decontamination solution.
4. Measure the FAC concentration of the bleach solution using the HACH kit (Section 2.4).
Record total digits in the laboratory notebook. Compare the FAC to the FAC threshold value
established for the 1:5 dilution in preliminary testing. If the % difference >10%, re-prepare
decontamination solution.
2.2.3 1:1 Dilution
(theoretical concentration of hypochlorite 4.13%)
1. Add 5000 ml_ of Dl water to sterilized (autoclaved) carboy of at least 15 L capacity.
2. Prepare 10 L of diluted bleach by adding 5000 ml_ of Clorox Concentrated Germicidal
Bleach® to a carboy. Cap the container. Mix solution (by hand) for 1 min. This will result in a
1:1 ratio solution (target concentration = 4.13 % sodium hypochlorite). Record the time and
date on the carboy and in the laboratory notebook.
3. Test the pH and temperature of bleach solution in a carboy. Record in the laboratory
notebook. Compare the pH to the pH threshold value established for the 1:1 dilution in
preliminary testing. If the % difference >10%, re-prepare decontamination solution.
4. Measure the FAC concentration of the bleach solution using the HACH kit (Section 2.4).
Record total digits in the laboratory notebook. Compare the FAC to the FAC threshold value
established for the 1:1 dilution in preliminary testing. If the % difference >10%, re-prepare
decontamination solution.
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2.3 Diluted Bleach with Surfactant Preparation
2.3.1 1:10 Dilution Equivalent with Surfactant
(theoretical concentration of hypochlorite 0.83%)
1. Add 7930 mL of Dl water to sterilized (autoclaved) carboy of at least 15 L capacity.
2. Prepare 10 L of diluted bleach with surfactant by adding 2070 mL of Clorox Splash-less
Concentrated Bleach® to a carboy. Cap the container. Mix solution (by hand) for 1 min. This
will result in an equivalent of 1:10 ratio regular bleach solution (final concentration of
hypochlorite 0.83%). Record the time and date on the carboy and in the laboratory notebook.
3. Test the pH and temperature of bleach solution in a carboy. Record in the laboratory
notebook. Compare the pH to the pH threshold value established for the 1:10 dilution with
surfactant in preliminary testing. If the % difference >10%, re-prepare decontamination
solution.
4. Measure the FAC concentration of the bleach solution using the HACH kit (Section 2.4).
Record in the laboratory notebook. Compare the FAC to the FAC threshold value
established for the 1:10 dilution with surfactant in preliminary testing. If the % difference
>10%, re-prepare decontamination solution.
2.3.2 1:5 Dilution Equivalent with Surfactant
(theoretical concentration of hypochlorite 1.65%)
1. Add 5860 mL of Dl water to sterilized (autoclaved) carboy of at least 15 L capacity.
2. Prepare 10 L of diluted bleach with surfactant by adding 4140 mL of Clorox Splash-less
Concentrated Bleach® to a carboy. Cap the container. Mix solution (by hand) for 1 min. This
will result in an equivalent of 1:5 ratio regular bleach solution (final concentration of sodium
hypochlorite 1.65 %). Record the time on the carboy and in the laboratory notebook.
3. Test the pH and temperature of bleach solution in a carboy. Record in the laboratory
notebook. Compare the pH to the pH threshold value established for the 1:5 dilution with
surfactant in preliminary testing. If the % difference >10%, re-prepare decontamination
solution.
-------
Assessment of Bacillus spore inactivation on
indoor surfaces using commercially-available
cleaning products
Draft Report
Appendix B
4. Measure the FAC concentration of the bleach solution using the HACH kit (Section 2.4).
Record in the laboratory notebook. Compare the FAC to the FAC threshold value established
for the 1:5 dilution with surfactant in preliminary testing. If the % difference >10%, re-prepare
decontamination solution.
2.3.3 1:1 Dilution Equivalent with Surfactant
(theoretical concentration of hypochlorite 4.125%)
NOTE: Concentration of sodium hypochlorite in Splash-less Clorox Concentrated
Bleach® is lower than 4.13% hence a 1:1 dilution equivalent with surfactant will
be prepared with 4.29% solution of Clorox Concentrated Regular Bleach®
added to Splash-less Clorox Concentrated Bleach® to adjust the concentration
of sodium hypochlorite.
