Expedient Approaches for the Management of Wastes

EPA 600/R-14/262 | September 2014 | www.epa.gov/research

Expedient Approaches for the Management of Wastes Generated from Biological Decontamination Operations in an Indoor EnvironmentEvaluation of WastE sampling and dEcontamination procEdurEs

Office of Research and DevelopmentNational Homeland Security Research Center

ii

EPA 600-R-14-262 September 2014

Expedient Approach for Decontamination of Biologicals – Indoor Environment -

Evaluation of Waste Decontamination Procedures

Assessment and Evaluation Report

U.S. Environmental Protection Agency

Research Triangle Park, NC 27711

jevans03

Typewritten Text

iii

Disclaimer

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

Questions concerning this document or its application should be addressed to:

M. Worth Calfee, Ph.D. Decontamination and Consequence Management Division National Homeland Security Research Center U.S. Environmental Protection Agency (MD-E343-06) Office of Research and Development 109 T.W. Alexander Drive Research Triangle Park, NC 27711 Phone: 919-541-7600 Fax: 919-541-0496 E-mail: [email protected]

mailto:[email protected]

iv

Acknowledgments

This effort was directed by the principal investigator from the Office of Research and Development’s (ORD) National Homeland Research Center (NHSRC), Decontamination and Consequence Management Division (DCMD) utilizing the support from the US Environmental Protection Agency’s (EPA’s) Chemical, Biological, Radiological, and Nuclear (CBRN) Consequence Management Advisory Division (CMAD) within the Office of Emergency Management (OEM). The contributions of the entire team are acknowledged.

Project Team:

Worth Calfee, Ph.D. (Principal Investigator) US EPA, Office of Research and Development, NHSRC, DCMD Research Triangle Park, NC 27711

Paul Lemieux, Ph.D. US EPA, Office of Research and Development, NHSRC, DCMD Research Triangle Park, NC 27711 Mario Ierardi, Ph.D. US EPA, Office of Solid Waste and Emergency Response, Office of Resource Conservation and Recovery, Materials Recovery and Waste Management Division, Water Compliance Branch Arlington, VA, 22202 Paul Kudarauskas US EPA, Office of Solid Waste and Emergency Response, OEM, CBRN CMAD Washington, DC, 20004 Jeanelle Martinez, Ph.D. US EPA, Office of Solid Waste and Emergency Response, OEM, CBRN CMAD Cincinnati, OH 45220 R. Leroy Mickelsen, M.S., P.E. US EPA, Office of Solid Waste and Emergency Response, OEM, CBRN CMAD Research Triangle Park, NC 27711

Randy Schademann US EPA, Federal On-Scene Coordinator, Region 7 Lenexa, KS, 66219

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 acknowledged.

Ramona Sherman (Quality Assurance) NHSRC, ORD, US EPA Cincinnati, OH 45268

v

The following peer reviewers of this report are also acknowledged for their input to this product:

Marshall Gray (EPA ORD), Cathy Young (EPA Region 1), John Martin (EPA Region 6).

vi

Table of Contents

Disclaimer ................................................................................................................................................... iii

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

List of Tables ............................................................................................................................................... x

List of Acronyms and Abbreviations ...................................................................................................... xii

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

Summary of Results ................................................................................................................................ xiii

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

1.1 Process ........................................................................................................................................... 2

1.2 Project Objectives ........................................................................................................................... 3

1.3 Experimental Approach .................................................................................................................. 3

1.3.1 Testing Sequence .................................................................................................................. 3

1.3.2 Decontamination Strategy ...................................................................................................... 4

1.3.3 Method Development for Neutralization ................................................................................. 4

2 Materials and Methods ........................................................................................................................ 5

2.1 Facility Design ................................................................................................................................ 5

2.2 Test Coupon Preparation ............................................................................................................... 5

2.2.1 Carpet and Upholstery ........................................................................................................... 5

2.2.2 Paper ...................................................................................................................................... 6

2.2.3 Nitrile Gloves .......................................................................................................................... 7

2.3 Spore Preparation .......................................................................................................................... 8

2.3.1 Coupon Inoculation and Test Preparation .............................................................................. 8

2.4 Decontamination Procedure ........................................................................................................... 8

2.5 Method Development for Neutralization ......................................................................................... 9

2.6 Test Matrix ...................................................................................................................................... 9

2.6.1 Neutralization Method Development Test Matrix ................................................................... 9

2.6.2 Test Matrix ............................................................................................................................ 10

3 Sampling and Analytical Procedures .............................................................................................. 12

3.1 Sampling Strategy ........................................................................................................................ 12

3.1.1 Sponge-Stick™ Sampling .................................................................................................... 12

3.1.2 Extractive Sampling .............................................................................................................. 12

3.1.3 Sample Preservation ............................................................................................................ 13

3.1.4 Sampling Points ................................................................................................................... 13

vii

3.1.5 Carpet and Upholstery ......................................................................................................... 13

3.1.6 Paper Samples ..................................................................................................................... 14

3.1.7 PPE Samples ....................................................................................................................... 14

3.2 Sampling Frequency ..................................................................................................................... 15

3.2.1 Sample Quantities ................................................................................................................ 15

3.3 Measurement Methods ................................................................................................................. 16

3.3.1 Decontamination Solutions .................................................................................................. 16

3.3.2 Microbiological Samples ....................................................................................................... 16

3.3.2.1 Sample Extraction ........................................................................................................ 17

3.3.2.2 Sample Analysis ........................................................................................................... 17

3.4 Data Analysis ................................................................................................................................ 17

3.4.1 Sampling Efficiency .............................................................................................................. 17

3.4.2 Surface Decontamination Efficacy ....................................................................................... 17

3.4.3 Statistical Analysis ................................................................................................................ 20

4 Results and Discussion .................................................................................................................... 22

4.1 Sampling Methods Evaluation ...................................................................................................... 22

4.1.1 Carpet Material ..................................................................................................................... 22

4.1.2 Upholstery Material .............................................................................................................. 24

4.1.3 PPE Material ........................................................................................................................ 26

4.1.4 Paper Material ...................................................................................................................... 27

4.1.5 Sampling Methods Test Synopsis ........................................................................................ 29

4.2 Neutralization Methods Evaluation ............................................................................................... 30

4.2.1 Optimization of Neutralizer Concentration ........................................................................... 30

4.2.2 Sample Hold Time Effects .................................................................................................... 31

4.2.3 Immersion Time Effects ........................................................................................................ 31

4.2.4 Neutralization Tests Synopsis .............................................................................................. 32

4.3 Dunking/Immersion Decontamination Test Results ..................................................................... 32

4.3.1 Carpet Decontamination Results ......................................................................................... 33

4.3.1.1 Sampling Methods Evaluation for Carpet ..................................................................... 33

4.3.1.2 Carpet Decontamination Effectiveness ........................................................................ 36

4.3.2 Upholstery Decontamination Results ................................................................................... 37

4.3.2.1 Sampling Methods Evaluation for Upholstery .............................................................. 37

viii

5.3 QA/QC Checks ............................................................................................................................. 50

5.4 Acceptance Criteria for Critical Measurements ............................................................................ 52

5.5 Data Quality Audits ....................................................................................................................... 56

5.6 QA/QC Reporting ......................................................................................................................... 56

6 Summary and Recommendations .................................................................................................... 57

Appendix A: Miscellaneous Operating Procedures .............................................................................A1

MOP 3120 VHP Operation July 2013 signed ..........................................................................................A2

MOP 3128 A pH Adjusted Bleach Dec 2013 signed .............................................................................A15

MOP 3148 Chlorine Dioxide and Chlorite by HACH Nov 2012 signed .................................................A19

MOP 3165 Sponge Sample Collection July 2013 signed ......................................................................A23

MOP 3194 Procedure for Fabricating 18" x 18" Upholstery Coupons for Liquid Innoculation ..............A31

MOP 3195 Immersion Decontamination Aug 2013 for Worth approval ................................................A36

MOP 6535a Bacterial Spore Plate Counting and Dilutions Jan 2013 signed .......................................A41

MOP 6562 Preparing Pre-Measured Tubes with Aliquoted Amounts of Phosphate Buffered Saline with Tween 20 (PBST) ...............................................................................................................A49

MOP 6565 Filter-Plate Method Feb 2013 signed ..................................................................................A55

MOP 6580 Recovery of Bacillus spores from 3M Sponge Stick Samples Feb 2013 signed ................A58

MOP 6584 Replating Bacteria Spore Plates Nov 2012 .........................................................................A65

ix

List of Figures Figure 2.1: Poly Hog Trough®. ................................................................................................................. 5

Figure 2.2: Carpet tile. .............................................................................................................................. 6

Figure 2.3: Front of assembled upholstered coupon. ............................................................................... 6

Figure 2.4: Paper material. ....................................................................................................................... 7

Figure 2.5: White disposable nitrile glove. ................................................................................................ 7

Figure 3.1: Material section shown with template during sampling with Sponge-Stick™ and extraction. ............................................................................................................................. 14

Figure 3.2: Sampling timeline. ................................................................................................................ 15

Figure 4.1: Effect of waste storage time on positive control recoveries (colony forming units [CFU]) from carpet for the extractive and the Sponge-Stick™ methods. ........................... 23

Figure 4.2: Effect of waste storage time on positive control recoveries (colony forming units [CFU]) from upholstered material for the extractive and the Sponge-Stick™ methods. ............................................................................................................................... 25

Figure 4.3: Effect of waste storage time on positive control recoveries (colony forming units [CFU]) from personal protective equipment for the extractive method. ............................... 26

Figure 4.4: Effect of waste storage time on positive control recoveries (colony forming units [CFU]) from paper for the extractive method. ...................................................................... 28

Figure 4.5: The effects of sampling method and waste storage duration on recoveries (colony forming units [CFU]) from carpet following decontamination with pH adjusted bleach. .................................................................................................................................. 33

Figure 4.6: The effects of immersion time in pH adjusted bleach on carpet decontamination efficacy (colony forming units [CFU] log reduction in recovery). .......................................... 37

Figure 4.7: The effects of sampling method and waste storage duration on recoveries (colony forming units [CFU]) from upholstery following decontamination. ....................................... 38

Figure 4.8: The effects of immersion time in pH adjusted bleach on upholstery decontamination efficacy (log reduction in colony forming units [CFU] in recovery). .......... 40

Figure 4.9: The effects of waste storage duration on recoveries (colony forming units [CFU]) from personal protective equipment following decontamination. ......................................... 42

Figure 4.10: Personal protective equipment decontamination efficacy by decontaminant type (colony forming unit [CFU] log reduction). ........................................................................... 44

Figure 4.11: Recoveries (colony forming units [CFU]) following a decontamination of paper with pH adjusted bleach (immersion time: 15 minutes). .............................................................. 45

Figure 4.12: Recoveries (colony forming units [CFU]) following decontamination of paper with diluted bleach (immersion time: 15 min). ............................................................................. 46

Figure 4.13: Decontamination efficacy of pH adjusted bleach and diluted bleach on paper (colony forming units [CFU] log reduction, immersion time: 15 min). .................................. 48

x

List of Tables

Table 2.1: Neutralization Methods Test Matrix ..................................................................................... 10

Table 2.2: Measurement and Neutralization Methods .......................................................................... 10

Table 2.3: Decontaminants and Accessibility ....................................................................................... 10

Table 2.4: Decontamination Procedures and Intensity ......................................................................... 11

Table 2.5: Decontamination Test Sequence Event ............................................................................... 11

Table 3.1: Coupon Types Used to Evaluate Waste Decontamination Procedures .............................. 13

Table 3.2: Number of Sample Types per Material Section per Sampling Sequence ........................... 16

Table 3.3: Number of Sample Types per Material Section per Sampling Sequence ........................... 21

Table 4.1: Effects of Waste Storage Time on Positive Control Recoveries from Carpet for the Extractive and Sponge-Stick™ Sampling Methods ............................................................. 23

Table 4.2: Two-Sample Independent T-test Performance Parameters for Effects of Waste Storage Time on Recoveries from Carpet by Sampling Method and Decontamination Procedure ................................................................................................. 24

Table 4.3: Effects of Waste Storage Time on Positive Control Recoveries from Upholstered Material for the Extractive and Sponge-Stick™ Sampling Methods .................................... 25

Table 4.4: Two-Sample Independent T-Test Performance Parameters for the Effects of Waste Storage Time on Recoveries from Upholstery by Sampling Method ................................... 26

Table 4.5: Effects of Waste Storage Time on Positive Control Recoveries from Personal Protective Equipment for the Extractive Sampling Method .................................................. 27

Table 4.6: Analysis of Variation Performance Parameters for Effects of Storage Time on Recoveries from Personal Protective Equipment by Sampling Method and Experiment ........................................................................................................................... 27

Table 4.7: Effects of Waste Storage Time on Positive Control Recoveries from Paper for the Extractive Sampling Method ................................................................................................ 29

Table 4.8: Analysis of Variance Performance Parameters for Effects of Waste Storage Time on Recoveries from Paper by Sampling Method and Experiment ....................................... 29

Table 4.9: Preliminary Neutralization Optimization ............................................................................... 30

Table 4.10: Effect of Sample Hold Time on Neutralizer Optimization ..................................................... 31

Table 4.11: Effect of Immersion Time on Spore Recovery (Colony Forming Units) from Neutralized pH Adjusted Bleach-Exposed Carpet Samples, High and Low Spore Concentrations ..................................................................................................................... 31

Table 4.12: Decontamination Test Sequence Event ............................................................................... 32

xi

Table 4-13: Post-Decontamination Recoveries (Colony Forming Units [CFU]) from Carpet for Extractive and Sponge-Stick™ Sampling Methods (Immersion Time: 15 min, Decontaminant: pH adjusted Bleach) .................................................................................. 34



Table 4.16: Analysis of Variance Performance Parameters for Effects of Post-Decontamination Storage Time on Recoveries (Colony Forming Units [CFU]) from Carpet ........................... 36

Table 4.17: Decontamination Efficacy versus Immersion Time (Colony Forming Units Log Reduction) for Carpet ........................................................................................................... 36

Table 4.18: Post-Decontamination Recoveries (colony forming units [CFU]) from Upholstery for Extractive and Sponge-Stick™ Sampling Methods (Immersion Time: 15 min) ................... 39

Table 4.19: Analysis of Variance Performance Parameters for Effects of Post-Decontamination Sample Storage Time on Recoveries (Colony Forming Units [CFU]) from Upholstery (immersion Time 15 min) ................................................................................... 39

Table 4.20: Decontamination Efficacy (Log Reduction in Recovery) for Upholstered Coupon Decontamination Efficacy ..................................................................................................... 40

Table 4.21: Recoveries (Colony Forming Units) Following Decontamination of Personal Protective Equipment with pH Adjusted Bleach (Immersion Time: 15 min) ........................ 42

Table 4.22: Recoveries Colony Forming Units Following Decontamination of Personal Protective Equipment with Diluted Bleach (Immersion Time: 15 min) ................................. 43

Table 4.24: Personal Protective Equipment Decontamination Efficacy (Log Reduction in Recovery) ............................................................................................................................. 44

Table 4.25: Recoveries (Colony Forming Units [CFU]) Following Decontamination of Paper with pH Adjusted Bleach (Immersion Time: 15 Min). .................................................................. 46

Table 4.26: Recoveries (Colony Forming Units [CFU]) Following Decontamination of Paper with Diluted Bleach (Immersion Time: 15 Min). ........................................................................... 47

Table 4.28: Paper Decontamination Efficacy (Log Reduction in Recovery, Immersion Time: 15 min) ...................................................................................................................................... 47

Table 5.1: Instrument Calibration Requirements .................................................................................. 49

Table 5.2: Quality Control Checks ........................................................................................................ 51

Table 5.3: Critical Measurement Acceptance Criteria ........................................................................... 53

Table 5.4: Data Quality Assessment ..................................................................................................... 54

Table 6.1: Portion of Samples with No Viable Spores Detected After Decontamination ...................... 58

xii

List of Acronyms and Abbreviations

ANOVA Analysis of variance

ATCC American Type Culture Collection

CBRN Chemical, Biological, Radiological, and Nuclear

CFU Colony forming units

CMAD Consequence Management Advisory Division

COC Chain of custody

DCMD Decontamination and Consequence Management Division

EPA U.S. Environmental Protection Agency

FAC Free available chlorine

FIFRA Federal Insecticide, Fungicide, and Rodenticide Act

HSRP Homeland Security Research Program

HSPD Homeland Security Presidential Directives

ISO International Organization for Standardization

MOP Miscellaneous Operating Procedure

NHSRC National Homeland Security Research Center

NIST National Institute of Standards and Technology

OEM Office of Emergency Management

OPP Office of Pesticides Programs

ORD Office of Research and Development

OSWER Office of Solid Waste and Emergency Response

pAB pH-adjusted bleach

PARTNER Program to Align Research and Technology with the Needs of Environmental Response

PBST Phosphate buffered saline with Tween®20

PPE Personal protective equipment

QA Quality assurance

QAPP Quality Assurance Project Plan

QC Quality control

RH Relative humidity

RTP Research Triangle Park

SD Standard deviation

STS Sodium thiosulfate

VHP® Vaporized hydrogen peroxide

xiii

Executive Summary

This project supports the mission of the U.S. Environmental Protection Agency’s (EPA) Office of Research and Development’s (ORD) National Homeland Security Research Center (NHSRC) by providing information relevant to the remediation of areas contaminated with biological agents.

