EPA 600/R-13/009 | March 2013 | www.epa.gov/ord
United States
Environmental Protection
Agency
Evaluation of Expedient
Decontamination Options
with Activated Peroxide-based
Liquid Sporicides
Office of Research and Development
National Homeland Security Research Center
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EPA/600/R-13/009
March 2013
Evaluation of Expedient Decontamination Options with
Activated Peroxide-based Liquid Sporicides
Assessment and Evaluation Report
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
Office of Research and Development
National Homeland Security Research Center, Decontamination and Consequence Management Division
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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 Contract
#EP-C-09-027 WA 3-08 with ARCADIS, U.S. This report has been peer and administratively reviewed and
has been approved for publication as an Environmental Protection Agency document. It does not
necessarily reflect the views of the Environmental Protection Agency. No official endorsement should be
inferred. The Environmental Protection Agency does not endorse the purchase or sale of any commercial
products or services. This report includes photographs of commercially available products. The photographs
are included for purposes of illustration only and are not intended to imply that the Environmental Protection
Agency approves or endorses the product or its manufacturer.
Questions concerning this document or its application should be addressed to the principal investigator on
this effort.
M. Worth Calfee, Ph.D.
Decontamination and Consequence Management Division
National Homeland Security Research Center
U.S. Environmental Protection Agency (MD-E343-06)
Office of Research and Development
109 T.W. Alexander Drive
Research Triangle Park, NC 27711
Phone:919-541-7600
Fax:919-541-0496
E-mail: Calfee.Worth@epamail.epa.gov
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Foreword
Following the events of September 11, 2001, addressing the critical needs related to homeland security
became a clear requirement with respect to EPA's mission to protect human health and the environment.
Presidential Directives further emphasized EPA as the primary federal agency responsible for the
country's water supplies and for decontamination following a chemical, biological and/or radiological
(CBR) attack. EPA's Homeland Security Research Program (HSRP) supports EPA's mission to assist in
and lead response and recovery activities associated with CBR incidents of national significance. The
National Homeland Security Research Center (NHSRC), within EPA's Office of Research and
Development (ORD), leads the HSRP by conducting research and delivering products that improve the
capability of the Agency and other federal, state and local agencies to carry out their homeland security
responsibilities.
One goal of the HSRP is to provide information on decontamination methods and technologies that can
be used in the response and recovery efforts resulting from a CBR release over a wide area. The
complexity and heterogeneity of the wide-area decontamination challenge necessitates the understanding
of the effectiveness of a range of decontamination options. In addition to effective fumigation approaches,
rapidly deployable or readily available surface decontamination approaches have also been recognized
as a tool to enhance the capabilities to respond to and recover from such an intentional CBR release.
Through working with ORD's program office partners (EPA's Office of Emergency Management and
Office of Chemical Safety and Pollution Prevention) and Regional on-scene coordinators, the HSRP is
attempting to understand and develop useful surface decontamination procedures for wide-area
remediation. This report documents the results of a laboratory study designed to better understand the
effectiveness of surface cleaning and decontamination methods in an attempt to develop a readily-
deployable treatment procedure for surfaces contaminated with, for example, Bacillus anthracis spores.
These results, coupled with additional information in separate HSRP publications (available at
www.epa.gov/nhsrc') can be used to determine whether a particular decontamination technology can be
effective in a given scenario. The HSRP is pleased to make this publication available to assist the
response community to prepare for and recover from disasters involving biological contamination. This
research is intended to move EPA one step closer to achieving its homeland security goals and its overall
mission of protecting human health and the environment while providing sustainable solutions to our
environmental problems.
Jonathan Herrmann, Director
Homeland Security Research Program
National Homeland Security Research Center
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Acknowledgments
This effort was initiated and funded through an Interagency Agreement (IA# RW-70-95814001) with the
Department of Homeland Security, under the Wide Area Recovery and Resiliency Program (WARRP). EPA
partnered with Sandia National Laboratories (SNL) to evaluate the sporicidal efficacy of activated hydrogen
peroxide, a Sandia-developed decontamination technology. The support and coordination from Lori Miller
(DHS), Chris Russell (DHS), Paula Krauter (SNL), Mark Tucker (SNL), Bill Ginley (ECBC), and Doug Hardy
(US Navy) are greatly appreciated.
This effort was managed by M. Worth Calfee from ORD's National Homeland Research Center (NHSRC).
This effort was completed under U.S. EPA contract #EP-C-09-027 with ARCADIS-US, Inc. The support
provided by Tanya Medley (U.S. EPA/ORD/NHSRC) in acquiring the vast quantities of supplies required for
the completion of this project is also acknowledged. In addition, quality assurance review of this report by
Ramona Sherman (NHSRC) and Eletha Brady-Roberts (NHSRC) are acknowledged, as their review
significantly strengthened this document.
Lastly, the authors would like to thank the peer reviewers for their significant contributions. Specifically, the
efforts of Shannon Serre (EPA NHSRC), Vipin Rastogi (ECBC), and Marissa Mullins (EPA OEM) are
gratefully acknowledged.
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Contents
Disclaimer i
Acknowledgments iii
Tables vi
Figures vi
Appendices vii
List of Acronyms and Abbreviations viii
Executive Summary x
1. Introduction 1
1.1 Process 2
1.2 Project Objectives 3
2. Experimental Approach 4
2.1 Preparation of Coupons 4
2.2 Inoculation of Coupons 6
2.3 Preparation of Decontamination Solution 7
2.4 Decontamination Procedures 8
2.5 Neutralization Tests 10
3. Determination of Sporicidal Effectiveness 12
4. Sampling and Analytical Procedures 14
4.1 Sampling Procedures 14
4.1.1 Wipe Sampling 14
4.1.2 Vacuum Sock Sampling 14
4.1.3 Swab Sampling 14
4.1.4 Runoff Sampling 14
4.1.5 Aerosol Sampling 15
4.2 Microbiological Analysis 15
4.3 Activated Peroxide Characterization Measurements 15
4.3.1 Measurements of pH and Temperature of AHP 15
4.3.2 Measurements of H2O2 Concentration in AHP 15
IV
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4.3.2.1 KMnO4 Titration Procedure: 15
4.3.3 Measurements of the AHP Application Rate 16
5. Results and Discussion 17
5.1 AHP Characterization 17
5.1.1 pH and Temperature 17
5.1.2 Hydrogen Peroxide Concentration 20
5.1.3 PAA Concentration 21
5.2 Application Characterization 21
5.3 Decontamination Results 22
5.3.1 Neutralization Test Results 22
5.3.2 Inoculation Results 23
5.3.3 Log Reduction Results 24
5.3.4 Fate of Spores 25
5.3.5 Summary of Results 27
6. Quality Assurance 28
6.1 Sampling, Monitoring, and Analysis Equipment Calibration 28
6.2 Data Quality 28
6.2.1 QA/QC Checks 29
6.3 Acceptance Criteria for Critical Measurements 31
6.4 Data Quality Audits 32
6.5 QA/QC Reporting 32
6.6 Amendment to Original QAPP 32
7. References 33
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Tables
Table 2-1. Descriptions of Test Materials 4
Table 2-2. AHP Decontamination Solution Recipes 8
Table 2-3. Decontamination Test Matrix 9
Table 2-4. Neutralization Test Matrix 10
Table 5-1. Activated Hydrogen Peroxide Characterization Parameters 17
Table 5-2. Sprayer Flow Rates 22
Table 5-3. Neutralization Test Results for Porous and Non-Porous Materials 22
Table 5-4. Inoculation Control Results 23
Table 5-5. Air Samples Results 26
Table 5-6. Runoff Samples Results 26
Table 6-1. Analysis Equipment Calibration Frequency 28
Table 6-2. QA/QC Sample Acceptance Criteria 29
Table 6-3. Critical Measurement Acceptance Criteria 31
Figures
Figure 2-1. Test Coupons 5
Figure 2-2. Decontamination Chamber for AHP-Based Decontamination Testing (Left) and
Application of AHP Using Backpack Sprayer (Right) 9
Figure 5-1. pH of the AHP Solution Over Time (Oto 120 min Post-Mixing); Results from
Optimization Testing 18
Figure 5-2. pH of AHP over Time (Combined Results for Ten Batches of Decontamination
Solution) 19
Figure 5-3. Temperature of AHP Over Time (Combined Results for Ten Batches of
Decontamination Solution) 20
Figure 5-4. Concentration of H2O2 in AHP over Time (Combined Results for Ten Batches of
Decontamination Solution) 21
Figure 5-5. Material Specific Log Reductions for Procedure 1 and Procedure 2 24
Figure 5-6. Average Log Reductions for Procedure 1 and Procedure 2 on Nonporous and
Porous Building Materials 25
VI
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Appendices
Appendix A: Process Parameters and Decontamination Efficiency Results
Appendix B: Miscellaneous Operating Procedures (MOPs)
Appendix C: Stock Chemicals for Preparation of AHP Solution
Appendix D: Coupon, Test Chamber and Equipment Cleaning and Sterilization Procedures
VII
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List of Acronyms and Abbreviations
ADA Aerosol Deposition Apparatus
AHP Activated Hydrogen Peroxide
ATCC American Type Culture Collection
8. Bacillus
BSC Biological Safety Cabinet
CBRN Chemical, Biological, Radiological, and Nuclear
CPU Colony Forming Unit(s)
cm centimeters
CMAT Consequence Management Advisory Team
COC Chain of Custody
DF decimal factor
DHS Department of Homeland Security
Dl Deionized
DPG Dugway Proving Ground
DQO Data Quality Objective
DTRL Decontamination Technologies Research Laboratory
ECBC Edgewood Chemical Biological Center
ft feet or foot
ft2 square feet or foot
FAC free available chlorine
FIFRA Federal Insecticide, Fungicide, and Rodenticide Act
HEPA High Efficiency Particulate Air
IBRD Interagency Biological Restoration Demonstration
in Inch(es)
INL Idaho National Laboratory
LR Log Reduction
MDI Metered Dose Inhaler
MOP Miscellaneous Operating Procedure
NHSRC National Homeland Security Research Center
NIST National Institute of Standards and Technology
OPP Office of Pesticide Programs
ORD Office of Research and Development
OSWER Office of Solid Waste and Emergency Response
PAA Peroxyacetic acid (or Peracetic acid)
pAB pH-Adjusted Bleach
PARTNER Program to Align Research and Technology with the Needs of Environmental Response
PBST Phosphate Buffered Saline with 0.05% TWEEN®20
PPE Personal Protective Equipment
ppmv parts per million by volume
psi pounds per square inch
QA Quality Assurance
QAPP Quality Assurance Project Plan
QC Quality Control
SD Standard Deviation
VIM
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SNL Sandia National Laboratories
SAR Supplied-Air Respirator
SI International System of Units
STS Sodium Thiosulfate
ISA Tryptic Soy Agar
TSP Trisodium Phosphate
UV Ultraviolet (light)
VHP Vaporized Hydrogen Peroxide
WA Work Assignment
WARRP Wide Area Recovery and Resiliency Program
IX
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Executive Summary
This project supports the mission of the U.S. Environmental Protection Agency's Office of Research and
Development's Homeland Security Research Program (HSRP) by providing information relevant to the
decontamination of areas contaminated with a biological agent.
This project evaluated "low tech" expedient decontamination options with an Activated Hydrogen Peroxide
(AHP)-based liquid sporicide. The AHP decontamination solution was generated in an alkaline environment,
but the final product reaches slightly alkaline to neutral pH rapidly, making it safer and easier to handle and
substantially less corrosive than bleach-based sporicides.
The efficacy of liquid AHP was evaluated on common building materials (stainless steel, painted and
unpainted plywood, concrete, carpet, linoleum, glass, and tile) experimentally inoculated with aerosolized
spores of Bacillus atrophaeus (surrogate of Bacillus anthracis). Evaluation exercised two operational
procedures: (Procedure 1) spray-apply AHP, maintain vertical position for 15 minutes (no rinse), and dry
overnight before surface sampling, and (Procedure 2) spray-apply AHP, maintain vertical position for 15
minutes (no rinse), reapply AHP by spraying, maintain vertical position for 15 minutes (no rinse), and dry
overnight before surface sampling. Estimates of the number of spores removed (e.g., aerosol or liquid
runoff) from the material surface during the decontamination procedures were also performed.
For the AHP single application procedure (Procedure 1), the average log reduction (LR) values ranged from
1.6 to 8.4. Typically, nonporous materials were easier to decontaminate than were porous. The exception,
carpet, was relatively easy to decontaminate. Of all the materials, concrete demonstrated the lowest LR (<
2 LR) with Procedure 1. Nonetheless, greater than 6 LR was achieved for 50% of materials
decontaminated with the one step procedure (Procedure 1). The AHP dual-application procedure
(Procedure 2) resulted in greater than 6 LR of seven on the eight materials tested (LR values from 6.8 to
8.4). High decontamination efficacy was not achieved for unpainted treated wood material, regardless of the
application procedure (mean LR = 4.2). Complete kill (recovery of no viable spores after decontamination)
was achieved on carpet and glass using Procedure 1, and on Tile, stainless steel, carpet, glass, and
linoleum using Procedure 2.
These data suggest that this "low-tech" decontamination process, when using the two-application procedure
described herein, can provide > 6 LR in the number of viable spores on the common building materials
tested, with the exception of unpainted treated wood. The longer exposure duration and increased volume
of AHP applied, associated with Procedure 2, afforded higher surface decontamination efficacy, especially
for porous materials. Materials with high organic demand (e.g., unpainted wood) that are typically more
difficult to decontaminate may present a challenge to the liquid AHP-based process. The preliminary fate of
spores estimates suggest that the AHP liquid sporicide-based decontamination process leads to physical
removal of spores from decontaminated surfaces, with a consequent transport of viable spores to air and to
the post-decontamination liquid waste. In several tests, considerable amounts (up to 7 logs) of viable
spores were recovered in the liquid runoff.
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1. Introduction
The Department of Homeland Security (DHS) and other appropriate Federal departments and agencies
have been tasked to develop comprehensive plans which "provide for seamless, coordinated Federal, state,
local, and international responses to a biological attack." As part of these plans, EPA, in a coordinated effort
with DHS, is responsible for "developing strategies, guidelines, and plans for decontamination of persons,
equipment, and facilities" to mitigate the risks of contamination following a biological weapons attack [1].
One goal of the EPA's Homeland Security Research Program (HSRP) is to provide expertise and guidance
on the selection and implementation of decontamination methods and provide the scientific basis for a
significant reduction in the time required for recovery and the cost of decontamination events. To meet this
goal, EPA's National Homeland Security Research Center (NHSRC) provides expertise and products that
can be widely used to help prevent, prepare for, and recover from public health and environmental
emergencies arising from terrorist threats and incidents.
Decontamination can be defined as the process of inactivating or reducing a contaminant in or on humans,
animals, plants, food, water, soil, air, areas, or items through physical, chemical, or other methods to meet a
cleanup goal. In terms of the surface of a material, decontamination can be accomplished by physical
removal of the contaminant or via inactivation of the contaminant with antimicrobial chemicals, heat,
ultraviolet (UV) light, etc. Physical removal could be accomplished via in situ removal of the contaminant
from the material or by physical removal of the material itself (i.e., disposal) [2-6]. Similarly, inactivation of
the contaminant can be conducted in situ or after removal of the material for ultimate disposal [2, 3, 5, 6].
Following the 2001 anthrax incidents, a combination of removal and in situ decontamination was used [2,
3]. The balance between the two procedures 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 decontamination technologies had been proven for use against spores of Bacillus
anthracis at the time. The cost of disposal proved to be significant and was complicated by the nature of the
waste (e.g., finding an ultimate disposal site). Since 2001, a primary focus for facility remediation has been
improving the effectiveness and practical application of in situ decontamination methods and evaluating
waste treatment options to be able to provide information necessary to optimize the
decontamination/disposal paradigm. This optimization has a significant impact on reducing the cost of and
time for the remediation effort.
The HSRP's decontamination-related efforts support the Office of Solid Waste and Emergency Response
(OSWER), with the Office of Pesticide Programs (OPP) as a key stakeholder. OSWER, through its Special
Teams which include the Chemical, Biological, Radiological, and Nuclear (CBRN) Consequence
Management Advisory Team (CMAT), supports the emergency response functions carried out by the
Regional Offices. OPP supports the decontamination effort by providing expertise on biological agent
inactivation and ensuring that the use of pesticides in such efforts is done in accordance with the Federal
Insecticide, Fungicide and Rodenticide Act (FIFRA). Close collaboration between the different program
offices having homeland security responsibilities is sought to rapidly increase the EPA's capabilities to help
the Nation recover from a terrorist event involving the intentional release of chemical, biological, and
radiological (CBR) materials.
Quick, effective and economical decontamination methods that have the capacity to be employed over wide
areas (outdoor and indoor) required to increase emergency preparedness are one specific focus of the
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HSRP. Numerous decontamination methods have been and continue to be evaluated under the HSRP.
These decontamination approaches span the spectrum from sophisticated technologies such as
fumigations; to more readily-available approaches such combined mechanical and chemical procedures
(vacuum, scrub/wash and bleach) for surface decontaminations. If proven effective, expedient approaches
involving washing and cleaning with readily-available equipment and off the shelf sporicides would
significantly increase EPA's readiness to respond to a wide-area contaminant release.
The research described in this report was conducted by EPA under the DHS-led Wide Area Recovery and
Resiliency Program (WARRP), which holistically aims to increase our Nation's ability to respond to and
recover from acts of chemical, biological, or radiological (CBR) terrorism. This work was a continuation of
previous decontamination studies with pH amended bleach (pAB)- based liquid sporicides. The
effectiveness of amended bleach has been demonstrated, yet several downsides of this technology have
been documented [2, 3, 7]. Bleach, and especially amended bleach, is known to be corrosive to metals and
its application often necessitates the use of specialized personal protective equipment (PPE) due to chlorine
off-gassing. This project evaluated an alternative to bleach, activated hydrogen peroxide (AHP), for'low-
tech' decontamination applications. Peroxides (-O-O-) are strong oxidants that offer an environmentally
friendly alternative to the toxic and corroding chlorine-based decontaminants [8, 9]. The sporicidal
effectiveness of hydrogen peroxide (H2O2) solutions is due to the peroxy anion (OOH~) and hydroxyl free
radicals (OH-) [9,10]. Another peroxy compound that is commonly used, and often added as a
supplemental oxidizing agent in mixtures with H2O2, is peracetic acid - PAA, CH3CO(OOH) [8]. PAA is
effective against all microorganisms, including bacterial spores, due to its high oxidizing potential. The
disinfectant composition mixtures of H2O2 and PAA have a synergistic sporicidal efficacy [8]. Mixed H2O2
and PAA liquid sporicides are most commonly prepared just prior to use by combining two or more
components. Due to stringent shipping and handling requirements, PAA is often generated in situ, prior to
decontamination, using activated H2O2 solutions. This method has many variations, but the foundation of
the chemistry is the same; generation of PAA through the perhydrolysis of an acetyl donor, with a
specifically formulated H2O2 solution as a peroxygen source [9,10].
1.1 Process
The general process investigated in this project was decontamination of building surfaces contaminated with
Bacillus spores (i.e., surrogates of 8. anthracis). Test coupons were decontaminated with AHP sprayed
using two different decontamination procedures. Positive control coupons (i.e., contaminated with spores
but not decontaminated) were used to determine the pre-treatment (pre-decontamination) loading on each
coupon type and were a reference for decontamination efficacy calculations (see Section 3).
The decontamination operational procedures were developed based on the previous EPA and Sandia
National Laboratories (SNL)/lnteragency Biological Restoration Demonstration (IBRD) efforts [11].
Decontamination procedures provided by SNL were refined and tested through pilot-scale evaluation in a
decontamination test chamber in the NHSRC testing facilities.
After decontamination, surface sampling of coupons was conducted with wetted wipes or vacuum socks,
depending upon material type. Liquid runoff samples and aerosol samples were also collected to determine
potential routes of contamination spread by the procedures. Quality control (QC) samples such as
procedural blank coupons (coupons that underwent the decontamination process but which were not
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inoculated) and negative controls (which were not inoculated and did not undergo the decontamination
process) were included in order to monitor for cross-contamination.
All samples were analyzed for the quantitative determination of viable spores recovered. Overall
decontamination effectiveness of AHP was determined as a function of the procedures and material types.
The fate of the spores (viable spores transferred to runoff or air) was also determined.
1.2 Project Objectives
The primary objectives of this study were:
1. To determine occurrence and potential reduction of viable bacterial spores (i.e., effectiveness) on
porous and nonporous building materials decontaminated with AHP-based liquid sporicides;
2. To determine the effect of varying the application procedure on sporicidal efficacy (i.e., the effect of
re-application and the increased contact time); and
3. To determine the fate of the spores during the decontamination process.
The operational parameters of decontamination (e.g., pH and temperature of decontamination solution
within the pot life of the decontamination solution, effectiveness of AHP delivery [as a function of the spray
apparatus flow rate and spray coverage/pattern]) were considered important to understand the sporicidal
activity of an AHP-based decontamination process and were characterized in addition to sporicidal
effectiveness.
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2. Experimental Approach
This section describes the test materials, test facilities and equipment, general decontamination approach
and test conditions, and the methods that were used to evaluate the data related to the project objectives.
Testing was conducted by EPA's Decontamination Technologies Research Laboratory (DTRL), in a spray
chamber described previously [5] and located at EPA's Research Triangle Park facility. Sampling and
analytical procedures are described in Section 4.
2.1 Preparation of Coupons
Coupons (14 inch by 14 inch) were prepared from eight types of porous and nonporous building materials
(Table 2-1, Figure 2-1) that are typical of the materials found in residential dwellings and meet industry
standards or specifications for indoor use. Documented procedures were established and followed for
coupon preparation (see Appendix B, DTRL MOP #3150) to ensure uniformity across samples and
experiments.
Table 2-1. Descriptions of Test Materials
Materials
Porous Materials
Painted treated plywood
Unpainted treated plywood
Carpet
Unsealed concrete
Nonporous Materials
Stainless steel
Glass
Linoleum
Tile
Source
Georgia Pacific
Atlanta, GA
Georgia Pacific
Atlanta GA
Home Depot
Durham, NC
Home Depot
Durham, NC
Dillon's Supply Co. Raleigh,
NC
Durham Glass, Inc.
Durham, NC
Home Depot
Durham, NC
Home Depot
Durham, NC
Description
23/32" Alkaline Copper
Quaternary Treated
Plywood
23/32" Alkaline Copper
Quaternary Treated
Plywood, painted with
BEHR Exterior Flat White
Beaulieu Loop Carpet
Quikrete Sand/Topping
Mix, trowel-smoothed
surface
304 stainless steel, 16-
gauge
1/4" (thickness) residential
glass, seamed edges
Armstrong Residential
Sheet Vinyl Flooring
MARAZZI Ceramic Floor
Tile; smooth, glazed finish
Part Number
"Plytanium® Sheathing"
"Plytanium® Sheathing"
BEHR Paint #4850
Home Depot #263-050
409-921
929-522
n/a
n/a*
243-154
538-570
*n/a = not applicable
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a. Unpainted treated plywood b.Glass
c. Painted treated plywood
e. Unsealed concrete
Figure 2-1. Test Coupons
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The stainless steel and glass coupons were sterilized prior to use by steam autoclave utilizing a gravity
cycle program (see Appendix B, BioLab MOP #6570). The remaining materials (painted and unpainted
treated plywood, carpet, linoleum, tile, and unsealed concrete) were sterilized using VHP® (1000 parts per
million volume * hours, ppm-hrs) generated using a STERIS VHP® 1000ED generator loaded with a 35%
H2O2 Vaprox® cartridge. The sterility of the coupons was verified through the use of laboratory blank control
samples.
2.2 Inoculation of Coupons
Inoculation of test and positive control coupons with spores of 8. atrophaeus was performed via aerosol
deposition using a metered dose inhaler (MDI).
