EPA/600/R-13/217 | October 2013 | www.epa.gov/ord
United States
Environmental Protection
Agency
Determination of the Efficacy
of Spore Removal from Carpets
using Commercially-available
Wet/Vacuum Carpet Cleaning
Systems
Assessment and Evaluation
Report
Office of Research and Development
National Homeland Security Research Center
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EPA600-R-13-217
October 2013
Determination of the Efficacy of Spore Removal
from Carpets using Commercially-available
Wet/Vacuum Carpet Cleaning Systems
Assessment and Evaluation Report
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
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Disclaimer
The United States Environmental Protection Agency, through its Office of Research and Development's
National Homeland Security Research Center, funded and managed this investigation through EP-C-04-
023 with ARCADIS U.S., Inc. This report has been peer and administratively reviewed and has been
approved for publication as an Environmental Protection Agency document. It does not necessarily reflect
the views of the Environmental Protection Agency. No official endorsement should be inferred. This report
includes photographs of commercially available products. The photographs are included for purposes of
illustration only and are not intended to imply that EPA approves or endorses the product or its
manufacturer. Environmental Protection Agency does not endorse the purchase or sale of any
commercial products or services.
Questions concerning this document or its application should be addressed to:
Shawn P. Ryan, Ph.D.
National Homeland Security Research Center
Office of Research and Development (E-343-06)
U.S. Environmental Protection Agency
109 T.W.Alexander Dr.
Research Triangle Park, NC 27711
(919)541-0699
ryan.shawn@epa.gov
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Acknowledgments
The authors would like to acknowledge the support of Alion Science and Technology, funded under
contract with EPA (EP-D-10-070) concerning statistical analysis of the core samples.
The contributions of the EPA peer reviewers listed below are also acknowledged:
M. Worth Calfee
Robert Vanderpool
Jason Weinstein
IV
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Table of Contents
Disclaimer iii
Acknowledgments iv
Table of Contents v
List of Figures vii
List of Tables vii
List of Appendices viii
List of Acronyms and Abbreviations ix
List of Units x
Executive Summary xi
1 Project Description and Objectives 1
1.1 Purpose 1
1.2 Process 1
1.3 Project Objectives 2
2 Experimental Approach 3
2.1 General Approach 3
2.1.1 Control Chamber (COMMANDER) 5
2.1.2 Material Surfaces 5
2.1.3 Chamber Setups 7
2.1.4 Material Sterilization 9
2.1.5 Spore Preparation 9
2.1.6 Controlled Contamination Procedure 10
2.1.7 Contamination Characterization 11
2.1.8 Decontamination Procedure 11
2.1.9 Final Sterilization 13
2.2 Test Matrix 13
2.3 Sampling Strategy 15
2.4 Sampling/Monitoring Points 16
2.5 Frequency of Sampling/Monitoring Events 18
2.6 Decontamination Event Sequence 18
3 Testing and Measurement Protocols 19
3.1 Methods 19
v
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3.1.1 Surface Sampling 19
3.1.1.1 Wipe Sampling 19
3.1.1.2 HEPA Vacuum Sampling 21
3.1.2 Core Sampling 24
3.1.3 Swab Sampling 25
3.1.4 Rinsate Collection and Sampling Procedures 26
3.2 Prevention of Cross-contamination of Sampling/Monitoring Equipment 26
3.2.1 Preventing Cross-Contamination during Execution of the Decontamination Process 27
3.2.2 Preventing Cross-Contamination during Sampling 28
3.2.3 Preventing Cross-Contamination during Analysis 29
3.3 Representativeness 29
3.4 Sample Quantities 29
3.5 Sample Containers for Collection, Transport, and Storage 29
3.6 Sample Identification 30
3.7 Sample Preservation 30
3.8 Sample Holding Times 31
3.9 Sample Handling and Custody 31
3.10 Sample Archiving 32
4 Testing and Measurement Protocols 33
4.1 Sample Analyses 33
4.1.1 Recovery from HEPA Vacuum Sample 33
4.1.2 Filter Plating 34
4.1.3 Recovery of Core Samples 35
4.2 Analysis Equipment Calibration 35
5 Quality Assurance 36
5.1 Data Quality 36
5.2 Quality Assurance/Quality Control Checks 36
5.3 Data Quality Objectives 37
5.4 Audits 37
6 Results and Discussion 38
6.1 Data Reduction and Validation 38
6.2 Test Results 39
VI
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6.2.1 Length of Decontamination Event 46
6.2.2 Physical Impact on Materials and Crew 46
6.2.3 Fate of Spores 46
6.2.4 Health and Safety Effects of Decontamination 46
References 47
List of Figures
Figure 2-1. Conceptual Flowchart for a Test 4
Figure 2-2. Carpet Coupon 6
Figure 2-3. Template for Creating 1' x 1' Carpet Areas 7
Figure 2-4. Carpet Setup in COMMANDER during Inoculation 8
Figure 2-5. Carpet Setup in COMMANDER during Decontamination 9
Figure 2-6. Diffusion Shield 10
Figure 2-7. Century 400 Ninja Carpet Cleaner 11
Figure 2-8. Judson Labs O2 Pre-Spray and Rinse System 12
Figure 2-9. Decontaminating Carpet B with Spor-Klenz Using the Carpet Extractor 13
Figure 2-10. Carpet Section Template and Sample Grid 17
Figure 3-1. HEPA Sock Sampling a Coupon Section for Viable Spores 22
Figure 6-1. Wipe Sampling Results for Test A 39
Figure 6-2. HEPA Sock Sampling Results for Test B 40
Figure 6-3. HEPA Sampling Results after Decontamination Attempts 41
Figure 6-4. Core Sampling Results after Decontamination Attempts 42
Figure 6-5. Spor-Klenz Applied to a New Carpet with a Backpack Ssprayer 43
Figure 6-6. Spor-Klenz Applied to a New Carpet with an Unheated Vacuum Cleaner 43
List of Tables
Table 2-1. Test Matrix 14
Table 3-1. Cleaning Methods and Frequency for Common Test Materials/Equipment 27
Table 3-2. Sample Quantities for Each Test Setup 30
Table 4-1. Instrument Calibration Frequency 35
VII
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Table 5-1. Quality Control Checks 36
Table 5-2. DQIs for Critical Measurements 37
Table 5-3. Acceptance Criteria for Critical Measurements 37
Table 6-1. Spor-Klenz Applied with a Carpet Cleaner* 44
Table 6-2. Spor-Klenz Applied with a Backpack Sprayer* 44
Table 6-3. Log reduction 45
List of Appendices
Appendix A Piping and Instrumentation Diagram of COMMANDER
Appendix B MSDS for Decontamination Solutions
Appendix C Miscellaneous Operating Procedure (MOP) 6535a
VIM
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List of Acronyms and Abbreviations
APPCD
ATCC
B.
CPU
CM
COC
COMMANDER
CT
DCMD
DQI(s)
DQO(s)
EPA
HEPA
H202
IDLH
INL
ISO
LOD
MOP
NOT
NHSRC
NIST
ORLS
OPP
ORD
OSWER
PBST
PPE
PVC
QA
QC
Air Pollution Prevention and Control Division
American Type Culture Collection
Bacillus
Colony Forming Unit(s)
Critical measurement(s)
Chain of Custody
Consequence ManageMent ANd Decontamination Evaluation Room
Concentration * time
Decontamination and Consequence Management Division
Data Quality Indicator(s)
Data Quality Objective(s)
U.S. Environmental Protection Agency
High Efficiency Particulate Air
Hydrogen peroxide
Immediately Dangerous to Life and Health
Idaho National Laboratory
International Organization for Standardization
Limit(s) of detection
Miscellaneous Operating Procedure
National Decontamination Team
National Homeland Security Research Center
National Institute for Standards and Technology
On-site Research Laboratory Support
Office of Pesticide Programs
Office of Research and Development
Office of Solid Waste and Emergency Response
Phosphate buffered saline with 0.05% TWEEN®-20
Personal protective equipment
Polyvinyl chloride
Quality Assurance
Quality Control
IX
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QAPP
RSD
RTP
TRIO
ISA
VHP®
VOC
Quality Assurance Project Plan
Relative Standard Deviation
Research Triangle Park
Task Force on Research to Inform and Optimize
Tryptic Soy Agar
Vaporous hydrogen peroxide
Volatile Organic Compound
List of Units
in
ft
ft2
mg/L
ml
ppm
rpm
Inch/Inches
Foot/Feet
Square feet
Milligrams per Liter
Milliliter
Part(s) per Million
Revolutions per Minute
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Executive Summary
This project supports the U.S. Environmental Protection Agency's (EPA) Homeland Security Research
Program (HSRP) to improve the capability to respond to terrorist attacks affecting buildings and the
outdoor environments. Given the impact that a few letters containing anthrax spores had on the U.S.
Postal Service system in 2001, critical public facilities contaminated following a wide area release could
quickly consume the Nation's entire remediation capacity, requiring years to clean up and resulting in
enormous economic impacts. Additional quick, effective and economical decontamination methods
having the capacity to be employed over wide areas (outdoor and indoor) are required to increase
preparedness.
Although some of the facilities in which these letters were processed or received in 2001 were heavily
contaminated, they were successfully remediated with approaches such as fumigation with chlorine
dioxide or vaporous hydrogen peroxide. In addition, other cleaning methods were used in secondarily
contaminated areas or primarily contaminated facilities showing a minimal presence of anthrax spores.
These methods included combinations of disposal of contaminated items, vacuuming, and the use of
liquid sporicides such as a pH-adjusted bleach solution. Additionally, a combined set of mechanical and
chemical procedures (vacuum, scrub/wash and bleach) was used successfully in the decontamination of
a small shed contaminated with anthrax spores originating from animal hides during a drum-making
process.1 If proven effective, any approach involving washing and cleaning with readily available
equipment would significantly increase EPA's readiness to respond to a wide area release.
This project investigated the decontamination of carpet surfaces contaminated with Bacillus spores (i.e.,
surrogates of 8. anthracis). Two types of wet/vacuum carpet cleaning systems - unheated (cold) and
steam/heated (hot) - were tested for efficacy. In addition, the sporicide Spor-Klenz® Ready to use (Spor-
Klenz) (STERIS, Mentor, OH) was used in a carpet cleaner instead of the typical surfactant. This
apparatus was compared to application of Spor-Klenz with a backpack sprayer. The goal was to provide
information to support the development, use, and/or statement of limitations of these lower-tech
decontamination procedures for surfaces.
This work measured the reduction in viable spores on and within the carpet surfaces (effectiveness) as a
function of the cleaning technique and duration applied to both new and used carpet. The size of the
carpet sections, roughly 4' x 4', was chosen as feasible yet representative of what will likely be
encountered in the field (e.g., walkways). Operational parameters such as time, physical impacts on
materials or the remediation crew, and the fate of the viable spores (e.g., contamination of equipment
carpet cleaner parts, rinsate) were also determined.
The major finding of this research is that spores are very difficult to recover from carpet once the carpet
has been wetted. The study suggests that carpet cleaners alone are not effective in completely removing
spores (to a non-detectable amount). Carpet cleaners may be effective if used in concert with sporicides
to decontaminate a carpet. Neither the High-Efficiency Particulate Air (HEPA) sock sampling nor wipe
sampling seemed capable of recovering spores from a wetted carpet, even after the carpet had dried.
This problem may have been compounded for used carpet, possibly due to the higher amounts of surface
area from dirt, debris, and worn fibers. Extractive techniques were deemed more reliable, but the
methods developed for this study need refinement to improve detection limit and recovery.
XI
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Test A - Wipe Samples
The first test was conducted with both the hot and cold vacuum cleaners on new carpet, using the
manufacturer-recommended pre-spray and extraction solutions, and four sequential decontaminations.
Wipe samples taken after the first decontamination seemed to suggest that both vacuum cleaners had
removed 99.99% of the spores.
Use of the wet carpet cleaners was not expected to kill the spores but to decontaminate by removing the
spores. In this way, the spores could be considered a material, rather than an organism, and a
rudimentary type of mass balance could be applied to them. Insufficient spray was used, however, to
actually effect such a dramatic log reduction for such a porous material and led to the consideration that
the spores had been pushed down within the carpet pile and inaccessible to the wipe sampling.
Test B - HEPA Samples
For Test B, conducted on old carpet, HEPA sock sampling was used instead of wipe sampling. The
HEPA sock results suggested that a significant log reduction could be obtained using either the heated or
unheated carpet cleaners. However, based on the ineffectiveness of the wipe samples, the data could
also suggest that the HEPA socks were no more effective than the wipe sample had been at recovering
spores from a wetted carpet.
Test C - HEPA and Core (Extractive) Samples
Results from Tests A and B left questions regarding the effectiveness of the decontamination process
based upon uncertainty in the sampling methods. Test C was designed to answer this question by
altering the carpet construction method to allow for the collection of the core (extractive) samples in
addition to the HEPA sock samples. This test was conducted on new carpet.
Each sample area was first sampled using HEPA socks, and extractive analysis was performed on a
small 18 mm diameter core taken from the center of the same sample area. These data show that while
HEPA sock sampling suggested that the decontamination methods were removing some amount of
spores, subsequent extractive samples showed no or minimal removal. The HEPA sock samples of
control areas (not decontaminated) showed minimal downward drift.
The act of decontaminating with the wet/dry vacuums seems to push the spores away from the surface
and into the carpet pile, where the HEPA socks are unable to sample effectively. Even though the
samples were allowed to dry overnight, residual moisture or detergent may have helped spores adhere to
carpet fibers. The apparent log reduction from the HEPA sock samples from Test B showed a greater log
reduction than the HEPA sock samples from Test C. The tamped down nature of the older, used carpet,
as well as the debris in the fibers, may further reduce recovery by binding to the spores or merely
retaining moisture.
Although the results from Tests A and B were inconclusive as to the efficiency of the carpet cleaners, all
three tests suggested that there was no statistical difference between the hot (heated) and cold
(unheated) cleaners. Test C indicated that the efficacy of the carpet cleaners per the manufacturer's
recommendations was poor. Extractive sampling for carpet using core samples appears to be the only
reliable sampling method following any dampening of the surface.
xii
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Test D - Spor-Klenz in Cold Vacuum versus Backpack Sprayer
A final test (Test D), using new carpet, was conducted to provide information about the benefits of using a
carpet cleaner with Spor-Klenz in place of the recommended cleaner versus the application of the
sporicide without any vacuuming.
Test D showed a very good log reduction from the use of Spor-Klenz, applied either from the carpet
cleaner or the backpack sprayer. The first decontamination showed a 6 log reduction using the HEPA
socks (no detection on the core samples, which have a higher detection limit than the HEPA sock
samples). Following the second application of Spor-Klenz, no spores were detected using either
sampling method, showing at least a 7 log reduction in the HEPA sock results.