The primary objective of this investigation was to determine the effectiveness of an expedient approach to waste decontamination. Such approaches were utilized in previous bioterror remediation, although their effectiveness has yet to be determined experimentally. To determine the effectiveness of decontamination approaches, the current study evaluated an immersion-based approach to decontaminate waste materials contaminated with Bacillus atrophaeus spores (surrogate for B. anthracis). The effectiveness of this decontamination approach was evaluated for high traffic commercial carpet tile, nitrile gloves (personal protective equipment [PPE]), books, and upholstered seat pans that are typical of porous material found in an indoor office or items expected to be generated during a sampling and remediation (i.e., PPE). The decontamination and sampling strategies utilized herein were selected by an EPA project team, which consisted of staff from EPA’s Office of Research and Development, EPA’s Office of Solid Waste and Emergency Response, and EPA’s Region 7. The methods utilized were chosen based on their expected effectiveness and ease of use during remediation.

Test materials were inoculated with Bacillus spores at known locations and concentrations, and subjected to prescribed decontamination procedures (i.e., immersion in decontaminant). After the decontamination procedure, a sub-set of the test materials were sampled immediately (Day 0), then the items were bagged and stored (to simulate waste handling/staging during a response). The simulated waste items were re-sampled in a waste staging area after a drying time of 1 day (at least 18 hours), 7 days, and 30 days. A subset of bagged, inoculated waste samples was left untreated and served as positive controls. The efficacies of two decontamination solutions (dilute bleach and pH-adjusted bleach (pAB)) were determined using immersion times varying from 15 minutes to 1 hour. Two sampling methods were used for carpet and upholstery: extractive and surface sampling with 3M Sponge-Stick™. Only extraction-based methods were utilized for PPE and books.

Summary of Results

Most waste materials were effectively decontaminated (greater than 6 log reduction) by a 15 minute immersion in pAB, with the exception of carpet. Longer immersion times increased the efficacy of the decontamination process on carpet, but a 60 minute immersion failed to provide more than a 4 log reduction in viable spores. Decontamination of spores inside closed books was sometimes difficult, as contact with the decontamination solution was not homogenous. Likewise, air pockets in gloves prevented contact with the decontamination solution and could randomly provide complete protection to spores on that surface. The pAB was found to achieve higher decontamination efficacies than diluted bleach for all of the materials tested in this study.

In addition to decontamination efficacy, the collection efficiency of the two sampling methods (extractive and Sponge-Stick™) used in this study were compared as a function of material and elapsed time from inoculation and the time when the sample was collected. Analysis showed no significant effect of sample storage time of up to 30 days on spore recovery, when using either sampling method. Results obtained when using the Sponge-Stick™ approach showed that this sampling method results in an overestimation

xiv

of the actual decontamination efficacy due to its lower recovery efficiencies compared to the extractive sampling technique. These data suggest that the extractive sampling approach should be used whenever wet porous materials are sampled.

1

1 Introduction

This project supports the mission of the U.S. Environmental Protection Agency’s (EPA) Office of Research and Development’s (ORD) Homeland Security Research Program (HSRP) by providing information relevant to the decontamination of areas contaminated as a result of an act of terrorism. Under Homeland Security Presidential Directives (HSPD)-5, 7, 8, and 10, the EPA, in a coordinated effort with other federal agencies, is responsible for “developing strategies, guidelines, and plans for decontamination of …equipment, and facilities” to mitigate the risks of contamination following a biological agent contamination incident.

EPA’s National Homeland Security Research Center (NHSRC) aims to help EPA address the mission of the HSRP by providing expertise and products that can be widely used to prevent, prepare for, and recover from public health and environmental emergencies arising from terrorist threats and incidents. One of NHSRC’s missions is to provide expertise and guidance on the selection and implementation of decontamination methods and provide the scientific basis for a significant reduction in the time, cost, and to address the complexity of decontamination activities. The NHSRC’s research supports the EPA’s Office of Solid Waste and Emergency Response (OSWER), Office of Pesticides Programs (OPP), and the Regions. Close collaboration between the different program offices having homeland security responsibilities is sought in order to rapidly increase EPA’s capabilities to help the nation recover from a terrorist event involving the intentional release of CBRN materials.

In 2001, the introduction of a few letters containing Bacillus anthracis (anthrax) spores into the U.S. Postal Service system resulted in the contamination of several facilities and the deaths of two postal employees. Although most of the facilities in which these letters were processed or received in 2001 were heavily-contaminated, they were successfully decontaminated with approaches such as fumigation with chlorine dioxide or vaporized hydrogen peroxide (VHP®). It is well agreed that additional quick, effective and economical decontamination methods with the capacity to be employed over wide areas (outdoor and indoor) are required to increase preparedness for such an incident. Fumigation was used in primarily contaminated facilities that were heavily-contaminated. Other cleaning methods were used in less heavily contaminated facilities such as those that were secondarily contaminated or those primarily contaminated facilities that showed a minimal presence of anthrax spores. These other “expedient” or “low-tech” methods included removal of contaminated items and/or on-site decontamination. For the surface of a contaminated material, decontamination can be accomplished by physical removal of the contaminant or by inactivation of the contaminant with antimicrobial chemicals. Physical removal could be accomplished by removing spores from the material (i.e., physical cleaning) or via disposal of the material. Inactivation of the contaminant can be done on-site (within the contaminated structure or on-site) or after removal of the contaminated material prior to ultimate disposal (i.e., incinerated off-site). The decision-makers’ selection of the balance between the on-site and off-site destruction of spores was facility-dependent and factored in many issues (e.g., physical state of the facility). One factor was that such decontamination was unprecedented for the United States government and no sporicidal technologies had been proven or registered for use against B. anthracis spores at the time. The cost of waste management proved to be very significant and was complicated by the nature of the waste.

Since 2001, the emphasis for facility decontamination has been to identify and characterize efficacious on-site decontamination methods and to optimize the decontamination/waste management paradigm; this optimization could reduce decontamination time and cost. If proven effective, a lower-tech approach to

2

decontaminating waste on-site could reduce overall decontamination costs by reducing the amount of waste treatment required by off-site, specialized facilities (e.g., medical waste incinerators) and the risk associated with transporting contaminated materials to such facilities. Developing and demonstrating lower cost waste management solutions could increase EPA’s readiness to respond to a wide area release that would generate contaminated waste volumes much larger than those previously managed. Response and remediation activities associated with this type of incident will require additional waste handling, segregation, and staging. These additional requirements illustrate the need for efficacy data and improved process knowledge to support assessments regarding decontamination operations and waste management.

Waste items (ceiling tile and carpet) generated during a recent facility-scale test (BOTE study1), that were decontaminated during the study by expedient methods (liquid bleach spray or spritz), bagged/managed in a manner typical of a real remediation effort, stored (~6 months), and subsequently sampled showed that significant quantities of the test organism (Bacillus atrophaeus) survived the treatment and subsequent 6 month storage duration. This finding indicates that current waste management techniques used during expedient decontamination efforts may generate waste items that have residual contamination. Since the willingness of waste disposal facilities to accept waste items may depend partly upon their contamination level, identification and demonstration of methodologies to effectively decontaminate waste on-site during low-tech decontamination activities are of significant need. This study evaluated several waste decontamination strategies that could be conducted on-site.

For waste generated during the decontamination of typical indoor office or indoor residential settings, no sampling methods have been standardized. Waste items generated from the decontamination of building interiors are expected to consist largely of porous materials1, which pose challenges to currently-available sampling methods. To provide the data necessary to standardize a waste sampling method, extraction-based and surface-wipe-based sampling methods were evaluated. Extraction-based sampling consisted of excision and subsequent extraction of a portion of the material. Surface-wipe-based sampling consisted of collection of the contaminant from the material surface with a Sponge-Stick™.

1.1 Process

This study investigated decontamination of selected materials by an immersion (dunking) approach of waste materials contaminated with Bacillus atrophaeus spore inoculum (i.e., surrogates for B. anthracis). The effectiveness of this decontamination approach was evaluated for high traffic commercial carpet tile, books, and upholstered seat pans that are typical of material found in indoor office, or items like nitrile gloves (personal protective equipment [PPE]) that would be expected to be generated during sampling and decontamination. Replicate sections of test materials were inoculated at known locations with a targeted number of Bacillus spores. After decontamination, sections of the test material were sampled immediately, and then bagged and stored (to simulate field waste handling procedures) in the waste staging area. The decontaminated test materials were re-sampled after a drying time of 1 day (at least 18 hours), 7 days, and 30 days to determine if storage duration could increase the effectiveness of the decontamination treatment (where decontamination treatment is defined, for the purpose of this study, as a prescribed decontamination procedure). A subset of inoculated bagged waste samples was left untreated and served as positive controls. The sampling strategies discussed herein have been selected and optimized to determine the survival of B. atrophaeus spores following decontamination treatment. The purpose of this study was to identify effective and efficient means to decontaminate waste on-site (i.e., not requiring transport of an infectious agent and treatment at remote, specialized, off-site facilities),

3

and to compare the efficiency of both detection methods used. The decontamination and sampling strategies utilized were selected by an EPA project team, which consisted of staff from EPA’s Office of Research and Development, EPA’s Office of Solid Waste and Emergency Response, and EPA’s Region 7.

1.2 Project Objectives The primary objective of this work was to estimate the efficacy of liquid-based decontamination approaches for on-site treatment of bundled or bagged waste items (contaminated indoor office items that would generally be placed in bags or bundled for transportation during the removal process) typically generated during an anthrax clean-up response for an indoor office setting. While there are no established decontamination procedures or performance criteria for B. anthracis-laden waste items, it is likely that waste disposal facilities will require post-decontamination sampling of waste items prior to acceptance. The criterion for waste acceptance is also not known, and may differ between facilities and states. Further, the amount of viable spore contamination within waste items is expected to vary widely. To this end, it is impossible to evaluate the exact conditions (spore load, waste acceptance criteria, etc.) expected to be encountered during an actual B. anthracis remediation. This challenge is not unlike that encountered in the evaluation of sporicidal decontaminant efficacies. It is impractical to evaluate all potential sporicides under all conditions (spore load, material type, environmental conditions, etc.). To address this challenge in the evaluation of sporicides and in the current waste decontamination evaluation, and allow comparison across products or methods (respectively), a consistent challenge is posed to evaluate effectiveness. For example, a 7 Log spore challenge (inoculation of simulated waste items with ~ 5 x 107 spores) was used across all tests and materials. Consistent with sporicidal efficacy tests used to register sporicides under FIFRA, the current study utilized the generally accepted criterion of 6 Log Reduction to consider an approach effective. Recovery of no viable spores following treatment was considered highly effective.

An additional objective was to assess the collection efficiency of the two waste sampling methods utilized. The collateral damage to materials during decontamination procedures was monitored. The ultimate objective was to provide data to support development of a step-wise procedure(s) for on-scene responders and decontamination teams to use for on-site waste treatment during responses involving the indoor environment. Demonstrated waste decontamination procedures could reduce the cost and time of a response by validation of decontamination method, in advance of an emergency incident, that could be used to justify reducing the number of waste characterization samples required and/or reducing the stringency of waste treatments required off-site.

1.3 Experimental Approach

1.3.1 Testing Sequence

The testing sequence used to meet the objectives of this project was:

1. Prepare material sections for each test material as described in Section 2.2.

2. Pre-punch material sections (carpet, upholstery, and book materials), and retain the excised sections for use as 18 mm coupons for extraction-based sampling procedure. The nitrile gloves did not require

4

the use of 18-mm punch coupons, but rather used the 1” tip of each finger for extraction-based analysis.

3. Assemble the material sections by re-inserting the 18 mm excised coupons into their respective void areas of the material section.

4. Sterilize the materials prior to inoculation using ethylene oxide or VHP sterilization. Wait a minimum of 7 days after sterilization before inoculating materials (See Section 2.3.1).

5. Inoculate 18 mm positive control and test material coupons (without removing from larger material section), each fingertip of each nitrile glove PPE, and the target page samples.

6. Allow inoculum carrier to dry for at least 18 hours. .

7. Apply the decontamination procedure to procedural blank material section batch then to the test material section batch.

8. Collect the samples from material sections immediately upon completion of the decontamination procedure.

9. After sample collection, bag the material batch such that the total weight does not exceed 35 lbs per bag.

10. Sample remaining bagged material sections in the waste staging area after a drying time of 1 day (at least 18 hours), 7 days, and 30 days.

1.3.2 Decontamination Strategy

It is preferred that an on-site decontamination procedure be effective, yet generate liquid and solid waste products that are easily disposable, and have minimal detrimental environmental impacts. Accordingly, the current study evaluated decontaminants in order of accessibility (most to least): diluted bleach (0.5% NaClO) and pH adjusted bleach (pAB). Material decontamination was initiated with diluted bleach. In those cases where diluted bleach proved ineffective (less than 6 log reduction in colony forming units of B. atrophaeus) when used in conjunction with the dunking/immersion procedure, pAB was used and the testing cycle repeated.

1.3.3 Method Development for Neutralization

The presence of decontamination solution components in the rinsate or extraction liquid (desorbed from the coupon) could negatively bias colony forming unit (CFU) quantification results. Prior to the decontamination testing sequence, neutralization tests were performed to determine the amount of neutralizer liquid needed to quench each decontaminant/application combination. Decontaminant neutralizers were added to liquid samples immediately after collection to quench their activity, resulting in precise chemical exposure durations and lower recovery bias.

5

2 Materials and Methods

2.1 Facility Design All decontamination activities were conducted inside the spray booth area located in the EPA’s Research Triangle Park (RTP) facility in High Bay Room H122A, a single access point room containing ventilation independent of the High Bay Building containing the room. The spray booth also served as the waste staging area.

The immersion tank was a 10 ft3 (0.28 m3) Poly Hog Trough® (EZ Grout Corp., Waterford, OH) made of virgin polyethylene with steel legs (http://www.ezgrout.com/products/masonry_products/hog-trough.php) (Figure 2.1). The overall dimensions of the tank were 26” x 54 ½” x 24” (66.04 cm x 138.43 cm x 60.96 cm).

Figure 2.1: Poly Hog Trough®.

2.2 Test Coupon Preparation All the coupons were sterilized prior to use to prevent any background organisms from confounding the tests; the books and the glove materials were fumigated with ethylene oxide using an Andersen (Haw River, NC) EO-Gas® 333 sterilization system, while the carpet and the upholstered coupons were fumigated via a Vaporous Hydrogen Peroxide (VHP) sterilization cycle according to Miscellaneous Operating Procedure (MOP) 3120 (Appendix A). To prevent cross contamination, on the day of testing, procedural blank coupons were moved into the spray chamber, and procedural blank decontamination occurred before decontamination of any inoculated coupons.

2.2.1 Carpet and Upholstery

The carpet coupons were ready-made 24” x 24” (0.61 m x 0.61 m) 100% nylon tiles, exuberant 00310 color type (Figure 2.2). Upholstered coupons (20” x 20” (0.51 m x 0.51 m)), were prepared according to Miscellaneous Operating Procedure (MOP) 3194, with layers of foam and fabric layer adhered together (Figure 2.3). MOP 3194 and other associated MOPs can be found in Appendix A.

http://www.ezgrout.com/products/masonry_products/hog-trough.php

6

Figure 2.2: Carpet tile.

Figure 2.3: Front of assembled upholstered coupon.

For both the carpet and upholstered coupons, a 20” x 20” (2581 cm2) template was used. This template (see Figure 3.1) was comprised of 4” x 4” (103 cm2) grid size sections to create a 5 by 5 sampling grid. An 18 mm-diameter coupon was excised from the center of each grid section for sample inoculation. For each sampling event, the 18 mm coupons were either removed for extraction, or left in place to be part of the grid section that was sampled using the surface sampling method.

2.2.2 Paper

Paper samples consisted on the entire front cover (inside cover inoculated) along with the first page of the Merck Manual of Medical Information (Second Home Edition, 2004) (Figure 2.4); and pages 955 (inoculated) plus one page before and two pages after (953-960), respectively. For sampling, the front pages, and the middle pages were removed, and processed separately. Each page measured 9” by 6.5”.

7

Figure 2.4: Paper material.

2.2.3 Nitrile Gloves

The material chosen to represent PPE waste consisted of powder free, 5.5 mils thick, 9" (23 cm) length, white disposable nitrile gloves (McMaster Part #52555T15, www.mcmaster.com) illustrated in Figure 2.5. The powder-free gloves are considered superior for applications where particulate contamination is a concern. Whole gloves were utilized, however, the inside tips of each finger were inoculated and served as replicates. Following decontamination, the terminal 1” (2.5 cm) of each finger was excised and collected as an individual sample.

Figure 2.5: White disposable nitrile glove.

8

2.3 Spore Preparation The test organism for this investigation was a liquid spore suspension of B. atrophaeus (strain: ATCC® 9372) in 29% ethanol solution. This bacterial species was formerly known as B. subtilis var Niger and subsequently B. globigii. The spores were purchased from Yakibou, Inc. (Holly Springs, NC), at a population of 1 x 109 colony forming units (CFU) per mL. The titer of the stock was confirmed at the start of each testing event by the procedure detailed in MOP 6535a.

2.3.1 Coupon Inoculation and Test Preparation

Inoculation of the 18 mm coupons (carpet and upholstery), and the front cover and the middle pages of the books were performed by aseptically applying 100 µL of a diluted spore solution to reach a target concentration of 5 x 107 CFU recoverable from each sample. Finger tips of the nitrile gloves were inoculated to reach the same target concentration. To simulate field conditions, where gloves are turned inside out during doffing procedures, tested gloves were inoculated on the exterior surface, allowed to dry for 18 – 24 hours, then turned inside out prior to use in testing.

2.4 Decontamination Procedure The general decontamination procedure consisted of “dunking” a batch of coupons in the immersion tank containing the decontamination solution for a prescribed immersion time. This decontamination procedure was performed according to MOP 3195 “General Procedure for Immersion Decontamination“, included in Appendix A, and is described below:

1. Prepared decontaminant bath in chemical resistant container. Performed all required quality control (QC) checks listed in Table 5-2.