The test organism for this work was a powdered spore preparation of B. atrophaeus American Type Culture
Collection (ATCC) 9372 and silicon dioxide particles. This bacterial species was formerly known as 8.
subtilis var. niger and subsequently 8. globigii. The preparation was obtained from the U.S. Army Dugway
Proving Ground (DPG) Life Science Division. The preparation procedure is reported in Brown et al. [11]
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 1 x
1011 viable spores per gram was prepared by dry blending and jet milling the dried spores with fumed silica
particles (Deguss, Frankfurt am Main, Germany). The powdered preparation was loaded into MDIs by
ECBC according to a proprietary protocol. QA documentation is provided by ECBC with each batch of MDIs.
Control checks for each MDI were included in the batches of coupons dosed with a single MDI.
Coupons (test and positive controls) were inoculated with ~2 x 108 spores of 8. atrophaeus from an MDI
using a modification to the procedure detailed in modified BioLab MOP #6561 (see Appendix B). Each
coupon was inoculated (dosed) independently by being placed into a separate dosing chamber designed to
fit one 14 in by 14 in coupon of any thickness. In accordance with modified BioLab MOP #6561, the MDI
was discharged a single time into the dosing chamber. The MDIs are claimed to provide 200 discharges per
MDI. The number of discharges per MDI was tracked so that use did not exceed this value. Additionally, in
accordance with modified BioLab MOP #6561, the weight of each MDI was determined after completion of
the contamination of each coupon.
Note: Due to problems with loading coupons observed during tests 1,6,8,9, and 14, an extra Quality
Assurance (QA) step was temporarily introduced to the inoculation procedure. Before and after each
stainless steel inoculation control coupon and every three test coupon samples, stainless stubs were
dosed and immediately processed by the on-site Biocontaminant Laboratory. A new procedure for
actuator cleaning between actuations was also developed (vertical actuator orifice was cleaned using
compressed air between each of the actuations). The latter procedure was used in all subsequent
inoculations.
The contamination control coupons (14 in by 14 in stainless steel coupons) were inoculated as the first,
middle, and last coupons within a single group of coupons inoculated by any one MDI within a single test.
Following dissemination, spores were allowed to settle onto the coupon surfaces for a minimum period of 18
hours.
A log was maintained for each set of coupons that were dosed via modified BioLab MOP #6561. Each
record in this log consisted of the unique coupon identifier, the MDI unique identifier, the date, the operator,
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the weight of the MDI before dissemination into the coupon dosing device, the weight of the MDI after
dissemination, and the difference between these two weights. The coupon codes were pre-printed on the
log sheet prior to the start of coupon inoculation (dosing).
After the minimum 18-hour period, the coupons were removed from the dosing chamber and moved to an
isolated cabinet (Test Coupon Cabinet) which contained all inoculated coupons for a single test. The
handling of the inoculated coupons, including movement to minimize or control spore dispersal, was done in
accordance with the modified BioLab MOP #6561. Procedural blank coupons were stored in a separate
isolated cabinet (Blank Coupon Cabinet).
2.3 Preparation of Decontamination Solution
The AHP decontamination formulation was developed by SNL under the WARRP research efforts. The
original recipe for preparation of AHP provided by SNL is given in Table 2-2. For preparation of large
batches of AHP, the original recipe was modified (Table 2-2) as follows:
• 15% H2O2 was used to prepare the decontamination solution; this change was due to facility-specific
Health and Safety regulations regarding alkalization of 50% H2O2;
• the discontinued alkyldimethylbenzyl ammonium chlorides (C12-C16)/isopropyl alcohol -based
surfactant (Variquat 80 MC) was replaced with a similar commercially available product;
(alkyldimethylbenzyl ammonium chlorides (C12-16)/alkyldimethylamines(C12-C16)/ethanol- based
surfactant; Maquat MC 1412-80%E; and
• the addition of potassium carbonate was slightly higher (160 g/5 L or 3.2 g/100 ml of final formulation)
than in the original recipe (3 g/100 ml of final formulation); this amount was optimized for mixing of
large batches of AHP and resulted in the pH values closest to the target pH of 9.5 immediately post-
mixing.
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Table 2-2. AHP Decontamination Solution Recipes
Part A
PartB
PartC
Original recipe*
Dl water [ml_]
K2C03 [g]
Variquat [ml_]
Ethanol [mL]
50% H2O2 [mL]
Triacetin [mL]
76.9
3
0.1
5
12
3
Modified recipe**
Dl water [mL]
K2C03 [g]
Maquat [mL]
Ethanol [mL]
15%H2O2[mL]
Triacetin [mL]
2435
160
5
250
2000
150
original recipe as developed by SNL for small scale testing.
** large batch (5000 mL) modified recipe as used in EPA pilot-scale testing; details on stock solutions of chemicals used for preparation
of decontamination solution are given in Appendix C.
The decontamination formulation was prepared according to DTRL MOP #3177 (see Appendix B) by mixing
Part A with Part B, and then adding Part C (the Activator) to the Part A/B mixture. The solution was ready to
use upon activation (combination of ingredients) and required no further dilution or manipulation of
components.
The target concentration of H2O2 (6% immediately post mixing) was verified by potassium permanganate
titration (see Appendix B, DTRL MOP #3177) and then monitored throughout testing (see Section 5.1.2).
The temperature and pH measurements were performed for each batch of the AHP solution prepared and
checked priorto each application of sporicide (see Section 5.1.1). Per communication with SNL, the pot life
of the AHP formulation was assumed to be two hours.
2.4 Decontamination Procedures
Decontamination procedures were developed in collaboration with SNL and are described in detail in DTRL
MOP #3177 (see Appendix B). An overview of the Decontamination Test Matrix is outlined in Table 2-3.
Briefly, sets of the building material coupons were inserted in vertical position in the test coupon holders of
the small spray test chamber (Figure 2-2). The chamber dimensions were 4 ft high by 4 ft wide by 4 ft deep.
The chamber was designed to accommodate three 14 x 14 inch coupons at a time in a horizontal or vertical
orientation. In this study, only the vertical orientation assembly was utilized. The chamber was constructed
of solid stainless steel with the exception of the front face and top which were both clear acrylic plastic. The
acrylic door was fitted with ports to allow the insertion of the backpack delivery system into the chamber.
Although the chamber is fitted with three ports (each port centered in front of one of the triplicate coupons),
to administer a seamless and rapid (15 seconds) application of the decontaminantto the set of coupons,
only the center-positioned port was used for this study. The other two ports remained in the closed position
during testing.
Decontamination solution was applied using a SHURflo ProPack™ SR600 Rechargeable Electric Backpack
Sprayer (SHURflo Inc., USA) equipped with a TeeJet spray wand with an adjustable tip (SHURflo Inc.,
-------
USA). The sprayer wand could be moved to accomplish the desired spray pattern. The reverse-pyramid
design of the chamber bottom allowed for collection of runoff from the coupons during the decontamination
procedure through a central (3 in diameter) drain. The bottom of the chamber has a 189 L (50 gallon)
collection capacity.
Figure 2-2. Decontamination Chamber for AHP-Based Decontamination Testing (Left) and Application of AHP
Using Backpack Sprayer (Right)
Table 2-3. Decontamination Test Matrix
Test ID
1R
2
3
4
5
6R
7
8R
9R
10
11
12
13
14R
15
16
Material
Painted treated plywood
Stainless steel
Carpet
Glass
Linoleum
Painted treated plywood
Stainless steel
Carpet
Glass
Linoleum
Concrete
Tile
Unpainted treated plywood
Concrete
Tile
Unpainted treated plywood
Decon Procedure
Procedure 1
Procedure 1
Procedure 1
Procedure 1
Procedure 1
Procedure 2
Procedure 2
Procedure 2
Procedure 2
Procedure 2
Procedure 1
Procedure 1
Procedure 1
Procedure 2
Procedure 2
Procedure 2
Note: "R" tests indicate repeated tests, as initial attempt was aborted due to failed inoculation
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The chamber was fitted with connections allowing filtered air to enter and filtered exhaust to exit via a readily
accessible connection to the facility's air handling system. Aerosol samples were collected from the
chamber using Via-Cell® BioAerosol Cassettes. The sampling point was approximately 5 cm above and 45
cm in front of the coupon surface (to approximate the head position of a decontamination worker).
After the Via-Cell® BioAerosol Cassettes and coupons were assembled, the decontamination sequence
continued as follows:
1. Verification of critical operational parameters (pH, concentration of H2O2 and temperature of AHP
decontamination solution, check of the backpack sprayer flow and spray pattern; results are described
in Section 4.3).
2. Application of a prescribed decontamination sequence. Two different decontamination procedures
(Procedure 1 and Procedure 2) were evaluated for each type of test material. The Procedure 1
decontamination sequence was: "spray once-wait 15 min-no rinse-dry overnight", while the Procedure 2
sequence was "spray-wait 15 min-reapply-wait 15 min-no rinse-dry overnight".
3. After the exposure time (15 min or 2 x 15 min) was reached, test coupons, procedural blanks, and
positive controls were moved to designated storage cabinets and allowed to dry overnight.
4. On the following day, coupons were sampled using sampling techniques described in Section 4.1 and
transferred to the on-site Biocontaminant Laboratory for microbiological analysis. Samples were
transferred in sterile primary independent packaging within sterile secondary containment containing
logical groups of samples for analysis. All samples were accompanied by a completed chain of custody
(COC) form.
5. After the on-site Biocontaminant Laboratory performed a quantitative assessment of viable spores for
each type of samples, the determination of surface decontamination efficacy (comparison of viable
spore concentrations from positive controls and test coupons) was performed (see Section 4.2).
2.5 Neutralization Tests
To test the effect of residual AHP on spore recovery, a set of preliminary tests was performed to investigate
the need for neutralization of samples post-collection. The test matrix for these neutralization tests is shown
in Table 2-4.
Table 2-4. Neutralization Test Matrix
Test
A1
A2
B1
B2
Decontaminated
Yes
No
Yes
No
Material Type
Plywood
Plywood
Stainless Steel
Stainless Steel
Total # of Coupons
5
5
5
5
Five replicate coupons of each type (sprayed and not sprayed) were sampled using wipes. Wipes were
then extracted with the sample extraction solution, and the extract solution was then spiked with 1 x 107
spores of 8. atrophaeus. Recoveries from the decontaminated coupons (sprayed) were compared to
recoveries from blank (not sprayed) coupons. If a statistically significant difference existed between the two
10
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populations (recovery from sprayed vs. recovery from not sprayed) for each coupon type, then neutralization
of samples post-collection would be needed. No statistical difference was observed between recoveries
from sprayed or not sprayed coupons, hence the use of neutralizer on coupons was not considered
necessary (see Section 5.3.1).
The runoff samples were neutralized using a 30% sodium thiosulfate (STS) solution. The target
concentration of STS in a single runoff sample needed to neutralize residual AHP without affecting recovery
assays was adopted from the results of liquid-based testing performed by SNL. In the liquid-based
experiments, 5 ml of 30% STS was added to 35 ml of total solution for each sample, resulting in the final
STS concentration of 4.3%. The maximum volume of the AHP runoff per 15 second spray was estimated as
approximately 200-250 ml depending on the backpack sprayer flow rate (in the preliminary testing, 210 ml
of the AHP solution runoff was collected from stainless steel per spray; no water rinse included). Hence, 42
ml of STS solution per single spray application was added to each runoff collection container (carboy) prior
to the runoff collection. Results for the concentration of H2O2 in the neutralized runoffs are given in Section
5.3.1.
11
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3. Determination of Sporicidal Effectiveness
The sporicidal effectiveness (efficacy) of a decontamination technique is a measure of the ability of the
method to inactivate and/or remove the spores from a contaminated material surface (i.e., represented by
coupons in this study). The sporicidal effectiveness is evaluated by measuring the difference in the
logarithm of the measured colony forming units (CPU) before decontamination (determined from sampling
the positive control coupons) and after decontamination (determined from sampling the test coupons) for the
same type of material. The number of viable spores recovered was measured as CPU and reported as a log
reduction on the specific material surface as defined in Equation 1-1.
Nr
where:
N.
(1-1)
Surface decontamination effectiveness; the average log
7? = reduction of spores on a specific material surface (surface
material designated by /)
NC The average of the logarithm (or geometric mean) of the
2_j log(CFC/Cj<.) _ number of viable spores (determined by CPU) recovered on the
— control coupons (C indicates control and Nc is the number of
^c control coupons)
The average of the logarithm (or geometric mean) of the
number of viable spores (determined by CPU) remaining on the
surface of a decontaminated coupon (S indicates a
decontaminated coupon and Ns is the number of coupons
tested).
When no viable spores were detected, a value of 0.5 CPU was assigned to the maximum plated volume to
determine the detection limit for CFUs,k, and the efficacy was reported as greater than or equal to the value
calculated by Eqn. 1-1.
12
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The standard deviation of the average log reduction of spores on a specific material (r\\) is calculated by
Eqn. 1-2:
where:
and
where:
k
Ns
_ Standard deviation of r\\, the average log reduction of spores on
a specific material surface
_
' *
The average log reduction of spores on a specific material
surface (surface material designated by /)
1.1.1.1.1.1 The average of the log reduction from the surface
of a decontaminated coupon (Equation 1-3)
= 1.1.1.1.1.2 Number of test coupons of a material surface type.
log(CFt/c) = —
CFU
s,k
s
Represents the "mean of the logs" (geometric mean), the
average of the logarithm-transformed number of viable
spores (determined by CPU) recovered on the control
coupons (C = control coupons, Nc = number of control
coupons, k = test coupon number and Ns is the number
of test coupons)
Number of CPU on the surface of the kth decontaminated
coupon
1.1.1.1.1.2.1.1 Total number (1,k) of decontaminated
coupons of a material type.
13
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4. Sampling and Analytical Procedures
4.1 Sampling Procedures
Within a single test, surface sampling of the materials was completed first for all procedural blank coupons
before sampling of any test material. Surface sampling was done either by wipe sampling or vacuum
sampling in accordance with the protocols documented below. Prior to the sampling event, all materials
needed for sampling were prepared using aseptic techniques. The materials specific for each protocol are
included in the relevant sections below. The general sampling supplies were purchased sterile or
sterilized/disinfected prior to each sampling event.
4.1.1 Wipe Sampling
Wipe sampling is typically used for small sample areas of nonporous smooth surfaces such as ceramics,
vinyl, metals, painted surfaces, and plastics [12]. Wipe sampling was done on all nonporous and some
porous materials according to DTRL MOP #3144 (see Appendix B). The general approach is that a
moistened sterile noncotton pad is used to wipe a specified area to recover bacteria, viruses, and biological
toxins.
4.1.2 Vacuum Sock Sampling
Vacuum socks were used to sample plywood and carpet coupons due to the difficulty and inefficiency of
implementing the wipe sampling procedure on these rough surfaces. Vacuum sock sampling was
conducted according to DTRL MOP #3145 (see Appendix B).
4.1.3 Swab Sampling
DTRL MOP #3135 (see Appendix B) was used for collecting swab samples. The general approach was to
use a moistened swab to wipe a specified area to recover bacterial spores. Swab samples were collected
from all decontamination procedure equipment and test materials prior to their use in experimentation to
confirm sterility.
4.1.4 Runoff Sampling
Prior to decontamination, the runoff collection vessel was charged with 30% STS neutralization solution to
neutralize all AHP solutions immediately upon collection, as the AHP solution drips from the sprayed
coupons. After all coupons from a single set were moved to the Decontaminated Coupon Cabinet or
Procedural Blank Cabinet, the chamber was rinsed with deionized (Dl) water. A pre-weighed, sterile runoff
collection carboy was used to collect the runoff. The total mass of liquid collected was recorded for
comparison of the final weight versus the initial weight value. For a given coupon set (material type or all
blanks) all runoff was pooled, homogenized (by shaking the carboy), and analyzed as a composite sample.
Triplicate 100 mL aliquots (subsamples) were collected from each carboy using aseptic technique. Following
collection, the runoff aliquots were triple-contained in sterile bags and transported to the on-site
Biocontaminant Laboratory for submission and analysis at the conclusion of the entire test. The runoff
samples were stored at 4 ± 2 °C until processed, which occurred within 24 hours.
14
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4.1.5 Aerosol Sampling
Aerosol samples were collected from the bulk environment of the spray chamber using Via-Cell® BioAerosol
Cassettes. These samples were used to estimate the occurrence and magnitude of fugitive emissions of
viable B. atrophaeus spores, during the decontamination process. The sampling point was approximately 5
cm above and 45 cm in front of the coupon surface (to approximate the head position of a decontamination
worker). Aerosol samples were collected according to DTRL MOP #3155 (see Appendix B). Samples were
not collected isokinetically, therefore the results should be used only to approximate the magnitude of 8.
atrophaeus spores aerosolized within the test chamber.
4.2 Microbiological Analysis
The on-site Biocontaminant Laboratory analyzed all samples for the presence (swab samples) of
contamination or to quantify the number of viable spores recovered per sample (vacuum, air, and surface
samples). For all sample types, phosphate buffered saline with 0.05% TWEEN®20 (PBST) was used as the
extraction buffer. The PBST was prepared according to BioLab MOP #6562 (see Appendix B). After the
appropriate extraction procedure, as described in the sections to follow, the buffer was subjected to a five
stage serial dilution (10"1 to 10"5) in accordance with BioLab MOP #6535a (see Appendix B). The resulting
samples were spread plated in triplicate onto tryptic soy agar (TSA) and incubated overnight (minimum of 18
hours) at 35 ± 2 °C. Following incubation, CFU were enumerated manually. Only data from plates with
between 30 and 300 CFU were used for calculations of recovery. When fewer than 30 CFU were observed
on plates from the least dilute samples, BioLab MOP #6565 (see Appendix B) was followed in an attempt to
detect spores at the lowest level possible.
The extraction procedure used to recover spores varied by sample type (wipes, filter socks, liquid, filter
cassette) and can be found in BioLab MOP #6572 for extraction of vacuum socks, BioLab MOP #6567 for
extraction of wipe samples, BioLab MOP #6563 for analyses of swab samples, and BioLab MOP #6571 for
air sample cassettes. These MOPs are included in Appendix B.
4.3 Activated Peroxide Characterization Measurements
4.3.1 Measurements of pH and Temperature of AHP
The pH and temperature measurements of the AHP solution were performed using an Oakton pH meter
(Oakton PC 510; Eutech Instruments Pte. Ltd., Singapore, Singapore) equipped with a pH probe and a
thermocouple probe. Calibration of the pH meter was performed daily.
4.3.2 Measurements of H2O2 Concentration in AHP
The concentration of H2O2 in the AHP solution was verified by analyzing with the potassium permanganate
(KMnO4) titration procedure described below.
4.3.2.1 KMnO4 Titration Procedure:
Reagents:
15
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• 5% Sulfuric acid (H2SO4)
1.0NKMn04
Procedure:
1. Transfer approximately 20 g (~20 ml) of the H2O2 sample to a tared weighing container and
weigh on an analytical balance. Record weight of the H2O2 sample.
2. Carefully wash the weighed sample into a 250 ml flask with distilled water, dilute to mark, and
mix thoroughly.
3. Pipet a 25 ml aliquot into a 400 ml beaker containing 250 ml of Dl water and 10 ml of H2SO4.
4. Titrate to the first permanent pink color with 1.0 N KMnO4.
Calculation:
% H202 = [(ml KMn04) x (N) x (0.01701) x (1000)]/(grams of H2O2 sample used)
where N = normality of the standard KMnO4.
4.3.3 Measurements of the AHP Application Rate
The spray pattern was tested by spraying at the appropriate distance (1 ft) onto a piece of 1.17 ft by 1.17ft
blue construction paper mounted in the orientation of the material section. The spray was discharged (1 sec)
into the center of the paper and the pattern was visually assessed for consistency, shape, and width.
The flow rate of the backpack sprayer was measured at the start and end of testing of each set of three
coupons on which the sprayer was being used. The flow rate was determined by discharging the AHP from
the backpack sprayer into a graduated cylinder for 10 sec. The volume of AHP dispensed was read from
the graduated cylinder and recorded. The results are presented in Table 5-2.
The time for spray application and exposure time (15 seconds per set of three coupons and 15 minutes or 2
x 15 minutes, respectively) was measured using an National Institute of Standards and Technology (NIST)-
traceable stopwatch. The exposure time, as defined here, was time at which the coupons were no longer
dripping decontaminant (runoff). This time was determined during preliminary experiments using a single set
of porous and nonporous material coupons and was utilized for all subsequent tests.
16
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5. Results and Discussion
This section discusses results of the AHP solution characterization tests (Section 5.1), results of application
characterization testing (Section 5.2), and results of the AHP-based decontamination of building materials
using Procedure 1 and Procedure 2 (Section 5.3).
5.1 AHP Characterization
The pH, temperature and concentration of H2O2 measurements in the working decontamination solution
were considered critical to understand dynamic changes of key decontamination agents (H2O2, PAA) in the
AHP solution. These three parameters were monitored throughout the entire testing (immediately after
mixing and before each application of decontamination solution). Test-specific results of the pH,
temperature and H2O2 concentration measurements are shown in Appendix A (Table A-1 - Procedure 1 and
Table A-2 - Procedure 2).
Table 5-1 shows cumulative results for the AHP critical characterization parameters. Sections 5.1.1 and
5.1.2 discuss these results in more detail.
Table 5-1. Activated Hydrogen Peroxide Characterization Parameters
Parameter measured
PH
H202 [%]
T[°C]
n
30
24
30
Average
8.5
6.0
28.4
SD
0.7
0.3
2.1
Min
7.4
5.3
27.1
Max
9.6
6.4
32.8
n - number of measurements.
5.1.1 pH and Temperature
In the small scale liquid-based decontamination testing, the pH value acceptance criterion for the working
AHP solution immediately post-mixing was set as 9.5 ± 0.1 pH units. The pH of decontamination solution
post-mixing was considered to be a good indicator of proper preparation and sporicidal effectiveness of the
AHP solution. This parameter was, therefore, also optimized for preparation of larger batches of AHP in the
pilot-scale study.
The optimization of the decontamination solution initial pH was performed in a series of small-volume (100
ml) preliminary tests (Figure 5-1). The optimum starting pH values (in the 9.5-9.6 range) were achieved by
a nominal increase of the ratio of potassium carbonate in the AHP recipe (see Section 2.3. for original recipe
as developed by SNL and modified recipe used in the pilot-scale study). The pH profiles of test AHP
solutions as a function of time were also investigated in these preliminary tests, with a drastic decline in pH
values observed during the first ten minutes post-mixing (Figure 5-1).
17
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-Test 1 072412 -B-Test 2 072412 -*-Test 3 072412
9.80
9.60
8.40
8.20 4
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140
Time post-mixing [min]
Figure 5-1. pH of the AHP Solution Over Time (Oto 120 min Post-Mixing); Results from Optimization Testing
Consequently, the strict pH acceptance criteria (9.5 ±0.1 pH units) used by SNL for the small scale liquid-
based testing was not considered practical for the pilot-scale testing decontamination experiments.
However, as stated before, the pH measurements were performed post-mixing and before each application
of AHP. Average pH of large batches of AHP (5000-10000 ml) prepared for pilot-scale decontamination
testing had shown starting pH ranges of 9.2-9.6 (average pH 9.4 ± 0.1 standard deviation (SD)). The pH of
the decontamination solution prior to application ranged from 7.4-9.0 (average pH 8.2 ± 0.6 SD). (see
Tables A-1 and A-2 in Appendix A).The pH overtime for all batches of AHP solutions used in the pilot scale
testing are shown in Figure 5-2. The downward trend of pH versus time observed in the initial AHP testing
(Figure 5-1) was also noted for all batches of decontamination solution used in the pilot-scale testing
experiments (Figure 5-2). The average pH drop rate was 1.4 pH units per hour.
