assessment of liquid and physical decontamination methods

EPA 600/R-12/025 | April 2012 | www.epa.gov/ord

Assessment of Liquid and Physical Decontamination Methods for Environmental Surfaces Contaminated with Bacterial SporesDevelopment and Evaluation of the Decontamination Procedural Steps

Office of Research and DevelopmentNational Homeland Security Research Center

EPA-600-R-12-025

Assessment of Liquid and Physical Decontamination Methods for Environmental Surfaces Contaminated with Bacterial Spores

Development and Evaluation of the Decontamination Procedural Steps

UNITED STATES ENVIRONMENTAL PROTECTION AGENCY CINCINNATI, OHIO

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Disclaimer

The United States Environmental Protection Agency, through its Office of Research and Development’s National Homeland Security Research Center, funded and managed this investigation through EP-C-09-027 WA 0-25 with ARCADIS-US, Inc. This report has been peer and administratively reviewed and has been approved for publication as an Environmental Protection Agency document. It does not necessarily reflect the views of the Environmental Protection Agency. No official endorsement should be inferred. The Environmental Protection Agency does not endorse the purchase or sale of any commercial products or services. This report includes photographs of commercially available products. The photographs are included for purposes of illustration only and are not intended to imply that 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.

Shawn P. Ryan, Ph.D. Division Director 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: 919-541-0699 Fax: 919-541-0496 E-mail: [email protected]

mailto:[email protected]

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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. To support 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) was established to conduct research and deliver 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 NHSRC’s research is to provide information on decontamination methods and technologies that can be used in the response and recovery efforts resulting from a 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 dispersion. 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, NHSRC 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 NHSRC publications (available at www.epa.gov/nhsrc) can be used to determine whether a particular decontamination technology can be effective in a given scenario. NHSRC has made this publication available to the response community to prepare for and recover from disasters involving chemical and/or 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 National Homeland Security Research Center

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Acknowledgments

This effort was initiated following discussions with the U.S. EPA’s Office of Solid Waste and Emergency Response’s Office of Emergency Management (OEM) on high-priority research needs to support response and recovery following incidents involving chemical, biological, or radiological (CBR) agents or materials. Due to their regulatory responsibilities under the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA), the U.S. EPA’s Office of Chemical Substances and Pollution Prevention (OCSPP) was also interested in this effort. The management support from both program offices towards the U.S. EPA’s Office of Research and Development (ORD) regarding the contribution that research and development makes towards the U.S. EPA’s preparedness in the homeland security area is greatly appreciated. Additional funding support by OCSPP to complete this effort is appreciated.

This effort was managed by the principal investigator from ORD’s National Homeland Research Center (NHSRC), utilizing the support of a project team consisting of staff from across the U.S. EPA. The contributions of the entire team, including those of Dr. Curtis Snook (formerly with U.S. EPA/OSWER/OEM/National Decontamination Team) while with the U.S. EPA, are acknowledged.

Project Team:

Shawn P. Ryan, Ph.D. (Principal Investigator) and M. Worth Calfee, Ph.D. Office of Research and Development, U.S. Environmental Protection Agency Research Triangle Park, NC 27711

R. Leroy Mickelsen Office of Solid Waste and Emergency Response, U.S. Environmental Protection Agency Research Triangle Park, NC 27711

Michael Nalipinski and Ted Bazenas U.S. Environmental Protection Agency - New England Boston, MA 02203

Carlton “Jeff” Kempter and Stephen Tomasino, Ph.D. Office of Chemical Safety and Pollution Prevention, U.S. Environmental Protection Agency Washington, DC 20460

This effort was completed under U.S. EPA contract #EP-C-09-027 with ARCADIS-US, Inc. The support and efforts provided by ARCADIS-US, Inc. are gratefully acknowledged. The support provided by Tanya Medley (U.S. EPA/ORD/NHSRC) in acquiring the vast quantities of supplies required for the completion of this project is also acknowledged.

Additionally, the authors would like to thank Joan Bursey, Ph.D., for her technical editing of this report and the peer reviewers for their significant contributions. Specifically, the efforts of Dave Rees (U.S. EPA/Region 10), Joe Wood (U.S. EPA/ORD/NHSRC), and Timothy Dean, Ph.D. (U.S. EPA/ORD/National Risk Management Research Laboratory) are recognized.

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Table of Contents

Disclaimer ............................................................................................................................................... ii

Foreword ................................................................................................................................................. iii

Acknowledgments ................................................................................................................................. iv

Table of Contents ................................................................................................................................... v

List of Tables ......................................................................................................................................... viii

List of Figures ....................................................................................................................................... viii

Acronyms and Abbreviations ................................................................................................................. x

Executive Summary .............................................................................................................................. xiii

1. Introduction....................................................................................................................................... 1

1.1 Objectives .......................................................................................................................... 2

1.2 Experimental Approach ..................................................................................................... 2

1.3 Definitions of Effectiveness ............................................................................................... 6

1.3.1 Surface Decontamination Efficacy ....................................................................... 7

1.3.1.1 Detection Limits ....................................................................................... 9

1.3.2 Overall Decontamination Effectiveness (Ultimate Fate of Spores) ................... 10

2. Materials and Methods .................................................................................................................. 11

2.1 Coupon Materials and Fabrication .................................................................................. 12

2.2 Material Inoculation Procedure ....................................................................................... 15

2.2.1 Bacillus Spore Preparation ................................................................................. 15

2.2.2 Coupon Inoculation Procedure ........................................................................... 16

2.3 Experimental Sequence .................................................................................................. 16

2.4 Decontamination Procedures.......................................................................................... 17

2.5 Test Matrix ....................................................................................................................... 20

2.6 Sampling and Analytical Procedures .............................................................................. 24

2.6.1 Factors Affecting Sampling/Monitoring Procedures .......................................... 25

2.6.2 Preparation for Sampling/Monitoring.................................................................. 25

2.6.3 Surface Sampling ................................................................................................ 26

2.6.3.1 Wipe Sampling ...................................................................................... 26

2.6.3.2 Vacuum Sampling ................................................................................. 26

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2.6.3.3 Swab Sampling ..................................................................................... 26

2.6.4 Rinsate Collection and Sampling ....................................................................... 27

2.6.5 Wet/dry Vacuum Collection Sampling Procedures ............................................ 27

2.6.5.1 Wet/dry Vacuum HEPA Filter Sampling Procedure ............................. 27

2.6.5.2 Wet/dry Vacuum Exhaust Sampling Procedure ................................... 27

2.6.6 Sample Analyses ................................................................................................ 28

2.6.7 Coupon, Material, and Equipment Cleaning and Sterilization ........................... 28

3. Results and Discussion ................................................................................................................. 31

3.1 Preliminary Testing of Sampling Procedures ................................................................. 31

3.1.1 Development of the Aerosolized Spore Deposition Method – Sampling Results for Positive Controls .............................................................. 31

3.1.2 Sampling Method Evaluation .............................................................................. 33

3.1.3 Rinsate Collection and Analysis Procedure Method Development .................. 35

3.2 Assessment of the Efficacy of Decontamination Steps and Procedures ...................... 36

3.2.1 Surface Sampling Results – Test Coupons ....................................................... 37

3.2.1.1 Surface Decontamination Efficacy Results by Decontamination Step or Procedure .................................................... 37

3.2.1.2 Surface Decontamination Efficacy Results by Decontamination Procedure and Material Type .............................................................. 37

3.2.2 Overall Decontamination Efficacy (Ultimate Fate of Viable Spores) ................. 42

3.2.2.1 Wet/Dry Vacuum Samples .................................................................... 42

3.2.2.2 Rinsate Samples ................................................................................... 44

3.2.2.3 Summary of Overall Decontamination Effectiveness (Ultimate Fate Analysis) ........................................................................ 48

3.3 Impact of pH-Adjusted Bleach Spray Parameters and Other Efficacy Test Method Parameters................................................................................................. 49

3.3.1 Evaluation of the pH-Adjusted Bleach Application Procedure .......................... 50

3.3.2 Effect of Recovery Methods, Coupon Contamination Methods, and Sample Loading Density on Decontamination Efficacy Determinations ........... 52

3.3.2.1 18 mm Coupons (stubs) ........................................................................ 53

3.3.2.2 14 Inch Coupons ................................................................................... 55

3.4 Assessment of Operational parameters ......................................................................... 58

3.4.1 Time ..................................................................................................................... 58

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3.4.2 Physical impact on materials .............................................................................. 59

3.4.3 Impact on the remediation crew ......................................................................... 59

4. Quality Assurance and Quality Control ......................................................................................... 60

4.1 Calibration of Sampling/Monitoring Equipment .............................................................. 60

4.2 Data Quality Indicator (DQI) Goals ................................................................................. 60

4.2.1 Free Available Chlorine (FAC) Measurements .................................................. 61

4.2.2 pH Measurements .............................................................................................. 61

4.2.3 Temperature Measurements .............................................................................. 61

4.2.4 Pressure Measurements .................................................................................... 62

4.2.5 Flow Measurements ........................................................................................... 62

4.2.6 CFU Counts ........................................................................................................ 62

4.3 Technical Systems Audit ................................................................................................. 62

4.4 Data Quality Audit............................................................................................................ 62

4.5 QA/QC Reporting ............................................................................................................ 62

4.6 Deviations from the QAPP .............................................................................................. 62

5. References ..................................................................................................................................... 67

Appendix A: Coupon Sterilization

Appendix B: Miscellaneous Operating Procedures (MOPs)

Appendix C: Spore Deposition and Handling Procedures

Appendix D: Decontamination Process

Appendix E: Sampling Procedures

Appendix F: Sampling Analyses

Appendix G: Test Chamber and Equipment Cleaning Procedures

Appendix H: Test Reports

Appendix I: Test 13 Preparation, Testing, and Sampling Procedures

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List of Tables

Table 2-1. Procedural step summary for each material type 19

Table 2-2. Test Matrix 21

Table 2-3. List of coupon material types by orientation 22

Table 2-4. Cleaning methods and frequency for common test materials/equipment 30

Table 3-1. Percent recoveries as a function of material type and sampling method 35

Table 3-2. Surface decontamination effectiveness 41

Table 3-3. Wet/dry vacuum sampling results 43

Table 3-4. Comparison of rinsate contamination of blanks to test coupons 44

Table 3-5. Wipe Sampling Results for Stainless Steel Coupons after Aerosol Deposition of Spores 54

Table 4-1. Instrument calibration frequency 60

Table 4-2. Acceptance criteria and test values for critical measurements 61

List of Figures

FIGURE 1-1. TYPICAL TIMELINE AND FLOW DIAGRAM FOR EACH TEST ................................................ 4

FIGURE 2-1. CARPET COUPON FRONT (LEFT) AND BACK (RIGHT) ........................................................ 12

FIGURE 2-2. WALLBOARD COUPON FRONT (LEFT) AND BACK (RIGHT) ................................................ 13

FIGURE 2-3. DECK WOOD COUPON ............................................................................................................. 14

FIGURE 2-4. BARN WOOD COUPON FRONT (LEFT) AND BACK (RIGHT) (NOTE OFFSET OF WOOD ORIENTATION) .............................................................................................................................. 14

FIGURE 2-5. CURING CONCRETE (LEFT) AND FINAL CONCRETE COUPON (RIGHT) .......................... 14

FIGURE 2-6. 18 MM STUB BARN WOOD COUPON ...................................................................................... 15

FIGURE 3-1. GEOMETRIC MEAN OF SPORES RECOVERED FROM THE POSITIVE CONTROL MATERIAL SURFACES AND RELATIVE STANDARD DEVIATION PER MATERIAL TYPE (NOTE: CARPET AND ROUGH-CUT WOOD COUPONS WERE VACUUM SAMPLED; ALL OTHER COUPONS WERE WIPE SAMPLED AS DISCUSSED IN SECTION 3.1.2.) ............................. 33

FIGURE 3-2. COMPARISON OF WIPE SAMPLING AND VACUUM SAMPLING ......................................... 34

FIGURE 3-3. TOTAL CFU IN THE RINSATE ................................................................................................... 36

FIGURE 3-5. AVERAGE SURFACE LOG REDUCTION ON MATERIAL SURFACES PER DECONTAMINATION STEP OR PROCEDURE (ERROR BARS INDICATED SD, EQN. 1-5) (V = VERTICAL, H = HORIZONTAL) ................................................................................................................. 39

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FIGURE 3-6. AVERAGE RINSATE VIABLE SPORE RESULTS (TOTAL CFU PER TOTAL RINSATE COLLECTED FOR ALL MATERIALS IN EACH TEST) NOTE: TEST 5-6 WERE VACUUMING ONLY AND DID NOT GENERATE RINSATE. THE RINSATE WAS NOT NEUTRALIZED IN TESTS 1 AND 2 BY ADDITION OF STS (ERROR BARS INDICATED SD) ............................................ 45

FIGURE 3-8. FATE OF SPORES PER DECONTAMINATION TECHNIQUE ................................................. 48

FIGURE 3-9. LOG REDUCTION IN SPORE LOADINGS BASED ON SURFACE SAMPLING (WIPE) ........ 51

FIGURE 3-10. TOTAL VIABLE SPORES IN THE RINSATE SAMPLES ........................................................ 52

FIGURE 3-11. AVERAGE LOG CFU/SAMPLE FOR 18 MM COUPONS ....................................................... 54

TABLE 3-5: WIPE SAMPLING RESULTS FOR STAINLESS STEEL COUPONS AFTER AEROSOL DEPOSITION OF SPORES ........................................................................................................................ 54

FIGURE 3-12. AVERAGE CFU/SAMPLE FOR 14-IN COUPONS .................................................................. 57

FIGURE 3-13. VIABLE SPORES IN THE RINSATE ........................................................................................ 58

FIGURE 4-1. VIABLE SPORES ON BLANK SAMPLES .................................................................................. 64

FIGURE 4- 2. VIABLE SPORES ON BLANK SAMPLES ................................................................................. 65

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Acronyms and Abbreviations

ADA Aerosol Deposition Apparatus

AOAC ATCC

AOAC International American Type Culture Collection

CBR chemical, biological, and radiological

CFR Code of Federal Regulations

CFU Colony Forming Unit(s)

CM Critical Measurements

COC DCMD

Chain of custody Decontamination and Consequence Management Division

DI Deionized

DPG Dugway Proving Ground

DQI Data Quality Indicator

DQO Data Quality Objective

dscm Dry Standard Cubic Meter

DTRL Decontamination Technologies Research Laboratory

ECBC Edgewood Chemical Biological Center

EPA U.S. Environmental Protection Agency

FAC Free Available Chlorine

FIFRA Federal Insecticide, Fungicide, and Rodenticide Act

ft foot, feet

G g

growth gram(s)

g/L gram(s) per liter

H2O2 Hydrogen Peroxide

HEPA High Efficiency Particulate Air

hp horsepower

hr hour

hrs hours

HSP Health and Safety Protocol

ID i.d. in

identification inner diameter inch(es)

INL Idaho National Laboratory

ISO International Organization For Standardization

L liter(s)

Lpm liter(s) per minute

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LOD Limit of Detection

LR MDI

Log Reduction Metered Dose Inhaler

min minute(s)

mL milliliter(s)

mm millimeter(s)

MOP Miscellaneous Operating Procedure

N NDT

normal National Decontamination Team

NG NHSRC

no growth National Homeland Security Research Center

NIST National Institute of Standards and Technology

OEM Office of Emergency Management

OPP Office of Pesticide Programs

OCSPP Office of Chemical Substances, and Pollution Prevention

o.d. ORD

outer diameter Office of Research And Development

OSC OSWER

On-scene Coordinator Office of Solid Waste And Emergency Response

OLS Onsite Laboratory Support

OSHA PBST

Occupational Safety and Health Administration Phosphate Buffered Saline with 0.05% TWEEN®-20

p/n PPE

Part number Personal Protective Equipment

ppm parts per million

ppmv parts per million by volume

psi pounds per square inch

QA Quality Assurance

QAPP Quality Assurance Project Plan

QC Quality Control

RSD Relative Standard Deviation

SD Standard Deviation

sec second(s)

SEM SOP

scanning electon microscope Standard Operating Procedure

SOW Scope of Work

SS stainless steel

sq square

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STS TBD

Sodium thiosulphate To Be Determined

TSA Trypticase Soy Agar

TSM TSP

Three-step method Trisodium Phosphate

U.S. United States [of America]

USG United States Government

USPS United States Postal Service

v volume

WAM Work Assignment Manager – not found in report

WAL Work Assignment Leader – not found in report

µm micrometer(s)

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Executive Summary

This project supports priorities established by the U.S. Environmental Protection Agency (EPA) National Homeland Security Research Center (NHSRC), to provide scientific expertise and evaluation on readily available, “low tech” decontamination methods which could be used to remediate and restore areas contaminated by biological threat agents such as Bacillus anthracis spores. These decontamination methods are combinations of rudimentary mechanical and chemical decontamination procedures (vacuum, scrub/wash and bleach). This project evaluated an eight-step sequential decontamination approach, similar to the process used by the U.S. EPA in Region 1 to decontaminate a wooden shed in Danbury, Connecticut, in 2007. The physical procedures used included vacuuming, washing, and rinsing of surfaces. The results of this effort were presented to the State of New Hampshire Department of Public Health in January 2010 as part of the technical support provided by U.S. EPA during the clean-up of a facility in Durham, NH, contaminated with naturally occurring Bacillus anthracis spores due to the use of drums made from untreated animal skins.

The chemical decontaminants used in this study included a prepared solution of pH-adjusted bleach (used in the procedure employed in Danbury, CT) or Clorox® Clean-Up® Disinfectant Cleaner with Bleach (used as received). The pH-adjusted bleach solution is prepared by diluting commercially available, off-the-shelf bleach with water and adjusting the pH to approximately neutral by adding acetic acid (white vinegar). Significant testing has been done to demonstrate the sporicidal properties of the pH-adjusted bleach (as opposed to undiluted or unadjusted bleach) and the solution has been granted several FIFRA crisis exemptions for use against Bacillus anthracis spores on pre-cleaned hard nonporous surfaces. Clorox® Clean-Up® Disinfectant Cleaner with Bleach is a commercially available, off-the-shelf product that is registered as a disinfectant (not a sporicide) to kill common household bacteria (not spore formers) and viruses on hard nonporous surfaces.

The objective of this study was to determine the effectiveness of individual or combined steps of the decontamination procedure(s) for “medium-sized (14 in by 14 in)” pieces (coupons) of selected building materials (carpet, wallboard, deck wood, rough cut barn wood, and concrete) inoculated via aerosol deposition with Bacillus atrophaeus spores (used as a surrogate for Bacillus anthracis spores). Coupons were placed in a testing chamber, in either a horizontal or a vertical orientation depending upon their likely orientation in an indoor or outdoor environment, and then the decontamination procedure was executed. The effectiveness of the decontamination procedure was defined in terms of surface decontamination efficacy (or effectiveness) and overall efficacy (or effectiveness). The overall effectiveness provides a measure of the ability to render the spores inactive. Overall effectiveness is generally referred to as sporicidal efficacy in the decontamination field. The surface decontamination effectiveness refers to the ability to remove viable spores from or inactivate them on surfaces; transfer of spores to other media via physical removal is included in this measure of effectiveness, but not in overall effectiveness.

This study demonstrated that the eight-step procedure, with two chemical decontamination steps employing pH-adjusted bleach (10 and 30 min surface contact) achieved surface decontamination effectiveness (measured as log reduction) of greater than 6 log reduction (>99.9999%), independent of material type or orientation. However, viable spores were found in the vacuum air exhaust (from background up to 284 colony forming units [CFU] per L of air) and in collected runoff (from background up to ~4E2 CFU) from some surfaces, indicating that surface decontamination was achieved by the combination of both chemical inactivation and physical removal.

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The use of just the first five decontamination steps (or four steps for horizontal surfaces), where the second 30-min chemical decontamination employing pH-adjusted bleach, second rinse, and third vacuuming steps were eliminated, achieved similar (> 6 log) reduction of spores on all the contaminated surfaces with the exception of concrete in the vertical position. In all instances, as with the full eight-step procedure, viable spores were found in the collected runoff (up to ~4E2 CFU) and the vacuum air exhaust. Hence, the full or modified procedure was successful at decontamination of the surfaces but did result in contaminated rinsate and some breakthrough/bypass of spores through wet/dry vacuum HEPA filters.

The vacuuming step alone resulted in less than one 1 log reduction on all surfaces. The addition of other physical spore removal steps such as rinsing with water, wiping with a sponge, and scrubbing with a detergent solution resulted in a range of 1-4 log reduction in viable spores from the surfaces. The spores removed from the surfaces using the washing and rinsing steps were found viable in the rinsate (~2E6 to ~7E6 CFU).

In general, either the eight-step or five-step procedure including the use of pH-adjusted bleach was effective (>6 log reduction) for surface decontamination on all material types (with the potential exception of vertical concrete noted above). However, the overall effectiveness was lower since the procedure did transfer viable spores to other media (air and rinsate) that would likely need to be collected and treated during implementation. The modified procedure using Clorox® Clean-up® instead of pH-adjusted bleach was effective on somewhat porous materials (carpet and painted wallboard) and less effective on more porous materials (concrete and wood). Again, the vacuuming steps did result in viable spores being detected (up to 68 CFU/L air) in the vacuum exhaust past the HEPA-rated filter. Viable spores were also detected in the rinsate (~5E3 to ~3E5 CFU), indicating that the application of Clorox® Clean-up® (as with the pH-adjusted bleach) was not sufficient to inactivate all spores on all material surfaces. Note that higher viable spore counts were found in the rinsate and on the surface of the rough cut wood after use of Clorox® Clean-up® compared to pH-adjusted bleach, suggesting that the former was less effective as a sporicide on most materials.

The surface decontamination effectiveness of each step in the decontamination procedure is not additive and is not necessarily proportional to effort. For example, repeating a step will not double the efficacy (e.g., increase from a 4 log reduction to an 8 log reduction), due to the log nature of the scale (i.e., an increase from a 4 log reduction to 8 log reduction is an actual increase of four orders of magnitude, not two-fold).

Additional testing was performed on surface and rinsate sampling and spore deposition to provide optimized protocols and to verify results relative to other studies. For example, an initial effort was conducted to determine the most appropriate method for sampling rough and porous materials. In general, the results showed that wipe sampling performed better on most tested materials in terms of higher spore recoveries and repeatability within material type. Based upon these results, the materials that were selected for wipe sampling were stainless steel (contamination control coupons), painted wallboard, concrete and sealed deck wood. Carpet and rough-cut wood were vacuum sampled, based upon current inter-agency guidance on sampling and the impact on the integrity of the wipe material during application to the rough wood. Surface sampling results from the positive control coupons of each material support the sampling methods by showing approximately a 7 log recovery of viable spores from the material surfaces and high repeatability of the sampling procedures with a relative standard deviation less than 4%.

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Some testing was done to examine the impact of changes to the volume or frequency of application of the pH-adjusted bleach solution. The tests showed that changes to the total volume or frequency of reapplication of the pH-adjusted bleach spray does not impact decontamination of the deck wood surface, provided the surface remains wet for the specified contact time. Additional testing is required to optimize the spray application procedure to ensure that the spray application procedure minimizes the time required for spraying and maximizes the decontamination efficacy.

Testing was also conducted to compare wipe sampling of coupon surfaces to coupon extraction and also to compare liquid inoculation of spores versus aerosol deposition. The recovery of spores from the wood surface was not significantly dependent upon sampling type (extraction, wipe, or vacuum) or coupon inoculation method (liquid inoculation or aerosol deposition of spores). Therefore, the methods used for spore recovery (consistent with those used in this study) do not impact the comparison of data to other studies using pH-adjusted bleach. A highly significant number of spores (i.e., same order of magnitude as initial sampling results) were still recoverable by additional wipe sampling or via extraction after initial sampling (wipe or vacuum). However, wood coupons inoculated via liquid suspension were considerably more difficult to decontaminate than the same material with spores deposited as an aerosol (> 6 log reduction (LR) for aerosol inoculated compared to <1 LR for liquid inoculated). Liquid inoculation methods may result in conservative estimates of log reduction for wood surfaces contaminated by aerosol deposition. In both cases (liquid inoculation and aerosol deposition), surface decontamination of wood was primarily due to physical removal of spores and not inactivation of the organism, resulting in a contaminated rinsate, wet/dry vacuum filters, and air exhaust.

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1. Introduction

This project supports the mission of the U.S. Environmental Protection Agency’s Office of Research and Development’s National Homeland Security Research Center (NHSRC) by providing information relevant to the decontamination of areas contaminated as a result of an act of terrorism. The project is relevant to the NHSRC program goal in which the Office of Solid Waste and Emergency Response (OSWER) and other clients use homeland security research program products and expertise to improve the capability to respond to terrorist attacks affecting buildings and the outdoor environments. This project addresses a direct need expressed by OSWER’s National Decontamination Team (NDT) and the U.S. EPA’s Regional On-Scene Coordinators (OSCs) resulting from the successful use of a multi-step decontamination procedure in the remediation of a Bacillus anthracis spore-contaminated shed in Danbury, CT, in 2007. In addition, the project is relevant to the U.S. EPA’s Office of Pesticide Programs’ (OPP’s) crisis exemption process and OPP’s regulatory function under the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA).

The availability of decontamination methods to support a timely response and recovery resulting from the wide-area release of a biological agent such as Bacillus anthracis spores (the causative agent of anthrax and often referred to as “anthrax spores”) is recognized as a critical need for preparedness. Such a release could potentially result in the contamination of a vast number of personal residences, businesses, public facilities (e.g., hospitals), and outdoor areas. In 2001, the introduction of a few letters containing anthrax spores into the U.S. Postal Service (USPS) system resulted in the contamination of several facilities.1 Although some of the facilities in which these letters were processed or received in 2001 were heavily contaminated, the facilities were successfully remediated with approaches such as fumigation with chlorine dioxide or hydrogen peroxide. 1 However, contamination of critical public facilities following a wide-area release could quickly consume the Nation’s entire remediation capacity, requiring years to clean up and resulting in enormous economic impact. Additional quick, effective and economical decontamination methods having the capacity to be employed over wide areas (outdoor and indoor) are required to increase preparedness for such a release.

In addition to fumigation procedures used primarily in heavily contaminated facilities, other cleaning methods have been used in secondarily contaminated areas (e.g., cross-contaminated letters potentially in contact with the anthrax spore-containing letters or tracked from primarily contaminated sites) or primarily contaminated facilities showing a minimal presence of anthrax spores.1 These other cleaning methods and combinations of methods include disposal of contaminated items, vacuuming, and the use of liquid sporicides such as a pH-adjusted bleach solution.1 Additionally, a combined set of mechanical and chemical procedures (vacuum, scrub/wash and bleach) was successfully used in the decontamination of a small shed contaminated with anthrax spores originating from animal hides used during a drum-making process. 2 If proven more widely effective (i.e., for additional materials and contamination types), such an approach involving washing and cleaning with readily available equipment would significantly increase EPA’s readiness to respond to a wide-area release. Currently, data to evaluate and quantify the effectiveness of such decontamination techniques for wide-area decontamination are not available. The testing of field-relevant application methods in a controlled manner, under an appropriate quality assurance process and plan, can provide for scientifically defensible data to support subsequent decontamination strategy guidance development.

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1.1 Objectives

The primary objective of this study was to address the decontamination method gaps that currently exist for preparedness to respond to and recover from a wide area contamination event (e.g., involving Bacillus anthracis spores). As a first step to understanding and further developing applicable methods, this study was initiated to measure the reduction in the number of viable spores (efficacy) on contaminated building material surfaces using a series of remediation approaches. The effectiveness of individual or combined steps of the decontamination procedure(s) was assessed for “medium-sized (14 in by 14 in)” pieces (coupons) of selected building materials. This effectiveness was defined in terms of surface decontamination efficacy (or effectiveness) and overall efficacy (or effectiveness). The overall effectiveness provides a measure of the ability to render the spores inactive. Overall effectiveness is generally referred to as sporicidal efficacy in the decontamination field. The surface decontamination effectiveness refers to the ability to remove viable spores from or inactivate them on surfaces; transfer of spores to other media via physical removal is included in this measure of effectiveness, but not in overall effectiveness (since the spores still exist in a viable state until treated by other measures when physically removed from a surface). Note that the terms effectiveness and efficacy are generally used interchangeably, including throughout this report. The coupons used in this testing were larger in size than the coupons commonly used in other decontamination testing,3-7 but smaller than the surfaces that would likely be encountered in the field (e.g., roadways, walkways, and walls). Operational parameters such as time, physical impacts on materials, and impact on the remediation crew (e.g., physical exertion) were also evaluated.

1.2 Experimental Approach

The general approach used to meet the objectives of this project was:

• use of controlled chambers, standardized coupons and spore inocula;

• inoculation of medium-sized pieces of materials (coupons) via aerosol deposition of bacterial spores;

• quantitative assessment of initial viable spore loading by sampling positive control coupons (coupons inoculated with the bacterial spores in the same manner as test coupons, but not subjected to the decontamination treatment being tested);

• application of a prescribed decontamination procedure to the test coupons and procedural blanks;

• quantitative assessment of residual viable spore loading on each material type, after application of combinations of decontamination procedures, by sampling test coupons and procedural blanks;

• quantitative and qualitative analysis of viable spores that survive the various decontamination procedures through transfer to rinsate, wet/dry vacuum filters and vacuum exhaust samples;

• determination of surface decontamination efficacy (comparison of viable spore concentrations from positive controls and test coupons);

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• determination of overall decontamination efficacy (accounting for viable spores transferred to other media [e.g., rinsate or air] during the decontamination process); and

• documentation of operational considerations (e.g., cross-contamination, procedural time, impacts on materials and personnel).

A test matrix was developed to allow different combinations of steps of the decontamination process (i.e., vacuuming, application of a pH-adjusted bleach solution, scrubbing, and rinsing) to be investigated independently for each surface type. This matrix included replication of test and control coupons within each test, but did not allow for replication of specific tests due to the scale of the testing (time and cost per test).

The inoculation of the coupons using aerosol deposition of viable spores, as opposed to the more traditional use of dispensing precise amounts of spore suspensions onto the material surface (liquid inoculation), was used in this study to more closely represent the nature of the contamination experienced in past “anthrax” incidents. Liquid inoculation has been the more commonly used method of inoculation for studies of decontamination efficacy due to the ease and acceptable precision of the application of the spore suspension. 3, 7-10 Recent advances in the ability to precisely deposit viable bacterial spores onto materials of relevant composition11 were utilized to allow for the inoculation method of aerosol deposition to be used in this study.

The projected timeline and flow diagram for the testing approach is shown in Figure 1-1. Details of the types and numbers of materials tested, as well as the procedures used for inoculation, decontamination, sampling and testing, are described in Section 2 and in the attached appendices.

Medium-sized coupons (14 in by 14 in; area of 196 in2) were fabricated (see Section 2) and sterilized (see Appendix B) in groups identified by sterilization batch number. This coupon size was chosen to be able to apply the physical decontamination methods (i.e., vacuuming, spraying, and brushing) and field sampling methods (i.e., wipe and vacuum sock) used in this current study. The materials used in this study included carpet (horizontal orientation), wallboard (primed and painted; horizontal orientation to represent a ceiling and vertical orientation to represent a wall), deck wood (sealed, pressure-treated; horizontal orientation with wood slats oriented parallel to the side of the chamber), rough cut barn wood (vertical orientation with wood slats oriented vertically), and concrete (horizontal and vertical orientation). All materials used are considered porous with the exception of the painted wallboard (a sealed surface). Prior to use, all test equipment intended to come in contact with coupons or samples was sterilized via autoclave sterilization at 121 °C and 30 psi, or using a STERIS VHP® 1000ED (STERIS Corporation, Mentor, OH) hydrogen peroxide (H2O2) generator cycle at 1000 ppmv H2O2 for 1 hour. All laboratory work surfaces were wiped with Dispatch® bleach wipes (Caltech, Midland, MI), rinsed with DI water, and dried with 70 percent ethanol (VWR, West Chester, PA).

Day 1 of testing involved coupon inoculation and preparation for testing on Day 2. Due to the time needed to take a coupon through the entire process from inoculation to laboratory analyses, coupons needed to be inoculated before the weekend in order to be ready to start the selected decontamination procedure on the following Monday morning. Therefore, the required number of test and positive control coupons were loaded

4

with the target spores at least two days, but no more than four days, prior to the decontamination procedure. The coupons remained isolated in independent deposition devices throughout this time.

On Day 2, inoculated coupons were removed from the deposition devices and loaded into their respective cabinets (positive controls and test coupons into the Test Coupon Cabinets and the procedural blanks into the Procedural Blank Cabinet). As indicated above, positive control and test coupons are both inoculated with the target number of viable spores; the test coupons are subjected to the decontamination procedure being tested while the positive control coupons are not (and are maintained under ambient laboratory conditions). The purpose of the positive controls is to determine the starting viable spore load on each coupon type for comparison to the viable spore load on the test coupons after decontamination. Since the sampling process removes spores from the material surfaces, the procedure of using positive controls and test coupons to determine effectiveness is common.3, 7-10 Procedural blank coupons (negative controls) were the same materials as the test and positive controls. However, they were intentionally not inoculated with spores. They were put through the same decontamination procedure as the test coupons for the purpose of elucidating any potential cross-contamination introduced during the testing procedure.