1. Add 4800 ml_ of Dl water to sterilized (autoclaved) carboy of at least 15 L capacity.
2. Prepare 10 L of diluted bleach at 4.29% sodium hypochlorite by adding 5200 ml_ of Clorox
Concentrated Germicidal Bleach® (8.25%). Mix solution (by hand) for 1 min.
3. Take 4750 ml_ of 4.29% solution of Clorox Concentrated Germicidal Bleach® just prepared
and add it to another sterilized (autoclaved) carboy of at least 15 L capacity.
4. Add 5250 ml_ of Clorox Splash-less Concentrated Bleach® to a carboy from step 3. Cap the
container. Mix solution (by hand) for 1 min. This will result in an equivalent of 1:1 ratio regular
bleach solution (final concentration of sodium hypochlorite 4.13 %). Record the time on the
carboy and in the laboratory notebook.
5. Test the pH and temperature of bleach solution in a carboy. Record in the laboratory
notebook. Compare the pH to the pH threshold value established forthe1:1 dilution with
surfactant in preliminary testing. If the % difference >10%, re-prepare decontamination
solution.
6. Measure the FAC concentration of the bleach solution using the HACH kit (Section 2.4).
Record in the laboratory notebook. Compare the FAC to the FAC threshold value established
for the 1:1 dilution with surfactant in preliminary testing. If the % difference >10%, re-prepare
decontamination solution.
2.4 Titration Using the HACH Kit
-------
Assessment of Bacillus spore inactivation on
indoor surfaces using commercially-available
cleaning products
Draft Report
Appendix B
1. Insert a clean delivery tube into the 2.26 N Thiosulfate Titrant Solution cartridge. Attach the
cartridge to the titrator body.
2. Flush the delivery tube by turning the delivery knob to eject a few drops of titrant. Reset the
counter to zero and wipe off the tip.
3. Fill the Erlenmeyer flask to about 150 mL with Dl water.
4. Add the contents of one potassium Iodide Power Pillow to the flask and stir to mix.
5. Add the contents of one Acid Reagent Powder Pillow to the flask and stir to mix.
6. Dispense 5 mL of the bleach sample below the solution level in the flask. The solution will turn
yellow - dark brown in color depending on the amount of chlorine present.
7. Place the delivery tube tip into the solution and stir the solution flask while titrating with the
thiosulfate titrant until the solution is light yellow.
8. Add 1 mL of starch indicator solution to the flask and stir to mix. The solution will turn a dark
blue color.
9. Continue titration until the solution becomes colorless.
10. Record the number of digits used.
2.5 Calculations
800 digits = 1 mL of 2.26 N STS dispensed
PPM Calculation:
Digits x 2.26 x 35453
ppm =
800 xV
-------
Assessment of Bacillus spore inactivation on
indoor surfaces using commercially-available
cleaning products
Draft Report
Appendix B
Where, V is volume (mL) of bleach sample used in titration.
PPM/1000 = g/L Chlorine
FINAL CHECKLIST:
~
~
~
Calibrate pH meter
Measure pH and temperature of solution
Measure FAC concentration of the solution
-------
Assessment of Bacillus spore inactivation on
indoor surfaces using commercially-available
cleaning products
Draft Report
Appendix B
MOP 6568
TITLE: ASEPTIC ASSEMBLY OF WIPE KITS
SCOPE: This MOP outlines the procedure for the aseptic assembly of wipe kits.
PURPOSE: To aseptically assemble kits that will be used to collect wipe samples from which
quantifiable data will be derived.
Materials:
• PPE (gloves, lab coat, safety goggles)
• Biological Safety Cabinet (Class II)
• pH-adjusted bleach
• Deionized water
• 70% solution of denatured ethanol
• Kim wipes
• Sterile, sealed Twirl-em bags in two sizes, 10"x15" and 5.5"x9"
• Sterile Kendall 4-ply all-purpose sponges
• Sterile, disposable thumb forceps
• 50mL conical tubes containing 5mL PBST tubes (MOP 6562)
• Sharpie
• Wire or foam rack for 50mL conical tubes
• Secondary containment such as a large Tupperware bin
• Lab notebook
• QAPP for project that is utilizing the wipe samples
-------
Assessment of Bacillus spore inactivation on
indoor surfaces using commercially-available
cleaning products
Draft Report
Appendix B
1.0 PROCEDURE
1.1 Preparation for Wipe Kit Assembly
Prior to wipe kit assembly, 50ml_ sterile conical tubes containing 5mL of sterile PBST and a sterile
2-ply sponge must be put together. They are assembled in the following manner:
1. Begin by donning PPE (gloves, lab coat, and protective eyewear).
2. Clean the workspace and biological safety cabinet by wiping surfaces with pH-adjusted
bleach, followed by deionized water, and then with a 70% solution of denatured ethanol. Wipe
the surfaces with a kimwipe to remove any excess liquid. Make sure the workspace is clean
and free of debris. Gather all necessary items to perform the task, place these items on a
clean cart beside the biological safety cabinet, within arm's reach so that, once the procedure
has begun, the task may be performed without interruptions.