18
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10.0
9.5
9.0
8.5
8.0
7.5
A
A
ApH
7.0
20
40 60 80
Minutes post-mixing
100
120
Figure 5-2. pH of AHP over Time (Combined Results for Ten Batches of Decontamination Solution)
Temperature was monitored for freshly-prepared batches of AHP and in samples taken from the backpack
sprayer prior to each spray. Average temperature prior to application was 29.6 °C ± 1.8 °C (range 27.1 to
32.8 °C) (see Tables A-1 and A-2 in Appendix A). The temperatures for all batches of AHP solutions used in
the pilot scale testing are shown in Figure 5-3. A slight increase in temperature overtime was observed for
each batch of the AHP solution tested (average AT between preparation and last application of AHP was
3.4 °C, with a 2.9 °C per hour average temperature increase rate).
19
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40.0
36.0
32.0
O
28.0
T[C]
24.0
20.0
20
40 60 80
Minutes post-mix ing
100
120
Figure 5-3. Temperature of AHP Over Time (Combined Results for Ten Batches of Decontamination Solution)
5.1.2 Hydrogen Peroxide Concentration
Concentration of H2O2 in the working decontamination solution was measured via permanganate titration in
24 samples of AHP taken from the backpack sprayer reservoir prior to each spray application and in
selected freshly prepared batches of AHP.
The concentration of H2O2in freshly prepared batches of AHP was 6.1% ± 0.2%. The average pre-spray
concentration of H2O2 in the working AHP solution was 6.0 ± 0.3% (range 5.3% to 6.2%) (see Tables A-1
and A-2 in Appendix A). Cumulative results for H2O2 concentration overtime measurements are shown in
Figure 5-4. Unlike pH and temperature values, the H2O2 concentration in the AHP solution did not show any
characteristic upward or downward trend (A%H2O2 in the AHP solution between preparation and last
application ranged from -0.6% to +0.1%).
20
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7.0
6.5
6.0
oi
o
CM
I
5.5
5.0
H202 [%]
4.5
20
40 60 80
Minutes post-mixing
100
120
Figure 5-4. Concentration of H2O2 in AHP over Time (Combined Results for Ten Batches of Decontamination
Solution)
5.1.3 PAA Concentration
Measurements of the PAA concentration in the AHP were outside the scope of this work. For screening
purposes, a semi-quantitative test using The EM Quant® Peracetic Acid Test test-strips (EMD Millipore
Chemicals, Billerica, MA,USA) was performed to determine PAA in the AHP working solution. This test is
suited for the selective determination of PAA concentration in solutions, also in cases in which H2O2 is
present. The PAA concentration measured in a 50x diluted aliquot of acidified AHP solution 1 h and 2.5
hours post-mixing was estimated as 10000 mg/L (1%) and 5000 mg/L (0.5%), respectively. A representative
PAA concentration profile over time in the AHP was not tested further.
5.2 Application Characterization
The diameter of the AHP spray was determined to be within acceptable limits (12 in to 16 in diameter at 1 ft)
for all decontamination tests.
21
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Sprayer flow rates varied significantly between test days (Table 5-2). The reason for this variability is
unknown, but the variability was suspected to have been caused by the foaming action of the
decontaminant (the sprayer was not designed for foam applications). The flow rate used was always the
maximum flow of the backpack sprayer, i.e., setting 4 (corresponding to 1 gallon/min flow rate according to
factory specifications). The liquid volume delivered per each sprayer setting was confirmed in the initial
testing with water (at 20 pounds per square inch (psi) and setting # 1,2,3 and 4, the sprayer delivered 800,
880, 960 and 1000 ml of water per minute, respectively).
There was no obvious correlation between flow rate and decontamination efficacy - samples with the
highest log reduction values were sprayed on days where medium backpack sprayer flow rates were noted
(080712 and 082212; see Tables A-1 and A-2 in Appendix A for details). A more systematic study would be
needed to investigate whether or not the backpack sprayer flow rates can affect the decontamination
efficacy directly.
Table 5-2. Sprayer Flow Rates
0801 12 test 11 1213
080712 test 1,2,3,4
082212 test 5,7,10,15,16
091812 test 1R,6R,8R,9R,14R
Average
SD
Min
Max
[mL/min]
521
512
587
970
102
131
110
114
372
372
408
780
696
780
780
1170
5.3 Decontamination Results
5.3.1 Neutralization Test Results
Table 5-3 shows results from neutralization tests for porous and nonporous materials.
Table 5-3. Neutralization Test Results for Porous and Non-Porous Materials
Test
A1
A2
B1
B2
Decontaminated
Yes
No
Yes
No
Material Type
Unpainted
Unpainted
Stainless Steel
Stainless Steel
n
5
5
5
5
Average
CPU/per
sample
3.71 E+07
3.06E+07
3.05E+07
3. 11 E+07
RSD
10%
11%
11%
20%
T-test p value*
0.004
0.90
*Probability associated with a Student's paired t-Test, with a two-tailed distribution.
The Student's t-test was used to compare recoveries between the two treatments (sprayed and nonsprayed
coupons). Analysis of the data with student's t-test suggested that the sprayed and unsprayed unpainted
plywood results were significantly different (p = 0.004). However, an average recovery from the AHP-
sprayed unpainted plywood coupons was higher than that from unsprayed samples. These data suggest
that the presence of residual AHP does not have a negative bias on sample recovery. There was no
22
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significant statistical difference (p = 0.90) between CPU recovery from sprayed and non-sprayed stainless
steel coupons. Based on these results, the neutralization of coupons or samples after the AHP
decontamination was determined to be unnecessary.
The H2O2 in runoff was neutralized using 30% STS. Neutralizer was added to the sterile runoff collection
container immediately prior to each decontamination test (42 ml per each 15- second spray application).
The average post-neutralization concentration of the residual H2O2 in the collected runoffs was 0.10% (±
0.049%). The microbiological results from the runoff samples that required re-analysis (filter plating) were
assessed for instabilities due to prolonged exposure to residual H2O2 in the solution. If such instabilities
were noted, the filter plating results were considered invalid and excluded from further data analysis and
processing. This phenomenon occurred during testing of Procedure 1 on concrete, tile, and unpainted
wood. For these samples, the initial estimates of runoff spore concentrations were utilized.
5.3.2 Inoculation Results
In this study, test and positive control coupons of porous and nonporous building materials were inoculated
with ~2 x 108 aerosol-deposited Bacillus spores. Test specific results for positive controls (CFU/sample) are
given in Table 5-4. The average CPU recovered from positive control coupons sets (n = 3) was 8.74 x 107,
with an average coefficient of variance of 33% (ranging from 9% RSD to 111 % RSD; see Tables A-1 and A-
2).
Reference control coupons were inoculated alongside test coupon sets. Control coupons were inoculated at
the beginning, in the middle and the end of the inoculation procedures. The recoveries from inoculation
control coupons are shown in Table 5-4 and demonstrate a repeatable inoculation procedure across all
tests.
Table 5-4. Inoculation Control Results
0801 12 test 11 1213
08071 2 test 1,2,3,4*
08221 2 test 5,7, 10, 15, 16
091812 test 1R,6R,8R,9R,14R
Average CFU/sample
1.53E+08
8.63E+07
1.88E+08
6.86E+07
% RSD
45%
49%
51%
25%
*One reference control rejected as non-representative of inoculation.
The positive controls and inoculation controls average recovery values (see Tables A-1 and A-2) indicate
that the initial spore loading allowed evaluation of decontamination efficacies with a dynamic range of
sporicidal effectiveness greater than 6 logs (i.e., > 6 logs reduction (LR)). The high variability among the
numbers of viable spores recovered from the positive control samples can be attributed to challenges
related to reproducible surface-sampling procedures. Presumably, sampling of test coupons was
analogously affected, hence the variability in the initial loading/CPU recovery of positive controls was not
considered a source of methodical bias in calculations of the log reduction values.
23
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5.3.3 Log Reduction Results
Tables A-1 and A-2 in Appendix A and Figure 5-5 summarize test-specific log reduction values. These data
suggest that AHP-based liquid sporicide provides greater than 6 LR of spores on most of the common
building materials tested, either with the one- or two-application procedure (Procedures 1 and 2,
respectively). The exception was unpainted treated wood, where decontamination with Procedure 2 resulted
in a 4.20 LR (3.24 LR with Procedure 1). The range in log reduction values for Procedure 1 was from 1.56
for concrete to 8.41 for glass (see Table A-1 of Appendix A for Procedure 1-specific results). The efficacy of
Procedure 2 ranged from 6.84 LR for concrete to 8.36 LR for stainless steel (see Table A-2 of Appendix A
for Procedure 2-specific results).
.o
"o
D5
O
10.00
8.00
6.00
4.00
2.00
0.00
Procedure 1
Procedure 2
Nonporous materials
Porous materials
Figure 5-5. Material Specific Log Reductions for Procedure 1 and Procedure 2
Decontamination efficacy was dependent upon material type and operational conditions (Figure 5-6). The
average LR for nonporous materials (stainless steel, glass, linoleum, and tile) was 6.62 ± 1.74 and 8.29 ±
24
-------
0.09 for Procedure 1 and 2, respectively. Complete kill was observed for all nonporous materials using
Procedure 2. The average LR for porous materials (painted treated plywood, unpainted treated plywood,
carpet and concrete) was 4.29 ± 1.96 and 6.53 ± 1.59 for Procedure 1 and 2, correspondingly.
Non-porous materials • Porous materials
Procedure 1
Procedure 2
Figure 5-6. Average Log Reductions for Procedure 1 and Procedure 2 on Nonporous and Porous Building
Materials
A comparison between 15-minute/no re-application (Procedure 1) and 2x15 minutes/re-application
(Procedure 2) exposures suggest that longer exposure time and re-application may provide higher sporicidal
efficacy, especially for porous materials. Lower variability in efficacies between materials was also observed
when Procedure 2 was used.
5.3.4 Fate of Spores
The reaerosolization of 8. anthracis during decontamination operations is a potential source of exposure for
personnel performing cleaning, and a mechanism for spreading contamination to previously uncontaminated
areas. The LD50 for 8. anthracis in humans is not definitively known but, based on animal data, is estimated
to be in the range of 4,100 -10,000 inhaled spores [13, 14]. The survival of spores in the post-
decontamination runoff that contains viable spores brings into consideration other possible routes of
accidental exposure of the personnel through cuts and scrapes, i.e., via cutaneous or gastrointestinal
pathways [15].
25
-------
Aerosol samples were collected using Via-Cell BioAerosols cassettes (Zefon International, Ocala, FL)
during application and exposure phases of each decontamination test run. Table 5-5 shows the recoveries
from the material/procedure-specific aerosol samples. These results suggest that the multi-step
decontaminations, like Procedure 2, may result in a greater spore reaerosolization. The highest number of
viable spores detected in all air samples was 540 CPU (equivalent of about 1 CPU per liter, for samples
collected during decontamination of concrete via Procedure 2). While this value may seem low (compared
to an LD50 of 4,100 to 10,000 spores), the value represents the reaerosolized spores from only three 14x14
inch coupons (total area of approximately 4.1 ft2). The decontamination cycle phase-related aerosolization
rates were not systematically investigated in this study, but the spraying stage is presumably the phase
when most of the spores were aerosolized.
Table 5-5. Air Samples Results
Stainless steel
Glass
Linoleum
Tile
Painted treated plywood
Un painted treated plywood
Carpet
Concrete
Procedure 1
Procedure 2
Total CPU/sample*
6.1E+00
3.3E+00
5.3E-01
4.3E+00
6.1E+00
5.6E-01
5.3E-01
2.2E+00
5.3E-01
1.2E+01
5.3E-01
2.0E+01
1.2E+01
5.3E-01
1.2E+01
5.4E+02
* Values in gray-shaded cells are based on detection limit.
Table 5-6 shows CPU recovered from the runoff samples, normalized per liter of runoff collected.
Table 5-6. Runoff Samples Results
Stainless steel
Glass
Linoleum
Tile
Painted treated plywood
Un painted treated plywood
Carpet
Concrete
Procedure 1
Procedure 2
Total CFU/L*
9.8E+00
6.0E+00
2.E+06
8.0E+03
1.2E+06
1.0E+06
6.E+02
1.0E+04
5.2.E+02
7.1.E+02
8.7.E+04
5.3.E+02
2.8.E+03
1.8.E+07
2.4.E+04
3.4.E+04
Values in gray-shaded cells are based on detection limit.
26
-------
The average number of spores detected in the Procedure 1 runoff samples was 5.5 x 105 CFU/L. The
Procedure 2 runoff samples averaged 2.2 x 106 CFU/L, with a maximum of 1.8.x 107 CPU per liter (collected
during decontamination of unpainted treated plywood via Procedure 2). The volumes of runoff collected
varied drastically between the tests (2.0 to 6.6 L; the AHP runoff plus the post-exposure water rinse).
Consequently, the CPU per liter estimates are influenced heavily by the post-decontamination chamber
rinse step (see Appendix B, DTRL MOP# 3177, section 3.4.3) duration/rinse water volume-dependant
variation. Procedure 2 appeared to result in a greater rate of physical removal of spores/higher rate of
viable spore transfer to the runoff. Nonetheless, both procedures resulted in significant amounts of viable
agent being transferred into the liquid runoff. While not directly comparable due to subtle differences in test
procedures, the amount of viable spores being transferred to the runoff appears slightly higher than that
achieved with similar pH-adjusted bleach procedures [16, 17]. Comparatively lower inactivation efficacies of
AHP could account for this difference.
5.3.5 Summary of Results
Decontamination efficacy of AHP was evaluated using two application procedures (Procedures 1 and 2,
respectively). The single application procedure (Procedure 1) resulted in surface LR values that ranged
from 1.6 to 8.4. Typically, nonporous materials were easier to decontaminate than were porous. The
exception, carpet, was relatively easy to decontaminate compared to other porous materials. Of all the
materials, concrete demonstrated the lowest LR (< 2 LR) with Procedure 1. Nonetheless, greater than 6 LR
was achieved for 50% of materials decontaminated with Procedure 1. The AHP two-application procedure
(Procedure 2) resulted in > 6 LR for seven of the eight materials tested (LR values from 6.8 to 8.4). Greater
than 6 LR was not achieved for unpainted treated wood material, regardless of the application procedure
(mean LR = 4.2). Complete kill (recovery of no viable spores after decontamination) was achieved on carpet
and glass using Procedure 1, and on Tile, stainless steel, carpet, glass, and linoleum using Procedure 2.
For most materials, surface decontamination efficacy of AHP was similar to that observed previously for pH-
adjusted bleach [16,17]. However, the data suggest that AHP may have lower efficacy than pH-adjusted
bleach when applied to concrete surfaces.
The longer exposure duration and increased volume of AHP applied, associated with Procedure 2, afforded
higher surface decontamination efficacy, especially for porous materials. Materials with high organic
demand (e.g., unpainted wood) that are typically more difficult to decontaminate may present a challenge to
the liquid AHP-based process. The preliminary fate of spores estimates suggest that the AHP liquid
sporicide-based decontamination process leads to physical removal of spores from decontaminated
surfaces, with a consequent transport of viable spores to air and to the post-decontamination liquid waste.
In some instances, considerable amounts (up to 7 logs) of viable spores were recovered in the liquid runoff.
Highest recoveries of spores in the runoff were observed during testing with painted and unpainted plywood.
When compared to pH-adjusted bleach testing results, AHP-based decontaminations appear to result in
substantially greater numbers of viable spores in the runoff fraction. Considering these findings, although
surface reduction efficacies for AHP were similar to pH-adjusted bleach, overall decontamination efficacy
may be lower for AHP.
27
-------
6. Quality Assurance
This project was performed under an approved Category III Quality Assurance Project Plan titled Evaluation
of Expedient Decontamination Options with Activated Peroxide-based Liquid Sporicides (June 2012).
All test activities were documented via narratives in laboratory notebooks and the use of digital photography.
The documentation included, but was not limited to, a record of time required for each decontamination step
or procedure, any deviations from the QAPP, and physical impacts on the materials. All the tests were
conducted in accordance with developed DTRL and BioLab miscellaneous operating procedures (MOPs),
listed in Appendix B, to ensure repeatability and adherence to the data quality validation criteria set for this
project.
6.1 Sampling, Monitoring, and Analysis Equipment Calibration
Standard operating procedures for the maintenance and calibration of all laboratory equipment were used.
All equipment was certified as being calibrated or having the calibration validated by EPA's on-site (RTP,
NC) Metrology Laboratory at the time of use. Standard laboratory equipment such as weigh balances, pH
meters, biological safety cabinets and incubators were routinely monitored for proper performance.
Calibration of instruments was performed at the frequency shown in Table 6-1. Any deficiencies were noted
in the laboratory notebooks. Instruments were adjusted to meet calibration tolerances and recalibrated
within 24 hours. If tolerances were not met after recalibration, additional corrective action was taken,
possibly including, recalibration and/or replacement of the equipment.
Table 6-1. Analysis Equipment Calibration Frequency
Equipment
pH meter
Stopwatch
Clock
Scale
Micropipettes
Calibration/Certification
Perform a 2-point calibration with standard buffers that
bracket the target pH before each use.
Compare against NIST Official U.S. time at
http://nist.time.gOV/timezone.cgi7Eastern/d/-5/java
once every 30 days.
Compare to office U.S. Time @ time.gov every 30
days.
Check calibration with Class 2 weights prior to
weighing
Once a year, tested with standards traceable to SI
through the NIST
Expected Tolerance
± 0.1 pH units
±1 min/30 days
±1 min/30 days
+.0.1% weight.
±5%
6.2 Data Quality
This section discusses the Quality Assurance/Quality Control (QA/QC) checks and acceptance criteria for
critical measurements considered critical to accomplishing the project objectives.
28
-------
6.2.1 QA/QC Checks
Uniformity of the test materials was a critical attribute for assuring reliable test results. Uniformity was
maintained by obtaining a sufficiently large quantity of material so that multiple material sections and
coupons could be constructed with presumably uniform characteristics. Samples and test chemicals were
maintained to ensure their integrity. Samples were stored away from standards or other samples which
could cross-contaminate them.
Supplies and consumables were acquired from reputable sources and were NIST-traceable when possible.
Supplies and consumables were examined for evidence of tampering or damage upon receipt and prior to
use, as appropriate. Supplies and consumables showing evidence of tampering or damage were not used.
All examinations were documented and supplies were appropriately labeled. Project personnel checked
supplies and consumables prior to use to verify that they met specified task quality objectives and did not
exceed expiration dates.
Quantitative standards do not exist for biological agents. Quantitative determinations of organisms in this
investigation did not involve the use of analytical measurement devices. Rather, the CPU were enumerated
manually and recorded. The QA/QC acceptance criteria were set at the most stringent level that could
routinely be achieved (Table 6-2). Positive controls and procedural blanks were included with the test
samples in the experiments so that well-controlled quantitative values were obtained. Background checks
were also included as part of the standard protocol. Replicate coupons were included for each set of test
conditions. MOPs executed by qualified, trained and experienced personnel were used to ensure data
collection consistency. The confirmation procedure, control, blank, and method validation efforts are the
basis of support for biological investigation results. When necessary, training sessions were conducted by
knowledgeable parties, and in-house practice runs were used to gain expertise and proficiency prior to
initiating the research.
Table 6-2. QA/QC Sample Acceptance Criteria
QC Sample
Procedural Blank
(coupon not inoculated
with biological agent)
Information Provided
Controls for sterility of
materials and methods
used in the procedure
Frequency
1 per test
from Table
2-1 or Table
2-2
Acceptance Criteria
No observed CPU
Corrective Action
Reject results upon
approval of WAM,
otherwise analyze data
with procedural blank
results as test
minimum,. Identify and
remove source of
contamination, if
possible.
29
-------
QC Sample
Positive Control
(Sample from material
coupon contaminated
with biological agent but
not subjected to the test
conditions)
Blank plating of
microbiological supplies
Blank Tryptic Soy Agar
Sterility Control
(plate incubated, but not
inoculated)
Decontamination
method checks
Spray pattern check
Exposed Field Blank
Samples. A wipe kit will
be handled, a vacuum
sock kit will sample
ambient air.
Unexposed Field Blank
Samples. A wipe kit will
be transferred without
being handled, a
vacuum sock kit will be
transferred without
switching on the
vacuum.
Background swabs
Information Provided
Initial contamination level
on the coupons; allows
determination of log
reduction; controls for
confounds arising from
history impacting
bioactivity; controls for
special causes.
Shows viability of
sampling technique and
plate's ability to support
growth.
Controls for sterility of
supplies used in dilution
plating.
Controls for sterility of
plates.
Details on the materials
and equipment used in
the decontamination.
Distribution of
decontamination solution
or rinse water.
The level of
contamination present
during sampling.
The level of
contamination present
during sampling.
Determine sterility of
materials and equipment
before use.
Frequency
3 or more
replicates
per test
3 of each
supply per
plating event
Each plate
As outlined
in MOP
3177
Peruse
1 per
sampling
event
1 per
sampling
event
1 per
material per
use
Acceptance Criteria
Target loading of 1 x
107CFU per sample
with a standard
deviation of < 0.5 log.
(5x106-5x107
CFU/sample);
Grubbs outlier test (or
equivalent).
No observed growth
following incubation
No observed growth
following incubation
As outlined in MOP
3177
Decontamination
solution sprayer: 12 in
to 16 in diameter at 1
foot.
Dl water:
12 into 16 in
dia mete rat 3 feet.
Non-detect
Non-detect
Non-detect
Corrective Action
Outside target range:
discuss potential impact
on results with EPA
WAM; correct loading
procedure for next test
and repeat depending on
decided impact.
Outlier: evaluate/exclude
value.
Sterilize or dispose of
source of contamination.
Replate samples.
All plates are incubated
prior to use, so any
contaminated plates will
be discarded.
As outlined in MOP 3177
Adjust nozzle
Clean up environment.
Sterilize sampling
materials before use.
Clean up environment.
Sterilize sampling
materials before use.
Clean up environment.
Sterilize sampling
materials before use.
30
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6.3 Acceptance Criteria for Critical Measurements
The Data Quality Objectives (DQOs) are used to determine the critical measurements (CMs) needed to
address the stated objectives and specify tolerable levels of potential errors associated with simulating the
prescribed decontamination environments.
The following measurements were deemed to be critical to accomplish part or all of the project objectives:
• Enumeration of spores on the surface coupon
• Plated volume
• Decontamination time
• Spray pattern.
After initial testing preparation of large batches of the AHP solutions, the H2O2 concentration and pH
measurements were deemed to be non-critical measurements and were used to characterize the AHP liquid
sporicide. The characterization of the AHP application was performed by measuring backpack sprayer flow
rates, the spray pattern and the duration of application measurements.
The critical measurement acceptance criteria and completeness values achieved in testing are listed in
Table 6-3.
Table 6-3. Critical Measurement Acceptance Criteria
Critical
Measurement
Plated volume
CFU/plate
Decontamination
Time (15 min, 2x
15 min)
Spray pattern (14-
16" from 1 ft)
Measurement
Device
Pipet
Hand counting
Timer
Measuring tape
Accuracy
±2%
±10% (between
2 counters)*
< 50% CV
between
triplicate plates
±1 second
1/32" over the
length of the
measuring tape
Precision
±1 %
±5
± 1 second
NA
Detection
Limit
NA
1 CPU
1 second
1/8"
Completeness
100%
100%*
94%**
100%
*25% of the plates within the quantification range (plates with 30 - 300 CPU) were counted by a second technician;
** For one test set of Procedure 1 (stainless steel, test ID # 2,) the spray application duration was 20 s instead of 15
seconds per set of three coupons.
31
-------
Plated volume critical measurement goals were met. All pipettes are calibrated yearly by an outside
contractor (Calibrate, Inc.).
Plates were quantitatively analyzed (CFU/plate) using a manual counting method. For each set of results
(per test), a second count was performed on 25 percent of the plates within the quantification range (plates
with 30 - 300 CPU). All second counts were found to be within 10 percent of the original count.
Many QA/QC checks were used to validate spore recovery measurements. These QA/QC checks include
samples that demonstrate the ability of the on-site Biocontaminant Laboratory to culture and grow spores as
well as to demonstrate that materials used in this effort do not themselves contain spores. The checks
included:
• Laboratory material coupons: includes all materials, individually, used by the on-site Biocontaminant
Laboratory in sample analysis; materials have been confirmed sterile.