2. Collected the material batch for immersion which consisted of 3 pre-punched sterile material sections (contains the test samples) and enough non-punched sterile material sections (does not contain test samples) to fill the waste storage bag (not to exceed 35 lb (16 kg) when wet; amounts vary per material). Of the material section batch, only three sections of the decontaminated material were inoculated with Bacillus spores. For example, of a 35 lb batch of carpet tiles, only 3 tiles contained inoculated 18 mm coupons.

3. The material batch (not to exceed 35 lb when wet) was submerged in the decontaminant bath and subjected to the prescribed decontamination procedure.

4. Removed material sections and allowed them to drain briefly (15 minutes) over the decontaminant bath. Immediately collected the post decontamination (T0) samples per material type.

5. Aseptically transfered decontaminated materials to a labeled material 55-60 Gallon Contractor’s storage bag (Uline Model S-19876) which remained closed in the waste staging area until the next sampling event. Material types were bagged separately and arranged such that one inoculated material section was located at the bottom of the batch, 1 was located in the middle, and one was located on the top.

This procedure was repeated for each material using a single immersion container. Therefore, the immersion container was sanitized between tests by removing all debris, wiping interior surfaces with

9

Dispatch® Hospital Cleaner Disinfectant Towels with Bleach wipes (Chlorox, Company, Oakland, CA), rinsing interior surfaces with deionized water, then drying with 70% ethanol prior to the start of each test.

Testing was performed using a “clean team/dirty team” technique. The “dirty team” was responsible for moving the material sections into and out of the immersion tank and performing the decontamination procedure. The “clean team” was used for procedural blank, control and test sampling. Only dirty team members handled contaminated items and only clean team members handled procedural blank coupons and samples. New disposable lab coats were worn for each new material or contamination level. Fresh gloves were donned prior to applying the decontamination procedure then changed before handling the material section after completion of the decontamination procedure. Dirty team members could become clean team members by donning a new set of protective garb (inner and outer gloves, lab coat, P95 mask, and hair net).

2.5 Method Development for Neutralization The presence of decontamination solution components in the rinsate or extraction liquid (desorbed from the sample) could negatively bias spore recoveries. Prior to the decontamination testing sequence, neutralization tests were performed to determine the amount of neutralizer liquid needed to quench residual decontaminant produced from each decontaminant/application combination.

HACH® Method 10100 (http://www.hachco.ca; Hach Company, Loveland, CO) for high range bleach was used to experimentally determine the amount of sodium thiosulfate (STS) required to neutralize in excess the active ingredient (i.e., free available chlorine [FAC]) in pAB and diluted bleach. Due to the variation of the amount of decontaminant solution on the coupons, excess of stoichiometric neutralization was evaluated to ensure that it did not hinder the recovery of the spores. Analyses of the spores in the optimized excess neutralizer solution was also evaluated at a 1 hour and a 24-hour hold time to see if the lag time for processing the samples had an effect on the viable spore recoveries. Finally, the effect of spore inoculation concentration and the decontamination time on the neutralization tests recoveries were also evaluated.

2.6 Test Matrix 2.6.1 Neutralization Method Development Test Matrix

Samples collected from wet, decontaminated material sections may contain enough decontamination liquid to confound enumeration analysis and therefore require neutralization. To determine the appropriate amount of neutralizer needed to quench residual decontaminant, two sets of 5 un-inoculated 18 mm coupons from each test material (carpet, PPE, upholstered furniture, and paper) were immersed in each fresh decontamination liquid (diluted bleach or pAB) for a prescribed soaking period corresponding to each test condition. Each soaked coupon was then placed into a 50 mL conical tube containing phosphate buffered saline with Tween® 20 (PBST) (ICI Americas Inc., Bridgewater, NH) solution used for extraction. One set of samples was spiked with 1 x 107 (High) and second set spiked with 1 x 102 (Low) B. atrophaeus spores to observe the effects of wet sample collection on both high and low B. atrophaeus concentrations that could be present on actual test samples. Two additional sets of coupons were used as controls; these samples were collected from materials not decontaminated, placed into PBST for extraction, and spiked with 1 x 107 and 1 x 102 spores, respectively.

http://www.hachco.ca/

10

The two populations (CFU recovered from decontaminated sample vs. CFU recovered from control spiked sample) for each coupon type and test condition were then analyzed to determine if there was a negative bias induced by the presence of the decontaminant solution in the extraction liquid. If such bias was statistically significant, then neutralization tests were performed using STS as the neutralizing agent. The amount of STS was determined based on the average STS required to neutralize the free available chlorine (FAC) of the diluted bleach or pAB solutions following extraction of pre-spiked decontaminated coupons, and the ability to obtain acceptable recoveries (within 0.5 log of the control coupon concentration). The neutralization methods Test Matrix is shown in Table 2.1.

Table 2.1: Neutralization Methods Test Matrix

Test ID Decontaminated Spike Control Spike Spike Amount Extraction Liquid

STS1 5 5 1 x 107 CFU PBST

STS2 5 5 1 x 107 CFU PBST + STS

STS3 5 5 1 x 102 CFU PBST

STS4 5 5 1 x 102 CFU PBST+ STS

CFU, colony forming units; PBST, phosphate buffered saline with Tween® 20; STS, sodium thiosulfate

Aliquots of the bulk decontamination solution were collected and analyzed for the active ingredients using the methods listed in Table 2.2 immediately (within 10 minutes) before use. Temperature readings of the bulk decontamination solution were also taken immediately (within 10 minutes) before use.

Table 2.2: Measurement and Neutralization Methods

Decontamination Solution Active Ingredient Measurement Method Proposed Neutralization Solution

Dilute Bleach (0.5% NaClO) Hypochlorite MOP 3128-A STS

pAB Hypochlorite MOP 3128-A STS

MOP, Miscellaneous Operating Procedures; pAB, pH-adjusted bleach; STS, sodium thiosulfate

2.6.2 Test Matrix

The test matrix was initially devised to optimize the on-site decontamination procedures by maximizing efficacy and minimizing the manual effort and hazard level of the procedure. The decontaminant optimization process was designed to test decontaminants in order of accessibility. The most accessible to the least, as shown in Table 2.3, were diluted bleach and pAB. The planned decontamination procedures in order of increasing intensity were spray, immersion, rigorous immersion, and immersion/spray, as shown in Table 2.4.

11

Table 2.3: Decontaminants and Accessibility

Decontaminant Decreasing Accessibility

Diluted Bleach

pH- Adjusted Bleach

Table 2.4: Decontamination Procedures and Intensity

Decontamination Procedure Increasing Intensity

Spray

Immersion

Rigorous Immersion

Immersion/Spray

Due to time constraints for each test (30 days to complete a full testing sequence), the testing approach consisted of starting with the two most readily available decontaminants (diluted bleach and pAB) and the immersion procedure. If the procedure were determined to be ineffective (<6 log reduction), a procedure with a higher intensity (rigorous immersion) would be applied. If the procedure was determined to be effective, then the procedure with a lower level of intensity would be applied (spraying). Tests were identified by combining the decontaminant and the application procedures (Tables 2.3 and 2.4). Table 2.5 lists the actual test matrix that was performed, analyzed and described in this report. Please note that the tests on carpet were performed at different dunking/immersion times of 15, 30, and 60 minutes, respectively, from the top to the bottom of Table 2.5.

Table 2.5: Decontamination Test Sequence Event

Decontamination

Procedure

Decontaminant

Solution

Material

Type

Exposure

Time

Test Date

(Day 0)

End Date (Day

30)

Immersion pAB Carpet 15 minutes Sept 25,

2013

Oct 23, 2013

Immersion pAB PPE 15 minutes Nov 5, 2013 Dec 4, 2013

Immersion Diluted Bleach PPE 15 minutes Nov 19,

2013

Dec 16, 2013

Immersion pAB Upholstery 15 minutes Dec 10, 2014

Jan 8, 2014

Rigorous Immersion pAB Carpet 30 Minutes Jan 23, 2014

Feb 20, 2014

Immersion Diluted Bleach Paper 15 minutes Feb 4, 2014

Mar 4, 2014

Immersion pAB Paper 15 minutes Feb 18, 2014

Mar 20, 2014

Rigorous Immersion pAB Carpet 60 minutes Mar 25, 2014

Apr 23, 2014

pAB, pH adjusted bleach; PPE, personal protective equipment

12

3 Sampling and Analytical Procedures

3.1 Sampling Strategy Prior to each sampling event, all materials needed for sampling were prepared using aseptic techniques and placed in a bin containing enough sampling kits, gloves and bleach wipes to accommodate all required samples for the specific test. The materials specific to each protocol are included in the relevant sections below.

In an effort to minimize the potential for cross-contamination during sampling, and in accordance with aseptic technique, a sampling team was utilized, made up of a “sampler” (handling only the sampling media, a “sample handler” (the only person to handle material coupons during the sampling event), and a “support person,” who in addition to being responsible for handing sterile templates to the sampler was also responsible for handling sealed samples and disinfecting outer bags and containers for transport.

Within a single test, sampling of the material sections was completed for all procedural blank coupons first before sampling of any test material or control sections. Sampling was done by collecting the coupons for extraction first, then sponge sampling the remaining coupons according to the protocols documented below. The surface area for all sponge samples was about 103 cm2 (16 in2) and the diameter for all extraction coupons was 18 mm (0.71 in). Once sampling was complete, material sections were returned to their original waste storage bag.

Sponge stick and stub sample integrity was maintained by storage of samples in triple containers (1 – sample collection container, 2 – sterile bag, 3 – disinfected container holding all samples from a test). All individual sample containers remained sealed while in the decontamination lab or in transport after the introduction of the sample. The sampling person did not handle any samples after they were relinquished to the support person during placement into the primary sample container.

Since the current sampling techniques are destructive, each coupon was sampled only once, however each test was replicated 3-5 times (See Table 3.2.1). Test coupons and positive controls were sampled in parallel for each sampling time sequence. Temperature, pH, and active ingredient measurements of each decontaminant solution were performed prior to each decontamination procedure. The temperature and relative humidity (RH) of the waste staging area were recorded by three strategically placed, calibrated HOBO® Data Loggers (Onset Computer Corporation, Bourne, MA) temperature and RH sensors. Additional measurements included quality control checks on the reagents and equipment used in the decontamination procedure.

3.1.1 Sponge-Stick™ Sampling

3M Sponge-Stick™ with neutralizing buffer (part number SSL10 NB; 3M, St. Paul, MN) were used to aseptically sample 103 cm2 (16 in2) areas on the carpet material sections and 79 cm2 (12.25 in2) areas on the upholstery and paper material sections using sampling templates with 25 sampling areas as a guide for Sponge-Stick™ sampling. Samples were collected according to MOP 3165.

3.1.2 Extractive Sampling

The 18 mm coupons for extractive sampling were removed from the sampling area and transferred in the waste staging area into 50 mL sterile vials containing 10 mL PBST and the predetermined amount of

13

neutralization liquid (STS). For PPE samples, excess pAB was captured in a separate vial for subsequent analysis.

3.1.3 Sample Preservation

After sample collection for a single test was complete, all biological samples were transported to the NHSRC Research Triangle Park (RTP) Biocontaminant Laboratory immediately, with appropriate chain of custody form(s) and stored at 4 °C ± 2 °C until extraction. All samples were allowed to equilibrate at room temperature for one hour prior to extraction and plating. Liquid samples were stored no longer than 24 hours prior to analysis. Samples of other matrices were stored no longer than 5 days before the primary analysis. A typical holding time, prior to analyses, for most biological samples was 2 days.

3.1.4 Sampling Points

All the samples were collected from wet materials immediately after application of the decontamination procedure, or after the required hold time (as bagged waste in the waste staging area), and neutralized immediately after sample collection Table 3.1 lists the coupon types and the respective sampling procedures.

Table 3.1: Coupon Types Used to Evaluate Waste Decontamination Procedures

Material Porous or Non-porous Material Description Coupon/Sample

Size Sampling Procedure(s)

Carpet Porous Building material, high traffic, commercial carpet tile, 24” x 24”

18 mm punch / 101 mm x 101 mm square

Extraction / Sponge-Stick™

Upholstered Furniture Porous Upholstered seat pan, 20” x 20” 18 mm punch / 89 mm x 89 mm square

Extraction / Sponge-Stick™

Paper Porous Book pages Whole Front and Middle pages/ 22.9 cm x 16.5 cm

Extraction

PPE materials Non-porous Nitrile, powder free, disposable exam gloves

2.5 cm tip of finger Extraction

PPE, personal protective equipment

3.1.5 Carpet and Upholstery

When sampling upholstery and carpet materials, a sterile template was placed on the coupon creating a grid with an inoculated 18 mm coupon within the center of each 4” x 4” area (Figure 3.1 shows a material section with template during sampling). Designated areas on the coupon were sampled by either Sponge-Stick™ or by removing a pre-punched 18 mm coupon for extraction sampling. When possible, Sponge-Stick™ and extraction samples were taken from areas representing different parts of the coupon (center, sides, and corners).

14

Sponge Stick18 mm coupon (removed)

18 mm coupon (installed)

Material section

Sampling template

Figure 3.1: Material section shown with template during sampling with Sponge-Stick™ and extraction.

3.1.6 Paper Samples

Paper samples, designated paper front (PF) and paper middle (PM), included the front cover and first page and middle pages, as described previously. The additional pages adjacent to those inoculated were collected to account for any spores being relocated (via capillary action, etc.) to the adjacent pages during inoculation and/or decontamination. Sterile razor cutters were used to excise the paper samples after decontamination testing. Once excised, the paper samples were put inside a sterilized pre-labeled stomacher bag along with 80 mL of PBST and a pre-determined volume of STS neutralizer, and mixed altogether. Eight books were used for each sampling sequence (3 books: front and middle pages) and inside cover page, 2 books for positive controls: front and middle pages, 2 books for field blank samples: front and middle pages, and a 1 book for laboratory blank: front and middle pages for a total of 32 books for the 4 test sequences for each decontamination method.

3.1.7 PPE Samples

Gloves were inoculated on the outside, and then aseptically turned inside-out to mimic removal and placement into a decontamination line waste stream. Three gloves were inoculated for each test (3 samples from 3 inoculated fingers (thumb, middle, pinky) for each glove, resulting in 9 samples), one glove for positive controls (5 samples from all 5 inoculated fingers), one glove for field blank sample (3 un-inoculated fingers), and one glove for laboratory blank sample (1 sample from 1 un-inoculated finger) for a total of 6 gloves per test sampling sequence, or 24 gloves for the 4 test sequences for each decontamination method.

15

3.2 Sampling Frequency After the waste decontamination procedure was executed, material sections were either sampled immediately or bagged and stored in the waste staging area for subsequent sampling. Sampling after storage occurred following several simulated waste storage durations, including 1 day (at least 18 hours), 7 days, and 30 days. Figure 3.2 outlines the sampling timeline for both sampling approaches (extraction and Sponge-Stick™). The indicated number of test samples was collected from each of the 3 inoculated material sections as well as from procedural blanks (un-inoculated coupons that were exposed to test procedures) and positive control sections (inoculated coupons that were not exposed to test procedures). This timeline was developed to model the hold times decontaminated materials may be subject to on-site prior to being transported off site for final disposal. Three positive control and 3 procedural blank samples were collected during each sampling event. Positive control coupons were inoculated concurrently with test coupons so they would have the same bacterial aging times as the samples. The synchronization of inoculation and sampling of positive control and test coupons was critical for accurate log reduction analysis. Bagged untreated positive control materials were sampled at the same time (within 8 hours) as their decontaminated counterparts.

Figure 3.2: Sampling timeline.

3.2.1 Sample Quantities

The number of samples collected for both the neutralization and on-site decontamination tests are outlined in Table 3.2. This table includes not only the biological samples, but also samples collected to describe the decontamination process for each test in the test matrix. Some tests required 18 mm samples, sponge samples, or both. For tests that indicate both 18 mm and sponge samples were collected, the sample quantities for sterility blanks, positive control samples, procedural blanks, and test samples for both sample types were identical.

Immediately After Decon (T0)

•3 test coupons removed for extraction

•3 test coupons Sponge- Stick™ sampled

•3 positive controls, 3 procedural blanks

1 Day After Decon (T1 day)




7 Days After Decon (T7 Days)




30 Days After Decon (T30 Days)




16

Table 3.2: Number of Sample Types per Material Section per Sampling Sequence

Test Type of Sample Material

Laboratory Blank

Positive Control Samples

Field Blanks

Test Samples

Extractive Samples

(Y/N)

Sponge Samples

(Y/N) E1 S2 E S E S E S

On-Site Decontamination Day (T0 Days,T1 Day, T7 Days, T30 Days)

STS 0 0 5 0 0 0 5 0 Y N

Carpet Coupons 1 1 3 3 3 3 3 3 Y Y

Upholstered Coupons 1 1 3 3 3 3 3 3 Y Y

PPE3 1 0 5 0 3 0 15 0 Y N

Paper 2 0 6 0 2 0 6 0 Y N N, no; PPE, Personal Protective Equipment; Y, yes 1Number of extractive samples; 2Number of Sponge-Stick™ samples; 3Each finger of a glove is considered one sample; note that for the gloves, runoff samples were also collected.

3.3 Measurement Methods In addition to the collection of material samples, temperature, pH, and active ingredient measurements of each decontaminant solution were performed prior to each decontamination procedure. The temperature and relative humidity (RH) of the waste staging area were recorded by three strategically placed, calibrated HOBO® Data Loggers temperature and RH sensors. Additional measurements included quality control checks on the reagents and equipment used in the decontamination procedure.