3. Discard gloves and replace with fresh pair.
4. Place the sterile 50ml_ conical tubes containing 5ml_ PBST tubes under the biological
safety cabinet in a foam or wire rack designed to hold 50ml_ conical tubes. Using two
sterile, disposable thumb forceps, aseptically transfer one half of a 4-ply, sterile, all-purpose
sponge to each of the tubes. Complete the transfer by using the two forceps together
to first separate the 4-ply sponge in half to create two 2-ply sponges. Then remove a cap from
one of the tubes, carefully fold one of the 2-ply sponges using the forceps together and
aseptically place it in the opening of the tube so that it sits at the top portion of the tube, while
the 5mL of PBST remain at the bottom of the tube. Replace the cap to the tube. Repeat this
process until all of the tubes have sponges in them. Once all of the tubes contain sterile
sponges, then label the tube rack appropriately with the action completed, the date and your
initials and place the tubes on the shelf. These tubes are shelf stable for up to three months.
1.2 Assembly of Wipe Kits
-------
Assessment of Bacillus spore inactivation on
indoor surfaces using commercially-available
cleaning products
Draft Report
Appendix B
Wipe kits are assembled in the following manner:
1. No more than 48 hours prior to testing or collecting samples, assemble the wipe kits. Wipe kits
can be assembled outside of the biological safety cabinet, in a dry, clean area. Make certain to
use proper PPE, including gloves, while handling all wipe kit materials. Gather all materials to
assemble the kits before assembly. These materials include:
- 50ml_ conical tubes containing both a sterile wipe sponge and 5ml_ PBST
- Twirl-em bags in two sizes, 10"x15" and 5.5"x 9"
- Sharpie
- Vortex mixer
2. Obtain a copy of the labeling scheme for the samples. This may be detailed in the QAPP. For
each wipe kit, use a Sharpie and label a large 10" x 15" Twirl-em bag and a 50ml_ conical tube
containing the sponge and PBST.
3. Once all of the tubes are labeled, use the vortex mixer on the highest setting to agitate the
tube. This will mix the sponge, which was placed at the top of the tube, with the 5ml_ of PBST.
4. Open the labeled, 10" x 15" Twirl-em bags one at a time. Place the labeled, agitated tubes in
the 10" x 15" Twirl-em bags that have the corresponding label (that matches the tube). Add a
non-labeled, sealed 5.5" x 9" Twirl-em bag into the 10"x 15" Twirl-em bag, along with the tube
containing the wipe sponge. This completes the wipe kit assembly. Record the time and date
in which the wipe kits were assemble in the lab notebook; include the labeling schematic for
the wipe kits.
5. Place the assembled wipe kits into a secondary containment, such as a large Tupperware bin.
Use within 48 hours. When moving the kits to a sampling location, always have them in
secondary containment.