Figure 1-1. Typical timeline and flow diagram for each test

5

After the coupons were appropriately stored, sets of three coupons were then positioned into the decontamination chamber and the selected decontamination procedure was applied. Coupons were placed in either a horizontal or vertical orientation according to their most likely use in construction. Procedural blank coupons were subjected to the decontamination procedure first, followed by the test coupons. The decontamination procedure was completed on all test coupons of one material type before moving on to the next material. After the decontamination procedure was applied to a coupon, the coupon was moved to the appropriate cabinet for drying (test coupons to the Decontaminated Coupon Cabinet and procedural blanks to the Procedural Blank Cabinet). The decontaminated coupons were allowed to dry for at least 24 hours. After the completion of each set of coupons, the test chamber was cleaned in accordance with the procedure described in Appendix G. A coupon set includes all blank coupons or all replicates of one material type.

Chemical neutralization of the decontaminant (when a liquid decontaminant such as pH-adjusted bleach or Clorox® Cleanup® was used) on the coupon after the desired application time was not performed in this study. The coupons were rinsed with water after the desired application time of the decontaminant which should have sufficiently ended the contact time of the material with the decontaminant. The intent was to utilize procedures that would be employed in the field; neutralization was not performed in past field use of pH-adjusted bleach. Rinsing has been performed in order to remove residual decontaminant from surfaces. The collected rinsate, however, was neutralized for all tests except Tests 1 and 2. This process is discussed in Section 2.6.4.

In addition, the temperature and pH of the pH-adjusted bleach solution and DI water were measured at the initiation of a test and prior to the start of each test set (i.e., material type). The free available chlorine content (FAC) of the pH-adjusted bleach solution was also measured (see Section 2 for method). The flow rate from the backpack sprayer (SRS-540 Propack, SHURflo, Cypress, CA) was measured at the start and end of testing of each set of three coupons on which the sprayer was being used. The spray pattern for the backpack sprayer and garden hose were confirmed (and adjusted as needed) prior to the start of a test. These measurements were made to ensure that such parameters were in accordance with the data quality objectives (DQOs) defined for the project (see Section 4). Adjustments were made as necessary to achieve the desired set-points, within the acceptable tolerances.

Although surface sampling of the coupons did not occur until Day 3, several other samples were collected to obtain additional information on the fate of the spores. To assess the potential for viable spores to be washed off of the surfaces, all liquid run-off (rinsate) generated in the decontamination process was collected and quantitatively analyzed. Rinsate samples were a composite of the rinsate from all replicate coupons of a particular material type per test. Quantitative analysis was conducted on rinsate samples so that the magnitude of spore relocation could be determined. The volume of run-off liquid collected for each coupon set was measured after collection.

• For each coupon set, a swab sample was collected from the HEPA-rated filter within the wet/dry vacuum that was used in each test and qualitatively analyzed to confirm contamination by the target organism. Such information is relevant to the treatment of the vacuum after use and the potential spread of contamination.

6

• Two or more exhaust samples were collected, one from the blank wet/dry vacuum and one or more from the test coupon wet/dry vacuums. These samples were collected because of the potential for the wet/dry vacuum to spread contamination via the presence of viable spores in the exhaust. This potential was assessed by collecting a composite sample from all vacuuming in a test.

On Day 3, after at least 24 hours of drying, sampling of the coupons was performed. A sampled area of 1.27 ft2 per coupon for this study was created by sampling the interior section of each coupon; a template was used to cover the exterior 0.25 in of each coupon leaving a square (13.5 in by 13.5 in) exposed for sampling. Surface sampling of each test coupon was conducted only once.

The analysis of the samples collected (coupon, filter, rinsate, and exhaust) occurred over a three-day period. In general, the NHSRC Microbiology Laboratory extracted the samples on the day of receipt, plated on the following day, and then counted colonies on the third day. However, instances occurred when it was possible to apply the decontamination procedure to only half of the coupons on the first day, with the remaining half decontaminated on the following day. In this case, the later samples were analyzed over a two-day period. Sample extraction and plating would occur on the day of receipt, with colony counting the following day.

Appendix A describes the procedure for coupon sterilization. Appendix B contains Miscellaneous Operating Procedures (MOPs), including the aerosol deposition of spores. Appendices C, D and E contain additional details of the inoculation, decontamination, sampling and analysis procedures, respectively. Appendix F describes the test chamber and equipment cleaning procedures.

1.3 Definitions of Effectiveness

The “overall effectiveness (efficacy)” of a decontamination technique is a measure of the ability of the method to inactivate and/or remove the spores from contaminated building material surfaces (i.e., represented by coupons in this study) while taking into account viable spores that may remain in rinsate, wet/dry vacuuming and air exhaust. The latter spores could result in secondary contamination of wastewater, air or other surfaces that would necessitate additional remediation strategies. The “surface decontamination effectiveness (efficacy)” of a decontamination technique is a measure of the ability to inactivate spores or remove spores from coupons. In this study, surface decontamination effectiveness was determined for each step of the surface remediation, for the complete procedure and for each specific material.

The surface decontamination efficacy of each decontamination step or method (combination of steps) was determined based on the number of viable spores collected from the surface of the decontaminated coupon, as compared to the number of viable spores collected from the surface of control coupons not subjected to decontamination procedures. The number of viable spores was measured as colony forming units (CFU).

The sporicidal effectiveness (efficacy) is a measure of the inactivation of the spores. An indication of the sporicidal efficacy in this study was determined by comparing the surface decontamination effectiveness to the overall effectiveness; e.g., viable spores found in the rinsate after application of a sporicide can be used to determine the sporicidal effectiveness by comparison to the number of viable spores recovered from the positive controls.

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1.3.1 Surface Decontamination Efficacy

The surface decontamination efficacy for each decontamination technique and surface material combination was evaluated by measuring the difference in the logarithm of the measured CFU before decontamination (determined from sampling the positive control coupons) and after decontamination (determined from sampling the test coupons) for that material. This value is reported as a log reduction on the specific material surface as defined in Equation 1-1.

s

N

kkS

C

N

kkC

i N

CFU

N

CFUtC

∑∑== −= 1

,1

, )log()log(η (1-1)

where:

η i =

Surface decontamination effectiveness; the average log reduction of spores on a specific material surface (surface material designated by i)

C

N

kkC

N

CFUC

∑=1

, )log( =

The average of the logarithm (or geometric mean) of the number of viable spores (determined by CFU) recovered on the control coupons (C indicates control and NC is the number of control coupons)

s

N

kkS

N

CFUt

∑=1

, )log( =

The average of the logarithm (or geometric mean) of the number of viable spores (determined by CFU) remaining on the surface of a decontaminated coupon (S indicates a decontaminated coupon and Ns is the number of coupons tested ).

When no viable spores were detected, a value of 0.5 CFU was assigned for CFUS,k (see Section 1.3.1.1) and the efficacy was reported as greater than or equal to the value calculated by Eqn. 1-1.

The standard deviation of the average log reduction of spores on a specific material (ηi ) is calculated by Eqn. 1-2:

( )

11

2

−

−=∑=

S

N

kik

i N

xs

SDη

η (1-2)

where:

8

iSDη =

Standard deviation of ηi, the average log reduction of spores on a specific material surface

η i =

The average log reduction of spores on a specific material surface (surface material designated by i)

xk = The average of the log reduction from the surface of a decontaminated coupon (Equation 1-3)

NS = Number of test coupons of a material surface type.

N s

N

kksc

k

s

CFUCFUx

∑=

−= 1

, ))log()log((( (1-3)

C

N

kkC

C N

CFUCFU

C

∑== 1

, )log()log( =

Represents the “mean of the logs” (geometric mean), the average of the logarithm-transformed number of viable spores (determined by CFU) recovered on the control coupons (C = control coupons, Nc = number of control coupons, k = test coupon number and Ns is the number of test coupons)

CFUs,k = Number of CFU on the surface of the kth decontaminated coupon

Ns = Total number (1,k) of decontaminated coupons of a material type.

The average surface decontamination effectiveness of the decontamination technique for spores recovered on the surface of building materials, independent of the type of material, was evaluated by comparing the difference in the logarithm of the CFU before decontamination (from sampling of the positive control coupons) and after decontamination (from sampling of the test coupons) for all the tested materials. These data are calculated by determining the arithmetic mean of η for all material types according to Equation 1-4 and reported as log reductions of spores for each decontamination technique.

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i

ii

T N

∑=

ηη (1-4)

where ηT is the overall surface log reduction efficacy for the technique, and Ni is the total number of

coupon material types tested with that technique (i indicates coupon material type).

The standard deviation of ηT is calculated by Eqn. 1-5:

( )

1

2

−

−=∑

i

iTi

T NSD

ηη

η (1-5)

TSDη =

Standard deviation of ηT, the overall surface log reduction efficacy for the technique

ηT = Overall surface log reduction efficacy for the technique

ηi = The average log reduction of spores on a specific material surface (surface material designated by i)

Ni = Number of coupon material types.

While this method of calculating surface decontamination efficacy is useful for comparing decontamination methods, the indoor clearance criterion for a facility following actual bioterrorism events has generally been no growth of the biocontaminant via culture for all environmental samples. Thus, clearance sampling after use of a particular decontamination method in which CFU of the biocontaminant were detected would indicate that the decontamination was not effective.

1.3.1.1 Detection Limits

The number of viable spores on material surfaces was determined according to MOP 6535a (Appendix B). This method calls for plating 0.1 mL of serial dilutions of the 20 mL extraction fluid in triplicate on Trypticase soy agar (TSA). CFU counts (30 to 300 colonies) were enumerated from the appropriate plate dilutions. The CFU per sample were calculated according to Equation 1-6. When fewer than 30 CFU (the suitable quantitation limit) were present on the primary (no dilution) plates, the extracts were filter plated as described in Section 2.6.6. When no detectable spores were found from the filter plating, a value of 0.5 CFU was assigned as the detection limit for efficacy determinations (calculation of log reduction). The use of this detection limit value for samples with less than 30 CFU on the primary plates is consistent with other published methods.4, 5, 9, 12 For the current effort, this detection limit was considered for the plating and, hence, the multiplier of 200 (20 mL divided by 0.1 mL) was applied for all non-filter sample results. This

10

procedure yielded an overall detection limit of 100 CFU/sample. The addition of filter plating lowered the overall detection limit to the stated 0.5 CFU/sample due to analysis of the entire sample extract.

(1-6)

For vacuum exhaust samples, the enumeration was done exactly as above, with an additional multiplier for the fraction of total exhaust captured by the aerosol spore sampling ([Via-Cell® Bioaerosol Sampling Cassette (Zefon International, Inc., Ocala, FL)]. For instance, if 30 L of the total 3000 L exhaust were sampled using the Via-Cell®, the CFU/sample would again be multiplied by 100 to represent the CFU/total exhaust. This multiplication would also apply to detection limit values.

The number of viable spores in the rinsate was calculated in a similar fashion when all of the rinsate was filtered. Spores from filters were also extracted with 20 mL of fluid. However, debris in the rinsate from the coupons was an impediment during the filtration step, so the filtration method was abandoned in favor of aliquot sampling. For aliquot sampling, a portion (typically 100 mL) of the rinsate was filtered and the filter was directly plated. The enumerated spores from this plate were then multiplied by the inverse fraction of the rinsate that was filtered. For instance, if 100 mL of the 20,000 mL rinsate (the total volume collected after one coupon set of a test) were plated, then the CFU counts on the filter would be multiplied by 20000/100 (or 200) to represent the total number of spores in the rinsate. If fewer than 30 spores were enumerated (i.e., results below the quantitation limit), then a value of 0.5 CFU was assigned as the detection limit (CFUS,k in Equation 1.1). This detection limit was still subject to the multiplier, resulting in a detection limit of 100 CFU for the above example.

1.3.2 Overall Decontamination Effectiveness (Ultimate Fate of Spores)

The surface decontamination efficacy, as calculated in accordance with Equation 1-4, is a measure of the effectiveness of the procedure to mitigate the contamination on the surface of the materials. The measure of effectiveness is an aggregate value due to inactivation of the spores on the materials (i.e., due to the application of a sporicide) and/or physical removal of the spores from the material (e.g., washed/rinsed off or removed by the vacuum process). When the spores are physically removed from the surface, viable spores may remain either in the rinsate, collected in the wet/dry vacuum, or exhausted from the wet/dry vacuum. Understanding the ultimate fate of the spores (or overall decontamination effectiveness) is critical to recognizing the utility or appropriate implementation of the specific decontamination process. Hence, the overall effectiveness of a decontamination procedure or method, including the fate of the spores, was calculated based on the sum of the numbers of viable spores collected from the surface of the coupon, in the rinsate, in wet/dry vacuum filters and in the air exhaust as compared to the number of viable spores collected from the surface of control coupons not subjected to decontamination procedures. This overall efficacy is reported as a log reduction as defined in Equation 1-7.

NCFUNCFUNCFUNCFU tEtRk

tSj

CCiLogLogLogLog /)(/)(/)(/)( −−−= ∑∑η

(1-7)

11

Where:

η i = The spore log reduction efficacy of the decontamination technique i

∑j

CC NCFULog /)( = The mean log CFU recovered from the control areas (C=control,

j = Coupon number, and NC is the number of coupons (1, j))

∑k

tS NCFULog /)( = The mean log CFU recovered from the surface of a

decontaminated coupon (S= sample from decontaminated carpet, k = coupon number, and Nt is the number of coupons tested (1, k))

)(CFU ELog = Log CFU recovered from the exhaust

)(CFU RLog = Log CFU recovered from the rinsate

2. Materials and Methods

Coupons consisting of carpet, wallboard, deck wood, rough cut barn wood, and concrete were prepared with dimensions of 14 in width by 14 in length (or approximately 1.17 ft width by 1.17 ft length). These dimensions provided an adequate edge for the spore deposition device (see MOP 6561 in Appendix B) to seal to the coupon surface and allow for preparation of a contaminated surface area of 1 ft by 1 ft. A sample area of 1 ft2 has been recommended for wipe samples and has been deemed an acceptable lower end for vacuum samples.13 While a validated wipe or vacuum sampling method does not currently exist, the methods used herein have been adopted from widely cited and used procedures (currently being considered for validation). The sampled area of 1.27 ft2 per coupon was used for this study by sampling the interior section of each coupon; a template covered the exterior 0.25 in of each coupon leaving a square (13.5 in by 13.5 in) exposed for sampling. The outer edge (0.25 in) was not sampled in order to avoid artifacts due to edges of the coupons. A larger area (13.5 in x 13.5 in) of each coupon than originally contaminated (12 in x 12 in) was sampled due to the expected contamination spread to these regions during the decontamination procedure application. The thickness of the coupons varied for each material based upon the fabrication procedures that were the most appropriate for each material type. However, thickness was uniform for all replicate coupons of each material type.

The only variation to this procedure was for Test 13, added to compare results from previous testing using circular “18 mm coupons” (diameter of 0.71 in) and a liquid inoculation procedure. More information on the 18 mm coupons and the liquid inoculation can be found in Sections 2.1 and 2.2.2, following the description of the other materials and the aerosol spore deposition method.

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2.1 Coupon Materials and Fabrication

This section describes each material and how each type of coupon and the 18 mm stub were fabricated.

1. Carpet (Figure 2-1): A 14 in by 14 in (196 in2) piece of Beaulieu Solutions Walnut Ridge loop carpet (Product Number 449-818, Home Depot, Durham, NC) was placed on a 14 in by 14 in piece of 6 pound (lb) carpet pad (Product Number 561-253, Home Depot, Durham, NC) and tacked to a 14 in by 14 in

piece of 15/32 in thick four-ply plywood sheathing (Product Number 231-355, Home Depot, Durham, NC) using 0.5 in staples (Product Number 6107A62, McMaster-Carr, Robbinsville, NJ). Black vinyl duct tape (3 in) (Product Number 76135A45, McMaster-Carr, Robbinsville, NJ) was used to seal the edges of the coupon. The tape extended about 0.5 in onto the surface of the carpet. The 0.5 in staples were then used to secure the tape.

2. Painted Wallboard (Figure 2-2): A 14 in by 14 in (196 in2) piece of 0.5 in thick wallboard was cut from a 4 ft by 8 ft sheet (Product Number 258-350, Home Depot, Durham, NC). The cut edges were sealed by applying a skim coat of joint compound (Product Number 258-725, Home Depot, Durham, NC) to about 1.5 in of the backside edge of the coupon. Using joint tape (2 in) (Product Number 430-684, Home Depot, Durham, NC), one half of the tape (utilizing the factory fold) was applied to the back of the coupon. A second skim coat of joint compound was applied over the first coat. After the joint compound was dry, the coupon was turned over and a skim coat of joint compound was applied to the cut edge and about 1 in of the front edge. The tape was folded over the edge extending 0.5 in over the front side of the coupon. A second skim coat was applied and allowed to dry. Using a sanding block (Product Number 733-336, Home Depot, Durham, NC), any rough spots of the joint compound were removed. One coat of KILZ Latex Primer (Product Number 317-390, Home Depot, Durham, NC) was applied to the front side of the coupon and allowed to dry. This coat of primer was covered with one coat of Behr Premium Plus interior flat white latex paint (Product Number 135-992, Home Depot, Durham, NC). The back side of the coupon received one coat of Behr interior enamel paint (no primer was used on the back) (Product Number 374-776, Home Depot, Durham, NC).

Figure 2-1. Carpet coupon front (left) and back (right)

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Figure 2-2. Wallboard coupon front (left) and back (right)

3. Deck Wood (Figure 2-3): The 14 in by 14 in (196 in2) coupons were made from 5/4 in by 6 in (nominal lumber size, 1 in by 5.625 in by actual measurement) pressure-treated decking board (Product Number 129-093, Home Depot, Durham, NC). The coupons were fabricated to 196 in2 by using two 14 in by 5.625 in pieces of the treated decking wood and one piece ripped to approximately 14 in by 2.75 in to allow a 1/8 in gap between each of the pieces. On the back side, a 1.5 in piece was placed in the center (perpendicular to the direction of the surface boards) and each piece was fastened with two screws with the exception of the ripped piece (which was fastened with one screw). Using the same material, a border was fabricated for the perimeter of the underside of the coupon. This border was attached from the underside using 2 in staples. The coupon was then sealed using one coat of Behr Waterproofing Wood Protector (Product Number 448-367, Home Depot, Durham, NC).

4. Rough-Cut Barn Wood (Figure 2-4): The material used to fabricate the 14 in by 14 in (196 in2) coupons of rough-cut wood was 1 in by 6 in (nominal lumber size, 0.5 in by 5.375 in, actual) pressure-treated Brazilian Pine Dog Ear Picket fence lumber (Product Number 884-831, Home Depot, Durham, NC). The coupons were assembled using two 14 in by 5.375 in pieces of lumber, plus one piece ripped to fabricate a 196 in2 coupon. The three pieces were assembled with no spaces between boards, attached with 1 in staples from the backside, using the same fence lumber material (perpendicular to the surface boards) as support.

5. Concrete (Figure 2-5): Quikrete® Sand/Topping (Product Number 10389, Home Depot, Durham, NC) mix was used to fabricate 1 in thick, 14 in by 14 in coupons. The mix was prepared and poured into forms and allowed to dry overnight. Once set, the coupons were removed and stacked on a pallet where they were wetted and covered with plastic to cure.

For the test involving the liquid inoculation technique (Test 13), 18 mm coupons were made of pressure treated Brazilian Pine Dog Ear Picket fence lumber (Figure 2-6). A core sample was taken from the board and cut 3/16 in thick. These coupons were affixed to a Scanning Electron Microscope (SEM) pin stub mount (aluminum, 18 mm [0.71 in] diameter, Ted Pella, Inc., Redding, CA) (henceforth, 18 mm stubs) using a carbon- based adhesive (carbon conductive tape, double-sided, Ted Pella, Inc., Redding, CA). The original finished surface of the wood was used as the inoculation surface. The coupons underwent sterilization by a

14

steam autoclave on a gravity cycle following an NHSRC Microbiology Laboratory internal MOP 6533 (Appendix B). Each sterilization batch included all coupons for a single test.

Figure 2-3. Deck wood coupon

Figure 2-4. Barn wood coupon front (left) and back (right) (Note offset of wood orientation)

Figure 2-5. Curing concrete (left) and final concrete coupon (right)

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Figure 2-6. 18 mm stub barn wood coupon

2.2 Material Inoculation Procedure

The investigation of the effectiveness of decontamination steps or procedures (combinations of steps) required that a target organism be applied to a “sterile” material surface (i.e., material inoculation) at a precise target loading (e.g., spores per piece of material [or coupon]). This section provides detail on the target organism and material inoculation procedures used for this investigation.

2.2.1 Bacillus Spore Preparation

The test organism for this work was a powdered spore preparation of Bacillus atrophaeus (ATCC 9372) and silicon dioxide particles. The bacterial species Bacillus atrophaeus was formerly known as B. subtilis var niger and subsequently B. globigii. The preparation was obtained from the U.S. Army Dugway Proving Ground (DPG) Life Science Division. The preparation procedure can be found in Brown et al.14 Briefly, after 80 – 90% sporulation, the suspension was centrifuged to generate a preparation of about 20% solids. A preparation resulting in a powdered matrix containing approximately 1 x 1011 viable spores per gram was prepared by dry blending and jet milling the dried spores with fumed silica particles (Deguss, Frankfurt am Main, Germany). The powdered preparation was loaded into metered dose inhalers (MDIs) by the U.S. Army Edgewood Chemical Biological Center (ECBC) according to a proprietary protocolNine MDIs were used in this study in accordance with the protocol developed to provide uniformity in precision despite the use of numerous MDIs (see Section 2.2.2). Each set of coupons in a test was inoculated using a single MDI; control checks were included in each set of coupons. The MDIs are stated by ECBC to provide a consistent dose of approx. 1 x 109 spores per puff. Quality assurance documentation was provided by ECBC with each batch of MDIs.

In a few specific tests discussed in detail in subsequent sections of this report, coupons were spiked with a liquid spore suspension for comparison to the use of aerosol deposition in this study (i.e., provide a cross-comparison to other studies that have relied on using liquid inoculation). The liquid inoculum was prepared with approximately 1 x 108 viable B. atrophaeus spores per mL, measured as CFU on TSA plates after an 18-24 hour incubation period at 37 oC. Approximately 1 gram of B. atrophaeus spores was added to 12 mL of 28.5% ethanol solution. The 12 mL solution was then vortexed and separated by placing 1 mL into each of 11 micro-centrifuge tubes (1.5 mL, clear, MIC104, Microstein, LLC, Central Point, OR).

16

2.2.2 Coupon Inoculation Procedure

Coupons were inoculated with spores of B. atrophaeus from an MDI using the procedure detailed in MOP 6561 (Appendix B). Briefly, each coupon was inoculated independently by being placed into a separate dosing chamber designed to fit one 14 in by 14 in (196 in2) coupon of any thickness. In accordance with MOP 6561, the MDI was discharged into the dosing chamber a single time. The spores were allowed to settle onto the coupon surfaces for a minimum period of 18 hrs. After the minimum 18-hr period, the coupons were then removed from the dosing chamber and moved to an isolated cabinet (Test Coupon Cabinet), which contained all loaded coupons for a single test. Coupons were inoculated in the same manner, irrespective of the orientation of the coupons in the decontamination testing. The target recovery range was 5 x 106 to 5 x 107 CFU per coupon.

For Test 13, 20 coupons were inoculated with spores from an MDI as described above. For comparison, ten additional coupons were spot-inoculated with the contents of the 1 mL micro-centrifuge tubes described in Section 2.2.1. The liquid inoculum was spread over an approximately 5 in by 5 in area of the couponsurface with a target loading consistent with the loading for coupons inoculated via aerosol deposition. In a select number of tests, small coupons (18 mm stubs) were used to provide a comparison to previous testing. The 18 mm stubs were inoculated with B. atrophaeus using the aerosol deposition method reported in Lee et al.11

The handling of the inoculated coupons, including movement to minimize or control spore dispersal, is described in Appendix C.

2.3 Experimental Sequence

The following steps describe the testing sequence:

• The coupons were sterilized and stored, one material type per container.

• All spores for the study were prepared, per the method discussed in Section 2.2.1, prior to the initiation of any testing.

• Two days prior to testing (at a minimum), all positive control and test coupons for a given test were inoculated per the method described in Section 2.2.2.

• On the decontamination procedure test day, the procedural blank, test and positive control coupons were placed into the appropriate cabinets. Three cabinets were used to contain the coupons prior to decontamination (one for the procedural blanks and two containing the inoculated [positive controls and test] coupons). One additional cabinet was used to store test coupons for drying after the decontamination procedure was applied. All materials and equipment necessary for the decontamination procedure were gathered and prepared (see Table D-1 in Appendix D). Measurements of temperature, pH, and concentrations of liquids were made and recorded. Any solutions falling outside the specified ranges were discarded and prepared again.

17

• The procedural blank coupons were subjected to the decontamination procedure first, followed by the test coupons.

• The decontamination procedure used in each test was conducted on test coupons of one material type in its entirety (e.g., all five replicate coupons) prior to moving on to the next material. Up to three coupons at a time were placed into the test chamber and the decontamination procedural steps were conducted, one directly after another.

• After completion of the test procedure, the test coupons were moved to the Decontaminated Coupon Cabinet for drying and the blank coupons were moved to the Procedural Blank Cabinet. The positive control coupons remained in their original locations, in the pre-decontamination inoculated coupon cabinet.

• The Decontaminated Coupon Cabinet was identical to the Test Coupon Cabinet. However, the Decontaminated Coupon Cabinet was located on the opposite side of the decontamination test chamber from the Test Coupon Cabinets. The Decontaminated Coupon Cabinet and Procedural Blank Cabinet were slightly positive-pressured in order to prevent contamination of the coupons from the laboratory environment and allow some air flow to promote drying.

• The rinsate from a coupon set was collected during the decontamination of that coupon set (see Section 2.6.4). After completion of a coupon set, the rinsate sample was removed and the container was capped until processed as described in Section 2.6.4.

• The test chamber was cleaned with the pH-adjusted bleach solution in accordance with Appendix F before the next set of coupons was loaded into the chamber.

• All used materials and equipment were replaced with the appropriate virgin materials (e.g., new sponges or brushes) or equipment (e.g., wet/dry vacuums) for the next coupon set.

• After decontamination test procedures were completed for all sets of coupons, samples were recovered from the wet/dry vacuum filters and exhaust (as applicable).

• After a minimum of 18 hours of storage in the Decontaminated Coupon Cabinet or Procedural Blank Cabinet (as appropriate), under slight positive pressure, and when all coupon surfaces were visibly dry, surface sampling was conducted (as discussed in Section 2.6).

2.4 Decontamination Procedures

The justification for the specific decontamination procedures evaluated here was a process employed by the U.S. EPA in Region 1 to decontaminate a wooden shed contaminated with B. anthracis spores in Danbury, CT. The decontamination process employed for the shed has been documented in the “After Action Report – Danbury Anthrax Incident (U.S. EPA Region 1, September 19, 2008)”15 and discussed by Snook et al. 2 The contamination resulted from the inhabitant’s processing untreated animal hides in the fabrication of African drums. The decontamination process that was the baseline for this project can be summarized in the following sequential procedural steps:

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1. Vacuum the surface with a wet/dry vacuum containing a HEPA-rated filter. The filter was not sterilized prior to testing.

2. Mist the surface with the pH-adjusted bleach solution until the surface remains wetted; reapply as necessary to keep surface wetted for a contact time of 10 minutes.

3. Scrub the surface with detergent solution using a brush. (Please refer to Appendix D regarding the protocol used for the brushes.)

4. Rinse the surface with water.

5. Vacuum standing water from horizontal surfaces with the wet/dry vacuum containing a HEPA-rated filter.

6. Completely cover the surface with the pH-adjusted bleach solution for the desired contact time (e.g., 30-60 min); reapply as necessary to keep the surface wet.

7. Rinse the entire surface with water.

8. Vacuum standing water from horizontal surfaces with the wet/dry vacuum containing a HEPA-rated filter.

Determining the surface and overall decontamination effectiveness of the steps in this procedure, individually and in combination, was the focus of this study, including the physical removal and the inactivation of spores.

This project employed wet/dry vacuums, sprayers, brushes, sponges, nozzles, garden hoses, pressure regulators, pumps, drying pans, bleach, vinegar, and detergent, as well as carboys, buckets for DI water, and containers for mixing the pH-adjusted bleach solution and holding the detergent solution. The specifications of the materials and equipment used for the decontamination procedural steps are detailed in Table D-1 of Appendix D.

The decontamination procedural steps for each material are summarized in Table 2-1.

It was critical for this project that each step in the decontamination procedure be implemented as uniformly as possible for all coupons and tests. Changes in technique during the study could lead to highly variable and/or biased data and lead to erroneous conclusions. Therefore, the methods for each step were documented in detail in order to provide as much standardization as possible. A mock Test 1 was performed to practice each step of the full eight-step procedure listed above. Staff performing the decontamination procedures practiced each step in advance and an attempt was made to add measurable controls. Additional details can be found in Appendix D.

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Table 2-1. Procedural step summary for each material type

Material Type Step 1 (Vacuum)

Step 2 (Bleach

mist)

Step 3 (Detergent

scrub) Step 4 (Rinse)

Step 5 (Vacuum standing

water)

Step 6 (Bleach)

Step 7 (Rinse)

Step 8 (Vacuum standing

water)

Concrete (horizontal) Yes Yes Yes Yes Yes Yes Yes Yes

Carpet (horizontal) Yes Yes No No Yes Yes Nod No

Sealed deck wood

(horizontal) Yes Yes Yes Yes Yes Yes Yes Yes

Primed/painted wallboard

(horizontalc) Yes Yes Yesa Yesb Noc Yes Yesb Noc

Concrete (vertical) Yes Yes Yes Yes No Yes Yes No

Primed/painted wallboard (vertical)

Yes Yes Yesa Yesb No Yes Yesb No

Rough cut barn wood (vertical) Yes Yes Yes Yes No Yes Yes No

a Using a detergent-soaked sponge instead of a brush.

b Using a water-soaked sponge instead of a garden hose. c The wallboard in the horizontal position was positioned to represent a ceiling; therefore, it was not vacuumed. d Carpet was not rinsed since rinsing did not seem feasible using the procedures defined for this study. Any residue that may be left

due to the decontamination procedure was not removed.

The results of the testing provide information to evaluate the surface decontamination and overall effectiveness of the full eight-step procedure for removing surface contamination. To assess the effectiveness of individual procedural steps or the potential for using a simplified procedure, six different combinations of selected steps were also tested. Additionally, the testing provided information on viable spore disposition for consideration in the development of remediation strategies (e.g., when/where the procedure might be considered for application, need for water collection and treatment, estimation of waste generation). This information could be useful to inform the development of alternative decontamination methods through understanding the importance of each procedural step(s), as well as how to use the steps effectively in combination.

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2.5 Test Matrix

The test matrix shown in Table 2-2 allowed different combinations of steps of the decontamination process to be investigated independently for each surface type, consistent with the sampling strategy. Completion of the test matrix provided information to support the development, use, and/or statement of limitations of the tested decontamination procedures for surfaces, such as those included in the test matrix, that can achieve a target cleanup goal while minimizing hazardous waste and the spread of contamination.

Twelve tests were performed to evaluate six different combinations of the steps of the decontamination process in two different coupon orientations. Table 2-1 identifies each procedural step for each material type.

As the study progressed, questions about methodology and efficacy arose that were not being addressed by the study design.Three additional tests were therefore added to the test matrix:

• Sodium Thiosulfate (STS) Tests: In order to quantify the number of viable spores washed from the surface of the coupons, elimination of any potential residual active ingredient in the rinsate (i.e., killing of spores in the collected rinsate) was necessary. Deactivation of the rinsate was critical to prevent the inactivation of the spores in the rinsate collection reservoir due to continued contact with the pH-adjusted bleach in the absence of the coupon material. The viability of the spores in the rinsate and sporicidal activity of the residual pH-adjusted bleach in that rinsate are expected to be highly dependent on where the rinsate was collected (e.g., surface material). However, investigating the parameters impacting the residual sporicidal activity of the rinsate was beyond the scope of this study. Elimination of any potential residua spore-killingl activity in the rinsate in this study was desired in order to provide a worst case scenario (i.e., no further residual sporicidal activity of the rinsate). Prior to Tests 1 and 2, based upon the method development testing, dilution of the pH-adjusted bleach in the collected rinsate by the rinse water and rapid filtration was expected to be sufficient to counteract the residual sporicidal activity of the rinsate (killing of spores in the rinsate). However, in Tests 1 and 2, rinsate samples could not be filtered as rapidly as the initial test procedure development work suggested due to clogging of the filters with debris removed from the coupons and collected in the rinsate during the decontamination procedure. Additionally, the dilution of the pH-adjusted bleach in the rinsate below a sporidical concentration was questioned after Tests 1 and 2. Viable spores in the rinsate were potentially inactivated by the residual activity of a further diluted bleach solution. Prior to testing involving pH-adjusted bleach (after Test 2) or Clorox® Cleanup®, a series of suspension tests was performed to determine the impact of the potential sporicidal activity of the diluted pH-adjusted bleach in the collected rinsate either to confirm that dilution was sufficient in Tests 1 and 2 to eliminate sporicidal activity of the rinsate or to determine that neutralization of the rinsate was required. In the suspension tests, spores were added directly to a solution and then recovery of viable spores was attempted and the spores were enumerated after a desired contact time. In these tests, spores were added to solutions of pH-adjusted bleach and/or STS (to neutralize the sodium hypochlorite in the pH-adjusted bleach).