3.3.1 Decontamination Solutions The pH-adjusted bleach was prepared as described in MOP 3128-A; in short, it consisted on diluting one part Clorox® concentrated germicidal bleach (Clorox Corp., Oakland, CA) with eight parts of deionized water and one part 5% (v/v) acetic acid (Fisher Scientific, Pittsburgh, PA; Part# 13025), or equivalent). The pH was adjusted to 6.5–7.0 with 5% acetic acid, and the free available chlorine content was adjusted to 6000–6700 ppm with deionized water after preparation. The pH-adjusted bleach was used within three hours of preparation. The diluted bleach was prepared fresh prior to testing by mixing one part Clorox® concentrated regular bleach with approximately 14 part of deionized water to reach a target FAC of about 6000 ppm. Safety precautions were taken to protect personnel from liberated chlorine gas produced as a result of pH reduction of the bleach solution.

The free available chlorine (FAC) concentration of bleach formulations was measured according MOP 3148 based on ASTM Method D2022-89. In short, a 5 mL aliquot was mixed with a buffered potassium iodide solution and iodometrically titrated with STS to a colorless end-point. The aliquot was taken and analyzed immediately after formulation and mixing. The validity of the FAC measurement equipment (Hach® High Range Bleach Test Kit, Method 10100 [Model CN-HRDT]) was confirmed through the titration of a chlorite ion standard. The pH of each solution was measured with an Oakton Acorn® Series pH 5 meter (Oakton Instruments, Vernon Hills, IL). This meter was calibrated daily. 3.3.2 Microbiological Samples

General aseptic laboratory technique to prevent cross-contamination was followed and was embedded in MOPs used by the NHSRC RTP Biocontaminant Laboratory to recover and plate samples. Additionally,

17

the order of analysis (consistent with the above) was as follows: (1) all blank coupons; (2) all decontaminated coupons; then (3) all positive control coupons. Both coupon and Sponge-Stick™ extracts samples were diluted, plated and manually enumerated. Details of the extraction and analytical procedures are provided below.

3.3.2.1 Sample Extraction

Extraction sample vials containing 18 mm coupons, phosphate-buffered saline with 0.05% TWEEN® 20 (PBST) (Sigma-Aldrich, Co, P/N P3563-10PAK [PBST]), and neutralizer were vortexed for 2 minutes to further dislodge any viable spores. Each vial was briefly re-vortexed immediately before any solution was withdrawn for dilution or filter plating. The Sponge-Stick™ samples were extracted according to the validated CDC cellulose Sponge-Stick™ procedure as outlined in MOP 6580 “Recovery of Bacillus Spores from 3M Sponge-Stick™ Samples”.

3.3.2.2 Sample Analysis

Experimental samples were subjected to up to five-stage serial dilutions (10-1 to 10-5) in accordance with MOP 6535a (a revision of MOP 6535 specifically for bacterial spores, attached in Appendix A), plated in triplicate and incubated overnight at 35 °C ± 2 °C. Following incubation, CFU were manually enumerated according to MOP 6535a. Samples that had fewer than the reportable limit of 30 CFU/plate of the undiluted sample underwent further analysis according to MOP 6565 and/or MOP 6584. These MOPs describe filter plating and re-plating, respectively.

3.4 Data Analysis The total spore recovery for each method, material and time point was calculated by multiplying the mean CFU counts from triplicate plates by the inverse of the volume plated (e.g., 1/0.1 mL or 10), by the dilution factor, and finally by the volume of the sample extract (X mL for Sponge-Stick™ samples and Y mL for extracted stubs).

3.4.1 Sampling Efficiency

To determine which of the two detection methods employed in the study was more efficient at detecting viable spores on the waste materials tested, the sampling efficiency (SE) for each detection, all time points and material types was calculated. SE is defined as the ratio of the measured mean sampled CFU (CFUm) to that of the inoculums (CFU0):

(3-1)

3.4.2 Surface Decontamination Efficacy

The efficacy of each decontaminant was assessed by determining the number of viable organisms remaining on each inoculated test coupon after decontamination and comparing this result to the number of viable organisms extracted from the positive control coupons, which were inoculated but not decontaminated. Excess decontamination solutions (in the form of rinsate) were also analyzed from PPE samples to determine if the representative decontamination application washed the spores from the surface of the PPE coupons or if the decontaminant inactivated the spores. These rinsate analysis results were calculated and evaluated, but not used in the decontamination efficacy calculation.

18

The surface decontamination efficacy is defined as the extent (as log10 reduction) by which viable spores extracted from test coupons after decontamination were less numerous than the viable spores extracted from positive control coupons. First, the logarithm of the CFU abundance from each coupon extract was determined, and then the mean of those logarithm values was determined for each set of control and associated test coupons, respectively. This value is reported as a log reduction on the specific material surface as defined in Equation 3-2.

s

N

kkS

C

N

kkC

i N

CFU

N

CFUtC

∑∑== −= 1

,1

, )log()log(η (3-2)

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 are detected, a value of 0.5 CFU was assigned for CFUS,k, and the efficacy was reported as greater than or equal to the value calculated by Equation 3-1.

The standard deviation of the average log reduction of spores on a specific material (ηi ) is calculated by Equation 3-3:

( )

11

2

−

−=

∑=

S

N

kik

i N

xs

SDη

η (3-3)

where:

iSDη = Standard deviation of ηi, the average log reduction of spores

on a specific material surface

19

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 3-4)

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

N s

N

kksc

k

s

CFUCFUx

∑=

−= 1

, ))log()log((( (3-4)

Where:

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 material, 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 3-5 and reported as log reductions of spores for each decontamination technique.

i

ii

T N

∑=

ηη (3-5)

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).

20

The standard deviation of ηT is calculated by Equation 3-6:

( )

1

2

−

−=

∑i

iTi

T NSD

ηη

η (3-6)

where:

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.

Decontamination procedures were considered effective if greater than 6 log reduction was achieved. A procedure was considered highly effective if no viable spores were recovered following decontamination.

3.4.3 Statistical Analysis

To determine if either the extraction or sponge sampling method was better for collecting spores, a 2 independent t-test (or 2-factor analysis of variance [ANOVA]) analysis of the recovery from two sampling methods was performed for each decontamination/material combination.

Single factor ANOVA was used to determine if time is a factor in the decontamination efficacy for each material and each sampling method individually (see Table 3.3).

The Shapiro-Wilk test was used to check if the data sets used in the 1 or 2-factor ANOVA statistical analysis came from normally distributed sample population. The Shapiro-Wilk test is designed to test for normality of small data-size population (n < 50). The null hypothesis of this test is that the population is normally distributed. In other word, if the p-value is less than 0.05 (95% confidence interval), the null hypothesis is rejected and there is evidence that the data tested are not from a distributed population. In contrary, if the p-value is greater than 0.05, then the population is normally distributed.

21

Table 3.3: Number of Sample Types per Material Section per Sampling Sequence Analysis Independent Variable Dependent Variable(s)

Paired t-test or 2 Factor ANOVA (To determine the better detection method for this application, over time). Performed for each material.

Detection method (extract and sponge)

Collection efficiency over time: calculated from known inoculum and analysis of positive controls at different time points

Single factor ANOVA (To determine if time is a factor in decon for each material). This would be a group of individual analyses whose results are compared.

Time points (for individual materials)

Decontamination efficacy over time

22

4 Results and Discussion

This section presents the results of the overall effectiveness of the dunking or immersion of the waste to reduce and/or inactivate spores of B. anthracis from a contaminated surface of different types and porosity. Effectiveness was determined by sampling the waste contents following decontamination and comparing to sampling controls, which did not undergo the decontamination treatment. A 7 Log spore challenge (inoculation of simulated waste items with ~ 5 x 107 spores) was used across all tests and materials. Consistent with sporicidal efficacy tests used to register sporicides under FIFRA, the current study utilized the generally accepted criterion of 6 Log Reduction to consider an approach effective. Recovery of no viable spores following treatment was considered highly effective.

The results of the comparison of effectiveness of each decontaminant/application combination for each material and the evaluation of the quantitative performance parameters are presented in Section 4-1. Evaluation of the two sampling methods (extraction versus Sponge-Stick™) is discussed in Section 4-2. The results for the neutralization tests performed prior to each decontamination sequence are presented in Section 4-3. The results of the decontamination approach that utilized dunking or immersion of the waste are reported in Section 4.4.The Shapiro-Wilk test was used to check if the data sets used in the 1 or 2-factor ANOVA statistical analysis came from normally distributed sample population. Only normally distributed data sets are used for the various analyses. If a dataset is not normally distributed, it will be discussed separately.

4.1 Sampling Methods Evaluation Independent of the decontamination assessment, the two sampling methods (extractive and Sponge-Stick™) were evaluated by comparing positive control recoveries at each sampling point (Day 0, Day 1, Day 7, and Day 30) for both carpet and upholstery. Comparing recoveries of the two methods at any particular time-point allowed a comparison of each method’s performance, comparison of recoveries to the starting inoculum shows the temporal effects on spore recovery through the duration of the experiment (30 Days). Such temporal effects on the extractive sampling method were also evaluated for both the PPE and the paper materials.

4.1.1 Carpet Material

Spore recoveries from carpet as a function of simulated waste storage time (sampling time delay) are shown in Figure 4.1, and summarized in Table 4.1. The averaged recoveries were 1.37 x 107 (Standard deviation, SD = 7.91 x 106, n= 33 samples) CFU using the extractive method (removal of a coupon from a larger sample), and 5.02 x 105 (standard deviation (SD) = 4.45 x 105, n= 33 samples) CFU using the Sponge-Stick™) sampling approach. The overall percent recovery of the extractive method when compared to the starting inoculum (26.7% + 3.56%) was much higher than that of the Sponge-Stick™ sampling method (0.94% + 0.27%). A two-sample independent t-test for these data show a p-value less than 0.001, confirming that the populations means between the 2 methods are significantly different. No significant effects of storage time on recoveries were detected for either sampling method (Table 4.2).

23

Figure 4.1: Effect of waste storage time on positive control recoveries (colony forming units [CFU]) from carpet for the extractive and the Sponge-Stick™ methods.

Table 4.1: Effects of Waste Storage Time on Positive Control Recoveries from Carpet for the Extractive and Sponge-Stick™ Sampling Methods

Waste Storage Time Statistic Recovery (CFU) Recovery (%)

Extraction Sponge-Stick™ Extraction Sponge-

Stick™

Inoculum Average 5.12 × 107

Reference Measurement SD 6.74 × 106

Day 0 Average 1.62 × 107 6.51 × 105 31.7 1.3 SD 9.66 × 106 4.49 × 105

Day 1 Average 1.42 × 107 4.81 × 105 27.8 0.9 SD 7.46 × 106 4.61 × 105

Day 7 Average 1.29 × 107 2.67 × 105 25.2 0.5 SD 9.98 × 106 1.85 × 105

Day 30 Average 1.13 × 107 5.32 × 105 22.0 1.0 SD 5.32 × 106 5.38 × 105

All Days (Combined) Average 1.37 × 107 5.02 × 105 26.7 0.94

SD 7.91 × 106 4.45 × 105 3.56 0.27

Inoculum Day 0 Day 1 Day 7 Day 30

105

106

107

Posit

ive C

ontro

ls Re

cove

ry (C

FU)

Waste Storage Time (Days)

Extractive Method Sponge Stick Method Inoculum

CFU, colony forming units; SD, standard deviation

24

Table 4.2: Two-Sample Independent T-test Performance Parameters for Effects of Waste Storage Time on Recoveries from Carpet by Sampling Method and Decontamination Procedure

Sampling Method (Waste Storage Time)

pH Adjusted Bleach Applied to Carpet

Immersion 15 min

Rigorous immersion 30 min

Rigorous immersion 60 min

Mean Variance Mean Variance Mean Variance

p-value

F-value

p-value

F-value

p-value

F-value

p-value

F-value

p-value

F-value

p-value

F-value

Extractive Method (Day 0, 1, 7, and 30) 0.70 0.38 0.93 0.07 0.17 2.14 0.54 0.78 0.15 2.29 0.86 0.25

Sponge-Stick™ Method (Day 0, 1, 7, and 30)

0.27 1.66 0.58 0.61 0.24 1.70 0.74 0.43 0.65 0.58 0.73 0.45

pAB, pH adjusted bleach

4.1.2 Upholstery Material

Spore recoveries from upholstery material as a function of simulated waste storage time (sampling time delay) are shown in Figure 4.2, and summarized in Table 4.3. The averaged recoveries were 1.73 x 107 (Standard deviation, SD = 1.16 x 107, n= 10 samples) CFU using the extractive method (removal of a coupon from a larger sample), and 1.08 x 107 (standard deviation (SD) = 7.21 x 106 n= 10 samples) CFU using the Sponge-Stick™ sampling approach. The overall percent recovery of the extractive method when compared to the starting inoculum (34.2% + 12.3%) was higher than the Sponge-Stick™ sampling method (18.9% + 10.37%) but within the same order of magnitude. A 2-sample independent t-Test showed that at the 95% confidence interval, the difference of the population means is not significant (p = 0.116). Recoveries for both sampling methods declined significantly (p < 0.05, ANOVA) over time (Table 4.3). Stain resistant coatings on the upholstery surface material may limit spore soaking into the fabric during inoculation, resulting in a large fraction of the spores remaining on the material surface and therefore explain the higher recoveries achieved from this material type.

25


104

105

106

107

Posit

ive C

ontro

ls Re

cove

ry (C

FU)


Extractive Method Sponge Stick Method Inoculum

Figure 4.2: Effect of waste storage time on positive control recoveries (colony forming units

[CFU]) from upholstered material for the extractive and the Sponge-Stick™ methods. Table 4.3: Effects of Waste Storage Time on Positive Control Recoveries from Upholstered

Material for the Extractive and Sponge-Stick™ Sampling Methods

Waste Storage Time Statistic

Recovery (CFU) Recovery (%)

Extraction Sponge-Stick™ Extraction Sponge-

Stick™ Inoculum Average 5.12 × 107 Reference Measurement

Day 0 Average 3.05 × 107 1.89 × 107 53.2 33.0 SD 6.95 × 106 1.52 × 106

Day 1 Average 2.06 × 107 1.37 × 107 35.9 23.9 SD 7.67 × 106 6.37 × 106

Day 7 Average 1.37 × 107 7.20 × 106 23.9 12.6 SD 6.37 × 106 2.11 × 106

Day 30 Average 4.29 × 106 7.91 × 106 23.9 6.04 SD 6.51 × 106 5.20 × 106

All Days (Combined)

Average 1.73 × 107 1.08 × 107 34.2 18.9

SD 1.16 × 107 7.21 × 106 12.0 10.4


26

Table 4.4: Two-Sample Independent T-Test Performance Parameters for the Effects of Waste Storage Time on Recoveries from Upholstery by Sampling Method


Mean Variance

p-value F-value p-value F-value

Extractive Method (Day: 0, 1, 7, 30) 0.0094 7.76 0.95 0.11

Sponge-Stick™ Method (Day: 0, 1, 7, 30)

0.0099 7.61 0.159 2.26

4.1.3 PPE Material

Spore recoveries from PPE material as a function of simulated waste storage time (sampling time delay) are shown in Figure 4.3, and summarized in Table 4.5. Due to the irregular shape and small size of the PPE samples only extractive sampling methods were used. The averaged CFU recoveries for two tests which included 4 sampling events, with 5 samples (each finger of nitrile glove is considered a single sample) are 2.88 x 107 (SD = 5.34 x 106, n= 40 samples). The overall percent recovery of the extractive method for the PPE materials is 42.3% ± 4.60%. Recoveries for the extractive sampling method used for the PPE samples were consistent over time ((p >0.05, ANOVA); Table 4.6).

Figure 4.3: Effect of waste storage time on positive control recoveries (colony forming units [CFU]) from personal protective equipment for the extractive method.


105

106

107

Posit

ive C

ontro

ls Re

cove

ry (C

FU)


Extractive Method Inoculum

27

Table 4.5: Effects of Waste Storage Time on Positive Control Recoveries from Personal Protective Equipment for the Extractive Sampling Method

Waste Storage Time Statistic Recovery(CFU) Recovery (%)

Inoculum Average 5.73 × 107 Reference Measurement

Day 0 Average 2.70 × 107 47.1

SD 5.99 × 106

Day 1 Average 2.68 × 107 46.7

SD 7.94 × 106

Day 7 Average 2.18 × 107 38.0

SD 5.80 × 106

Day 30 Average 2.15 × 107 37.4

SD 4.59 × 106

All Days (Combined) Average 2.88 × 107 42.3

SD 5.34 × 106 4.60


Table 4.6: Analysis of Variation Performance Parameters for Effects of Storage Time on Recoveries from Personal Protective Equipment by Sampling Method and Experiment


Mean Variance


Extractive Method (Day: 0, 1, 7, 30) 0.077 2.47 0.29 1.31

4.1.4 Paper Material

Spore recoveries from the front page paper “PF” and the middle page paper “PM” as a function of simulated waste storage time (sampling time delay) are shown in Figure 4.4, and summarized in Table 4.7). Due to the highly porous and absorptive nature of paper, spores were expected to migrate from the original location to subsequent pages in the book. As such, extraction-based methods were used on the inoculated page, and the adjacent pages to optimize spore recovery. The averaged CFU recoveries for the “PF” and “PM” are 7.68 x 106 (SD = 4.30 x 106, n= 23 samples) and 8.05 x 106 (SD = 3.95 x 106, n= 25 samples), respectively. The recoveries achieved by the extractive method for the “PF” and “PM” sample materials were 17.3% + 7.5% and 18.3% + 5.5%, respectively. A two-sample independent t-Test showed that at the 95% confidence interval, the difference of the spore recovery population means between the ”PF” Samples and the “PM” is not significantly (p = 0.75). Recoveries for the “PF” samples seem to be less consistent (p= 0.032, ANOVA)) than the recoveries for the “PM” samples (p=0.267 ANOVA) over the 30 days sampling period (Table 4.8).

28

Figure 4.4: Effect of waste storage time on positive control recoveries (colony forming units [CFU]) from paper for the extractive method.