• Positive control coupons: coupons inoculated but not decontaminated; results are discussed in Section
5.3.2.
• Inoculation control coupons: stainless steel coupons dosed at beginning, middle, and end of each
inoculation campaign, not decontaminated, to assess the variability of the inoculation operation; results
are discussed in Section 5.3.2
• Procedural blank coupons; spores were detected on some blank and procedural blank coupons. Test -
specific results for procedural controls are given in Tables A-1 and A-2; the potential impact on the
decontamination efficacy results was considered negligible.
6.4 Data Quality Audits
This project was assigned QA Category III and did not require technical systems, performance evaluation or
data quality audits by EPA or contractor QA personnel.
6.5 QA/QC Reporting
QA/QC procedures were performed in accordance with the QAPP for this investigation.
6.6 Amendment to Original QAPP
The DTRL MOP #3177 (see Appendix B) was prepared as an Amendment to the QAPP by the EPA WAM
upon receipt of the final recipe for AHP from SNL.
32
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7. References
1. U.S. Department of Homeland Security. National Response Framework. 2008 [cited 2012 August
27, 2012]; Available from: http://www.fema.gov/national-response-framework.
2. Canter, D.A., et al., Source reduction in an anthrax-contaminated mail facility. Biosecurity and
bioterrorism : biodefense strategy, practice, and science, 2009. 7(4): p. 405-12.
3. Canter, D.A., et al., Remediation of Bacillus anthracis Contamination in the U.S. Department of
Justice Mail Facility. Biosecurity and Bioterrorism: Biodefense Strategy, Practice, And Science,
2005. 3(2): p. 119-127.
4. Canter, D.A., Remediating anthrax-contaminated sites: Learning from the past to protect the future.
Chemical Health and Safety, 2005. 12(4): p. 13-19.
5. Calfee, M.W., et al., Laboratory evaluation of large-scale decontamination approaches. Journal of
Applied Microbiology, 2012.112(5): p. 874-82.
6. U.S. Environmental Protection Agency, After Action Report - Danbury Anthrax Incident, National
Decontamination Team, Editor 2008: Erlanger, KY.
7. Calfee, M.W., et al., Lab-Scale Assessment to Support Remediation of Outdoor Surfaces
Contaminated with Bacillus anthracis Spores. Journal of Bioterrorism and Biodefense, 2011. 2(3): p.
1-8.
8. Rutala, W., Disinfection, Sterilization and Antisepsis: Principles, Practices, Current Issues, and New
Research, ed. W. Rutala. 2007, Washington, D.C.: Association for Professionals in Infection Control
and Epidemiology. 281.
9. Heisig, C.C., et al., Low odor, hard surface sporicide. Inorganic active ingredient containing
peroxide or compositions of or releasing gaseous oxygen or ozone hydrogen peroxide, U.S. P.
Office, Editor 2010: United States.
10. Gupta, A., et al., Strategy for on-site in situ generation of oxidizing compounds and application of
the oxidizing compound for microbial control, U.S. Patent Office, Editor 2012: United States.
11. Krauter, P. and M. Tucker, A biological decontamination process for small, privately owned
buildings. Biosecur Bioterror, 2011. 9(3): p. 301-9.
12. Brown, G.S., et al., Evaluation of a Wipe Surface Sample Method for Collection of Bacillus Spores
from Nonporous Surfaces. Appl. Environ. Microbiol., 2007. 73(3): p. 706-710.
13. Peters, C.J. and D.M. Hartley, Anthrax inhalation and lethal human infection. Lancet, 2002.
359(9307): p. 710-1.
14. Classman, H.N., Industrial inhalation anthrax. Bacteriol. Rev., 1965. 30: p. 657-659.
15. U.S. Department of Health and Human Services. ESF-8 Aerosolized Anthrax Playbook. 2012 [cited
201212-5-2012].
16. U.S. Environmental Protection Agency, Effectiveness of Physical and Chemical Cleaning and
Disinfection Methods for Removing, Reducing or Inactivating Agricultural Biological Threat Agents
2011, US EPA: Research Triangle Park, NC.
17. U.S. Environmental Protection Agency, Assessment of Liquid and Physical Decontamination
Methods for Environmental Surfaces Contaminated with Bacterial Spores: Evaluation of Spray
Method Parameters and Impact of Surface Grime, 2012: RTP, NC.
33
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Evaluation of Expedient Decontamination Options
with Activated Peroxide-based Liquid Sporicides
Appendix A
Appendix A: Process Parameters and Decontamination Efficiency Results
Table A-1. Process Parameters and Decontamination Efficiency Results for Procedure 1
Test
ID
1R
2
3
4
5
11
12
13
Date
9/18/2012
8/7/2012
8/7/2012
8/7/2012
8/22/2012
8/1/2012
8/1/2012
8/1/2012
Material
type
Painted
treated
plywood
Stainless
steel
Carpet
Glass
Linoleum
Concrete
Tile
Unpainte
d treated
plywood
Log
reduction
4.35
8.26
>8.04
58.41
3.53
1.56
6.27
3.24
Positive Controls
Avg. CPU
recovered
8.69E+07
1.49E+08
6.24E+07
1.59E+08
1 .46E+08
3.49E+07
1 .31 E+07
5.22E+07
RSD
(%)
28
15
57
17
9
59
111
73
Test Coupons
Avg. CPU
recovered
3.90E+03
8.26E-01
5.72E-01
6.20E-01
4.32E+04
9.71 E+05
7.05E+00
3.03E+04
RSD
(%)
172
45
3
1
162
117
159
158
Procedural
Blank
Coupons
(CPU)
not
performed
1 .81 E+03
2.05E+03
2.35E+02
6.45E-01
2.00E+01
5.88E-01
5.62E-01
Application
frequency
At 0 min, no
rinse
At 0 min, no
rinse
At 0 min, no
rinse
At 0 min, no
rinse
At 0 min, no
rinse
At 0 min, no
rinse
At 0 min, no
rinse
At 0 min, no
rinse
Application
duration
(sec)
15
20
15
15
15
15
15
15
Decon
duration
(hh:mm)
0:16
0:15
0:15
0:15
0:15
0:15
0:15
0:15
AHP
PH
(pH
units)
7.39
8.74
8.16
7.80
7.52
7.41
8.87
8.11
AHP
Temp
(°C)
32.8
27.3
29.8
29.7
30.7
29.50
27.90
28.7
H202
cone
(%)
5.3
6.1
5.8
5.9
6.0
5.9
6.1
6.1
-------
Evaluation of Expedient Decontamination Options
with Activated Peroxide-based Liquid Sporicides
Appendix A
Table A-2. Process Parameters and Decontamination Efficiency Results for Procedure 2
Test
ID
6R
7
8R
9R
10
14R
15
16
Date
9/18/2012
9/18/2012
9/18/2012
9/18/2012
8/22/2012
9/18/2012
8/22/2012
8/22/2012
Material
type
Painted
treated
plywood
Stainless
steel
Carpet
Glass
Linoleum
Concrete
Tile
Unpainte
d treated
plywood
Log
reduction
7.42
>8.36
>7.67
58.16
>8.34
6.84
>8.28
4.20
Positive Controls
Avg. CPU
Recovered
8.69E-KD7
1 .51 E+08
2.99E+07
8.51 E+07
1 .46E+08
2.69E+07
1 .24E+08
4.55E-KD7
RSD
(%)
28
14
15
28
9
22
13
25
Test Coupons
Avg. CPU
Recovered
3.33E+00
6.52E-01
6.40E-01
5.83E-01
6.64E-01
3.87E+00
6.55E-01
2.90E+03
RSD
(%)
141
2
4
2
1
145
3
100
Procedural
Blank
Coupons
(CPU)
7.14E-01
6.29E-01
5.71 E-01
6.25E-01
6.06E-01
6.71 E-01
1 .25E+01
1.13E+01
Application
frequency
At 0 min, re-
apply after 15
min, no rinse
At 0 min, re-
apply after 15
min, no rinse
At 0 min, re-
apply after 15
min, no rinse
At 0 min, re-
apply after 15
min, no rinse
At 0 min, re-
apply after 15
min, no rinse
At 0 min, re-
apply after 15
min, no rinse
At 0 min, re-
apply after 15
min, no rinse
At 0 min, re-
apply after 15
min, no rinse
Application
duration
(sec)
15
15
15
15
15
15
15
15
Decon
duration
(hh:mm)
2x0:15
2x0:15
2x0:15
2x0:15
2x0:15
2x0:15
2x0:15
2x0:15
AHP
PH
(pH
units)
8.87
8.93
7.65
7.46
7.99
8.99
8.92
8.01
AHP
Temp
(°C)
28
28.2
31.8
31.5
31.2
27.1
28.5
31.3
H202
cone
(%)
5.9
6.1
6.2
5.5
6.2
6.0
NA
6.0
-------
Appendix B
Appendix B: Miscellaneous Operating Procedures (MOPs)
Note: MOPs are dynamic documents. For the testing described in this report, the following MOPs were followed as presented.
DTRL Procedures
• MOP 3177 Activated Hydrogen Peroxide-based decontamination for WA 3-08: Procedure 1 and
Procedure 2
• MOP 3135 Procedure for WA 1 -25 Sample Collection using BactiSwab™ Collection and Transport
Systems
• MOP 3144 Procedure for Wipe Sampling of Coupons
• MOP 3145 Procedure for Vacuum Sampling of Large and Small Coupons
• MOP 3150 Procedure for Fabrication of 14" x 14" Material Coupons
• MOP 3155 Procedure for Via-Cell® Air Sampling
BioLab Procedures
• MOP 6535a: Serial Dilution: Spread Plate Procedure to Quantify Viable Bacterial Spore
• MOP 6561: Aerosol Deposition of Spores onto Material Coupon Surfaces Using the Aerosol Deposition
• MOP 3128: Procedure for Preparing pH-Adjusted Bleach Solution with Trisodium Phosphate Substitute
• MOP 6562: Preparing Pre-Measured Tubes with Aliquoted Amounts of Phosphate Buffered Saline with
Tween 20 (PBST)
• MOP 6563: Swab Streak Sampling and Analysis
• MOP 6565: Filtration and Plating of Bacteria from Liquid Extracts
• MOP 6567: Recovery of Bacillus Spores from Wipe Samples
• MOP 6570: Use of STERIS Amsco Century SV 120 Scientific Prevacuum Sterilizer
• MOP 6571: Recovery of Bacillus Spores from Via-Cell® Aerosol Sampling Cassettes
• MOP 6572: Recovery of Spores from Vacuum Sock Samples
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MOP 3177
TITLE: ACTIVATED HYDROGEN PEROXIDE-BASED DECONTAMINATION FOR WA 3-08:
PROCEDURE 1 AND PROCEDURE 2
SCOPE: This MOP outlines the setup, operation, and timeline schedule for conducting
decontamination testing in the H-130 small test chamber.
PURPOSE: To provide a standardized and repeatable procedure for all activated hydrogen peroxide-
based decontamination tests to be conducted under WA 3-08 using Decontamination
Procedure 1 and Procedure 2.
1.0 INTRODUCTION
Preparations for each test will be conducted according to the schedule listed in this procedure. Any
deviations will be noted in the laboratory notebook, along with the reason for the deviation.
Section 2.0 lists the Day 1 preparation steps that need to be taken the day before decontamination testing.
Sections 3.0 details the Day 2 steps for the Decontamination Procedure 1 (spray-no rinse-dry). Section 4.0
discusses sampling and other post-decon procedures to be conducted on Day 3. Section 5.0 provides a
summary of the tasks to be completed each day.
2.0 PREPARATION (Day 1)
NOTE: The test organism for this work will be a powdered spore preparation of B. atrophaeus
(ATCC 9372) and silicon dioxide particles, pre-loaded in metered dose inhalers (MDI).
2.1 Materials for Inoculation Check
• Three (3) sets of sterilized 14 in by 14 in Material Coupons in VHP bags (8 coupons per set)
• Three (3) 14 in by 14 in reference coupons (stainless steel)
• Supplies for inoculation as listed in MOP 6561 (Aerosol Deposition of Spores onto Material Coupon
Surfaces using the Aerosol Deposition Apparatus).
2.2 Inoculation of Coupons per MOP 6561
a. Print the coupon codes prior to the start of coupon inoculation. These are unique identifiers described in
Section 2.13 of the WA 3-08 QAPP. These coupon codes will be needed in Step c below.
b. The inoculation team will assemble the aerosol deposition apparatus (ADAs) and clamp everything
down before any inoculation begins (the day before inoculation, if possible). Pictures should be taken of
the completed ADAs.
c. After a coupon is dosed via MOP 6561, the coupon will be labeled with a unique identifier (printed out in
Step a above). Write the sample ID code the label on the side of the coupon using a permanent marker
(e.g., black or silver Sharpie®).
d. Maintain a log for each set of coupons that are dosed via MOP 6561. Each record in this log will include
the unique coupon identifier, the MDI unique identifier, the date, the operator, the weight of the MDI
before dissemination into the coupon dosing device, the weight of the MDI after dissemination, and the
difference between these two weights.
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e. After the minimum 18-hour settling period, remove coupons from the ADA and move to an isolated
storage cabinet, which will contain all inoculated coupons fora single test. The handling of the
contaminated coupons, including movement to minimize or control spore dispersal, will be done in
accordance with MOP 6561. One person will be tasked with removing the clamps holding the dosing
chamber to the coupon and the removal of the dosing chamber and gasket from the coupon. A second
person, wearing new, sterile gloves for each coupon, will then be tasked with moving the coupon to the
proper location (e.g., test and positive control coupons to the Test Coupon Cabinet and blank coupons
to the Blank Coupon Cabinet).
NOTE: The contamination control coupons will be stainless steel coupons (14 in by 14 in) that
will be contaminated in accordance with MOP 6561, then sampled and analyzed in
accordance with Sections 2.6.1 and Section 3.1.1 of the WA 3-08 QAPP, respectively.
The stainless steel coupons will be placed on a clean cookie sheet which will be labeled
with the unique identifier described in Section 2.13 using a black Sharpie®. If the results
from the contamination controls are outside the acceptance criteria, the results will be
discussed with the EPA WAM immediately to determine the corrective action.
3.0 DECONTAMINATION
3.1 Preparation of the Decontamination Formula and Neutralizer
3.1.1 Decontamination Formula
Chemicals:
• Hydrogen peroxide (50%): Fisher Scientific, CAS: 7722-84-1
• Triacetin, Acros Organics CAS: 102-76-1
• Maquat® MC1412-80%, Mason Chemical Company, CAS: 68424-85-1
• Ethanol (99.5%), Acros Organics, CAS: 64-17-5
• Potassium Carbonate: Sigma Aldrich, CAS: 584-08-7
The decontamination formula (modification of formula # 10 developed by Sandia Laboratories) will be
prepared in three parts (A, B, and C):
• Part A = water, buffer, Maquat 1412 and ethanol
• Part B = 15% hydrogen peroxide (H202) solution
• Part C = the activator, Triacetin
NOTE: Decontaminant should be prepared fresh each day (the formulation must be used within
2 hours after mixing).
A Safety Requirements:
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A PPE Required: Safety glasses, lab coat, Nitrile gloves
A All work performed in a chemical fume hood
To make 10 liters of decontamination solution:
1. Prepare Part A: In 4870 ml of water, mix in 320 g potassium carbonate, 10 ml of Maquat, and 500 ml of
ethanol. Mix by hand in a carboy.
2. Prepare 5000 ml of Part B, 15% H202 solution, by adding 1500 ml of 50% H202 to 3500 ml of
deionized (Dl) water. Measure the pH and temperature of the Part B solution.
3. Prepare the decontamination solution as follows:
a. Add 4000 ml of Part B to all of Part A and mix by hand for 30 sec.
b. Add 300 ml of Part C (the activator, Triacetin) to the Part A/B mixture and stir for 1 min.
c. Record the time the decontamination solution was prepared in the notebook and on the carboy.
Solution shelf-life is two hours.
d. Test the pH and temperature of each formulation, which should be around 9.5 ± 0.1. This is the
target pH immediately after Parts A, B, and C are first mixed. Record the pH and temperature in
the logbook.
NOTE: The pH will drop rapidly overtime and the temperature of the solution will increase;
hence, both parameters must be recorded before and after each decontamination step.
e. Verify the H202 concentration by analyzing with the KMnO4 titration procedure described below:
KMnOa Titration Procedure
Reagents:
• 5% Sulfuric acid (H2SO4)
• 1 .ON Potassium permanganate
1. Transfer approximately 20 g (~20 ml) of H2O2 sample to a tared weighing container and weigh on an
analytical balance. Record weight of H2O2 sample.
2. Carefully wash the weighed sample into a 250 ml flask with distilled water, dilute to mark, and mix
thoroughly.
3. Pipet a 25 ml aliquot into a 400 ml beaker containing 250 ml of distilled water and 10 ml of sulfuric
acid.
4. Titrate to the first permanent pink color with 1 .ON potassium permanganate.
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Calculation:
% H2O2 (by weight) = [(ml KMnO4) x (N) x (0.01701) x (1000)]/(grams of H2O2 sample used)
where N is the normality of the standard potassium permanganate.
3.7.2 Neutralizer
The neutralizer used to neutralize the runoff is 30% sodium thiosulfate (STS). Prior to decontamination, add
14 ml of the neutralizer per coupon to the carboy (42 ml per 3 coupons). This volume was based on the
average volume of the runoff collected per test, as determined during the preliminary experiments.
3.2 Preparation of the Sprayer
NOTE: The ShurFlo 4 ProPack Rechargable Electric Backpack Sprayer SRS-600 will be used
for decontamination testing.
1. Rinse the sprayer with Dl water.
2. Discard the Dl water. Fill the sprayer and record the pH and temperature of the decontamination
solution. Take a sample of solution to verify the concentration of of H2O2 in the decon solution in the
backpack via titration.
3. Check the flow rate and the spray pattern of the activated peroxide (decontamination) solution:
• Verify the flow rate prior and post each test using a 1 L graduated cylinder and a stopwatch; time the
spray for 10 seconds to verify the flow rate. Record in the logbook.
• The spray pattern will be tested by spraying at the appropriate distance (1 ft) onto a piece of 14 in by 14
in blue construction paper mounted in the orientation of the material section. The spray shall be
discharged into the center of the paper and the pattern will be visually assessed for consistency with
that shown in Figure 1.
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Figure 1. Spray Pattern from Nozzle 1 Foot Away
4. Determine the diameter of the spray to ensure that is within the acceptable limits (12 in to 16 in diameter
at 1 foot). Record the spray pattern size.
3.3 Cleaning of the Decontamination Chamber
NOTE: The decontamination chamber is to be cleaned between each material type and
before/after each test.
A A personal chlorine monitor must be worn by the employee cleaning the chamber. If the monitor
alarms, all decon work will cease and additional ventilation brought in. If the alarm continues,
leave the area and contact H&S.
a. Using the backpack sprayer, spray the interior surfaces with adjusted bleach solution comprised of 10%
germicidal bleach, 10% acetic acid (5% v/v) and, 80% germicidal bleach.
A Leave the sprayer on a cart or table to prevent muscle strain.
b. After 15 minutes, rinse the surfaces with Dl water; the runoff is to be collected in a carboy for proper
disposal.
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3.4 Decontamination Procedures using Procedure 1 (spray once-wait 15 min-no rinse-dry
overnight) and Procedure 2 (spray-wait 15 min-reapply-wait 15 min-no rinse- dry overnight)
NOTE: The time for application of each procedural step and time between procedural steps on
each coupon will be measured using an NIST-traceable stopwatch and recorded in the
laboratory notebook.
NOTE: The time after the coupon is no longer dripping runoff (wait time post-application) was
determined to be 15 minutes in preliminary experiments for a single set of porous and
non-porous material coupons.
3.4.7 Sterility checks
Swab sampling, as described in MOP 6563 (Swab Streak Sampling and Analysis), will be used for sterility
checks on coupons and equipment prior to use in the testing. Each item to be used in the decontamination
process will be sampled before use. The randomly selected procedural blank coupon (from the same
sterilization batch as the rest of the coupons of the same type) will also be swab sampled before the puffing
operations begin. The multiple swab samples may be needed for the sterilization checks to be completed.
3.4.2 Procedural Blank Coupon Decontamination
1. Place a sterile pre-weighed carboy (with neutralizer - if deemed necessary as detailed in Section 3.1.2)
under the drain of the chamber to collect the runoff from the coupons throughout the entire
decontamination procedure.
2. Collect sterility check samples according to MOP 6563 (as detailed in Step 3.4.1 above).
3. Using the pH meter, record pH and temperature of the decontamination solution in the backpack
sprayer. Take a sample of decon solution to verify the concentration of H2O2 via titration.
4. Move three procedural blank (not-inoculated) coupons to the decontamination chamber.
5. Start application of the decontamination solution; record the time.
6. Spray each set of three coupons for 15 seconds using the Z pattern specified for 14 in by 14 in
coupons. Verify the post-decon flow rate of the backpack sprayer.
7. Using the pH meter, record pH and temperature of the decontamination solution in the backpack
sprayer.
8. After 15 minutes record the time and carefully move the coupons to the Procedural Blank Cabinet for
overnight drying.
9. After all coupons have been moved to the Procedural Blank Cabinet, rinse the chamber with Dl water.
Label the carboy and record the mass of liquid collected. Take a sample of runoff to verify the
concentration of H2O2 in the runoff sample via titration. Sample three 100 ml aliquots of the runoff in
120ml_ sterile, prelabeled specimen cups and place into double sterile bags and send to the on-site
Biocontaminant Laboratory for analysis.
10. Clean the decontamination chamber using the procedure described in Section 3.3.
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3.4.3 Test Coupon Decontamination
3.4.3.1 Procedure 1
1. Place a sterile pre-weighed carboy with neutralizer under the drain of the chamber to collect the runoff
from the coupons throughout the entire decontamination procedure.
2. Using the pH meter, record pH and temperature of the decontamination solution in the backpack
sprayer. Take a sample of decon solution to verify the concentration of H2O2 via titration.
3. Move three test coupons from the Contaminated Coupons Cabinet to the decontamination chamber.
4. Start application of the decontamination solution.
5. Spray each set of three coupons for 15 seconds using the Z pattern specified for 14 in by 14 in
coupons.
6. After 15 minutes, carefully move coupons to the Decontaminated Coupon Cabinet for overnight drying.
7. After all coupons have been moved to the Decontaminated Test Coupon Cabinet, rinse the chamber
with Dl water. Label the carboy and record the mass of liquid collected. Take a sample of runoff to verify
the concentration of H2O2 in the runoff sample via titration. Sample three 100 ml aliquots of the runoff in
120ml_ sterile, prelabeled specimen cups and place into double sterile bags and send to the ON-SITE
Biocontaminant Laboratory for analysis.
Clean the decontamination chamber using the procedure described in Section 3.3. Repeat the
decontamination procedure for the remaining sets of inoculated coupons.
3.4.3.2 Procedure 2
1. Place a sterile pre-weighed carboy with neutralizer to collect the runoff from the coupons throughout the
entire decontamination procedure.
2. Using the pH meter, record pH and temperature of the decontamination solution in the backpack
sprayer. Take a sample of the decon solution to verify the concentration of H2O2 via titration.
3. Move three test coupons from the Contaminated Coupons Cabinet to the decontamination chamber.
4. Start application of the decontamination solution.
5. Spray each set of three coupons for 15 seconds using the Z pattern specified for 14 in by 14 in
coupons.
6. After 15 minutes, re-apply the activated H2O2 solution - spray each set of three coupons for 15 seconds
using the Z pattern specified for 14 in by 14 in coupons.
7. After 15 minutes, carefully move coupons to the Decontaminated Coupon Cabinet for overnight drying.
8. After all coupons have been moved to the Decontaminated Test Coupon Cabinet, rinse the chamber
with Dl water. Label the carboy and record the mass of liquid collected. Sample three 100 mL aliquots of
the runoff in 120mL sterile, prelabeled specimen cups and place into double sterile bags and send to
the ON-SITE Biocontaminant Laboratory for analysis.