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Table 2-2. Test Matrix

Test ID

Decontamination Procedure Coupon Orientation Number of

material types Total

coupons 1a 1b

Complete decontamination process (Steps 1 -8) with a 30 minute contact time in

Step 6

Horizontal (Test 1a,1b) 4 44

2 Vertical (Test 2)

3 33

3a 3b

Steps 1-4

Horizontal (Test 3a, 3b)

4 44

4 Vertical (Test 4)

3 33

5 Step 1

Horizontal (Test 5)

4 44

6 Vertical (Test 6)

3 33

7 Steps 3, 4, and 5 combined

Horizontal (Test 7)

4 44

8 Vertical (Test 8)

3 33

9 Steps 4 and 5 combined

Horizontal (Test 9)

4 44

10 Vertical

(Test 10) 3 33

11a 11b

Steps 1, 3, 6*, 7, 8

Horizontal (Test 11a, 11b)

4 44

12 Vertical

(Test 12) 3 33

13a Steps 2 and 4

Vertical (18 mm stubs)

1 30

13b Vertical

(14 inch coupons) 1 32

STS Tests of STS effect on spores (neutralization study) N/A N/A N/A

T1 One 4-second bleach spray

Vertical (14 inch coupons)

1 5

T2 Three 4-second bleach sprays 1 5

T3 One 12-second bleach spray 1 5 T4 Positive Controls 1 5

N/A = not applicable, no coupons were used (suspension testing).

*pH-Aadjusted bleach was used in Steps 2 and 6 except in Tests 11(a/b) and 12 where Clorox® Cleanup® was used in Step 6.

Note: Each test (1-12) included five positive control coupons, five test coupons, and one procedural blank (negative control) for

each material (hence, a total of 11 coupons for each material type).

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• T Tests: These tests (T1-T4) were added to determine whether the frequency or length of spraying significantly affected the overall (i.e., sporicidal) efficacy of the pH-adjusted bleach solution on the surface of the material.

• Test 13: This test was intended to provide a means of comparison of data obtained in Tests 1-12 to previously reported findings for pH-adjusted bleach sporicidal efficacy using liquid inoculation of spores on smaller coupons, different pH-adjusted bleach application methods (fine spray or immersion), and different spore recovery procedures (extraction of spores from surfaces).

Each test was run independently, primarily to manage the number of samples submitted to the NHSRC Microbiology Laboratory per test. The odd-numbered tests (in Tests 1-12 series) involved coupons in a horizontal orientation; the even-numbered tests (in Tests 1-12 series) utilized coupons in a vertical orientation. Table 2-3 lists the orientation (horizontal or vertical) of each material type during the decontamination process. These orientations were chosen to reflect the most likely orientation of each material as it would be found in construction.

Table 2-3. List of coupon material types by orientation

Horizontal Orientation Vertical Orientation

Concrete Concrete

Sealed pressure-treated deck wood (oriented with slats parallel to the side

of the chamber)

Rough-cut wood (oriented with slats vertical)

Primed and painted wallboard (horizontal and inverted, to represent

a ceiling) Primed and painted wallboard

Carpet

• A single test included the completion of all material types within that orientation (hence, four materials for the horizontal orientation and three for the vertical orientation).

• Procedural blanks (coupons of each material not inoculated with the target spore) were run first, followed by the test coupons of each material type. A maximum of three coupons were run at a single time in the decontamination chamber. Only one material type was run at a time.

• Cleaning of the chamber was performed in accordance with Appendix G after the completion of each material type per test.

• Each test required five test inoculated coupons, one procedural blank, and five positive control coupons of each material type. A total of 11 coupons was required for each material type.

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• Wipe sampling was used on concrete, painted wallboard and sealed deck wood. Vacuum sampling was used on rough-cut wood and carpet. These methods were chosen to be consistent with preliminary findings (see Section 3.1.2), to minimize bias and to allow comparison of results.

In Tests 1 and 2, the full eight-step decontamination procedure was used with a 30-minute contact time for the bleaching step (Step 6). In Test 1a, two of the four horizontal material types were tested; Test 1b incorporated the remaining two horizontal material types. The decontamination procedure for Tests 3 and 4 included Steps 1-4 (initial vacuuming, bleach mist, detergent scrub and rinse). In Test 3a, two of the four horizontal material types were tested; Test 3b incorporated the remaining two materials.

In Tests 5 and 6, the efficacy of the wet/dry vacuuming step was evaluated. In Tests 7 and 8, the efficacy of Steps 3, 4 and 5 (detergent scrub, rinse, and vacuuming the standing water from the horizontal surfaces) was evaluated. In Tests 9 and 10, the assessment of the efficacy of removing contamination by rinsing with water (Step 4) and vacuuming standing water from horizontal surfaces (Step 5) was completed. In Tests 11 and 12, the efficacy of a modified procedure was tested. In the modified procedure, wet/dry vacuuming of surfaces (Step 1) was followed by scrubbing with the detergent solution (Step 3), maintaining the surface wetted with Clorox® Clean-Up® Cleaner with Bleach (Step 6), rinsing with water (Step 7) and vacuuming standing water from horizontal surfaces (Step 8). In Test 11a, two of the four horizontal material types were tested; Test 11b incorporated the remaining two materials.

The pH-adjusted bleach solution was prepared as described in Appendix D. The detergent solution used was prepared using TSP-substitute (DAP® T.S.P. Substitute Heavy Duty Cleaner, DAP Inc., Baltimore, MD), per the manufacturer’s directions.

In Test 13, further discussed in detail in Section 3.1.2 and Appendix I, the bleach mist and rinse step (Steps 2 and 4) remained unchanged so that the effect of the spore loading method (aerosol vs. liquid inoculation) and sampling technique (extraction vs. vacuum or wipe sampling) could be studied. The liquid- and aerosol- inoculated test coupons (14 in by 14 in) were sampled using the vacuum sock method, then re-sampled using the wipe method. The aerosol-inoculated smaller coupons (18 mm stubs) were wipe sampled only. For the positive control coupons, the liquid- and aerosol-inoculated coupons were vacuum-sock sampled and the aerosol-inoculated stubs were wipe-sampled.

The STS Tests (neutralizer confirmation studies) were conducted to determine whether any viable spores were being inactivated by contact with the further diluted pH-adjusted bleach in the rinsate prior to being plated (in Tests 1 and 2). Additionally, a method for neutralization of the rinsate that would not interfere with the culture analysis procedure was also developed. To test spore survival in a typical bleach-laden rinsate, 1 x 107 Bacillus atrophaeus spores were added to the following mixtures which were agitated to insure homogeneous mixing and held 10 minutes before three 100 mL aliquots were collected.

• Mixture 1 (S1): DI Water + Spores. ~1 x 107 spores were added to 11.5 L of DI water , a volume equal to that collected during a typical five-replicate coupon vertical test.

• Mixture 2 (S2): DI Water + Spores + STS. STS (214 mL of 1N solution) was added to 1.3 L of DI water, followed by ~1 x 107 spores and then an additional 10 L of DI water.

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• Mixture 3 (S3): DI Water + Spores + Bleach. The pH-adjusted bleach solution (1.2 L) was added to 10.3 L of DI water to which ~1 x 107 spores were then added.

• Mixture 4 (S4): DI Water + Spores + Bleach + STS. ~1 x 107 Spores were added to the 10 L of DI water containing 1.2 L of pH-adjusted bleach solution and 214 mL of 1N STS.

The T Tests were added to determine whether the frequency or length of spraying significantly affected the sporicidal efficacy of the pH-adjusted bleach solution on the surface of the material. Twenty 14 by 14 in barn wood coupons were loaded with spores in accordance with MOP 6561 (Appendix B). Five coupons were decontaminated using one of three test procedures and the final five were designated as positive controls. Prior to decontaminating the inoculated test coupons, each test procedure was applied to one blank coupon. Each rinsate carboy contained 400 mL of 1 N STS to neutralize the bleach in the runoff and prevent inactivation of any remaining viable spores. This procedure was used to simulate a worst case field situation where the residual killing power of the pH-adjusted bleach solution would be removed (i.e., due to material demand from the collection surface [e.g., soil, concrete, carpet, wood]). The rinsate from all test coupons within a single test was collected in a single carboy, and three 100 mL rinsate aliquots were then taken for each test. Likewise, the rinsate from all blanks was in a single carboy (separate from the carboy used for the test coupon rinsate) per test. Coupon surfaces were sampled in increasing order of the likely contamination level: first, blank coupons; then, test coupons; and finally, positive controls.

• Test 1 (T1): Apply one 4-second spray and then rinse with DI water after 10 min.

• Test 2 (T2): Apply three 4-second sprays at t = 0, 5, and 10 minutes, and then rinse with DI water.

• Test 3 (T3): Apply one 12-second spray and then rinse with DI water after 10 minutes.

• Test 4 (T4): Positive controls (no pH-adjusted bleach application).

2.6 Sampling and Analytical Procedures

Four types of samples were included in this project.

• Surface sampling procedures were used to collect samples from the coupon materials. The sampling procedures included wipe sampling or vacuum sampling for quantitative determination of viable spores on coupon surfaces. Additionally, wet swab sampling was done to qualitatively determine the presence of the target organism on wet/dry vacuum filters and for sterility checks of materials and equipment prior to testing.

• The rinsate generated during the decontamination procedure was collected for each material type.

• HEPA filters from the wet/dry vacuums used for each coupon set were removed and sampled by swabbing.

• A single exhaust sample was collected from the blank wet/dry vacuum during the application of the decontamination procedure to the blank coupons. Additionally, a single exhaust sample from all wet/dry vacuums was collected for the test coupons within a test.

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A sampling event log sheet was maintained for each sampling event (or test). The names of sampling team members, date, run number, and all sample codes with corresponding coupon codes were recorded on each sheet. The coupon codes were pre-printed on the sampling event log sheet prior to the start of sampling. The materials and equipment used as well as the sampling protocols for sampling are detailed in Table E-1 in Appendix E.

2.6.1 Factors Affecting Sampling/Monitoring Procedures

Sampling of coupon surfaces was conducted after coupons that were wetted during the decontamination procedure had become visibly dry. Drying was allowed to occur in the Decontaminated Coupon Cabinet or Procedural Blank Cabinet (as appropriate), facilitated by a slight air flow through the cabinet due to a constant positive pressure from a compressed air flow. The flow is from a facility air compressor, with flow filtered through a 5 μm compressed air regulator and filter (part number 9891K43, McMaster-Carr, Atlanta, Georgia). All coupons were allowed to dry for at least 18 hours. The actual time that each coupon was allowed to dry was recorded.

The recovery of spores from the material surfaces by the sampling procedure can be affected by the material type. The material surface of the rough cut wood allowed for only vacuum sampling. Preliminary sampling tests revealed that the texture of the wood caused the wipes to tear with fragments remaining on the sampling surface. In this case, wipe sampling would downwardly bias the data.

2.6.2 Preparation for Sampling/Monitoring

Sampling kits for wipes or vacuum socks were prepared as specified in the following sections. All laboratory surfaces intended for use during sampling were wiped with Dispatch® bleach wipes (Caltech Industries, Inc., Midland, MI). Precut 20 in by 20 in sheets of absorbent bench liner (Fisher p/n 14-127-47) were used to cover all work surfaces and were replaced after each phase of a test (e.g., coupon contamination is considered one phase, decontamination another, and surface sampling a third). Sampling was conducted on only one coupon at a time. One coupon was moved from the Decontaminated Coupon Cabinet (test coupons), Test Coupon Cabinet (positive controls), or Procedural Blank Coupon Cabinet (procedural blanks) to the sampling space located immediately outside (to the front) of each cabinet. All coupons were placed horizontally for sampling, regardless of their orientation during the decontamination.

Within a single test, surface sampling of the coupons was performed starting with coupons from the lowest level of contamination and ending with the highest level of contamination (i.e., all procedural blank coupons first, followed by all test coupons, and then all positive control coupons). Surface sampling was performed either by wipe sampling or vacuum sampling in accordance with the protocols included in Appendix E. The surface area for all samples was 1.27 ft2, except for carpet, which was approximately 1 ft2 because of the taped edges.

A template was used to cover the exterior 0.25 in of each coupon leaving a square (13.5 in by 13.5 in) exposed for sampling for all coupons except carpet. Vacuum sampling was performed within the taped border around the edge of the coupons which extended 0.5 in over the carpet surface. The outer 0.25 in of each coupon was not sampled in order to avoid unrepresentative edge effects.

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A sampling material bin was stocked with all appropriate items (consistent with the protocols in Appendix E) for each sampling event. The bin contained enough wipe sampling and vacuum sock sampling kits to accommodate all required samples for the specific test. An additional kit of each type was also included for backup. Enough gloves and bleach wipes needed to complete the test were available. Templates (1.17 ft by 1.17 ft with an interior opening of 13.5 in by 13.5 in) were wrapped in aluminum foil, autoclave-sterilized, and packaged in sterile bags (ten templates per bag). These bags of templates were also included with the sampling kits. A sample collection bin was used to transport samples back to the NHSRC Microbiology Laboratory. The exterior of the transport container was decontaminated by wiping all surfaces with a Dispatch® bleach wipe prior to transport from the sampling location to the NHSRC Microbiology Laboratory.

2.6.3 Surface Sampling

To assess the efficacy of the decontamination procedure, several surface sampling methods were utilized consistent with methods that have been used and are anticipated to be used in the field. These methods included the use of wipe sampling (wet wipes), vacuum sampling and swab sampling.

2.6.3.1 Wipe Sampling

Wipe sampling is typically used for small sample areas and is effective on nonporous smooth surfaces such as ceramics, vinyl, metals, painted surfaces and plastics.13 The general approach is that a moistened sterile noncotton pad is used to wipe a specified area to recover bacteria, viruses, and biological toxins.13 The protocol that was used in this project is described in Appendix E and has been adapted from the protocol provided by Busher et al.,13 Brown et al.,16 and documented in the INL 2008 Evaluation Protocols.17 None of these references provides a validated wipe procedure for Bacillus spores, as a validated sampling procedure does not currently exist. However, the procedures used and described herein have been adopted from widely used or cited sources.

2.6.3.2 Vacuum Sampling

Vacuum (sock) sampling is typically used for large, porous areas.13 A vacuum is used to entrain particulate matter in an air stream and a collection sock is used to trap these particulate materials.13 The protocol that was used in this project is described in Appendix E and has been adapted from that provided by Busher et al., 13 Brown et al.,16 and documented in the INL 2008 Evaluation Protocols.17 None of these references provides a validated vacuuming procedure for Bacillus spores, as a validated sampling procedure does not currently exist. However, the procedures used and described herein have been adopted from widely used or cited sources.

2.6.3.3 Swab Sampling

Swab sampling was used for sterility checks on coupons and equipment prior to use in the testing. The protocol that was used in this project is described in Appendix E and was adapted from the protocol provided by Busher et al.,13 Brown et al.,16 and documented in the INL 2008 Evaluation Protocols.17 None of these references provides a validated vacuuming procedure for Bacillus spores, as a validated sampling procedure does not currently exist. However, the procedures used and described herein have been adopted from widely used or cited sources. Additionally, swabbing of the wet/dry vacuum HEPA-rated filters was also performed after testing to indicate contamination with the target organism.

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2.6.4 Rinsate Collection and Sampling

The runoff from the coupons throughout the entire decontamination procedure being tested was collected for a given coupon set (material type or all blanks). After all coupons from a single set were moved to the Decontaminated Coupon Cabinet or Procedural Blank Cabinet, the chamber was rinsed with DI water using the garden hose. The sterile runoff collection carboy was labeled and the volume of liquid collected was recorded. The runoff was not neutralized for Tests 1 and 2. However, the remaining tests had 400 mL of 1N STS added to the carboy prior to collecting the runoff. Neutralization was done in order to standardize the results from all tests, i.e., any sporicidal activity of the runoff was eliminated once the runoff was captured in the carboy preventing run-to-run variability due to differences in the runoff composition. Neutralization of the rinsate was used to simulate a worst case field situation where the residual killing power of the pH-adjusted bleach solution would be removed (i.e., due to material demand from the collection surface [e.g., soil, concrete, carpet, wood]). Investigating the residual sporicidal propensity of the rinsate and the parameters impacting this residual sporicidal propensity was beyond the scope of this effort. Not neutralizing the rinsate in Tests 1 and 2 impacted the ability to determine the overall effectiveness, but not the surface decontamination effectiveness in these tests. However, the overall effectiveness could be determined from Test 3 and 4, so the project was not adversely impacted.

After collection, the rinsate was filtered immediately or 100 mL aliquots were taken using aseptic technique according to the protocol described in Appendix E. The filter-collected spores were then rinsed with DI water to remove any residual decontaminant. The filters were then submitted to the NHSRC Microbiology Laboratory for analysis at the conclusion of each entire set of tests.

2.6.5 Wet/dry Vacuum Collection Sampling Procedures

Samples from the wet/dry vacuums were taken for two purposes: to confirm contamination after use and to investigate the potential release of spores from the vacuum exhaust. To confirm contamination, the HEPA filter was sampled using a swab as described in Section 2.6.5.1. This procedure was not used to confirm a negative result since the entire vacuum was not sampled (nor was sampling the entire vacuum considered practical). To investigate the potential for release of spores in the vacuum exhaust, air samples were taken during vacuum operation as described in Section 2.6.5.2.

2.6.5.1 Wet/dry Vacuum HEPA Filter Sampling Procedure

The purpose of sampling the wet/dry vacuum after use was to confirm contamination of the unit with the target organism. The most logical place to sample was the High Efficiency Particulate Air (HEPA) filter. The filter was sampled using the swab protocol discussed in Section 2.6.3.3 and discussed in detail in Appendix E. All pleats of the filter were sampled with a single swab.

2.6.5.2 Wet/dry Vacuum Exhaust Sampling Procedure

Sampling for the target bacterial spores was done by drawing a sample from the exhaust duct connected to the vacuum only during the operation of the wet/dry vacuums. The sampling pump was turned on when the wet/dry vacuum was on and off when the wet/dry vacuum was off. This operating scheme minimized the potential for confounding organisms to accumulate on the collection filter. Sampling was done using a Via-Cell® Bioaerosol Sampling Cassette (VIA010, Zefron International, Inc., Ocala, FL). The sampling pump flow

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rate was 0.53 ft3 per min (15 Lpm) through a sterile nozzle, the size of which was determined based on the exhaust duct flow rate according to the guidelines in 40 CFR 60 (http://www.eti-usepa.com/testing_methods.htm). The total sample flow was calculated using the difference between the totalized volume reading on the dry gas meter of the sampling console before and after sample collection. Upon completion of a test, the sampling cassette was placed in a labeled sterile bag. This bag was sealed and placed inside an outer unlabeled sterile bag. This bag was sealed and then the outside surface was wiped with a Dispatch® bleach wipe. After the bleach wipe was discarded, the sample was placed inside another unlabeled sterile bag and that bag was sealed. The sample was then placed into the sample collection/transport bin to be stored prior to transport to the NHSRC Microbiology Laboratory.

2.6.6 Sample Analyses

Analyses of all samples were performed in the on-site NHSRC Microbiology Laboratory. For all sample types, phosphate buffered saline with 0.05% TWEEN®-20 (PBST) was used as the extraction buffer. After the appropriate extraction procedure, as described in Appendix E, the buffer was subjected to a four stage serial dilution (10-1 to 10-4), plated, incubated, and analyzed (CFU enumerated) in accordance with MOP 6535a (see Appendix B).

In addition to the analysis in MOP 6535a, two additional analysis procedures were used for samples resulting in less than 30 CFU/sample in the undiluted sample extract (e.g., wipe or vacuum sock in the extraction buffer). These analyses were used in order to lower the current detection limit associated with MOP 6535a. First, 1 mL of the extract was filter-plated in accordance with MOP 6565 (see Appendix B). Next, the remainder of the sample was then filter-plated in accordance with MOP 6565. The undiluted extract was refrigerated when held between any of the above mentioned analyses.

The PBST was prepared according to the manufacturer’s directions and in accordance with an internal NHSRC Microbiology Laboratory MOP 6562 (see Appendix B), dissolving one packet in one liter of sterile water. The solution was then vacuum-filtered through a sterile 0.22 µm filter unit to sterilize.

The extraction procedure used to recover spores varied depending upon the different matrices (wipes, filter socks, wet/dry vacuum filter, liquid, filter cassette). The procedures are described in Appendix E.

2.6.7 Coupon, Material, and Equipment Cleaning and Sterilization

Several management controls were put in place to prevent cross-contamination. This project was labor intensive and required that many activities be performed on coupons that were intentionally contaminated (test coupons and positive controls) and not contaminated (procedural blanks). The treatment of these three groups of coupons (positive control, test, and procedural blank) varied for each group. Hence, specific procedures were put in place in an effort to prevent cross-contamination among the groups.

Due to the amount of waste and reusable items (requiring decontamination after use) generated during this testing (e.g., sterilization bags, sampling templates, brushes, sponges, etc.), creation of a rigid plan to segregate such items was imperative. Reusable items were clearly distinguished and separated from waste items after use and put in distinct, segregated and clearly labeled locations within the testing area.

http://www.eti-usepa.com/testing_methods.htm


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During the decontamination procedure, one person (sample handler) was tasked with moving the coupons to the decontamination chamber. A different person was tasked with moving the treated coupon to the appropriate drying cabinet. Disposable laboratory coats were used by the sample handler (tasked with moving the coupons) to further minimize the potential of cross-contamination. The sample handler donned a new disposable laboratory coat after moving a complete set (i.e., 3) of test samples from the test coupon cabinet to the decontamination chamber.

All bins, buckets, and containers remained closed or covered unless in use (e.g., material being placed into or extracted from the bin, bucket or container). Adequate cleaning of all common materials and equipment was critical in preventing cross-contamination.

Each test in the experimental matrix included four primary activities. These primary activities were:

• preparation of the coupons;

• execution of the decontamination process (including sample recovery);

• sampling; and

• analysis.

Specific management controls for each of these activities are shown in Table 2-4. Appendix A details the coupon sterilization procedures and Appendix G describes the test chamber and equipment cleaning procedures.

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Table 2-4. Cleaning methods and frequency for common test materials/equipment

Material/Equipment Use Cleaning Method Frequency

Decontamination Procedure Chamber

Contain coupons during the application of the decontamination procedure being tested

Washing with the pH-adjusted bleach solution for 10 min and rinsing with DI water

Before/after each test and between test materials

Coupon Cabinets Store coupons prior to testing and/or sampling

pH-Adjusted bleach solution or wiping with Dispatch® bleach wipes for 10 min, followed by isopropanol and air drying

Before/after each test

Wet/dry vacuums Part of the decontamination procedure

Fumigation with hydrogen peroxide per product manual instructions

Before/after each test

Heads of wet/dry vacuums Part of the decontamination procedure

Fumigation with hydrogen peroxide per product manual instructions.

After each use within a test (e.g., after initial vacuuming of a coupon and again after final vacuuming of a coupon)

All work surfaces Throughout each test Wiping with isopropyl alcohol, followed by maintaining the surface wet with a pH-adjusted bleach solution for 10 minutes before wiping dry with a clean towelette; tables used for spore deposition and sampling were covered with disposable pre-cut covers.

Before/after each use (cleaning of surfaces between handling of replicate coupons during sampling; cleaning before/after moving all contaminated coupons)

31

3. Results and Discussion

The investigation of the effectiveness of decontamination steps and procedures (combinations of steps) required some initial method development prior to commencement of the execution of the test matrix shown in Table 2-2. This preliminary work included:

• development of the aerosolized spore deposition method;

• determining the appropriate surface sampling methods; and

• developing a rinsate collection, neutralization confirmation, and analysis protocol for viable spores (e.g., rinsate filtration and aliquot samples).

The results of these efforts are discussed in Section 3.1, as the data were used to develop the decontamination testing procedure. The results of the decontamination testing are reported and discussed in Section 3.2. Some additional tests were designed and conducted to aid in the explanation or comparison of current test results with prior studies using pH-adjusted bleach. The results from these tests are discussed in Section 3.3.

3.1 Preliminary Testing of Sampling Procedures

Prior to the assessment of the efficacy of decontamination steps and procedures, the primary objective of this effort, some method development was essential. Two areas requiring development were the utilization of field sampling procedures (wipe or vacuum sampling) and the capture and analysis of run-off. The test results, conclusions, and decisions impacting the subsequent decontamination testing are discussed in this section. In addition, one of the novelties of the current study is the use of aerosolized spores deposited onto the surface of materials as the target for decontamination. All prior standard or widely-used methods have relied on liquid inoculation of materials using a suspension containing the target organism at a defined/determined concentration. Prior to initiation of this effort, the development of a deposition method for aerosolized spores was required. The results of the use of this method for testing are also described in this section.

3.1.1 Development of the Aerosolized Spore Deposition Method – Sampling Results for Positive Controls

Most standard or widely-used laboratory methods to test the sporicidal efficacy of decontamination products rely on the inoculation of carriers (i.e., uniform pieces of materials, also referred to as coupons) with the target organism using a liquid suspension.18 Such methods offer the ability to inoculate the material precisely in order to maintain intra- and inter-test consistency. While there are substantial benefits to using liquid inoculation-based test methods in the laboratory measure of efficacy, questions remain as to the representativeness of the laboratory results relative to field decontamination of materials contaminated with spores via aerosol deposition. A study was undertaken to investigate this relationship for four common material types and two decontamination products (pH-adjusted bleach and fumigation with chlorine dioxide).19 That study required the development of a novel method to deposit aerosolized spores precisely onto materials at a target loading consistent with the loading used in liquid inoculation-based methods in order to determine at least a six-log reduction due to the decontamination process. The new deposition method and that study were the predecessors for the methods used for the current effort.

32

The method reported in Ryan et al.19 was modified for application to the larger coupons (14 in by 14 in) required for the current study. The procedure is described in Section 2.2.2. The target loading, based upon recovery from the positive controls, was 1E7 spores per coupon with a standard deviation (SD) of 0.5. The sampling methods used for each material were based on the results of the above-mentioned preliminary comparison test along with consultation with the Project Team.

Surface sampling results from the positive control coupons of each material demonstrate the ability of the deposition and sampling methods to meet the target criteria. Results shown in Figure 3-1 confirm approximately a 7-log recovery (on average) of viable spores from the material surfaces of the positive controls. The low RSD values for each material (Figure 3-1) indicate consistency and are well below the targeted SD values of 0.5.

Three stainless steel coupons were incorporated into each test as material control coupons. The smooth surface of stainless steel allows for optimal recovery of viable spores. The average spore recovery from the positive controls of each material fell within 1 log of the stainless steel controls with painted wallboard having the smallest deviation and concrete the largest. For each test (1-12), stainless steel coupons were loaded with spores before any other coupons -- one coupon in the middle of the contamination series and one coupon at the end. Thus, these material control coupons also verified the operation of the spore dispersion apparatus. This procedure was followed as a precaution in case anomalies became apparent in the positive controls for a particular test; no such anomalies were observed.

Spore recovery from concrete demonstrated the highest RSD of all the materials. Observation during the wipe sampling of concrete showed some complications that may have contributed to the large RSD. Despite efforts to wipe loose debris from the concrete coupons prior to sterilization, fine particles were present on the surfaces prior to sampling. During sampling, fine particles on the surface of the concrete coupon clustered together, forming larger masses that stayed behind on the surface of the coupon, presumably containing target organisms. Debris from concrete wipe samples clogged the 200 µL pipette tips used for dilution plating. These tips were graduated to allow for a visual check that the micropipettor dispensed the correct volume. While tips with a larger orifice did allow passage of concrete debris, the tips with the larger orifice were not graduated and may therefore not have provided the same replicate volumes during plating.

33

Figure 3-1. Geometric mean of spores recovered from the positive control material surfaces and relative standard deviation per material type

(Note: Carpet and rough-cut wood coupons were vacuum sampled; all other coupons were wipe sampled as discussed in Section 3.1.2.)

3.1.2 Sampling Method Evaluation

For rough and/or porous surfaces, vacuum sampling is generally considered the preferred method.13 In the current study, such surfaces include carpet, rough cut barn wood, and concrete. Limits of detection (LOD) and sensitivities derived from the comparison of application of wipes on nonporous surfaces and the vacuum on nonporous and porous surfaces indicate that vacuuming has an LOD of 400-600 CFU per sample area, comparable to wipe sampling with an order of magnitude greater sensitivity. 20 Of the literature reviewed, however, only one reference provided a direct comparison between vacuuming and wipe sampling for a porous surface (carpet).21 While wipe sampling of carpet resulted in higher collection efficiency, the level of detection was lower for the vacuuming due to the larger sampling area. For consistency with current interagency guidance in development, vacuuming was used for the carpet in this study.

Due to the lack of definitive study results or guidance for sampling the other porous materials in the test matrix, a preliminary study was conducted in which ten 14 in x 14 in coupons of each material type were fabricated, sterilized, and inoculated via aerosol deposition in accordance with the study procedures.

0

1

2

3

4

5

6

7

8

Stainless Steel Carpet Concrete Deck Wood PaintedWallboard

Rough-cut Wood

Material

Ave

rage

log 1

0 CFU

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0S

tandard Deviation

Log CFU SD of Log CFU

34

Stainless steel coupons were also included in this comparison test. Five coupons of each material type were then sampled using the wipe method and five were sampled using the vacuum sampling method. The results are shown graphically in Figure 3-2.

The results of the preliminary sampling comparison demonstrated that wipe sampling performed better by recovering higher average numbers of spores from each material type and showing lower standard deviations within material types. As a note, both methods resulted in average recovered viable spores above the targeted 1E6 spores/sample; however, values less than 1E6 were observed from some of the replicate vacuum samples as noted by the SDs indicated in Figure 3-2. By consensus of the Project Team, wipe sampling was chosen as the preferred sampling method for surface sampling for all materials with the exception of rough-cut wood and carpet. The wipe matrix was torn during the sampling of rough-cut wood and there was a concern that tearing of the wipe could cause anomalies in the data and difficulties if applied to large areas in a field decontamination event. While wipe sampling performed well on carpet, the Project Team decided that vacuum sampling would be used for large areas of carpet in a field event. Additionally, this recovery test featured spores deposited on carpet without subsequent action to move the spores into the material (e.g., washing, scrubbing, walking); i.e., this test is unrepresentative of recovery of spores after decontamination (based upon unpublished data). Therefore, vacuum sampling was chosen for rough cut wood and carpet.

Figure 3-2. Comparison of wipe sampling and vacuum sampling

1.E+00

1.E+01

1.E+02

1.E+03

1.E+04

1.E+05

1.E+06

1.E+07

1.E+08

1.E+09

stainless steel painted wallboard carpet rough-cut wood sealed deck wood concrete

Aver

age

CFU

/sam

ple

Material Type

Wipe samplingVacuum sampling

35

Another way to analyze the above data is to look at percent recovery. However, examination of percent recovery requires that the number of viable spores inoculated onto each material type be known for comparison to the number recovered by sampling. For the aerosol deposition approach, the exact amount inoculated is not precisely known, although there was some effort expended to determine this quantity (e.g., dispensing into a liquid medium). Such determination remained questionable due to potential losses (e.g., back-splashing, adherence to tubes, etc.). An alternative approach to determining the percent recovery is comparison to the recovery from a standard material, e.g., stainless steel. Percent recoveries based upon the wipe sampling results for the stainless steel plates are reported in Table 3-1.

Table 3-1. Percent recoveries as a function of material type and sampling method (referenced to the wipe sampling from the stainless steel plates)

Sampling Method Concrete Carpet Deck

Wood Painted

Wallboard Rough-Cut

Wood

Wipe 9 19 22 57 16

Vacuum 4 4 3 3 3

Typical percent recoveries for viable spores using coupon extraction (e.g., in PBST) used in other published methods have been shown to range from 31-73%, based largely upon material type. 3, 7, 22 Typical acceptable (per the study quality assurance documentation) recoveries from some materials can be as low as 1%. 23 The recoveries from these citations are for liquid-inoculated spores. The recoveries of aerosol-deposited spores by wipe or vacuum sampling are within the range of the recoveries cited above for liquid inoculation of spores onto materials recovered via extraction procedures.

3.1.3 Rinsate Collection and Analysis Procedure Method Development

Rinsate samples were composited from all replicate coupons of a particular material type per test. Quantitative analysis was performed on rinsate samples so that the magnitude of spore relocation could be assessed. The volume of run-off liquid collected for each coupon set was measured after collection. For Tests 1 and 2, all rinsate collected in the chamber was rinsed out with deionized water (using the garden hose) into the collection vessel. Due to the debris in the rinsate (from scrubbing and pressure washing the coupons), filtration took much longer than anticipated, increasing the time that the spores were subjected to the decontamination action of any residual bleach in the rinsate. STS tests (Section 2.5) were then performed to determine a better procedure for rinsate CFU enumeration in a scenario where residual bleach could be absorbed by natural sources (such as dirt or concrete). The results for these “STS” Tests are shown in Figure 3-3. The impact of the increased contact time for Tests 1 and 2, compared to subsequent tests, is discussed in Section 3.2.2.2.

36

Figure 3-3. Total CFU in the rinsate

The data presented in Figure 3-3 demonstratethat STS did not significantly impact the recovery of viable spores (DI water + STS). Left un-neutralized, the presence of pH-adjusted bleach resulted in a significant inactivation of spores in the sample (i.e., about 5 logs reduction). An identical volume of pH-adjusted bleach was neutralized by adding 1 N STS to the solution prior to adding spores. Neutralizing the bleach resulted in an increase in CFU recovered (i.e.,4 logs recovered) . However, the viable spores recovered were still only 1% of those with DI only or with STS and DI water, indicating that there is still significant inactivation of spores attributed to bleach (i.e., 3 logs reduction). Based on the results from the STS test, STS solution was added to the rinsate collection vessel before collection of rinsates after decontamination with pH-adjusted bleach to maximize the recovery of viable spores from the rinsate and better determine the fate of the spores.