-------
Evaluation of Bio Agent Decontamination Options
For Owner/Occupants
Draft Report
Appendix C
Appendix C. Decontamination Efficiency Results
Table C-1. Decontamination Efficiency Results for Phase I
Test ID
Material Type
Decontamination
Agent
Application
mode
Log Reduction
(surface)
% Non-
detect
(n=3)
Positive Controls
Test Coupons
Wiping Medium Step 1
(bleach solution/rag or sponge)
Runoff
Wiping MediumSstep 2
(water rinse/rag or sponge)
Rinsate
Aerosol
Procedural
Blank
Negative
control
Average LR
STD
Average CFU
STD
RSD
Average
CFU
Median
CFU
STD
RSD
Average CFU
STD
RSD
Average CFU
STD
RSD
Average
CFU
STD
RSD
Average
CFU
STD
RSD
CFU/ft3
CFU/sample
CFU/sampI
e
1
Finished wood
flooring
Bleach 1:10
Rag
5.30
0.57
0%
1.95E-K)7
6.56E-K)6
34%
1.52E-K)2
7.50E-K)1
1.74E-K)2
110%
4.22E-K)0
8.79E-02
2%
2.23E-K)3
1.76E-K)3
79%
/
4.06E-K)1
3.24E-K)1
80%
<1
<1
<1
2
Glass
Bleach 1:10
Rag
6.78
1.93
33%
1.60E-K)7
1.00E-K)6
6%
1.45E-K)3
1.47E-K)1
2.50E-K)3
170%
5.00E-K)4
3.34E-K)4
67%
9.73E-K)2
4.08E-K)2
42%
/
1.48E-K)2
2.24E-K)2
150%
<1
<1
<1
3
Painted wallboard
Bleach 1:10
Rag
5.85
0.21
33%
2.64E-K)7
1.08E-K)7
41%
3.68E-K)1
3.37E-K)1
1.14E-K)1
31%
8.46E-K)4
8.62E-K)4
102%
2.78E-K)3
7.02E-K)2
25%
/
5.10E-K)0
1.45E-01
2.8%
1.0E-KK)
<1
<1
4
Grouted ceramic
tile
Bleach 1:10
Rag
3.57
0.39
0%
2.33E-K)7
7.93E-K)6
34%
7.62E-K)3
4.70E-K)3
6.50E-K)3
85%
6.74E-K)4
6.40E-K)4
95%
6.53E-K)3
5.53E-K)3
85%
/
4.53E-K)1
1.79E-K)1
39%
<1
<1
<1
5
Finished wood
flooring
Bleach 1:5
Rag
6.51
1.01
33%
2.49 E-K)7
1.16E-K)7
47%
1.98E-K)1
1.61 E-K)1
2.14E-K)1
108%
4.21 E-K)0
6.60E-02
2%
6.54E-K)2
2.89E-K)2
44%
/
4.98E-K)0
9.14E-02
1.8%
<1
<1
<1
6
Glass
Bleach 1:5
Rag
7.52
0.13
100%
1.90E-K)7
5.12E-K)6
27%
<1
<1
0.00E-K)0
0%
3.65E-K)3
6.29E-K)3
172%
1.82E-K)2
2.60E-K)2
140%
/
2.34E-K)0
2.75E-02
1.2%
1.2E-K)0
<1
<1
7
Painted wallboard
Bleach 1:5
Rag
6.08
0.70
0%
1.39E-K)7
1.09E-K)7
78%
7.12E-K)0
7.14E-K)0
2.67E-K)0
37%
2.17E-K)5
1.71E-K)5
79%
6.40E-K)3
6.22E-K)3
97%
/
3.72E-K)0
4.38E-02
1.2%
1.4E-KK)
<1
<1
8
Grouted ceramic
tile
Bleach 1:5
Rag
6.17
1.27
0%
2.64E-K)7
4.56E-K)6
17%
1.31 E-K)2
1.28E-K)1
2.15E-K)2
160%
4.68E-K)4
4.23E-K)4
90%
6.70E-K)0
5.71E-K)0
85%
/
5.74E-K)0
6.67E-02
1.2%
<1
2.3E-K)0
<1
17
Finished wood
flooring
Bleach 1:5
Sponge
7.43
0.37
67%
2.48 E-K)7
6.25E-K)6
25%
1.14E-K)0
1.14E-K)0
9.84E-01
87%
1.37E-K)4
1.79E-K)4
130%
2.10E-K)3
3.56E-K)2
17%
1.77E-K)2
1.98E-K)2
112%
4.74E-K)0
1.16E-01
2.4%
2.9E-K)0
<1
<1
18
Glass
Bleach 1:5
Sponge
5.71
1.85
33%
2.95E-K)7
1.15E-K)7
39%
7.72E-K)2
1.34E-K)2
1.22E-K)3
160%
1.87E-K)3
2.64E-K)3
141%
3.03E-K)3
2.53E-K)3
84%
1.62E-K)2
2.23E-K)2
137%
6.96E-K)1
8.91 E-K)1
130%
3.8E-K)0
<1
<1
19
Painted wallboard
Bleach 1:5
Sponge
4.87
0.69
0%
2.05E-K)7
3.75E-K)6
18%
4.75E-K)2
5.10E-K)2
4.14E-K)2
87%
3.30E-K)3
3.62E-K)3
110%
1.17E-K)3
6.20E-K)2
53%
9.60E-K)3
1.58E-K)4
164%