105

106

107

Posit

ive C

ontro

ls re

cove

ry (C

FU)


PM Samples PF Samples Inoculum

29

Table 4.7: Effects of Waste Storage Time on Positive Control Recoveries from Paper for the Extractive Sampling Method

Waste Storage Time Statistic

Sample Location

Front Page Middle Page

Recovery (CFU) Recovery (%) Recovery (CFU) Recovery (%)

Inoculum Average

Day 0 Average 5.10 × 106

12.1 5.98 × 106

14.2 SD 1.91 × 106 8.00 × 105


12.3 7.71 × 106

18.3 SD 2.36 × 106 2.04 × 106


16.6 6.09 × 106

14.5 SD 4.77 × 106 4.51 × 106


28.1 1.09 × 107

26.0 SD 4.58 × 106 5.85 × 106

All Days (Combined) Average 7.68 × 106 17.3 8.05 × 106 18.3

SD 4.30 × 106 7.5 3.95 × 106 5.5


Table 4.8: Analysis of Variance Performance Parameters for Effects of Waste Storage Time on Recoveries from Paper by Sampling Method and Experiment

Sample Location Mean Variance


Front Page 0.032 3.61 0.423 0.979

Middle Page 0.267 1.41 0.018 4.21

4.1.5 Sampling Methods Test Synopsis

The two sampling methods (extractive and Sponge-Stick™) were evaluated for overall and temporal recoveries (Day 0, Day 1, Day 7, and Day 30) for both the carpet material and the upholstered coupons using the results of the positive controls for each sampling event. The mean recoveries of the extractive method, when compared to the reference inoculum plating, were much higher than that achieved by the Sponge-Stick™ sampling method for both materials that were sampled by both methods. There were no significant temporal effects on mean recoveries or variance for these two materials and sampling methods. For the upholstered materials, the difference in the overall mean recoveries for both sampling methods was found to be not significant; however, recoveries for both sampling methods declined significantly over time. The overall and temporal recoveries for the PPE and paper materials were found to be consistent over time.

4.20 × 107

30

4.2 Neutralization Methods Evaluation The presence of decontamination solution components in the rinsate or extraction liquid (desorbed from the sample) could negatively bias spore recoveries via residual decontamination. Prior to each decontamination testing sequence, neutralization tests were performed to determine the optimal neutralization concentration (neutralizer to decontaminant), the effect of holding time between the time the sample is collected and the time when the sample is analyzed, and the effect of the immersion time on the amount of neutralizer required for full neutralization

4.2.1 Optimization of Neutralizer Concentration

To determine the optimal amount of neutralizer (STS) for each material/decontaminant, preliminary neutralization tests were conducted. The samples were neutralized at different stoichiometric ratio of STS to decontaminant (X); then the solutions (with the samples) were spiked with either ~2 x 102 spores (low concentration) or ~5 x 107 spores (high concentration) before analysis. The results are presented in Table 4.9. The data collected showed that complete spore recovery can be obtained when the required amount of STS, based on stoichiometric ratio, is applied for each material/decontaminant combination.

Table 4.9: Preliminary Neutralization Optimization

Decontaminant Spore Inoculum [CFU]

Stoichiometric Ratio

Material Type

Spore Recovery [CFU]

Mean CFU SD

pH Adjusted Bleach

~5 x 107

1.3-1.5X

Carpet 2.57 x 107 8.84 x 106

Upholstery 4.46 x 107 2.94 x 106

Paper 4.79 x 107 3.13 x 106

PPE 5.16 x 107 6.60 x 106

2.5X Carpet 3.89 x 107 7.79 x 106

PPE 4.88 x 107 2.13 x 106

~2 x 102

1.3-1.5X

Carpet 2.78 x 102 4.91 x 101

Upholstery 3.24 x 102 4.42 x 101

Paper 1.45 x 102 6.76 x 101

PPE 9.97 x 101 1.66 x 101

2.5X Carpet 2.40 x 102 1.75 x 101

PPE 3.91 x 101 3.58 x 10-1

Diluted Bleach

~5 x 107 1.3-1.5X

Carpet 3.39 x 107 2.95 x 106

Upholstery 4.56 x 107 4.11 x 106

Paper 1.11 x 107 7.87 x 106

PPE 4.83 x 107 2.15 x 106

~2 x 102 1.3-1.5X

Carpet 2.78 x 102 4.91 x 101

Upholstery 3.12 x 102 7.71 x 101

Paper 1.77 x 102 5.78 x 101

PPE 1.62 x 102 4.43 x 101

31

4.2.2 Sample Hold Time Effects

The effect of sample hold time on the neutralizer efficacy was evaluated for both the pAB and diluted bleach at two different spore concentrations: ~2 x 102 CFU (low concentration) or ~5 x 107 CFU (high concentration). The processing lag time at 24 hours after sampling compared to within 1 hour of a sampling event did not show any bias in the spores’ recoveries for both types of decontaminants and spore concentrations (Table 4.10).

Table 4.10: Effect of Sample Hold Time on Neutralizer Optimization

Decontaminant Spore Inoculum [CFU] Hold Time

[Hours] Spore Recovery [CFU]

Mean CFU SD

pH Adjusted Bleach

~5 x 107 1 h 3.89 x 107 7.79 x 106

24 h 3.87 x 107 8.12 x 106

~2 x 102 1 h 2.64 x 102 2.87 x 101

24 h TNC

Diluted Bleach

~5 x 107 1 h 3.39 x 107 2.95 x 106

24 h 5.25 x 107 8.62x 106

~2 x 102 1 h 2.78 x 102 4.91 x 101

24 h 1.69 x 102 1.17 x 102

CFU, colony forming units; pAB, pH adjusted bleach; SD, standard deviation; TNC, Tests Not Conducted

4.2.3 Immersion Time Effects

Two neutralization tests, at two spore concentrations (~ 2 × 102 and ~ 5 × 107 CFU), were completed to determine the effect of an extended immersion time (60 min instead of 15 min) on the neutralization effectiveness. The amount of neutralizer liquid needed for each decontaminant/application combination was found to be dependent on the immersion time for porous materials, meaning that the carpet material was not saturated at the 15 min immersion time; thus requiring more neutralizer volume. The results presented in Table 4.11 show that when the required amount of STS is added, based on stoichiometric ratio, complete neutralization is achieved independently of immersion time.

Table 4.11: Effect of Immersion Time on Spore Recovery (Colony Forming Units) from Neutralized

pH Adjusted Bleach-Exposed Carpet Samples, High and Low Spore Concentrations

Decontaminant Spore Inoculum [CFU] Immersion Time

[Minutes] Spore Recovery [CFU]

Mean CFU SD

pH Adjusted Bleach

~5 x 107 15 min 3.89 x 107 7.79 x 106 60 min 1.46 x 107 1.35 x 106

~2 x 102 15 min 2.40 x 102 1.75 x 101 60 min 1.57 x 102 1.85 x 101


32

4.2.4 Neutralization Tests Synopsis

The neutralization tests determined that the excess STS stoichiometric neutralization is dependent on the type of material used; full recoveries of the spores inoculated on material spores were obtained with 2.5 X (stoichiometric ratio) for the carpet/pAB combination, and 1.3 X was sufficient for the carpet/diluted bleach combination. The ratio of 1.5 X was found adequate for upholstered material with either pAB or diluted bleach as decontaminant, and the same ratio was found for PPE/diluted bleach and paper/pAB decontaminants. Time lag between the time the sample was neutralized and the time it was processed over a 24 hour period, immersion time, and spore inoculum concentration did not appear to bias the spore recoveries if adequately neutralized.

4.3 Dunking/Immersion Decontamination Test Results The results presented in this section report the overall effectiveness of the decontamination treatment for each material/decontaminant/procedure combination. Material sections of the test materials were sampled immediately after the decontamination treatment, and were bagged and re-sampled and analyzed after a drying time of 1 day (at least 18 hours), 7 days, and 30 days. A subset of bagged waste samples was left untreated and served as positive controls. The test matrix for the dunking/immersion evaluation is presented in Table 4.12.

Table 4.12: Decontamination Test Sequence Event

Decontamination Procedure

Decontaminant Solution

Material Type Exposure / Hold

Time Test Date

(Day 0) End Date (Day

30)

Immersion pH Adjusted Bleach Carpet 15 minutes Sept 25, 2013 Oct 23, 2013

Immersion pH Adjusted Bleach PPE 15 minutes Nov 5, 2013 Dec 4, 2013

Immersion Diluted Bleach PPE 15 minutes Nov 19, 2013 Dec 16, 2013

Immersion pH Adjusted Bleach Upholstery 15 minutes Dec 10, 2014 Jan 8, 2014

Rigorous Immersion pH Adjusted Bleach Carpet 30 Minutes Jan 23, 2014 Feb 20, 2014

Immersion Diluted Bleach Paper 15 minutes Feb 4, 2014 Mar 4, 2014

Immersion pH Adjusted Bleach Paper 15 minutes Feb 18, 2014 Mar 20, 2014

Rigorous Immersion pH Adjusted Bleach Carpet 60 minutes Mar 25, 2014 Apr 23, 2014

pAB, pH adjusted bleach; PPE, personal protective equipment

33

4.3.1 Carpet Decontamination Results

Material sections (coupons) simulating carpet wastes were subjected to various immersion-based decontamination procedures. The results presented in this section report the effectiveness of these procedures as a function of waste storage time, decontamination method, and sampling method.

4.3.1.1 Sampling Methods Evaluation for Carpet

The two sampling methods (extractive and Sponge-Stick™) were evaluated for overall recoveries and for recoveries following various simulated waste storage durations (Day 0, Day 1, Day 7, and Day 30). The evaluation was performed for each decontaminant type following each decontamination event (15, 30, and 60 minutes) using 3 carpet samples for each sampling method at each time point. A multiple-population ANOVA statistical analysis was used to determine if recoveries differed significantly as a function of storage time, the amount of time between decontamination and sample collection.

Spore survival on carpet following each decontamination approach, and after various waste storage duration are shown in Figure 4.5, and summarized in Table 4.13, 4.14, and 4.15 for immersion times of 15, 30, and 60 minutes, respectively (note that samples were not collected for Day 7 [15 minutes immersion time] for logistical reasons).

Figure 4.5: The effects of sampling method and waste storage duration on recoveries (colony forming units [CFU]) from carpet following decontamination with pH adjusted bleach.

The mean combined spore recoveries (CFU recovered) calculated for the 15, 30, and 60 minutes immersion times are respectively 3.36 x 104 + 2.39 x 104 (n= 27 samples), 1.27 x 104 + 9.55 x 103 (n= 36 samples), and 1.44 x 103 + 1.67 x 103 (n= 36 samples) using the extractive method (removal of a coupon

Day 0 Day 1 Day 7 Day 30100

102

104

106

Immersion Time: 15 min

Post

Dec

onta

min

atio

n Sa

mpl

e Re

cove

ry (C

FU)


100

102

104

106


100

102

104

106

Extractive Sampling Sponge-Sticks™


34

from a larger sample). For the Sponge-Stick™ sampling approach, the corresponding results are respectively 5.78 x 102 + 9.78 x 102 (n= 27 samples), 2.50 x 102 + 6.30 x 102 (n= 36 samples), and 1.03 x 101 + 2.78 x 102 (n= 36 samples). The recoveries obtained with the extraction-based sampling was up to 2 orders of magnitude higher than the Sponge-Stick™ sampling method, which may have biased the calculation of the decontamination efficiency with the latter sampling technique. No viable spores were detected in some replicate samples using the Sponge-Stick™ method, while spores were detected in all the samples collected using the extraction-based method.

Table 4-13: Post-Decontamination Recoveries (Colony Forming Units [CFU]) from Carpet for

Extractive and Sponge-Stick™ Sampling Methods (Immersion Time: 15 min, Decontaminant: pH adjusted Bleach)

Recoveries (Immersion Time: 15 min, Decontaminant: pH adjusted Bleach)

Coupon ID Statistic

Extraction Sampling Method Sponge-Stick™ Sampling Method

Waste Storage Time

0 Day 1 Day 30 Day 0 Day 1 Day 30 Day

C01 Average 1.62 × 104 3.80 × 104 3.46 × 104 6.04 × 10-1 2.72 × 103 1.19 × 102

SD 8.62 × 103 1.38 × 104 1.50 × 104 2.75 × 10-2 1.69 × 103 4.89 × 101

C02 Average 5.13 × 104 2.91 × 104 4.97 × 104 3.53 × 102 6.80 × 102 3.86 × 102

SD 5.15 × 104 1.12 × 104 1.47 × 104 1.59 × 102 5.40 × 102 3.99 × 102

C03 Average 1.85 × 104 1.62 × 104 4.89 × 104 4.44 × 101 8.38 × 102 5.98 × 101

SD 9.20 × 103 2.37 × 103 3.23 × 104 7.28 × 101 5.75 × 102 5.70 × 101

All Coupons

Average 2.87 × 104 2.31 × 104 4.89 × 104 1.33 × 102 1.41 × 103 1.88 × 102

SD 3.15 × 104 2.46 × 104 3.23 × 104 1.88 × 102 1.35 × 103 2.53 × 102

SD, standard deviation

35

Table 4-14: Post-Decontamination Recoveries (Colony Forming Units [CFU]) from Carpet for Extractive and Sponge-Stick™ Sampling Methods (Immersion Time: 30 min, Decontaminant: pH adjusted Bleach)


Coupon ID Statistic


Waste Storage Time

0 Day 1 Day 7 Day 30 Day 0 Day 1 Day 7 Day 30 Day

C01 Average 2.59 × 104 1.46 × 104 3.55 × 103 1.50 × 104 4.38 × 102 2.23 × 102 1.03 × 102 8.74 × 100

SD 1.28 × 104 8.14 × 103 7.43 × 102 5.47 × 103 6.91 × 102 2.47 × 102 6.11 × 101 4.91 × 100

C02 Average 1.29 × 104 5.82 × 103 2.20 × 104 1.79 × 100 1.11 × 103 2.13 × 102 1.97 × 101 1.79 × 100

SD 7.63 × 103 3.18 × 103 1.40 × 104 7.47 × 10-1 1.92 × 103 1.78 × 102 1.46 × 101 7.47 × 10-1

C03 Average 1.82 × 104 1.56 × 104 7.07 × 103 2.76 × 103 6.64 × 101 6.10 × 102 2.05 × 102 3.25 × 100

SD 9.02 × 103 8.97 × 103 3.26 × 103 1.36 × 103 9.15 × 101 8.87 × 102 3.02 × 102 3.94 × 100

All Coupons

Average 1.90 × 104 9.81 × 103 1.45 × 104 8.91 × 103 5.38 × 102 3.49 × 102 1.09 × 102 4.59 × 100

SD 1.04 × 104 2.68 × 103 1.11 × 104 6.48 × 103 1.12 × 103 5.08 × 102 1.74 × 102 4.49 × 100


Table 4-15: Post-Decontamination Recoveries (Colony Forming Units [CFU]) from Carpet for Extractive and Sponge-Stick™ Sampling Methods (Immersion Time: 60 min, Decontaminant: pH adjusted Bleach)


Coupon ID Statistic


Waste Storage Time


C01 Average 1.69 × 103 3.19 × 103 8.68 × 101 9.75 × 102 6.47 × 10-1 1.58 × 101 6.47 × 101 3.02 × 100

SD 1.53 × 103 4.14 × 103 9.08 × 101 8.75 × 102 2.00 × 10-2 1.72 × 101 8.59 × 101 1.04 × 10-

1

C02 Average 3.90 × 103 2.07 × 103 8.93 × 102 3.95 × 100 6.77 × 10-1 5.18 × 100 1.67 × 101 3.95 × 100

SD 2.28 × 103 4.08 × 102 8.75 × 102 1.78 × 100 1.51 × 10-2 7.21 × 100 2.01 × 101 1.78 × 100

C03 Average 1.01 × 103 1.43 × 103 4.51 × 102 8.99 × 102 8.99 × 10-1 5.05 × 100 3.86 × 100 3.07 × 100

SD 2.76 × 102 4.76 × 102 3.70 × 102 7.53 × 102 3.83 × 10-1 6.09 × 100 2.10 × 100 1.15 × 10-

1

All Coupons

Average 2.0 × 103 1.36 × 103 6.72 × 102 8.61 × 102 7.41 × 10-1 8.67 × 100 2.84 × 101 3.34 × 100

SD 1.90 × 103 1.01 × 103 5.92 × 102 6.66 × 102 2.26 × 10-1 1.12 × 101 5.21 × 101 1.00 × 100


36

At the 95% confidence level, no significant effects of sample waste storage time on recoveries were detected for the extraction-based sampling method. At least one significant interaction (15 minute) was noted for the Sponge-Stick™ sampling approach (Table 4.16, significant is shown in bold).

Table 4.16: Analysis of Variance Performance Parameters for Effects of Post-Decontamination Storage Time on Recoveries (Colony Forming Units [CFU]) from Carpet


Immersion Time

15 min 30 min 60 min

Mean Variance Mean Variance Mean Variance

p- value

F- value

p- value

F- value

p- value

F- value

p- value

F- value

p-value

F-value

p-value

F-value

Extractive Method (Day: 0, 1, 7, 30) 0.25 1.48 0.76 0.28 0.122 2.083 0.897 0.198 0.13 2.029 0.224 1.534

Sponge-Stick™ Method (Day: 0, 1, 7, 30)

0.014 5.17 0.026 4.25 0.278 1.342 0.279 1.339 0.14 1.982 0.171 1.778

4.3.1.2 Carpet Decontamination Effectiveness

The results of the carpet decontamination tests are presented in Table 4.17 and in Figure 4.6. The decontamination effectiveness is presented as the mean Log10 reduction in CFU recovered, from all samples within a particular material and treatment. For example, recoveries following sampling at all storage times were averaged to yield one estimate of recovery for that particular treatment. This aggregate approach was utilized since the ANOVA indicated no significant interaction between sample storage time and recovery.