9. Clean the decontamination chamber using the procedure described in Section 3.3. Repeat the
decontamination procedure for the remaining sets of inoculated coupons.
3.5 Sample Load per Test
These decontamination tests should results in the following number and type of samples:
• Numerous swab samples to satisfy a sterility check (see Section 3.4.1 for description).
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4 to 5 Procedural blanks (decontaminated procedural blanks stored in the Procedural Blanks Cabinet),
12 to 15 Test samples (decontaminated test coupons stored in the Decontaminated Test Coupons
Cabinet),
12 to 15 Test controls (non-decontaminated positive control coupons stored in the Contaminated
Coupons Cabinet),
5 to 6 Triplicate samples of runoff collected to a carboy with neutralizer for each decontamination
sequence,
4.0 SAMPLING AND EXTRACTION
4.1 Wipe Sampling and Extraction
The procedure for wipe sampling of coupons in described in MOP 3144 (Procedure for Wipe Sampling of
Coupons). The recovery of the spores from the wipe samples is adapted from the Idaho National Laboratory
(INL) 2008 Evaluation Protocols and described in MOP 6567 (Recovery of Bacillus Spores from Wipe
Samples). This procedure may be modified to include an extractive step before plating to eliminate extra
debris. By eliminating the debris, filter plating using MOP 6565 (Filtration and Plating of Bacteria from Liquid
Extracts) will be more effective. Dilution plating will follow MOP 6535a (Serial Dilution: Spread Plate
Procedure to Quantify Viable Bacterial Spores).
4.2 Vacuum Sock Sampling and Extraction
The procedure for Vacuum Sock sampling of coupons is described in MOP 3145 (Procedure for Vacuum
Sampling of Large and Small Coupons). The extraction from vacuum sock samples is described in MOP
6572 (Recovery of Spores from Vacuum Sock Samples). This procedure may be modified to include an
extractive step before plating to eliminate extra debris. By eliminating the debris, filter plating using MOP
6565 will be more effective.
4.3 Swab Samples
Swab sampling will be used for sterility checks on coupons and equipment prior to use in the testing. The
protocol that will be used in this project is described in MOP 6563. Each item to be used in the
decontamination process will be sampled before use. The randomly selected procedural blank coupon (from
the same sterilization batch as the rest of the coupons of the same type) will also be swab sampled before
the puffing operations begin.
4.4 Runoff Collection
During application of the decontamination procedure for each set of coupons, a sterile pre-weighed carboy
with neutralizer added (if needed) will be placed under the drain (described in Section 3.4). The neutralizer
added will be sufficient to neutralize an amount of H202 equal to that to be sprayed during decontamination.
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The runoff from the coupons throughout the entire decontamination procedure being tested will be collected
fora given coupon set (material type or all blanks). Microbiological analysis of the aliquots will be done by
dilution plating as described in MOP 6535a and by filtration of the liquid as described in MOP 6565.
4.5 Sampling Decontamination Equipment
The purpose of sampling the decontamination equipment after use is to confirm contamination of the unit
with the target organism. The most logical place to sample to confirm contamination is the part of the
sampling equipment that stays in contact with a decontamination solution or can potentially touch the
coupon surface (such as outside of the nozzle). These parts will be sampled using the swab protocol
described in MOP 3135 (Procedure for Sample Collection using BactiSwab™ Collection and Transport
Systems). Swab analysis (growth/no growth) will be performed in accordance with MOP 6563.
4.6 Aerosol Samples
Aerosol samples collected with the Via-Cell® according to MOP 3155 (Procedure for Via-Cell® Air Sampling)
will be processed according to MOP 6571 (Recovery of Bacillus Spores from Via-Cell® Aerosol Sampling
Cassettes). The total flow in the duct will be measured using an S-type pitot tube as described in EPA
Method 2. This measurement will be done at the beginning and end of each day of testing.
4.7 Activated Peroxide Measurement
The H202 concentration will be measured via KMnO4 titration as described in MOP 3136.
4.8 pH Measurement
The pH of the solution will be measured with an Oakton pH5 pH meter. This meter will be calibrated daily.
5.0 TIMELINE SCHEDULE
The test cycle will start on Monday and end on Wednesday. Samples will be sent to the ON-SITE
Biocontaminant Laboratory for analysis as they become available. Below is an outline of the tasks to be
completed each day:
• Inoculate coupons in H-130 (4 sets of coupons)
• Move test coupons to the blank and positive coupons storage cabinets
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Prepare decontamination solution
Check sprayer
Clean the decontamination chamber before tests
Perform sterility checks
Perform decontamination in the H-130 small test chamber and move coupons to the appropriate
storage cabinets for overnight drying
Collect aerosol samples during decontamination
Collect runoff samples during decontamination
Clean the decontamination chamber post-last test
• Sample blanks
• Sample test coupon and positives
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WA 3-08 Coupon Deposition Log
Page.
of
Test ID
Date
Personnel /Title
MDI ID:
Initial Weight (g):
Final Weight (g):
Time
Coupon ID
Vortex Interval (s)
Puff Number
MDI
Weight (g)
Comments
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MOP 3135
TITLE: Procedure forWA 1-25: Procedure for Sample Collection using BactiSwab™ Collection and
Transport Systems
SCOPE: This MOP describes the procedure for collecting swab samples for Low Tech
Decontamination Technique Testing
PURPOSE: The purpose of this MOP is to ensure all swab sampling is performed in a consistent
manner.
Equipment/Reagents
• Disposable lab coat
• Nitrile examination gloves
• P95 Respirator
• Shoe covers
• Bouffant cap
• Safety glasses
• BactiSwab™ Collection and Transport System
1.0 PROCEDURE
1. Enter the COMMANDER airlock wearing appropriate, project-specific PPE (at a minimum gloves, lab
coat, and safety glasses), making sure the airlock door is closed.
2. Through the sleeve, crush the BactiSwab™ ampule at midpoint.
3. Hold BactiSwab™ tip end up for at least five seconds to allow the medium to wet the swab.
4. Open the package and remove the BactiSwab™.
5. Label the plastic tube appropriately using the following scheme:
X-Y-N where,
X is the test number,
Y is the material abbreviation, and
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N is the material number.
6. Remove the cap-swab from the plastic tube.
7. Swab the surface following the recommend guidelines for each material while spinning the cap-swab
between the thumb and index fingers.
a. Brushes (B).
Pull the cap-swab through the brush bristles using one continuous stroke moving top to bottom and
left to right.
b. Nozzles (N).
Swab around the squeegee, inside the divisions, and inside the opening for the hose attachment.
c. Buckets (P).
Swab the sides and the bottom surfaces in an "S" pattern.
d. Brush Handles (BH).
Swab the top quarter of the handle top the bottom then bottom to top turning the handle as you go.
e. Hoses (VH).
Swab inside and outside the hose opening that attaches to the nozzle.
f. Vacuums (V).
Randomly swab the folds of the High Efficiency Particulate Air (HEPA) filter, swab the bottom of the
vacuum lid, them swab the walls and bottom of the canister. Swab the inside of the exhaust port.
8. Return cap-swab to tube.
9. Date and initial each sample tube. Enter this information into the lab notebook.
10. Complete the chain of custody form and relinquish the samples to the BioLab.
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MOP-3144
TITLE: PROCEDURE FOR WIPE SAMPLING OF COUPONS
SCOPE: This MOP describes the procedure for wipe sampling both small and large coupons.
PURPOSE: The purpose of this MOP is to ensure consistent and representative sampling of such
coupons.
EQUIPMENT (quantities are per sampling kit)
• Sterile sampling bag (10" x 14") - outer bag
• Sterile sampling bag (5.5" x 9 ") - inner "sample collection sterile sampling bag"
• Two sterile 50 mL Falcon Blue-Max™ Polypropylene Conical Tubes
• Sterile Kendall (ref. # 8402) 4-ply all-purpose sponge
• Sterile phosphate buffered saline with 0.005% TWEEN®-20, prepared according to
MOP -6562
• Pipette or other method for aseptic dispensing of 5 mL liquid
• Sterile Posi-grip® forceps
• P-95 Particulate Respirators - to prevent contamination and for respiratory protection. (Specific
projects may require additional respiratory protection and will be addressed in the project Quality
Assurance Project Plan (QAPP), e.g., SAR)
• Powder-free Nitrile gloves (support person) and Kimtech Pure G3 Sterile Nitrile gloves (sampler)
• Dispatch® bleach wipes
1.0 PREPARATION
All materials needed for collection of each sample will be prepared in advance using aseptic technique. A
sample kit for a single wipe sample will be prepared as follows:
1. Two sterile sampling bags (10" x 14", 5.5" x 9 ") and a 50 mL conical tube, capped, will be uniquely
labeled as specified in the project QAPP. These bags and conical tube will have the same label. The
5.5" x 9" labeled sterile sampling bag will be referred to as the sample collection sampling bag.
2. A sterile all-purpose sponge will be placed in an unlabeled sterile 50 mL conical tube using sterile
forceps and aseptic technique. The all-purpose sponge will be moistened by adding 2.5 mL of sterile
phosphate buffered saline with 0.005% TWEEN®-20. The tube will then be capped.
3. The labeled 50 mL conical tube (capped), the unlabeled conical tube containing the pre-moistened all-
purpose sponge, and the 5.5" x 9" labeled sampling bag will be placed into the 10" x 14" labeled
sampling bag. Hence, each labeled sampling bag will contain a labeled 50 mL conical tube (capped), an
unlabeled capped conical tube containing a pre-moistened all-purpose sponge, and an empty labeled
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sampling bag.
4. Each prepared bag is one sampling kit.
2.0 SAMPLING PROCEDURE FOR SMALL 14"x14" COUPONS
1. A three person team will be used, employing aseptic technique throughout. The team will consist of a
sampler, sample handler, and support person.
2. Throughout the procedure, the support person will log anything they deem to be significant into the
laboratory notebook.
3. In general, the team works from the least contaminated sample set (i.e., control blanks) towards the
most contaminated sample set (i.e., positive controls).
4. The sampling team will each don a pair of sampling gloves (a new pair per sample, non-sterile, as they
will only be handling non-sterile items); the sampler's gloves shall be sterile sampling gloves as they are
the only member of the team in contact with the sample. All members shall wear dust masks to further
minimize potential contamination of the samples. Depending on the situation, respiratory protection
beyond a dust mask may be required to protect the sampling team (e.g., SAR; this will be specified in
the project QAPP). New disposable lab coats are required for the sample handler when changing
between different types of materials or when direct contact between the coupon and lab coat occurs.
5. The sample handler will remove the coupon from the appropriate cabinet and place it on the sampling
area, being careful to handle the coupon only around the edges.
6. The support person will record the coupon code on the sampling log sheet.
7. The support person will remove a template from the bag and aseptically unwrap it such that the sampler
may grab it wearing sterile gloves.
8. The sampler will place the template onto the coupon surface and align it such that the edges of the
coupon are visible through the holes on the template.
9. The support person will remove a sample kit from the sampling bin and record the sample tube number
on the sampling log sheet next to the corresponding coupon code just recorded.
10. The sampler and support person will verify the sample code and ensure that the correct coupon and
location are being sampled.
11. The support person will:
a. Open the outer sampling bag touching the outside of the bag.
b. Touching only the outside of the (10"x 14") bag, remove and open the unlabeled conical tube
and pour the pre-moistened all-purpose sponge onto the sample or into the sampler's hands.
c. Discard the unlabeled conical tube.
d. Remove the sample collection sample bag (5.5" x 9"), being careful to not touch the inside of
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the outer sampling bag, and open it touching only the outside.
e. Maneuver the labeled 50 ml conical tube to the end of the outer sterile sampling bag and
loosen the cap.
f. Remove the cap from 50 ml conical tube immediately preceding the introduction of the sample
into the tube.
12. The sampler will:
a. Wipe the surface of the sample horizontally using S-strokes to cover the entire sample area of
the coupon using a consistent amount of pressure.
b. Fold the all-purpose sponge concealing the exposed side and then wipe the same surface
vertically using the same technique.
C. Fold the all-purpose sponge over again and roll up the folded sponge to fit into the conical tube.
d. Carefully place the all-purpose sponge into the 50 ml conical tube that the support person is
holding, being careful not to touch the surface of the 50 ml conical tube or plastic sterile
sampling bag.
13. The support person will then immediately close and tighten the cap to the 50 ml conical tube and slide
the tube back into the sample collection sampling bag and seal it.
14. The support person will then wipe the sample collection sampling bag with a Dispatch® bleach wipe
and place it into the outer sampling bag.
15. The support person will then seal the outer sample collection bag now containing the capped 50 ml
conical tube (containing the all-purpose sponge) inside a sealed 5.5" x 9" sample collection bag.
16. The support person will then decontaminate the outer sample bag by wiping it with a Dispatch® bleach
wipe.
17. The support person will then place the triple contained sample into the sample collection bin.
18. All members of the sampling team will remove and discard their gloves.
19. Steps 2 - 18 will be repeated for each sample to be collected.
3.0 SAMPLING METHOD FOR LARGE (4'x4' or larger) COUPONS
3.1 Sample Layout
The sampling of large coupons is carried out using a sample grid to divide the large coupons into
representative sections. These sections are then numbered and selected to be sampled at different times
during the course of the experiment as a blank, a control, or an experimental group sample. This selection
grid is pre-determined and the Project Quality Assurance Project Plan (QAPP) may overrule the template
shown in Figure 1 if otherwise specified.
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As in the example below, the first cell is sampled as a before contamination. Starting in cell 3, every
third cell is sampled as a positive Control. This sample is to be taken post-contamination and before
decontamination. Every cell directly following a Control cell is sampled as and is taken
following decontamination. The sample kit labeling will be based on this grid and the sampling team must
ensure to correctly sample the coupons based on this template.
1
Blank
5
9
Control
13
Experimental
2
6
Control
10
14
3
Control
7
11
15
Control
4
Experimental
8
12
Control
16
Experimental
Figure 1. 4' x 4' Material Section Template and Sample Grid
3.2 Sampling Procedure
1. A two-person team will be used, employing aseptic technique throughout. The team will consist of a
sampler and a support person.
2. Throughout the procedure, the support person will log anything they deem to be significant into the
laboratory notebook.
3. The sampling team will each don a pair of sampling gloves (a new pair per sample, non-sterile, as they
will only be handling non-sterile items); the sampler's gloves shall be sterile sampling gloves as they are
the only member of the team in contact with the sample. All members shall wear dust masks to further
minimize potential contamination of the samples. Depending on the situation, respiratory protection
beyond a dust mask may be required to protect the sampling team (e.g., SAR; this will be specified in
the project QAPP).
4. The support person will record the coupon code on the sampling log sheet.
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5. The sampler will place the template onto the coupon surface (using clamps as necessary).
6. The support person will remove a sample kit from the sampling bin and record the sample tube number
on the sampling log sheet next to the corresponding coupon code just recorded.
7. The sampler and support person will verify the sample code and ensure that the correct coupon and
location (cell) is being sampled.
8. The support person will:
a. Open the outer sampling bag touching the outside of the bag.
b. Touching only the outside of the (10"x 14") bag, remove and open the unlabeled conical tube
and pour the pre-moistened all-purpose sponge onto the sample or into the sampler's hands.
c. The unlabeled conical tube is retained for Step 9.
d. Remove the sample collection sample bag (5.5" x 9") being careful to not touch the inside of the
outer sampling bag and open it touching only the outside.
e. Maneuver the labeled 50 ml conical tube to the end of the outer sterile sampling bag and
loosen the cap.
f. Remove the cap from 50 ml conical tube immediately preceding the introduction of the sample
into the tube.
9. The sampler will:
a. For a vertical coupon, the sampler will squeeze excess moisture from the sampling sponge to
prevent dripping down the sampling surface. The excess moisture is caught in the unlabeled
conical tube from Step 8c and is then discarded.
b. Wipe the surface of the sample using S-strokes to cover the entire sample area of the coupon
(inside the grid) using a consistent amount of pressure.
c. Fold the all-purpose sponge concealing the exposed side and then wipe the same surface
vertically using the same technique.
d. Fold the all-purpose sponge over again and roll up the folded sponge to fit into the conical tube.
e. Carefully place the all-purpose sponge into the 50 ml conical tube that the support person is
holding being careful not to touch the surface of the 50 ml conical tube or plastic sterile
sampling bag.
10. The support person will then immediately close and tighten the cap to the 50 ml conical tube and slide
the tube into the sample collection sampling bag and seal it.
11. The support person will then wipe the sample collection sampling bag with a Dispatch® bleach wipe and
place it into the outer sampling bag.
12. The support person will then seal the outer sample collection bag now containing the capped 50 ml
conical tube (containing the all-purpose sponge) inside a sealed 5.5" x 9" sample collection bag.
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13. The support person will then decontaminate the outer sample bag by wiping it with a Dispatch bleach
wipe.
14. The support person will then place the triple contained sample into the sample collection bin.
15. All members of the sampling team will remove and discard their gloves.
16. Steps 2 - 15 will be repeated for each sample to be collected.
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MOP-3145
TITLE: PROCEDURE FOR VACUUM SAMPLING OF LARGE AND SMALL COUPONS
SCOPE: This MOP describes the procedure for vacuum sampling of porous areas.
PURPOSE: The purpose of this MOP is to ensure consistent and representative sampling of such
areas.
EQUIPMENT (quantities are per sampling kit)
• 2 - Fisherbrand bags with round wire enclosure, 5.5" x 15" (Fisher Scientific, p/n 14-955-181)
• 1 - Fisherbrand bag with round wire enclosure, 10" x 14" (Fisher Scientific, p/n 01-002-53)
• 1 - Vacuum sock filtration kit (Midwest Filtration, p/n FAB-20-01-001 A)
• Permanent marker
• Nitrile gloves
1.0 PREPARATION
All materials needed for each sample to be collected will be prepared in advance. A sample kit for a single
vacuum sock sample will be prepared using the following procedure (as in MOP- 3141: Procedure for
Assembling Vacuum Sock Sampling Kits):
1. Don nitrile gloves.
2. Remove the sock from the manufacturer's kit, place it in a 5.5" x 15" bag so that it is oriented tapered
side down, and seal the bag by rolling the top down and folding the tabs. Discard the remains of the
manufacturer's kit.
3. Label a 10"x 14" bag and a 5.5"x 15" bag with the sample ID as instructed by the project QAPP.
4. Open the labeled 10" x 14" bag and insert:
- the labeled 5.5" x 15" bag,
- an unlabeled 5.5" x 15" bag, and
- the 5.5" x 15" bag containing the sock.
5. Close the bag and store in a clean dry location.
2.0 VACUUM SAMPLING OF SMALL (14" by 14") COUPONS
The following procedure will be used in this study for vacuum sampling of each coupon surface:
1. A two person team will be used, employing aseptic technique. The team will consist of a sampler and a
support person.
2. Both members of the sampling team will each don a pair of sampling gloves (a new pair per sample);
the sampler's gloves shall be sterile sampling gloves as they are the only member of the team in contact
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with the sample. Both members shall wear dust masks to further minimize potential contamination of the
samples. Further respiratory protection beyond a dust mask may be required to protect the sampling
team (e.g., Supplied-Air Respirator (SAR); this will be specified in the project QAPP).
3. The sampler will plug in the vacuum power cord and then don his/her sterile gloves.
4. The vacuum will be maintained on a rolling cart for easy movement into place.
5. The support person will aseptically unwrap a template from the bag and present it to the sampler, taking
care to not touch the template.
6. The sampler will place the template onto the coupon surface.
7. The sampler will hold the vacuum nozzle for the support person to place the vacuum sock assembly
onto the nozzle.
8. The support person will open the sampling supply bin and remove the vacuum sock sample kit from the
bin.
9. The support person will record the sample collection bag ID number on the sampling log sheet or in the
laboratory notebook.
10. The sampler and support person will ensure that the correct sample coupon has been selected,
referencing the coupon code on the sampling bag.
11. The support person will record the coupon code on the sampling log sheet next to the corresponding
vacuum sock collection bag number that was just recorded.
12. The support person will:
a. Open the vacuum sock sample kit outer bag and remove the unlabelled vacuum sock assembly
bag.
b. Open the small unlabelled sampling bag containing the vacuum sock assembly and, working
from the outside of the bag, maneuver the assembly from the bottom to expose the cardboard
applicator tube opening.
c. Firmly place the vacuum sock assembly onto the nozzle of the vacuum tube, using the bag to
handle the sock assembly, while the sampler holds the vacuum nozzle.
13. The sampler will:
a. Ensure that the sock is correctly placed on the nozzle and adjust, if necessary. Care must
be taken to not puncture or tear the sock.
b. Turn on the vacuum.
c. Vacuum "horizontally" using S-strokes to cover the entire area of the material surface not
covered by the template, while keeping the vacuum nozzle angled so that the tapered
opening of the vacuum sock is flush with the sample surface.
d. Vacuum the same area "vertically" using the same technique.
e. Turn off the vacuum when sampling is completed.
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14. The support person will open the labeled 5.5" x 15" bag and remove the vacuum sock assembly
from the nozzle with the inner sterile sampling bag, using care not to touch the sock.
NOTE: It is very important that the vacuum sock not be inverted after the sample
is taken. During Steps 14 thru 17, keep the white "sock" that contains the
sample in an upright position to ensure sample integrity.
15. The support person will then seal the inner sterile sampling bag, wipe it with a Dispatch® wipe, and
place it into the outer sterile sampling bag.
16. The support person will then seal the outer sterile sampling bag and wipe it with a Dispatch® wipe.
17. The support person will then seal the 10" x 14" overpack sample bag now containing the outer and
inner bags, the inner containing the vacuum sock assembly. The outermost bag will then be wiped
with a Dispatch® wipe.
18. The sampler will wipe down the nozzle (inside and out) and end of the tubing with a Dispatch® wipe.
19. The support person will then place the triple contained sample into the sample collection bin.
20. All members of the sampling team will remove and discard their gloves.
21. Steps 2-20 will be repeated for each sample to be collected.
3.0 SAMPLING METHOD FOR LARGE (4'x4' or larger) COUPONS
3.1 Sample Layout
The sampling of large coupons is carried out using a sample grid to divide the large coupons into
representative sections. These sections are then numbered and selected to be sampled at different times
during the course of the experiment as a a Control, or an group sample. This selection
grid is pre-determined and the Project QAPP may overrule the template shown in Figure 1 if otherwise
specified. The first cell is sampled as a (negative control) before contamination. Starting in cell 3,
every third cell is sampled as a positive Control. This sample is to be taken post contamination and before
decontamination. Every cell directly following a Control cell is sampled as and is taken
following decontamination. The sample kit labeling will be based on this grid and the sampling team must
ensure to correctly sample the coupons based on this template.
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1
5
9
Control
13
2
6
Control
10
14
3
Control
7
11
15
Control
4
8
12
Control
16
Figure 1. 4' x 4' Material Section Template and Sample Grid
3.2 Sampling Procedure
The procedure detailed below will be used in this study for vacuum sampling of each coupon surface. In
addition to these collected samples, one vacuum sock would be left unopened and used as a Trip or
vacuum sock negative control. The purpose of this Trip blank is to verify that the vacuum socks - as
received from the manufacturer - were not defective and/or contaminated, nor are the socks being
contaminated simply by traveling through the collection process with the real samples. The vacuum sock
negative control results should always come back negative.
1. A two person team will be used, employing aseptic technique. The team will consist of a sampler and a
support person. Both members of the sampling team will wear task specific PPE as specified in the
project QAPP. P95 respirators are the minimum requirement for sample protection.
2. The sampler and support person will don sterile gloves and position the sampling grid ono the coupon,
using clamps if necessary.
3. The support person will plug in the vacuum power cord. The vacuum will be maintained on the floor with
easy access to the sample. The exhaust of the vacuum should be directed directly into the air handling
return duetto help prevent cross-contamination.
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4. The sampler will don a new pair of sterile gloves and position the vacuum nozzle in anticipation of the
support person.
5. The support person will don a new pair of nitrile gloves, open the sampling supply bin, and remove one
vacuum sock sample kit from the bin.