Due to the quantity of spores in the small size of the core samples, the core samples could only show a
greater than 3 log reduction. The core samples taken for the controlled contamination had lower recovery
than expected, possibly due to the rigorous method that was used to extract the core samples causing re-
aerosolization of many spores and the high detection limit induced by the small size of the core samples.
The length of the decontamination event was very short using the carpet cleaners (approximately 5
seconds per square foot). Test D with Spor-Klenz suggested that performing the decontamination twice
may yield no recoverable spores. If these decontaminations are performed immediately (back-to-back),
then there is minimum impact on the remediation crew. If, on the other hand, the carpet is allowed to dry
between successive applications, then an extra day is involved. Though it was not a consideration for this
test, the ultimate disposal of the carpet cleaners after use in an event may contribute to the cleanup time
and expense.
The backpack sprayer method as performed does require an extended duration, allowing for treatment of
an extrapolated 192 square feet in 30 minutes, or 6.4 square feet per minute, significantly longer per
square foot than when a carpet cleaner is used.
No physical impact on the carpet was noted for any of the decontamination methods. While neither was
physically strenuous, any activity inside Level C suits (even with cooling vests) leads to heat stress.
Moreover, the use of Spor-Klenz in an area without very high air exchanges could lead to levels of
hydrogen peroxide or acetic acid above IDLH (Immediately Dangerous to Life and Health) conditions.
Determining the ultimate fate of spores has proven very complicated due to sampling difficulties. The
rinsate recovered from the carpet cleaners used in Tests A, B, and C was very contaminated with
organisms of unknown origin, obscuring enumeration of the spores of interest, suggesting that the
likelihood our target organism was in the rinsate was very high. The data suggest that only a fraction of
the spores were removed from the carpet during Tests A, B, and C, so any spores not removed may be
viable, and viable spores may be present in carpet, carpet cleaner parts, and rinsate. The spores in Test
D may all be inactivated due to the presence of Spor-Klenz in all locations, but some doubt lingers due to
difficulties of sampling the rinsate.
XIII
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1 Project Description and Objectives
This project supports the mission of U.S. Environmental Protection Agency's (EPA) Homeland Security
Research Program (HSRP) by providing relevant information pertinent to the decontamination of
contaminated areas resulting from an act of terrorism.
A significant gap in preparedness is in the ability to effectively respond to a wide area release of a
biological agent such as 8. anthracis spores (the causative agent of anthrax and often referred to as
such, or anthrax spores). Such a release could potentially result in the contamination of a vast number of
personal residences, businesses, public facilities (e.g., hospitals), and outdoor areas. In 2001, the
introduction of a few letters containing anthrax spores into the U.S. Postal Service system resulted in the
contamination of several facilities. Although some of the facilities in which these letters were processed or
received in 2001 were heavily contaminated, they were successfully remediated with approaches such as
fumigation with chlorine dioxide or vaporous hydrogen peroxide. However, it is believed that critical public
facilities contaminated following a wide area release would quickly consume the Nation's entire
remediation capacity, requiring years to clean up and resulting in enormous economic impacts.
Additional quick, effective and economical decontamination methods having the capacity to be employed
over wide areas (outdoor and indoor) are required to increase preparedness for such a release.
Fumigation has primarily been used in heavily contaminated facilities, while other cleaning methods have
been used in secondarily contaminated areas or primarily contaminated facilities showing a minimal
presence of anthrax spores. These methods included combinations of disposal of contaminated items,
vacuuming, and the use of liquid sporicides such as a pH-adjusted bleach solution. Additionally, a
combined set of mechanical and chemical procedures (vacuum, scrub/wash and bleach) was used
successfully in the decontamination of a small shed contaminated with anthrax spores originating from
animal hides during a drum-making process.1 If proven effective, any approach involving washing and
cleaning with readily available equipment would significantly increase EPA's readiness to respond to a
wide area release. Data to quantify the effectiveness of such decontamination techniques are not
available.
1.1 Process
The general process being investigated in this project is the decontamination of carpet surfaces
contaminated with Bacillus spores (i.e., surrogates of 8. anthracis). 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 contamination or
via inactivation of the contaminant with antimicrobial chemicals, heat, UV light, etc. Physical removal
could be accomplished via in situ removal of the contamination from the material or 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.
During the decontamination activities following the results of the 2001 anthrax incidents, a combination of
removal and in situ decontamination was used. The balance between the two was facility-dependent and
factored in many issues (e.g., physical state of the facility). 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 very significant and was complicated by the nature of the
1
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waste (e.g., finding an ultimate disposal site). Since 2001, a primary focus for facility remediation has
been on 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 very significant impact on reducing the cost
of and time for the remediation effort.
In this research, the basis for the specific decontamination procedure is the use of wet/vacuum carpet
cleaning systems. Two types of carpet cleaning systems - unheated (cold) and steam/heated (hot) -
were tested for efficacy. Completion of the test matrix was expected to provide information to support the
development, use, and/or statement of limitations of this lower-tech decontamination procedure for
surfaces that can achieve a target cleanup goal while minimizing hazardous waste and the spread of
contamination.
1.2 Project Objectives
This work measured the reduction in viable spores on and within the carpet surfaces (effectiveness) as a
function of the cleaning technique and duration of application to various carpet types. The size of the
carpet sections, roughly 4' x 4', was chosen as feasible yet representative of what will likely be
encountered in the field (e.g., walkways). Operational parameters such as time, physical impacts on
materials or the remediation crew, and fate of the viable spores (e.g., contamination of equipment, wash
water, filters) were also determined.
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2 Experimental Approach
This section documents the general approach, test conditions, test equipment, and the methods that were
used to evaluate the data related to the project objectives.
2.1 General Approach
The general approaches to meet the objectives of this project were:
1. Use of controlled chambers, standardized sections and spore inoculums;
2. Contamination of large sections of materials via aerosol deposition of bacterial spores;
3. Quantitative assessment of spore contamination by sampling representative sections of carpet
sections before decontamination;
4. Application of a prescribed decontamination procedure to the test sections;
5. Quantitative assessment of residual contamination by sampling test sections;
6. Quantitative and qualitative analysis of decontamination procedure residues (e.g., waste water,);
7. Determination of decontamination effectiveness (comparison of results from positive control
samples and test sections); and
8. Documentation of operational considerations (e.g., cross-contamination, procedural time, impacts
on materials and personnel).
Testing was conducted in the consequence ManageMent ANd Decontamination Evaluation Room
(COMMANDER) located in H130-A of EPA's Research Triangle Park, NC, facility. For the purposes of
this project, effectiveness of a procedure was measured by generating a quantitative estimate of log
reduction of viable spores on a surface - a 6 log reduction would be considered very successful.
However, factors such as spread of viable spores due to the decontamination procedure itself and
recovery were factored into the overall measure of effectiveness. Additionally, procedures showing less
than a 6 log reduction may be deemed effective depending upon the circumstantial need (e.g., treatment
of scant contamination or repeated treatment of hot spots). Thus, while a log reduction value was
reported and may be termed effective, the effectiveness related to use of this procedure is less tangible
without the context of the need.
The general test approach is depicted graphically in the flow chart shown in Figure 2-1. The following
sections provide details on the approach used to complete the testing.
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Material
Sample
Fabrication
and Set-up
Decon
Equipment
Sterilization
Material Sample
and Chamber
Sterilization
Blank
Sampling to
Confirm
Sterilization
Decontamination
Procedure (wet
vacuuming or
backpack spray
application)
Controlled
Contamination
(control)
Sampling
Sampling to
Confirm
Sterilization
Test Area
Sampling
Decontamination Procedure
(wet vacuuming or backpack
spray application)
Chamber Purge
and clean up (2
days)
Controlled
Contamination
(2 hours)
Decon
Equipment
Sterilization
Sampling to
Confirm
Sterilization
Have
enough Decon
attempts been
made?
Chamber and
Sample
Sterilization and
Disassembly
Figure 2-1. Conceptual Flowchart for a Test
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2.1.1 Control Chamber (COMMANDER)
All testing was done in the COMMANDER, an enclosed single-access-point chamber (henceforth, may
also be referred to as the chamber). COMMANDER meets the following criteria:
1. Supports repeated fabrication of an environment (e.g., furnished office room; outdoor setting)
contained within the chamber;
2. Allows for release of biological organisms or chemicals into the chamber;
3. Allows for application of a decontamination technology (including fumigation with toxic corrosive
gases);
4. Supports entry into the chamber during all of the above mentioned activities (in appropriate
personal protective equipment or PPE);
5. External dimensions of 9 ft. x 12 ft. x 10 ft. high;
6. Contains one air-tight entry/exit port with a window;
7. Contains a 6 ft. x 6 ft. x 8 ft. high airlock with single entry/exit port with a window;
8. Contains entry/exit ports in line with the enclosure double door to allow for large materials to be
brought into or out of the chamber; and
9. Complies with all relevant local and national codes.
A piping and instrumentation diagram of COMMANDER is attached in Appendix A.
2.12 Material Surfaces
Carpet sections for both the new and "old" carpets were prepared using Sherwood carpet tiles with a
Shaw Contract Group Ecoworx® Backing System. These carpet sections were made of 100% nylon
woven on a 100% polyvinyl chloride (PVC)-free recyclable backing system with recycled content (made
from thermoplastic polyolefin compound with a reinforcing layer). The tiles were manufactured using a
multilevel loop construction.
Four individual 2 ft. by 2 ft. carpet tiles were glued directly on a 4 ft. x 4 ft piece of 15/32 in. four-ply
plywood, mounted atop a 4 ft. by 4 ft. frame of commercial grade 2 ft. x 3 ft. lumber. A 1 in. x 4 in. border
was then attached to the edge of the frame, creating a slight lip to the coupons. A photo of a carpet
coupon is shown in Figure 2-2.
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Figure 2-2. Carpet Coupon
The used carpet tile came from EPA's Research Triangle Park facility, and was taken from the south
corridor adjacent to the elevator lobby of Building C, 6th Floor. The carpet had been in place for
approximately eight years.
The 4 ft. x 4 ft. carpet sections were then sectioned into sixteen 1 ft. x 1 ft. sample areas by using the
template shown in Figure 2-3. The template was positioned starting at a corner marked on the frame of
the carpet section. The resulting 1 ft. x 1 ft. areas were numbered 1 through 16. The only deviation from
this sample grid was for the first test, where an 8 x 8 grid of 6 in. squares was used. The change in
design for subsequent tests was made both to simplify the sampling process and to provide more
information about the homogeneity of the deposition.
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Figure 2-3. Template for Creating 1' x 1' Carpet Areas
2.1.3 Chamber Setups
Each test consisted of two carpet sections of the same carpet type. During the inoculation phase, the
carpet coupons were centered as shown in Figure 2-4 to allow for a more uniform deposition of
aerosolized spores during the spore release.
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COMMANDER
chamber
JtL
Airlock
Figure 2-4. Carpet Setup in COMMANDER during Inoculation
The carpet sections were shifted during the decontamination phase to allow a more natural range of
motion using the long vacuum cleaner hoses in the relatively small space within COMMANDER. The
setup during decontamination is shown in Figure 2-5.
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COMMANDER
chamber
Airlock
Figure 2-5. Carpet Setup in COMMANDER during Decontamination
2.1.4 Material Sterilization
After the sections were assembled inside COMMANDER, the carpets were sterilized using STERIS
vaporous hydrogen peroxide (VHP®). Hydrogen peroxide (H2O2) vapor concentration within the chamber
was monitored using an Analytical Technology Corp. (Collegeville, PA) H2O2 electrochemical sensor
(Model B12-34-6-1000-1) to provide real-time concentration readings and control through a feedback
loop. A minimum concentration* time (CT) of 1000 parts per million (ppm)*hours was required for the
materials to be considered sterile. One test area of each carpet section was sampled (as described in
Section 3.1.1.1.2) to test sterility. All sterility checks were negative for growth.
2.1.5 Spore Preparation
The test organism for this work was a powdered spore preparation of 8. atrophaeus (American Type
Culture Collection [ATCC] 9732) and silicon dioxide particles. This bacterial species was formerly known
as 8. subtilis var niger and subsequently 8. globigii. The preparation was obtained from the U.S. Army
Dugway Proving Ground Life Science Division. The preparation procedure is reported in Brown et al.2
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
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1x10 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 nominal particulate size was 1 micron. The
estimated quantity of spores to be dispersed was approximately 2x1010 per event.
2.1.6 Controlled Contamination Procedure
The controlled contamination procedure was required to deliver a contamination of 1x106to 1x107
recoverable viable spores per square foot to the material samples. This was done by releasing 0.2 g of
the spore preparation using a TSI (Shoreview, MN) Model 3400A Fluidized Bed Aerosol Generator. The
Model 3400A Fluidized Bed Aerosol Generator was placed in the center of the COMMANDER exposure
chamber at a height of 4 feet. A perforated diffusion shield, as shown in Figure 2-6, was placed over the
fluidized bed. The shield was made of type 304 stainless steel with 0.25 in. holes and an open area of 58
percent.
Fans inside COMMANDER during the spore release created significant turbulence, forcing spores onto all
the surfaces. The real-time concentration of aerosols was monitored using a Dekati ELPI® (Tampere,
Finland) Once the aerosol concentration began to subside (indicating that most spores had been
released), the fans were turned off (to prevent the turbulence from beginning to remove the spores). The
chamber was aerated until no aerosol was detected inside the chamber (typically approximately two
hours).Spores were then cleaned from all surfaces except the carpet coupons.
For the first three tests, the walls, floors, and ceiling of COMMANDER were decontaminated using pH-
amended bleach to reduce chances of cross-contamination. For the fourth test, this method was not
available to use, so the walls, floors, and ceiling were decontaminated with Dispatch® (Caltech Industries,
Inc., Midland, Ml) wipes.
2ft.
_"_"_" _" J _"_"_"_"_"
J J J J w _"_"_"_"_"
J J J If J J J J J
J J J JV JJJJJJJJJ
j j j j* j^j j j j j j j
j j j j jj^j-fs-3-j j j
«j j j j j j j*i~3Ts * * w
•2 ft. diameter
Figure 2-6. Diffusion Shield
10
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2.1.7 Contamination Characterization
The "control" areas (described in Section 2.4 and shown in Figure 2-5 in that section) were sampled (as
described in Section 3.1.1.1.2). These "control" areas were the basis for any log reduction as a result of
decontamination procedures. This characterization sampling took place on the same day as the
decontamination procedure was carried out.