4.52E-K)2
1.13E-K)2
25%
7.3E-K)0
<1
<1
20
Grouted ceramic
tile
Bleach 1:5
Sponge
6.45
0.99
33%
2.30E-K)7
1.43E-K)6
6.2%
2.54E-K)1
1.21 E-K)1
3.33E-K)1
130%
2.58E-K)4
2.64E-K)3
10%
6.58E-K)2
6.12E-K)2
93%
2.11 E-K)3
3.62E-K)3
172%
4.54E-K)0
2.88E-02
0.63%
3.8E-K)0
<1
<1
21
Finished wood
flooring
Bleach with
surfactant 1:5
Rag
7.78
0.11
100%
3.40E-K)7
8.11 E-K)6
24%
<1
<1
1.03E-02
1.9%
4.15E-K)0
8.02E-02
2%
3.60E-K)0
7.70E-02
2%
3.51E-K)0
8.61E-02
2%
2.41 E-K)0
4.97E-02
2.1%
<1
<1
<1
22
Glass
Bleach with
surfactant 1:5
Rag
7.74
0.13
100%
3.19E-K)7
9.74E-K)6
31%
<1
<1
1.65E-02
2.9%
4.20E-K)0
1.81E-01
4%
4.60E-K)0
3.22E-02
1%
3.51 E-K)0
1.80E-02
1%
4.60E-K)0
3.27E-02
0.71%
<1
<1
<1
23
Painted wallboard
Bleach with
surfactant 1:5
Rag
6.22
2.02
33%
1.90E-K)7
3.00E-K)6
16%
7.94E-K)2
1.18E-K)0
1.37E-K)3
170%
1.60E-K)3
2.11 E-K)3
131%
7.28E-K)1
5.74E-K)1
79%
5.52E-K)0
1.84E-K)0
33%
4.23E-K)0
2.97E-02
0.70%
<1
<1
<1
24
Grouted ceramic
tile
Bleach with
surfactant 1:5
Rag
7.00
0.90
67%
2.04E-K)7
6.34E-K)5
3.1%
7.83E-K)0
<1
1.25E-K)1
160%
8.13E-K)2
7.97E-K)2
98%
3.86E-K)0
4.65E-02
1%
3.44E-K)0
9.88E-02
3%
4.88E-K)0
0.00E-K)0
0.00%
<1
<1
<1
25
Finished wood
flooring +grime
Bleach with
surfactant 1:5
Rag
7.26
0.35
67%
1.42E-K)7
3.66E-K)6
26%
<1
<1
3.30E-01
41%
4.14E-K)1
9.09E-01
2%
6.60E-K)1
2.86E-K)1
43%
3.44E-K)0
1.41E-01
4%
3.55E-K)1
0.00E-K)0
0.00%
3.8E-K)0
<1
<1
-------
Evaluation of Bio Agent Decontamination Options
For Owner/Occupants
Draft Report
Appendix C
Table C-2. Decontamination Efficiency Results for Phase II
Test ID
Material type
Decon agent
Application Mode
LogRreduction (surface)
%Non-detect
(n = 5)
Positive Controls
Test Coupons
Wiping Medium Step 2
(water rinse rag)
Runoff
Rinsate
Aerosol
Procedural Blank
Negative
Control
Average LR
STD
Average CFU
STD
RSD
Average CFU
Median CFU
STD
RSD
Average CFU
STD
RSD
Average CFU
STD
RSD
Average CFU
STD
RSD
CFU/ft3
CFU /
Sample
CFU
/Sample
1
Finished wood
flooring
Lysol® Mold & Mildew
Blaster
Spray/Rag
6.93
0.72
20%
3.40E-K)7
2.64E-K)7
78%
8.67E-K)0
1.33E-KJ0
1.11E-+01
130%
1.08E-+03
1.77E-KJ3
160%
3.68E-K)0
4.55E-02
1.2%
1.04E-+02
9.89E-+01
95%
7.32E-K)0
<1
3.85E-K)0
2
Glass
Lysol® Mold & Mildew
Blaster
Spray/Rag
7.55
0.14
80%
2.94E-K)7
1.61E-+07
55%
<1
<1
3.01 E-01
39%
3.04E-K)2
5.67E-K)2
190%
5.12E-+00
9.64E-02
1.9%
6.70E-K)0
1.95E-01
2.9%
3.01E-+00
<1
<1
3
Painted wallboard
Lysol® Mold & Mildew
Blaster
Spray/Rag
7.78
0.16
60%
4.55E-K)7
2.69E-K)6
5.9%
<1
<1
3.07E-01
39%
3.43E-K)0
1.19E-KJ0
35%
4.44E-K)0
8.56E-02
1.9%
6.46E-K)0
4.56E-02
0.71%
3.53E-K)0
<1
<1
4
Grouted ceramic
tile
Lysol® Mold & Mildew
Blaster
Spray/Rag
8.02
0.008
100%
6.05E-K)7
8.28E-K)6
14%
<1
<1
1.00E-02
1.7%
3.40E-K)0
6.68E-02
2.0%
8.44E-K)0
5.99E-02
0.71%
5.56E-K)0
6.86E-02
1.2%
6.80E-K)0
<1
<1
5
Finished wood
flooring
Tilex® Mold & Mildew
Remover
Spray/Rag
7.55
0.023