6. The support person will record the sample collection bag number on the sampling log sheet.
7. The sampler and support person will confirm the correct sampling location has been selected by
referencing the coupon code on the sampling bag.
8. The support person will:
a. Open the vacuum sock sample kit outer bag and remove the unlabelled vacuum sock assembly
bag.
b. Open the small unlabelled sampling bag containing the vacuum sock assembly and, working
from the outside of the bag, maneuver the assembly from the bottom to expose the cardboard
applicator tube opening.
c. Firmly place the vacuum sock assembly onto the nozzle of the vacuum tube, using the bag to
handle the sock assembly, while the sampler holds the vacuum nozzle.
9. The sampler will:
a. Turn on the vacuum.
b. Vacuum "horizontally" using S-strokes to cover the entire area of the material surface not
covered by the template, while keeping the vacuum nozzle angled so that the tapered opening
of the vacuum sock is flush with the sample surface.
c. Vacuum the same area "vertically" using the same technique.
d. Turn off the vacuum when sampling is completed.
10. The support person will open the labeled 5.5" x 15" bag and remove the vacuum sock assembly from
the nozzle using the inner sterile sampling bag.
11. The support person will then seal the inner sterile sampling bag, wipe it with a Dispatch® wipe, and
place it into the outer sterile sampling bag.
12. The support person will then seal the outer sterile sampling bag and wipe it with a Dispatch® wipe.
13. The support person will then seal the 10" x 14" overpack sample bag now containing the outer and inner
bags, the inner containing the vacuum sock assembly. The outermost bag will then be wiped with a
Dispatch® wipe.
14. The sampler will wipe down the nozzle (inside and out) and end of the tubing with a Dispatch® wipe.
15. The support person will then place the triple contained sample into the sample collection bin.
16. All members of the sampling team will remove and discard their gloves.
17. Steps 2-16 will be repeated for each sample to be collected.
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MOP-3150
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MOP-3150
TITLE: PROCEDURE FOR FABRICATION OF 14" x 14" MATERIAL COUPONS
SCOPE: This MOP describes the procedure for construction of 14" x 14" material coupons.
PURPOSE: The purpose of this MOP is to ensure consistent manufacturing of these coupons.
INTRODUCTION
This MOP is split into five sections, each section being dedicated to one material type. Sections 1.0 to 5.0
present the methods for fabricating coupons from carpet, drywall, concrete, deck wood, and rough-cut barn
wood, respectively.
1.0 CARPET
1.1 Equipment
• Beaulieu Solutions Walnut Ridge Loop Carpet
• 6 Ib carpet padding
• 15/32" - Four Ply Plywood Sheathing
• V-i staples
• Safety Glasses
• Cut-Resistant Gloves
• Staple Gun
• Safety Razo r Uti I ity Kn ife
• Table saw
• Tape measure
• Straight edge
• %" Duct Tape
1.2 Procedure
1. Don safety glasses and cut-resistant gloves.
2. Cut a 14" x 14" square of 15/32" thick Four Ply Plywood Sheathing using a table saw.
3. Cut a 14" x 14" square of 6 Ib carpet padding using a safety razor utility knife.
4. Cut a 14" x 14" square of Beaulieu Solutions Walnut Ridge Loop Carpet (or carpet type specified by the
project QAPP) using a safety razor utility knife.
5. Align the carpet padding on top of plywood.
6. Align the Beaulieu Solutions Walnut Ridge Loop Carpet on top of carpet padding.
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7. Carefully secure carpet and carpet padding to plywood sheathing using 1/4" staples and a staple gun
around the outside edges (no more than 1/8" from coupon edge).
8. Seal the edges of the coupon with duct tape by taping the outer 1/8" edges of the front and stapling.
Fold the tape over and staple to the back. This creates a sampling area of 13.5" square.
Figure 1. Carpet Coupon Front
2.0 DRYWALL
Figure 2. Carpet Coupon Back
2.1 Equipment
• Safety Glasses
• Cut-Resistant Gloves
• Table saw
• One sheet of 1/2"drywall
• Joint Compound
• Putty Knife
• Joint Tape
• Sanding Block
• KILZ latex primer
• Behr Interior Enamel Paint
• Paint brushes
• Tape measure
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2.2 Procedure
1. Don safety glasses and cut-resistant gloves.
2. Cut a 14" x 14" section of Drywall from a drywall sheet using a table saw.
3. Apply a skim coat of joint compound about 1.5" from each cut edge of the coupon to the cut edge on the
front side using a putty knife.
4. Using two inch joint tape, apply one half of the tape (utilizing the factory fold) to the front side of the
coupon over the joint compound.
5. Apply a second skim coat of joint compound over the tape using a putty knife.
6. Allow to dry.
7. After the compound has dried, apply a skim coat of joint compound to the back side of the coupon about
1" from the cut edge of the coupon to the cut edge using a putty knife.
8. Fold the joint tape over the cut edge to the backside of the coupon (it should extend about 1/2" over the
back).
9. Apply a second skim coat of joint compound over the tape using a putty knife.
10, Allow to dry.
11. Remove any rough spots in the joint compound using a sanding block.
12. Allow to dry.
13. Smooth out all rough spots in the joint compound using a sanding block.
14. Apply one coat of KILZ latex primer to the front side of the coupon.
15. Allow to dry.
16. Apply one coat of Behr Premium Plus Interior Flat White Latex Paint to the front side of the coupon.
17. Allow to dry.
18. Seal the back side of the coupon with any non-white latex or enamel paint.
Figure 3. Drywall Coupon Front
Figure 4. Drywall Coupon Back
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3.0 CONCRETE
3.1 Equipment
Quikrete Sand/Topping Mix
Water Source
Mixing trough
Trowel
Leveling board
Plastic Covering for Curing Process
3.2 Procedure
1. Custom 14" x 14" forms have been manufactured for these coupons.
2. Prepare concrete mix according to indications on package using a trough and garden hose for the water
supply.
3. Pour concrete mix into custom manufactured forms.
4. Use a trowel to smooth coupon surface and allow form to dry overnight.
5. Repeat for each coupon to be manufactured.
6. Lay plastic over the coupons and allow to cure for at least 5 days.
Figure 5. Concrete Curing
Figure 6. Finished Concrete Coupon
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4.0 DECKWOOD
4.1 Equipment
• 5/4"x 6" Pressure treated decking board, allowed to dry at least 1 week after purchase.
• 2" Exterior Deck Screws
• 1-1/4" Common Nails
• Safety Glasses
• Cut resistant Gloves
• Circular Saw
• Impact wrench
• Behr Waterproofing Wood Protector
• Natural bristle brush
• Tape measure
4.2 Procedure
1. Don safety glasses and cut-resistant gloves.
2. Using a table saw, cut enough of the decking wood into %" wide strips to fabricate a 14" x 14" frame.
3. Cut an additional piece 1.5" wide to be used to simulate a floor joist.
4. Construct framing as shown in Figure 7, securing with 1-1/4" common nails.
Figure 7. Deck Wood Coupon Back
Figure 8. Deck Wood Coupon Front
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5. Carefully cut 5/4" x 6" board into 14" sections using a table saw. Two full size boards will be used. A
third board will be needed to be ripped to size to completely cover the frame, allowing a 1/8" gap in
between.
6. Mount the boards to framing from left to right leaving a 1/8" gap between each pair of boards.
7. Mount each board using two deck screws in the center strut.
8. Cut the last board to approximately 2.5" or as needed to completely cover the framing.
9. Mount the last board using one deck screw.
10. Secure the outer coupon edges to the framing using 1-1/4" Common Nails.
11. Once the assembly is complete, seal the front of the coupon using one coat of Behr Waterproofing
Wood Protector.
5.0 ROUGH CUT BARN WOOD
5.1 Equipment
• 1" X 6" pressure treated Brazilian Pine Dog Ear Picket Fence Lumber
• Safety Glasses
• Cut resistant Gloves
• 1" Staples
• Staple Gun
• Circular Saw
• Tape measure
5.2 Procedure
1. Don safety glasses and cut-resistant gloves.
2. Using a radial arm saw, cut six 14" x 6" pieces of rough cut wood.
3. Lay three of the pieces, butted together, face down on a bench. Lay the other three pieces
perpendicular to the first three to cover the butt joints of the wood. Use 1" staples to fasten the boards
together from the back so that no staples show on the front.
4. Use a table saw to trim the coupon to 14" square.
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6.0 PLYWOOD
6.1 Equipment
• 23/32" Alkaline Copper Quaternary Treated Plywood, allowed to dry at least 1 week after purchase.
• Safety Glasses
• Cut resistant Gloves
• Circular Saw
• Tape measure
6.2 Procedure
1. Don safety glasses and cut-resistant gloves.
2. Cut a 14" x 14" square of 15/32" thick Four Ply Plywood Sheathing using a table saw.
7.0 PAINTED PLYWOOD
7.1 Equipment
• 23/32" Alkaline Copper Quaternary Treated Plywood, allowed to dry at least 1 week after purchase.
• Safety Glasses
• Cut resistant Gloves
• Circular Saw
• Tape measure
• Exterior Paint (i.e., BEHR Exterior Enamel, white)
• Paint brush and paint tray
7.2 Procedure
1. Don safety glasses and cut-resistant gloves.
2. Cut a 14" x 14" square of 15/32" thick Four Ply Plywood Sheathing using a table saw.
3. Using a paint brush, apply two coats of exterior paint, allowing at least 2 hours for drying between coats
(or per the paint manufacturer's label instructions)
8.0 GLASS
Ordered to size from Durham Glass, Inc.
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9.0 LINOLEUM
9.1 Equipment
• Vinyl linoleum flooring, in 12'-wide roll
• Safety Glasses
• Cut resistant Gloves
• Sharp Knife / Box-blade
• Tape measure
• Construction adhesive (i.e., liquid nails)
• Heavy-duty staple gun, with staples
9.2 Procedure
1. Don safety glasses and cut-resistant gloves.
2. Cut linoleum sheets into 14" x 14" squares
3. Cut 14" x 14" squares of 15/32" thick Four Ply Plywood Sheathing using a table saw, one for each
coupon.
4. Apply a thin bead of construction adhesive around the perimeter of the rear of the sheet, and as a "z"
within the middle of the rear of the sheet.
5. Place the linoleum square onto the plywood, rub the surface to distribute the adhesive. Fasten the vinyl
to the plywood using heavy-duty staples, three staples along each side.
10.0 CERAMIC TILE
10.1 Equipment
• Ceramic tile (18" x 18")
• Safety Glasses
• Cut resistant Gloves
• Tape measure
• Tile saw
10.2 Procedure
1. Don safety glasses and cut-resistant gloves.
2. Cut tile into 14" x 14" squares by cutting 4" from each of two sides.
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MOP-3155
Revision 1
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MOP-3155
TITLE: PROCEDURE FOR VIA-CELL® AIR SAMPLING
SCOPE: This MOP describes the procedure for air sampling using the Via-Cell® Bioaerosol
Sampling Cassette.
PURPOSE: The purpose of this MOP is to ensure consistent and representative air sampling.
EQUIPMENT
• Zefon Via-Cell® Bioaerosol Sampling Cassette (p/n VIA010)
• EPA Method 5 Dry Gas meter box within annual calibration
• 1/4" I.D. vacuum tubing
• Labeled 5.5" x 15" sterile bag for tertiary containment
• Writing pen
• Project notebook
1.0 PREPARATION
1. Verify from the package that the Via-Cell® cassette is within its expiration date. If not, discard.
2. Label the Via-Cell® cassette with the appropriate sample ID according to the test plan or Quality
Assurance Project Plan (QAPP).
3. In the project laboratory notebook, record the cassette sample ID, lot number, and expiration date.
2.0 AIR SAMPLING
There are two areas of operation for Via-Cell® air sampling: 1) at the cassette and 2) at the meter box.
These two areas of operation may be performed by the same person or different people depending on the
situation. When air sampling inside COMMANDER while occupied, for instance, a two-person team will be
necessary; one person inside to connect the cassette and the other outside to start and stop the dry gas
meter.
1. Wearing nitrile gloves, tear open the foil package using the tear strip on top. Use care when
opening, as this package is re-sealable and is required to be used after sampling for transport to the
laboratory for analysis.
2. Remove Via-Cell® from the package (see Figure 1).
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sm»»-i»
10 i .„
1 »8« ( Illliillll!-""'
,. ,1-,,.
0,1V
Figure 1. Disassembled Via-Cell , showing cap (far left), package, and cassette with outlet
plug.
3. Remove the blue outlet plug from the cassette and place it into the foil for safe keeping (seen at the
bottom of the green Via-Cell® cassette in Figure 1).
4. Connect the Via-Cell® sampler outlet to the dry gas meter using vacuum tubing and position the
cassette in the desired location. The Via-Cell® sampler is capable of operating in any vertical or
horizontal orientation and in confined spaces.
5. Perform a leak-check on the cassette by pulling a vacuum on the inlet cap. The meter box flow rate
should be zero.
6. Remove the large blue inlet cap for the cassette and place into the foil package for safekeeping.
7. Record the dry gas meter's initial volume.
8. At the meter box, turn on the pump and set the sampling pump to a flow rate of 15 Lpm. Over the
pressure drop of the Via-Cell ®cassette, this is at a AH of 1.1" water as read on the front of the
meter box. Pull a sample for the desired amount of time, monitoring the AH every ten minutes.
When sampling is completed, and with new gloves, replace the blue plug in the outlet and the blue
cap over the inlet.
9. Record the sampling time and final volume. Check to ensure flow rate was 15 Lpm.
10. Place the Via-Cell® cassette into the special foil bag and zip it closed. Apply the red safety seal label
over the top of the foil bag opening to ensure sample integrity until analysis.
11. Place foil bag containing cassette inside a pre-labeled 5.5" x 15" sterile bag for tertiary containment.
12. Submit the cassette to the Biocontaminant Laboratory along with a Chain-of-Custody (COC); the
cassette should be analyzed according to MOP 6571 within 24 hours of collection. The COC form
should include the collection time and the analysis by time and date in the comments section.
13. Each sample should be associated with a laboratory blank (plain, unused Via-Cell® cassette) and a
field blank of at least 150 liters of clean air in the same area as samples are collected.
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MOP-6535a
Revision 2.0
April 8, 2009
Page 1 of 2
MOP 6535a
Title: SERIAL DILUTION: SPREAD PLATE PROCEDURE TO QUANTIFY VIABLE BACTERIAL SPORES
Scope: Determine the abundance of bacterial spores in a liquid extract
Purpose: Determine quantitatively the number of viable bacterial spores in a liquid suspension using the spread
plate procedure to count colony-forming units (CPU)
Materials:
• Liquid suspension of bacterial spores
• Sterile centrifuge tubes
• Diluent (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 micro-centrifuge tubes as follows: 10"1,10"2,10"3,10"
4,10~5,10~6... (The number of dilution tubes will vary depending on the concentration of spores in the
suspension. Aseptically, add 900 uL of sterile diluent to each of the tubes.
2. Label three Trypticase Soy Agar plates for each dilution that will be plated. These dilutions will be plated in
triplicate.
3. Mix original spore suspension by vortexing thoroughly for 30 seconds. Immediately after the cessation of
vortexing, transfer 100 uL of the stock suspension to the 10~1 tube. Mix the 10~1 tube by vortexing for 10
seconds, and immediately pipette 100 uL to the 10~2 tube. Repeat this process until the final dilution is made.
It is imperative that used pipette tips be exchanged for a sterile tip each time a new dilution is started.
4. To plate the dilutions, vortex the dilution to be plated 10 seconds, immediately pipette 100 uL of the dilution
onto the surface of a TSA plate, taking care to dispense all of the liquid from the pipette tip. If less than 10
seconds elapses between inoculation of all replicate plates, then the initial vortex mixing before the first
replicate is sufficient for all replicates of the sample. Use a new pipette tip for each set of replicate dilutions.
5. Carefully pour the sterile glass beads onto the surface of the TSA 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 to 37 °C (incubation conditions will vary depending on the organism's
optimum growth temperature and generation time.)
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7. Enumerate the colony forming units (CPU) on the agar plates by manually counting with the aid of a plate
counting lamp and a marker (place a mark on the surface of the Petri dish over each CPU when counting, so
that no CPU is counted twice).
Since each dilution was tested in triplicate, determine the average of the triplicate plate abundances. Plates
suitable for counting must contain between 30 - 300 colonies.
Calculations
Total abundance of spores (CPU) within extract:
(Avg CPU / volume (ml) plated) X (1 / tube dilution factor) X extract volume
For example:
Tube Dilution
10-3
10-3
10-3
Volume Plated
100uL(0.1 ml)
100 ML (0.1 ml)
100uL(0.1 ml)
Replicate
1
2
3
CPU
150
250
200
Extract total volume = 20 ml
(200 CPU/ 0.1 ml) x (1/1 0"3) x 20 ml
(2000) x (1000) x 20 4.0 xio7
Note: The volume plated (ml) and tube dilution can be multiplied to yield a 'decimal factor' (DP). DP can be used
in the following manner to simplify the abundance calculation.
Spore Abundance per ml = (Avg CPU) x (1 / DP) x extract volume
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MOP- 6555
TITLE: PETRI DISH MEDIA INOCULATION USING BEADS
SCOPE: This MOP outlines the procedure for cleaning, assembling, and using beads to inoculate
agar plates.
PURPOSE: To provide an easily repeatable method for spreading liquid inoculation onto agar plates.
1.0 EQUIPMENT
1. #13 test tubes
2. 6x12 test tube racks (which hold 72 tubes)
3. Beads of various sizes (glass)
4. Glass autoclavable trays (stainless steel is eventually corroded by the bleach and autoclaving
processes)
5. Bleach
6. Dl water
7. Hot gloves
8. Amber bottle for collecting hazardous waste (with hazardous waste label)
9. Funnel
10. Aluminum foil
11. Label tape
12. Chemical hood
13. Autoclave
14. Oven
15. Labeled bead container or cup (All mold and bacteria beads must be kept separately)
2.0 CLEANING BEADS
1. When a sufficient number of beads have been collected, or at least once a day when beads are
being used to spread colonies, place the used beads into a tray with a 1:5 ratio of bleach to
deionized water solution.
Add the bleach to the beads first, under the protection of a chemical safety cabinet. Then add the
deionized water. Cover the pan with aluminum foil and label it with the contents (for example:
"bacteria beads in 1:5 bleach to Dl water solution"). Soak the beads 12-24 hours (usually
overnight) in a chemical fume hood.
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2. After soaking, take a bottle brush and thoroughly scrub the beads.
3. Decant the bleach solution (collect the bleach for proper disposal) and rinse with deionized water 6
to 8 times, collecting the runoff after the first rinse for disposal (subsequent rinses can be discarded
in the lab sink). Rinse until the decanted liquid is clear. Use a funnel to add the bleach waste to a
labeled amber waste bottle. These liquids must be labeled "hazardous waste" and can then be
stored, collected or disposed of properly.
4. Cover the beads with deionized water and autoclave for 1 hour on the liquid cycle.
5. Decant the deionized water and place the tray of beads in the Thelco lab oven at 121° C until dry
(a minimum of 3 hours).
6. Remove the beads from the oven using proper safety equipment (heat gloves) and cover with
clean aluminum foil to prevent contamination. Label each tray with the following information:
"Clean bacteria (or mold) beads," the date beads were cleaned, initials of the person who
cleaned them.
7. These beads are then ready for use as described in "PLACING BEADS IN TUBES".
3.0 PLACING BEADS IN TUBES
1. Fill a 6x12 rack with tubes.
2. Place clean beads into a shallow pan, and then manually fill each tube with 7-15 beads/tube.
Note: Beads vary in size and will therefore fill the tubes to different heights.
3. Tightly attach a cap to each tube
4. Autoclave for using 1 hour gravity sterilization cycle (see MOP 6570). Autoclave tape must be
placed on the top of each rack to provide evidence that the beads have been sterilized
4.0 SPREADING BEADS
1. To spread inoculum on the agar surface, one tube of beads should be used for each individual
plate.
2. After the beads have been added, the plates can be stacked up to six plates high. The plates are
then shaken 10 times from side-to-side. Turn the stack of plates % turn, and again shake 10 times
from side-to-side. Repeat this procedure (% turn and 10 shakes) two more times, so that the beads
are shaken a total of forty times.
3. Turn the plates over (upside down), and tap the beads into the lid.
4. Aseptically dump the beads into a labeled bead container (mold and bacteria beads must be
labeled and collected separately), which should be considered contaminated. This should be done
one plate at a time, replacing the lid as quickly as possible to prevent contamination.
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1. http://serc.carleton.edu/microbelife/k12/LIMW/dilution.html
2. http://www.qbioqene.com/businessdivisions/platformnews/news0005-b.shtml
3. http://www.qenlantis.com/obiects/cataloq/Product/extras/C400050.pdf
4. EPA - SHEM Chemical Hygiene Plan.
MOP-6555
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MOP-6561
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MOP 6561
TITLE:
SCOPE:
PURPOSE:
Materials:
AEROSOL DEPOSITION OF SPORES ONTO MATERIAL COUPON SURFACES USING
THE AEROSOL DEPOSITION APPARATUS
This MOP outlines the procedure for assembly and usage of the Aerosol Deposition
Apparatus (ADA).
Precise and highly repeatable aerosol deposition of bacterial spores onto material surfaces
for detection, sampling, and/or decontamination studies.
Aerosol Deposition Apparatus (ADA) (shown in Figure 1)
Metered Dose Inhaler (MDI) preloaded with a bacterial spore suspension of known concentration (i.e.
1 x 109 spores per puff)
Vertical MDI Actuator (shown in Figure 2)
Material coupon (with dimensions at least that of the ADA)
ADA-coupon gasket (1 per ADA) (see Figure 1)
Clamping devices (i.e., medium-size steel binder clips, C-clamps (8 per ADA))
Vortex mixer (shown in Figure 4)
Aerosol trap (described in Appendix A and shown in Figure 4)
Personal Protective Equipment (PPE) (gloves, lab coat, safety goggles)
pH-adjusted bleach (pAB) (MOP 3128-A)
0.22um pore-size syringe filters (shown in Figure 1)
PVC tubing (3/8" OD, 1/4" ID)
Mass balance (with 0.01 gram accuracy)
Bench liner
1.0 STERILIZATION OF MATERIALS
Prior to the start of any experiment, all components must be sterilized and stored in a sterile environment
until usage. Sterilization is not necessary for binder clips, MDI, vortex, or the aerosol trap.
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ADAs can be sterilized by autoclave, VHP, or by wiping with pH-adjusted bleach (pAB) with subsequent
deionized (Dl) water and ethanol rinse/wipes. The ADA lid should be attached and in the closed position
during the sterilization.
ADA with lid in
closed position
Figure 1. ADA apparatus
The MDI actuator, with attached MDI adaptor, can be wiped with pAB then rinsed with Dl water.
Figure 2. MDI and vertical actuator
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Sterilization requirements for coupons vary by material. Regardless of the sterilization method, quality
control (QC) checks (typically by collecting a swab sample per MOP 3135) should be administered to
ensure the effectiveness of the sterilization method.
Gasket sterilization may also vary by material. Care should be taken to thoroughly degas gaskets if
sterilized via fumigation.
2.0 PROCEDURE
1. Begin by donning PPE (gloves, lab coat, and protective eyewear).
2. Clean the workspace by wiping with pAB, next with Dl water, and lastly with a 70-90% solution of
denatured ethanol. Alternatively, new, clean bench liner may be placed on the work surfaces. Make
sure the workspace is clean and free of debris.
3. Discard gloves and replace with fresh pair.
4. Using aseptic techniques (when possible) assemble the coupon/ADA by first placing the sterilized
material coupon onto the clean lab bench or workspace, next place the sterilized gasket on top of the
coupon, and lastly seat the ADA on the coupon + gasket. Orient each component so that it fits squarely
with the previously placed item. Take care not to touch the inside of the ADA or the coupon surface.