2.1.8 Decontamination Procedure
For Tests A, B and C, two different Century 400 (Chandler, AZ) Ninja Carpet Extractors (Figure 2-7) were
used for the decontamination procedure, the notable difference being that one carpet cleaner used a
heated surfactant (Century 400 Ninja 150 PSI, 411-22AHMO) while the other did not include a heater
(Century 400 Ninja 150 PSI, 411-22AMO). The same pre-treatment and surfactant (Judson Labs
[Greenville, S.C.] O2 Pre-Spray and Rinse System) were used for both carpet cleaners (Figure 2-8). Both
cleaners include a 12 in. dual jet head wand. The pretreatment was applied for a target time of 20
seconds at approximately 660 mL/minute before use of the carpet cleaner. The surfactant was placed in
the carpet cleaner reservoir and allowed to heat up (if the heater was present). The surfactant is sprayed
from the wand when the operator opens a trigger valve on the wand. During the decontamination
procedure, the wand was placed in the left corner closest to the operator and the surfactant was applied
along the full length of the carpet. The surfactant was then extracted by pulling the wand back in a
straight line using a single stroke left to right across the entire coupon. This pattern was repeated at a 90
degree angle to the first pass. Completion of the second pass was considered the end of a procedure.
Figure 2-7. Century 400 Ninja Carpet Cleaner
For Test D, Coupon A was saturated with Spor-Klenz from a backpack sprayer, back and forth across the
carpet with 50 percent overlap on each pass. Spor-Klenz was reapplied in this manner at 20 and 40
minutes from initial application.
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The carpet extractor was used to decontaminate Coupon B (see Figure 2-7). During the first
decontamination, the wand was placed in the center of the coupon on one side. The operator sprayed the
Spor-Klenz RTU solution (Steris Corporation, St. Louis, MO) while moving the wand toward the opposite
end of the coupon. The wand was then pulled back toward the operator, extracting the Spor-Klenz from
the carpet left to right across the top half of the coupon. The procedure was then repeated across the
bottom half of the coupon, with an overlap on the top half of approximately 10 percent.
Figure 2-8. Judson Labs O2 Pre-Spray and Rinse System
For Test D, Coupon A was saturated with Spor-Klenz from the backpack sprayer (ShurFlo 4 ProPack
Rechargable Electric Backpack Sprayer, SHURFLO, LLC., Elkhart, Indiana) back and forth across the
carpet with 50 percent overlap on each pass. Spor-Klenz was reapplied in this manner at 20 and 40
minutes from initial application.
The carpet extractor was used to decontaminate Coupon B (see Figure 2-9). During the first
decontamination, the wand was placed in the center of the coupon on one side. The operator sprayed the
Spor-Klenz RTU solution while moving the wand toward the opposite end of the coupon. The wand was
then pulled back toward the operator, extracting the Spor-Klenz from the carpet left to right across the top
half of the coupon. The procedure was then repeated across the bottom half of the coupon, with an
overlap on the top half of approximately 10 percent.
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Figure 2-9. Decontaminating Carpet B with Spor-Klenz using the Carpet Extractor
The second decontamination with the carpet extractor was slightly different. Due to the awkward nature
of the first decontamination (the COMMANDER was too narrow to pull the wand to the edge of the carpet
hence decontamination was performed in halves), the carpet was placed against the wall opposite the
operator to allow for more room. The wand was placed in the left corner closest to the operator and Spor-
Klenz was applied along the full length of the carpet. The Spor-Klenz was extracted by pulling the wand
back in a straight line using a single stroke left to right across the entire coupon.
2.1.9 Final Sterilization
Once all decontamination procedures had been completed (up to four repeat cleanings or until the
decontamination procedure was deemed successful), the material sections and COMMANDER were
sterilized again with VHP®. After this final sterilization, the material sections were discarded.
2.2 Test Matrix
To fulfill the project purpose (Section 1.1) and meet the objectives (Section 1.3), the test matrix shown in
Table 2-1 was developed. In the presentation of results (Section 6), the reasoning behind the change in
surface sampling methods is discussed.
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Table 2-1. Test Matrix
Test
A
B
C
D
Carpet Type
New
Old
New
New
Protocol
Hot vs. cold carpet cleaners
Hot vs. cold carpet cleaners
Hot vs. cold carpet cleaners
Backpack sprayer vs. cold
carpet cleaner
Carpet Cleaner Liquid
Judson Laboratories
Oxygen 2 Pre-Spray and
Extraction
Judson Laboratories
Oxygen 2 Pre-Spray and
Extraction
Judson Laboratories
Oxygen 2 Pre-Spray and
Extraction
Spor-Klenz
Sampling Techniques
Wipe Samples
HEPA sock samples
HEPA sock samples and
core samples
HEPA sock samples and
core samples
The decontamination procedure was repeated (up to four times) on materials to determine the number of
cleanings necessary to remove the spores as completely as possible. These tests were performed
according to the following parameters:
1. Each test was run independently.
2. A single test included the completion of all carpet cleaner types within that setup.
3. A material section blank was taken from each carpet section.
4. Following controlled contamination and decontamination, samples were collected for the test
sections of each material type.
5. Cleaning of COMMANDER and all equipment used during testing was performed as described in
Section 2.1.9 after the completion of each test.
6. Each 4 ft. x 4 ft. carpet section required three positive control samples and three test samples of
each carpet section for each of the consecutive decontamination procedures (up to four), as well
as vacuum liquid samples from the decontamination step for each carpet section. Hence, if four
consecutive decontamination procedures were conducted, a total of 16 vacuum samples was
generated for each carpet section (one vacuum sample for each 1 ft. x 1 ft. area). The exception
to this procedure was Test 1, using wipe sampling, which had six samples instead of three at
each sampling time.
7. The general testing sequence was shown in Figure 2-1. The following steps describe the testing
sequence:
a. All material sections needed for this project were prefabricated before any testing was
begun.
b. All spores for the study were prepared, per the method discussed in Section 2.1.5, prior
to the initiation of any testing.
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c. The material sections were installed in COMMANDER and sterilized using VHP®.
d. All material sections for a given test were contaminated in accordance with Section 2.1.6.
e. After air purging, personnel wearing appropriate PPE entered the COMMANDER. Initial
cleaning of the walls to reduce chances of cross-contamination was conducted.
f. All materials and equipment necessary for the decontamination procedure were gathered
and prepared as documented in Section 2.1.8.
g. Sampling of each test area was done according to Section 3.1.1.
h. Decontamination according to Section 2.1.8 was completed on one material section. All
decontamination steps were completed before moving on to the next material.
i. After all decontamination was complete, samples were recovered from the wet/dry
vacuums in accordance with Sections 3.1.2.
j. After a minimum of 18 hours and when all coupon surfaces were visibly dry, surface
sampling was done in accordance with Section 3.1.1.
k. Decontamination procedures were repeated up to four times, followed each time by
sampling.
I. Sample analysis was performed as described in Section 4.1. Data reduction and
validation were conducted as described in Section 6.1.
2.3 Sampling Strategy
The objective of the study was to assess the effectiveness of a decontamination procedure to
decontaminate the surfaces. The effectiveness was measured by the determination of the log reduction
calculated per Section 6.1. Hence, surface sampling of the test areas before and after decontamination
was required to determine the log reduction after application of the procedure. Because current surface
sampling techniques are intrusive, they will also remove viable spores from the surface of the section.
Sampling of positive control areas was required to compare to post-decontamination sampling of test
sections for this study. Positive controls and test areas are subsections of the carpet section. Positive
control areas were sampled in accordance with Section 4, before the decontamination procedure. The
entire carpet section, including test areas, is carried through the decontamination procedure, allowed to
dry, and subsequently sampled in accordance with Section 4.
The effectiveness of removing contamination from the surface of the sections provides critical information
regarding the potential of the procedure; however, field applicability is also dependent upon several other
factors including the ultimate disposition (or fate) of the spores. This latter information is required to
provide information pertinent to the development of a comprehensive site-specific remediation strategy.
For example, if viable spores are washed off materials, remediation field strategies might require rinsate
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collection and treatment. Hence, it is important to understand the fate of the spores resulting from the
application of the decontamination procedure on the section surface.
To obtain the additional critical information on the fate of the spores, several samples in addition to the
surface sampling of the sections was collected. To assess the potential for viable spores to be washed off
the surfaces, all liquids used in the decontamination process were collected and quantitatively analyzed
as a composite sample for the entire decontamination procedure on a particular carpet section.
Quantitative analysis was done on these rinsate samples to provide for an order of magnitude
determination of the disposition of viable spores in this media.
There are currently no validated methods for sampling biological agents from porous materials. Hence,
results from past field practices1 and recent studies2"5were used to define the surface sampling strategy.
For rough and/or porous surfaces, HEPA vacuum sampling is the preferred method.36 Limits of detection
(LOD) and sensitivities determined from the comparison of wipes on nonporous surfaces and the HEPA
vacuum on nonporous and porous surfaces indicate that HEPA vacuuming has a comparable LOD (400-
600 colony forming units [CPU] per sample area) to wipe sampling and an order of magnitude greater
sensitivity.2 Of the literature reviewed, however, only one reference provided a direct comparison between
HEPA vacuuming and wipe sampling for a porous surface (carpet).5 While wipe sampling had a higher
collection efficiency, the level of detection was lower for the HEPA vacuuming.
2.4 Sampling/Monitoring Points
The front face of each carpet section was the only surface of the sections that was sampled in this study.
Two 4 ft. x 4 ft. sampling templates of welded stainless steel wire were made for each carpet section, one
for before decontamination and one for post-decontamination sampling. The template, with 16 areas of 1
square foot, is shown in Figure 2-10. This template was placed against the material during the sampling
events. The only deviation from this sample grid was for the first test, where an 8 x 8 grid of 6 in. squares
was used. Six areas were sampled, rather than the three replicates described here. The change in
design simplified the sampling process and provided more information about the homogeneity of the
deposition.
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Figure 2-10. Carpet Section Template and Sample Grid
One area was sampled as a blank after sterilization. This area was "randomly" selected based on the last
digit of the date the blank sample was taken. A small indelible mark was placed on the sample area to
mark it as the starting point for the subsequent samples. As an example, blank sample testing on April
28th would indicate a blank sample taken from area 8. This area has been highlighted yellow in Figure
2-10.
After the controlled contamination, three areas were sampled as controls, i.e., indicative of spore counts
before decontamination. Starting from the blank sample area, every fifth area was designated as one of
these controls. In our example, this would mean that areas 13, 2, and 7 would be control sample areas
(highlighted red in Figure 2-10). A small mark in indelible ink was made on each area of the material
surface after sampling.
After decontamination, three more areas were sampled. The decontaminated samples were taken from
areas with preceding numbers to the control areas. In our example, these would be Areas 12, 1, and 6
(highlighted green in Figure 2-10). A small mark in indelible ink was made on each of these areas of the
material surface after sampling.
The decontamination procedure was repeated up to three more times, with successive rounds of
sampling being conducted on the sampling areas with preceding numbers to the first post-
decontamination sampling areas. In our scenario of blank testing starting on June 28th, areas 11, 16 and 5
would be sampled following the second round of decontamination; areas 10, 15, and 4 after the third
round of decontamination; and areas 9, 14, and 3 after the fourth and final round of decontamination. No
area was sampled twice.
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The liquid in each carpet cleaner recovery tank was analyzed independently. The COMMANDER
chamber was cleaned as detailed in Section 2.1.9 after the final sampling round.
2.5 Frequency of Sampling/Monitoring Events
Three surface samples of each carpet section were collected before decontamination (control samples)
and three samples after each of the following decontaminations (up to four) and the appropriate drying
time post-decontamination. The liquid collected by the cleaners from the carpet surfaces was filtered and
labeled appropriately (see Section 3.6) after the conclusion of decontamination of each carpet section.
2.6 Decontamination Event Sequence
For Tests A, B and C, the pre-spray and the solution used in the hot and cold vacuums were those
recommended by the manufacturer (Judson Laboratories Oxygen 2/Pre-Spray and Oxygen 2/Extraction,
respectively). The STERIS Spor-Klenz used in Test D was ready-to-use as provided by the manufacturer.
Material Safety Data Sheets (MSDS) for each decontamination solution are included in Appendix B.
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3 Testing and Measurement Protocols
Several types of samples were included in this project. Surface sampling procedures were used to collect
samples from the coupon materials. These samples included wipe samples as well as HEPA socks. Core
(punch or hole saw) samples were taken for spore enumeration via extractive analysis. In addition, swab
sampling was done for each sterilization batch for all equipment used during decontamination (vacuum
nozzles, etc), as well as to identify spores captured in the any filter associated with the wet vacuum
cleaners used during the decontamination. The rinsate generated during the decontamination procedure
was collected for each material type. Details of the sampling procedures are provided below. A laboratory
notebook was used to document the details of each sampling event (or test).
3.1 Methods
3.1.1 Surface Sampling
Prior to the sampling event, all materials needed for sampling were prepared. The materials specific to
each protocol are included in the relevant sections below. In addition, general sampling supplies were
also needed. A sampling material bin was stocked for each sampling event, using the information
included in Section 3.4 (Table 3-2). The bin contained enough sampling kits to accommodate all required
samples for the specific test. Additional kits of each type were also included for back-up. Sufficient
prepared packages of gloves and bleach wipes were also included in the bin. 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 bleach wipe ortowelette moistened with a solution of
pH-adjusted bleach prior to transport from the sampling location to the Microbiology Laboratory.
3.1.1.1 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.7 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.7 The protocol that was used in this project is described below and has been adapted
from that provided by Busher et al.7 and Brown et al.8, and documented in the INL 2008 Evaluation
Protocols.9 None of these references provides a validated wipe procedure for Bacillus spores, as a
validated sampling procedure does not currently exist.
The following procedure was used in this study for wipe sampling of each coupon surface:
1. A two-person team was used, employing aseptic technique throughout. The team consisted of a
sampler and a support person.
2. All materials needed for collection of each sample were prepared in advance using aseptic technique.
A sample kit for a single wipe sample was prepared as follows:
a. Two sterile sampling bags 10 in. x 15 in. (Fisherband Twirl'Em Sterile Sampling Bags, item
14-955-196, Pittsburg, PA) 5.5 in. x9 in. (Fisherbrand Sterile Sampling Bags, item 14-955-
185, Pittsburgh, PA) and a 50 mL conical tube (Becton Dickson Labware, item 352098,
Franklin Lakes, NJ), capped, were labeled. These bags and conical tube had the same label.
The 5.5 in. x 9 in. labeled sterile sampling bag was referred to as the sample collection sterile
sampling bag.
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b. A dry sterile wipe was placed in an unlabeled sterile 50 ml conical tube using sterile forceps
and aseptic technique. The wipe was moistened by adding 5 ml of sterile phosphate buffered
saline with 0.05% TWEEN®-20 (item P-3563, SIGMA, St. Louis, M.O., USA). The tube was
then sealed.
c. The labeled 50 ml conical tube, capped, the unlabeled conical tube containing the pre-
moistened wipe, and the 5.5 in. x9 in. labeled sampling bag were placed into the 10 in. x 15
in. labeled sterile sampling bag. Hence, each labeled sterile sampling bag contained a
labeled 50 ml conical tube (capped), an unlabeled capped conical tube containing a pre-
moistened wipe, and an empty labeled sterile sampling bag.
d. Each prepared bag was one sampling kit.