100%
3.58E-K)7
2.88E-K)7
80%
<1
<1
3.21 E-02
5.6%
4.16E-K)0
1.58E-KJ0
38%
6.72E-K)0
9.77E-K)0
150%
3.39E-K)0
4.04E-02
1.2%
4.89E-K)0
<1
1.11E-+00
6
Glass
Tilex® Mold & Mildew
Remover
Spray/Rag
7.80
0.009
100%
4.07E-K)7
4.01E-KJ6
10%
<1
<1
1.41E-02
2.2%
4.16E-K)0
1.54E-KJ0
37%
1.39E-+00
1.67E-02
1.2%
6.70E-K)0
1.71 E-01
2.6%
1.04E-KJ1
3.41E-+00
<1
7
Painted wallboard
Tilex® Mold & Mildew
Remover
Spray/Rag
6.72
0.53
20%
1.94E-+07
2.25E-K)6
12%
5.96E-K)0
4.69E-K)0
5.39E-K)0
90%
2.06E-K)3
2.04E-K)3
99%
3.65E-01
1.38E-02
3.8%
1.10E-KJ0
4.49E-02
4.1%
<1
1.54E-+00
<1
8
Grouted ceramic
tile
Tilex® Mold & Mildew
Remover
Spray/Rag
7.33
0.013
100%
1.67E-+07
1.53E-+06
9.2%
<1
<1
2.31 E-02
3.0%
1.64E-KJ2
3.32E-K)2
200%
5.78E-K)0
4.97E-02
0.86%
1.22E-+01
2.18E-15
0.0%
<1
1.38E-KJ1
1.71E-KJ1
9
Finished wood
flooring
Clorox® Clean-up Cleaner
+ Bleach
Spray/Rag
7.12
0.55
20%
4.82E-K)7
1.71E-KJ7
35%
5.68E-K)0
6.33E-K)0
4.57E-K)0
81%
5.12E-+02
6.31E-+02
120%
9.11E-+02
3.43 E-K)2
38%
3.28E-K)2
4.59E-K)2
140%
1.48E-KJ0
<1
1.28E-KJ0
10
Glass
Clorox® Clean-up Cleaner
+ Bleach
Spray/Rag
7.85
0.11
80%
5.23E-K)7
1.87E-KJ7
36%
<1
<1
2.25E-01
31%
2.00E-+01
3.70E-+01
190%
3.12E-KJ0
4.06E-02
1%
7.50E-K)0
1.07E-01
1.4%
2.75E-K)0
<1
<1
11
Painted wallboard
Clorox® Clean-up Cleaner
+ Bleach
Spray/Rag
7.89
0.019
100%
5.10E-+07
4.41E-KJ6
8.6%
<1
<1
2.82E-02
4.3%
4.70E-K)0
1.87E-KJ0
40%
3.72E-K)0
1.63E-KJ0
44%
6.45E-K)0
0.00E-K)0
0.0%
<1
<1
<1
12
Grouted ceramic
tile
Clorox® Clean-up Cleaner
-•-Bleach
Spray/Rag
7.79
0.13
80%
4.80E-K)7
2.76E-K)6
5.8%
<1
<1
2.87E-01
36%
1.17E-KJ1
1.62E-+01
140%
1.34E-KJ2
2.27E-K)2
170%
1.11E-KJ1
4.89E-K)0
44.1%
<1
<1
1.33E-KJ0
13
Finished wood
flooring
Clorox® Disinfecting Bleach
Foamer
Spray/Rag
7.81
0.007
100%
4.36E-K)7
6.93E-K)6
16%
<1
<1
1.05E-02
1.6%
3.35E-K)0
5.62E-02
1.7%
8.85E-K)0
7.66E-K)0
87%
1.77E-KJ1
0.00E-K)0
0.0%
<1
<1
<1
14
Glass
Clorox® Disinfecting Bleach
Foamer
Spray/Rag
7.93
0.008
100%
5.57E-K)7
8.18E-KJ6
15%
<1
<1
1.25E-02
1.9%
9.60E-K)0
1.38E-KJ1
140%
1.63E-KJ1
0.00E-K)0
0.0%
1.89E-KJ1
0.00E-K)0
0.0%
<1
<1
<1
15
Painted wallboard
Clorox® Disinfecting Bleach
Foamer
Spray/Rag
7.35
0.94
60%
4.71E-+07
8.16E-KJ6
17%
1.95E-+01
6.76E-01
4.18E-+01
210%
3.46E-K)0
5.58E-02
1.61%
1.28E-KJ1
3.08E-01
2.4%
2.71E-+00
3.87E-02
1.4%
<1
<1
<1
16
Grouted ceramic
tile
Clorox® Disinfecting Bleach
Foamer
Spray/Rag
7.77
0.13
80%
4.67E-K)7
2.65E-K)6
5.7%
<1
<1
3.04E-01
36%
3.42E-K)0
2.15E-02
0.63%
9.16E-+00
5.85E-02
0.64%
7.00E-K)0
4.48E-02
0.64%
<1
5.26E-K)0
1.33E-+00
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Appendix D: Quality Assurance
This project was performed under an approved Category III Quality Assurance Project Plan (QAPP) titled
Evaluation of Bioagent Decontamination Options for Owner/Occupants (January 2014) and Amendment 1
to this QAPP.