3.2 Assessment of the Efficacy of Decontamination Steps and Procedures

The primary objective of this study was to measure any reduction in the number of viable spores (surface decontamination efficacy) remaining on the surface of contaminated building materials as a function of the decontamination remediation approach (individual steps and combinations of steps). In addition to reduction of contamination of material surfaces, determining the overall efficacy (accounting for viable spores transferred to other media, i.e., the ultimate fate of the spores) was also a critical measurement objective.

1.E+00

1.E+01

1.E+02

1.E+03

1.E+04

1.E+05

1.E+06

1.E+07

DI water only DI water+ STS DI water + pH-adjusted bleach

DI water+ pH-adjusted bleach +

STS

Via

ble

Spo

res

Rec

over

ed

(Mea

sure

d as

CFU

)

37

Combined, these pieces of information can inform selection or further development of appropriate situation- specific decontamination procedures. This section discusses the results from the assessment of the surface decontamination, the overall effectiveness of individual decontamination steps and procedures (combination of steps) and the ultimate fate of the spores after the steps or procedures.

3.2.1 Surface Sampling Results – Test Coupons

One of the primary objectives of this study was to assess the surface decontamination efficacy of the individual or combined decontamination procedure steps. The results are discussed first focusing on the decontamination steps or procedures across material types. An in-depth discussion of surface decontamination efficacy as a function of material type follows.

3.2.1.1 Surface Decontamination Efficacy Results by Decontamination Step or Procedure

Findings from this study show effective surface decontamination (> 6 log reduction) of material for some procedures (combinations of decontamination steps). Results for individual decontamination steps ranged from less than 1 LR to approximately 4 LR, depending on material type and process. Figure 3-4 shows the surface decontamination efficacy of the decontamination technique averaged for all material surfaces for each test (cf. Table 2-2), as calculated in accordance with Equation 1-4.

Of the procedures tested, only those incorporating pH-adjusted bleach (Tests 1-4) were highly effective (> 6 log reduction) for decontaminating all surface types tested. However, this high degree of decontamination was accomplished by a combination of both removal (viable spores transferred to another medium) and inactivation of spores (sporicidal effectiveness). Viable spores were found in both rinsate and some wet/dry vacuum exhaust samples (discussed below). When Clorox® Clean-Up® (Tests 11 and 12) was used in place of the pH-adjusted bleach, the surface decontamination effectiveness (measured as log reduction) was comparable to the surface decontamination effectiveness observed with pH-adjusted bleach on some materials; the lower average log reductions (across all material types) compared to Test 1-4 (incorporating pH-adjusted bleach) were due to decreased effectiveness on some materials (i.e., < 6 log reduction).

Vacuuming the surface of the coupons (Tests 5 and 6) resulted in less than 1 log reduction. Rinsing the coupons with deionized water using the garden hose (or sponge for wallboard) was more effective at removing viable spores from the surfaces for vertically oriented materials (Test 10) than for horizontally oriented materials (Test 9). The addition of the scrubbing procedure resulted in no change for the vertical materials (Test 8) and approximately an additional 1 log reduction of surface spores for horizontal materials (Test 7).

3.2.1.2 Surface Decontamination Efficacy Results by Decontamination Procedure and Material Type

The surface decontamination efficacy of a decontamination procedure can be dependent largely upon the specific material being treated.8 The results presented in Figure 3-4 are separated by material type in Figure 3-5. This separation allows for a thorough investigation of the impact of different material surfaces (rough and/or porous) and orientations (horizontal or vertical) that may impact the decontamination results. The data in the figure were calculated in accordance with Equation 1-1.

38

0

1

2

3

4

5

6

7

8

9

Test 1

Test 2

Test 3

Test 4

Test 5

Test 6

Test 7

Test 8

Test 9

Test 10

Test 11

Test 12

Aver

age

Log

Red

uctio

n in

Sur

face

Loa

ding

(Log

10C

FU)

Figure 3-4. Avergae surface decontamination effectiveness (measured as log reduction) across all material types for each decontamination test (error bars indicate SD, Eqn. 1-5)

39

Figure 3-5. Average surface log reduction on material surfaces per decontamination step or procedure (error bars indicated SD, Eqn. 1-5)

(v = vertical, h = horizontal)

In some instances, no viable spores were detected on the material surfaces. When no viable spores were detected, a value of 0.5 CFU was assigned as the detection limit, and the efficacy reported as greater than or equal to the value calculated by Equation 1-1. Table 3-2 reports the efficacies of the decontamination procedures on the specific material surfaces. The asterisk denotes that no viable spores were recovered from any replicate test coupons of that material type within a test. The reported log reduction therefore incorporates the substitution of the above-defined detection limit (0.5 CFU).

In Figure 3-5 and Table 3-2, not all material types were evaluated for each test (i.e., some were tested in only the horizontal or vertical orientation as documented in Table 2-3). Tests that did contain both vertically and horizontally oriented coupons of the same material type showed comparable trends for each decontamination procedure.

0

1

2

3

4

5

6

7

8

9

Test 1 & 2 Test 3 & 4 Test 5 & 6 Test 7 & 8 Test 9 & 10 Test 11 & 12

Log

Red

uctio

nConcrete-v Concrete-hPainted Wallboard-v Painted Wallboard-hRough-cut Wood-v Carpet-hDeck Wood-h

40

Step 1 (vacuuming) alone demonstrated less than a one log reduction for all material types (Tests 5 and 6). Rinsing (Steps 4-5, Tests 9 and 10) and simple washing (Steps 3-5, Tests 7 and 8) produced a 2-4 log reduction of viable surface spores. The addition of scrubbing (Step 3) increased the efficacy of decontamination only for certain materials, i.e., painted wallboard (vertical) and deck wood (horizontal). For the wallboard, the sponge was used (not the brush) and the process of wiping with the sponge was essentially done twice (once with detergent solution, once with water for rinsing). Hence, the impact of the addition of washing with the detergent (compared to doubling the rinse step) is not discernible from the testing.

In general, tests including use of pH-adjusted bleach (Tests 1-4) showed effective decontamination (>6 log reduction) on all material types. However, viable spores were still recovered on some materials for some replicate coupons as indicated in the figure (by the standard deviation) or denoted in Table 3-2. Tests using Clorox® Clean-up® (Tests 11 and 12) were effective on somewhat porous materials (carpet and painted wallboard) and less so on more porous materials (concrete and wood).

The efficacies of each decontamination technique are not additive and not necessarily proportional to effort. Scrubbing painted wallboard (Steps 3-5) twice will not boost the efficacy from 4 to 8, due to the log nature of the scale. Repeating individual steps is not expected to improve the efficacy tremendously although repetition of individual steps was not tested directly in the current effort.

41

Table 3-2. Surface decontamination effectiveness [log reduction (standard deviation)] on material surfaces per decontamination technique (listed in Test Description)

Test Number

Test Description Concrete Carpet Deck

Wood Painted

Wallboard Rough-Cut

Wood

1 Steps 1-8, Horizontal

≥ 6.57 (0.02 )

≥ 6.85 (0.13 )

≥ 7.13 (0.01 )

≥ 7.81 (0.35 )

2 Steps 1-8, Vertical

≥ 6.95 (0.01 ) ≥ 8.01

(0.01 ) ≥ 7.24 (0.01 )

3 Steps 1-4, Horizontal

≥ 7.01 (0.01 )

≥ 6.99 (0.02 )

≥ 7.22 (0.16 )

≥ 7.35 (0.81 )

4 Steps 1-4, Vertical

≥ 6.39 (0.39 ) ≥ 7.35

(0.28 ) ≥ 6.71 (0.71 )

5 Step 1, Horizontal 0.58 (0.25 ) 0.64

(0.13 ) 0.01

(0.08 ) 0.08 (0.14 )

6 Step 1, Vertical 0.31 (0.41 ) 0.20 (0.17 ) 0.78 (0.19 )

7 Steps 3-5, Horizontal 1.45 (0.37 ) 3.08

(0.29 ) 1.75 (0.17 )

8 Steps 3-5, Vertical 1.87 (0.3 ) 3.79 (0.1 ) 1.86 (0.2 )

9 Steps 4-5, Horizontal 1.92 (0.26 ) 0.87

(0.2 ) 0.91 (0.14 )

10 Steps 4-5, Vertical 2.03 (0.55 ) 2.54 (0.11 ) 2.23 (0.17 )

11 Steps 1,3-8 with Clorox® Clean-up®, Horizontal

≥ 6.23 (1.09 )

≥ 7.14 (0.13 )

≥ 5.67 (0.58 )

≥ 7.85 (0.01 )

12 Steps 1,3-8 with Clorox® Clean-up®, Vertical

≥ 6.60 (0.01 ) ≥ 7.82

(0.01 ) 5.70 (1.06 )

Note: Orange cells denote LR values that are equal to or greater than the number listed. These values are based on detection limit of 0.5 CFU when no viable spores were recovered from the test coupons. Gray cells (with no values) indicates materials that were not included in the tests indicated for that row.

42

3.2.2 Overall Decontamination Efficacy (Ultimate Fate of Viable Spores)

An overall assessment of the decontamination procedural steps not only considered the viable spores recovered from the surface of the materials, but also the viable spores recovered from the wet/dry vacuums and the rinsate.

3.2.2.1 Wet/Dry Vacuum Samples

For the wet/dry vacuums, samples collected included swab samples of the HEPA filters and samples from the air exhaust of each vacuum. Iindividual wet/dry vacuums were used for each material type (i.e., swabs from filters can be resolved by material type), but only one exhaust sample was taken from the vacuums used for the test coupons. Therefore, results are from the entire use of the wet/dry vacuum for a material type within a test, i.e., which may have included multiple vacuuming steps as detailed in Tables 2-1 and 2-2. One exhaust sample was also taken for the wet/dry vacuum used for the blank coupons. The results of the testing are reported in Table 3-3. One wet/dry vacuum was used for the blanks and one per each material type; each HEPA filter was therefore swab-sampled. The swab samples indicated the presence of contamination on the HEPA-filter with the target organism following use of the vacuum. Air samples from the exhaust were taken from each wet/dry vacuum used for the blank coupons and a cumulative sample was collected from all wet/dry vacuums used for test coupons within each test. Data are reported as CFU per liter of air sampled. The wet/dry vacuum was not used in Tests 8 and 10 (vertical coupons) or for the horizontal painted wallboard coupons (oriented as a ceiling) in Tests 7 and 9. The carpet material was not incorporated into Tests 7 and 9.

Contamination was confirmed on all HEPA filters used for test coupons with the exception of the wet/dry vacuum used on deck wood in Test 7. The log reduction from deck wood by using the wet/dry vacuum was extremely low (Table 3-2, Test 5), showing that the method was ineffective on this surface. Two of the ten HEPA filters used for the blank coupons were also confirmed as contaminated. Since the swab analysis is not precisely specific for the target spore, contamination from other background organisms cannot be ruled out.

More than half (six out of ten) of the air exhaust samples from the wet/dry vacuums showed concentrations of viable spores higher than those from the vacuums used for the blanks. In one test (Test 6), the HEPA filter was either installed incorrectly or had dislodged in the wet/dry vacuum that was used for concrete. The results generally indicate that the use of the wet/dry vacuum does have the potential to reaerosolize spores, in addition to providing minimal effectiveness at reducing target spore surface contamination.

43

Table 3-3. Wet/dry vacuum sampling results

Test Number Material

Vacuum HEPA Filter

Vacuum Exhaust (CFU/L)

Test Number Material

Vacuum HEPA Filter

Vacuum Exhaust (CFU/L)

1

Blank NG 0.012

7

Blank NG 0.043

Carpet G

0.012

Carpet NT

0.059 Concrete – h G Concrete – h G

Deck Wood G Deck Wood NG

Painted Wallboard – h G Painted

Wallboard – h NA

2

Blank NG 16

8

Blank NA NA

Concrete – v G

9

Concrete – v NA

NA Painted

Wallboard – v G Painted Wallboard – v NA

Rough-cut Wood G Rough-cut

Wood NA

3

Blank G 0.76

9

Blank NG 0.062

Carpet G

3.3

Carpet NT


Deck Wood G Deck Wood G


Wallboard – h NA

4

Blank NG 0.53

10

Blank NA NA

Concrete – v G

214

Concrete – v NA

NA Painted

Wallboard – v G Painted Wallboard – v NA


Wood NA

5

Blank G 0.085

11

Blank NG 0.027

Carpet G

0.11

Carpet G


Deck Wood G Deck Wood G


Wallboard – h G

6

Blank NG NS

12

Blank NG 0.77

Concrete – v G

284

Concrete – v G

68 Painted

Wallboard – v G Painted Wallboard – v G


Wood G

NT: Material not included in the test; NA: Not applicable NG = No Growth. G = Growth.

44

3.2.2.2 Rinsate Samples

The results for the analyses of the rinsates are shown in Figure 3-6, averaged over all material types for each test. A breakout of results by material is shown later in this section. Viable spores in the rinsate were observed in all tests (except Test 1), above background (or detection limit) levels. These values represent the total viable spores collected in the entire volume of rinsate for each material.

The rinsate was either analyzed by filtering the entire volume of collected rinsate for each coupon set within each test or by taking aliquots of those samples for analysis. The overall detection limits for these samples varied based upon the ratio of the volume sampled to total volume of rinsate collected. Sampling/analysis of rinsate is further discussed in Section 1.3.1.1.

Table 3-4 puts the detection limit or background contamination into perspective, presenting the percentage of the detection limit or background level compared to the average value for all test coupons. The tests are listed in the order in which they were performed. Most of the blank samples were at or close to the detection limit (as discussed in Section 1.3.1.1 and Section 4.6). Test 1 presented the biggest problem because the number of spores found in the rinsate from the test coupons was in the same range as the detection limit-based value from the blank samples. The difference between the blank rinsate and the test coupons drops to less than 25% in Tests 2-4. For Tests 7-12, the comparison between the blank rinsate and the test coupons is less than 3%. Additionally, for Tests 1 and 2, the rinsate was not neutralized, thereby further confounding the data analysis. For all other tests, the rinsate was neutralized in order to provide a determination of the potential of the rinsate to contain viable spores without any confounding untested variables (e.g., the material onto which the rinsate is collected).

Table 3-4. Comparison of rinsate contamination of blanks to test coupons

Test Numberc Blanks Test

Coupons Percentage

8 4.01E03b 2.63E06 0.15 10 2.67E02a 5.33E06 0.01 7 1.00E03a 2.09E06 0.05 9 7.80E03b 7.19E06 0.11 1 1.70E02* 4.31E01 395 2 2.00E01* 1.22E02 16 3 3.56E01* 2.43E02 15 4 1.01E02* 4.17E02 24

11 1.44E02* 5.03E03 2.85 12 1.12E02* 3.47E05 0.03

Notes: Tests 5-6 were vacuuming only and did not generate rinsate. *Blank values based upon detection limit of 0.5 CFU and multiplied by the ratio of volume sampled to total volume of rinsate collected. aBlank values based upon analysis of the entire collected rinsate volume and no detection above the quantitation limit. bBlank values based upon analysis of the entire collected rinsate volume and detection above the quantitation limit only for filter plate

analysis. cListed in order in which the tests were conducted.

45

Figure 3-6. Average rinsate viable spore results (total CFU per total rinsate collected for all materials in each test)

Note: Test 5-6 were vacuuming only and did not generate rinsate. The rinsate was not neutralized in Tests 1 and 2 by addition of STS (error bars indicated SD)

Figure 3-6 shows that, on average, nearly 1E7 CFU were present in the rinsate from Tests 7-10. These tests employed rinsing (Tests 9 and 10) and washing and rinsing (Tests 7 and 8) and no use of a sporicidal product (i.e., pH-adjusted bleach). As expected, the surface decontamination observed (see Table 3-2 or Figure 3-4) was a result of physically removing the spores from the materials. Washing methods without sporicide left a considerable number of viable spores on the material surfaces and transferred the contamination to the rinsate. Hence, without complete capture of the rinsate, the use of these methods can result in the spread of contamination.

Tests 3 and 4 are the most meaningful relative to the use of the procedures that included the incorporation of the pH-adjusted bleach. The surface sampling results (see Table 3-2 or Figure 3-4) demonstrate that surface decontamination was effective after Step 4 (vertical materials) or 5 (horizontal materials). The additional pH-adjusted bleach application (Step 6), rinsing (Step 7), and vacuuming (Step 8 for horizontal materials) did not increase the log reduction in surface contamination. In other words, no detectable viable spores remained on the material surfaces after Step 4 or 5 in these tests. The rinsate results from Tests 3 and 4 indicate that surface decontamination was likely not complete after Step 2. The viable spore load

0

1

2

3

4

5

6

7

8

9

Test 1 Test 2 Test 3 Test 4 Test 7 Test 8 Test 9 Test10

Test11

Test12

Ave

rage

Log

10 C

FU R

ecov

ered

in R

insa

te

Test Coupons

Blanks

46

was probably reduced on the surface due to contact with the sporicides, with residual contamination washed/rinsed off the material surfaces in the subsequent steps. Therefore, the rinsate results from these tests can be used to determine the effective inactivation of the spores on the material surfaces by the pH-adjusted bleach application, as discussed in more detail below. In general, the rinsate results suggest that 3-4 log of viable spores remain on the surface and end up in the rinsate during the wash/rinse step after the vacuum/pH-adjusted bleach application (10 minutes).

Comparing the results discussed above for Tests 3 and 4 with the rinsate results for Tests 11 and 12 indicates the lower effectiveness of Clorox® Clean-up® as a sporicide relative to pH-adjusted bleach. In the latter tests, a significantly greater number of viable spores were found in the rinsate. For some materials, the number is within one log for the use of washing/rinsing only (no sporicides). The rinsate data indicate that the primary surface decontamination process in Tests 11 and 12 was physical removal, and decontamination was not significantly due to spore inactivation.

Additional conclusions can be drawn from analyzing the rinsate data as a function of material type. This breakdown is shown in Figure 3-7 for Tests 3-4 and 7-12. In Tests 3 and 4, the carpet, painted wallboard (vertical) and rough cut wood had rinsate contamination levels at or below background. The combination of the vacuuming (Step 1) and 10-minute pH-adjusted bleach (Step 2) steps appeared to be highly effective at reducing the surface loading of viable spores. Since vacuuming has been shown to be minimally effective on these (and all) material surfaces (Table 3-2), these results indicate that the 10-minute pH-adjusted bleach step was highly effective at inactivating spores on these three surfaces. For the other materials, rinsate values for total viable spores were above the background level, showing partial effectiveness of the 10-minute pH-adjusted bleach step on these surfaces (i.e., viable spores remained after the pH-adjusted bleach step and were washed/rinsed off the surface). Of these four materials, the 10-minute pH-adjusted bleach step was least effective on the deck wood and vertical concrete surfaces. The most significant quantities of viable spores were detected in the rinsate for these two materials. Thus, the order of decreasing effectiveness of the combination of vacuuming and pH-adjusted bleach step was:

• carpet = rough cut wood = vertical painted wallboard

• horizontal painted wallboard = horizontal concrete

• deck wood

• vertical concrete.

For comparison, prior work testing the effectiveness of spray-applied pH-adjusted bleach for materials contaminated with Bacillus anthracis Ames or Bacillus subtilis spores observed that raw wood and unpainted porous surfaces were more difficult to decontaminate than non-porous and/or sealed (e.g., painted) surfaces.12 The cited work reported log reduction values of 0.81 (± 0.40), 4.99 (± 1.67), and 7.31 (± 1.27) for bare pine wood, unpainted concrete, and painted cinder block, respectively, after maintaining the materials wetted for 60 minutes using a pH-adjusted bleach spray (hand-pumped garden sprayer). No vacuuming or rinsing steps were done in the cited effort. Another study that investigated the efficacy of spray-applied pH-adjusted bleach on outdoor materials contaminated with B. anthracis Ames spores reported efficacy values of 1.90 (± 0.79), 3.60 (± 0.47), and 6.27 (± 1.49) for treated wood, asphalt, and unpainted concrete (all horizontal).3

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Tests 11 and 12 show a similar trend based upon the same analysis. If spores are viable in the rinsate, then they were not removed by the vacuuming or deactivated by the contact with the decontaminant on the coupon surface. With Clorox® Clean-up®, the least inactivation was observed for deck wood, rough cut wood, and vertical painted wallboard. The removal of the vast majority of the spores from these materials was due to a physical process (leaving the spores viable) and not inactivation via sporicidal chemical action. Rough cut wood and deck wood also exhibited the lowest overall surface log reductions in Tests 11 and 12 (see Figure 3-5). Some minimal inactivation was observed on horizontal and vertical concrete. The predominant mechanism for the observed decrease in viable spore loadings on the carpet and horizontal painted wallboard was inactivation; these materials also exhibited some of the largest log reductions in surface contamination within these two tests.

All surface log reductions exhibited in Tests 7-10 were due to physical removal, so the spores removed from the surfaces were found viable in the rinsate. There was good agreement between the quantity of spores removed from the surfaces (based upon the surface loading determined from the positive controls) and the number of viable spores found in the rinsate samples.

Figure 3-7. Rinsate viable spore results (total CFU per total rinsate collected) for each material type

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3.2.2.3 Summary of Overall Decontamination Effectiveness (Ultimate Fate Analysis)

Figure 3-8 presents the data discussed above related to surface sampling, rinsate, and air sampling in a summary format. Within the limits of the sampling analysis, this depiction provides an accounting of the fate of the spores as a percentage based on a starting loading determined from the positive controls. This summary for each test encompasses all materials and does not provide information specific to each individual material type. A summary such as this is not warranted for each material type, since vacuum exhaust samples were cumulative across all material types within a test. In accordance with Equation 1-4, four categories related to ultimate spore disposition are considered:

• Active on coupon – viable spore recovered during the surface sampling of the materials after decontamination;

• Active in rinsate – viable spores found in the rinsate from the decontamination process;

• Vacuum exhaust – viable spores collected in the post HEPA-filter vacuum exhaust sample (i.e., spores that may be re-emitted into the environment due to the vacuuming process);

• Vacuum/Inactivated – spores that have been either chemically inactivated due to the sporicides application or collected in the vacuum.

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Figure 3-8. Fate of spores per decontamination technique Values on the yellow bars represent the percentage of viable spores removed via the vacuum step and/or inactivated (as

relevant) based upon comparing amount recovered on test coupons after treatment to amount recovered from positive controls not subjected to the treatment process.

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One desired result of the decontamination process is for all viable spores to be captured in the last category (vacuum/deactivated), no longer remaining a contamination concern. However, successful decontamination can be accomplished by taking additional remediation steps to capture/treat the remaining viable spores (on surfaces, in rinsate, and in air). Data are reported in percentages in Figure 3-8 (referenced to the average of the positive controls for each test). A 90% reduction in spores is equivalent to one log reduction; a 99% reduction is equivalent to a 2-log reduction. A 6-log reduction equates to a 99.9999% reduction in viable spores. Due to the scale required in the figure to represent all four categories, the important and fine details of actual vacuum/deactivated category cannot be discerned (i.e., all tests show 100% vacuum/deactivated spores). However, the values for this category are transcribed on the bars. Again, these are average values and not necessarily reflective of the actual log reduction for each material type.

In Tests 1-4, less than 1.6E-5% (on average) of the initial viable spores remain on the surface of the material. As reported above, this value equates to greater than a 6-log reduction in surface spore loading. However, not all the spores are inactivated by the pH-adjusted bleach application(s), as viable spores are found in the vacuum exhaust samples (i.e., re-entrained in the environment) and in the rinsate. The rinsate for Tests 3 and 4 more accurately reflects the viable spores removed from the surface to the rinsate due to the use of the neutralization procedure in those tests.

Vacuuming alone (Tests 5 and 6) resulted in less than a 1-log reduction in surface spore loading, reflected in the high percentage of spores found “active on the coupon.” Similarly, the (on average) 1-2 log reduction in surface spore loading observed for rinsing (Tests 7 and 8) and washing/rinsing (Tests 9 and 10) is also best reflected by the 0.5-11% of active spores found “active on the coupon.” The transfer of viable spores to the rinsate is reflected in the 2.5 - 61% of the initial spore load found in the rinsate. The vacuum/inactivated values indicate the magnitude of the uncertainty of this kind of analysis for biological systems; attempting to account for spores in all analysis fractions in order to determine the fate of the spores is only approximate (e.g., within approximately one log) and only used for gross comparison. For example, the actual percentages of spores unaccounted for via surface sampling or rinsate analysis (hence, reported as “vacuum/inactivated”) for Tests 7-10 were 96, 96, 61, and 90, respectively, so 96% (or 1.4 log) of the initial spore load is unaccounted for using the mass balance approach presented in Equation 1-7. Some of this inaccuracy is due to the use of average values, but a finer level of analysis (i.e., by material type) is not supported by the test plan.

The decreased activity of the Clorox® Clean-up® as a sporicide compared to pH-adjusted bleach can readily be observed in Figure 3-7, Tests 11 and 12. A considerably higher percentage of viable (“active”) spores in the rinsate and viable spores on the surface were found when Clorox® Clean-up® was used, hence Clorox® Clean-up® showed less inactivation.

3.3 Impact of pH-Adjusted Bleach Spray Parameters and Other Efficacy Test Method Parameters

In order to understand the results more universally and compare these results with data from prior testing, some additional testing was performed. One question related to a more universally applied understanding of the data was the impact of changing the pH-adjusted bleach spray parameters. Several novel approaches were used in the current study to provide a more directly visible tie of laboratory efficacy testing to field application of decontamination methods (e.g., use of aerosol spore deposition instead of a liquid inoculation, use of field sampling methods instead of coupon extraction methods, and use of large coupons).

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To compare the current results with prior data from laboratory testing, an understanding of the impact of some of these novel approaches on efficacy testing needed to be developed. This section details the results and conclusions drawn from tests incorporated into the study to address some of the questions just discussed.

3.3.1 Evaluation of the pH-Adjusted Bleach Application Procedure

The T Tests were added to the test matrix in order to determine whether the frequency or length of application significantly affected the efficacy of the decontamination process using spray-applied pH-adjusted bleach. The test parameters were as follows:

• Test 1 (T1): Apply one 4-sec pH-adjusted bleach spray and then rinse with DI water using the garden hose after 10 minutes.

• Test 2 (T2): Apply three 4-sec pH-adjusted bleach sprays at t = 0, 5, and 10 minutes, and then rinse with DI water using the garden hose.

• Test 3 (T3): Apply one 12-sec pH-adjusted bleach spray and then rinse with DI water using the garden hose after 10 minutes.

• Test 4 (T4): Positive controls (no pH-adjusted bleach spay or rinsing).

Deck wood coupons were prepared consistently with the preparation procedures detailed in Section 2.1 and Bacillus globigii spores were deposited on the surface as described in Section 2.2 (in accordance with MOP 6561). For each test (T1 – T4), five positive control and five test coupons were used. One blank coupon was included in each test. For the pH-adjusted bleach, the same pressure (and, hence, flow rate), distance, and pattern were used for the spaying as discussed in Appendix D. The coupons were utilized in the vertical orientation for the tests.

Each decontamination procedure tested (T1 – T3) resulted in at least a 6-log reduction from the surface of the deck wood coupons when compared to the positive controls (T4). The results are presented in Figure 3-9. A minor impact of spray volume on effectiveness may have been observed (T1 resulted in the lowest log reduction). No impact of spray frequency was observed (compare T2 and T3).

Further, there were no notable differences in the total viable spores recovered in rinsate from the tests (Figure 3-10). The volume of spray or frequency of reapplication of the pH-adjusted bleach had no effect on the concentration of viable spores observed in the runoff after rinsing with DI water. The spores in the runoff thus may be the result of a physical removal from the surface by the rinse with DI water. However, the total number of spores recovered in the rinsate from the blank coupons was approximately 5E3 CFU. The source of this contamination could not be determined. Since this contamination is of the same order of magnitude as the number of viable spores in the test coupon rinsate, the rinsate data from these tests are questionable. This contamination would impact the ultimate fate of the spores (i.e., an attempt to determine inactivation versus physical removal due to the decontamination process), but the contamination of the blanks does not impact the conclusion that the volume of spray or frequency of reapplication had no effect on the concentration of viable spores in the runoff.

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In summary, the tests have shown that changes to the total volume or frequency of reapplication of the pH-adjusted bleach spray do not impact decontamination of the deck wood surface. Additional testing is required to optimize the spray application procedure to ensure that the procedure for application of the spray minimizes the time required for spraying and maximizes the decontamination efficacy.

Figure 3-9. Log reduction in spore loadings based on surface sampling (wipe)

(The numbers on the bars are the actual Surface Log Reduction Values determined for each Test [T1-T3])

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Figure 3-10. Total viable spores in the rinsate samples

3.3.2 Effect of Recovery Methods, Coupon Contamination Methods, and Sample Loading Density on Decontamination Efficacy Determinations

Several differences between the current tests using larger coupons (14 in by 14 in, 196 in2) and previous testing using smaller coupons (e.g., 18 mm stubs) contributed to difficulties in comparing data. Some notable differences are:

1. Standard or most common test methods use a liquid suspension of spores in which known volumes of suspension of a determined titer (CFU/mL) of spores are inoculated onto the coupon surface, compared to the use of aerosol deposition in this current study.

2. All standard laboratory methods use extractive techniques for recovery of spores from coupons compared to the wipe or vacuum sampling methods used in this study. Extractive methods utilize small-size test coupons (e.g., 0.71 in diameter of the 18 mm stubs used in a recent NHSRC study), while larger test coupons (e.g., 14 in by 14 in used here) are more logical for methods employing field sampling methods.

3. Differences in spore density (spores per area) can result from the choice of contamination procedures. In order to measure a six-log reduction, methods utilizing smaller coupons require a more concentrated spore loading (e.g., 1E7 spores over 0.4 in2 for the 18 mm coupons) compared to the loading used in this study (i.e., 1E7 spores over 196 in2 for the 14 in by 14 in coupons).

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Test 13 was subsequently added to the test matrix in an effort to provide a means to compare results from the current testing with prior results for pH-adjusted bleach application on smaller coupons. This test (Test 13) was comprised of two sub-tests. In the first sub-test, rough-cut wood coupons on the 18 mm stubs were used. In the second test, 14 in by 14 in rough-cut wood coupons were used. For both sets of coupons, pH-adjusted bleach was spray applied, followed by rinsing with DI water using the garden hose at the end of the desired contact time. See Appendix I for information on test preparation, test procedure and sampling methods.

3.3.2.1 18 mm Coupons (stubs)

The first sub test of Test 13 was added to investigate the potential impact of the spore recovery method (e.g, extraction versus wipe sampling) on log reduction determinations.

All 18 mm rough-cut wood coupons were dosed by aerosol deposition with approximatly 1E7 B.g. spores (See Appendix B – MOP 3113). These coupons underwent decontamination by spraying with pH-adjusted bleach and then rinsing with DI water after the target contact time. The pH-adjusted bleach was applied to the smaller coupons in a manner consistent with that used for the larger coupons, i.e., with the backpack sprayer with reapplicaiton every two minutes for a total of 10 minutes. The DI water rinse was done using the garden hose. Seven test coupons were directly extracted; the remaining seven coupons were wipe sampled first and then extracted. The method of extraction was consistent with a previous study conducted with the 18 mm coupons investigating the impact of liquid inoculation versus aerosol deposition of spores on decontamination studies.24

One order of magnitude higher spores were recovered from surface sampling the 18 mm coupons using wipe sampling, compared to extraction (see Figure 3-11). The positive control results for extraction resulted in an average of 1.9E6 viable spores per coupon while wipe sampling yielded a value of 2.6E7. Note that the method used to contaminate the coupons with the spores has been approximated as depositing around 1E8 spores per coupon. Extracting the stubs after the wipe sampling still resulted in a recovery of approximately 1.5E6 spores per coupon on average, a value representing approximately 24% of the viable spores collected from wipe sampling. Hence, even after wipe sampling, considerable numbers of recoverable viable spores may remain on the coupon surface. Prior results, obtained during aerosol deposition method development, also resulted in considerable numbers of viable spores being recovered in a second wipe sample of stainless steel (after an initial wipe sampling of the surface). The results for those samples are shown in Table 3-5.

54

Figure 3-11. Average Log CFU/sample for 18 mm coupons

Table 3-5: Wipe Sampling Results for Stainless Steel Coupons after Aerosol Deposition of Spores

Stainless Steel Coupon 1 Stainless Steel Coupon 2

Sampling Average CFU/sample Sampling Average

CFU/sample

First wet wipe of 14 in x 14 in

coupon 6.9E6

First wet wipe of 14 in x 14 in

coupon 7.4E6

Second wet wipe of same

coupon 1.8E6

Second wet wipe of same

coupon 1.3E6

The decontamination procedure resulted in comparable numbers of viable spores being recovered after decontamination for both the extraction and wipe sampling methods. The spore recovery methods used

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55

here did not impact the decontamination efficacy test results; i.e., comparison of data from this study can be done with other studies using consistent spore recovery methods (e.g., extraction of coupons).

3.3.2.2 14 Inch Coupons

The second sub-test of Test 13 was added ito investigate the potential impact of spore loading concentration (spores per area) and method (aerosol deposition versus liquid inoculation) on log reduction determinations.

The target Bacillus spores were deposited onto twenty 14 in by 14 in rough-cut wood coupons following the procedure outlined in MOP 6561 – Aerosol Deposition of Spores onto Material Coupons Surfaces using the Aerosol Deposition Apparatus (see Appendix B). In addition, ten 14 in by 14 in rough-cut wood coupons were each spot-inoculated with 1 mL of a liquid suspension containing approximately 1E8 viable Bacillus globigii spores per mL. Based upon the stainless steel recovery data (see Figure 3-10), approximately 5E8 viable spores (measured as CFU/mL) were deposited on the coupons by following MOP 6561. Hence, the number of viable spores initially loaded onto each coupon was approximately the same irrespective of the deposition or inoculation method used for this study.