3. The sampler and support person placed the template onto the coupon surface.
4. Each member of the sampling team donned a pair of sampling gloves (a new pair per sample); the
sampler's gloves were sterile sampling gloves. All members wore N95 dust masks to further minimize
potential contamination of the samples.
5. The support person removed a sample kit from the sampling bin and confirmed sample ID to sampler.
6. The support person:
a. Opened the outer sterile sampling bag touching the outside of the bag.
b. Touching only the outside of the outer bag, maneuvered the unlabelled conical tube until it
was at the opening of the bag. The sampler then grabbed the tube, removed it from the bag,
opened the tube, and poured the pre-moistened wipe into the glove of the sampler.
c. Discarded the unlabelled conical tube.
d. Maneuvered the labeled 50 ml conical tube to the end of the outer sterile sampling bag and
loosen the cap.
e. Removed the cap from 50 ml conical tube immediately preceding the introduction of the
sample into the tube.
7. The sampler:
a. Squeezed out excess moisture from the wipe before approaching sample area.
b. Confirmed the area ID of the carpet to sample.
c. Wiped the surface of the sample horizontally using S-strokes to cover the entire sample area
of the coupon using a consistent amount of pressure.
d. Folded the wipe concealing the exposed side and then wiped the same surface vertically
using the same technique.
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e. Folded the wipe over again and rolled the folded wipe to fit into the conical tube.
f. Carefully placed the wipe into the 50 ml conical tube that the support person was holding
being careful not to touch the surface of the 50 ml conical tube or plastic sterile sampling
bag.
8. The support person immediately closed and tightened the cap to the 50 ml conical tube and slid the
tube back into the sample collection sterile sampling bag.
9. The support person then put the 50 ml conical tube into the empty labeled 5.5 in. x 9 in. sampling
bag and sealed the bag.
10. The support person then sealed the outer sample collection bag containing the capped 50 ml conical
tube (containing the sample wipe) inside a sealed 5.5 in. x 9 in. sample collection bag.
11. The support person then decontaminated the outer sample bag by wiping it with a Dispatch® bleach
wipe.
12. The support person then placed the triple-contained sample into the sample collection bin.
13. All members of the sampling team removed and discarded their gloves.
14. Steps 3-13 were repeated for each sample collected.
3.1.1.2 HEPA Vacuum Sampling
HEPA vacuum sampling is typically used for large porous areas. The general approach is that a collection
sock is used to trap dust material. The protocol that was used in this project is depicted below and has
been adapted from that provided by Busher et al.2 and Brown et al.3 and documented in the INL 2008
Evaluation Protocols9. None of these references provides a validated HEPA vacuuming procedure for
Bacillus spores, as a validated sampling procedure does not currently exist.
The following procedure, shown in Figure 3-1, was used in this study for HEPA vacuum sampling the
surfaces of each area of carpet section or blank coupon:
1. A two person team was used. The team consisted of a sampler and a support person.
2. All materials needed for each sample to be collected were prepared in advance. A sample kit for a
single HEPA vacuum sample was prepared as follows:
a. Two sterile sampling bags were labeled in accordance with Section 3.6. These bags had the
same label. An additional unlabeled bag was utilized.
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Figure 3-1. HEPASock Sampling a Coupon Section for Viable Spores
b. A HEPA sock assembly was placed into one of the unlabeled sterile sampling bags.
c. The bag containing the HEPA sock assembly was placed into a labeled sterile sampling bag; the
second unlabeled bag was also placed into the labeled bag. The label was clearly distinguishable
through the unlabeled bag.
d. Each prepared bag was one sampling kit.
3. The sampler and support person placed the template onto the coupon surface.
4. Each member of the sampling team donned a pair of sampling gloves (a new pair per sample).
5. The sampler plugged in the HEPA vacuum power cord and then donned his/her gloves.
6. The sampler placed the HEPA vacuum onto a convenient surface and held the vacuum nozzle for the
support person to place the HEPA vacuum sock assembly onto the nozzle.
7. The support person opened the sampling supply bin and removed one HEPA vacuum sock sample kit
from the bin.
8. The support person recorded the sample collection bag number on the sampling log sheet or
laboratory notebook.
9. The support person recorded the coupon code on the sampling log sheet or laboratory notebook next
to the corresponding sample collection bag number that was just recorded.
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10. The support person:
a. Opened the outer sampling bag containing the HEPA vacuum sock assembly.
b. Opened the bag within the outer sterile sampling bag and pushed the HEPA vacuum sock
assembly from the bottom to expose the cardboard applicator tube opening.
c. Placed the HEPA vacuum sock assembly onto the nozzle of the vacuum tube, using the inner
sampling bag to handle the HEPA sock assembly, while the sampler held the vacuum nozzle.
11. The sampler:
a. Turned on the vacuum.
b. Vacuumed "horizontally" using S-strokes to cover the 1.0 sq. ft. sample area of the template,
while keeping the vacuum nozzle perpendicular to the sample surface.
c. Vacuumed the same area "vertically" using the same technique.
d. Turned off the vacuum when sampling was completed.
12. The support person opened the labeled sterile sampling bag and removed the HEPA sock assembly
from the nozzle by sliding the sampling bag over the HEPA sock assembly and gripping the sock from
the outside of the bag.
13. The support person then sealed the inner sterile sampling bag and placed it into the outer sterile
sampling bag.
14. The support person then sealed the outer sterile sampling bag and wiped the outer bag with a bleach
wipe.
15. The support person then placed the outer sample bag into the remaining labeled sterile sampling bag
and disposed of the bleach wipe.
16. The sampler wiped the nozzle (inside and out) and ends of the tubing with bleach wipe, then
disposed of the bleach wipe.
17. The support person then placed the triple-contained sample into the sample collection bin.
18. If sampling from the carpet section was completed, the sample handler marked the tested areas as
having been sampled and moved the template to the appropriate location for decontamination.
19. All members of the sampling team removed and discarded their gloves.
20. Steps 3-19 were repeated for each sample to be collected.
A sterilized stainless steel nozzle was used between the HEPA sock and the vacuum hose. A separate
nozzle was used for each carpet cleaning technique.
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3.1.2 Core Sampling
The following procedure was used in this study for core sampling of each coupon surface. This procedure
involved a two-person team, referred to below as the sampler and the assistant. Punch sampling (utilizing
a punch and hammer) was employed for Tests C and D. Because of the difficulty in obtaining core
samples with this procedure, it was modified to use a hole saw. The resulting minor modifications to this
procedure are shown in italics.
1. Kit Assembly
a. PPE for non-HazMat situations was donned.
b. Sample IDs were determined.
c. A sterile 50 ml vortex tube (or 120 mL sample cup) was labeled with each sample ID.
d. An over pack bag was labeled with each sample ID.
e. The kits were assembled ahead of time. Each kit included a clean, sterile 50 ml tube (or
cup), a small bag, and a pair of sterile tweezers, all packed into the larger bag.
2. Sample Collection
This procedure followed HEPA Sock sampling.
a. The assistant donned clean gloves.
b. The assistant opened the over pack bag and maneuvered the tweezers from the outside of the
over pack until the assistant could grab the package.
c. The sampler recorded the time and the sample ID (and the size of the hole saw and the pilot bit).
d. The sampler donned sterile gloves.
e. The assistant opened the tweezers package so that the sampler could grab the sterile tweezers.
f. The assistant opened the bag holding the sterilized punch (or the bag holding the sterilized hole
saw and attaches it to the drill using the outside of the autoclave bag to maintain sterility).
g. The assistant identified the sample or sample area as indicated by the sample ID on the over
pack bag.
h. The assistant used the punch and the hammer (or drill) to collect the extractive sample from the
center of the carpet sampling area identified in the previous Step g. This often required repeated
blows.
i. Once the carpet had been cut, the core sample typically was lodged inside the punch (or hole
saw). The assistant held the punch such that the sampler could use the tweezers to grab the
carpet piece (or the assistant used the Allen wrench to loosen the pilot bit. The sampler removed
24
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the bit. The sampler then used the tweezers to grab the carpet piece). If the carpet core did not
lodge inside the punch (or hole saw), but remained on the carpet surface, the sampler used the
tweezers to collect as much of the core as possible. While the sampler was holding the circle of
carpet, the assistant maneuvered the 50 ml vial (or 120 mL sterile cup) from the outside of the
over pack.
j. The assistant opened the vial (or cup), touching only the cap.
k. The sampler put the carpet into the vial (or cup).
I. The assistant aseptically returned the cap to the vial (or cup) and closed the vial (or cup).
m. The assistant placed the vial (or cup) in the small inner bag and wiped the outside of the bag with
a Dispatch® wipe.
n. The inner bag was placed inside the over pack bag, and was wiped with a Dispatch® wipe. (The
assistant then removed the used hole saw from the drill and placed in a bag for eventual re-
sterilization.)
3.1.3 Swab Sampling
Two types of swab samples were collected, though a similar procedure was used for each. Swab
sampling was done for three items of each sterilization batch for all equipment used during
decontamination (vacuum nozzles, etc). The process for this sampling is described below.
1. The sampler donned a P95 respirator, bouffant cap, gloves, disposable lab coat, and safety glasses
2. The sampler:
a. Opened the package and removed the BactiSwab™ (Remel, item 12100/12110, Lenexa, KS).
b. Labeled the plastic tube appropriately using the scheme detailed in Section 3.6.
c. Removed the cap-swab from the plastic tube.
d. Swabbed the surface while spinning the cap-swab between the thumb and index fingers.
e. Returned cap-swab to tube.
f. Through the sleeve, crushed the BactiSwab™ Transport System ampoule at midpoint.
g. Held BactiSwab™ tip end to allow the medium to wet the swab.
h. The sampler dated and initialed each sample tube.
3. All BactiSwab™ Transport Systems were placed in a 5 in. x 9 in. sterile sampling bag.
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4. A chain of custody form was completed and the samples relinquished to the Microbiology Laboratory.
This procedure was modified slightly for the final Test D. Disposable booties were added to the PPE to
help prevent re-contamination of the COMMANDER during subsequent decontamination and sampling
events. Second, the BactiSwab™ ampoule was broken prior to taking the swab sample, as it was
determined that a moistened swab increased the collection efficiency. The process for this sampling is
described below.
1. The sampler donned a P95 respirator, bouffant cap, gloves, disposable lab coat, disposable booties,
and safety glasses
2. The sampler:
a. Through the sleeve, crushed the BactiSwab™ Transport System ampoule at midpoint.
b. Held BactiSwab™ tip end up for at least five seconds to allow the medium to wet the swab.
c. Opened the package and removed the BactiSwab™.
d. Labeled the plastic tube appropriately using the scheme detailed in Section 3.6.
e. Removed the cap-swab from the plastic tube.
f. Swabbed the surface while spinning the cap-swab between the thumb and index fingers.
g. Returned cap-swab to tube.
3. All BactiSwab™ Transport Systems were placed in a 5 in. x 9 in. sterile sampling bag.
4. A chain of custody form was completed and the samples relinquished to the Microbiology Laboratory.
3.1.4 Rinsate Collection and Sampling Procedures
The liquid collected by the carpet cleaners during the decontamination procedure was collected fora
given carpet section. After all steps of the decontamination process had been completed, aliquots of the
collected liquid in the cleaner were filtered immediately to remove the spores from the liquid according to
the protocol described in Section 4.1.2 below. The filtered spores were then rinsed with Dl water to
remove any residual surfactant.
3.2 Prevention of Cross-contamination of Sampling/Monitoring Equipment
Several management controls were put in place to prevent cross-contamination. This project was labor
intensive and required that many activities be performed on carpet sections or coupons that were
intentionally contaminated (test coupons and positive controls). The treatment of these two groups of test
areas (positive control and test) varied for each group. Hence, specific procedures were put in place in
the effort to prevent cross-contamination among the groups. Adequate cleaning of all common materials
26
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and equipment was critical in preventing cross-contamination. Cleaning methods for this purpose are
listed in Table 3-1.
There were four primary activities for each test in the experimental matrix. These activities were
preparation of the coupons, execution of the decontamination process (including sample recovery),
sampling, and analysis. Coupons were fumigated with VHP® prior to the contamination process. Specific
management controls for each of the three following activities are described below.
3.2.1 Preventing Cross-Contamination during Execution of the Decontamination
Process
The decontamination process was labor intensive; it required that a multistep procedure be executed
repeatedly for each coupon. Additionally, the process occurred using a single test chamber. Hence,
controlling the order of processing and actions taken to minimize cross-contamination were essential.
The following management controls were followed in an effort to minimize the potential for cross-
contamination:
Table 3-1. Cleaning Methods and Frequency for Common Test Materials/Equipment
Material/Equipment
COMMANDER
Wet vacuums
Heads of wet vacuums
Other Bulk equipment (fans,
templates, etc)
Use
Contain carpet sections during the
application of the decontamination
procedure being tested
Part of the decontamination
procedure
Part of the decontamination
procedure
Various
Cleaning Method
Fumigation with VHP® or washing
with pH-adjusted bleach solution in
accordance with the wet/dry vacuum
cleaning procedure.*
pH-adjusted bleach solution
pH-adjusted bleach solution
Fumigation with VHP or washing
with pH-adjusted bleach solution in
accordance with the wet/dry vacuum
cleaning procedure.*
Following use in a test, the wet/dry vacuum (including head assembly) was cleaned by being fumigated with a
STERIS VHP® sterilization cycle. This cycle entailed the use of a STERIS VHP® ARD H2O2 generator and
exposure of all components of the wet/dry vacuum for a minimum concentration * time (CT) of 1000 ppm*hours.
27
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• COMMANDER was cleaned prior to the start of each test in Table 2-2 via fumigation with VHP .
• COMMANDER was cleaned with a pH-adjusted bleach solution as described above following the
controlled contamination procedure and before decontamination of the carpet sections began.
• COMMANDER was cleaned after completion of each test in Table 2-2 via fumigation with VHP®.
• All cleaner wands were decontaminated after use by soaking in a pH-adjusted bleach solution for at
least one hour. Wands were put in the solution immediately after use to eliminate any accumulation
of contaminated equipment in the test area. The bucket of pH-adjusted bleach solution for test
equipment clean-up was clearly identified and maintained separate from the cleaning solutions being
used for the test coupon decontamination process.