Table 4.17: Decontamination Efficacy versus Immersion Time (Colony Forming Units Log Reduction) for Carpet


Immersion Time

15 min 30 min 60 min

Average SD Average SD Average SD

Extractive Method (Day: 0, 1, 7, 30) 2.72 0.32 3.37 0.34 3.91 0.75

Sponge-Stick™ Method (Day: 0, 1, 7, 30) 3.99 1.15 4.34 1.05 4.72 0.61


37

Figure 4.6: The effects of immersion time in pH adjusted bleach on carpet decontamination efficacy (colony forming units [CFU] log reduction in recovery).

The mean combined Log10 reduction in spore recoveries for the 15 , 30, and 60 minutes immersion times are respectively 2.72 + 0.32 (n= 27 samples), 3.37 + 0.34 (n= 36 samples), and 3.92 + 0.76 (n= 36 samples) using the extractive method (removal of a coupon from a larger sample). For the Sponge-Stick™ sampling approach, the corresponding results are respectively 3.99 + 1.15 (n= 27 samples), 4.34 + 1.05 (n= 36 samples), and 4.72 + 0.61 (n= 36 samples). These results suggest that increasing the immersion time increases the efficacy of the decontamination technique. As mentioned previously, results obtained using the Sponge-Stick™ approach may result in overestimation of the actual decontamination efficacy due to the relatively lower recovery of this sampling technique. Interestingly, complete decontamination of carpet was not achieved with pAB, even when the 60 minute immersion procedure was rendered. Viable spores were recovered by both sampling approaches, for at least one replicate sample from all immersion times.

4.3.2 Upholstery Decontamination Results

Samples simulating upholstered waste were subjected to a 15 minutes pAB immersion-based decontamination procedure. The results presented in this section report the effectiveness of these procedures, as a function of waste storage time, decontamination method, and sampling method.

4.3.2.1 Sampling Methods Evaluation for Upholstery

The two sampling methods (extractive and Sponge-Stick™) were evaluated for overall recoveries and recoveries following various simulated waste storage durations (Day 0, Day 1, Day 7, and Day 30). The evaluation was performed for pAB decontaminant following each a 15 minutes decontamination time using 3 carpet samples for each sampling method at each time point. A multiple-population ANOVA statistical analysis was used to determine if recoveries differed significantly as a function of storage time, the amount of time between decontamination and sample collection.

15 min 30 min 60 min0

1

2

3

4

5

6Ef

ficac

y (L

og10

CFU

Red

uctio

n)

Immersion Time


38

Spore survival on upholstery material the 15 minutes decontamination approach, and after various waste storage duration are shown in Figure 4-7, and summarized in Table 4.18. The mean combined spore recoveries (CFU recovered) for the 15 minutes immersion times in pAB are respectively 8.58 x 101 + 1.78 x 102 (n= 36 samples), and 4.22 x 101 + 1.46 x 1032 (n= 36 samples) for the extraction-based method and the Sponge-Stick™ method. A two sample Paired t-Test showed that at the 95% confidence interval, the difference in the spore recovery populations means between the 2 sampling techniques is not significant (p = 0.75). The average values were based partially upon detection limit, as no viable spores were detected in some replicate samples.

Figure 4.7: The effects of sampling method and waste storage duration on recoveries (colony forming units [CFU]) from upholstery following decontamination.


100

200

300

400

Post

Dec

onta

min

atio

n Sa

mpl

e Re

cove

ry (C

FU)



39

Table 4.18: Post-Decontamination Recoveries (colony forming units [CFU]) from Upholstery for Extractive and Sponge-Stick™ Sampling Methods (Immersion Time: 15 min)

Recoveries (Immersion Time: 15 min, Decontaminant: pH Adjusted Bleach*)

Coupon ID Statistic


Sampling Interval


C01 Average 1.08 × 100 5.40 × 100 4.56 × 101 7.12 × 10-1 8.48 × 10-1 9.02 × 10-1 6.81 × 10-1 6.50 × 10-1

SD 3.63 × 10-1 7.70 × 100 7.74 × 101 1.39 × 10-2 5.04 × 10-2 6.06 × 10-2 2.21 × 10-2 9.14 × 10-3

C02 Average 5.57 × 100 1.17 × 101 6.25 × 10-1 2.92 × 102 9.09 × 10-1 8.56 × 10-1 1.20 × 100 2.92 × 102

SD 8.41 × 100 1.91 × 101 7.81 × 10-3 5.05 × 102 1.14 × 10-1 2.35 × 10-2 1.03 × 100 5.05 × 102

C03 Average 2.11 × 102 3.30 × 102 5.92 × 100 6.62 × 100 1.05 × 100 8.20 × 10-1 6.17 × 10-1 6.37 × 10-1

SD 3.38 × 102 5.63 × 102 7.60 × 100 9.00 × 100 5.19 × 10-1 1.56 × 10-2 2.10 × 100 1.15 × 10-1

All Coupons

Average 7.24 × 101 1.15 × 102 3.27 × 100 2.68 × 100 9.37 × 10-1 8.59 × 10-1 2.84 × 101 3.34 × 100

SD 1.98 × 102 1.92 × 102 4.43 × 101 5.38 × 100 2.82 × 10-1 1.12 × 101 5.21 × 101 1.00 × 100

SD, standard deviation * Note: values based partially upon detection limit, as no viable spores were detected in some replicate samples.

At the 95% confidence level, no significant effects of sample hold times on recoveries were detected for either sampling method (Table 4.19).

Table 4.19: Analysis of Variance Performance Parameters for Effects of Post-Decontamination Sample Storage Time on Recoveries (Colony Forming Units [CFU]) from Upholstery (immersion Time 15 min)

Immersion Time: 15 min, Decontaminant: pH Adjusted bleach *


Mean Variance

p-value F-value p-value F-value Extractive Method (Day: 0, 1, 7, 30) 0.58 0.67 0.58 0.67 Sponge-Stick™ Method (Day: 0, 1, 7, 30) 0.41 0.995 0.41 0.997

* Note: values based partially upon detection limit, as no viable spores were detected in some replicate samples.

4.3.2.2 Upholstered Coupon Decontamination Effectiveness

The results of the upholstery decontamination tests are presented in Table 4.20 and in Figure 4.8. The decontamination effectiveness is presented as the mean Log10 reduction in CFU recovered, from all samples within a particular material and treatment. The decontamination effectiveness is presented as the mean Log10 reduction in CFU recovered, from all samples within a particular material and treatment. For example, recoveries following sampling at all storage times were averaged to yield one estimate of

40

recovery for that particular treatment. This aggregate approach was utilized since the ANOVA indicated no significant interaction between sample storage time and recovery.

The averaged combined Log10 CFU decontamination efficacy for the 15 minutes immersion time is 6.4 + 0.6 (n= 36 samples) using the extractive method (removal of a coupon from a larger sample). For the Sponge-Stick™ sampling approach, the corresponding result is 6.8 + 0.7 (n= 36 samples). Both sampling techniques seem to lead to the same decontamination efficiency suggesting that the 15 minutes immersion pAB procedure on upholstered coupons is very effective (>6 log reduction). The higher efficacy values observed for upholstery, as compared to carpet, might be explained by the lower absorption of the inoculum by the upholstery material during inoculation. This phenomenon likely resulted in more spores remaining on the upholstery coupon surface, and might have been more easily accessed, and therefore killed, by the decontaminant.

Table 4.20: Decontamination Efficacy (Log Reduction in Recovery) for Upholstered Coupon Decontamination Efficacy

Immersion Time: 15 min, Decontaminant: pH Adjusted Bleach

Sampling Method (Waste Storage Time) Mean LR SD

Extractive Method (Day: 0, 1, 7, 30) 6.4 0.6 Sponge-Stick™ Method (Day: 0, 1, 7, 30) 6.8 0.7

LR, log reduction; SD, standard deviation

Figure 4.8: The effects of immersion time in pH adjusted bleach on upholstery decontamination efficacy (log reduction in colony forming units [CFU] in recovery).

15 min0

1

2

3

4

5

6

7

8

Effic

acy

(Log

10 C

FU R

educ

tion)

Immersion time


41

4.3.3 PPE Decontamination Results

Material sections (coupons) simulating PPE waste were subjected to various immersion-based decontamination procedures, and stored for up to 30 days to simulate waste staging during an actual anthrax incident. Samples (glove sections) were collected at each time point, and spore survival was determined. The results presented in this section report the effectiveness of these procedures, as a function of waste storage time, and decontamination method. PPE material coupons were immersed in either pAB or diluted bleach for an overall immersion time of 15 minutes.

4.3.3.1 The Effects of Waste Storage Duration on Recoveries from PPE following Decontamination

For each experimental trial, the PPE material sections were bagged following the decontamination treatment, and samples (glove tips) were collected at various days (Day 0, Day 1, Day 7, and Day 30) during simulated waste storage. Spore survival was determined at each time point. The PPE sampling technique consisted of collecting 3 inoculated fingers (1st, 3rd and 5th fingers starting with the thumb) from each glove separately. Spores were recovered by extraction of individual glove fingers in PBST. Five gloves were used to produce 15 test samples (3 samples from 3 inoculated fingers for each glove) one glove for positive controls (5 samples from 5 inoculated fingers), one glove for field blank sample (3 un-inoculated fingers), and one glove for laboratory blank sample (1 sample from 1 un-inoculated finger) for a total of 8 glove per test sampling sequence, or 32 gloves for the 4 test sequences for each decontamination procedure. Field blanks were not inoculated, but were subjected to the decontamination treatment. Lab blanks were not inoculated, and were not subjected to a decontamination treatment. ANOVA was used to determine if recoveries differed significantly as a function of storage time.

Recoveries (CFU) following a decontamination treatment as a function of waste storage time are shown in Figure 4.9 and summarized in Table 4.21 and 4.23 for pAB and diluted bleach, respectively. Note that for the pAB decontamination procedure, the decontamination consistently achieved greater than 6 Log reductions, but for one sample (1 sample from one finger among the 15 samples (CO5) showed little or no decontamination. As mentioned previously, this low efficacy was presumably due to incomplete contact of the decontaminant within the glove. The pAB was found to be more effective than the diluted bleach using the immersion decontamination procedure. One data set, which included the finger that was not decontaminated, did not pass the Shapiro-Wilk test for normality since the null hypothesis is rejected and there is evidence that the data tested are not from a normally distributed population. Therefore, the difference in the spore recovery populations means between the two sampling techniques was not assessed.

42

Figure 4.9: The effects of waste storage duration on recoveries (colony forming units [CFU])

from personal protective equipment following decontamination.

Table 4.21: Recoveries (Colony Forming Units) Following Decontamination of Personal Protective Equipment with pH Adjusted Bleach (Immersion Time: 15 min)

Recoveries (Immersion Time: 15 min, Decontaminant: pAB)

Coupon ID Statistic Sampling Interval

0 Day 1 Day 7 Day 30 Day

C01 Average 5.70 × 10-1 5.76 × 10-1 1.32 × 100 5.53 × 10-1

SD 2.43 × 10-2 2.25 × 10-2 1.11 × 100 3.48 × 10-3

C02 Average 5.57 × 10-1 5.94 × 10-1 6.33 × 10-1 5.57 × 10-1

SD 1.31 × 10-2 5.88 × 10-3 4.59 × 10-3 1.27 × 10-2

C03 Average 5.42 × 10-1 5.92 × 10-1 6.62 × 10-1 7.66 × 10-1

SD 5.62 × 10-3 8.89 × 10-3 5.78 × 10-2 3.66 × 10-1

C04 Average 5.54 × 10-1 5.81 × 10-1 6.45 × 10-1 7.66 × 10-1

SD 7.78 × 10-3 1.97 × 10-2 2.94 × 10-2 3.66 × 10-1

C05 Average 5.49 × 10-1 5.90 × 10-1 6.53 × 10-1 7.43 × 105

SD 2.89 × 10-3 1.20 × 10-2 2.28 × 10-2 1.29 × 106

All coupons Average 5.55 × 10-1 5.87 × 10-1 7.83 × 10-1 1.49 × 105

SD 1.48 × 10-2 1.48 × 10-2 5.06 × 10-1 5.75 × 105 SD, standard deviation Note: values based partially upon detection limit, as no viable spores were detected in some replicate samples.

43

Table 4.22: Recoveries Colony Forming Units Following Decontamination of Personal Protective

Equipment with Diluted Bleach (Immersion Time: 15 min) Recoveries (Immersion Time: 15 min, Decontaminant: Diluted Bleach)

Coupon ID Statistic Sampling Interval

0 Day 1 Day 7 Day 30 Day

C01 Average 6.16 × 103 6.15 × 10-1 6.17 × 10-1 1.45 × 100

SD 1.03 × 104 8.67 × 10-2 4.33 × 10-3 9.27 × 10-1

C02 Average 1.12 × 104 4.09 × 106 1.24 × 100 1.24 × 100

SD 1.94 × 104 7.09 × 106 1.08 × 100 1.06 × 100

C03 Average 1.94 × 106 2.35 × 104 1.50 × 102 3.15 × 105

SD 1.80 × 106 4.06 × 104 2.59 × 102 5.46 × 105

C04 Average 1.47 × 103 4.39 × 106 6.38 × 10-1 3.15 × 105

SD 2.53 × 103 3.66 × 106 1.59 × 10-2 5.46 × 105

C05 Average 2.61 × 104 1.28 × 104 5.19 × 104 1.30 × 106

SD 1.52 × 104 2.22 × 104 8.99 × 104 2.26 × 106

All coupons Average 3.97 × 105 1.70 × 106 1.04 × 104 3.25 × 105

SD 1.05 × 106 3.70 × 106 4.02 × 104 1.02 × 106 SD, standard deviation 4.3.3.2 PPE Decontamination Effectiveness

The results of the PPE decontamination tests are presented in Table 4.24 and in Figure 4.10. The decontamination effectiveness is presented as the mean Log10 reduction in recoveries (CFU), from all samples within a particular material and treatment. The decontamination effectiveness is presented as the mean Log10 reduction in CFU recovered, from all samples within a particular material and treatment. For example, recoveries following sampling at all storage times were averaged to yield one estimate of recovery for that particular treatment. Only extraction-based sampling methods were utilized for PPE.

The mean combined Log10 CFU decontamination efficacy for the 15 minutes immersion time in pAB was 7.57 + 0.86 (n= 60 samples), and 5.45 + 2.51 (n= 60 samples) for the 15 minutes immersion time in diluted bleach. The pAB/15 minutes immersion procedure did achieve greater than 6 log reduction for all samples, with the exception of one sample (1 finger of the glove). The decontamination efficacy with diluted bleach ranged from 0.22 to 7.58 log reduction. These data suggest that achieving complete coverage of the decontaminant with all PPE surfaces is challenging. This is not unexpected, as PPE such as gloves may have trapped air inside that may prevent decontaminant from contacting all interior surfaces. Since gloves are inverted when doffed and therefore contaminants are likely concentrated on the inside of the glove, decontamination of PPE waste with this method may have difficulty in achieving complete kill throughout the entire contents of the waste. Full decontamination with either decontaminant is more probable if the immersion time is greater than the permeation time for these decontaminant/PPE material decontamination procedures. The permeation time of bleach through nitrile gloves is greater than 480 minutes.

44

Table 4.24: Personal Protective Equipment Decontamination Efficacy (Log Reduction in Recovery)

Decontaminant

Immersion Time 15 min

Mean Log Reduction

Standard Deviation

pH adjusted bleach 7.57 0.86 Diluted Bleach 5.45 2.51

Figure 4.10: Personal protective equipment decontamination efficacy by decontaminant type

(colony forming unit [CFU] log reduction).

4.3.4 Paper Decontamination Results

Material sections (coupons) simulating Paper waste were subjected to various immersion-based decontamination procedures, and stored for up to 30 days to simulate waste staging during an actual anthrax incident. Samples were collected at each time point, and spore survival was determined. The results presented in this section report the effectiveness of these procedures, as a function of waste storage time, decontamination method, and sampling method. Paper material coupons were immersed in either pAB or diluted bleach for an overall immersion time of 15 minutes.

4.3.4.1 The Effects of Waste Storage Time on Paper Decontamination Efficacy

For each experimental trial, batches of contaminated books and non-contaminated books were subjected to a 15-minutes decontamination treatment, and samples were collected using the extractive method at various days (Day 0, Day 1, Day 7, and Day 30) during simulated waste storage. Spore survival was determined at each time point. The paper samples consist of paper front “PF”” that included the

15 min0

1

2

3

4

5

6

7

8

Effic

acy

(Log

10 C

FU R

educ

tion)

Immersion time

pAB Diluted Bleach

45

inoculated front cover of the Merck Manual of Medical along with the first page, and paper middle “PM” samples that included the inoculated page 955, and one page before and two pages after (pages 953-960).

Spore survival data for the Paper are shown in Figures 4.11 and 4.12, and summarized in Tables 4.25 and 4.26 for pAB and diluted bleach, respectively. The middle pages showed lower decontamination efficacies than the front pages for the pAB decontamination procedures. This may have been due to the protection of the spores inside the books when they were closed. Diluted bleach decontamination was almost negligible for either front or middle pages as shown for Day 0 and Day 1 sampling events. The residual bleach in the papers seems to continue its decontamination over time suggesting that the off-gassing of the paper continued to be efficacious.

The pAB and the diluted bleach spore survival data did not pass the Shapiro-Wilk test for normality since the null hypothesis is rejected and there is evidence that the data tested are not from a normally distributed population. Therefore, the effect of storage time on spore recoveries, and the difference in decontamination efficiency between the two decontaminants, could not be assessed.

Figure 4.11: Recoveries (colony forming units [CFU]) following a decontamination of paper with pH adjusted bleach (immersion time: 15 minutes).