The 14 in by 14 in coupons were decontaminated in the same manner as the 18 mm coupons (i.e., sprayed with pH-adjusted bleach every 2 minutes for a total contact time of 10 minutes and then rinsed with DI water using the garden hose). For the positive controls:

1. Five of the ten liquid-inoculated coupons were sampled using the vacuum sock method, then re-sampled using the wipe method.

2. Five of the twenty aerosol-deposited coupons were vacuum sock sampled, then re-sampled using the wipe method.

3. An additional five of the twenty aerosol-deposited coupons were sampled using only the wipe method.

The test coupons were also sampled in this manner after the decontamination treatment:

1. The remaining five of the ten liquid-inoculated coupons were sampled using the vacuum sock method after drying under ambient conditions overnight (after decontamination), then re-sampled using the wipe method.

2. Five of the remaining ten aerosol-deposited coupons were vacuum sock sampled after drying at ambient conditions (after decontamination), then re-sampled using the wipe method.

3. An additional five of the remaining ten aerosol deposited coupons were sampled using only the wipe method after drying at ambient conditions (after decontamination).

For liquid inoculation, with a stock enumeration of 5E8 CFU/mL (i.e., correspondiong to 5E8 CFU per coupon), the vacuum sock positive controls returned viable spore counts of approximately 2E8 (Figure 3-12) corresponding to approximately 40% recovery. Positive control data are available to correlate to the initial

56

sampling of a coupon, but not subsequent sampling. Therefore, as noted in the figure, positive controls are not available for “wipe after vacuum” samples.

For the aerosol-deposited coupons, vacuum and wipe sampling of the positive controls resulted in averages of 1.04E7 (23% RSD)and 3.94E6 (36% RSD) CFU per coupon, respectively. Since the number of spores deposited on the surface cannot be determined exactly (unlike with the liquid inoculation procedure), calculation of recoveries cannot be made. In this case, the results from the vaccum sampling were statiscally significantly higher than those of the wipe sampling for rough cut wood.

The data from the positive controls confirmed that intial loadings on the coupons (based upon recovered spores by vacuum sampling) were roughly equivalent for the liquid and aerosol deposited coupons. This equivalence allows for a comparison of decontamination via spray-applied pH-adjusted bleach as a function of aerosol deposition of spores onto surfaces versus liquid incoculation of materials. The latter method has been a key protocol of laboratory efficacy test methods due to the ease of application, precision, and repeatability. Until the development of the spore deposition method in the current work, viable alternatives to liquid inoculation providing comparable precision and repeatability necessary for efficacy test methods did not exist.

Data from the 14 in by 14 in coupons that were inoculated with the liquid spore suspension demonstrate a 1.1 log reduction of viable spores for the liquid inoculated coupons using vacuum sampling (Figure 3-12: compare positive control versus test coupons for vacuum sampling, liquid inoculation). However, the same decontamination procedure performed on the aerosol deposited coupons resulted in a much greater reduction in viable spores on the surface of the coupon, for either wipe or vacuum sampling. For wipe sampling, a 5.8 log reduction was observed; for vacuum sampling a 6.7 log reduction was determined. The lower log reduction for wipe sampling may be due to the higher efficiency of spore recovery for that sampling method compared to the use of the vacuum (although no data are available to support this hypothesis). However, since the difference is relatively small (e.g., within a log), this hypothesis cannot be determined definitively without considerable repeat testing (i.e., to overcome other factors contributing to variability).

For the liquid inoculated coupons, wipe sampling of the test coupons after vacuum sampling resulted in the recovery of a number of viable spores (measured as CFU) comparable to the number of viable spores recovered from the preceding vacuum sock samples. These results again demonstrate the inefficiency of current field sampling methods and the high loadings of spores that may remain after initial sampling.

The vacuum sock and wipe samples from aerosol-inoculated positive control coupons yielded results that were comparable to the positive control 18 mm coupons (also loaded with spores via aerosol deposition). These data suggest that spore loading, concentrated or dilute, may have no discernible impact on spore recovery efficency for either wipe or vacuum sock sampling methods. However, this study was limited to two high loadings. A significant impact of coupon size on decontamination efficacy was observed. The log reductions on the 14 in by 14 in coupons were notably higher than the 4 log reduction oberserved for the 18 mm coupons. Note that the 18 mm and 14 in by 14 in coupons had approximately equivalent starting spore loads based upon positive control recoveries (independent of sampling method). The concentrated spore loading on the 18 mm coupons may have enabled the exposed layer of spores to protect the underlying layers during the pH-adjusted bleach application, thereby reducing the overall efficacy of the decontamination procedure.

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The rinsate data for both sub-tests of Test 13 indicate that approximately 1E7 viable spores are relocated from the coupon surface into the rinsate (Figure 3-13). These quantites are comparable to the numbers of viable spores recovered from surface sampling the positive control coupons, i.e., the starting loading on the surface of the coupons. Therefore, the observed surface decontamination discussed above for both the 18 mm and 14 in by 14 in coupons was largely due to washing the spores off the surface and not due to inactivation of the organism, an important factor to consider in use or further development of this procedure. Failure to handle the rinsate appropriately may present the potential for contamination. The rinsate was neutralized immediately during collection to remove any confounding parameters of inactivation of spores by residual decontaminant in rinsate (e.g., material onto which the rinsate settles). Hence, the rinsate provides the maximum potential for contamination that may be expected in the rinsate in this experiment. Additionally, the 18 mm blank rinsate sample had a minor amount of contamination consistent with the target organism. However, this contamination is less than 0.09% of the total CFU found in the test coupon rinsate sample.

Figure 3-12. Average CFU/sample for 14-in coupons

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Figure 3-13. Viable spores in the rinsate

In summary, the recovery of spores from the wood surface was not significantly dependent upon sampling type (extraction, wipe, or vacuum) or coupon contamination method (liquid inoculation or aerosol deposition of spores). Therefore, the methods used for spore recovery (consistent with those used in this study) do not impact the comparison of data to other studies using pH-adjusted bleach. Highly significant numbers of spores (i.e., same order of magnitude as initial sampling results) were still recoverable by additional wipe sampling or via extraction after initial sampling (wipe or vacuum). However, wood coupons contaminated via liquid inoculation were considerably more difficult to decontaminate than the same material with spores deposited as an aerosol (> 6 LR for aerosol inoculated compared to <1 LR for liquid inoculated). Liquid inoculation methods may result in considerably conservative estimates of log reduction for material surfaces contaminated by aerosol deposition. In both cases (liquid inoculation and aerosol deposition) in this study, surface decontamination was primarily due to physical removal of spores and not inactivation of the organism, resulting in a contaminated rinsate.

3.4 Assessment of Operational parameters

3.4.1 Time

The time required to decontaminate a batch of coupons depended on the decontamination procedure being applied. The full 12-step procedure with the 30 minute pH-adjusted bleach treatment was the most time consuming, requiring approximately 1 hour per batch or 1 hour per 4 ft2. A typical decontamination day

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would start with about 1.5 hours of preparation that included triple-rinsing then filling the DI water reservoir, decontaminating the decontamination chamber, and preparing the pH-adjusted bleach solution. Since the pH-adjusted bleach solution had to be used within 3 hours of preparation, a second batch, taking approximately 20 minutes to prepare, was usually necessary. The full 12-step procedure with the 30 minute pH-adjusted bleach treatment allowed for a maximum of two materials (13.6 ft2) to be decontaminated per day.

Since special care was taken to prevent cross-contamination and produce repeatable and documented decontamination steps, the procedures used in this study may provide an underestimate of field-scale productivity per person. Field scale personnel, though, may be hampered by Level C suits and supplied air respirators (see Section 3.4.3). The PPE used in the field may perhaps be more restrictive than the PPE used in this study.

3.4.2 Physical impact on materials

Wallboard, barn wood, and concrete showed no signs of physical changes after being decontaminated. The combination of bleaching and scrubbing caused the seal on the deck wood coupons to weaken in places. The bleach treatment slightly faded the color of the carpet coupons.

3.4.3 Impact on the remediation crew

For this study, the decontamination steps were performed by personnel outside the chamber housing the coupons because of the high concentration of chlorine (Cl2) gas generated by the pH-amended bleach. Based upon chlorine concentrations during a field-scale event, respiratory protection may be required. The measured chlorine concentrations in the space may determine whether OSHA Level C air purifying respirators or supplied air (e.g., Self Contained Breathing Apparatus) might be required. The full 12-step procedure with the 30 minute pH-adjusted bleach treatment, while laborious at times, required only a moderate level of exertion from the test remediation crew. The team member applying the decontamination procedure experienced the highest level of physical exertion. However, there were ample opportunities for rest between applications. Even so, all crew members were exhausted at the end of a 10-hour day. Contrast this level of exhaustion to a field event where there is the added impact of wearing a respirator and Level C suit, and decontamination showers would have to be used for each break. An upcoming study using medium sized coupons will give much more representative data on remediation crew fatigue.

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4. Quality Assurance and Quality Control

This project was performed under an approved Category III Quality Assurance Project Plan titled Assessment of Liquid and Physical Decontamination Methods for Environmental Surfaces Contaminated with Bacterial Spores, Part 1-Development and Evaluation of the Decontamination Procedures Steps (July 2009).

4.1 Calibration of Sampling/Monitoring Equipment

Standard operating procedures were availabe for the maintenance and calibration of all laboratoryoratory equipment. All equipment was verified as being certified calibrated or having the calibration validated by the EPA on-site (RTP, NC) Metrology Laboratory at the time of use. Calibration of instruments was done at the frequency shown in Table 4-1. Any deficiencies were noted. The instrument was adjusted to meet calibration tolerances and recalibrated within 24 hrs. If tolerances were not met after recalibration, additional corrective action was taken, possibly including the replacement of the equipment.

Table 4-1. Instrument calibration frequency

Equipment Calibration/Certification Expected Tolerance

Thermometer

Compare to independent NIST thermometer value once per quarter (the NIST thermometer is recertified annually by either NIST or an International Organization for Standardization (ISO)-17025 facility)

±1 °C

pH meter Perform a 2-point calibration with standard buffers that bracket the target pH daily. ± 0.1 pH units

Stopwatch Compare against NIST Official U.S. time at (http://nist.time.gov/timezone.cgi?Eastern/d/-5/java) monthly.

±1 second/min

Clock Compare to office U.S. Time @ www.NIST.time.gov at the start of each test (before coupon loading).

±1 min/30 days

Pressure Gauge Compare to independent NIST pressure gauge annually. +2 psi

Sampling Pump Flow Rate

Compare to a NIST certified and calibrated soap bubble meter monthly + 1 Lpm

4.2 Data Quality Indicator (DQI) Goals

Target acceptance criteria for the critical measurements are shown in Table 4-2 with precision goals.

http://nist.time.gov/timezone.cgi?Eastern/d/-5/java

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Table 4-2. Acceptance criteria and test values for critical measurements

Measurement Parameter

Target Value Test Value

Target Precision RSD (%)

Test Precision RSD (%)

FAC in PH-adjusted

bleach solution

6000-6700 ppm

6010-6671 ppm ± 1.6 ± 4

pH of pH-adjusted

bleach solution

6.5 < pH < 7.0

6.18 < pH < 6.99 ± 1 ± 2

Temperature of liquids 18 – 24 oC 23.6 – 27.2

oC ± 2 ± 5

Head pressure of rinse water 60 psi 60 psi ± 3.3 0

Pressure of backpack

sprayer used for pH-

adjusted bleach

spraying

35 psi 35 psi ± 6 0

Flow rate of pH-adjusted bleach spray

900 mL/min 1269 mL/min ± 5 ± 4

Positive control CFU

1E7 CFU/coupon

1E7 CFU/coupon 50 6

Test coupon CFU

30-300 CFU per plate

30-300 CFU per plate 20 < 20

4.2.1 Free Available Chlorine (FAC) Measurements

The Hach High Range Bleach Test Kit was used to titrate a standard solution of 1000 ppm NaClO2. The Hach test kit returned a value within 10% of the standard.

4.2.2 pH Measurements

The Oakton pH probe was calibrated with certified pH 7.0 buffer solution per manufacturer’s instructions at the start of each test day.

4.2.3 Temperature Measurements

Temperature measurements were taken at least once during a test day. Initially, the DI H2O reservoir was filled 3 days prior to testing. The DI water would be left over the weekend, during which time the water would reach a room temperature of approximately 27 °C. Additions to contamination prevention protocol required

62

the DI water reservoir to be filled the day of testing. Therefore, the water temperature was allowed to remain outside specification.

4.2.4 Pressure Measurements

All pressure measurements were consistently within specification.

4.2.5 Flow Measurements

The backpack sprayer did not allow the required flow rate and spray diameter to be achieved simultaneously. Therefore, the flow rate was adjusted to approximately 1270 mL/min to allow for a 10” spray diameter at the material surface.

4.2.6 CFU Counts

CFU enumerations by a second individual fell within 10% of the initial count.

4.3 Technical Systems Audit

A technical systems audit (TSA) was conducted by the EPA QA Manager and her designees to ensure that the tests were being performed in accordance with the QAPP. As part of the audit, the EPA QA Manager and her designee reviewed the reference sampling and analysis methods used, compared actual test procedures with those specified in the test/QA plan, and reviewed data acquisition and handling procedures. All comments and concerns were recorded by the EPA QA Manager and her designee. ARCADIS staff compiled written responses to the EPA QA Manager’s comments and included descriptions of how the concerns would be addressed.

ARCADIS also conducted an internal assessment during a mock run prior to testing.

4.4 Data Quality Audit

At least 10% of the data acquired during the investigation was audited. The ARCADIS QA Manager traced the data from the initial acquisition through reduction to final reporting, to ensure the integrity of the reported results. All calculations performed on the data undergoing the audit were checked.

4.5 QA/QC Reporting

QA/QC procedures were performed in accordance with the QAPP and the test/QA plan for this investigation

4.6 Deviations from the QAPP

The clogging of filters proved to be a recurring problem during the rinsate filtration process. Particulate material would clog the filters so that they would either become entirely clogged and filtration would cease or they would become partially clogged causing the filtration of one sample to take an excessive amount of time (e.g., 4 hours). In response, the alternative method of filter plating three 100 mL aliquots of the rinsate samples was implemented.

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The addition of STS to the rinsate carboys was employed to prevent further deactivation of viable spores in the rinsate caused by excess pH-adjusted bleach. This step was used for all tests including pH-adjusted bleach with the exception of Tests 1 and 2.

Drying pans were omitted during the coupon drying process due to space restrictions in the cabinets. The pans were used only for the stainless steel positive control coupons as a means of transportation without handling the surfaces.

Tests 3 and 4 employed Steps 1-4 instead of the full decontamination procedure with a 60 minute contact time for pH-adjusted bleach as originally planned. In Tests 1 and 2, the full procedure, which included a contact time of 30 minutes for the pH-adjusted bleach step (Step 6), resulted in complete decontamination of all material surfaces. Hence, testing with a longer contact time did not make sense. In response to these findings, Steps 1-4 only were used as the procedure for Tests 3 and 4.

The 70 µL screen was omitted during rinsate collection. Initially, the rinsate was collected in the chamber. Then, using a vacuum pump, the rinsate was transferred through sterile vacuum tubing into a collection carboy. The purpose of the screen was to prevent large particulate from settling in the tubing during this transfer. However, prior to testing, the decontamination chamber was raised to allow the carboys to fit directly under the drain, thus rendering the screen and vacuum tubing superfluous.

During the tests that did not include pH-adjusted bleach, Spore-Klenz® was used to decontaminate the decontamination chamber between materials. The ventilation system needed for the use of pH-adjusted bleach had not yet been installed in the chamber and Spore-Klenz® proved to be a safer alternative.

Stainless steel coupons were included in all tests to serve as material controls and as indicators of spore loading consistency.

All reusable items (e.g., buckets, gaskets, vacuums, etc.) were decontaminated using STERIS VHP® sterilization.

The pH-adjusted bleach was applied at a rate of approximately 1270 mL/min, not the 900 mL/min stated in the QAPP. This change was due to the limitations of the backpack sprayer, which was incapable of reaching the rate of 900 mL/min while maintaining a spray diameter of 10 in at a distance of one foot from the material surface.

Test 13 was added as the final test to provide data to be used for a comparative analysis on coupons of differing dimensions and inoculation methods.

The DI water temperature of Tests 1-2 and 7-10 was not within the specified 23.6 °C to 27.2 °C range. This variation was due to the DI water being allowed to reach room temperature by filling the water reservoir 2-3 days prior to use. Contamination prevention measures were enacted that required the reservoir to be filled the day of testing, thereby preventing the increase in temperature.

Samples taken from the blank (uninoculated) coupons were expected to yield counts at the detection limit, i.e., exhibit no growth of target organism to provide evidence that any growth of spores on test coupons was not a result of contamination. In fact, all blank surface samples resulted in non-detectable or CFU counts

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less than the stated analytical detection limit (30 CFU per plate) at the lowest spread plate dilution. The non-detectable spore count invokes the use of the defined detection limit of 0.5 CFU, which equates to 100 CFU/sample when multiplied by the dilution factor (0.1 mL of a 20 mL sample, see Section 3.1.1.1).

However, an attempt was also made to lower this detection limit for this work by filter plating all samples resulting in less than 30 CFU on the lowest dilution spread plate. This revised analytical procedure resulted in the entire sample being analyzed and not just a fraction of the sample extract (as in other standard or published methods).4, 5, 9, 12 Using this revised procedure, contamination with the target organism was sometimes detected on blank coupons. The results from this analysis are reported in the data reports shown in Appendix G. Figure 4-1 shows the level of contamination per material for each test.

Figure 4-1. Viable spores on Blank samples

While undesirable, viable spores on blank samples are strictly significant only when the contamination is on the same order of magnitude as the test coupons or when spores are more numerous on the blank samples than on the test coupons. Figure 4-2 shows a foreshortened graph of the ratio of viable spores on test coupons to viable spores from the blank coupons. Contamination on blank coupons is shown to be significant for Test 3, Test 11, and Test 12, for painted wallboard samples. For these tests, the presence of viable spores on the blank coupons could be a result of cross-contamination, rather than remaining after decontamination. This contamination prevents stating that residual contamination on the test coupons, if

1.00E-01

1.00E+00

1.00E+01

1.00E+02

1.00E+03

1.00E+04

Test 1 Test 2 Test 3 Test 4 Test 5 Test 6 Test 7 Test 8 Test 9 Test 10 Test 11 Test 12

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detected, is due to ineffective decontamination of the surface. The reported LR for these tests should be considered a minimum for that surface.

Figure 4- 2. Viable spores on Blank samples

Additionally, as discussed in Section 3.2.2.2, the rinsate collected from the decontamination of the blank coupons had some contamination with the target organism for some tests. Average detection limit or background values ranged from 20 (Test 2) to 7800 (Test 9) CFU. In no instances was the target organism found to be present at levels above the quantitation limit of 30 CFU for dilution plating of aliquots of samples of rinsate collected from the blank coupons. For blank samples in which the entire rinsate volume was filtered (Tests 7-10), all samples resulted in no detection of viable spores or of values for detection of viable spores below the quantitation limit for dilution plating. As discussed in Section 4.6, an attempt was made in this work to reduce the detection limit over prior published methods4, 5, 9, 12 by the use of filter plating samples with values below the quantitation limit for dilution plating. Filter plating samples with values below the quantitation limit resulted in the detection of contamination of some blank samples with the target organism. Results for sampling of DI water, blank coupon surfaces, materials and equipment, the test chamber, and laboratory controls suggested that such materials were not the source of the background contamination. Cross-contamination from other samples was also not a likely contributor, confirmed by a targeted investigation for background contamination.

1.00E-01

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1.00E+01

Test 1 Test 2 Test 3 Test 4 Test 5 Test 6 Test 7 Test 8 Test 9 Test 10 Test 11 Test 12

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Specifically, low level contamination was observed in Tests 8 and 9 where the entire collected rinsate volume was filtered and filter plating analysis was used (i.e., non-detects were reported for dilution plating of the extracted filters). As discussed in Section 3.2.2.2, this low level contamination was orders of magnitude lower than the test coupons for those tests. Therefore, the detected spores in the blank rinsate did not impact the quality of the test data for these tests.

67

5. References

1. Sharp RJ, Roberts AG. Anthrax: the challenges for decontamination. Journal of Chemical Technology and Biotechnology 2006;81:1612-1625.

2. Snook C, Cardarelli J, Mickelsen R, et al. Medical Toxicology and Public Health: National Decontamination Team, U.S. Environmental Protection Agency (EPA). Journal of Medical Toxicology 2008;4:289-291.

3. Chick H. The Process of Disinfection by Chemical Agencies and Hot Water. Journal of Hygiene 1910;10:237-286.

4. ASTM International. Standard Test Method E-2414-05. Quantitative sporicidal three-step method (TSM) to determine sporicidal efficacy of liquids, liquid sprays, and vapor or gases on contaminated carrier surfaces. West Conshohocken, PA: American Society for Testing and Materials; 2005.

5. AOAC International. Method 2008.05 Determining efficacy of liquid sporicides against spores of Bacillus subtilis on a hard nonporous surface using the quantitative three step method (TSM). In; 2008.

6. Rogers JV, Richter WR, Choi YW, et al. Evaluation of spray-applied sporicidal decontamination technologies. Washington, D.C.: U.S. Environmental Protection Agency; 2006 September. Report No.: EPA 600-R-06-146.

7. Rastogi VK, Wallace L, Smith S, et al. Quantitative method to determine sporicidal decontamination of building surfaces by gaseous fumigants, and issues related to laboratory-scale studies. Applied and Environmental Microbiology 2009;75:3688-3694.

8. Rogers JV, Sabourin CLK, Choi YW, et al. Decontamination assessment of Bacillus anthracis, B. subtilis, and Geobacillus stearothermophilus spores on indoor surfaces using a hydrogen peroxide gas generator. Journal of Applied Microbiology 2005;99:739-748.

9. Tomasino SF, Pines RM, Cottrill MP, et al. Determining the efficacy of liquid sporicides against spores of Bacillus subtilis on a hard nonporous surface using the quantitative three step method: Collaborative study. Journal of AOAC International 2008;91:833-852.

10. Sagripanti J-L, Carrera M, Insalaco J, et al. Virulent spores of Bacillus anthracis and other Bacillus species deposited on solid surfaces have similar sensitivity to chemical decontaminants. Journal of Applied Microbiology 2006;102:11-21.

11. Lee SD, Ryan SP, Snyder EG. Development of Aerosol Surface Inoculation Method for Bacillus Spores. Applied and Environmental Microbiology 2010;77:1638-1645.

12. Wood JP. Evaluation of liquid and foam technologies for the decontamination of B. anthracis and B. subtilis spores on building and outdoor materials. Washington, D.C.: U.S. Environmental Protection Agency; 2009 November. Report No.: EPA/600/R-09/150.

13. Busher A, Noble-Wang J, Rose L. Surface sampling. In: Emanuel P, Roos JW, Niyogi K, eds. Sampling for biological agents in the environment. Washington, DC: ASM Press; 2008:95-131.

14. Brown GS, Betty RG, Brockmann JE, et al. Evaluation of a wipe surface sample method for collection of Bacillus spores from nonporous surfaces. Applied Environmental Microbiology 2007;73:706-710.

15. Nalipinski M. After Action Report – Danbury Anthrax Incident (U.S. EPA Region 1, September 19, 2008). Boston, MA: U.S. EPA Region 1; 2008.

16. Brown GS, Betty RG, Brockmann JE, et al. Evaluation of a wipe surface sample method for collection of Bacillus spores from nonporous surfaces. Appl Environ Microbiol 2007;73:706-710.

17. Ryan SP, Calfee MW, Wood JP, et al. Reserach to Support the Decontamination of Surfaces and Buildings Contaminated wiht Biothreat Agents. In: William A. Rutala PD, M.P.H., ed. Disinfection, Sterilization, and Antisepsis. Washington, DC: APIC; 2010:260-306.

18. U.S. EPA. Determining the efficacy of liquids and fumigants in systematic decontamination studies for Bacillus anthracis using multiple test methods. Washington, D.C. : U.S. Environmental Protection Agency,; 2010. Report No.: EPA/600/R-10/088.

19. Ryan SP. Comparison of the decontamination of biological threat agents on building materials (in preparation. Washington, D.C.: U.S. Environmental Protection Agency; 2010. Report No.: publication pending.

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20. Brown G. Evaluation of surface sample collection methods for Bacillus Spores on porous and nonporous surfaces. In: Agency USEP, editor. Workshop on Decontamination, Cleanup and Associated Issues for Sites Contaminated with Chemical, Biological, or Radiological Materials; 2006; Washington, DC: EPA/600/R-06/121; 2006.

21. Estill CF, Baron PA, Beard JK, et al. Recovery efficiency and limit of detection of aerosolized Bacillus anthracis Sterne from environmental surface samples. Applied and Environmental Microbiology 2009; 75:4297-4306.

22. Rogers JV, Richter WR, Choi YW, et al. Technology Evaluation Report: Evaluation of sporicidal decontamination technology, Saber Technology services chlorine dioxide gas generator. Washington, D.C.: U.S. EPA; 2006. Report No.: EPA/600/R-06/048.

23. U.S. EPA. Systematic Investigation of Liquid and Fumigant Decontamination Efficacy against Biological Agents Deposited on Test Coupons of Common Indoor Materials. Washington, D.C.: U.S. Environmental Protection Agency,; 2011. Report No.: EPA/600/R-11/076.

24. Ryan SP, Lee S, Betancourt D, et al. Comparative sporicidal efficacy testing using liquid inoculation and aerosol deposition. In: Biothreat Agent Workshop. Chapel Hill, NC; 2009.

25. Anthrax spore decontamination using bleach (sodium hypochlorite). (Accessed January 26, 2010, at http://www.epa.gov/opp00001/factsheets/chemicals/bleachfactsheet.htm.)

26. Sanderson WT, Stoddard RR, Echt AS, et al. Bacillus anthracis contamination and inhalational anthrax in a mail processing and distribution center. Journal of Applied Microbiology 2004;96:1048-1056.

27. Brazis AR, Leslie JE, Kabler PW, et al. The Inactivation of Spores of Bacillus globigii and Bacillus anthracis by Free Available Chlorine. Appl Environ Microbiol 1958;6:338-342.

28. Babb J, Bradley C, Ayliffe G. Sporicidal activity of glutaraldehydes and hypochlorites and other factors influencing their selection for the treatment of medical equipment. Journal of Hospital Infection 1980;1:63-75.

29. Cousins C, Allan C. Sporicidal properties of some halogens. Journal of Applied Bacteriology 1966;30:168-174.

30. Brown GS, Betty RG, Brockmann JE, et al. Evaluation of vacuum filter sock surface sample collection method for Bacillus spores from porous and non-porous surfaces. Journal of Environmental Monitoring 2007;9:666-671.

31. Brown GS, Betty RG, Brockmann JE, et al. Evaluation of rayon swab surface sample collection method for Bacillus spores from nonporous surfaces. Journal of Applied Microbiology 2007;103:1074-1080.

http://www.epa.gov/opp00001/factsheets/chemicals/bleachfactsheet.htm.

Appendix A – Page 1

Appendix A

Coupon Sterilization

Coupon Sterilization The coupons underwent sterilization using a STERIS VHP®

sterilization cycle. This cycle entails the use of a STERIS VHP®

ARD hydrogen peroxide (H2O2) generator. The coupons were individually enclosed in H2O2

vapor-permeable sterilization bags (General Econopak, Inc.; Steam Component Autoclave Bag, White, 20" X 20"; Item # 62020TW) and exposed to H2O2 at 1000 ppmv for 60 minutes by maintaining this constant concentration in a 47 ft3 PVC chamber containing the coupons. This chamber was previously used for material and equipment testing and is described in the QAPP entitled, “Compatibility of Material and Electronic Equipment with Chlorine Dioxide Fumigation” (Approved July 2007). The coupons were sterilized in batches. The number of coupons per batch was determined so that all coupons in the chamber were exposed to the vapor without shielding (e.g., no coupons were physically on top of others) and appropriate mixing of the H2O2 occurred in the chamber. After sterilization, coupons of the same type were placed in a sterile container for storage prior to use and transport to the testing location. The container was marked with the contents, including the date of sterilization. One coupon of each material type and sterilization cycle was sampled according to the sterilization sampling procedure described in Appendix D. The samples from each material were analyzed qualitatively for the presence of any potentially confounding contamination. Batches found to have the presence of contamination were re-sterilized. If after a second sterilization cycle the batch was determined to still be contaminated, all coupons from the batch were discarded.

Test parameters such as temperature, relative humidity and concentration were monitored and recorded to ensure STERIS’s defined quality standards were met. The quality of the cycle was considered in compliance with STERIS’s label as long as all parameters were within the manufacturer’s specifications.

The STERIS VHP® sterilization cycle described above was determined to be inadequate for the sterilization of the concrete coupons. These concrete coupons were sterilized by steam autoclave utilizing a gravity cycle program consistent with an NHSRC Microbiology Laboratory internal MOP 6533 (included in Appendix B). Confirmation of sterilization was done as described above with respect to the coupons sterilized using the STERIS VHP® sterilization cycle.

Trial coupons were prepared and sterilized as preliminary checks of the method for each material. After sterilization, coupons were assessed to determine if any subsequent changes had occurred (e.g., color, brittleness, noticeably weakened). No changes occurred.

In addition to the test materials, MDI control check coupons made of stainless steel (1.17 ft by 1.17 ft) were also used. These coupons were sterilized prior to use by steam autoclave utilizing a gravity cycle program consistent with an NHSRC Microbiology Laboratory internal MOP 6533. Confirmation of sterilization was done by sampling.

Appendix B – Page 1

Appendix B

Miscellaneous Operating Procedures (MOPs)

MOP 3113: Procedure for Deposition of Bacillus subtilis Spores Using a Metered Dose Inhaler

MOP 6570: Use of Steris Amsco Century SV 120 Scientific Prevacuum Sterilizer

MOP 6533: Equipment (Non-Liquid Materials) Sterilization Using an Autoclave

MOP 6535a: Serial Dilution: Spread Plate Procedure to Quantify Viable Bacterial Spores

MOP 6561: Aerosol Deposition of Spores onto Material Coupon Surfaces Using the Aerosol Deposition Apparatus

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 6568: Aseptic Assembly of Wipe Kits


MOP 3113

TITLE: PROCEDURE FOR DEPOSITION OF BACILLUS SUBTILIS SPORES USING A METERED DOSE INHALER

SCOPE: This MOP describes a process for deposition of aerosolized spores on different types of building materials to achieve a reproducible number of deposited spores.

PURPOSE: This MOP describes the procedure for deposition of aerosolized spores in a reproducible manner onto small coupons of different types of building materials using a metered dose inhaler (MDI) in laboratory H106. Concentrations and distances mentioned in this MOP are specific for a deposition of between 3 x 109 and 7 x 109 spores onto 18 mm stubs. Different stub sizes, concentrations, and distances may be used to achieve a different desired concentration.

1 Reagents

1.2 Clorox® Germicidal bleach

Mixed with DI water to make an amended bleach solution to clean and disinfect surfaces.

1.3 Hydrochloric Acid Solution, 6N; Fisher Chemical [7647-01-0] or equivalent

Used to adjust the pH of the amended bleach solution.

1.4 Ethyl alcohol: Sigma-Aldrich, 459844 or equivalent

Used to wipe down and disinfect surfaces following amended bleach wipe down.

1.5 Methanol: Sigma-Aldrich, 676780 or equivalent

Used to sterilize grippers and forceps.

2 Equipment

2.1 Metered Dose Inhaler


A small can that contains Bacillus subtilis spores in ethanol solution and propellant gas designed to deposit 10E9 spores each time the MDI can is depressed inside an actuator. Each MDI can be actuated (puffed) a maximum150 times before it is considered expired and should be replaced.

2.2 MDI Actuator

A small plastic tube in which the MDI is inserted. The actuator is permanently mounted onto one of the lids of the deposition chamber.

2.3 Deposition Chamber or Substrate Holder

A clear acrylic tube 12 cm long and 5 cm inner diameter with caps on both ends. At one end there are two interchangeable caps. A threaded rod extends through the other end and into the chamber. A knob on the end of the rod outside the chamber adjusts the distance from the opposite end of the chamber to the tip of the threaded rod. The tip has a hole in the end for holding the stubs (substrates) that receive the spores when the actuator is depressed. One of the two caps houses a laser pointer for center-aligning the threaded rod and the second lid has the MDI actuator permanently attached. The chamber has three screws threaded into the sides that are used, with the laser pointer, to center the rod.

2.4 Vortex Shaker (Fisher Mini-Shaker Model 58 (115 volt, 0.45 amp); manufactured by Fisher in Pittsburgh, PA 1527)

Used to shake the MDI for 10 seconds before the MDI is depressed (puffed). It is necessary to vortex the MDI before every puff, including the puffs meant to purge the MDI.

2.5 Depth Micrometer

Used to adjust the distance from the end of the chamber to the surface of the substrate to 0.592”. This adjustment is done each time the type of substrate is changed due to differing heights of the substrates (carpet, wallboard paper, wood, etc.)