• Testing was done using a "clean team/dirty team" technique. One dirty team was responsible for
chamber wipe-down and moving the carpet sections into the decontamination shower. A clean team
(with clean and dirty members) was used for control sampling. A dirty team performed the
decontamination procedure. A clean team (with clean and dirty members) was used for test
sampling. Only dirty members handled contaminated items and only clean members handled
samples.
3.2.2 Preventing Cross-Contamination during Sampling
Sampling poses an additional significant opportunity for cross-contamination of samples. In an effort to
minimize the potential for cross-contamination, several management controls were implemented.
• In accordance with aseptic technique, a sampling team made up of a "sampler" and a "support
person" was utilized.
• The sampler handled only the sampling media and the support person handled all other supplies.
The sampler sampled the surface according to the appropriate procedure described in Section 3.1.1.
• The collection medium (e.g., HEPA filter) was then placed into a sample container that was opened,
held and closed by the support person.
• The sealed sample was handled only by the support person.
• All of the following actions were performed only by the support person, using aseptic technique:
.1.1 The sealed bag with the sample was placed into another sterile plastic bag that was then sealed;
that bag was then decontaminated using a bleach wipe.
.1.2 The double-bagged sample was then placed into a third sterile bag that was sealed and then
placed into a sterile sample container for transport.
.1.3 The exterior of the transport container was decontaminated by wiping all surfaces with a bleach
wipe ortowelette moistened with a solution of pH-adjusted bleach prior to transport from the
sampling location to the Microbiology Laboratory.
28
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• The sampling crew then changed their gloves in preparation for working with the next sample.
3.2.3 Preventing Cross-Contamination during Analysis
Aseptic laboratory technique was followed per the standard operating procedures (SOPs) and
miscellaneous operating procedures (MOPs) of the Microbiology Laboratory. The SOPs and MOPs
document the aseptic technique employed to prevent cross-contamination. Additionally, the order of
analysis (consistent with the above) was as follows: (1) all blank coupons; and (2) all positive control or
decontaminated coupons.
3.3 Representativeness
The representativeness of the test material, decontamination procedure, and equipment used were
critical attributes to assure reliable test results. Representativeness of the test materials means that the
materials used are typical of such materials used in buildings in terms of quality, surface characteristics,
structural integrity, etc. The materials chosen for this study (carpet) are representative of surfaces that are
likely to contribute significantly to the overall decontamination challenge in the event of a wide area
release of 8. anthracis spores. The particular carpet chosen for this study is representative of modern low
Volatile Organic Compound (VOC) carpets used in large government institutions; this carpet is being
used by US EPA in the RTP, NC facility. Representativeness was assured by selection of test materials
that met government procurement specifications and by obtaining those materials from appropriate
suppliers. The material coupons were fabricated to be representative of the bulk surfaces. The size of
the carpet sections was chosen to be representative of a large surface area yet manageable within the
confines of COMMANDER. The sampling strategy for the 1 ft. x 1 ft. sample areas analyzed 18% of the
carpet section both before and after each decontamination. The equipment used in the decontamination
procedures was also representative of the equipment actually used in the field. The only minor exception
is that the equipment was chosen with a preference to allow for as much quality assurance as possible.
During analysis, samples were homogenized to ensure that any aliquots taken were representative of the
bulk titer (e.g., viable spores per ml).
3.4 Sample Quantities
For each carpet section, and assuming that four decontaminations were performed, a total of 29 samples
were generated. The total numbers of samples of each type for each test are listed in Table 3-2. As
discussed in Section 3.3, the samples from each test were analyzed in six batches, represented by "Test
Day" in Table 3-2.
3.5 Sample Containers for Collection, Transport, and Storage
For each wipe, the primary containment was an individual sterile 50 ml conical tube. Secondary and
tertiary containment were sterile sampling bags. The primary, secondary, and tertiary containment of
each HEPA vacuum sock consisted of separate sterile sampling bags. All samples from a single test
were then placed in a sterilized plastic bin. Sterilization of all containers prior to their use for the test
samples was via autoclaving on a gravity cycle or via use of pH-adjusted bleach solution. After samples
were placed in the container for storage and transport to the Microbiology Laboratory, the container was
wiped with a towelette saturated with a pH-adjusted bleach solution. A single plastic bin was used for
storage in the decontamination laboratory during sampling and for transport to the Microbiology
Laboratory.
29
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Table 3-2. Sample Quantities for Each Test Setup
Test
Day
1
2
3
5
7
9
Description
Carpet Sterility
Sampling
Post-Controlled
Contamination
Control Sampling
Decontamination 1
Sampling
Decontamination 2
Sampling
Decontamination 3
Sampling
Decontamination 4
Sampling
Sterility
Blank
2
Control Samples
3 Samples x 2
Decontamination Types
(Test A- 6 samples)
Test Samples
3 Samples x 2
Decontamination Types
3 Samples x 2
Decontamination Types
3 Samples x 2
Decontamination Types
3 Samples x 2
Decontamination Types
Liquid Filter
samples
2
2
2
2
3.6 Sample Identification
Each carpet section was identified by a description of the material and a unique sample number. The
sampling team maintained an explicit laboratory log which included records of each unique sample
number and its associated test number, contamination application, any preconditioning and treatment
specifics, and the date treated. Each carpet section test area sample was marked with only the material
descriptor and unique code number. The wet/dry vacuum samples from each test were identified with an
associated test number and carpet section type. The sample codes eased written identification. Once
the coupons were transferred to the Microbiology Laboratory for plate counts, each sample was
additionally identified by replicate number and dilution. The Microbiology Laboratory also included on
each plate the date it was placed in the incubator.
3.7 Sample Preservation
After sample collection, sample integrity was maintained by storage of samples in quadruple containers
(1 - sample collection container, 2 - sterile bag, 3 - sterile bag with exterior sterilized during sample
packaging process, 4 - sterile container holding all samples from a test). All individual sample containers
remained sealed while in the decontamination laboratory or in transport after the introduction of the
sample. The locking lid on the container holding all samples remained closed except for the brief period it
was opened for sample introduction by the support person of the sampling team. The sampling person
did not handle any samples after they were relinquished to the support person during placement into the
primary sample container.
In the Microbiology Laboratory, all samples were stored in the refrigerator at approx. 4 °C±2°C until they
were analyzed. All samples were allowed to stabilize at room temperature for 1 hour prior to analysis.
30
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3.8 Sample Holding Times
After sample collection for a single test was complete, all samples were transported to the Microbiology
Laboratory immediately, with appropriate chain of custody form(s). The samples were stored in
accordance with Section 3.7 and no longer than ten days before they were analyzed. A typical holding
time for most samples was a maximum of two days.
3.9 Sample Handling and Custody
Careful coordination with the Microbiology Laboratory is required to arrange for successful transfer of
uncompromised samples in a timely manner for analysis. Test schedules were confirmed with the
Microbiology Laboratory prior to the start of each test. To ensure the integrity of samples and to maintain
a timely and traceable transfer of samples, an established and proven chain of custody or possession is
mandatory. It is imperative that accurate records be maintained whenever samples are created,
transferred, stored, analyzed, or destroyed. The primary objective of these procedures is to create an
accurate written record that can be used to trace the possession of the sample from the moment of its
creation through the reporting of the results. A sample is in custody if it is in any one of the following
states:
• In actual physical possession.
• In view, after being in physical possession.
• In physical possession and locked up so that no one can tamper with it.
• In a secured area, restricted except to authorized personnel.
• In transit.
Laboratory test team members received copies of the test plans prior to each test. Pre-study briefings
were held to apprise all participants of the objectives, test protocols, and chain of custody procedures to
be followed. These protocols were required to mesh with any protocols established by EPA.
In the transfer of custody, each custodian signed, recorded, and dated the transfer on the Chain of
Custody (COC). Sample transfer could be on a sample-by-sample basis or on a bulk basis. The following
protocol was followed for all samples as they were collected and prepared for distribution:
• A chain of custody record accompanied the samples. When turning over possession of samples, the
transferor and recipient signed, dated, and noted the time on the record sheet. This record sheet
allowed transfer of custody of a group of samples from H130-A to the Microbiology Laboratory.
• If the custodian had not been assigned, the laboratory technician had the responsibility of packaging
the samples for transport.
• Samples were carefully packed and hand-carried between on-site laboratories.
• The chain of custody record showing the identity of the contents accompanied all packages.
31
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3.10 Sample Archiving
All samples and diluted samples were archived for two weeks following completion of analysis. This time
allowed for all data to be processed according to quality control requirements and allowed for the data to
be reviewed. Samples were archived by maintaining the primary extract at 4 °C+/-2°C in a sealed
extraction tube. Two weeks post-analysis, all samples were discarded.
32
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4 Testing and Measurement Protocols
The primary results from this study were from the analysis of samples in the Microbiology Laboratory,
resulting in recovered CPUs per sample expressed on a log-10 scale. This analysis for each sample type
is detailed in Section 4.1.
Additional measurements prior to or during the decontamination procedure application were also required
to ensure quality control in the testing.
The time for application of each procedural step and time between procedural steps on each coupon was
measured using a stopwatch (Table 3-1) and recorded in the laboratory notebook.
4.1 Sample Analyses
The Microbiology Laboratory analyzed all samples for presence (swab samples) or to quantify the number
of CPU per sample (wipe, vacuum, or core samples), which were used as per Section 6.1. For all sample
types, phosphate buffered saline with 0.05% TWEEN®-20 (PBST) were used as the extraction buffer.
After the appropriate extraction procedure (as described in the sections to follow), the buffer was
subjected to a four stage serial dilution (10° to 10~4) in accordance with MOP 6535a (a revision of MOP
6535 specifically for bacterial spores; attached as Appendix C). The resulting samples were plated in
triplicate and incubated overnight. CPU were counted as detailed in MOP 6535a.
The PBST was prepared according to an internal MOP. The extraction procedure used to recover spores
will be varied depending upon the different matrices (HEPA filter socks, wipe samples, core samples).
The procedures are described in the following subsections.
4.1.1 Recovery from HEPA Vacuum Sample
The recovery of the spores from the HEPA socks was done as follows, as adopted from the Idaho
National Laboratory (INL) 2008 Evaluation Protocols:9
1. The analyst donned a fresh pair of gloves. Gloves were changed periodically (at least between
batches) or after direct contact with a sample to reduce contamination.
2. Sterile 3 oz. specimen cups were pre-labeled as per the sample log corresponding to the batch of
samples being processed.
3. The 3 oz. specimen cup sample containers were loaded with 20 mL of PBST.
4. Both sterile sample bags were opened without removing the inner bag from outer bag. The HEPA
sock assembly was moved to the opening of the bag using the bag.
5. The HEPA sock was removed from the assembly using sterile forceps while holding the cardboard
applicator from the outside of the bag. The HEPA sock was placed into the corresponding pre-
labeled specimen cup containing 20 mL PBST. The plastic bag with cardboard applicator was
discarded. A new pair of sterile forceps was used for each sample.
33
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6. After use, forceps were placed in a container of pH-adjusted bleach solution. The forceps were
soaked for at least one hour before being autoclaved using a gravity cycle in preparation for use with
the next sample batch.
7. The HEPA sock was wetted by holding the upper blue portion of the HEPA sock and dipping the
lower 1 inch of the HEPA sock into the PBST. The HEPA sock was allowed to soak up the PBST for
a few seconds.
8. After the soaking, the HEPA sock was lifted up just above the opening of the specimen bottle. A 1-
inch vertical slit was cut up the center from the bottom of the sock using sterile scissors. A new pair
of scissors was used for each sample.
9. The HEPA sock was cut horizontally from side to side about 1 inch from the bottom, allowing the two
pieces to fall into the specimen bottle. The HEPA sock was only cut where the sock had been wetted
(dip, wet, look, cut).
10. Steps 7-9 were repeated until the entire white portion of the HEPA sock was cut.
11. The upper top blue portion of the HEPA sock was then discarded.
12. After use, scissors were placed in a container of pH-adjusted bleach solution. The scissors were
soaked for at least one hour before being autoclaved using a gravity cycle in preparation for use with
the next sample batch.
13. Gloves were changed between samples.
14. Steps 4-12 were repeated for each sample in the batch.
15. Twelve samples at a time were loaded into the well plate of the incubator shaker (Lab-Line, Melrose
Park, IL).
16. The samples were agitated in the shaker incubator at 300 revolutions per minute (rpm) for 30 minutes
with the heat off.
17. The samples were then removed from the shaker incubator and brought to the BioSafety Cabinet
(NuAire, Inc., Plymouth, MN) for dilution plating as described in Section 4.1.
4.1.2 Filter Plating
Filter plating was done by the Microbiology Laboratory for the Spor-Klenz tests as well as for the rinsate
samples. Three 100 mL aliquots of Spor-Klenz or rinsate were delivered to the Microbiology Laboratory.
One of the 100 mL aliquots was filtered by pouring through a 0.2 micron Nalgene filter unit (Thermo-
Scientific, Waltham, MA). The filter was then rinsed by pouring 10 mL of sterile deionized water over the
filter. The filter was removed from the filtration unit aseptically with disposable thumb forceps and placed
onto a Tryptic Soy Agar (TSA) plate, filter side up. The plate was then placed in a 35 °C+/-2°C incubator
(Thermo-Scientific, Waltham, MA), for at least 18 hours. Filter plating was also done for any samples for
which there were fewer than 30 CFU in the primary dilution sample.
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4.1.3 Recovery of Core Samples
The smaller core punches used in Test C were processed in 20 ml PBST tubes. They were sonicated for
10 minutes, vortexed for two continuous minutes, and then vortexed immediately prior to dilution plating
as described in Section 4.1. The larger hole saw cores used in Test D were processed in 40 ml PBST
specimen cups. The cups were placed in the orbital shaker incubator for 30 minutes at ambient
temperatures and were vortexed immediately prior to dilution plating as described in Section 4.1.
4.2 Analysis Equipment Calibration
Standard laboratory equipment such as biological safety cabinets and incubators were routinely
monitored for proper performance. All equipment was verified as being certified calibrated or having the
calibration validated by the Metrology Laboratory at the time of use. Calibration of instruments was done
at the frequency shown in Table 4-1. Any deficiencies were noted. The instrument was adjusted to meet
calibration tolerances and recalibrated within 24 hrs. If tolerances were not met after recalibration,
additional corrective action was taken, possibly including the replacement of the equipment.
Table 4-1. Instrument Calibration Frequency
Equipment
Thermometer
Micropipettes
Clock
Biological
Cabinet
Calibration/Certification
Compared to independent National Institute for Standards and Technology
(NIST) thermometer (this is a thermometer that is recertified annually by either
NIST or an International Organization for Standardization (ISO)-17025 facility)
value once per quarter.
All micropipettes were verified to be within the calibration date at time of use.