Secure these components by attaching medium-size binder clips, one at each corner, and one at the
midpoint of each of the four sides of the ADA. The binders should firmly secure the coupon to the ADA,
and apply sufficient pressure to the gasket to seal the union. If material coupons are too large to use
binder clips other methods may be used to secure the coupon and gasket to the ADA (i.e., larger
clamps, weight added to the ADA, etc.). Lastly, attach 0.2 urn syringe filters to each vent tube on all
ADAs (4 per ADA). Syringe filters can be attached using PVC tubing (3/8" OD, 1/4" ID).
5. Determine the weight of the MDI canister using a balance. Record the MDI ID number and the weight
(to the nearest 0.01g) in lab notebook. In addition, keep a record of the total number of 'puffs' dispensed
for each MDI canister.
NOTE: The MDI canister full is approximately 15 g, an empty canister is approx 9.5 g. To ensure the
canister contains adequate spore suspension for dosing, canisters should be retired from use
when their weight falls below 10.5 g.
6. Next, assemble the MDI and actuator by inserting the MDI into the actuator, taking care not to activate
the MDI.
7. Vortex the MDI/actuator assembly for 30 seconds (the MDI canister should be in direct contact with the
vortex mixer).
8. Holding the MDI/actuator assembly upright (Figure 3), with a swift, firm motion, dispense three test
'puffs' into the aerosol trap to prime the MDI. It is important to vortex the assembly 10 seconds before
every puff (the exception being 30 seconds prior to the initial puff of the experiment, as prescribed in
Step 7).
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Figure 3. MDI orientation while dispensing test puffs into the aerosol trap.
9. Vortex the assembly for 10 seconds and then attach to the ADA lid by mating the ADA adaptor to the
hole in the ADA lid. Loosen the lid screws enough to allow the lid to be slid into the 'open' position.
Secure the lid in the open position by tightening the lid screws.
NOTE: The 'open' position is achieved when the hole in the lid aligns with the hole in the top of the
ADA.
10. With a swift, firm motion, dispense the spores by activating the MDI. Hold the MDI in the activated
position for 3 seconds before releasing. Activation is best achieved by grasping the MDI/actuator with
two hands, and using a thumb to press the bottom of the MDI canister.
11. Follow the reverse order of the lid opening procedure to close the ADA lid.
12. Determine the weight of the actuator-MDI using a balance, and record the weight in lab notebook.
NOTE: If the dosing puff is faulty, return to Step 9 and attempt a second puff on the current coupon.
Do not proceed to the next coupon until a 'successful' puff has been delivered. A 'successful'
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puff is achieved when the weight of the actuator-MDI assembly has a 0.04 g to 0.07 g loss.
Familiarity and professional judgment will be needed to determine the success of a puff.
13. Vortex the assembly for 10 seconds, then proceed to dosing the next coupon (Step 9).
14. Repeat Steps 9 through 13 until all coupons have been dosed.
15. Once all coupons have been puffed, remove the MDI from the actuator and weigh. Record the final
weight and total number of puffs.
16. Allow spores to settle onto the coupon surface for at least 18 hours. Settling time should not exceed 26
hours.
17. Carefully remove binder clips (or other attachment device), and remove aerosol deposition apparatus
(ADA) and gasket from coupon surface, taking care not to disturb the surface of the coupon.
18. Test coupon is now ready for use.
19. Decontaminate the ADA and associated components with the same procedures utilized during the initial
sterilization.
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APPENDIX A - Aerosol Trap
Purpose: This device allows test puffs of the MDI to be deployed without contamination of the surrounding
area. Spores are pulled into the trap, contained, and inactivated.
This device consists of a suction source, a trap (containing pAB), and an inlet funnel. Aerosolized spores
are pulled into the funnel, and forced into the trap. The spores are collected and inactivated as the aerosol
flows through the pAB solution. The effluent air traveling toward the suction device is spore-free
downstream of the trap. See Figure 4.
The aerosol trap should be assembled inside a biological safety cabinet (BSC) or chemical fume hood.
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Figure 4. Aerosol trap
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MOP-3128
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MOP-3128
TITLE: PROCEDURE FOR PREPARING pH-ADJUSTED BLEACH SOLUTION WITH TRI-
SODIUM PHOSPHATE SUBSTITUTE
SCOPE: This MOP describes a procedure for reproducibly preparing the pH-adjusted bleach
solution with trisodium phosphate (TSP) substitute.
PURPOSE: The purpose of this MOP is to ensure the solution meets QA specifications for each test.
Equipment/Reagents:
. Draeger or personal chlorine (CI2) monitor [Oakton Acorn Series pH 5 meter or equivalent]
. Funnel
. Triple-rinsed container suitable for transporting hazardous solutions
Oakton pH 7 (pH = 7.00 ± 0.01 @ 25°C) buffer or equivalent
DAP® Trisodium Phosphate (TSP) Substitute (Lowe's p/n 224908)
. Clorox Commercial Solutions Germicidal Bleach (Lowe's p/n 33692), less than 1 year old
. 5% v/v Acetic Acid (Ricca Chemical, p/n 7732-18-5 or equivalent)
1.0 PROCEDURE
1.1 Calibrate pH Meter
1. Turn meter on (Figure 1). Meter will automatically enter pH mode.
2. Rinse electrode thoroughly with Dl water. DO NOT wipe the electrode.
3. Dip both the electrode and temperature sensor into pH 7.00 buffer solution. The glass bulb must be
completely immersed into the sample. Stir gently, and wait for the reading to stabilize (about 40
seconds).
4. Press CAL key to enter the calibration mode. The display will momentarily flash "CA" to indicate
Calibration. The display will show the current noncalibrated reading, blinking while in calibration
mode.
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Figure 1. Oakton pH meter
5. Allow the reading to stabilize. The meter will automatically recognize 7.00, 4.01, or 10.00 buffers.
6. Press Enter key once to confirm calibration. The LCD displays "CO" to indicate the calibration point
has been confirmed. The meter exits calibration mode and returns to measurement mode.
7. Record the pH buffer measurement and temperature (Press MODE key to select parameter) in the
appropriate lab notebook.
1.2 Bleach + TSP Preparation
1 . Prepare TSP solution by dissolving % cup per 1 L of deionized water (Dl H2O).
2. The pH-adjusted bleach + TSP solution should consist of 9.8% germicidal bleach, 23.8% acetic acid,
26.4% TSP solution, and 40% Dl H2O. For example, to prepare 1 gallon (3.785 L) of solution, combine
370 ml of germicidal bleach, 1L of TSP solution, 900 ml of acetic acid, and 1515 ml of Dl H2O in that
order. Record the total volume as V^rt in the lab notebook.
3. Measure the pH of the solution (target pH = 6.8). If pH is above 7.0, add acetic acid. If below 6.5, add
germicidal bleach. Record the volume required for adjustment as Vadd. Calculate Vtotai as V^rt + Vadd in
the lab notebook.
4. Measure the free available chlorine (FAC) per MOP 3148. The target FAC is 6350 mg/L. The
acceptable range is 6000 mg/L< FAC < 6700 mg/L.
a) If FAC exceeds the acceptable range, dilute the total volume with TSP solution by the percent
difference between the target FAC and the actual FAC.
Dilution volume = [(actual - target) •*• target] x (Vtotai)
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b) If the FAC is less than the acceptable range, add bleach and trisodium phosphate (TSP) solutions
according to the following equations:
Additional volume of bleach = (target - actual)/ target x Vtotai x 0.098
Additional volume of TSP solution = (target - actual)/ target x Vtotai x 0.264
5. Recalculate Vtotai according to the all additions and repeat steps 3 and 4 until both parameters are met.
Record the final FAC, pH, temperature, and time in the lab notebook.
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MOP-6562
TITLE: PREPARING PRE-MEASURED TUBES WITH ALIQUOTED AMOUNTS OF PHOSPHATE
BUFFERED SALINE WITH TWEEN® 20 (PBST)
SCOPE: This MOP provides the procedure for preparing PBST.
PURPOSE: This procedure will ensure that that the PBST is prepared correctly and that all measured
tubes are filled aseptically.
1.0 PREPARING STERILE PHOSPHATE BUFFERED SALINE WITH TWEEN® 20 (PBST)
Phosphate Buffered Saline with Tween® 20 (PBST) is prepared 1 L at a time in a 1 L flask.
1 . Add 1 packet of SIGMA Phosphate Buffered Saline with Tween® 20 (P-3563) to 1 L of deionized
(Dl) water.
2. Shake vigorously to mix until dissolved.
3. Label bottle as "non-sterile PBST" and include date and initials of person who made PBST.
4. Filter sterilize into two 500 mL reagent bottles using 1 50 mL bottle top filter (w/ 33 mm neck and .22
urn cellulose acetate filter) for sterilization. Complete this by pouring the liquid into the non-sterile
PBST into the top portion of the filtration unit 1 50 mL at a time, while using the vacuum to suck the
liquid through the filter. Continue to do this until 500 mL have been sterilized into a 500 mL bottle.
Change bottle top filter units between each and every 500 mL bottle.
5. Change label to reflect that the PBST is now sterile. Include initials and date of sterilization. The
label should now include information on when the PBST was initially made and when it was
sterilized and by whom.
6. Each batch of PBST should be used within 90 days.
2.0 PREPARING 20 ML/5 ML PBST TUBES FOR USE DURING EXPERIMENTATION
Twenty (20) mL or five (5) mL of the prepared PBST will be added to each sterile 50-mL conical tube as
detailed below. Each flat of conical tubes contains 25 tubes, so one 500 mL sterile bottle of PBST should fill
approximately one flat when 20 mL tubes are needed and four flats when 5 mL tubes are needed.
1 . Prepare the hood by wiping down with ethanol, followed by bleach, followed by Dl water and a clean
Kimwipe orTechwipe. Then stock the hood with the following items if they are not already there:
The flats of sterile conical tubes you need to fill with PBST.
Sufficient bottles of sterile PBST to fill these tubes.
Ample 25 mL serological pipettes (at least 3 per flat) for 20 mL transfers and 1 0 mL
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serological pipettes for the 5 ml transfers.
Serological pipetter (automatic, hand-held pipette).
Burner and striker.
2. Light the burner and adjust the flame for a width adequate to flame the lips of the PBST bottles.
3. Take one flat of sterile conical tubes and loosen each cap on the outside edges (about 1/4 turn).
4. Open a serological pipette and insert into the serological pipetter, taking care to not touch the tip to
any surface.
5. Hold the pipetter with the first three fingers of your right (or dominant) hand. With your left hand (or
non-dominant hand), pick up a bottle of the PBST and use the bottom of your right hand to unscrew
the lid. Place the lid upside down on the benchtop and quickly flame the lip of the bottle. Turn the
bottle and repeat, taking care to thoroughly flame the lip without getting the glass so hot that it
shatters.
6. Inset the tip of the pipette into the bottle and fill to the 20 ml line. Flame the bottle lip and place the
bottle on the benchtop.
Note: If the tip of the pipette touches the outside of the bottle or any other surface in the hood,
consider it contaminated. Discard the pipette and reload a new one.
7. Quickly pick up one of the tubes that you have loosened the cap on, and use the bottom of your
right hand to remove the cap. Completely discharge the entire pipette into the tube, taking care not
to touch anything with the tip of the pipette. Recap the tube and place back into the flat (the lid does
not have to be tight - you will tighten the lids after you have completed filling the 10 outside tubes).
Note: If the tip touches the outside or rim of the tube (or any other surface in the hood), consider
the tube and pipette contaminated. Discard both the tube and the pipette.
8. Pick up the PBST bottle and flame the lip. Repeat Steps 6 and 7 until all 10 of the tubes on the
outside of the flat have been filled. Flame the lip of the PBST bottle and replace the cap. Slide the
used pipette back into the plastic sleeve and put to the side of the hood for disposal. Then tighten
the lid of each tube you just filled. But rather than placing it back into its original spot in the flat,
switch it for the empty tube from the next row. When this has been completed, go around the
outside of the flat again and loosen the lids of these 10 tubes. Repeat steps 4 through 7 to fill and
cap these tubes.
9. This same procedure is used to fill the middle row of tubes from the flat, and if more than one flat of
tubes is being filled, can be done at the same time as the outside rows of a second flat.
10. When all tubes have been filled, label each flat as follows, and place on the shelf in room E390B:
"PBST Tubes (20 ml or 5 ml)"
Date prepared
Your initials
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11. These tubes should be made at least 14 days before they need to be used so that they can be
verified as sterile. Any tubes that are cloudy or that have any floating matter/turbidity should be
discarded. The tubes are stable for and should be used within 90 days.
3.0 CLEANUP FOR 20 ML/5 ML PBST TUBES
1. Dispose of the used pipettes in the nonregulated waste.
2. Plug in the serological pipetter so that it can recharge.
3. Replace any unused PBST in the liquid containment on the shelf. Make sure that the bottle is
labeled as having been opened (date opened and initials of whoever used it).
4. Turn off the burner.
5. Wipe down the hood benchtop with ethanol, followed by bleach, followed by Dl water and a clean
Kimwipe orTechWipe.
4.0 PREPARING 900 |jL PBST TUBES FOR USE DURING EXPERIMENTATION
1. Prepare the hood by wiping down with ethanol, followed by bleach, followed by Dl water and a clean
Kimwipe or Techwipe. Then stock the hood with the following items if they are not already there:
A sterile beaker of microcentrifuge tubes.
Sufficient tubes of sterile PBST to fill these tubes (PBST may be aseptically transferred to 50
ml conical tubes for an easier aseptic transfer to the microcentrifuge tubes- it is easier than working
from a 500 ml reagent bottle. Make certain that these 50 ml conical tubes are labeled to when
the PBST was made, sterilized, etc.).
1000 uL micropipette.
1000 uL sterile pipette tips.
Microcentrifuge tube racks.
Labeled beaker or waste container used to hold non-regulated waste, such as tips, under
the hood.
2. Carefully remove the microcentrifuge tubes one at a time from the beaker and close the top on each
one before placing it in the tube rack. Place the tubes in the rack skipping every other row. Fill up
two racks doing this.
3. Add 900 uL of PBST to the microcentrifuge tubes by aseptically transferring the PBST from the
sterile 50 mL conical tube containing the PBST. Do this by using the 1000 uL micropitte and tips.
Change tips whenever two rows of tubes are completed or whenever a contamination event (such
as touching the outside of the 50 mLtube or the microcentrifuge tube) occurs. Put the dirty tips in
the beaker or container used to contain waste (tips, tubes) in the hood. If any 900 uL tubes are
contaminated during the transfer, dispose of them in the waste container used to hold tips under the
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hood. If a new box of tips has to be opened, make certain the date it was opened and initials of the
person who opened it are clearly labeled on the box.
4. After both racks are full, carefully move all the tubes from one rack to fill in the empty rows on the
other rack. In this manner, one rack should be completely filled with tubes at this point.
5. Label the rack of tubes as "Sterile 900 uL PBST Tubes", along with the name of the person who
completed the transfer, along with the date. Also, include the date that the original stock of PBST
was made and the date it was sterilized, along with the initials of the person who completed those
steps.
5.0 CLEANUP FOR 900 |jL PBST TUBES
1. Dispose of the waste that was put in the labeled beaker or waste container (micropipette tips and
tubes) in the nonregulated waste. Then, place this beaker in the "To be decontaminated via
sterilization- contaminated glassware" bin or if it is a disposable container, then it can be put in the
non-regulated waste container.
2. Put the unused sterile tips and the micropipetter back in its original location.
3. Replace any unused 50 ml conicals of PBST in the liquid containment on the shelf. Make sure that
the tube is labeled as having been opened (date opened and initials of whomever used it). If the
tube could possibly be contaminated in any way, dispose of it in non-regulated waste.
4. Wipe down the hood benchtop with ethanol, followed by bleach, followed by Dl water and a clean
Kimwipe orTechWipe.
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MOP- 6563
TITLE: SWAB STREAK SAMPLING AND ANALYSIS
SCOPE: This MOP provides the procedure for the process of completing a swab streak plate and
subsequent qualitative analysis for samples being analyzed for Bacillus species.
PURPOSE: This procedure will ensure that the swab streak plate sampling and analysis methods are
standardized and that the collection and plating of samples are free from contamination.
These methods are specific to Bacillus species as the target organism.
1.0 PREPARING THE MATERIALS
There are two types of prepared swabs that can be used in this procedure:
Environmental Transport Swabs - purchased swabs that are individually packaged and pre-sterilized.
In-house Sterilized Swabs -swabs placed into autoclave pouches and sterilized using a 1-hour gravity
cycle.
This procedure requires the following materials and equipment:
Tryptic soy agar (TSA) media plates
35 °C incubator
Nitrile (non-sterile) gloves
Sharpie for writing on plates
2.0 COLLECTING AND PLATING SAMPLES
The procedure for collecting and plating samples is dependent on the type of swab being used. Appropriate
PPE should be worn at all times and include a lab coat, nitrile gloves and safety glasses.
2.1 Environmental Transport Swabs
2.1.1 Collection of Environmental Transport Swab Sample
1. Break the seal on the individually packaged and sterile swab. Remove the cap, collect the specimen
with the swab applicator by touching the swab tip to the area in question, and then replace the cap
on the swab.
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2. Label the tube with the sample ID, the date, time, and initials of the person performing the
procedure.
3. Place the swab into a secondary container, such as a sterile bag, and label the bag with the same
information placed on the tube label.
4. Transport the sample(s) to the Microbiology Laboratory for processing.
2.1.2 Plating of Environmental Transport Swab Sample
1. When the sample is received in the Microbiology Laboratory, label three ISA plates with a Sharpie
with the information from the swab packaging. Verify that the sample ID and date match.
2. Place label plates and swab samples under the biological safety cabinet. Remove the sample swab
from the secondary container and the tube. Press onto the first plate in an S-stroke motion, turning
the swab as it is plated to ensure that all of the surface area of the swab touches the plate. Press
firmly, but not so hard that the surface of the media is broken.
3. Perform Step #2 on the remaining two plates.
4. Replace the swab into its tube and discard in the non-regulated waste container.
5. Repeat steps #1 through #4 for each sample.
6. Label three ISA plates as Swab Blank A, Swab Blank B, and Swab Blank C. These plates will
serve as negative controls for the swabs.
7. Open a new/unused Environmental Transport Swab and use it to plate the three blank plates as
detailed in Step #2.
8. Stack the triplicate plates media side up and place in a 35 °C ± 2 °C incubator for at least 18 hours.
Note the time the plates were placed in the incubator.
2.2 In-house Sterilized Swabs
When In-house Sterilized Swabs are being used to collect samples, they need to be plated immediately
(unlike the Environmental Transport Swabs that are transported to the Microbiology Laboratory for plating).
Therefore, prior to travelling to the sample site, collect the following materials and supplies which will be
needed:
• A minimum of three TSA media plates (in a media bag) per sample to be collected plus three
additional plates to be used as negative controls for swab blanks.
• One In-house Sterilized Swab (in their autoclave pouches) per sample to be collected, one swab for
the control plates, plus a few extras.
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Sharpie for labeling plates.
Use the following procedure to collect and plate samples.
1. Once at the sample collection site, take the ISA plates out of the media bag and label three plates
for each sample with what is being swabbed (sample ID), date, time, and initials of the person
performing the procedure.
2. As carefully and as aseptically as possible, remove the swab from the autoclave pouch by the stick
end. Be sure not to touch the swab end to anything but the sample. If the swab's sterility is
compromised, dispose of the swab and use one of the extras.
3. Collect the specimen with the swab applicator as detailed in the specific test protocol.
4. Press onto the first plate in an S-stroke motion, turning the swab as it is plated to ensure that all of
the surface area of the swab touches the plate. Press firmly, but not so hard that the surface of the
media is broken. Because these samples are being plated in the open air and not in a biological
safety cabinet, be certain to limit the time that the lid is removed from the ISA plate.
5. Perform Step #2 on the remaining two plates.
6. Replace the swab into the autoclave pouch it came in and discard in the non-regulated waste
container.
7. Repeat steps #1 through #6 for each sample.
8. Label three ISA plates as Swab Blank A, Swab Blank B, and Swab Blank C. These plates will
serve as negative controls for both the swabs and the ISA.
9. Open another in-house sterilized swab from the autoclave pouch and use it to plate the three
"Blank" plates as detailed in Step #2.
10. Put the ISA plates back into the media bag and transport to the Microbiology Laboratory.
11. When received by the laboratory, the triplicate plates will be stacked media side up and placed in a
35 °C ± 2 °C incubator for at least 18 hours. Note the time the plates were placed in the incubator.
3.0 ANALYZING THE SAMPLES
The Swab Results Template, which follows this section, is used to record the results of the sampling. Some
quantities of samples may require more than one form. Make certain that the data are filled in completely on
each page. The analyst will use the information on the TSA plates to fill in the following blanks at the top of
the form:
• Swab samples taken on: (date)
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• Swabbed by: (person)
• Plating completed on: (date)
• Plated by: (person)
The following procedure is used to analyze the samples and complete the remainder of the Swab Results
Template form.
1. Fill in the final two sections at the top of the form: Plate results read on and Results read by.
2. Take the first set of triplicate plates and note the sample IDs on the first three lines in the Sample
column.
3. For each plate, check whether there was growth (G) or no growth (NG). No growth is indicative that
the sample is sterile. Growth is indicative of an organism(s) being present, and should be described
on the form. Be as detailed as possible, noting colony morphology (size, shape, color and any other
distinctive things that can be seen concerning the growth).
4. The Swab Results Template form serves as the sample report and should be provided to the Project
Manager.
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Swab Results Template
Swab samples taken on: Swabbed by:
Plating completed on: Plated by:
Plate results read on: Results read by:
Sample Name
Controls
Swab blank A
Swab blank B
Swab blank C
Result
cnision
cnision
cnision
cnision
cnision
cnision
cnision
cnision
cnision
cnision
GniSIGn
cnision
GONGn
GONGn
GONGn
GONGn
cnision
Result
GONGn
GONGn
GONGn
If growth, describe
If growth, describe
Key G = Growth. NG = No Growth.
All plates are plated in triplicate resulting in sample identification of "A", "B", and "C"
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MOP 6565
Title: FILTRATION AND PLATING OF BACTERIA FROM LIQUID EXTRACTS
Scope: This MOP outlines the procedure for filtration and subsequent cultivation of bacterial spores
from a liquid extract.
Purpose: This method is deployed when results from spread-plate methods yield less than 30 colony-
forming units (CFU) per plate. This method allows a lower limit of detection for bacterial
recovery/survivorship assays.
Materials
• Petri dishes with appropriate agar
• 0.2 urn pore-size disposable analytical filter units (2 per sample)
• P1000 pipette and sterile tips
• Sterile forceps
• Pipettman and sterile serological pipettes
Procedure
1. For each liquid sample to be analyzed, gather two disposable analytical filter units and two Petri dishes
containing the desired sterilized/QC'd media.
2. Label plates
3. Vortex liquid extract vigorously for 2 minutes, using 10 second bursts.
4. Using a P1000 sterile tip and aseptic techniques, immediately following vortexing, pipette 1 mLofthe
extract into one of the filter units.
5. Apply vacuum to the filter unit to pull the liquid through the filter and collect the spores on the surface of
the filter.
6. Using a sterile serological pipette, rinse the filter unit by pipetting 10 mL of sterile Dl H2O along the inner
sides of the unit while it is under vacuum.
7. Aseptically remove the filter from the filter apparatus using sterile forceps and lay the filter onto the agar
surface within the Petri dish (spore side up).
8. Vortex the liquid extract vigorously for 10 seconds.
9. Using the appropriate volume serological pipette, collect the remainder of the liquid and dispense in the
second filter unit.
10. Note and record the volume.
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11. Apply vacuum to the filter unit, to pull the liquid through the filter and collect the spores on the surface of
the filter.
12. Using a sterile serological pipette, rinse the filter unit by pipetting 10 ml of sterile Dl H2O along the inner
sides of the unit while it is under vacuum.
13. Aseptically remove the filter from the filter apparatus using sterile forceps, and lay the filter onto the agar
surface within the Petri dish (spore side up).