Quality control (QC) samples such as procedural blank coupons (coupons that underwent the fumigation
process but which were not inoculated) and negative controls (which did not undergo the fumigation
process) were included to monitor for cross-contamination. Results are given in Appendix C (Table C-1 and
C-2).
All test activities were documented via narratives in laboratory notebooks and the use of digital photography.
The documentation included, but was not limited to, a record of time required for each decontamination step
or procedure, any deviations from the QAPP, and physical impacts on the materials. All the tests were
conducted in accordance with developed Decontamination Technologies Research Laboratory (DTRL) and
BioLab MOPs, listed in Appendix B, to ensure repeatability and adherence to the data quality validation
criteria set for this project.
D-1. Calibration Procedures
All equipment was verified as being certified calibrated or having the calibration validated by the Metrology
Laboratory prior to use. Calibration of instruments was done at the frequency shown in Table D-1. Any
deficiencies were noted, and the instrument was adjusted to meet calibration tolerances and recalibrated
within 24 h. No deviations were noted.
Table D-1. Instrument Calibration Frequency
Equipment
Calibration/Certification
Expected Tolerance
Frequency
pH meter/pH calibration
Perform a 3-point calibration with non-expired standard
buffers that bracket the target pH before each use.
± 0.1 pH units
Daily
pH meter/temperature
calibration
Per manufacturer's instructions, compare displayed
value to a NIST certified thermometer
±0.5 °C
Monthly
Stopwatch
Compare against NIST Official U.S. time per MOP
±1 min/30 days
Monthly
Scale
Calibration by the EPA Metrology Laboratory/Check
calibration with Class 2 weights prior to weighing
±0.1% weight
Yearly/Daily
Micropipettes
Certified as calibrated at time of use/Recalibrated by
gravimetric evaluation of pipette performance to
manufacturer's specifications every year
±5%
Yearly
Burettes
Manufacturer certified
0.1 mi-
NA
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D-2. QA/QC Checks
D-2.1. Data Quality Objectives & Data Quality Indicators
The data quality objectives (DQOs) were used to identify the critical measurements needed to address the
objectives of the test program, and specify tolerable levels of potential errors associated with data collection
as well as the limitations of the use of the data. Data quality indicators (DQIs) for these critical
measurements were used to determine if the collected data met the quality assurance (QA) objectives. A list
of these data quality indicators can be found in Table D-2. Failure to provide a measurement method or
device to meet these goals resulted in a rejection of results derived from the critical measurement, no
results were rejected.