2.6 Impinger/Funnel Assembly

Assembly filled with amended bleach used to catch the spores from the MDI blanks. The actuator is depressed 5 times into the funnel before each inoculation to clear or purge the MDI spore path. Before depressing the actuator the house air attached to the impinger exit should be on to create suction inside the funnel and capture the spores in the amended bleach. The amended bleach should be prepared fresh once per week to replaced the bleach in the impinger.

2.7 Stubs

A 18 mm diameter flat surface that serves as a platform on which the substrates are mounted. The substrates will be prepared on the stubs in advance then autoclaved by the BioLab Pick up the substrates and the glassware from the BioLab immediately before beginning inoculation and take them to Laboratory H106.


2.8 Stub Stages

Used to hold the stubs after inoculating. Stub stages have holes labeled A, B, C, D, E, F, and G. Grippers are used to handle the stubs while placing them into the substrate holder, removing from the substrate holder and placing them in the holes on the stages.

2.9 Forceps (SEM pin stub grippers, #1661/#1662; manufactured by Ted Pella, Inc in Redding, CA 96049)

Helpful to prevent the stubs from rolling around when picking them up from the Petri dish before placing them in the substrate holder.

2.10 Glassware

Used to hold the stages and the inoculated stubs. The stages are placed in 6” Petri dishes after inoculating. If the substrate is carpet then the normal Petri dish lid can’t be used because it can touch the inoculated carpet due to the height of the carpet in the stage. The BioLab should include a tall lid to be used in place of the normal lid.

2.11 Torch

Used to sterilize the grippers and the tweezers. The forceps need to be sterilized only once before inoculating, the grippers should be sterilized each time they are used. The forceps only need sterilizing only once because they are used only to hold the sterilized stubs still and never come into close proximity to the MDI or the spores.

2.12 Fume hood

Used when inoculating the substrates. The only equipment located in the hood during deposition should be the impinger/funnel assembly, the vortex shaker, the MDI and its actuator, and the substrate holder. This limitation of equipment in the hood minimizes possible unintended deposition of spores on equipment and minimizes efforts when cleaning after deposition.

2.13 Sharpie Marker

Used to label the glass Petri dishes.

2.14 Plastic Storage Bin

Used to isolate the stub-holding Petri dishes during transport to and from the BioLab.

3 Set-up Procedures


3.1 Pre-inoculation Procedures:

• Print six blank chain of custody forms, one for each Petri dish, and obtain the WA-4-3 laboratory notebook. The COC forms are to be filled out after inoculating and returned to the BioLab with the inoculated samples where they will be signed as received. Notes are to be made in the laboratory notebook recording the Petri dish IDs, the substrate being inoculated and the hole in which the stubs are placed. Note that one puff was completed on the stub or if zero puffs, indicate that the stub was a negative control. After completing the inoculation return the laboratory notebook to H226 where it will be used during testing the next day.

• Go to the BioLab and pick up the sterile glassware, stages, and substrates that should be inside autoclave bags.

• Verify the number of Petri dishes and substrates that have been prepared and are on the cart: Petri dishes -6; Stages -6; Tall Petri dish covers (for carpet) – 6; and 35 or 23 substrates depending on the test.

3.2 Aseptic procedures:

• Prepare 500 mL of amended bleach fresh each week by mixing DI water with germicidal bleach at 10:1. HCl should be added to achieve a pH=~7.0. Label beaker Amended Bleach Solution with the date prepared. About half of the Amended Bleach solution will be used in the impinger assembly to catch the spores during the purge puffs; the rest will be used to wipe down the work area and equipment.

• The following are to be wiped down first with amended bleach (to be made fresh once each week), then with ethyl alcohol before and after each deposition in laboratory H106 where deposition takes place: the hood bottom, sides, and back, the hood sash and any exterior components of the hood that may come into contact with the person’s gloves such as the hood light switch. Gloves are recommended.

• The laboratory countertop, storage bin, and the cart that will be used during deposition should also be wiped down. The substrate holder, the MDI and its dispenser, the vortex shaker and any other equipment in the hood during deposition should be wiped down. All other equipment should not be located in the hood during deposition.

3.3 Depth alignment:

• Place one substrate (not sterilized) in the substrate holder. Whenever the coupon substrate is changed to a different type (e.g., aluminum (naked) to carpet, carpet to wood, etc.), the distance needs to be readjusted.

• Adjust the distance from the coupon surface to the nozzle of the MDI actuator to 0.592” using the depth micrometer. Set the micrometer to 0.575” and then turn to the number 17 on the dial to get 0.592”.

• Rest the micrometer on the open end of the substrate holder and turn the threaded rod in or out so that the micrometer tip just touches the surface of the substrate.


• A special aluminum “blank coupon” is used to adjust the distance for the carpet because the carpet substrate’s top surface is not hard and gives.

3.4 Center alignment:

• After the depth alignment, close the one end of the chamber with the laser pointer cover.

• Turn on the laser pointer by plugging it into the AC outlet.

• Align the center of stub holder to the laser point using three screws on the middle side of the chamber.

• Turn off the laser pointer. Remove the laser pointer cover from the chamber.

4 Loading/Inoculation Procedures

4.1 Coupon Loading

All deposition should be conducted under a hood. Gloves, eye protection and laboratory coat are recommended.

• Position the cart used to transport the materials from the BioLab next to the hood on the left to minimize the distance the inoculated substrates must travel outside the hood before being placed in one of the stage holes.

• Remove all items from the inside of the hood except the amended bleach-filled impinger/funnel assembly, the vortex shaker, and the MDI and the MDI actuator.

• Place the torch and a beaker with about 100 mL of methanol on the counter opposite the hood to be used for sterilizing the grippers and forceps.

• Aseptically clean and wipe down the hood, laboratory counter, the cart top, the storage bin, the vortex shaker, the substrate holder and the MDI.

• First set the depth for the appropriate material in the substrate holder using the depth micrometer, then center-align the substrate holder using the cap with the laser pointer.

• Carefully tear open the autoclave pouch containing an empty glass Petri dish and place it in the bin on the cart. Label the bottom and top of this Petri dish “A1” with a sharpie. If inoculating carpet, use and label the TALL glass covers and DO NOT use the normal Petri dish covers.

• Carefully tear open an autoclave pouch containing a glass Petri dish with the samples inside and place it on the cart next to the Petri dish from the previous step. Leave the Petri dish remaining on top of the sterile opened pouch. The pouch will serve as a clean place mat on which to place the Petri dishes with the samples and the sterilized grippers.


• Using the torch outside the hood, sterilize the forceps and the grippers by rinsing them in methanol and burning them. Place the sterilized grippers on the pouch next to the Petri dishes containing the samples.

• Note the total number of puffs thus far made using the MDI currently in the actuator and if the number will exceed 150 when inoculations for the day are completed then a new MDI should be used. A tally of the puff count is kept in the laboratory notebook.

• Carefully tear the stub stage autoclave pouch and put a stage in the Petri dish labeled A1. Do not touch the stages. Use the pouch as a mitt to place the stage in the Petri dish.

• Make sure the vacuum source for the impinger is attached and that there is amended bleach inside the impinger. Turn on the vacuum.

• Shake the actuator with the MDI for10 seconds using the vortex shaker.

• Press the MDI for purging puffs while holding it inside the funnel inlet allowing the spores to go into the amended bleach-filled impinger. The MDI should be held in a vertical position.

• Repeat shaking the MDI followed by a purge puff into the funnel a total of 5 times. Note in the lab notebook that five purge puffs were completed so they will be included in the MDI total puff count.

• Place a substrate on the stub holder inside the chamber using a gripper. The forceps are useful to prevent the substrates from rolling around.

• Stubs are already grooved on their sides and the gripper should touch only the grooved side of a stub while holding it.

• Shake the actuator with MDI in it using the vortex shaker in the hood for 10 seconds.

• Hold the body of the chamber with one hand and use the other hand to hold the MDI actuator in a vertical position with the thumb positioned on the bottom of the MDI.

• Press the MDI firmly and hold it for 3 seconds. There shouldn’t be any hesitant motion to push the MDI.

• Remove the cover and take the loaded stub with the gripper and put it in the appropriate stub stage hole.

• After putting the inoculated stub on a stage, the used gripper needs to be sterilized by dipping the gripper tip in methanol and putting it in the torch flame until the yellow flame goes away.

• With a sterilized gripper, grab a new coupon and put it into the chamber.

• When a total of 35 substrates is used: Repeat the process filling holes A-E with inoculated coupons and then a negative control substrate (not inoculated) goes into Hole F of the stage. Repeat this process


filling holes A-F for the first five stages. The 6th stage does not receive the negative control so holes A-E only will be filled with inoculated substrates. (Note: The substrates are not labeled, only the Petri dish is labeled). The locations of the inoculated substrates are recorded in the laboratorynotebook as having received one puff and placed into holes A-E while the location of the un-inoculated substrate will be recorded in the laboratory notebook as having received 0 puffs and placed into Hole F.

• When a total of 23 substrates is used: Repeat the process filling holes A-C with inoculated coupons and then a negative control substrate (not inoculated) goes into Hole D of the stage. Repeat this process filling holes A-D for the first five stages. The 6th stage does not receive the negative control so holes A-C only will be filled with inoculated substrates. Record the number of puffs and the stage hole locations as describe.

4.2 Counting puffs

MDI has a number of puffs limited to 150. Therefore, it is necessary to track the number of puffs used for each set of experiments. This tracking is accomplished by recording the number of purge puffs and inoculation puffs in the laboratory notebook.

5 Quality Assurance/Quality Control

The Petri dish labeled A6 will contain the positive controls as indicated in the last steps of the coupon loading section. This stage will have one less substrate than stages A1 thru A5 so holes A-E will be occupied. Stages A1 thru A5 will all have one negative control as indicated in the last part of the coupon loading section. The negative control is inserted in hole F. Substrates in stages A1-A5 will occupy holes A-F. The positive controls (Stage A6) are not inserted into the fumigation chamber during testing but do accompany the other stages through the rest of the process. The negative controls will experience the same exposure time as the inoculated substrates on the same stage during testing. Negative controls are expected to have no bacteria colonies after incubation while the positive controls will have a number of colonies consistent with inoculation by 10E9 spores per puff.


MOP 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

• De-Ionized (DI) water

1.0 PROCEDURE

1.1 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”


1.2 Basic Operation

1. Prepare any items that need to be sterilized. The items must be carefully wrapped or sealed in sterilization pouches in order to maintain sterility when removed from the autoclave. Examples of this 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, then they must have secondary containment (usually a large plastic autoclave-safe tray) to contain any fluids in the event of a leak, spill or boil-over. 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 ampuole 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 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 in which they were autoclaved and the initials of the individual that 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).

10. Place the contents of the autoclave in an appropriate place to cool, and close the autoclave door using the foot pedal.


1.3 Cycles

1.3.1 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.

1.3.2 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 2L total). When attempting to sterilize any volume larger than 2L, 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 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 of 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.


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 (CFU).

Materials:

• Liquid suspension of bacterial spores

• Sterile centrifuge tubes

• Diluent (sterile deionized water, buffered peptone water or phosphate buffered saline)

• Trypticase Soy Agar (TSA) plates

• Microliter pipettes with sterile tips

• Sterile beads placed inside a test tube (will be used for spreading samples on the agar surface)

• Vortex mixer

Procedure: (This protocol is designed for 10-fold dilutions.)

1. For each bacterial spore suspension to be tested, label microcentrifuge tubes as follows: 10-1, 10-2, 10-3, 10-4, 10-5, 10-6... (The number of dilution tubes will vary depending on the concentration of spores in the suspension. Aseptically, add 900 μL 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 µL of the stock suspension to the 10-1 tube. Mix the 10-1 tube by vortexing for 10 seconds, and immediately pipette 100 µL 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 for 10 seconds, immediately pipette 100 µL 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.)


7. Enumerate the colony forming units (CFU) 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 CFU when counting, so that no CFU 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 (CFU) within extract:

(Avg CFU / volume (mL plated) X (1 / tube dilution factor) X extract volume

For example:

Tube Dilution

Volume Plated Replicate CFU

10-3 100 µL (0.1 mL) 1 150

10-3 100 µL (0.1 mL) 2 250

10-3 100 µL (0.1 mL) 3 200

Extract total volume = 20 mL

(200 CFU / 0.1 mL) × (1/10-

3) × 20 mlL =

(2000) × (1000) × 20 = 4.0 × 107

Note: The volume plated (mL) and tube dilution can be multiplied to yield a ‘decimal factor’ (DF). DF can be used in the following manner to simplify the abundance calculation.

Spore Abundance per mL = (Avg

CFU) × (1 / DF) × extract

volume


MOP 6561

TITLE: AEROSOL DEPOSITION OF SPORES ONTO MATERIAL COUPON SURFACES USING THE AEROSOL DEPOSITION APPARATUS

SCOPE: This MOP outlines the procedure for assembly and use of the Aerosol Deposition Apparatus.

PURPOSE: Precise and highly repeatable aerosol deposition of bacterial spores onto material surfaces for detection, sampling, and/or decontamination studies.

Materials

• Aerosol Deposition Apparatus (ADA)

• Metered Dose Inhaler (MDI) preloaded with a bacterial spore suspension of known concentration (i.e., 1 x 109 spores per puff).

• MDI Actuator with ADA adaptor (adaptor required for proper attachment of actuator to ADA lid).

• Material coupon (with dimensions at least that of the ADA).

• ADA-coupon gasket (1 per ADA).

• Clamping devices (i.e., medium-size steel binder clips, C-clamps (8 per ADA)).

• Vortex mixer.

• Aerosol trap (described in Appendix A).

• PPE (gloves, laboratory coat, safety goggles).

• pH-Adjusted bleach

• 0.22 µm pore-size syringe filters.

• PVC tubing (3/8” o.d., 1/4” i.d.).

• Mass balance (with 1 mg accuracy).

Sterilization of Materials

• Prior to the start of any experiments, all components must be sterilized and stored in a sterile environment until use. (Sterilization is not necessary for binder clips, MDI, vortex, or the aerosol trap)

• ADAs can be sterilized by autoclave, VHP, or pH-adjusted bleach wiping with subsequent DI H2O and ethanol rinse/wipes. The ADA lid should be attached and in the closed position during the sterilization.

• MDI actuator with attached MDI adaptor can be wiped with pH-adjusted bleach then rinsed with DI H2O. DO NOT expose to ethanol, as ethanol will shatter the ADA adaptor.

• Sterilization requirements for coupons vary by material. Regardless of sterilization method, QC checks should be administered to ensure the effectiveness of the sterilization method.


• Gasket sterilization may also vary by material.

Procedure

1. Begin by donning PPE (gloves, laboratory coat, and protective eyewear) .

2. Clean the workspace by wiping with pH-adjusted bleach, next with DI H2O, and last with a 70-90 % solution of denatured ethanol. 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 laboratory 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 secure the coupon firmly 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.). Last, attach 0.2 um syringe filters to each vent tube on all ADAs (4 per ADA). Syringe filters can be attached using PVC tubing (3/8” o.d., 1/4” i.d.).

5. Determine the weight of the MDI canister using a balance. Record the weight (to the nearest mg) in laboratory notebook. (The MDI canister full is approximately 15 grams, an empty canister is approx 9.5 grams. To ensure the canister contains adequate spore suspension for dosing, canisters should be retired from use when their weight falls below 10.5 grams.) In addition, keep a record of the total number of ‘puffs’ dispensed for each MDI canister.

6. Next, assemble the MDI and actuator (with ADA adaptor) 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 (MDI canister on top, MDI nozzle pointing down, and actuator + adaptor outlet pointing horizontally towards the aerosol trap inlet, Figure A-1), dispense three test ‘puffs’ into the aerosol trap to prime the MDI. It is important to vortex the assembly 10 seconds before every puff (except for 30 seconds prior to the initial puff of the experiment, as prescribed in Step 7).

9. Vortex the assembly 10 seconds and attach it 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. (The ‘open’ position is achieved when the hole in the lid aligns with the hole in the top of the ADA.). Secure the lid in the open position by tightening the lid screws.


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.

Note: If the dosing puff is faulty, return to Step 8 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’ puff is achieved when the plume can be seen through the clear ADA adaptor immediately following the puff, and the puff results in audible characteristics typical of a successful puff. Familiarity and professional judgment will be needed to determine the success of a puff.

11. Follow the reverse order of the lid opening procedure to close the ADA lid. Detach the MDI/actuator assembly from the lid.

12. Determine the weight of the MDI using a balance, record the weight in laboratory notebook.

13. Vortex the assembly for 10 seconds, holding the assembly upright (MDI canister on top, MDI nozzle pointing down, and actuator + adaptor outlet pointing horizontal, towards the aerosol trap inlet, Figure A-1) dispense a test puff into the aerosol trap (expect a faulty puff, as the MDI requires recharging after a puff in the non-upright orientation). Repeat once more (vortex 10 seconds, puff). Expect the subsequent puff to be characteristic (visually and audibly) of a successful puff. If the second test puff is successful, proceed to dosing the next coupon (Step 9), if the second puff is faulty, repeat Step 13.

14. Repeat Steps 9 through 13 until all coupons have been dosed.

15. Allow spores to settle onto the coupon surface for at least 18 hours. Settling time should not exceed 26 hours.

Figure A-1. MDI orientation while dispensing test puffs into the aerosol trap.


16. Carefully remove binder clips (or other attachment device) and remove ADA and gasket from coupon surface, taking care not to disturb the surface of the coupon.

17. Test coupon is now ready for use.

18. Decontaminate the ADA and associated components with the same procedures utilized during the initial sterilization.


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 (DI) water.

2. Shake vigorously to mix until dissolved.

3. Label bottle as “nonsterile PBST” and include date and initials of person who made PBST.

4. Filter sterilize into two 500 mL reagent bottles using 150 mL bottle top filter (w/ 33mm neck and .22 µm cellulose acetate filter) for sterilization. Complete this procedure by pouring the liquid into the non-sterile PBST into the top portion of the filtration unit 150 mL at a time, while using the vacuum to suck the liquid through the filter. Continue to do this until 500 mL have been sterilized into a 500 mL bottle. Change bottle top filter units between each and every 500 mLbottle.

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 tubes 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 1:10 diluted bleach, followed by DI water and a clean Kimwipe or Techwipe. Then stock the hood with the following items if they are not already there: - The flats of sterile conical tubes you need to fill with PBST. - Sufficient bottles of sterile PBST to fill these tubes. - Ample 25 mL serological pipettes (at least 3 per flat) for 20 mL transfers and 10 mL serological pipettes for the 5 mL transfers. - Serological pipetter (automatic, hand-held pipette). - Burner and striker.

2. Light the burner and adjust the flame for a width adequate to flame the lips of the PBST bottles.

3. Take one flat of sterile conical tubes and loosen each cap on the outside edges (about ½ turn).

4. Open a serological pipette and insert into the serological pipetter, taking care not to 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 dominant hand to unscrew the lid. Place the lid upside down on the benchtop and quickly flame the lip of the bottle. Turn the bottle and repeat, taking care to thoroughly flame the lip without getting the glass so hot that it shatters.

6. Inset the tip of the pipette into the bottle and fill to the 20 mL line. Flame the bottle lip and place the bottle on the benchtop. Note: If the tip of the pipette touches the outside of the bottle or any other surface in the hood, consider it contaminated. Discard the pipette and reload a new one.

7. Quickly pick up one of the tubes that you have loosened the cap on and use the bottom of your right hand to remove the cap. Completely discharge the entire pipette into the tube, taking care to not touch anything with the tip of the pipette. Recap the tube and place back into the flat (the lid does not have to be tight – you will tighten the lids after you have completed filling the 10 outside tubes). Note: If the tip touches the outside or rim of the tube (or any other surface in the hood), consider the tube and pipette contaminated. Discard both the tube and the pipette.

8. Pick up the PBST bottle and flame the lip. Repeat Steps 6 and 7 until all 10 of the tubes on the outside of the flat have been filled. Flame the lip of the PBST bottle and replace the cap. Slide the used pipette back into the plastic sleeve and put to the side of the hood for disposal. Then tighten the lid of each tube you just filled. But rather than placing it back into its original spot in the flat, switch it for the empty tube from the next row. When this has been completed, go around the outside of the flat again and loosen the lids of these 10 tubes. Repeat steps 4 through 7 to fill and cap these tubes.

9. This same procedure is used to fill the middle row of tubes from the flat and, if more than one flat of tubes is being filled, can be done at the same time as the outside rows of a second flat.

10. When all tubes have been filled, label each flat as follows and place on the shelf in room E390B:


“PBST Tubes (20 mL or 5 mL)” Date prepared Your initials

11. These tubes should be made at least 14 days before they need to be used so that they can be verified as sterile. Any tubes that are cloudy or that have any floating matter/turbidity should be discarded. The tubes are stable for and should be used within 90 days.

3.0 CLEANUP FOR 20 ML/5 ML PBST TUBES

1. Dispose of the used pipettes in the nonregulated waste.

2. Plug in the serological pipetter so that it can recharge.

3. Replace any unused PBST in the liquid containment on the shelf. Make sure that the bottle is laboratoryeled as having been opened (date opened and initials of whomever used it).

4. Turn off the burner.

5. Wipe down the hood benchtop with ethanol, followed by bleach, followed by DI water and a clean Kimwipe or TechWipe. 4.0 PREPARING 900µL PBST TUBES FOR USE DURING EXPERIMENTATION

1. Prepare the hood by wiping down with ethanol, followed by bleach, followed by DI 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 mLconical 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 as to when the PBST was made, sterilized, etc.).

- 1000 µL micropipette. - 1000 µL sterile pipette tips - Microcentrifuge tube racks. - lLabeled 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 µL 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 µL micropipette and tips. Change


tips whenever two rows of tubes are completed or whenever a contamination event (such as touching the outside of the 50 mL tube or the microcentrifuge tube) occurs. Put the dirty tips in the beaker or container used to contain waste (tips, tubes) in the hood. If any 900 µL tubes are contaminated during the transfer, dispose of them in the waste container used to hold tips under the hood. If a new box of tips has to be opened, make certain the date it was opened and initials of the person who opened it are clearly labeled on the box.

4. After both racks are full, carefully move all the tubes from one rack to fill in the empty rows on the other rack. In this manner, one rack should be completely filled with tubes at this point.

5. Label the rack of tubes as “Sterile 900 µL 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 µL 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 nonregulated waste container.

2. Put the unused sterile tips and the micropipetter back in its original location.

3. Replace any unused 50 mL conical vials 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 whoever used it). If the tube could possibly be contaminated in any way, dispose of it in nonregulated waste.

4. Wipe down the hood benchtop with ethanol, followed by diluted (1:10) bleach, followed by DI water and a clean Kimwipe or TechWipe.


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 which is 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 laboratory 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, then replace the cap on the swab.

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 NHSRC Microbiology Laboratory for processing.

2.1.2 Plating of Environmental Transport Swab Sample

1. When the sample is received in the NHSRC Microbiology Laboratory , label three TSA plates with a Sharpie with the information from the swab packaging. Verify that the sample ID and date match.

2. Place labeled 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 medium is broken.

3. Perform Step #2 on the remaining two plates.

4. Replace the swab into its tube and discard in the nonregulated waste container.

5. Repeat steps #1 through #4 for each sample.

6. Label three TSA 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 medium 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 which are transported back to the NHSRC 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 per sample to be collected (in their autoclave pouches), one swab for the control plates, plus a few extras.

• Sharpie for labeling plates.

Use the following procedure to collect and plate samples.


1. Once at the sample collection site, take the TSA 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 medium 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 TSA 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 nonregulated waste container.

7. Repeat steps #1 through #6 for each sample.

8. Label three TSA 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 TSA.

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 TSA plates back into the media bag and transport to the NHSRC 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) • 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 (NG) indicates that the sample is sterile. Growth (G) indicates that an organism(s) is present, and the organism 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.


Swab Results Template

Swab samples taken

Swabbed by:

Plating completed on: Plated by:

Plate results read on: Results read

Sample Name Result If growth, describe G NG

G NG

G NG

G NG

G NG

G NG

G NG

G NG

G NG

G NG

G NG

G NG

G NG

G NG

G NG

G NG

G NG

Controls Result If growth, describe Swab blank A G NG

Swab blank B G NG

Swab blank C G NG

Key G = Growth. NG = No Growth. All plates are plated in triplicate resulting in sample identification of “A”, “B”, and “C”.


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 µm 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, , pipette 1 mL of the extract into one of the filter units immediately following vortexing.

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 DI H20 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.


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 DI 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 for 16 – 28 hours at the optimal growth temperature for the organism used.

15. Enumerate and record the number of CFU on each plate

Data Calculations

Utilize the following equation to determine the total abundance of recovered spores:

filtered

Extract

VV

CFUN ×=

where N is the total number of spores recovered in the extract, CFU is the abundance of colonies on the agar plate, VExtract is the total volume of the extract (before any aliquots were removed), VFiltered is the volume of the extract filtered.


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, laboratory coat, safety goggles)

• Biological Safety Cabinet (Class II)


• Deionized water

• 70% solution of denatured ethanol

• Kimwipes

• Dispatch® bleach wipes

• Non-regulated waste container

• 50 mL sterile conical tubes containing 20mL 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 μL tubes of sterile PBST

• Pipettor and pipette tips for dilutions

• Incubator set to appropriate growth temperature for target organism (35 °C or 55 °C)

• Light box for counting colonies

• Laboratory notebook

• QAPP for project that is utilizing the wipe samples

2.0 Procedure

1. Begin by donning PPE (gloves, laboratory coat, and protective eyewear).

2. Obtain wipe samples that may contain Bacillus spores. Wipe samples should be received as one wipe/sponge in a sterile 50 mL conical tube delivered in secondary containment. Make certain that all of the samples are labeled. Review any chain of custody forms that may accompany the samples to


ensure that all of the labels are consistent and that there is no notable variation in the samples. If variation has occurred, make a note of it in the notebook.

3. Clean the workspace (Biological Safety Cabinet) by wiping surfaces with pH-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.


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 nonregulated 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 appropriately 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 at 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 6535a, and the 900 uL tubes should be made with sterile PBST to stay consistent with materials/solutions. Plating in this manner should be repeated for all samples, with any changes in protocol noted in the laboratory notebook.

10. Once the dilution plating has been completed, the plates are to be placed in an incubator. For non-thermophilic Bacillus species, the plates should be placed at 35 °C ± 2 °C for 12-24 hours. For thermophilic 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 for a sufficient amount of time (12-24 hours) and the growth from any Bacillus colonies is quantifiable, the colonies should be manually counted using the light box and the


data should be properly recorded as dictated per project by the QAPP. All results will be checked for quality assurance and all data will be reported to the proper personnel as listed in the QAPP.


MOP 6568

TITLE: ASEPTIC ASSEMBLY OF WIPE KITS

SCOPE: This MOP outlines the procedure for the aseptic assembly of wipe kits.

PURPOSE: To aseptically assemble kits that will be used to collect wipe samples from which quantifiable data will be derived.

1.0 MATERIALS

• PPE (gloves, laboratory coat, safety goggles)

• Biological Safety Cabinet (Class II)


• Deionized water

• 70% solution of denatured ethanol

• Kimwipes

• Sterile, sealed Twirl-em bags in two sizes, 10” x 15” and 5.5” x 9”

• Sterile Kendall 4-ply all-purpose sponges

• Sterile, disposable thumb forceps

• 50 mL conical tubes containing 5 mL PBST tubes (MOP 6562)

• Sharpie

• Wire or foam rack for 50 mL conical tubes

• Secondary containment such as a large Tupperware bin

• Laboratory notebook

• QAPP for project that is utilizing the wipe samples


2.0 PROCEDURE

2.1 Preparation for Wipe Kit Assembly

Prior to wipe kit assembly, 50 mL sterile conical tubes containing 5 mL of sterile PBST and a sterile 2-ply sponge must be put together. The sterile conical tubes are assembled in the following manner:

1. Begin by donning PPE (gloves, laboratory coat, and protective eyewear).

2. Clean the workspace and Biological Safety Cabinet by wiping surfaces with pH-adjusted bleach, followed by deionized water, and then with a 70% solution of denatured ethanol. Wipe the surfaces with a Kimwipe to remove any excess liquid. Make sure the workspace is clean and free of debris. Gather all necessary items to perform the task, place these items on a clean cart beside the biological safety cabinet, within arm’s reach so that, once the procedure has begun, the task may be performed without interruptions.


4. Place the sterile 50 mL conical tubes containing 5 mL PBST tubes under the Biological Safety Cabinet in a foam or wire rack designed to hold 50 mL conical tubes. Using two sterile, disposable thumb forceps, aseptically transfer one half of a 4-ply, sterile, all-purpose sponge to each of the tubes. Complete the transfer by using the two forceps together to first separate the 4-ply sponge in half to create two 2-ply sponges. Then remove a cap from one of the tubes, carefully fold one of the 2-ply sponges using the forceps together and aseptically place it in the opening of the tube so that it sits at the top portion of the tube, while the 5 mL of PBST remain at the bottom of the tube. Replace the cap to the tube. Repeat this process until all of the tubes have sponges in them. Once all of the tubes contain sterile sponges, then label the tube rack appropriately with the action completed, the date and your initials and place the tubes on the shelf. These tubes are shelf-stable for up to three months. 2.2 Assembly of Wipe Kits

Wipe kits are assembled in the following manner:

1. No more than 48 hours prior to testing or collecting samples, assemble the wipe kits. Wipe kits can be assembled outside the Biological Safety Cabinet, in a dry, clean area. Make certain to use proper PPE, including gloves, while handling all wipe kit materials. Gather all materials to assemble the kits before assembly. These materials include: - 50 mL conical tubes containing both a sterile wipe sponge and 5 mL PBST

- Twirl-em bags in two sizes, 10” x 15” and 5.5” x 9”

- Sharpie

- Vortex mixer


2. Obtain a copy of the labeling scheme for the samples. This labeling scheme may be detailed in the QAPP. For each wipe kit, use a Sharpie and label a large 10” x 15” Twirl-em bag and a 50 mL conical tube containing the sponge and PBST.

3. Once all of the tubes are labeled, use the vortex mixer on the highest setting to agitate the tube to mix the sponge, which was placed at the top of the tube, with the 5 mL of PBST.

4. Open the labeled, 10” x 15” Twirl-em bags one at a time. Place the labeled, agitated tubes in the 10” x 15” Twirl-em bags that have the corresponding label (that matches the tube). Add a nonlabeled, sealed 5.5” x 9” Twirl-em bag into the 10” x 15” Twirl-em bag, along with the tube containing the wipe sponge to complete the wipe kit assembly. Record the time and date when the wipe kits were assembled in the laboratory notebook; include the labeling schematic for the wipe kits.

5. Place the assembled wipe kits into a secondary containment, such as a large Tupperware bin. Use within 48 hours. When moving the kits to a sampling location, always have them in secondary containment.

Appendix C – Page 1

Appendix C

Spore Deposition and Handling Procedures

The handling of the contaminated coupons, including movement to minimize or control spore dispersal, was done in accordance with the MOP 6561. One person was tasked with removing the clamps holding the dosing chamber to the coupon and removing the dosing chamber and gasket from the coupon. A second person was then tasked with moving the coupon to the proper location (e.g., test and positive control coupons to the Test Coupon Cabinet and blank coupons to the Blank Coupon Cabinet).

The Test Coupon Cabinet was a steel cabinet (48 inches wide by 24 inches deep by 78 inches high) with twelve shelves each 6 inches apart. Each cabinet held a total of 36 coupons, so two Test Coupon Cabinets were needed for a test. These two cabinets were labeled as Test Coupon Cabinet 1 and Test Coupon Cabinet 2. Test and positive control coupons were arranged in each cabinet according to material types. A single material type was not split among cabinets. Procedural blank coupons of each material/orientation to be used in a single test were contained in a separate isolated cabinet (Blank Coupon Cabinet) of similar construction. The dimensions of the Blank Coupon Cabinet were 48 inches wide by 24 inches deep by 36 inches high with 3 shelves.

Each of the MDIs was claimed to provide 150 discharges. The number of discharges per MDI was tracked so that use did not exceed this value. Additionally, in accordance with MOP 6561, the weight of each MDI was recorded after completion of the contamination of each coupon. If an MDI weighed less than 10.5 g at the start of the contamination procedure described in MOP 6561, the MDI was retired and a new MDI used. For quality control of the MDIs, a contamination control coupon was run as the first, middle, and last coupon contaminated with a single MDI in a single test. The contamination control coupon was a stainless steel coupon (1.17 feet by 1.17 feet) that was contaminated in accordance with MOP 6561, sampled, and analyzed.

A log was maintained for each set of coupons that was dosed via the method of MOP 6561. Each record in this log recorded a unique coupon identifier (see Table C-1), the MDI unique identifier, the date, the operator, the weight of the MDI before dissemination into the coupon dosing device, the weight of the MDI after dissemination, and the difference between these two weights. The coupon codes were pre-printed on the log sheet prior to the start of coupon contamination (dosing).