Pipettes were recalibrated by gravimetric evaluation of pipette performance to
manufacturer's specifications every year by supplier (Rainin Instruments,
Oakland, C.A., Ovation, VistaLabs, Brewster, NY).
Compared to office U.S. Time @ www.NIST.time.gov every 30 days.
The biological cabinets were verified to be within certification dates at the time of
use. Biological Cabinets are adjusted yearly to be within flow tolerances
established by the manufacturer.
Expected
Tolerance
±rc
±5%
±1 min/30
days
35
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5 Quality Assurance
5.1 Data Quality
The objective of this study was to investigate the reduction in viable spores on and within the carpet
surfaces (effectiveness) as a function of the cleaning technique and duration of application to various
carpet types. This section discusses the Quality Assurance/Quality Control (QA/QC) checks (Section 6.2)
and Data Quality Objectives (DQOs; Section 6.3) considered critical to accomplishing the project
objectives.
The QAPP10 in place for this testing was followed with several deviations, many of which were
documented in the text above. Deviations included incorporating HEPA sock samples in place of the
inefficient wipe samples, and the addition of the core (extractive) samples and the use of Spor-Klenz.
These deviations did not substantially affect data quality and were necessitated by the test results
themselves.
5.2 Quality Assurance/Quality Control Checks
Quantitative standards do not exist for biological agents. Quantitative determinations of organisms in this
investigation did not involve the use of analytical measurement devices. Rather, the CPU were
enumerated manually and recorded. Critical QC checks are shown in Table 5-1, with acceptance criteria
set at the most stringent level that could routinely be achieved and that were consistent with the DQOs.
Table 5-1. Quality Control Checks
QC Sample
Positive Control
(sample from carpet
section area contaminated
with biological agent but
not subjected to the test
conditions)
Blank ISA, Sterility Control
(plate incubated, but not
inoculated)
Stability Control
Information Provided
Initial contamination level
on the coupons; allows for
determination of log
reduction (see Section
5-1); controls for
confounds arising from
history impacting
bioactivity; controls for
special causes.
Controls for sterility of
plates.
Verifies that the spores are
stable for the length of time
required for multiple
decontamination cycles
Acceptance Criteria
Target loading of 1E7 CPU
per sample with a standard
deviation of
<0.5. (5E6-5E7
CPU/sample);
No observed growth
following incubation.
No downward trend over
time
Corrective Action
Outside target range:
discuss potential impact on
results with EPAWAM;
correct loading procedure
for next test and repeat
depending on decided
impact.
Incubate additional ten
plates. If any additional
growth is observed, reject
results from the lot.
Re-evaluate efficacy
results based on natural
decay.
The positive control samples did not meet acceptance criteria for Tests A and B, but were deemed high
enough to continue with the testing. Extractive samples did not meet the criteria because of their smaller
size.
All sterility control samples met the acceptance criteria.
36
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The stability control samples did show a downward trend overtime, on the order of 0.5 log reduction. This
downward trend has not been factored into the log reduction values.
5.3 Data Quality Objectives
The DQOs define the critical measurements (CMs) needed to address the stated objectives and specify
tolerable levels of potential error associated with simulating the prescribed decontamination
environments. The following measurements were deemed to be critical to accomplish part or all of the
project objectives:
• time
• CPU counts.
The Data Quality Indicators (DQIs) listed in Table 5-2 are specific criteria used to quantify how well the
collected data meet the DQOs. Detection limits were defined by the QAPP as 50% of the minimum
number of detectable spores, or 0.5 CPU.
Table 5-2. DQIs for Critical Measurements
Measurement
Parameter
Counts of CPU
Streak Plate
Analysis Method
Visual counting,
See Section 4.1
Visual detection
of growth
Accuracy
±10% of
CPU count
N/A*
Detection
0.5 CPU
0.5 CPU
Completeness Goals
100%
100%
Actual Completeness
100%
100%
*N/A= not applicable
The quantitative acceptance criteria in terms of precision (%Relative Standard Deviation, %RSD) for each
critical measurement are shown in Table 5-3. Tests with conditions falling outside these criteria were
rejected and repeated. Decisions to accept or reject tests were based upon engineering judgment used
to assess the likely impact of the parameter on the conclusions drawn from the data.
Table 5-3. Acceptance Criteria for Critical Measurements
Measurement Parameter
Target Value
Precision RSD
Test coupon replicates
Negative control CPUs
30 - 300 CPU per
quantifiable plate
0-1 CPU per
sample area
40%
±0.5 CPU
5.4 Audits
This project was assigned QA Category III and did not require technical systems or performance
evaluation audits.
37
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6 Results and Discussion
This work measured the reduction in viable spores on and within the carpet surfaces (effectiveness) as a
function of the cleaning technique (hot versus cold vacuum) and duration of application to various carpet
types (up to four subsequent decontaminations). The size of the carpet sections, roughly 4' x 4', was
chosen as feasible yet representative of what will likely be encountered in the field (e.g., walkways).
Operational parameters such as time, physical impact on materials or the remediation crew, and fate of
the viable spores (e.g., contamination of equipment, carpet cleaner parts, and rinsate) were also
determined. The data reduction and validation procedures used are presented in Section 6.1, followed in
Section 6.2 by the results for each of the tests from the test matrix (Table 2-2).
6.1 Data Reduction and Validation
Data reduction was performed to tabulate all results from each test. The data reduction included the total
CPU recovered from each replicate sample area, the average recovered CPU and standard deviation for
each group of sample areas, log reductions, and total recovered CPU for each wet vacuum liquid sample.
The coupons included the following, for each combination of material type and decontamination type:
• Positive control areas (three replicates, average, standard deviation)
• Test areas (three replicates, average, standard deviation)
CPU counts per coupon were calculated according to the equation shown in MOP 6535a (Appendix C).
Efficacy is defined as the extent (by log reduction) to which the agent extracted from the coupons after
the treatment with the decontamination procedure is reduced below that extracted from positive control
areas (not exposed to the decontamination procedure). Efficacy was calculated for each test coupon
within each combination of decontamination procedure (i) and test material as:
//, = I>L°8(CFUc)/Nc-I,Lo8(CFUs)'Nt
j k
where :
~ = the spore log reduction efficacy of decontamination technique i
the mean log CPU recovered from the control areas (C= control,
j = coupon number, and Nc is the number of coupons (1 , j))
the mean log CPU recovered from the surface of a
)/7V( = decontaminated coupon (S= sample from decontaminated
carpet, k = coupon number, and Nt is the number of coupons
tested (1 , k))
When no viable spores were detected, then a value of 0.5 CPU was assigned as the detection limit, and
the efficacy was reported as greater than or equal to the value calculated by Eqn. 6-1.
For the recovered liquid samples, the results were reported as CPU per area cleaned.
38
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At least 10% of the data generated during sample analysis from each test was reviewed. This review
occurred within one week after the analysis was completed. The review included an independent
verification of CPU per plate and the calculation of CPU per sample (per Equation 6-1).
6.2 Test Results
The first test (Test A) was conducted on new carpet using wipe samples. Use of the wet carpet cleaners
was not expected to kill the spores but to decontaminate by removing the spores. In this way, the spores
could be considered a material, rather than an organism, and a rudimentary type of mass balance could
be applied to them.
Figure 6-1 shows the wipe sampling recovery following spore inoculation (loading) and after each of the
four subsequent decontaminations. When wipe sampling was used to determine spore concentration for
Test A, the results failed to satisfy the mass balance.
o
S5
o-
4-
3-
2-
1-
n
4-
3-
2-
1-
n-
T
1
1 1 Heated Cleaner
T , I , T
1 1
i
i • i • i • i
1
I | Unhetaed Cleaner
I
i T i
1 X 1
Carpet Decontamination Steps
Figure 6-1. Wipe Sampling Results for Test A
The wipe results seemed to suggest that a single use of a carpet cleaner (whether heated or unheated)
removed 99.99% of the spores after the first decontamination. This observation would further suggest that
the recovery of the surfactant was also 99.99%, if the spores were carried away by the surfactant. This
value was beyond the anticipated (or believable) efficacy of the vacuum cleaner itself. For example, the
flow rate of the surfactant was 26.6 (±3%) mL/sec for the heated carpet cleaner and 39.8 (±0.5%) mL/sec
for the unheated carpet cleaner. The surfactant was applied for approximately 45 seconds. For the
unheated carpet cleaner, only 560 ml of the approximately 1200 ml was recovered, much below the
99.99% recovery suggested by the spore results. This observation led to the consideration that the
spores were not inactivated or removed at all, but had been pushed down within the carpet pile and made
inaccessible to the wipes.
39
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For Test B, conducted on old carpet, HEPA sock sampling was used instead of wipe sampling, and core
samples were also intended to be collected for extraction. Because core samples were a late addition to
the test plan, the carpet construction method (gluing the carpet down) was not designed to allow for the
collection of these samples. The core samples could not be collected for Test B.
Figure 6-2 shows the results after loading and three subsequent decontaminations. The HEPA sock
results suggested that a significant log reduction could be obtained with repetition of the decontamination
process using either the heated or unheated carpet cleaners. However, based on the ineffectiveness of
the wipe samples (and without the core samples for verification), the data would suggest that the HEPA
socks were no more effective than the wipe sample had been.
o
K-
5-
4-
3-
2-
1-
Q
7-
6-
5-
4-
3-
2-
1-
Ł
1 1 Heated Cleaner
T
1
I
J_
I
1
1 ' 1 ' 1 ' 1
I
1 Unheated Cleaner |
I
1
T
Carpet Decontamination Steps
Figure 6-2. HEPA Sock Sampling Results for Test B
Results from Tests A and B suggested that neither of the surface sampling methods - wipe sampling or
HEPA sock sampling - was sufficiently efficient at collecting spores once the wet/dry vacuuming
operation had been performed. Test C was designed to answer the question of spore recovery by altering
the carpet construction method to allow for the collection of the core (extractive) samples in addition to
the HEPA sock samples. This test was conducted on new carpet. Figure 6-3 shows the logarithm of the
CFU recovered per square foot following multiple decontamination attempts using the HEPA sock
samples.
40
-------
o
D)
3
7-
6-
5-
4-
3-
2-
1-
n '
7-
6-
5-
4-
3-
2-
1-
n '
|_[ 1 Heated vani n im
i
1
i • i • i • i
1 1 1 Unheated Vacuum
T T
1
Carpet Decontamination Steps
Figure 6-3. HEPA Sampling Results after Decontamination Attempts
Each sample area was first sampled using HEPA socks, then extractive analysis was performed on a
small 18 mm diameter core taken from the center of the same sample area. These data show that while
HEPA sock sampling suggested that the decontamination methods were removing some spores,
subsequent extractive samples showed no or minimal removal (less than 1 log reduction, see Figure 6-4).
The HEPA sock samples of control areas (not decontaminated) showed minimal downward drift.
41
-------
7-
6-
5-
4-
3-
2-
^ 1~
3 0-
± 8
5 ^
-1 6-
5-
4-
3-
2-
1-
n.!
|_[ 1 Heated vacuum
T
1
I
1
i • i • i • i
T
J
:
1 1 i Unheated Vacuum
T
1
T
1
Carpet Decontamination Steps
Figure 6-4. Core Sampling Results after Decontamination Attempts
The act of decontaminating with the wet/dry vacuums seems to push the spores away from the surface
and into the carpet pile where the HEPA socks were unable to sample effectively. Even though the
samples were allowed to dry overnight, residual moisture or detergent may have helped spores adhere to
carpet fibers. Because the apparent log reduction from the HEPA sock samples from Test B (Figure 6-2)
showed a greater log reduction that the HEPA sock samples from Test C (Figure 6-3), the tamped down
nature of the older used carpet as well as the debris in the fibers may further reduce recovery by binding
to the spores or merely retaining moisture.
Although the results from Tests A and B were inconclusive regarding the efficiency of the carpet cleaners,
all three tests suggested that there was no statistical difference between the hot (heated) and cold
(unheated) cleaners. Test C indicated that the efficacy of the carpet cleaners per the manufacturer's
recommendations was poor. For carpet, extractive sampling appears to be the most reliable method
following any dampening of the surface, especially for used carpet.
One final test (Test D), using new carpet, was conducted to provide information about the benefits of
using a carpet cleaner with a sporicide (Spor-Klenz) in place of the recommended cleaner versus the
simple application of the sporicide without any vacuuming. These results are shown in Figure 6-5 and
Figure 6-6 using a back pack sprayer and an unheated vacuum cleaner, respectively.
42
-------
7-
6-
5-
4-
3-
2 7-
0 6-
5-
4-
3-
2-
0-
-1-
-21
1
Positive control dec
1 1 Hepa Socks Samples |
Detecti9n Limits
1 T
>n 1 Decon 2
Carpet Decontamination Steps
Figure 6-5. Spor-Klenz Applied to a New Carpet with a Backpack Sprayer
6-
4-
3-
2-
"c i J
,9 o-
o UH
LL 7:
O ' -
6-
5-
4-
3-
2-
1-
0-
-1-
}
1
i
^
Positive control
1 1 I Core samples |
Detection Limits
T ^_^ \
1
1 1 1 Hepa Socks Samples
Detection Limits
1
T
decon 1 Decon 2
Carpet Decontamination Steps
Figure 6-6. Spor-Klenz Applied to a New Carpet with an Unheated Vacuum Cleaner
Test D showed a very good log reduction from the use of Spor-Klenz, applied either from the carpet
cleaner (Table 6-1) or the backpack sprayer (Table 6-2). The first decontamination showed a 6 log
reduction from the HEPA socks (no detection on the core samples, which have a higher detection limit
than the HEPA sock samples). Following the second application of Spor-Klenz, no spores were detected
using either sampling method, a 7 log reduction in the HEPA sock results.
43
-------
The core samples could show only a 3 log reduction, in part because of the higher detection limit. The
core samples taken for the controlled contamination had lower recovery than expected, possibly due to
the rigorous method that was used to extract the core samples causing re-aerosolization of many spores.
The summary of the results for the spore counts log reductions for Test B through Test D are presented in
Table 6-3.
Table 6-1. Spor-Klenz Applied with a Carpet Cleaner*
Carpet Status
Controlled
Contamination
After 1st
Decontamination
After 2nd
Decontamination
Date
8/25/2010
8/30/2010
9/1/2010
HEPA socks
Mean of log
CFU/ft2
7.2
1.5
-0.03
Error (95%
Confidence Interval)
0.11
1.82
0.10
Core Samples
Mean of
log CFU/ft2
5.29
2.5
1.8
Error (95%
Confidence
Interval)
1.09
0.30
0.04
Detection Limit values are in red.