Table D-2. Data Quality Indicators for the Critical Measurements
Measurement
Parameter
Analysis Method
Accuracy
Precision/
Repeatability
Dilution ratio
Volumetric
0.01%
NA
FAC
Titration
±2%
NA
PH
pH meter/NIST-traceable buffer
solutions
± 0.01 pH units
NA
Temperature drift
pH meter/NIST-traceable
thermometer
±0.5 °C
NA
Time
NIST calibrated stopwatch
± 1 minute per hour
NA
Neutralizer volume
Volumetric
1 mL
NA
Volume
Volumetric
1 mL
NA
Plated Volume
Pipette
2%
1%
Temperature of
Incubation Chamber
NIST traceable thermometer (daily)
+2 °C
NA
Volumes
Serological pipette tips
0.1 mL
NA
Burettes
0.1 mL
NA
Counts of CFU/Plate
Manual counting
±10% of all CFUs per
plate between first
and second count
100%RSD between
triplicates for each plate
The quantitative acceptance criteria for each critical measurement are shown in Table D-3. Tests with
conditions falling outside of these criteria were rejected and repeated upon approval by the EPA WAM.
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Table D-3. Acceptance Criteria for Critical Measurements
Measurement Parameter
Target
Values
Acceptance criteria
Decontamination solution FAC between batches*
6500-15,000
ppm
± 10% of target value
Decontamination solution pH between batches*
11.4-11.8
± 10% of target value
Decontamination solution temperature drift (Task 1)
< 5°C
< 5°C between mixing and application of
decontaminant
Volume of neutralizer
varied
±1 mL
Interaction/processing time for Task 1
10min
± 1 min
*FA C and pH of all decontamination solutions were established experimentally prior to testing in a series of preliminary experiments.
Triplicate measurements of these parameters were performed for each liquid decontaminant to be tested. Measurements were performed
per MOP 3198. The averages from these measurements were then used as the target threshold for FAC and pH for each
decontamination formulation (Table A-2).
An additional set of biological data quality indicators was applied to the laboratory blanks, procedural blanks,
positive controls, test coupons and supplies used for microbiological analyses. These data quality indicators
are listed in Table D-4. The acceptance criteria were all met.
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Table D-4. Additional Data Quality Indicators Specific to Microbiological Data
Coupon or Sample Type
Acceptance Criteria
Information Provided
Corrective Action
Positive Control Coupons
Sample from material coupon contaminated
with biological agent but not subjected to the
test condition
Target recovery of 1 107 CFU per coupon
(sample) with a standard deviation of < 0.5. (5
106 - 5 107 CFUs/coupon); 90% recovery of
inoculum; Grubbs outlier test (or equivalent).
Initial contamination level on the coupons; allows
for determination of log reduction; controls for
confounds arising from history impacting
bioactivity; controls for special causes.
Shows viability of sampling technique and ability
of the plate to support growth.
Outside target range: discuss potential impact on
results with EPA WACOR; check inoculum and
prepare new inoculum if necessary.
Outlier: evaluate/exclude value
Procedural Blank
Coupon without biological agent
Non-detect
Controls for sterility of materials and methods
used in the procedure
Reject results upon approval of WACOR,
otherwise analyze data with procedural blank
results as test minimum, identify and remove
source of contamination if possible.
Blank Plating of Microbiological Supplies
No observed growth following incubation
Controls for sterility of supplies used in dilution
plating
Sterilize or dispose of source of contamination.
Replate samples.
Blank Tryptic Soy Agar (TSA) Sterility
Control
Plate incubated, but not inoculated
No observed growth following incubation
Controls for sterility of plates.
All plates are incubated prior to use, so any
contaminated plates will be discarded.
Exposed Field Blank Samples
A wipe kit will be handled, a vacuum sock kit
will sample ambient air.
Non-detect
The level of contamination present during
sampling
Clean up environment.
Sterilize sampling materials before use.
Unexposed Field Blank Samples
A wipe kit will be transferred without being
handled, a vacuum sock kit will be transferred
without switching on the vacuum
Non-detect
The level of contamination present during
sampling
Clean up environment.
Sterilize sampling materials before use.
Background Swabs
Non-detect
Determines sterility of materials and equipment
before use
Clean up environment.
Sterilize sampling materials before use.
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vvEPA
United States
Environmental Protection
Agency
PRESORTED STANDARD
POSTAGE & FEES PAID
EPA
PERMIT NO. G-35
Office of Research and Development (8101R)
Washington, DC 20460
Official Business
Penalty for Private Use
$300
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