14. Incubate the plates at the optimal growth temperature for the organism used for 16 -28 hours.
15. Enumerate and record the number of CPU on each plate
Data Calculations
Utilize the following equation to determine the total abundance of recovered spores:
V,
= CFUx-
Extract
' filtered
Where N is the total number of spores recovered in the extract, CPU is the abundance of colonies on the
agar plate, VExtract is the total volume of the extract (before any aliquots were removed), \/F/?teredis the volume
of the extract filtered.
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MOP 6567
Title: RECOVERY OF BACILLUS SPORES FROM WIPE SAMPLES
Scope: This MOP outlines the procedure for recovering Bacillus spores from wipe samples
Purpose: To aseptically extract and quantify Bacillus spores from wipe samples in order to determine
viability and obtain quantifiable data
1.0 MATERIALS
• PPE (gloves, lab coat, safety goggles)
• Biological Safety Cabinet (Class II)
• pH-Adjusted bleach
• Deionized water
• 70% solution of denatured ethanol
• Kimwipes
• Dispatch bleach wipes
• Non-regulated waste container
• 50 ml sterile conical tubes containing 20 ml of sterile phosphate buffered saline with Tween® 20
solution (PBST) (MOP 6562)
• Vortex mixer
• Cart
• Wire or foam rack for 50 ml conical tubes
• Tryptic soy agar plates
• 900 uL tubes of sterile PBST
• Pipettor and pipette tips for dilutions
• Incubator set to appropriate growth temperature for target organism (35 °C or 55 °C)
• Light box for counting colonies
• Lab notebook
• QAPP for project that is utilizing the wipe samples
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2.0 Procedure
1. Begin by donning PPE (gloves, lab coat, and protective eyewear).
2. Obtain wipe samples that may contain Bacillus spores. Wipe samples should be received as one
wipe/sponge in a sterile 50 ml conical tube delivered in secondary containment. Make certain that all of
the samples are labeled. Review any chain of custody forms that may accompany the samples to
ensure that all of the labels are consistent and that there is no notable variation in the samples. If
variation has occurred, make a note of it in the notebook.
3. Clean the workspace (biological safety cabinet) by wiping surfaces with pH-adjusted bleach, next with
deionized water, and lastly with a 70-90 % solution of denatured ethanol. Wipe with a kimwipe to
remove any excess liquid. Make sure the workspace is clean and free of debris. Gather all necessary
items to perform the task, place these items on a clean cart beside the biological safety cabinet, within
arm's reach so that, once the procedure has begun, the task may be performed without interruptions.
4. Discard gloves and replace with fresh pair.
5. One at a time, under the biological safety cabinet, remove the sample tube containing the wipe sample
from the secondary containment bag in which it arrived. Using the Dispatch bleach wipes, wipe each
sample tube with one wipe, and then wipe it with a clean kimwipe. Discard the used bleach wipe and the
used kimwipe in the secondary containment bag and place them in the non-regulated waste container.
Remove gloves and don a fresh pair of gloves. Repeat this procedure for every sample. After each
sample has been cleaned, place the tubes containing the wipe samples in an appropriate sized wire or
foam rack to hold the tubes in an upright, vertical position.
6. Leaving the tubes in the rack underneath the biological safety cabinet, aseptically add 20 ml of PBST
solution (this should be in a pre-measured, sterile conical tube, per MOP 6562) to each sample tube
containing a wipe, one a time. Remove the rack containing wipe samples from hood when all samples
have had the PBST added. Place the rack with the samples on the cart.
7. Using the procedure to clean the biological safety cabinet, as found in Step 3, clean the biological safety
cabinet again. Afterwards don a fresh pair of gloves.
8. Using a vortex mixer, agitate the wipe samples, four at a time, in a biological safety cabinet, for ten
second bursts for two minutes total. Make certain to clean the biological safety cabinet after each set of
four samples and change gloves between each set of samples.
Note: The reason that four samples are done at one time is to limit the time between agitation
and plating. The samples need to be processed immediately after agitation, and agitation of
more than four samples at a time leaves too much time between agitation and spread plating.
9. Using tryptic soy agar media plates that are appropriately labeled with the sample number, dilution set
and date, complete dilution plating for the wipe samples immediately after the two minute agitation step
is completed. The samples should also be agitated again for ten seconds directly prior to removing an
aliquot from the sample tube. Each dilution tube should also be agitated for ten seconds prior to removal
of aliquots. Dilutions should be completed using the techniques and methodology as described in MOP
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6535a, and the 900 |jl_ tubes should be made with sterile PBST to stay consistent with
materials/solutions. Plating in this manner should be repeated for all samples, with any changes in
protocol noted in the lab notebook.
10. Once the dilution plating has been completed, the plates are to be placed in an incubator. For non-
thermophilic Bacillus species, the plates should be placed at 35 °C ±2 ° C for 12-24 hours. For
thermophilc Bacillus species, such as Geobacillus stearothermophilis, the plates should be incubated at
55 °C ±2 °C for 12-24 hours. The target Bacillus organism that will be used for the wipe samples will be
specific to the project and noted in the QAPP.
11. After the plates have incubated fora sufficient amount of time (12-24 hours) and the growth from any
Bacillus colonies is quantifiable, the colonies should be counted manually using the light box and the
data should be properly recorded as dictated per project by the QAPP. All results will be checked for
quality assurance and all data will be reported to the proper personnel as listed in the QAPP.
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MOP NO. 6570
TITLE: Use of STERIS Amsco Century SV 120 Scientific Prevacuum Sterilizer
SCOPE: Basic instructions for use of the large STERIS autoclave
PURPOSE: To outline proper procedural use of the autoclave, using preprogrammed cycles, to
effectively sterilize items, while complying with quality control standards.
Materials:
• Amsco Century SV 120 Scientific Prevacuum Sterilizer
• Items to be sterilized (liquids, solids, waste, etc)
• Pouches to contain materials during sterilization and maintain sterility until use
• Aluminum foil
• Autoclave indicator tape
• Sterilization verification ampoules (such as Raven ProSpore Ampoules)
• Thermally resistant gloves
• Deionized (Dl) water
Procedure:
Start up:
1. Turn on the autoclave. The power switch is located behind the door in the top right corner. The
digital touch screen on the front of the unit will power up and indicate that a memory test is in
progress.
2. After the memory test is complete, the device will request that it be flushed. This should be
conducted daily to minimize scaling inside the boiler. The flush valve is located behind the door on
the bottom, left of the device (yellow handle). Move the valve to the open position and then press
the "Start Timer" button on the touch screen. The flush will run for 5 minutes and will alert at
completion with a single chime.
3. Once the flush is complete, close the flush valve and press the "Continue" button on the touch
screen. The screen should then return to its default menu which has 2 choices "Cycle Menu" and
"Options"
Basic Operation:
1. Prepare any items that need to be sterilized. The items must be carefully wrapped or sealed in
sterilization pouches in order to maintain sterility when removed from the autoclave. Examples of
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this include: wrapping any orifices with aluminum foil, placing whole items in autoclave pouches,
loosely applying a cap on a bottle (to allow for the pressure changes inside).
2. Once prepared, each item should be outfitted with a sterility indicator such as a small piece of
autoclave indicator tape; or by utilizing an autoclave pouch with a built-in sterility indicator strip.
These indicators provide a visual verification that the sterilizing temperature (121 °C) was reached.
3. To add items to the autoclave, open the autoclave door by pressing down on the foot pedal on the
bottom right corner on the front of the device.
4. Place items that need to be sterilized into the autoclave, adding or moving racks to accommodate
the load. If liquids are being autoclaved, they must have secondary containment (usually a large
plastic autoclave-safe tray) to contain any fluids in the event of a leak, spill or boil-over. Add an
indicator ampoule to the first autoclave cycle of the day, regardless of the type of cycle.
5. Once the autoclave is loaded, press the foot pedal to close the autoclave door.
6. Once the door is sealed, a menu of the cycles can be seen by pressing the button on the touch
screen labeled "Cycle Menu". Then choose the appropriate cycle by touching the corresponding
button. If the cycle chosen is the one desired for the sterilization process, press the "Start Cycle"
button. Otherwise, press "Back" to return to the prior menu screen.
7. After the cycle has started, the type of cycle, the number of the cycle, the items placed in the
autoclave during the cycle, the time, whether or not an indicator ampoule was included in the load,
and the initials of the person starting the cycle must be recorded in the autoclave log book, located
in the drawer across form the unit labeled "Autoclave Supplies."
8. Quality control (QC) indicator ampoules, usually Raven ProSpore Ampoules with Geobacillus
stearothermophilus (at a concentration of 10E6), are added to one cycle each day to ensure that the
autoclave is functioning properly. These ampoules are used according to manufacturer's
instructions. These ampoules must be properly labeled with the date when they were autoclaved
and the initials of the individual who completed the cycle. At the beginning of each week, a positive
control ampoule must be processed, where the ampoule is placed directly into the 55 °C water bath
without being autoclaved. The positive control indicator ampoule should change from purple to
yellow in color, indicating growth. All test ampoules should be placed in a water bath following the
end of the cycle in which they are run. These ampoules should not change color (from purple to
yellow but instead should remain a purple to purple-brown color). Ampoules should be checked at
both 24 and 48 hour intervals for growth and then finally recorded and disposed of after 48 hours. All
QC information concerning ampoules should be recorded in the autoclave notebook.
9. Upon completion of any cycle, the autoclave will alarm with a repeating beep for approximately one
minute. Any time after this alarm starts, it is safe to open the main door (take caution because the
steam escaping the chamber will be very hot when the door is opened). The contents from the
autoclave will be very hot; use protection to remove items from the autoclave (thermally resistant
gloves).
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10. Place the contents of the autoclave in an appropriate place to cool, and close the autoclave door
using the foot pedal.
Cycles:
Gravity Cycles:
Gravity cycles are used to sterilize glassware and other utensils, which are not submerged in nor contain
any volume of liquid. These cycles are typically used for "dry" materials.
Currently there are two different gravity cycles programmed for daily operations: a 1-hour cycle and a 30-
minute cycle. The time that the chamber is held at the sterilization temperature (121 °C) is the only
difference between these two cycles. The different sterilization times allow for the compensation of the
various sizes of materials and more resilient organisms. The 30-minute cycle is primarily used for a small
quantity of material. The 1 hour cycle is used for large loads or items containing a large amount of
contamination. The 1 hour cycle is recommended for inactivation of Gram-positive spore-forming bacteria.
Liquid Cycles:
Liquid cycles are used to sterilize a variety of liquids and solutions. The solutions are typically mixed prior to
sterilization. It is important to have secondary containment to contain any fluids in the event of a leak, spill or
boil-over. The 30-minute liquid cycle is used to sterilize small volumes of liquid (usually less than 2 L total).
When attempting to sterilize any volume larger than 2 L, the 1-hour liquid cycle should be used to ensure
complete sterilization. The 1-hour liquid cycle is the preferential cycle used as the destruction cycle for
waste. In the event of materials (liquid or otherwise) being contaminated/exposed to microorganisms, the 1-
hour liquid cycle will be used as the initial means of decontamination. When completing a decontamination
cycle, if there is no liquid inside a container, then deionized water must be added to the container or the item
must be submerge prior to the start of the cycle. Only items that are being decontaminated can go in
destruction cycles. Decontamination cycles cannot be mixed with sterilization cycles.
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MOP 6571
Title: RECOVERY OF BACILLUS SPORES FROM VIA-CELL® AEROSOL SAMPLING
CASSETTES
Scope: This MOP outlines the procedure for recovering Bacillus spores from Via-Cell® aerosol
sampling cassettes
Purpose: To aseptically extract and quantify Bacillus spores from Via-Cell® samples in order to
determine viability and obtain quantifiable data
1.0 MATERIALS
• Via-Cell® Bioaerosol Sampling Cassettes (Zefon International, Ocala, FL, Part# VIA010)
• PPE (gloves, lab coat, safety goggles)
• Biological Safety Cabinet (Class II)
• pH-Amended bleach
• Deionized water
• 70% solution of denatured ethanol
• Kimwipes
• Dispatch bleach wipes
• Non-regulated waste container
• 50 mL sterile conical tubes containing appropriate volume of buffer
• Vortex mixer
• Cart
• Wire or foam rack for 50 mL conical tubes
• Sterile blade
• Sterile, disposable forceps
• Tryptic soy agar plates
• 900 uL tubes of sterile PBST
• Pipettor and pipette tips for dilutions
• Incubator set to appropriate growth temperature for target organism (35 °C or 55 °C)
• Light box for counting colonies
• Lab notebook
• QAPP for project that is utilizing the wipe samples
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2.0 PROCEDURE
1. Begin by donning fresh PPE (gloves, lab coat, and protective eyewear).
2. Obtain Via-Cell® samples that may contain Bacillus spores. Via-Cell® samples should be received as
one Via-Cell® cassette delivered in secondary containment. Make certain that all of the samples are
labeled. Review any chain of custody forms that may accompany the samples to ensure that all of the
labels are consistent and that there is no notable variation in the samples. If variation has occurred,
make a note of it in the notebook.
3. Clean the workspace (biological safety cabinet) by wiping surfaces with pH-amended bleach, next with
deionized water, and lastly with a 70-90 % solution of denatured ethanol. Wipe with a kimwipe to
remove any excess liquid. Make sure the workspace is clean and free of debris. Gather all necessary
items to perform the task, place these items on a clean cart beside the biological safety cabinet, within
arm's reach so that, once the procedure has begun, the task may be performed without interruptions.
4. Discard gloves and replace with fresh pair.
5. One at a time, under the biological safety cabinet, remove the sample cassette. Using a sterile blade,
cut through the tape around the outside of the cassette. Twist apart the cassette and discard of the top
portion not containing the sample slide (portion of the cassette where the sample is collected). Using
sterile, disposable forceps, remove the slide and place into the appropriate amount of buffer solution.
Repeat this procedure for every sample.
6. Using the procedure to clean the biological safety cabinet, as found in Step 3, clean the biological safety
cabinet again. Afterwards don a fresh pair of gloves.
7. Using a vortex mixer, agitate the Via-Cell® samples, four at a time, in a biological safety cabinet, for ten
second bursts for two minutes total. Make certain to clean the biological safety cabinet after each set of
four samples and change gloves between each set of samples.
Note: The reason that four samples are done at one time is to limit the time between agitation
and plating. The samples need to be processed immediately after agitation, and agitation
of more than four samples at a time results in excessive lag-time between agitation and
plating.
8. Using tryptic soy agar (or other appropriate growth media) media plates that are appropriately labeled
with the sample number, dilution set and date; conduct dilution plating for the Via-Cell® samples
immediately after the two minute agitation step is completed. The samples should also be agitated
again for ten seconds directly prior to removing an aliquot from the sample tube. Each dilution tube
should also be agitated for ten seconds prior to removal of aliquots. Dilutions should be completed using
the techniques and methodology as described in MOP 6535a, and the 900 uL tubes should be made
with the appropriate buffer to stay consistent with materials/solutions. Plating in this manner should be
repeated for all samples, with any changes in protocol noted in the lab notebook.
9. Once the dilution plating has been completed, the plates are to be placed in an incubator. For non-
thermophilic Bacillus species, the plates should be placed at 35 °C ± 2 °C for 18-24 hours. For
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thermophilc Bacillus species, such as Geobacillus stearothermophilis, the plates should be incubated at
55 °C ± 2 °C for 18-24 hours. The target Bacillus organism that will be used for the wipe samples will be
specific to the project and noted in the QAPP.
10. After the plates have incubated for a sufficient amount of time (18-24 hours) and the growth from any
Bacillus colonies is quantifiable, the colonies should be manually counted using the light box and the
data should be properly recorded as dictated per project by the QAPP. All results will be checked for
quality assurance and all data will be reported to the proper personnel as outlined in the QAPP.
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MOP 6572
Title: RECOVERY OF SPORES FROM VACUUM SOCK SAMPLES
Scope: This MOP outlines the procedure for recovering spores from vacuum sock samples
Purpose: To aseptically extract and quantify spores from vacuum sock samples in order to determine
viability and obtain quantifiable data
1.0 MATERIALS
• PPE (gloves, lab coat, safety goggles)
• Biological Safety Cabinet (Class II)
• pH-Amended bleach
• Deionized water
• 70% solution of denatured ethanol
• Kimwipes
• Dispatch bleach wipes
• Non-regulated waste container
• 3 oz. sterile specimen cup containing 20 ml of sterile phosphate buffered saline with Tween® 20
solution (PBST) (MOP 6562)
• Sterile scissors
• Vortex mixer
• Cart
• Tryptic soy agar plates
• 900 uL tubes of sterile PBST
• Pipettor and pipette tips for dilutions
• Incubator set to appropriate growth temperature for target organism (35 °C or 55 °C)
• Light box for counting colonies
• Lab notebook
• QAPP for project that is utilizing the vacuum sock samples
2.0 PROCEDURE
1. Begin by donning PPE (gloves, lab coat, and protective eyewear).
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2. Obtain vacuum sock samples that may contain Bacillus spores. Vacuum sock samples should be
received as one vacuum sock in a sterile 5.5' x 9 bag secondarily contained in a 10' x 15' bag. Make
certain that all of the samples are labeled. Review any chain of custody forms that may accompany the
samples to ensure that all of the labels are consistent and that there is no notable variation in the
samples. If variation has occurred, make a note of it in the notebook.
Clean the workspace (biological safety cabinet) by wiping surfaces with pH-amended bleach, next with
deionized water, and lastly with a 70-90% solution of denatured ethanol. Wipe with a kimwipe to remove
any excess liquid. Make sure the workspace is clean and free of debris. Gather all necessary items to
perform the task, place these items on a clean cart beside the biological safety cabinet, within arm's
reach so that once the procedure has begun the task may be performed without interruptions.
3. Discard gloves and replace with fresh pair.
4. Label a 3 oz. specimen cup to match the vacuum sock sample ID. The specimen cup contains 20 ml of
sterile PBST.
5. When extracting samples, handle one sample at a time from start to finish. Begin by removing the inner
bag from the outer bag. Discard the outer bag in the non-regulated waste container. Place the inner bag
containing the vacuum sock under the hood. Loosen the cap on the 3 oz. specimen cup and open a
pack of sterile scissors. Open the bag and remove the sock, careful not to touch the white part. Roll the
non-sterile blue portion of the vacuum sock onto the smaller cardboard ring. Dispose of the larger
cardboard ring. Wet the vacuum sock by holding the upper blue portion of the vacuum sock (around the
smaller cardboard ring) and dipping the lower 1-inch of the vacuum sock into the PBST. The vacuum
sock will be allowed to absorb the PBST for a few seconds. After wetting, the vacuum sock will be lifted
up just above the opening of the specimen bottle, and a 1-inch vertical slit will be cut up the center from
the bottom of the sock using sterile scissors (a new pair of scissors should be used for each sample).
The vacuum sock is then cut horizontally from side to side about 1 inch from the bottom, allowing the
two pieces to fall into the specimen bottle. The vacuum sock should be cut only where the sock has
been wetted. Repeat the dip/cutting procedure until the entire collection portion of the sock has been
excised. The upper top blue portion of the vacuum sock will then be discarded. Place used scissors in a
discard pan. After samples are all extracted, scissors will immediately be autoclaved using a one hour
gravity destruction cycle in preparation for use with the next sample batch. Remove gloves and don a
fresh pair of gloves. Repeat the extraction procedure for every sample, while maintaining aseptic
technique.
6. After cutting all vacuum sock samples, all specimen cups (up to sixty samples at a time) should be
loaded into the sample cup holder of the orbital shaker-incubator. The samples are then agitated in the
shaker incubator at 300 rpm for 30 minutes at room temperature. The samples are then removed from
the shaker incubator and brought to the Biological Safety Cabinet for dilution plating.
7. Using the procedure to clean the biological safety cabinet, as found in Step 3, clean the biological safety
cabinet again. Afterwards don a fresh pair of gloves.
8. Using tryptic soy agar media plates that are appropriately labeled with the sample number, dilution set
and date, complete dilution plating for the vacuum sock samples immediately after the thirty minute
agitation step is completed. The samples should also be agitated again for ten seconds directly prior to
removing an aliquot from the specimen cup. Each specimen cup should also be agitated for ten
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MOP-6572
Revision 0
August 2010
Page 3 of 3
seconds prior to removal of aliquots. Dilution-plating should be carried out according to MOP 6535a.
Dilution tubes used in MOP 6535a should contain PBST to stay consistent with materials/solutions.
Repeat procedure for all samples.
9. Once the dilution plating has been completed, the plates should be incubated. For non-thermophilic
Bacillus species, the plates should be placed at 35 °C ± 2 °C for 18-24 hours. Forthermophilc Bacillus
species, such as Geobacillus stearothermophilis, the plates should be incubated at 55 °C
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Evaluation of Expedient Decontamination Options
with Activated Peroxide-based Liquid Sporicides
Appendix C
Appendix C: Stock Chemicals for Preparation of AHP Solution
1) Hydrogen peroxide (50%): Fisher Scientific, CAS: 7722-84-1
2) Triacetin, ACROS Organics, , CAS 102-76-1
3) Ethanol (99.5%), ACROS Organics, CAS 64-17-5
4) Potassium Carbonate, Sigma Aldrich, CAS 584-08-7
5) MAQUAT® MC1412-80%E, Mason Chemical Company, no CAS available
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Evaluation of Expedient Decontamination Options
with Activated Peroxide-based Liquid Sporicides
Appendix D
Appendix D: Coupon, Test Chamber and Equipment Cleaning and Sterilization
Procedures
The pH-adjusted bleach solution to be used for cleaning surfaces in equipment in both the decontamination
and microbiology laboratories is prepared as a 1:10 dilution of bleach in Dl water, pH-adjusted to ~6.8 using
glacial acetic acid.
The following steps are followed for cleaning the decontamination chamber between each material type and
before/after each test:
1. Using the back sprayer, the interior surfaces are kept wet with solution for 10 minutes.
2. With the drain open, the surfaces will then be rinsed with Dl water. The runoff is collected in a
carboy and ultimately discarded.
3. After ensuring all runoff is removed from the chamber, the valve is closed in preparation for the next
test.
4. A mop assembly with a disposable pad is used to wipe down the interior of the chamber with
isopropyl alcohol or ethanol.
5. The pad is then removed and placed in a bucket of pH-adjusted bleach solution for decontamination
prior to disposal.
The following steps are followed for cleaning the buckets after use in a test:
1. Fill the buckets with pH-adjusted bleach and leave them covered for at least 60 minutes.
2. Rinse all buckets five times with Dl water.
3. Air dry prior to re-use.
The following steps are followed for cleaning the work surfaces before and after use:
1. Wet all surfaces with pH-adjusted bleach solution or using Dispatch® bleach wipes.
2. Rinse with Dl water.
3. Wet and wipe surfaces with isopropyl alcohol or ethanol.
4. Air dry prior to re-use.
5. Alternatively, cover paper can be used and replaced before/after each use.
The sampling templates are autoclaved before/after each use.
The following steps are followed for cleaning the coupon cabinets before and after use:
1. Wet and wipe all surfaces with pH-adjusted bleach solution or using Dispatch® bleach wipes.
2. Rinse with Dl water.
3. Wet and wipe surfaces with isopropyl alcohol or ethanol.
4. Air dry prior to re-use.
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Evaluation of Expedient Decontamination Options
with Activated Peroxide-based Liquid Sporicides
Appendix D
The gaskets used in MOP 6561 during the contamination procedure will be cleaned via fumigation with the
STERIS VHP® sterilization cycle. This cycle entails the use of a STERIS VHP® ARD hydrogen peroxide
(H2O2) generator at 250 parts per million by volume (ppmv) for 4 hours by maintaining this constant
concentration in a decontamination chamber.
The carboys will be autoclaved according to MOP 6570.
Cement/concrete, stainless steel, and glass coupons will be autoclaved according to MOP 6570. Other
types of coupons will be cleaned via fumigation with the STERIS VHP® sterilization cycle. This cycle entails
the use of a STERIS VHP® ARD hydrogen peroxide (H2O2) generator at 250 ppmv for 4 hours by
maintaining this constant concentration in a decontamination chamber.
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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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