Table C-1: Sample Coding

Coupon Identification: T-S-M-NN

Code

T 1 – 13, T1-T4 Test Number (Table 3-4)

S

C Coupon

V Wet/dry vacuum

E Wet/dry vacuum exhaust sample

R Rinsate

M

(Material)

CH Concrete (horizontal orientation)

CV Concrete (vertical orientation)

A Asphalt

BH Brick (horizontal orientation)

BV Brick (vertical orientation)

BW Rough-cut barn wood

DW Water-sealed, pressure-treated deck wood

PH Painted wallboard (horizontal orientation)

PV Painted wallboard (vertical orientation)

C Carpet

SS Stainless Steel (for QC purposes)

0 None (exhaust samples)

NN

(Sample Number)

NN 01 – ## (typically, ## was 11; however, exceptions may apply where >11 were needed)

X blanks

NHSRC Microbiology Laboratory Plate Identification: T-S-M-NN-R-D

Replicate R A – C

Dilution D 1E1-1E5


Additionally, after a coupon was dosed via the above procedure, the coupon was labeled with the unique identifier using the coding outlined in Table C-1. The label was printed on the side of the coupon using a permanent marker (e.g., black or silver Sharpie). The sampling team maintained an explicit laboratory log which included records of each unique sample number and its associated test number, contamination application, any preconditioning and treatment specifics, and the date treated. Each coupon was marked with only the material descriptor and unique code number. The wet/dry vacuum samples and exhaust samples from each test were identified with an associated test number and coupon set. Once the coupons were transferred to the NHSRC Microbiology Laboratory for plate counts, each sample was additionally identified by replicate number and dilution.

Appendix D – Page 1

Appendix D

Decontamination Process

1 Material and equipment

The materials and equipment used for the decontamination procedure were standardized as much as possible and are listed in Table D-1. Decontamination steps are described in the subsequent sections of this Appendix.

Table D-1. Material and Equipment Used in the Decontamination Procedural Steps

Material/Equipment Description

Wet/dry vacuum (Total of 7 vacuums and 39 head attachments) (Filters are used as disposable, i.e., used for one test only)

RIDGID 14 Gallon Pro Vac WD1450: http://www.homedepot.com/webapp/wcs/stores/servlet/ProductDisplay?storeId=10051&langId=-1&catalogId=10053&productId=100081216&N=10000003+90401+524502+1600 Head attachment: RIDGID 2-1/2 In. Wet Nozzle (Squeegee) Accessory: http://www.homedepot.com/webapp/wcs/stores/servlet/ProductDisplay?storeId=10051&langId=-1&catalogId=10053&productId=100046467&N=10000003+90401+524502+1600 Filter: RIDGID 5-Layer Vacuum HEPA Filter: http://www.homedepot.com/webapp/wcs/stores/servlet/ProductDisplay?storeId=10051&langId=-1&catalogId=10053&productId=100022800

Sprayer (Total of 2 units)

Agri Supply Backpack Sprayer, 4 Gallon, 12 Volt: http://www.agrisupply.com/backpack-sprayer-gallon-volt-/p/59540/cn/2600071/

Bleach Ultra Clorox® Regular Bleach (EPA Reg. No. 67619-8): http://www.clorox.com/products/overview.php?prod_id=clb 6.15% sodium hypochlorite; <1% sodium hydroxide: http://www.thecloroxcompany.com/products/msds/bleach/cloroxregularbleach0505_.pdf

Vinegar 5% v/v technical grade acetic acid

Container for mixing pH-adjusted bleach solution

Bucket of detergent solution

3 gallons in a 5-gallon plastic pail

Detergent Klean-Strip TSP Substitute: http://www.wmbarr.com/product.aspx?catid=32&prodid=84

http://www.homedepot.com/webapp/wcs/stores/servlet/ProductDisplay?storeId=10051&langId=-1&catalogId=10053&productId=100081216&N=10000003+90401+524502+1600








http://www.homedepot.com/webapp/wcs/stores/servlet/ProductDisplay?storeId=10051&langId=-1&catalogId=10053&productId=100022800



http://www.agrisupply.com/backpack-sprayer-gallon-volt-/p/59540/cn/2600071/

http://www.agrisupply.com/backpack-sprayer-gallon-volt-/p/59540/cn/2600071/

http://www.clorox.com/products/overview.php?prod_id=clb

http://www.thecloroxcompany.com/products/msds/bleach/cloroxregularbleach0505_.pdf

http://www.thecloroxcompany.com/products/msds/bleach/cloroxregularbleach0505_.pdf

http://www.wmbarr.com/product.aspx?catid=32&prodid=84



Brush (Total of 38 brushes)

Rubbermaid Floor Scrubber: http://www.homedepot.com/webapp/wcs/stores/servlet/ProductDisplay?storeId=10051&langId=-1&catalogId=10053&productId=100644166

Sponge (Sponges are used as disposable, i.e., used for one test only)

QEP Extra Large Grouting Sponge, 7-1/2 x 5-1/2 x 2 In., Rectangle with Rounded Corners http://www.homedepot.com/webapp/wcs/stores/servlet/ProductDisplay?storeId=10051&langId=-1&catalogId=10053&productId=100173109

Nozzle Standard Adjustable-Flow Garden Hose Nozzle, Standard Brass, 4" Length

Garden hose 75 ft.; 5/8 inch diameter

Pressure regulator Bronze Pressure Regulator-Plumbing-Code Rated Standard, 3/4" NPT Female, 25-75 PS

Bucket of DI water 3 gallons in a 5-gallon plastic pail

Carboy container (Total of 9)

Carboys; Nalgene; Heavy Duty; polypropylene; Autoclavable; Leakproof; For full vacuum applications up to 8 Hours; USP class VI, vacuum rated for intermittent vacuum use only; 83B Closure size; capacity: 5.25 gal. (20 L)

Pump NSF-Certified Rotary Vane Pump for Water with Motor, Brass, 4.3 Max GPM, 3/4 Horsepower

Drying Pans KitchenAid Slider Cookie Sheet - 15"








Wet/dry Vacuuming

For each material type, a single wet/dry vacuum was used and clearly identified with a label indicating the test number and coupon set (blanks or material type). A single wet/dry vacuum was used for all blanks (combined). Before beginning the decontamination procedure for each material type, the wet/dry vacuum designated for that material type was moved to the vacuum exhaust sampling stand. The vacuum exhaust sampling stand was designed to capture the wet/dry vacuum exhaust for sampling. The sampling stand consists of a 12 in long stainless steel pipe with a 4 in outer diameter (O.D.). The pipe was modified with an opening 1-1/2 in in diameter located 4 in from the outlet of the pipe. A reducing elbow connected the inlet of the pipe to the wet/dry vacuum exhaust port. A 6-in duct was used to transport exhaust from the vacuum to the facility air handling system. The wet/dry vacuum used a 6.0 hp unit with a 14-gallon capacity. The head attachment that was used in this study is the 2-1/2 in wet nozzle (squeegee) shown in Figure D-1.

Figure D-1. 2-1/2 Inch Wet Nozzle (Squeegee) Accessory

For vertical surfaces, the vacuuming action started at the top of the coupon and the vacuum head was firmly moved down the coupon surface. This action was repeated three times. For horizontal surfaces, the same procedure was performed, starting first from the side of the coupon farthest from the operator. The filter inside the wet/dry vacuum was a five-layer HEPA-rated filter (shown in Figure D-2) that can be purchased as an accessory replacement for the standard pleated filter (non-HEPA-rated) provided with the vacuum as received by the consumer. The details of the filter can be found in the link provided in Table C-1 in Appendix C.


Figure D-2. Five-Layer HEPA Filter

During application of the procedure, the time was recorded during each step. The recorded times included both the duration of the step and the time of day that the step was started for each coupon. Times were recorded to provide information on the consistency and repeatability of each of the procedures. After use of the vacuum, the head assembly (wet nozzle shown in Figure D-1) was sterilized using the STERIS VHP® sterilization cycle. An independent head assembly was used for each coupon set. The head assemblies were replaced with new head assemblies after each coupon set in the decontamination chamber. Swab samples were taken from each vacuum canister and head assembly and from six wet nozzles per batch to verify sterility prior to re-use.

After completion of the decontamination testing, all wet/dry vacuums (including head assemblies) were cleaned in accordance with Appendix F. The Five-Layer HEPA filters were discarded and replaced after use in a decontamination test. All other wet/dry vacuum components were cleaned and re-used in subsequent decontamination tests.

pH-Adjusted Bleach Solution and Application

Sodium hypochlorite (bleach) is a registered antimicrobial pesticide under the authority of the FIFRA for use as a sanitizer or disinfectant to kill bacteria, fungi, and viruses in households, food processing plants, agricultural settings, animal facilities, hospitals, and human drinking water supplies. However, bleach is not a registered sterilant under FIFRA, so bleach does not have a registration claim to inactivate bacterial spores (including Bacillus anthracis). Published scientific data demonstrated that pH-adjusted bleach reduced bacterial spore populations under specific conditions including concentration, pH, and contact time. Hence, EPA issued several crisis exemptions at different times permitting the limited sale, distribution, and use of EPA-registered bleach products against Bacillus anthracis spores at a number of contaminated facilities such as: Capitol Hill, USPS Processing and Distribution Centers at Brentwood (Washington, D.C.) and Hamilton (Trenton, NJ), Department of State, General Services Administration, and Broken Sound Boulevard, Boca Raton, FL.25, 26

The application of bleach under crisis exemptions was limited to specific buildings or treatment sites and the following specific conditions applied:

• Only hard nonporous surfaces could be treated;


• A bleach solution close to but not above pH 7 (neutral), as tested with a paper test strip, and at a concentration of 5,000 to 6,000 parts per million (ppm) was prepared by mixing:

A one part bleach (with a 5.25 percent - 6.00 percent sodium hypochlorite concentration);

B one part white vinegar, and;

C eight parts water.

• Bleach and vinegar were not combined together directly. Water was first added to the bleach (e.g., two cups water to one cup of bleach), then vinegar (e.g., one cup), and then the remaining water (e.g., six cups).

• Treated surfaces had to remain in contact with the bleach solution for 60 minutes. Repeated applications were necessary to keep the surfaces wet.

• Treated PPE and containers being removed from an area required only 10 minutes contact time with the pH-adjusted bleach solution.

While the chlorine content of the solution also has an impact on the time required for inactivation or overall effectiveness, the pH of the solution has a much greater impact.27-29 Hence, the comparative effectiveness of alternative formulations (e.g., use of outdoor bleach having a higher sodium hypochlorite concentration) to the above is not easily predictable.

The concentration of household bleach and the strength of white vinegar can vary by batch and storage time. Hence, following the above mixing directions can result in varied pH and chlorine concentrations depending upon the starting reagents. This source of variation can complicate a laboratory study such as this by skewing data, potentially leading to erroneous conclusions.

To reduce the impact of “natural” variations in the bleach solution in this study, the pH and chlorine content were measured at the start and monitored throughout each test. The frequency of pH measurement was at least at the start of each coupon set. Additional Data Quality Indicators (DQIs) and Data Quality Objectives (DQOs) for the bleach solution are discussed in Section 4. The solution had a mean pH close to, but not above, neutral (>6.5 and <7.0) and a mean total chlorine content of 6,000-6,700 ppm. The temperature of the solution was between 18 – 24 oC (64 – 75 oF). Any solution having a pH, chlorine content or temperature falling outside of this range at any time was discarded and a fresh pH-adjusted bleach solution prepared. The chlorine content was measured by titrating 2.5 mL of solution with a Hach high range bleach test kit (Method 10100). The pH and temperature were measured using an Oakton pH probe (OKPH502; pH5). DI water was used as the base for all solutions.

The pH-adjusted bleach solution was prepared just prior to the initiation of testing on a particular day and was used within a window of 3 hours from the time of preparation. After 3 hours, the bleach solution was discarded and a fresh pH-adjusted bleach solution was prepared. However, a single preparation was used within a single coupon set. Additionally, technical grade acetic acid (5% v/v) was used instead of off-the-shelf white vinegar with the expectation of reducing the variability in the pH-adjusted bleach solution for the purpose of this study.


The pH-adjusted bleach solution was applied to each coupon using a backpack sprayer (see Table C-1 in Appendix C). For each coupon, the spray wand was inserted into the port on the chamber corresponding to the coupon to which the bleach was being applied. The bleach was applied by spraying in a full square around the coupon ensuring the entire coupon was wetted. The time for this action was recorded by stopwatch and was consistently 4 seconds across all coupons. This recorded value included both the duration of the step and the time of day when the step was started for each coupon.

A constant spray having a diameter of about 16 in at the coupon surface was used; the surface was completely flooded to wet the surface every 2 minutes for the first ten minutes, then every five minutes for the remainder of the specified contact time. The spray nozzle was held a constant distance of approximately one foot (± 2 inches) from the coupon surface. This distance was maintained by inserting the spray wand a certain distance into the port, indicating the correct distance. A constant pressure spray of 35 psi was applied throughout the application and was maintained by the internal pump in the sprayer. At this constant pressure, the flow rate was maintained at approximately 0.34 gallons/min (1.3 L/min) (1.8% RSD) with a cone spray pattern of 16 inches in diameter at one foot from the surface. The flow rate was checked at the start and end of each test and before and after use on each coupon set to ensure proper operation of the sprayer. The coupons were sprayed to completely wet the surface of the materials. During the bleach application, the start time, duration, and application frequency were recorded. This measurement and the recording of flow rate were used for a determination of the amount of pH-adjusted bleach applied to each coupon.

For Test 13, the blank coupons were mounted vertically inside the chamber and sprayed with pH-adjusted bleach every 2 minutes for a total of 10 minutes. Once the chamber was cleaned, five liquid inoculated coupons and ten aerosol inoculated coupons were decontaminated as before in sets of three.

Detergent Solution

The detergent solution used in this study was initially Klean-Strip® TSP-substitute (W.M. Barr & Co., Memphis, TN), a phosphate-free detergent. However, Klean-Strip® TSP-substitute was discontinued prior to completion of testing. In its place, DAP® T.S.P Substitute (DAP® T.S.P. Substitute Heavy Duty Cleaner, DAP Inc., Baltimore, MD) was used. The major differences between the two products were Klean-Strip® TSP-substitute was in liquid form while DAP® T.S.P Substitute was availlable only as a powder. Both TSP-substitutes were used per the directions on their labels. Three gallons of TSP-substitute were contained in a 5-gallon plastic bucket. The solution was applied by dipping the entire brush (see below for brush details) into the mixture before the brushing procedure began for each coupon, so that the brush was dripping with the detergent solution. The brush was dipped into the solution only once per coupon and no brush was re-used within a single test. No more than five seconds elapsed between the time the brush was removed from the bucket of the solution and the time the brush was applied to the coupon.

A fresh detergent solution was prepared as required. Buckets were reused but only after STERIS VHP® sterilization between tests. Details of the equipment cleaning can be found in Appendix F.


Brushing

The brush type that was used in this project was a synthetic fiber brush designed for heavy-duty rough floor scrubbing and adsorbing cleaning solution (see Figure D-3). The dimensions of the brush were 10 in wide by 3 in deep. The brush was dipped into the appropriate solution per the test matrix. For vertical surfaces, the scrubbing action started at the upper right corner of the coupon. Using a “W” shaped action, the brush was firmly moved over the coupon surface. The brushing was then done again starting from the lower right corner of the coupon, using an “M” shaped action, moving the brush firmly over the coupon surface. For the horizontal surfaces, the same procedure was used starting first from the right corner farthest from the operator and then from the nearest right corner. The brush was dipped into the solution only once per coupon. A new brush was used for each coupon within a single test. The start time of the brushing application for each coupon was recorded.

Figure D-3. Brush for Scrubbing (handle not shown)

Used brushes were sterilized using a STERIS VHP® sterilization cycle. After sterilization, six brushes were randomly selected and sampled using the swab procedure (Appendix D) to ensure sterility. Viable target organisms were not found on any sampled brushes after sterilization.

The procedure described above was used for all coupon types except the painted wallboard and carpet. For the painted wallboard, the brush was replaced with a sponge (described in Table C-1 in Appendix C). The sponge was squeezed to remove excess liquid prior to application to the coupon surface. Starting from the top right corner, a block-shaped “5” was used to wipe the coupon, ending in the bottom left corner. The sponge was then flipped over to expose a clean side and the wiping done again, starting from the top left corner and wiping in a block-shaped “2”, ending in the bottom right corner. A sponge was applied only to a single coupon, and then the sponge was sterilized using a STERIS VHP® sterilization cycle before being discarded. Neither brushing nor sponging was performed on the carpet coupons.

Rinsing with Water

Rinsing of the coupons was done using a standard garden hose nozzle (described in Table C-1 in Appendix C). The water was supplied to the nozzle through a 75 foot garden hose of 5/8 in diameter. The head pressure was maintained constant at approximately 60 psi using a pressure regulator (see Table C-1). The water was supplied via a closed loop system having a modified plastic 55 gallon drum as the reservoir and a pump to provide a pressurized stream and continual recirculation. A 36-inch thermocouple probe was


maintained in the water reservoir to monitor the temperature continually. The water used in this study was DI water. Between tests, two gallons of germicidal bleach were added to the full DI water reservoir to prevent bacterial growth in the reservoir and to disinfect the reservoir prior to use. Via adjustment of the nozzle, the spray pattern was controlled to a one foot diameter measured at three ft from the nozzle. The coupons were sprayed with three up/down strokes starting first from the top left of the coupon, then at the top center and top right of the coupon. The start time and duration for this action was recorded and was performed at approximately 5 seconds per coupon.

For the painted wallboard and carpet, rinsing with the hose was performed. For wallboard, a sponge identical to that used for the detergent solution procedural step was used. However, the sponge was soaked with DI water from a 5-gallon bucket. Two rinses of each coupon via this method were performed, one with one side of the sponge and the other with the opposite side of the sponge. A sponge was applied only to a single coupon and then sterilized using a STERIS VHP® sterilization cycle before discarding.

Rinsing was not performed on the carpet.

Appendix E – Page 1

Appendix E

Sampling Procedures

E.1 Sampling Material and Equipment

The materials and equipment used for sampling are listed in Table E-1.

Table E-1. Material and equipment used in sampling


Nonpowdered, sterile surgical gloves

KIMTECH PURE* G3 Sterile Nitrile Gloves, Kimberly-Clark (VWR p/n HC61110 for extra-large; VWR p/n HC61190 for large; VWR p/n HC61180 for medium)

Nonpowdered, non-sterile surgical gloves

Exam gloves (Fisherbrand Powder-Free Nitrile Exam Gloves, Fisher p/n 19-130-1597D (for large);19-130-1597C (for medium))

Phosphate Buffered Saline

Phosphate Buffered Saline with TWEEN® 20 (Sigma Aldrich, p/n: P3563-10PAK)

50 mL conical tubes BD Falcon® BlueMax Graduated Tubes, 15 mL (Fisher Scientific p/n 14-959-70C)

Sterile sampling bags

Fisherbrand Sterile Sampling Bags (wirlL'em) Overpack Size : 10” x 14” Inner bag size: 5.5" x 9" (wipe); 10” x 14” (HEPA vacuum) Sample Bag Size: 5.5” x 9 “

Bleach wipes Dispatch® bleach wipes

Wipes Kendall Curity Versalon absorbent gauze sponge 2'' x 2'' sterile packed (rayon/polyester blend) http://www.mfasco.com/

Vacuum filter sock collection kit

Midwest Filtration (FAB-20-01-001A)

HEPA vacuum Euroclean 0.8 L HEPA hip vacuum

Swabs Bacti-Swab® http://www.remelinc.com/Industrial/CollectionTransport/BactiSwab.aspx

Filtration Carboy 20 L Filtration Carboy (Fisher Cat# 02-960-15)

Carboys (2) Nalgene autoclavable carboys with tabulation (20 L) (Fisher Cat# 02-690-23)

Carboy Filtration fittings

Nalgene Filling/Venting Carboy Closure ½” I.D. (Fisher Cat# 02-923-15)

In-line filter holder Nalgene in-line filter holder (Fisher Cat# 09-748)

Membrane filters 47 mm Millipore membrane filters (0.6 μm DTTP) (Fisher Cat# DTTP04700)

Vacuum pump Gast oil-free vacuum pump with adjustable suction (Fisher Cat# 01-092-25)

http://www.mfasco.com/

http://www.remelinc.com/Industrial/CollectionTransport/BactiSwab.aspx



Tubing Fisher PVC clear tubing (1/2“ i.d., 1/16” thickness) (Fisher Cat# 14-169-7J) Fisher PVC clear tubing (3/8” i.d., 1/16” thickness) (Fisher Cat# 14-169-7G) Fisher PVC clear tubing (vacuum tubing) (3/8” i.d., 1/8” thickness) (Fisher Cat# 14-169-7H)

Filter cassettes Via-Cell® Bioaerosol Sampling Cassette (http://www.zefon.com/store/via-cell-bioaerosol-sampling-cassette.html)

Sampling pump Isokinetic Method 5 Source Sampling Console http://www.apexinst.com/products/consoles.htm

E.2 Sampling Protocol for Wipe Sampling

E.2.1 Coupon Wipe Sampling

Wipe sampling has typically been used for small sample areas and is considered most effective on nonporous, smooth surfaces such as ceramics, vinyl, metals, painted surfaces, and plastics.13 The general approach for wipe sampling has been the use of a moistened sterile noncotton pad to wipe a specified area to recover bacteria, viruses, and biological toxins.13 The protocol that was used in this project is described below and has been adapted from that provided by Busher et al.13, Brown et al.16, and documented in the INL 2008 Evaluation Protocols.17 None of these references provides a validated wipe procedure for Bacillus spores, as a validated sampling procedure does not currently exist.

The following procedure was used in this study for wipe sampling of each coupon surface:

1. A three-person team was used, employing aseptic technique throughout. The team consisted of a sampler, sample handler, and support person.

2. All materials needed for collection of each sample were prepared in advance using aseptic technique. A sample kit for a single wipe sample was prepared as follows:

a. Two sterile sampling bags (10” x 14”, 5.5” x 9 “) and a 50 mL conical tube, capped, were labeled. These bags and conical tube had the same label. The 5.5” x 9” labeled sterile sampling bag was referred to as the sample collection sterile sampling bag.

b. A dry sterile wipe was placed in an unlabeled sterile 50 mL conical tube using sterile forceps and aseptic technique. The wipe was moistened by adding 5 mL of sterile phosphate buffered saline with 0.05% TWEEN®-20. The tube was then sealed.

c. The labeled 50 mL conical tube, capped, the unlabeled conical tube containing the pre-moistened wipe, and the 5.5” x 9” labeled sampling bag were placed into the 10” x 14” labeled sterile sampling bag. Hence, each labeled sterile sampling bag contained a labeled 50 mL conical tube (capped), an unlabeled capped conical tube containing a pre-moistened wipe and an empty labeled sterile sampling bag.

d. Each prepared bag was one sampling kit.

http://www.apexinst.com/products/consoles.htm


3. All members of the sampling team donned a pair of sampling gloves (a new pair per sample); the sampler’s gloves were sterile sampling gloves. All members wore N95 dust masks to further minimize potential contamination of the samples.

4. The sample handler removed the coupon from the appropriate cabinet and placed it in the sampling area.

5. The support person recorded the coupon code on the sampling log sheet.

6. The support person removed a template from the bag and handed it to the sampler.

7. The sampler placed the template onto the coupon surface.

8. The support person removed a sample kit from the sampling bin and recorded the sample tube number on the sampling log sheet next to the corresponding coupon code just recorded.

9. The support person:

a. Opened the outer sterile sampling bag touching the outside of the bag.

b. Touching only the outside of the outer bag, removed and opened the unlabeled conical tube and poured the pre-moistened wipe onto the sample.

c. Discarded the unlabeled conical tube.

d. Maneuvered the labeled 50 mL conical tube to the end of the outer sterile sampling bag and loosened the cap.

e. Removed the cap from the 50 mL conical tube immediately preceding the introduction of the wipe sample into the tube.

10. The sampler:

a. Wiped the surface of the sample horizontally using S-strokes to cover the entire sample area of the coupon using a consistent amount of pressure.

b. Folded the wipe concealing the exposed side and then wiped the same surface vertically using the same technique.

c. Folded over again and rolled the folded wipe to fit into the conical tube.

d. Carefully placed the wipe into the 50 mL conical tube that the support person was holding being careful not to touch the surface of the 50 mL conical tube or plastic sterile sampling bag.

11. The support person immediately closed and tightened the cap to the 50 mL conical tube and slid the tube back into the sample collection sterile sampling bag.


12. The support person then put the 50 mL conical tube into the empty labeled, 5.5” x 9” sampling bag and sealed the bag.

13. The support person then sealed the outer sample collection bag containing the capped 50 mL conical tube (containing the sample wipe) inside a sealed 5.5” x 9” sample collection bag.

14. The support person then decontaminated the outer sample bag by wiping it with a Dispatch® bleach wipe.

15. The support person then placed the triply contained sample into the sample collection bin.

16. If sampling from the coupon was completed, the sample handler moved the coupon and template to the appropriate location for archival or discarding.

17. All members of the sampling team removed and discarded their gloves.

18. Steps 3 – 17 were repeated for each sample collected.

E.2.1 Wipe Sampling for 18 mm Coupons (Stubs)

The following steps were performed in order as the wipe sampling procedure for the 18 mm coupons:

1. The Biosafety Cabinet was cleaned by wiping with a Dispatch® bleach wipe, rinsing with DI water, and then rinsing with 70% ethanol.

2. The coupons were removed from the blank coupon cabinet and placed under the biosaftey hood located in H-130.

3. Donning examination gloves, the sampling kit was removed for the sample kit container. The contents of the kit were removed and placed under the hood.

4. Changed into sterile sampling gloves.

5. Using one hand, the cap was removed from the tube, then, with the other hand, the excess PBST was pressed from the wipe.

6. Using the same hand, sampled the coupon by gently wiping the surface 10 times with the wipe.

7. With the same hand used to open the tube containing the wipe, picked up and opened the empty sterile sample tube then, inserted the wipe sample and replace the cap.

8. Replaced gloves with a new pair of examination gloves.

9. Placed the tube containing the wipe sample inside the empty sample collection sterile bag and then placed the sample collection bag containing the tube with the wipe sample into the large sample bag.

10. Placed the kit containing the wipe sample into a clean transport container.


Care was taken to handle the wipe sample with only one hand and the collection materials with the other while performing Steps 3-7.

The two blank 18 mm coupons were sampled and the stubs transferred to a sterile tube for extraction prior to handling the test and positive control coupons. Seven contaminated 18 mm coupons were directly extracted (extraction positive controls) with 20 mL of PBST used as the extraction buffer. Seven contaminated 18 mm coupons were wipe-sampled (wipe positive controls) then extracted (extraction after wipe positive controls).

E.3 Sampling Protocol for Vacuum Sock Sampling

Vacuum sock sampling has typically been used for large, porous areas.13 The general approach has been to use a collection sock to trap dust material. 13 The protocol that was used in this project, described below, has been adapted from that provided by Busher et al. 13, Brown et al.30, and documented in the INL 2008 Evaluation Protocols.17 None of these references provides a validated vacuuming procedure for Bacillus spores, as a validated sampling procedure does not currently exist.

The following procedure was used in this study for vacuum sampling of each coupon surfaces:

1. A three-person team was used, employing aseptic technique. The team consisted of a sampler, sample handler, and support person.

2. All materials needed for each sample that was collected were prepared in advance using aseptic technique A sample kit for a single vacuum sample contained the following:

a. Two sampling bags (10” x 14”, 5.5” x 9”) were labeled per the sampling plan. These bags had the same label. An additional unlabeled bag contained the vacuum sock collection assembly. The label was clearly distinguishable through the unlabeled bag.

b. The two sterile labeled sampling bags and the vacuum sock assembly bag were placed inside a second 10” x 14” unlabeled bag.

c. Each prepared bag was one vacuum sock sampling kit.

3. All members of the sampling team donned a pair of sampling gloves (a new pair per sample); the sampler’s gloves were sterile sampling gloves. All members wore dust masks to further minimize potential contamination of the samples.

4. The sampler plugged in the HEPA vacuum power cord and then donned his/her sterile gloves.

5. The HEPA vacuum was maintained on a rolling cart for easy movement into place.

6. The sampler held the vacuum nozzle for the support person to place the vacuum sock assembly onto the nozzle.

7. The support person opened the sampling supply bin and removed one vacuum sock sample kit from the bin.


8. The support person recorded the sample collection bag number on the sampling log sheet.

9. The sample handler removed the coupon from the appropriate cabinet and placed it on the sampling area.

10. The support person removed a template from the bag and handed it to the sampler.

11. The sampler placed the template onto the coupon surface.

12. The support person recorded the coupon code on the sampling log sheet next to the corresponding vacuum sock collection bag number that was just recorded.


a. Opened the vacuum sock sample kit outer bag and removed the unlabeled vacuum sock assembly bag.

b. Opened the small unlabeled sampling bag containing the vacuum sock assembly and pushed the assembly from the bottom to expose the cardboard applicator tube opening.

c. Placed the vacuum sock assembly onto the nozzle of the vacuum tube, using the bag to handle the sock assembly, while the sampler held the vacuum nozzle.

14. The sampler:

a. Securely held the outer edge of the sock onto the tube.

b. Turned on the vacuum with her foot.

c. Did not release the filter sock while the vacuum was turned on in order to prevent the sock from being sucked into the vacuum

d. Vacuumed “horizontally” used S-strokes to cover the entire area of the material surface not covered by the template, while keeping the vacuum nozzle perpendicular to the sample surface.

e. Vacuumed the same area “vertically” using the same technique.

f. Turned off the vacuum when sampling was completed.

15. The support person removed the vacuum sock assembly from the nozzle, using the inner sterile sampling bag.

16. The support person then sealed the inner sterile sampling bag and placed it into the outer sterile sampling bag.

17. The support person then sealed the outer sterile sampling bag.


18. The support person then sealed the 10” x 14” overpack sample bag now containing the outer and inner bags, the inner containing the vacuum sock assembly. The 3rd size bag was then wiped with a bleach wipe and then the bleach wipe was discarded.

19. The sampler wiped down the nozzle (inside and out) and the end of the tubing with a bleach wipe, then disposed of the bleach wipe.

20. The support person will then place the triple contained sample into the sample collection bin.

21. If sampling from the coupon was completed, the sample handler moved the coupon and template to the appropriate location for archival or disposal.


23. Steps 3 – 21 were repeated for each sample collected.

E.4 Sampling Protocol for Swab Sampling

Swab sampling was used for sterility checks on coupons and equipment prior to use in the testing. The protocol that was used in this project, described below, was adapted from that provided by Busher et al.13, Brown et al. 31, and documented in the INL 2008 Evaluation Protocols.17

Collecting Swab Samples:

1. A three-person team was used, employing aseptic technique throughout. The team consisted of a sampler, sample handler, and support person.

2. All materials needed for collection of each sample were prepared in advance using aseptic technique. A sample kit for a single wipe sample was prepared as follows:

a. Two sterile sampling bags (10” x 14”, 5.5” x 9”) and a 50 mL conical tube, capped, were labeled in accordance with the sampling plan. These bags and conical tube had the same label. The 5.5” x 9” labeled sterile sampling bag was referred to as the sample collection sterile sampling bag.

b. A dry sterile swab was placed in an unlabeled sterile 50 mL conical tube using sterile forceps and aseptic technique. The swab was moistened by adding 1 mL of sterile PBST. The tube was then sealed.

c. The, capped, labeled 50 mL conical tube, the unlabeled, capped, conical tube containing the pre-moistened swab, and the 5.5” x 9” labeled sampling bag were placed into the 10” x 14” labeled sterile sampling bag. Each labeled sterile sampling bag contained a labeled 50 mL conical tube (capped), an unlabeled capped conical tube containing a pre-moistened swab, and an empty labeled sterile sampling bag.

d. Each prepared bag was one sampling kit.

3. All members of the sampling team donned a pair of sampling gloves (a new pair per sample). All members wore dust masks to further minimize potential contamination of the samples.


4. The sample handler removed the item to be sampled from the appropriate location and held it for the sampler to sample.

5. The support person recorded the coupon or item code on the sampling log sheet.

6. The support person removed a sample kit from the sampling bin and recorded the sample tube number on the sampling log sheet next to the corresponding coupon code just recorded.


a. Opened the outer sterile sampling bag touching the outside of the bag.

b. Touching only the outside of the overpack bag, removed and opened the unlabeled conical tube and removed the pre-moistened swab.

c. Discarded the unlabeled conical tube.

d. Maneuvered the labeled 50 mL conical tube to the end of the outer sterile sampling bag and loosened the cap.

e. Removed the cap from 50 mL conical tube immediately preceding the introduction of the sample into the tube.

8. The sampler:

a. Swabbed the surface of the sample horizontally using S-strokes to cover as much of the surface of the item as possible with one swab, using a consistent amount of pressure.

b. Carefully placed the swab into the 50 mL conical tube that the support person was holding being careful not to touch the surface of the 50 mL conical tube or plastic sterile sampling bag.

9. The support person then immediately closed and tightened the cap to the 50 mL conical tube and slid the tube back into the sample collection sterile sampling bag.

10. The support person then put the 50 mL conical tube into the empty, labeled, 5.5” x 9” sampling bag and sealed the bag.

11. The support person then sealed the outer sample collection bag now containing the capped 50 mL conical tube (containing the sample swab) inside a sealed 5.5” x 9” sample collection bag.

12. The support person then decontaminated the outer sample bag by wiping it with a Dispatch® bleach wipe.

13. The support person then placed the triply contained sample into the sample collection bin.

14. If sampling from the item was completed, the sample handler moved the item to the appropriate location for storage.



Steps 3 – 15 were repeated for each sample collected.

E.5 Rinsate Collection and Sampling Procedures

The runoff from the coupons throughout the entire decontamination procedure being tested was collected for a given coupon set (material type or all blanks). After all coupons from a single set were moved to the Decontaminated Coupon Cabinet or Procedural Blank Cabinet, the chamber was rinsed with DI water. The sterile runoff collection carboy was labeled and the total volume of liquid collected was recorded. The runoff was not neutralized for Test 1; however, the remaining tests had 400 mL of 1N STS added to the carboy prior to collecting the runoff. After collection, the entire rinsate sample was filtered immediately and/or 100 mL aliquots were taken using aseptic technique. The aliquot collection procedure was performed as follows:

1. Sampler donned a face mask, pair of examination gloves, disposable laboratory coat, and bouffant cap.

2. The contents of the carboy were agitated to ensure homogeneous mixing.

3. The carboy cap was removed.

4. Using a new 50 mL sterile pipette tip, 100 mL of sample was aseptically pipetted into a sterile 100 mL container. Repeated Step 4 until triplicate samples were obtained.