Table 6-2. Spor-Klenz Applied with a Backpack Sprayer*
Carpet Status
Controlled
Contamination
After 1st
Decontamination
After 2nd
Decontamination
Date
8/25/2010
8/30/2010
9/1/2010
HEPA socks
Mean of log
CFU/ft2
7.07
0.95
-0.13
Error (95%
Confidence Interval)
0.20
2.07
0.10
Core Samples
Mean of
log CFU/ft2
6.12
2.49
1.81
Error (95%
Confidence
Interval)
0.41
0.3
0
Detection Limit values are in red.
44
-------
Table 6-3. CPU Log Reduction with Spor-Klenz Application
Decontamination procedure
Spor-Klenz from backpack
sprayer
Spor-Klenz application with
carpet cleaner
HEPA Sock Sampling
Decon 1
1.7
-------
6.2.1 Length of Decontamination Event
The decontamination procedure using the carpet cleaners is very short, if only performed once,
approximately five seconds per square foot. Test D with Spor-Klenz suggested that performing the
decontamination twice would yield no recoverable spores. If the decontaminations are performed
immediately (back-to-back), then there is minimum impact on the remediation crew. If, on the other hand,
the carpet is allowed to dry between successive applications, then an extra day is involved. Though it was
not a consideration for this test, the ultimate disposal of the carpet cleaners after use in an event may
contribute to the cleanup time and expense.
The backpack sprayer method as performed does require an extended duration, allowing for treatment of
an extrapolated 192 square feet in 30 minutes, or 6.4 square feet per minute, significantly higher than
when a carpet cleaner is being used.
6.2.2 Physical impact on Materials and Crew
No physical impact on the carpet was noted for any of the decontamination methods. While neither
decontamination procedure was physically strenuous, any activity inside Level C suits (even with cooling
vests) leads to heat stress. Moreover, the use of Spor-Klenz in an area without very high air exchanges
could lead to levels of hydrogen peroxide or acetic acid above IDLH conditions.
6.2.3 Fate of Spores
Determining the ultimate fate of spores has proven very complicated due to sampling difficulties. The
rinsate recovered from the carpet cleaners used in Tests A, B, and C was very contaminated, obscuring
enumeration of the spores of interest. This level of contamination does suggest that conditions are such
that the likelihood our target organism was in the rinsate was very high. No data suggest that all spores
were removed from the carpet during Tests A, B, and C, so the ultimate fate of those spores is probably
viable and present in carpet, carpet cleaner parts, and rinsate. The fate of spores in Test D is probably
deactivated due to the presence of Spor-Klenz in all locations, but some doubt lingers due to difficulties
sampling the rinsate.
6.2.4 Health and Safety Effects of Decontamination
There were no noxious fumes or gases detected from the use of the carpet cleaner solutions (Judson
Laboratories Oxygen 2 Pre-Spray and Extraction) or the Spor-Klenz.
46
-------
References
1 After Action Report - Danbury Anthrax Incident, U.S. EPA Region 1, September 19, 2008. (Available
upon request from Mike Nalipinski, Nalipinski.Mike@epa.gov, Region 1 On-Scene Coordinator)
2 Brown, G.S.; Betty, R.G.; Brockmann, J.E.; Lucero, D.A.; Souza, C.A.; Walsh, K.S.; Boucher, R.M.;
Tezak, M.S.; Wilson, M.C. Evaluation of Surface Sample Collection Methods for Bacillus Spores on
Porous and Non-porous Surfaces. In 2006 Workshop on Decontamination, Cleanup and Associated
Issues for Sites Contaminated With Chemical, Biological, or Radiological Materials, Proceedings of the
2006 NHSRC Decontamination Workshop, Washington, DC, April 26 - 28, 2006.
3 Busher, A.;Noble-Wang; J.; Rose, L. Surface Sampling. In Sampling for Biological Agents in the
Environment; Emanual, P., Roos, J.W., Niyogi, K., Eds.; ASM Press: Washington, DC, 2008; pp 95 -
131
4 Brown, G.S.; Betty, R.G.; Brockmann, J.E.; Lucero, D.A.; Souza, C.A.; Walsh, K.S.; Boucher, R.M.;
Tezak, M.; Wilson, M.C.; Rudolph, T. Evaluation of a Wipe Surface Sample Method for Collection of
Bacillus Spores from Nonporous Surfaces. Appl Environ Microbiol 2007, 73 (3), 706 - 710
5 [FOUO] Estill, C.F.; Baron, P.A.; Beard, J.K.; Hein, M.J.; Larsen, L.D.; Rose, L.; Schaefer III, F.W.;
Noble-Wang, J.; Hodges, L.; Lindquist, A.; Deye, G.J.; Arduino, M.J. Recovery Efficiency and Limit of
Detection of Aerosolized B. anthracis Sterne from Environmental Surface Samples. Submitted to Appl
Environ Microbiol.
6 Mattorano, D. U.S. EPA/OSWER/OEM/National Decontamination Team, Erlanger, KY. Personal
communication (regarding the current sampling strategies under development by the Interagency
Workgroup for the Validated Sampling Plan for B. anthracis), December 12, 2008.
7 Busher, A.;Noble-Wang; J.; Rose, L. Surface Sampling. In Sampling for Biological Agents in the
Environment; Emanual, P., Roos, J.W., Niyogi, K., Eds.; ASM Press: Washington, DC, 2008; pp 95 -
131.
8 Brown, G.S.; Betty, R.G.; Brockmann, J.E.; Lucero, D.A.; Souza, C.A.; Walsh, K.S.; Boucher, R.M.;
Tezak, M.; Wilson, M.C.; Rudolph, T. Evaluation of a Wipe Surface Sample Method for Collection of
Bacillus Spores from Nonporous Surfaces. Appl Environ Microbiol 2007, 73 (3), 706 - 710.
9 [FOUO] INL 2008 Evaluation Protocols.
10ARCADIS G&M, Inc. Quality Assurance Project Plan for the Determination of the Efficacy of Spore
Removal from Carpets using Commercially-available Wet/Vacuum Carpet Cleaning Systems. Prepared
under Contract No. EP-C-04-023, Work Assignment No. 4-35. U.S. Environmental Protection Agency,
National Homeland Security Research Center, Research Triangle Park, NC. May 2009. (Available upon
request from Shawn Ryan, Ryan.Shawn@epa.gov, DCMD Division Director)
11 ARCADIS U.S., Inc. Compatibility of Material and Electronic Equipment with Chlorine Dioxide
Fumigation, Assessment and Evaluation Report. Prepared under Contract No. EP-C-04-023, Work
Assignment No. 4-50 for U.S. Environmental Protection Agency, National Homeland Security Research
47
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Center, Office of Research and Development, Research Triangle Park, NC. July 2009. (Available upon
request from Shawn Ryan, Ryan.Shawn@epa.gov, DCMD Division Director)
48
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Appendix A: COMMANDER Piping and Instrumentation Diagram
49
-------
Appendix B: MSDS for Decontamination Solutions
• Judson Laboratories Oxygen 2 / Pre-Spray
• Judson Laboratories Oxygen 2 / Extraction
• Steris Spor-Klenz® Ready To Use
50
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JUDSON LABORATORIES
P.O. BOX 4388
GREENVILLE, SC 29608
(864) 233-6442
MATERIAL SAFETY DATA SHEET
I. IDENTIFICATION
PRODUCT NAME: OXYGEN 2 / PRE-SPRAY
CHEMICAL NAME: ALKALINE DEGREASER
CHEMICAL FAMILY: ALKALI BLENDED/OXYGEN BRIGHTENER/D-LLV1QNENE SOLVENT/PH-93 to 9.9
FORMULA: TRADE SECRET
SYNONYM'S: NONE
CAS # AND NAME: TRADE SECRET
II. PHYSICAL DATA
BOILING POINT: 220 F
FREEZING POINT: 32 F
PH: 10.8
SPECIFIC GRAVITY (H2O=1): 1.0002
VAPOR DENSITY (AIR=1): NA
VAPOR PRESSURE AT 20'C: NA
SOLUBILITY IN WATER BY WT: NA
EVAPORATION RATE
-------
O2PRE-SPRAY /PG. 2
IV. FIRE AND EXPOSURE HAZARD DATA
FLASH POINT: COMPLETELY NON COMBUSTIBLE, NOT A FIRE HAZARD
FLAMMABLE LIMITS IN AIR, % BY VOLUME: LOWER: NA / UPPER NA
EXTINGUISHING MEDIA: THIS MATERIAL IS NOT COMBUSTIBLE
SPECIAL FIRE FIGHTING PROCEDURES: N/A
UNUSUAL FIRE AND EXPLOSION HAZARDS: NONE
V. HEALTH HAZARD DATA
EXPOSURE LIMITS: NONE
EFFECTS OF ACCUTE OVER EXPOSURE:
SWALLOWING: INGESTION OF THE PRODUCT CAN CAUSE GASTRIC WALL IRRITATION AND POSSIBLE RED
BLOOD CELL IIEMOTOSIS
SKIN ABSORFIION: MODERATE SKIN PENETRATION
INHALATION: INHALATION OF AIR CONTAMINATED WITH LARGE AMOUNTS OF MIST SPRAY OR ATOMIZED
PRODUCTS WILL IRRITATE MUCUS MEMBRANE
SKIN CONTACT: LONG TERM CONTAMINATION OF SKIN MAY CREATE EXCESSIVE SKIN DRYNESS AND MILD
SKIN DERMATITIS BECAUSE OF THE BREAKDOWN OK NATURAL OILS
EYE CONTACT: FLUSH WITH COPIUS AMOUNTS OF WATER
EFFECTS OF REPEATED OVEREXPOSURE: WILL CAUSE THE BREAD DOWN OF NATURAL SKIN OILS CAUSING
CONTACT DERMATITIS
MEDICAL CONDITIONS AGGRAVATED BY OVEREXPOSURE: SKIN DISEASES AND CONTACT DERMATITIS AS
WELL AS PERSONS HAVING CHRONIC RESPIRATORY DISEASES
EMERGENCY AND FIRST AID PROCEDURES:
SWALmWING: WASH OUT MOUTH, DRINK LARGE QUANTITIES OF CITRUS JUTCE,(EXAMPLE: GRAPEFRUIT
JUICE) AND THEN INDUCE VOMITING
SKIN: WASH CONTAMINATED AREA WITH DILUTED VINEGAR AND WATER RINSE
INHALATION: THIS PRODUCT HAS NO HARMFUL VAPOR CHARACTERISTICS BUT DROPLETS FROM SPRAY
APPLICATION CAN CAUSE RESPIRATORY IRRITATION. EVACUATE AREA TO FRESH AIR
EYES: FLUSH WITH COPIUS AMOUNTS OF WATER FOR 15 .MINUTES, WASH EYES WITH 5% BORIC ACID SOLUTION
THEN, CONSULT A PHYSICIAN IMMEDIATELY
NOTES TO PHYSICIAN: TREAT ALL CONTACT PROBLEMS BY NEUTRALIZATION WITH MILD ACIDIC WASH THEN
TREAT FOR CHEMICAL BURNS
52
-------
O2 PRE-SPRAY / PG, 3
VI. REACTIVITY DATA
STABILITY: STABLE
CONDITIONS TO AVOID: CONTACT WITH STRONG ACIDS
HAZARDOUS COMBUSTION OK DECOMPOSITION PRODUCTS: THIS PRODUCT SHOULD NEVER I3L; STORED IN
ALUMINUM CONTAINERS
HAZARDOUS POLYMERIZATION: NONE
CONDITIONS TO AVOID: FURNITURE, WALLS ECT.
VII. SPILL OR LEAK PROCEDURES
STEPS TO BE TAKEN IF MATERIAL IS RELEASED OR SPILLED: SMALL SPILLS: ABSORB WITH ABSORBENT
MATERIAL AND DISCARD IN TRASH RECEPTACLE. LARGE SPILLS: PUMP INTO DRUMS FOR RECOVERY
WASTE DISPOSAL METHOD: SPILL, THEN NEl TTRALI7.E WASTE WITH HYDROCHLORIC ACID SOLUTION.
DILUTE WITH WATER BEFORE DISCHARGING TO SEWER SYSTEM
MIL SPECIAL PROTECTIVE INFORMATION
RESPIRATORY PROTECTION: THE APPROVED RESPIRATORY MASK SHOULD BE USED IN HIGH PRESSURE
SPRAY MIST ENVIRONMENTS, IF SPRAYED INTO THE AIR.
VENTILATION: CLOSE AREAS SHOULD BE PROVIDED WITH FRESH AIR
PROTECTIVE GIXWES: IT IS SUGGESTED RUBBER NEOPRENE GLOVES TO BE WORN
EYE PROTECTION: GOGGLES OR MASK SHOULD BE WORN IF PRODUCT BECOMES IRRITATING
OTHER PROTECTIVE EQUIPMENT: IT IS SUGGESTED ALL SKIN AREAS TO BE COVERED WHEN USING THIS
PRODUCT
IX. SPECIAL PRECAUTIONS
STATUS ON SUBSTANCE: THE CONCENTRATIONS SHOWN ARE MAXIUM OR CEILING LEVELS (WEIGHT %) TO BE
USED FOR CALCULATIONS FOR REGULATIONS. TRADE SECRETS ARE INDICATED BY "TS"
FEDERAL EPA: COMPREHENSIVE ENVIRONMENTAL RESPONSE, COMPENSATION, AND LIABILITY ACT OF 1980
(CERCLA) REQUIRES NOTIFICATION OF THE NATIONAL RESPONSE CENTER OF RELEASE OF QUANTITIES OF
HAZARDOUS SUBSTANCES EQUAL TO OR GREATER THAN THE REPORT ABLE QNANTIT1ES (RQ'S) IN 40 CFR 302.4
THE OPINIONS ARE THOSE OF QUALIFIED SCIENTIST WITHIN JUDSON LABORATORIES. WE BELIEVE THE
INFORMATION CONTAINED IS CURRENT AND VALID. SINCE USE OF THIS INFORMATION, OPINIONS AND THE
CONDITIONS OF USE OF THE PRODUCT ARE NOT WITHIN THE CONTROL OF JUDSON LABORATORIES, IT IS THE
USERS OBLIGATION TO DETERMINE THE CONDITIONS OF SAFE USE OF THE PRODUCT.