1x103

2x103

3x103

4x103

5x103

6x103

Post

Dec

onta

min

atio

n Sa

mpl

e Re

cove

ry (C

FU)

Waste Storage Time


46

Table 4.25: Recoveries (Colony Forming Units [CFU]) Following Decontamination of Paper with pH Adjusted Bleach (Immersion Time: 15 Min).

Coupon ID

Front Page Sample Type Middle Page Sample Type

Waste Storage Time


C01 7.09 × 10-1 7.22 × 10-1 7.09 × 10-1 4.04 × 101 5.96 × 103 6.74 × 10-1 1.46 × 102 4.04 × 102

C02 7.09 × 10-1 7.35 × 10-1 7.22 × 10-1 7.70 × 10-1 1.03 × 102 7.22 × 10-1 7.63 × 10-1 4.04 × 101

C03 6.74 × 10-1 7.63 × 10-1 7.77 × 10-1 7.70 × 10-1 7.09 × 10-1 1.37 × 101 7.93 × 10-1 8.08 × 101

Average 6.97 × 10-1 7.40 × 10-1 7.36 × 10-1 1.40 × 101 2.02 × 103 5.05 × 100 4.90 × 101 1.75 × 102

SD 2.05 × 10-2 2.09 × 10-2 3.63 × 10-2 2.29 × 101 3.41 × 103 7.53 × 100 8.36 × 101 1.99 × 102

SD, standard deviation Note: values based partially upon detection limit, as no viable spores were detected in some replicate samples

Figure 4.12: Recoveries (colony forming units [CFU]) following decontamination of paper with diluted bleach (immersion time: 15 min).


1x106

2x106

3x106

4x106

5x106


Post

Dec

onta

min

atio

n Sa

mpl

e Re

cove

ry (C

FU)

Waste Storage Time

47

Table 4.26: Recoveries (Colony Forming Units [CFU]) Following Decontamination of Paper with Diluted Bleach (Immersion Time: 15 Min).

Coupon ID

Front Page Sample Middle Page Sample


C01 7.09 × 100 1.65 × 106 2.02 × 100 4.04 × 100 1.84 × 106 1.92 × 104 8.08 × 101 3.31 × 103

C02 8.08 × 101 7.63 × 10-1 8.60 × 10-1 2.02 × 100 6.74 × 10-1 4.99 × 106 7.52 × 101 4.04 × 100

C03 2.91 × 106 7.35 × 10-1 9.19 × 10-1 2.69 × 100 6.74 × 10-1 1.39 × 102 7.09 × 10-1 1.12 × 103

Average 9.70 × 105 5.52 × 105 1.27 × 100 2.92 × 100 6.12 × 105 1.67 × 106 5.23 × 101 1.48 × 103

SD 1.68 × 106 9.55 × 105 6.54 × 10-1 1.03 × 100 1.06 × 106 2.87 × 106 4.47 × 101 1.68 × 103 SD, standard deviation Note: values based partially upon detection limit, as no viable spores were detected in some replicate samples

4.3.4.2 Paper Decontamination Effectiveness

The results of the Paper decontamination tests are presented in Table 4.28, and illustrated in Figure 4.13. The decontamination effectiveness is presented as the mean Log10 reduction in recoveries (CFU), from all samples within a particular material and treatment. The decontamination effectiveness is presented as the mean Log10 reduction in CFU recovered, from all samples within a particular material and treatment. For example, recoveries following sampling at all storage times were averaged to yield one estimate of recovery for that particular treatment.

The mean combined decontamination efficacies (Log10 CFU Reductions) for the front and middle pages after a 15 minutes immersion time in pAB was 6.6 + 0.5 (n= 24 samples) and 5.4 + 1.3 (n= 24), respectively. Following a 15 minute immersion in diluted bleach, the respective decontamination efficacies were 6.0 + 1.8 (n= 24 samples) and 4.5 + 2.4 (n= 24). The data suggest that a 6 Log reduction in recoverable spores is more easily obtained for front pages than those in the middle of the books. Full decontamination may have been achieved by using a longer immersion time and/or by opening the books during the immersion process.

Table 4.28: Paper Decontamination Efficacy (Log Reduction in Recovery, Immersion Time: 15 min)

Decontaminant

Sample Location

Front Page

Middle Page

Average SD Average SD pH Adjusted Bleach 6.6 0.5 5.4 1.3

Diluted Bleach 6.0 1.8 4.5 2.4


48

Figure 4.13: Decontamination efficacy of pH adjusted bleach and diluted bleach on paper (colony forming units [CFU] log reduction, immersion time: 15 min).

Front Page Middle Page0

1

2

3

4

5

6

7

8Ef

ficac

y (L

og10

CFU

Red

uctio

n)

Paper Sample Type

pAB Diluted Bleach

49

5 Quality Assurance

This project was performed according to an approved Category III Quality Assurance Project Plan (QAPP). Sufficient detail of the methods outlined in the QAPP are provided within this report.

5.1 Sampling, Monitoring, and Analysis Equipment Calibration Operating procedures for the maintenance and calibration of all laboratory and NHSRC RTP Biocontaminant Laboratory equipment were prepared. All equipment was verified as being certified calibrated or having the calibration verified by EPA’s Air Pollution Prevention and Control Division on-site (Research Triangle Park, NC) Metrology Laboratory at the time of use. Standard laboratory equipment such as balances, pH meters, biological safety cabinets and incubators were routinely monitored for proper performance. Data gathered with the HOBO thermistors were processed using the factory calibration. Calibration of instruments was done at the frequency shown in Table 5.1. Any deficiencies were noted. The instrument was adjusted to meet calibration tolerances and recalibrated within 24 hours. If tolerances were not met after recalibration, additional corrective action was taken, possibly including, recalibration or/and replacement of the equipment.

Table 5.1: Instrument Calibration Requirements Equipment Calibration/Certification Expected Tolerance

Thermometer

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

±1°C

pH Meter Perform a 2 point calibration with standard buffers that bracket the target pH before each use. ± 0.1 pH units

HOBO® RH Sensor Compare to calibrated RH sensor prior to use. ± 5%

Stopwatch Compare against NIST Official U.S. time at http://nist.time.gov/timezone.cgi?Eastern/d/-5/java once every 30 days.

±1 min/30 days

Micropipettes

All micropipettes will be certified as calibrated at least once per year. RaininTM Pipette liquid handling devices are recalibrated by gravimetric evaluation of pipette performance to manufacturer's specifications every six months by the supplier (Rainin Instruments, Mettler Toledo, Greifensee, Switzerland).

± 5%

Clock Compare to office U.S. Time <www. time.gov> every 30 days. ±1 min/30 days

Scale Check calibration with Class 2 weights + 0.1% weight

NIST, National Institute of Standards and Technology; RH, relative humidity 5.2 Data Quality The primary objective of this project (Task 1: Evaluation of Waste Decontamination Procedures) was to estimate the efficacy of liquid-based decontamination approaches for on-site treatment of bundled or bagged waste items typical of an indoor office setting that had been contaminated with B. anthracis

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

http://www.nist.time.gov/

50

spores. The QAPP in place for this project was followed with deviations that have been documented in the laboratory notebook. These deviations did not affect data quality.

5.3 QA/QC Checks Uniformity of the material sections was a critical attribute to assure reliable test results. Uniformity was maintained by obtaining a large enough quantity of material that multiple material sections and coupons could be constructed with presumably uniform characteristics. Samples and test chemicals were maintained to ensure their integrity. Samples were stored away from standards or other samples which could cross-contaminate them.

Supplies and consumables were acquired from reputable sources and were NIST traceable when possible. Supplies and consumables were examined for evidence of tampering or damage upon receipt and prior to use, as appropriate. Supplies and consumables showing evidence of tampering or damage were not used. All examinations were documented and supplies were appropriately labeled. Project personnel checked supplies and consumables prior to use to verify that they met specified task quality objectives and did not exceed expiration dates.

Quantitative standards do not exist for biological agents. Quantitative determinations of organisms in this investigation did not involve the use of analytical measurement devices. Rather, the CFU were enumerated manually and recorded. QC checks for critical measurements/parameters are shown in Table 5.2. These checks also served as data quality indicator goals. The acceptance criteria were set at the most stringent level that can be routinely achieved. Positive controls and procedural blanks were included along with the test samples in the experiments so that well-controlled quantitative values could be obtained. Background checks were also included as part of the standard protocol. Replicate coupons were included for each set of test conditions. Standard operating procedures using qualified, trained and experienced personnel were used to ensure data collection consistency. The confirmation procedure, controls, blanks, and method validation efforts were the basis of support for biological investigation results. If necessary, training sessions were conducted by knowledgeable parties, and in-house practice runs were used to gain expertise and proficiency prior to initiating the research.

Tests with conditions falling outside of these criteria were rejected and repeated upon approval by the EPA project team.

Background contamination was controlled by sterilization of test materials and use of aseptic technique, procedural blank controls, and a pure initial culture. Aseptic technique was used to ensure that the culture remains pure. Procedural blank controls were run in parallel with the contaminated materials. Assuming that the procedural blank controls showed no CFU, the observed colonies from inoculated coupons indicated surviving spores from the inoculated organisms provided they were consistent with the expected colony morphology (i.e., orange color, round form, flat elevation, rough texture, and undulate margin).

51

Table 5.2: Quality Control Checks

QC Sample Information Provided Frequency Acceptance

Criteria Corrective Action

Procedural Blank (coupon without biological agent)

Controls for sterility of materials and methods used in the procedure.

1 per test No observed colony forming units (CFU)

Reject results of test coupons on the same order of magnitude, Identify and remove source of contamination.

Positive Control (Sample from material coupon contaminated with biological agent but not subjected to the test conditions)

Initial contamination level on the coupons; allows for determination of log reduction; controls for confounds arising from history impacting bioactivity; controls for special causes. Shows plate’s ability to support growth.

3 or more replicates per test

For high inoculation, target loading of 1 x 107 CFU per sample with a standard deviation of < 0.5 log. (5 x 106 – 5 x 107 CFU/sample); For low inoculation, target loading of 1 x 102 CFU per sample with a standard deviation of < 0.25 log. (56 – 177 CFU/sample); Grubbs outlier test (or equivalent).

Outside target range: correct loading procedure for next test and repeat depending on decided impact. Outlier: evaluate/exclude value.

Blank plating of microbiological supplies

Controls for sterility of supplies used in dilution plating

3 of each supply per plating event

No observed growth following incubation

Sterilize or dispose of source of contamination. Re-plate samples.

Blank Tryptic Soy agar Sterility Control (plate incubated, but not inoculated)

Controls for sterility of plates. Each plate No observed growth

following incubation.

All plates are incubated prior to use, so any contaminated ones will be discarded.

Chlorine concentration

Concentration of free available chlorine (FAC) in the fresh pH adjusted bleach or diluted bleach solution

1 per use 6000-6700 ppm for fresh pH adjusted bleach or diluted bleach

Reject solution, replace reagents and prepare a new solution

pH Effective concentration of hydrogen ions in solution

1 per use >6.5 and <7.0 for fresh pH adjusted bleach

Reject solution, replace reagents and prepare a new solution

Field blank samples

The level of contamination present during sampling

1 per sampling event Non-detect

Clean up environment. Sterilize sampling materials before use.

Several management controls were put in place in order to prevent cross-contamination. This project was labor intensive and required that many activities be performed on material sections or coupons that were intentionally contaminated (test samples and positive controls) or intentionally not-contaminated (procedural blanks). The treatment of these three groups of test areas (positive control, test, and procedural blank) varied for each group. Hence, specific procedures were put in place to prevent cross-contamination among the groups. Adequate cleaning of all common materials and equipment was critical

52

in preventing cross-contamination; therefore, all common materials were fumigated using a VHP or ethylene oxide sterilant, then swab sampled for sterility prior to each use.

There are four primary activities for each test in the experimental matrix. These activities are preparation of the coupons, execution of the decontamination process (including sample recovery), sampling, and analysis. Specific management controls for each of these activities are described below.

5.4 Acceptance Criteria for Critical Measurements The data quality objectives define the critical measurements needed to address the stated objectives and specify tolerable levels of potential errors associated with simulating the prescribed decontamination environments. The following measurements were deemed to be critical to accomplish part or all of the project objectives:

• Chlorine concentration, determined by measuring FAC in decontaminant solutions.

• pH

• Temperature

• RH

• Time

• Decontamination time

• Plated volume

• Spore log reduction

Data quality indicators for the critical measurements were used to determine if the collected data met the data quality objectives. The critical measurement acceptance criteria are shown in Table 5.3. The target values and actual test parameters for each run are shown in Table 5.4.

The tests were conducted so that all the critical parameters are within the measurements accepted criteria listed in Table 5.4. When one of the test parameters did not meet the test target value, the test method was repeated or modified to reach test target values and therefore achieve 100% completeness for the task. For example, if the target FAC concentration in the bleach (decontaminant) solution was not met, the solution was either re-prepared or adjusted according to the procedure in MOP 3128-A. Test RH values were adjusted with data from calibrated RH sensors. Similarly, if the CFU count for bacterial growth didn’t fall under the target range, the sample was either filtered or re-plated.

53

Table 5.3: Critical Measurement Acceptance Criteria

Critical Measurement Measurement Device Accuracy Detection Limit Completeness

Plated volume Pipette ±2 % NA 100 %

CFU/plate Hand counting ±10 % (between 2 counters) 1 CFU 100 %

FAC HACH® Method 10100 – Digital

Titrator ± 1 % 1 Digit (0.5 g/L

Chlorine) 100 %

Exposure time Timer ±1 second 1 second 100 %

pH Oakton® pH Meter ± 0.01 pH NA 100%

RH/temp of chamber HOBO® U12 Sensor ± 2.5% from 10% to 90% NA 60 %

CFU, colony forming units; FAC, free available chlorine; NA, Not applicable

Plates were quantitatively analyzed (CFU/plate) using a manual counting method. For each set of results (per test), a second count was performed on 25 percent of the plates within the quantification range (plates with 30 - 300 CFU). All second counts were found to be within 10 percent of the original count.

There are many QA/QC checks used to validate microbiological measurements. These checks include samples that demonstrate the ability of the NHSRC RTP Biocontaminant Laboratory to culture the test organism, as well as to demonstrate that materials used in this effort do not themselves contain spores. The checks include:

• Negative control coupons: sterile coupons that underwent the decontamination process

• Field blank coupons: sterile coupons carried to the decontamination location but not decontaminated

• Laboratory blank coupons: sterile coupons not removed from NHSRC RTP Biocontaminant Laboratory

• Laboratory material coupons: includes all materials, individually, used by the NHSRC RTP Biocontaminant Laboratory in sample analysis

• Positive control coupons: coupons inoculated but not fumigated

54

Table 5.4: Data Quality Assessment

Decontaminant Decontamination

Procedure Material

Type

Chlorine Concentration (FAC) - per 5 mL Bleach titrated

pH Chamber Parameters (HOBO®)

Target Value (ppm)

Test Value (ppm)

Frequency Target Value

Test Value

Frequency RH (%)

Temp oF

Frequency

pH Adjusted Bleach

Immersion Carpet 6000 – 6700 6650 Once before

testing 6.5 - 7.0

6.61 Once before testing

46 71

Data recorded at 5 min intervals for the duration

of the test

pH Adjusted Bleach

Immersion Personal

Protective Equipment

6000 – 6700 6610 Once before

testing 6.5 - 7.0


42 64


of the test

Diluted Bleach Immersion Personal

Protective Equipment

5700- 6300 6049 Once before

testing ~ 11 11.09

Once before testing

31 61


of the test

pH Adjusted Bleach

Immersion Upholstery 6000 – 6700 6390 Once before

testing 6.5 - 7.0


25 61


of the test

pH Adjusted Bleach

Rigorous Immersion

Carpet 6000 – 6700 6470 Once before

testing 6.5 - 7.0


20 62


of the test

55

Diluted Bleach Immersion Paper 5700- 6300 5989 Once before

testing ~11 11.39

Once before testing

21 63


of the test

pH Adjusted Bleach

Immersion Paper 6000 – 6700 6209 Once before

testing 6.5 - 7.0


38 62


of the test

pH Adjusted Bleach

Rigorous Immersion

Carpet 6000-6700 6550 Once before

testing 6.5 - 7.0


HOBO® Data Not Available this test

FAC, free available chlorine

56

5.5 Data Quality Audits This project was assigned QA Category III and did not require technical systems or performance evaluation audits.

5.6 QA/QC Reporting Quality Assurance (QA)/QC procedures were performed in accordance with the QAPP for this investigation.

57

6 Summary and Recommendations

The pAB immersion-based waste decontamination procedure, when performed on carpet material coupons, showed that decontamination efficacy increases with increasing immersion time. The mean combined Log10 CFU reductions for the 15, 30, and 60 minutes immersion times were respectively 2.72 + 0.32, 3.37 + 0.34, and 3.91 + 0.51 using the extraction-based sampling method (removal of a coupon from a larger sample). As mentioned, for this study, a log reduction of 6.0 was considered effective. The same pAB immersion-based procedure, when applied to upholstered coupons, resulted in a much higher decontamination efficacy (6.4 + 0.6 Log Reduction) for an equal immersion time of 15 minutes, and using the same sampling method.

The mean combined Log10 CFU reductions for PPE materials immersed for 15 minutes in pAB and diluted bleach are respectively 7.57 + 0.86 and 5.45 + 2.51. The pAB/15 minutes immersion procedure did achieve greater than 6 log reduction for all but one sample. When PPE waste decontamination was attempted with immersion in diluted bleach for 15 minutes, the efficacy ranged from 0.22 to 7.58 log reduction. Difficulties in wetting all interior surfaces of PPE materials may explain the wide range of decontamination efficacies observed for this material. One potential explanation for why this effect was less evident during the attempted decontamination with pAB, is that the higher volatility of pAB (compared to diluted bleach) may have resulted in higher exposures of spores to chlorine gas regardless of whether these spores were located on wetted areas.