The rinsate aliquots were triply-contained and transported to the NHSRC Microbiology Laboratory for submission and analysis at the conclusion of the entire test.

The protocol for rinsate filtration was as follows:

1. The filtration tubing and filter housing were sterilized using a 1 hour autoclave gravitation cycle. Using aseptic techniques, the NHSRC Microbiology Laboratory staff inserted a clean 47 mm (0.6 μm pore-size) filter into the sterile housing. This assembly was put into a sterile bag for transport to H130.

2. The rinsate was stored (no more than 10 minutes) with the 5-gallon carboy lid tightened until filtered (Step 3).

3. To sample the runoff, the inlet-end of the tubing was attached to the outlet of the sample carboy. The volume of sample water inside the carboy was noted and recorded prior to sampling. The valve on the sample carboy was opened and the vacuum pump was activated. The entire sample volume was passed through the filter. The vacuum pump was then turned off.

4. Using aseptic techniques, the in-line filter holder containing the filter was removed and placed in a pre-labeled sterile sample bag.

5. The filter holder containing the filter was then stored in a closed secondary container until the extraction was performed.


6. The decontaminated contents of the vacuum-trap carboy were then discarded. The contents of the vacuum-trap carboy were adequately decontaminated by the end of the sample filtration due to the presence of the pH-adjusted bleach solution collected during the initial system decontamination procedure.

The filtered spores were then rinsed with DI water to remove any residual decontaminant.

The samples from the test were triple-contained and transferred to the NHSRC Microbiology Laboratory for analysis at the conclusion of the entire test.

E.6 Wet/dry Vacuum Collection Sampling Procedures

E.6.1 Wet/dry Vacuum HEPA Filter Sampling Procedure

The purpose of sampling the wet/dry vacuum after use was to confirm contamination of the unit with the target organism. The most logical place to sample to confirm contamination was the HEPA filter. The filter was sampled using the swab protocol discussed in Section E.4. All pleats of the filter were sampled with a single swab.

E.6.2 Wet/dry Vacuum Exhaust Sampling Procedure

Wet/dry Vacuum Exhaust Sampling Procedure

Sampling for the target bacterial spores was done by drawing a sample from the exhaust duct of the vacuum containment chamber only during the operation of the wet/dry vacuums. The sampling pump was turned on when the wet/dry vacuum was on and off when the wet/dry vacuum was off. This operating scheme minimized the potential for confounding organisms to accumulate on the collection filter. Sampling was done using a Via-Cell® Bioaerosol Sampling Cassette. The sampling pump flow rate was 0.53 ft3 per min. (15 liters per min.) through a sterile nozzle, the size of which was determined based on the exhaust duct flow rate according to the guidelines in 40 CFR 60 (http://www.eti-usepa.com/testing_methods.htm). The total sample flow was calculated using the difference between the start and stop total values on the sampling console. Upon completion of a test, the sampling cassette was placed in a labeled sterile bag. This bag was sealed and placed inside an outer unlabeled sterile bag. This bag was sealed and then wiped with a Dispatch® bleach wipe. After the bleach wipe was discarded, the sample was placed inside another unlabeled sterile bag and that bag sealed. The sample was then placed into the sample collection/transport bin to be stored prior to transport to the NHSRC Microbiology Laboratory .


Appendix F – Page 1

Appendix F

Sampling Analyses

F.1 Sample Analyses

The EPA NHSRC Microbiology Laboratory, located in E-288 of the RTP, NC campus facility, analyzed all samples to quantify the number of CFU per sample. For all sample types, PBST was used as the extraction buffer. After the appropriate extraction procedure (see below), the buffer was subjected to a four-stage serial dilution (10-1 to 10-4), plated, incubated, and read (CFU counts) in accordance with MOP 6535a (see Appendix B)

In addition to the analysis in MOP 6535a, two additional analysis procedures were used for samples resulting in less than 30 CFU/sample in the zero tube (undiluted sample, e.g., wipe or vacuum sock in the extraction buffer). These analyses were tested in order to lower the current detection limit associated with MOP 6535a. First, 1 mL of the extract was filter plated in accordance with MOP 6565 (see Appendix B). Then, the remainder of the sample was filter plated in accordance with MOP 6565.

The PBST was prepared according to the manufacturer’s directions and in accordance with an internal NHSRC Microbiology Laboratory MOP 6562, dissolving one packet in one liter of sterile water. The solution was then vacuum filtered through a sterile 0.22 µm filter unit to sterilize.

The extraction procedure used to recover spores varied depending upon the matrix (wipes, filter socks, wet/dry vacuum filter, liquid, filter cassette). The procedures are described in the following subsections.

F.1.1. Recovery from Wipe Samples

The recovery of the spores from the wipe samples was done as follows, as adapted from the INL 2008 Evaluation Protocols17:

1. The analyst donned a fresh pair of gloves. Gloves were changed periodically (at least between batches) or after direct contact with a sample to reduce contamination.

2. The 50 mL conical tube containing the sample wipe was removed from the double sterile bag and wiped with a bleach wipe. The analyst changed gloves after the wipe step.

3. 20 mL of PBST was added to each 50 mL conical tube by aseptically pouring a pre-measured volume.

4. The sample was then vortexed for 2 minutes in 10 second bursts, leaving the wipe in the same tube.

5. If the sample sat for more than one minute after Step 4, the sample was vortexed briefly to homogenize. The conical tube was then uncapped and the cap placed underside up on the Biosafety Cabinet surface.


6. Steps 1-5 were repeated for each sample in the batch.

7. The samples were then brought to the Biosafety Cabinet for dilution plating.

F.1.2 Recovery from Vacuum Sock Samples

The recovery of the spores from the vacuum socks was done as follows, as adapted from the INL 2008 Evaluation Protocols17:

1. The analyst donned a fresh pair of gloves. Gloves were changed between each sample and after direct contact with a sample to reduce contamination.

2. Sterile 3-ounce specimen cups were pre-labeled as per the sample log corresponding to the batch of samples being processed.

3. The 3-ounce specimen cup sample containers were then loaded with 20 mL of PBST.

4. Both sterile sample bags were opened, without removing the inner bag from outer bag. The vacuum sock assembly was manipulated to the opening of the bag using the bag.

5. The vacuum sock assembly was removed from the bag by handling only the cardboard portions.

6. The nonsterile blue portion of the vacuum sock assembly was flipped from the larger cardboard ring to the smaller ring.

7. The larger cardboard ring was removed, exposing the vacuum sock.

8. The vacuum sock was wetted by holding the upper blue portion of the sock (around the smaller cardboard ring) and dipping the lower inch of the sock into the PBST. The sock was allowed to soak up the PBST for a few seconds.

9. After the soaking, the vacuum sock was lifted up just above the opening of the specimen bottle. A 1-inch vertical slit was cut up the center from the bottom of the sock using sterile scissors. A new pair of scissors was used for each sample.

10. The vacuum sock was 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 was only cut where the sock has been wetted.

11. Steps 8-10 were repeated until the entire white portion of the vacuum sock was cut.

12. The upper top blue portion of the vacuum sock was then discarded.

13. After use, scissors were autoclaved using a one hour gravity destruction cycle in preparation for use with the next sample batch.

14. Gloves were changed between samples.


15. Steps 4 – 12 were repeated for each sample in the batch.

16. All samples (up to sixty samples at a time) were loaded into the sample cup holder of the orbital shaker incubator.

17. The samples were agitated in the shaker incubator at 300 rpm for 30 minutes with the heat off.

18. The samples were then removed from the shaker incubator and brought to the Biosafety Cabinet for dilution plating.

F.1.3 Analysis of Wet/dry Vacuum Filter Samples

The wet/dry vacuum sampling was done using the swab sampling procedure. Qualitative analysis (growth/no growth) with organism identification was performed in accordance with MOP 6563.

F.1.4 Recovery from Liquid

The extraction of the spores from the filters was done by adding 20 mL of PBST to the 50 mL conical tube containing the sample filter and vortexed for 2 minutes (in 10 second burst). The samples were then dilution plated. The concentration of spores in the original runoff water was determined by dividing the total abundance of spores by the total runoff volume.

F.1.5 Recovery from Air Sample

The extraction of the spores from the filters was done according to MOP 6565 (see Appendix B). In short, the filter housing allows for in-situ extraction using 20 mL PBST. This suspension was then dilution plated in triplicate. The concentration of spores in the air was determined by dividing the total abundance of spores by the total sampled air volume.

Appendix G – Page 1

Appendix G

Test Chamber and Equipment Cleaning Procedures

The pH-adjusted bleach solution to be used for cleaning surfaces in equipment in both the decontamination and NHSRC Microbiology Laboratoryoratory was prepared as a 1:10 dilution of bleach in DI water, pH-adjusted to ~6.8 using glacial acetic acid.

The following steps were followed for cleaning the decontamination chamber between each material type and before/after each test:

1. Using the back sprayer, the interior surfaces were kept wet with pH-adjusted bleach solution for 10 min.

2. With the drain open, the surfaces were then rinsed with DI water. The rinsate was collected in a carboy and ultimately discarded.

3. After ensuring all rinsate was removed from the chamber, the valve was closed in preparation of the next test.

4. A mop assembly with a disposable pad was used to wipe down the interior of the chamber with isopropyl alcohol or ethanol.

5. The pad was then removed and placed in a bucket of pH-adjusted bleach solution for decontamination prior to disposal.

The following steps were followed for cleaning the wet/dry vacuum head assemblies between uses on a specific coupon within a test:

1. Soak the head assembly in pH-adjusted bleach for at least 10 min.

2. Rinse with DI water.

3. Air dry prior to re-use.

The following steps were followed for cleaning the wet/dry vacuum (including head assembly) after use in a test:

1. Soak the head assembly in pH-adjusted bleach for at least 60 min.

2. Spray the wet/dry vacuum drum with pH-adjusted bleach and maintain wetted for at least 60 min.

3. Soak the hoses in pH-adjusted bleach for at least 60 min.

4. Rinse all parts with DI water.


6. Alternatively, the wet/dry vacuums may be fumigated with a STERIS VHP® sterilization cycle. This cycle entails the use of a STERIS VHP® ARD hydrogen peroxide (H2O2) generator and exposure of all


components of the wet/dry vacuum to H2O2 at 1000 ppmv for 60 min by maintaining this constant concentration in a decontamination chamber.

7. Replace HEPA filters.

The following steps were 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 min.

2. Rinse all buckets five times with DI water.


The following steps were followed for cleaning the brushes after use in a test:

1. Soak the brushes in pH-adjusted bleach for at least 60 min.



The following steps were followed for cleaning the work surfaces before and after use:

1. Wet all surfaces with pH-adjusted bleach solution or using Dispatch® bleach wipes.


3. Wet and wipe surfaces with isopropyl alcohol or ethanol.


5. Alternatively, cover paper can be used and replaced before/after each use.

The sampling templates were autoclaved before/after each use.

The following steps were 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.


3. Wet and wipe surfaces with isopropyl alcohol or ethanol.


The gaskets used in MOP 6561 during the contamination procedure were cleaned via fumigation with the STERIS VHP® sterilization cycle. This cycle entails the use of a STERIS VHP® ARD hydrogen peroxide (H2O2) generator and exposure of all components of the wet/dry vacuum to H2O2 at 1000 ppmv for 60 min by maintaining this constant concentration in a decontamination chamber.

Bins used in the study were either filled with pH-adjusted bleach and left covered for at least 60 min, rinsed with DI water, and air dried or cleaned by the following procedure:


1. Wet and wipe all surfaces with pH-adjusted bleach solution or using Dispatch® bleach wipes.



Appendix H – page 1

Appendix H

Test Reports

Appendix H – Page 2

Test Date: 8/17-18/2009, 8/24-8/25 Sampling Date: 8/19-20/2009, 8/26-8/27/09 Analysis Date: 8/21-23/2009, 8/26-8/27-09Test Number: 1A, 1B Sampling Team: Analyst: C. Slone, J. Novak

Coupon Orient.: HorizontalTest Team: S.Payne, R.Delafield,C.Whitfield

Blank CouponsMaterial Sample Type Avg. CFU/sample RSD (%) CFU/sample Avg. CFU/sample RSD (%) LR SDStainless Steel wipe 5.33E+07 51%Carpet HEPA 5.60E+06 43% 6.3E-01 7.92E-01 35% 6.85 0.13Concrete wipe 2.65E+06 43% 6.7E-01 6.52E-01 4% 6.57 0.02Deck Wood wipe 8.09E+06 31% 5.9E-01 5.80E-01 3% 7.13 0.01Painted Wallboard wipe 6.17E+07 29% 1.2E+00 1.42E+00 87% 7.81 0.35

Rinsate VacuumMaterial (Total CFUs) (growth/no growth)Blanks 1.70E+02 no growth Blank Coupons 1.22E+01Carpet 2.93E+02 growth Test Coupons 6.47E+01Concrete 7.75E+01 growthDeck Wood 2.22E+00 growthPainted Wallboard 6.82E+01 growth

Observations/Comments:

Vacuum Air Exhaust (CFU/L)

Test Coupons

DCMD 3.41: Assessment of Liquid and Physical Decontamination Methods for Environmental Surfaces Contaminated with Bacterial Spores

Part 1 - Development and Evaluation of the Decontamination Procedural Steps

>>> Test Report <<<

Positive ControlsSurface Samples

S.Payne, R.Delafield, C.Whitfield

Deck wood rinsate results are based on filter plating the remainder of the 20 mL sample.


Test Date: 8/3109-9/1/09 Sampling Date: 9/2-3/09 Analysis Date:Test Number: 2 Sampling Team: Analyst: C. Slone, J. Novak

Coupon Orient.: VerticalTest Team: S.Payne, R.Delafield, C.Whitfield

Blank CouponsMaterial Sample Type Avg. CFU/sample RSD (%) CFU/sample Avg. CFU/sample RSD (%) LR SDSS Wipe 3.88E+07 35%Concrete Wipe 5.88E+06 30% 6.25E-01 6.33E-01 3% 6.95 0.01Dry Wall Wipe 5.74E+07 31% 6.25E-01 5.44E-01 3% 8.01 0.01Rough-cut Wood HEPA 1.17E+07 35% 5.41E-01 6.37E-01 3% 7.24 0.01

Rinsate VacuumMaterial (Total CFUs) (growth/no growth)Blanks 2.00E+01 no growth Blank Coupons 1.61E+01Concrete 7.02E+02 growth Test Coupons 9.36E+00Dry Wall 5.10E+01 growthRough-cut Wood 4.12E+02 growth





>>> Test Report <<<


M.Clayton, R.Delafield, C.Whitfield

Test Coupons

9/3-4/09

Collected rinsate from both the 10 min and the 30 min pH-adjusted bleach spray stepsRinsate from only 10 min bleach step is reported.


Test Date: 9/28/09-9/29/09 Sampling Date: 9/30/09-9/3/09 Analysis Date:Test Number: 3 Sampling Team: Analyst: C. Slone, J. Novak


Blank CouponsMaterial Sample Type Avg. CFU/sample RSD (%) CFU/sample Avg. CFU/sample RSD (%) LR RSD (%)Stainless Steel wipe 4.81E+07 13%Carpet HEPA 1.19E+07 83% 6.3E-01 6.31E-01 4% 6.99 2%Concrete wipe 7.30E+06 57% 6.3E-01 6.35E-01 3% 7.01 1%Deck Wood wipe 1.49E+07 28% 3.4E+02 9.89E-01 30% 7.22 16%Painted Wallboard wipe 4.85E+07 5% 8.9E+00 1.19E+00 1854% 7.35 81%

Rinsate VacuumMaterial (Total CFUs) (growth/no growth)Blanks 3.56E+01 growth Blank Coupons 7.61E-01Carpet 2.95E+01 growth Test Coupons 3.27E+00Concrete 1.72E+02 growthDeck Wood 3.34E+03 growthPainted Wallboard 2.05E+02 growth

Observations/Comments:Deck wood rinsate values are based on 10 mL filter plates and the rest on 89 mL filter plates.



>>> Test Report <<<


S.Payne, R.Delafield, S. Terll

Test Coupons

10/1/09-10/2/09



Test Date: 10/5-6/09 Sampling Date: 10/7-8/09 Analysis Date:Test Number: 4C Sampling Team: Analyst: C. Slone, J. Novak


Blank CouponsMaterial Sample Type Avg. CFU/sample RSD (%) CFU/sample Avg. CFU/sample RSD (%) LR SDSS Wipe 4.07E+07 19%Concrete Wipe 3.21E+06 42% 6.25E-01 1.62E+00 89% 6.39 0.39Dry Wall Wipe 3.60E+07 18% 2.29E+00 1.66E+00 48% 7.35 0.28Rough-cut Wood HEPA 1.44E+07 41% 6.25E-01 8.05E+00 171% 6.71 0.71

Rinsate VacuumMaterial (Total CFUs) (growth/no growth)Blanks 1.01E+02 no growth Blank Coupons 5.26E-01Concrete 2.32E+04 growth Test Coupons 2.14E+02Dry Wall 5.59E+01 growthRough-cut Wood 1.07E+05 growth



This test was a repeat of Test 4 however, 300 mL of sodium thiosulfate were added to the concrete, rough cut wood, and dry wall carboys prior to collecting the rinsate.



>>> Test Report <<<


S.Payne, R.Delafield, S. Terll

Test Coupons

10/8-9/09


Test Date: 7/6/2009 Sampling Date: 7/8/2009 Analysis Date:Test Number: 5 Sampling Team: Analyst: C. Slone, J. Novak

Coupon Orient.: HorizontalTest Team: S.Payne, R.Delafield, M. Clayton

Blank CouponsMaterial Sample Type Avg. CFU/sample RSD (%) CFU/sample Avg. CFU/sample RSD (%) LR SDStainless Steel wipe 7.12E+07 27%Carpet HEPA 7.42E+06 28% 1.0E+03 1.70E+06 25% 0.64 0.13Concrete wipe 1.68E+07 29% 2.8E+02 4.95E+06 64% 0.58 0.25Deck Wood wipe 1.50E+07 30% 2.4E+02 1.42E+07 18% 0.01 0.08Painted Wallboard wipe 6.73E+07 20% 1.9E+02 5.68E+07 32% 0.08 0.14

Rinsate VacuumMaterial (Total CFUs) (growth/no growth)Blanks NA growth Blank Coupons 8.46E-02Concrete NA growth Test Coupons 1.14E-01Rough Cut Wood NA growthDry Wall NA growth





>>> Test Report <<<



7/9/2009

Test Coupons




Blank CouponsMaterial Sample Type Avg. CFU/sample RSD (%) CFU/sample Avg. CFU/sample RSD (%) LR SDSS Wipe 4.26E+07 3%Concrete Wipe 2.44E+06 61% 5.00E+02 1.34E+06 58% 0.31 0.41Dry Wall Wipe 5.10E+07 18% 6.00E+01 3.37E+07 36% 0.20 0.17Rough-cut Wood HEPA 1.67E+07 32% 1.00E+03 2.85E+06 38% 0.78 0.19

Rinsate VacuumMaterial (Total CFUs) (growth/no growth)Blanks NA no growth Blank Coupons naConcrete NA growth Test Coupons 2.84E+02Dry Wall NA growthRough-cut Wood NA growth

Observations/Comments:CV vacuum filter not correctly installed.



>>> Test Report <<<

Positive Controls Test CouponsSurface Samples


6/19/2009





Blank CouponsMaterial Sample Type Avg. CFU/sample RSD (%) CFU/sample Avg. CFU/sample RSD (%) LR SDStainless Steel wipe 9.71E+07 12.78%

Concrete wipe 3.51E+06 66% 5.9E-01 1.31E+05 81% 1.45 0.37Deck Wood wipe 1.73E+07 28% 1.0E+02 1.68E+04 66% 3.08 0.29Painted Wallboard wipe 4.95E+07 24% 5.6E-01 9.13E+05 33% 1.75 0.17

Rinsate VacuumMaterial (Total CFUs) (growth/no growth)Blanks 1.00E+03 no growth Blank Coupons 4.28E-02Concrete 5.79E+06 growth Test Coupons 5.88E-02Deck Wood 2.32E+06 no growthPainted Wallboard 6.75E+05 NA


8/6/2009

vacuum filtered entire rinsate sample



>>> Test Report <<<







Blank CouponsMaterial Sample Type Avg. CFU/sample RSD (%) CFU/sample Avg. CFU/sample RSD (%) LR SDSS Wipe 3.24E+07 45%Concrete Wipe 1.07E+07 66% 2.82E+02 1.28E+05 53% 1.87 0.30Dry Wall HEPA 6.04E+07 33% 2.71E+01 9.65E+03 25% 3.79 0.10Rough-cut Wood Wipe 2.18E+07 36% 5.88E-01 3.08E+05 50% 1.86 0.20

Rinsate VacuumMaterial (Total CFUs) (growth/no growth)Blanks 4.01E+03 NA Blank Coupons naConcrete 1.26E+07 NA Test Coupons naDry Wall 5.55E+05 NARough-cut Wood 2.59E+06 NA



6/5/2009



>>> Test Report <<<



Rough cut wood, dry wall rinsate filters were torn which may have led to breakthroughsvacuum filtered entire rinsate sample




Blank CouponsMaterial Sample Type Avg. CFU/sample RSD (%) CFU/sample Avg. CFU/sample RSD (%) LR SDStainless Steel wipe 4.59E+07 15%

Concrete wipe 8.57E+06 13% 4.3E-01 1.17E+05 59% 1.92 0.26Deck Wood wipe 1.03E+07 23% 2.2E+02 1.49E+06 42% 0.87 0.20Painted Wallboard wipe 3.65E+07 7% 1.1E+02 4.65E+06 31% 0.91 0.14

Rinsate VacuumMaterial (Total CFUs) (growth/no growth)Blanks 7.80E+03 no growth Blank Coupons 6.21E-02Concrete 3.73E+07 growth Test Coupons 7.75E-03Deck Wood 3.82E+07 growthPainted Wallboard 2.61E+05 NA



8/13/2009



>>> Test Report <<<






Blank CouponsMaterial Sample Type Avg. CFU/sample RSD (%) CFU/sample Avg. CFU/sample RSD (%) LR SDSS Wipe 4.09E+07 4%Concrete Wipe 4.06E+06 133% 1.41E+01 4.15E+04 148% 2.03 0.55Dry Wall Wipe 4.47E+07 12% 2.07E+02 1.32E+05 24% 2.54 0.11Rough-cut Wood HEPA 1.60E+07 49% 2.00E+03 8.93E+04 34% 2.23 0.17

Rinsate VacuumMaterial (Total CFUs) (growth/no growth)Blanks 2.67E+02 NA Blank Coupons NAConcrete 4.03E+06 NA Test Coupons NADry Wall 6.13E+06 NARough-cut Wood 2.25E+07 NA


6/26/2009

Rough Cut Wood rinsate data is from 2 coupons only.vacuum filtered entire sample



>>> Test Report <<<





Test Date: 11/2-3/2009 Sampling Date: 11/4-5/2009 Analysis Date:Test Number: 11 Sampling Team: Analyst: C. Slone, J. Novak

Coupon Orient.: HorizontalTest Team: S.Payne, R.Delafield, S.Terll

Blank CouponsMaterial Sample Type Avg. CFU/sample RSD (%) CFU/sample Avg. CFU/sample RSD (%) LR SDStainless Steel wipe 5.22E+07 15%Carpet HEPA 1.07E+07 29% 6.5E-01 7.74E-01 37% 7.14 0.13Concrete wipe 3.85E+06 63% 6.3E-01 3.55E+01 220% 6.23 1.09Deck Wood wipe 9.92E+06 29% 6.1E-01 4.01E+01 127% 5.67 0.58Painted Wallboard wipe 3.95E+07 17% 1.2E+01 5.56E-01 2% 7.85 0.01

Rinsate VacuumMaterial (Total CFUs) (growth/no growth)Blanks 1.44E+02 no growth Blank Coupons 2.71E-02Carpet 7.36E+01 growth Test Coupons 5.82E+00Concrete 2.42E+04 growthDeck Wood 3.71E+06 growthPainted Wallboard 9.69E+01 growth


11/5-6/2009

Clorox Clean-Up was applied every 2 min for 10 min for step 6



>>> Test Report <<<


S.Payne, R.Delafield, S.Terll




Coupon Orient.: VerticalTest Team: S.Payne, R.Delafield, T.Terll

Blank CouponsMaterial Sample Type Avg. CFU/sample RSD (%) CFU/sample Avg. CFU/sample RSD (%) LR SDSS Wipe 4.56E+07 34%Concrete Wipe 2.69E+06 47% 6.25E-01 6.14E-01 2% 6.60 0.01Dry Wall Wipe 3.69E+07 19% 1.00E+02 5.47E-01 2% 7.82 0.01Rough-cut Wood HEPA 1.75E+07 21% 6.25E-01 1.08E+02 106% 5.70 1.06

Rinsate VacuumMaterial (Total CFUs) (growth/no growth)Blanks 1.12E+02 no growth Blank Coupons 7.70E-01Concrete 4.75E+04 growth Test Coupons 6.80E+01Dry Wall 5.34E+05 growthRough-cut Wood 1.65E+06 growth



10/29-30/2009



>>> Test Report <<<



Clorox Clean-Up was administered every 2 minutes for 10 minutes during step 6



Coupon Orient.: VerticalTest Team: S.Payne, R.Delafield, S.Terll

Blank Coupons

Material Coupon Size Sample Type Avg. CFU/sample RSD (%) CFU/sampleAvg.CFU/ sample

RSD (%) LR SD

SS 14 inch aerosol wipe 3.98E+07 13%Rough-cut Wood 18 mm aerosol extract 1.90E+06 124% 9.09E-01 8.23E+05 247% 1.63 1.49Rough-cut Wood 18 mm aerosol wipe 2.62E+07 23% 1.03E+01 3.00E+03 93% 4.10 0.44Rough-cut Wood 18 mm aerosol extract after wipe 1.49E+06 94% 1.82E+05 130% 1.01 0.63Rough-cut Wood 14 inch liquid vacuum 2.52E+08 58% 2.72E+07 64% 0.96 0.25Rough-cut Wood 14 inch liquid wipe after vacuum 0 3.56E+07 50%Rough-cut Wood 14 inch aerosol vacuum 1.04E+07 23% 6.25E-01 1.65E+01 215% 6.78 0.94Rough-cut Wood 14 inch aerosol wipe 3.94E+06 36% 5.31E+00 6.08E-01 3% 6.79 0.01Rough-cut Wood 14 inch aerosol wipe after vacuum 0 5.92E-01 1%

RinsateMaterial (Total CFUs)18 mm aerosol inoc.wipe Blank Coupons NA18 mm aerosol inoc. Extractio Test Coupons NA14 inch liquid inoc. HEPA14 inch aerosol inoc. HEPA14 inch aerosol inoc. Wipe




>>> Test Report <<<

Positive Controls Test Coupons

Loading Method

Surface Samples

10/29-30/2009


14 inch rinsate results were based on a too numerous to count results from 10 microliter aliquotCoupons were sprayed with pH-adjusted bleach every 2 minutes for 10 minutes, then rinsed with DI water


4.58E+07

6.18E+11

Blank Rinsate (Total CFUs)

4.29E+04

3.74E+04

Appendix I – Page 1

Appendix I

Test 13 Preparation, Testing, and Sampling Procedures

Test 13

Test 13 was comprised of two smaller tests. The first test used rough-cut wood coupons 0.71 inches (18 mm) in diameter (13A) and the other used 14 in by 14 in rough-cut wood coupons (13B).

Preparation

The 18 mm stubs were fabricated on the Scanning Electron Microscope (SEM) stubs (aluminum, 18 mm [0.71 in] diameter, Ted Pella, Inc., Redding, CA) using assembly methods consistent with prior work.11 The coupons underwent sterilization by a steam autoclave on a gravity cycle following the NHSRC Microbiology Laboratory internal MOP 6565. Each sterilization batch included all coupons for a single test. Positive control and test coupons were contaminated with Bacillus globigii (B.g.) using methods consistent with MOP 3133.

A preparation resulting in a liquid mix containing approximately 1E8 viable B.g. spores per mL was prepared by adding approximately 1 gram of B.g. to 12 mL of 28.5% ethanol solution. The 12 mL solution was vortexed then separated by placing 1 mL into 11 micocentrifuge tubes. Ten 14 in by 14 in coupons were each spot inoculated with the contents of the 1 mL microcentrifuge tubes over an approximate 5 in by 5 in area of the coupon surface.

Twenty 14 in by 14 in sterile coupons were inoculated following the procedure outlined in Section 2.2 of the report: Material Inoculation Procedure and per MOP 6561 – Aerosol Deposition of Spores onto Material Coupon Surfaces using the Aerosol Deposition Apparatus.

Test Procedure

Two blank and 14 contaminated 18 mm coupons were aseptically transferred to 1 and 3 custom stainless steel stages, respectively, fabricated for use with the 4 ft by 4 ft decontamination spray chamber described in Section 2 of the report. After cleaning the chamber with pH-adjusted bleach then rinsing with DI water, the blank coupons were mounted vertically inside the chamber. The coupons were then sprayed with pH-adjusted bleach every 2 min for a total of 10 min. Rinsing was then done using DI water from the garden hose. The blanks were then removed and the chamber cleaned with pH-adjusted bleach and rinsed with DI water. After cleaning the chamber, the test coupons were mounted vertically in the chamber and decontaminated as described for the blanks (pH-adjusted bleach solution application followed by rinsing with DI water).

After cleaning the chamber, two blank 14 in by 14 in coupons were aseptically transferred to the decontamination chamber. The coupons were sprayed with pH-adjusted bleach every 2 minutes for a 10 min duration. Rinsing was then done using DI water from the garden hose. The blanks were removed and placed in their designated cabinet to dry. Once the chamber was cleaned, five liquid inoculated coupons and


ten aerosol inoculated coupons were decontaminated as described for the blanks (pH-adjusted bleach solution application followed by rinsing with DI water), in sets of three. The coupons were then removed from the chamber and placed in their designated cabinets.

All decontaminated coupons were allowed to completely dry for approximately 48 hours prior to sampling.

Sampling Procedure

For both tests, the rinsate from the blank coupons and test coupons was collected in two separate carboys containing 400 mL of 1 N STS each. The rinsates for both the liquid and aerosol inoculated 14 in by 14 in coupons were combined in one carboy. Three 100 mL aliquots were aseptically pipetted from the rinsate samples, then submitted to the NHSRC Microbiology Laboratory for analysis.

Sampling was performed starting with coupons from the lowest level of contamination (blanks) and ending with the highest level of contamination (positive controls).

The following steps were performed in order as the wipe sampling procedure for the 18 mm coupons:

1. The working surfaces of the Biosafety Cabinet were cleaned by wiping with a Dispatch® bleach wipe, rinsing with DI water, then rinsing with 70% ethanol.

2. The coupons were removed from the blank coupon cabinet and placed under the Biosaftey hood located in H-130.

3. Donning exam gloves, the sampling kit was removed for the sample kit container. The contents of the kit were removed and placed under the hood.

4. Sterile sampling gloves were donned. 5. Using one hand, the cap was removed from the tube. With the other hand, the excess PBST was pressed

from the wipe. 6. Using the same hand, the coupon was sampled by gently wiping the surface 10 times with the wipe. 7. With the same hand used to open the tube containing the wipe, picked up and opened the empty sterile

sample tube then, inserted the wipe sample and replaced the cap. 8. Replaced gloves with a new pair of exam gloves. 9. Placed the tube containing the wipe sample inside the empty sample collection sterile bag, and then placed

the sample collection bag containing the tube with the wipe sample into the large sample bag. 10. Placed the kit containing the wipe sample into a clean transport container 11. Care was taken to handle the wipe sample with only one hand and the collection materials with the other

hand while performing steps 3-7. The two blank 18 mm coupons were sampled and the stubs transferred to a sterile tube for extraction prior to handling the test and positive control coupons. Seven contaminated 18 mm coupons were directly extracted (extraction positive controls) with 20 mL of PBST used as the extraction buffer. Seven contaminated 18 mm coupons were wipe sampled (wipe positive controls) then extracted (extraction after wipe positive controls).

The 14 in by 14 in coupons were sampled following the wipe and vacuum sock sampling procedures outlined in Appendix E.

Before handling the test or positive control coupons, the two blank 14 in by 14 in coupons were vacuum sampled, then sampled using the wipe method.


Of the test coupons, five liquid inoculated test coupons were sampled using the vacuum method, and then re-sampled using the wipe method. Five aerosol inoculated test coupons were vacuum sampled, and then re-sampled using the wipe method. Finally, five aerosol inoculated coupons were sampled using only the wipe method.

Of the positive control coupons, five liquid inoculated coupons were vacuum sock sampled (liquid inoculated, vacuum sock positive controls), five aerosol inoculated coupons were vacuum sock sampled (aerosol inoculated, vacuum sock positive controls), and five aerosol inoculated coupons were wipe sampled (aerosol inoculated, wipe positive controls).

Office of Research and Development (8101R) Washington, DC 20460

Official Business Penalty for Private Use $300

PRESORTED STANDARDPOSTAGE & FEES PAID

EPAPERMIT NO. G-35

assessment of liquid and physical decontamination methods

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