53
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JUDSON LABORATORIES
P.O. BOX 4388
GREENVILLE, SC 29608
(864) 233-6442
MATERIAL SAFETY DATA SHEET
I. DENTIFICATION
PRODUCT NAME: OXYGEN 2 / EXTRACTION
CHEMICAL NAME: CLEANER / OXYGEN BRIGHTNER / NEUTRAL AGENT / IPA DRYING AGENT
CHEMICAL FAMILY: NEUTRALIZING AGENT
FORMULA: TRADE SECRET
PH: 7.0
SYNONYMS: NONE
CAS # AND NAME: TRADE SECRET WITH NO CAS NUMBERS ASSIGNED
II. PHYSICAL DATA
BOILING POING: 220 F
FREEZING POINT: 32' F
SPECIFIC GRAVITY (H2O=1): 1.0002
VAPOR DENSITY (AIR=1): NA
SOLUBILITY IN WATER BY WT: COMPLETE
EVAPORATION RATE: 1
APPEARANCE & ODOR: AQUA-BLUE LIQUID WITH CLEAN ODOR
EMERGENCY PHONE NUMBERS:
MONDAY - FRIDAY: 9:OOAM - 6:OOPM (864) 233-6442 / OTHER TIMES (864) 787-3038
****HAZARD RATING SCALE****
0=MINIMAL 3=HIGH
1=SLIGHT 4=SERIOUS
2=MODERATE 5=SEVERE
III. INGREDIENTS
MATERIAL % EXPOSURE LIMITS HAZARD - 1
SODIUM BORATE 3-8% 25ppm
SOppm (SKIN) OSHA
ORAL 1.48gm/kg (RAT)
INHALATION LD 50-700ppm / 7 HOURS (RAT)
54
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O2 EXTRACTION /PG. 2
IV. FIRE AND EXPOSURE HAZARD DATA
FLASH POINT: COMPLETELY NON COMBUSTIBLE, NOT A FIRE HAZARD
FLAMMABLE LIMITS IN AIR, % BY VOLUME: LOWER: NA UPPER: NA
EXTINGUISHING MEDIA: NONE REQUIRED
SPECIAL FIRE FIGHTING PROCEDURES: N/A
UNUSUAL FIRE AND EXPLOSION HAZARDS: NONE
V. HEALTH HAZARD DATA
EXPOSURE LIMITS: MODERATE
EFFECTS OF ACCUTE OVER EXPOSURE:
SWALLOWING: INGESTION OF THE PRODUCT CAN CAUSE GASTRIC WALL IRRITATION
SKIN ABSORPTION: MODERATE SKTN PENETRATION
INHALATION: INHALATION OF AIR CONTAMINATED BY THE HIGH PRESSURE SPRAY MIST OF THIS PRODUCT
MAY CAUSE RESPIRATORY IRRITATION
SKIN CONTACT: LONG TERM CONTAMINATION OF SKIN MAY CREATE EXCESSIVE SKIN DRYNESS AND MILD
SKIN DERMATITIS BECAUSE OF THE BREAKDOWN OF NATURAL OILS
EYE CONTACT: FLUSH WITH COPIUS AMOUNTS OF WATER
EFFECTS OF REPEATED OVEREXPOSURE: MAY CAUSE DRY IRRITATED SKIN DUE TO REMOVAL OF OILS
MEDICAL CONDITIONS AGGRAVATED BY OVEREXPOSURE: CHRONIC SKIN DISEASE PERSONS WITH ALLERGY
PROBLEMS AND CHRONIC RESPIRATORY DISEASES MAY EXPERIENCE COMPLICATIONS
EMERGENCY AND FIRST AID PROCEDURES:
SWALLOWING: WASH OUT MOUTH. DRINK LARGE QUANTITIES OF CITRUS JUICE. GRAPEFRUIT AND THEN
INDUCE VOMITING
SKIN: WASH CONTAMINATED AREA WITH DILUTED VINEGAR AND WATER
INHALATION: THIS PRODUCT HAS NO HARMFUL VAPOR CHARACTERISTICS BUT MIST FROM HIGH PRESSURE
SPRAY MAY BE IRRITAING TO RESPIRATORY SYSTEM SEEK FRESH AIR
EYES: FLUSH WITH COPIUS AMOUNTS OF WATER FOR 15 MINUTES
NOTES TO PHYSICIAN: NONE
55
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O2 EXTRACTION /PG. 3
VI. REACTIVITY DATA
STABILITY: STABLE
CONDITIONS TO AVOID: STRONG OXIDIZING AGENTS
HAZARDOUS COMBUSTION OR DECOMPOSITION PRODUCTS: NONE
HAZARDOUS POLYMERIZATION: NONE
CONDITIONS TO AVOID: NONE
VII. SPILL OR LEAK PROCEDURES
STEPS TO BE TAKEN IF MATERIAL IS RELEASED OR SPILLED: CONTAIN SPILL VACUUM OR BLOT UP WITH
MOP OR ABSORB WITH WATER ABSORBING MATERIAL CONTAMINATED SURFACES MAY BE WASHED WITHOUT
ADVERSE ENVIRONMENTAL IMPACT
WASTE DISPOSAL METHOD: SPILLED MATERIAL MAY BE RECLAIMED DEPENDENT UPON OTHER
CONTAMINATS. WASTE DISPOSAL (INSURE CONFORMITY WITH ALL APPLICABLE DISPOSAL REGULATIONS)
INCINERATE OF OTHERWISE MANAGE IN A RCRA PERMITTED WASTE MANAGEMENT FACILITY
VIII. SPECIAL PROTECTIVE INFORMATION
RESPIRATORY PROTECTION: GENERALLY NOT REQUIRED
VENTILATION: USE ADEQUATE VENTILATION
PROTECTIVE GLOVES: SUGGESTED USE OF RUBBER GLOVES IS A GOOD PRACTICE
EYE PROTECTION: FOR SPLASH PROTECTION USE PROTECTIVE GOGGLES OR FACE SHIELD
OTHER PROTECTIVE EQUIPMENT: NO SPECIAL GARMENTS REQUIRED AVOID UNNECCARY SKIN
CONTAMINATION WITH MATERIAL
IX. SPECIAL PRECAUTIONS
STATUS ON SUBSTANCE: THE CONCENTRATIONS SHOWN ARE MAXIUM OR CEILING LEVELS (WEIGHT %) TO BE
(CERCLA) REQUIRES NOTIFICATION OF THE NATIONAL RESPONSE CENTER OF RELEASE OF QUANTITIES OF
HAZARDOUS SUBSTANCES EQUAL TO OR GREATER THAN THE REPORTABLE QUANTITIES (RQS) IN 40 CFR 302,4
THE OPINIONS ARE THOSE OF QUALIFIED SCIENTIST WITHIN JUDSON LABORATORIES. WE BELIEVE THE
INFORMATION CONTAINED IS CURRENT AND VALID. SINCE USE OF THIS INFORMATION. OPINIONS AND THE
CONDITIONS OF USE OF THE PRODUCT ARE NOT WITHIN THE CONTROL OF JUDSON LABORATORIES, IT IS THE
USERS OBLIGATION TO DETERMINE THE CONDITIONS OF SAFE USE OF THE PRODUCT.
56
-------
Material Safety Data Sheet
May be used to comply with OSHA's
Hazard Communication Standard,
29 CFR 1910.1200. Standard must be
consulted for specific requirements.
product Name
SPOR-KLENZ
READY TO USE
Kroouct uescnption
COLD STERILANT
ALIPHATIC CARBOXYLIC ACID,
INORGANIC PEROXIDE.
ORGANIC PEROXIDE
(STRONG OXIPIZERj
product ID
6525
STERIS'
Manufacturers Name
STERIS Corporation
Address
P.O. Box 147
St. Louis, Missouri 63166
tmergency leiepnone Numoer
1-314-535-1395 (STERIS); 1-800-424-9300 (CHEMTREC)
Telephone Number for Information
1-800-444-9009 (Customer Service - Scientific Products)
Date Prepared: July 1,1999
Prepared by: M. Ebers
Health
Fire
Reactivity
2
0
0
Least Sfcght Mtoersie H^h Ex:fe-ra
0 2 34
SECTION II - HAZARDOUS INGREDIENTS/IDENTITY INFORMATION
Hazardous Components (Specific Chemical
Identity; Common Name(s))
Hydrogen Peroxide (7722-84-1)
Peracetic Acid (79-2 1 -0)
Acetic Acid (64-19-7)
% by Wt,
0.80
0.06
Proprietary
ACGIH
TLV
1ppm
N/E
10ppm
OSHA PEL
1ppm
N/E
10ppm
Other Limits
Recommended
SECTION III - PHYSICAL/CHEMICAL CHARACTERISTICS
Solubility in
Water
Freezing Point F
pH @ Solution
Appearance
Complete
N/A
N/A
Clear, colorless liquid
Specific Gravity (HZ0=1 @
25C)
% Volatile by wt @ 105C/1 hr
pH as Distributed
Odor
1.01
N/A
~ 1.87
Mild vinegar odor
SECTION IV - FIRE AND EXPLOSION HAZARD DATA
Flash Point (Method Used) N/A
Flammable Limits N/A LEL
UEL
Extinguishing Media
Use water, foam, C02, dry chemicals.
Special Fire Fighting Procedures
As with any chemical fire, the use of a self-contained breathing apparatus and full protective equipment is
recommended. Dilute with large volumes of water.
Unusual Fire and Explosion
Hazards
N/A
57
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SECTION V - REACTIVITY DATA
Stability
Unstable
Stable
Incompatibility (Materials to Avoid)
Hazardous Decomposition or
Byproducts
Hazardous
Polymerization
May Occur
Will Not
Occur
X
Conditions to Avoid
Avoid temperatures above 75F. Avoid contact with combustible
materials.
Heavy metals including iron, copper, copper alloys, brass and aluminum,
salts, alkalis, caustics, and formaldehyde.
Oxygen and heat Do not mix with chlorinated products as this could
liberate toxic corrosive chlorine gas.
X
Conditions to Avoid
None known.
SPOR-KLENZ READY TO USE
SECTION VI - HEALTH HAZARD DATA
A. ACUTE (Primary Route of Exposure)
EYES: Contact with eyes may cause burns.
SKIN; May cause moderate skin irritation, including oxidation (i.e., whitening of the skin). LD--0 Rat; > 20 g/kg.
INHALATION: May cause irritation to mucous membranes. LDS2 Rat: >13,439 mg/m3.
B. SUBCHRONIC, CHRONIC, OTHER: None known
SECTION VII - EMERGENCY AND FIRST AID PROCEDURES
EYES: In case of contact, immediately flush with water for 15 minutes and get medical attention.
SKIN: For skin contact, wash with soap and water Get medical attention if irritation persists. Remove and launder
contaminated clothes before reuse.
INHALATION: Remove to fresh air.
INGESTION: Do not induce vomiting. Drink large quantities of water. Get immediate medical attention.
SECTION VIII - PRECAUTIONS FOR SAFE HANDLING & USE
58
-------
Spill Management:
Waste Disposal Methods:
Precautions to be taken in handling
and storage:
Shipping information:
Flush with large quantities of water; mop thoroughly. Do not use absorbent
Dispose of in accordance with all local, state and federal regulations.
Store in a cool, dry place below 75F. Do not expose to sunlight Read
observe all labeled use instructions.
and
Not restricted
SECTION IX - PROTECTION INFORMATION/CONTROL MEASURES
Respiratory:
Eyes:
Use only in well-ventilated area
Safety glasses/goggles/full face
shield
Other Clothing and Equipment:
Ventilation;
Use local exhaust
Gloves:
Rubber or plastic
Rubber apron
U.S. FEDERAL REGULATIONS:
All components are listed in the Toxic Substance Control Act (TSCA) Chemical Substance Inventory. OSHA
Hazardous Communication Standard (29 CFR 1910.1200) Hazardous Class: Corrosive.
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Appendix C: Miscellaneous Operating Procedure (MOP) 6535a
BL MOP NO. 6535a M. Worth Calfee
4-8-2009, rev. 2.0
Title: SERIAL DILUTION: SPREAD PLATE PROCEDURE TO QUANTIFY VIABLE
BACTERIAL SPORES
Scope: Determine the abundance of bacterial spores in a liquid extract
Purpose: Determine quantitatively the number of viable bacterial spores in a liquid suspension
using the spread plate procedure to count colony-forming units (CPU)
Materials:
• Liquid suspension of bacterial spores
• Sterile microcentrifuge tubes
• Diluent (sterile deionized water, buffered peptone water or phosphate buffered saline)
• Trypticase Soy Agar plates
• Microliter pipettes with sterile tips
• Sterile beads placed inside a test tube (will be used for spreading samples on the agar surface)
• Vortex mixer
Procedure: (This protocol is designed for 10-fold dilutions.)
1. For each bacterial spore suspension to be tested, label the microcentrifuge tubes as follows: 10~1,
10"2, 10"3, 10"4, 10"5, 10"6... (The number of dilution tubes will vary depending on the concentration
of spores in the suspension). Aseptically, add 900 uL of sterile diluent to each of the tubes.
2. Label three Trypticase Soy Agar (TSA) plates for each dilution that will be plated. These dilutions
will be plated in triplicate.
3. Mix original spore suspension by vortexing thoroughly for 30 seconds. Immediately after the
cessation of vortexing, transfer 100 uL of the stock suspension to the 10"1 tube. Mix the 10"1 tube
by vortexing for 10 seconds, and immediately pipette 100 uL to the 10"2 tube. Repeat this
process until the final dilution is made. It is imperative that used pipette tips be exchanged for a
sterile tip each time a new dilution is started.
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4. To plate the dilutions, vortex the dilution to be plated for 10 seconds, immediately pipette 100 uL
of the dilution onto the surface of a ISA plate, taking care to dispense all of the liquid from the
pipette tip. If less than 10 seconds elapses between inoculation of all replicate plates, then the
initial vortex mixing before the first replicate is sufficient for all replicates of the sample. Use a
new pipette tip for each set of replicate dilutions.
5. Carefully pour the sterile glass beads onto the surface of the ISA plate with the sample and
shake until the entire sample is distributed on the surface of the agar plate. Aseptically remove
the glass beads. Repeat for all plates.
6. Incubate the plates overnight at 32 °C - 37 °C (incubation conditions will vary depending on the
organism's optimum growth temperature and generation time.)
7. Enumerate the colony forming units (CPU) on the agar plates by manually counting with the aid of
a plate counting lamp, and with a marker (place a mark on the surface of the Petri dish over each
CPU when counting, so that no CPU is counted twice).
Since each dilution was tested in triplicate, determine the average of the triplicate plate abundances.
Plates suitable for counting must contain between 30 - 300 colonies.
Calculations
Total abundance of spores (CPU) within extract:
(Avg CPU / volume (ml_) plated) x (1/tube dilution factor) X extract volume
For example:
Tube Dilution Volume plated Replicate CPU
10'3 100 uL (0.1 ml) 1 150
10'3 100uL (0.1 ml) 2 250
10-3 100uL (0.1 ml) 3 200
Extract total volume = 20 ml
(200 CPU/0.1 ml) x (1/10'3) X 20 ml = (2000) x (1000) x 20 = 4.0X107CFU
Note: The volume plated (ml) and tube dilution can be multiplied to yield a 'decimal factor' (DP). DP can
be used in the following manner to simplify the abundance calculation.
Spore Abundance per ml_ = (Avg CPU) X (1 / DP) X extract volume
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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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