The mean combined Log10 CFU reductions for the front and middle pages of a book material, when a 15 minutes immersion time in pAB was utilized are 6.6 + 0.5 and 5.4 + 1.3, respectively. When the paper material decontamination was attempted with a 15 minute immersion in diluted bleach 15, the respective efficacies were 6.0 + 1.8 and 4.5 + 2.4. These data suggest that decontamination of paper materials fully exposed to the decontaminant (i.e., cover and front pages of a book) is more efficient than of those materials shielded from the liquid decontaminant (i.e., middle of the book pages). Agitation methods designed to expose all pages of books or similar waste items may increase the efficacy of treatment for this type of waste.

For the material/decontamination procedures that achieved more than 6 Log reductions, there were still instances in which full decontamination was not achieved. The residual spores detected in the samples were due to the inability of the decontamination solution to have full contact with all the surface areas of the materials tested as shown in Table 6.1. Carpet data is not included in this table since it never achieved 6 Log reductions.

Overall, the data from these tests demonstrate that immersion time (contact time) is a critical parameter in waste decontamination, as demonstrated in this study for carpet material. The pAB decontaminant achieved greater spore reductions than diluted bleach for all of the materials tested in this study. Materials such as carpet may prove difficult to achieve complete spore inactivation with these techniques, especially in a field setting where large quantities of the materials are requiring treatment. Such large-scale application of this method may prove logistically challenging.

The two sampling methods (extractive and Sponge-Stick™) were evaluated for both carpet and upholstered materials. For these materials, the extraction-based method consistently achieved higher recoveries. As a consequence of its lower recoveries, the Sponge-Stick™ method resulted in over-

58

estimation of the decontamination efficacies. If possible, utilization of extraction-based methods for waste sampling provides improved sensitivity of detection. However, these methods might not be easily deployed in the field, and could generate samples that are not as easily analyzed in the laboratory.

Representative extraction samples can be difficult to collect for certain materials. For example, it was found, during preliminary exercises, that carpet (specifically, the backing) and upholstery materials were difficult to cut with simple hand tools. For aseptic sampling purposes specific to this study, these materials were pre-cut for extractive sampling during coupon material preparation however; this would not be the case during a field sampling event. Damaged material samples and poor sample collection technique are a few of the issues to be encountered with extractive sampling. Furthermore, preliminary testing showed that the required laboratory analysis method was specific to the material. Variations in neutralizer volumes, extraction solution volumes, extraction vessels, etc. were necessary for each material during analysis. An effective surface sampling technique that could be utilized for the majority of materials would result in an efficient and economical sample analysis process. More work is needed to develop and characterize effective waste sampling methods.

Table 6.1: Portion of Samples with No Viable Spores Detected After Decontamination

Sampling Method Material Type Post-Decon Samples with No Viable Spores

Detected / Total Number of Samples Collected

pAB Diluted Bleach

Extractive method

Upholstery

19/36

Paper Front Page 12/13 9/12

Paper middle Page 5/11 4/12

PPE 58/60 28/60

Carpet (15-min Immersion Time) 0/27



Sponge-Stick™

Upholstery

34/36




Overall, numerous knowledge gaps and capability gaps were identified during the current study. Some of these gaps include:

• What are the logistical challenges in scaling up waste decontamination and sampling methods for a wide area release?

• What are the most efficient waste sampling methodologies for various waste streams? • Do current waste management approaches affect contaminant resuspension or aerosol

generation? • What are the likely waste acceptance criteria at waste management facilities that may be

able to handle these wastes? How will that impact sampling and decontamination methods (e.g., liquids, residual chemicals, spore loading, etc.)?

59

• What are the likely volumes of waste liquids being generated and how those will need to be managed as a result of these methods?

• Will sample processing labs accept these sample types? Are the sample types optimized for preferred analytical methods?

Within this list are many research gaps for which future studies are needed.

References

1. U.S. EPA. Technical Brief - Bio-response Operational Testing and Evaluation (BOTE) Project . U.S. Environmental Protection Agency, Washington, DC, EPA/600/S-12/001, 2012.

Appendix A: Miscellaneous Operating Procedures

MOP 3120 VHP® Operation

MOP 3128-A Procedure for preparing pH-Adjusted Bleach Solution

MOP 3148 Iodometric Method for the Determination of Chlorine Dioxide and Chlorite using the HACH Test Kit

MOP 3165 Sponge Sample Collection Protocol

MOP 3194 Procedure for Fabricating 18” x 18” Upholstery Coupons for Liquid Inoculation

MOP 3195 General Procedure for Immersion Decontamination

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

MOP 6562 Preparing Pre-Measured Tubes with Aliquoted Amounts of Phosphate Buffered Saline with Tween 20 (PBST)

MOP 6565 Filtration and Plating of Bacteria from Liquid Extracts

MOP 6580 Recovery of Bacillus Spores from 3M Sponge-Stick™ Samples

MOP 6584 Procedure for Replating Bacteria Spore Extract Samples

A1

MOP-3120

Revision 5

June 2013

Page 1 of 13

Miscellaneous Operating Procedure (MOP) 3120:

VHP Operation

Prepared by: __________________________________________ Date: 6/26/2013

Rob Delafield, ARCADIS Technical Lead Author

Reviewed by: __________________________________________ Date: 6/26/2013

Dahman Touati, ARCADIS Project Manager

Approved by: __________________________________________ Date: 6/26/2013

Worth Calfee, EPA Work Assignment Manager

Prepared for

National Homeland Security Research Center

Office of Research and Development



Prepared by

ARCADIS U.S., Inc.

4915 Prospectus Drive, Suite F

Durham, NC 27713

A2

MOP-3120

Revision 5

June 2013

Page 2 of 13

MOP 3120

TITLE: VHP Operation

SCOPE: Outlines setup and operation of the VHP 1000-ED.

PURPOSE: Ensure the decontamination and/or sterilization of the COMMANDER chamber

and/or airlock contents.

1.0 OVERVIEW

The VHP has no method to control the concentration generated in the chamber. Therefore the

target concentration must be achieved and maintained through the set up of the operating

parameters. These values may need to be adjusted as material type and volume vary.

2.0 SAFETY

Ensure all personnel in the room are aware that VHP is going to be dispensed in

COMMANDER or the airlock.

Activate the warning lights outside the door from the control room and outside the

exterior door.

Verify that the enclosure air monitor is calibrated and functioning properly.

3.0 SETUP

Starting parameters may be as follows:

PHASE TIME (hr:min:sec) INJECTION RATE (g/min)

(DH)de-humidification 00:00:00

(CD) conditioning 00:30:00 12.0 (Maximum rate)

(DC) de-contamination 06:00:00 12.0

(AR) aeration 04:00:00

(AA) aux aeration 00:00:00

The times and injection rates can be adjusted after start-up if needed. Airflow is set and

maintained at 17scfm for all cycles.

4.0 PRE-START UP

a. Use the chamber SCADA system to verify the supply and exhaust blowers for the

chamber are off. On the 485 Com screen, the buttons for SC 101 and SC 102 blowers

A3

MOP-3120

Revision 5

June 2013

Page 3 of 13

should be red (Figure 1). Click them with the mouse to change them from green to red

if necessary.

Figure 1. 485 Com screen showing interface for the SC 101 and SC 102 blowers

b. Check that the filtered exhaust valves on top of the chamber are closed (the two valves

labeled Chamber Exhaust (Filtered) in Figure 2), and that the center valve is closed

(the valve labeled Chamber Exhaust Bypass). This Bypass circuit is not longer

functional and has been blanked off.

A4

MOP-3120

Revision 5

June 2013

Page 4 of 13

Figure 2. Top of the chamber showing the valve locations

c. Check that the chamber supply valve is closed (labeled Chamber Supply Air in Figure 3

and shown open).

d. Ensure that the ATI sensor is in the chamber and not covered.

A5

MOP-3120

Revision 5

June 2013

Page 5 of 13

Figure 3. Top of the chamber showing the Chamber Supply Air valve location

e. Securely close the chamber door.

f. Ensure the supply and return lines of the VHP are connected to the chamber (Figure 4).

Keep in mind that the supply line gets hot during operation which softens the tubing. This

makes it susceptible to kinking if stressed.

g. Ensure the chamber P-trap is filled with water (Figure 4).

h. Connect the Emergency Stop cable to the back of the Steris 1000ED (Figure 5).

A6

MOP-3120

Revision 5

June 2013

Page 6 of 13

Figure 4. Locations of VHP return and supply ports and the COMMANDER P-trap

i. The Enclosure ATI is wired through the E-Stop switch to shut down the VHP operation

in the event the concentration in the enclosure reaches 5 ppm. This is a latching action

and will require pressing the alarm reset (A\R) on the ATI monitor to unlatch. Of course,

check the concentration on the SCADA to ensure it is safe to enter the Enclosure.

VHP Return

VHP Supply P-trap

A7

MOP-3120

Revision 5

June 2013

Page 7 of 13

Figure 5. Emergency Stop connection at the DC INPUT

5.0 VHP OPERATION

a. Fill out a VHP safety checklist and insert in the Steris Log book.

b. Log in to the Steris 1000ED. The VHP has a touch screen. A pen works well for keying

in selections. Select Run.

c. From OPERATOR menu, enter Username= 3, Password= 3. Select H2O2 Fill. Fill

reservoir to 1850 grams.

d. It may be necessary to fill or refill the supply bottle. If so, then:

A8

MOP-3120

Revision 5

June 2013

Page 8 of 13

1) Turn the knob above the bottle door to Replace (This knob only turns clockwise).

Wearing a lab coat, nitrile gloves and safety glasses, remove the bottle by sliding

forward.

2) Fill the bottle from the 5 gal container, replace the bottle and slide all the way back.

3) Turn the knob to Engage. Unless you manually stopped the fill process, it will

automatically resume. If manually stopped, press Start to resume.

e. Press X to return to the operator menu and select run cycle.

f. Choose the cycle named chamber (or airlock).

g. Start the cycle by pressing the green icon.

h. Answer YES to run regeneration after completion of cycle. Record dryer and scale values

in the “Steris log” notebook.

i. Monitor the conditioning phase for desired concentration. (typically 250 or 400 ppm).

j. If necessary, press Cycle Setup and increase the time and/or the injection rate to achieve

target (use the right arrow key to advance to the second screen). Press X to exit and save

changes.

NOTE: Frequent changes to injection rates can cause injection rate deviation trips.

k. Monitor decontamination phase and adjust injection rate and/or time if necessary to

achieve set point conditions. Typical targets are 250 ppm for 4 hours and 400 ppm for 6

hours.

6.0 CYCLE COMPLETION

Open chamber supply and filtered exhaust valves, then turn on the chamber supply and

exhaust blowers to aerate the chamber. Chamber is safe to enter when the concentration falls

below 1.0 ppm. (See Section 9.0 on chamber entry).

7.0 DECON of the AIRLOCK

a. Place a fan inside the airlock and make sure it is turned on.

b. Ensure the VHP hoses are connected to the airlock ports. The VHP supply hose should

always be as short as possible.

A9

MOP-3120

Revision 5

June 2013

Page 9 of 13

c. A short extension tube is used on the supply port inside the airlock to separate the

supply and return.

d. Check the H2O2 sensor location to be sure it is attached to the hanger on the left-hand

side wall.

e. Check with one other person that all items are in airlock, especially BIs and bin lids.

Also review that the VHP connection is correct (Figure 6).

Figure 6. VHP connections

f. At the SCADA 485 Com screen (Figure 7), ensure the blower is off (SC-211). Mouse

click to turn the blower off (red) if necessary.

VHP Supply & Return Sampling port

A10

MOP-3120

Revision 5

June 2013

Page 10 of 13

Figure 7. 485 Com Screen showing SC-211 Blower interface

g. Turn supply damper off (shown off in Figure 8).

A11

MOP-3120

Revision 5

June 2013

Page 11 of 13

Figure 8. Airlock Supply Damper shown in the off position

h. Close exhaust dampers/valves (Airlock Exhaust (Filtered) shown open in Figure 9).

i. Put up the WARNING sign on the airlock door.

j. Verify the airlock drain is closed.

k. Seal bottom of the airlock door with duct tape.

l. Run the COMMANDER program per Section 5.0.

m. Close Enclosure doors.

A12

MOP-3120

Revision 5

June 2013

Page 12 of 13

Figure 9. Filtered Airlock Exhaust shown in the open position

8.0 AERATION OF AIRLOCK

a. Open filtered exhaust valve near the floor (Figure 9).

b. Open supply air damper at waist level (Figure 8).

c. Turn on airlock blower from the SCADA “485 Com” screen (Figure 7). SC-211 will be

red – make it green.

9.0 CHAMBER ENTRY

Check the ATI enclosure sensor reading on the SCADA. The reading should be less than 1 ppm

for entry into the enclosure.

A13

MOP-3120

Revision 5

June 2013

Page 13 of 13

Use 0.1 to 3 ppm H2O2 Dräger tubes (P/N 81010414) per manufacturer’s directions to monitor

concentration for safe entry (MOP 3187). If the measured concentration is above 1 ppm, more

aeration is required before opening the door.

10.0 RH PROBE FAILURE TRIP

A common problem with the VHP is a RH probe failure trip. If under certain conditions moisture

gets on the probe, the alarm cannot be cleared by usual methods. Should this happen these steps

can be taken to clear the alarm (this procedure is not outlined in the manual).

a. Enter the service mode (use the same ID and password).

b. Select “Calibration”.

c. Select “Cycle Airflow”. You should here the fan start and the flow rate ramp-up to set

point. This dries the sensor. Allow it to run for 30 seconds.

d. Selecting the red X in the bottom right corner will terminate the fan.

e. Continue selecting the red X to return to the operation screen. You should now be able to

reset the alarm.

A14

A15

MOP 3128-A Revision 2 Dec 2013

Page 2 of 4 MOP 3128-A TITLE: PROCEDURE FOR PREPARING pH-ADJUSTED BLEACH SOLUTION SCOPE: This MOP describes a procedure for reproducibly preparing the pH-adjusted

bleach solution. PURPOSE: The purpose of this MOP is to ensure the solution meets QA specifications for

each test. Equipment/Reagents:

Draeger or personal chlorine (Cl2) monitor [Oakton Acorn Series pH 5 meter or equivalent

Plastic or glass funnel

Triple rinsed container suitable for transporting hazardous solutions

Oakton pH 7 (pH = 7.00 +/- 0.01 @ 25°C) buffer or equivalent

Concentrated Clorox Commercial Solutions Germicidal Bleach (LOWE’S p/n 174273 ), less than 1 year old

5% v/v Acetic Acid (Ricca Chemical, p/n 7732-18-5 or equivalent)

Deionized water

1.0 PROCEDURE 1.1 Calibrate pH Meter

1. Turn meter on (Figure 1). Meter will automatically enter pH mode.

2. Rinse electrode thoroughly with DI water. DO NOT wipe the electrode.

3. Dip both the electrode and temperature sensor into pH 7.00 buffer solution. The glass bulb must be completely immersed into the sample. Stir gently, and wait for the reading to stabilize (about 40 seconds).

4. Press CAL key to enter the calibration mode. The display will momentarily flash “CA” to indicate Calibration. The display will show the current uncalibrated reading, blinking while in calibration mode.

A16


Page 3 of 4

Figure 1. Oakton pH meter

5. Allow the reading to stabilize. The meter will automatically recognize 7.00, 4.01, or 10.00 buffers.

6. Record the uncalibrated value in the laboratory notebook. Press Enter key once to confirm calibration. The LCD displays “CO” to indicate the calibration point has been confirmed. The meter exits calibration mode and returns to measurement mode.

7. Record the pH buffer measurement and temperature (Press MODE key to select parameter) in the appropriate lab notebook.

1.2 Bleach Preparation

1. Dilute concentrated germicidal bleach to regular germicidal bleach by making a 2:1 dilution

with deionized water. For example, to prepare 750 ml of regular germicidal bleach, add 250 ml (1 part) of deionized water to 500 ml (2 parts) of concentrated germicidal bleach.

2. The pH-adjusted bleach should consist of 80% deionized water, 10% germicidal bleach (prepared in Step 1) and, 10% acetic acid. For example, to prepare 10 L of solution, combine 1 L of prepared regular germicidal bleach, 2 L of deionized water, 1 L of acetic acid, and 6 L of deionized water in that order. Prepare the solution in a container that accommodates the total volume of solution and a funnel if necessary. Record the total volume as Vstart in the lab notebook.

3. Seal the mixing container and gently agitate for mixing. Place the pH probe into the solution and measure the pH (target pH = 6.8). If pH is above 7.0, add small increments of acetic acid.

A17


Page 4 of 4

If below 6.5, add germicidal bleach. Refer to the QAPP to determine if adjustments are permitted. Record the volume required for adjustment as Vadd. Calculate Vtotal as Vstart + Vadd in the lab notebook.

4. Measure the free available chlorine (FAC) per MOP 3148. The target FAC is 6350 mg/L. The acceptable range is 6000 mg/L< FAC < 6700 mg/L.

a) If FAC exceeds the acceptable range, dilute the total volume with deionized water by the percent difference between the target FAC and the actual FAC.

Dilution volume = [(actual - target) ÷ target] x (Vtotal)

b) If the FAC is less than the acceptable range, add bleach according to the following equations:

Additional volume of bleach = (target – actual)/ target x Vtotal

Recalculate Vtotal according to the all additions and repeat steps 3 and 4 until both parameters are met. Record the final FAC, pH, temperature, and time in the lab notebook.

A18

MOP-3148Revision 2

November 2012Page 1 of 4


Iodometric Method for the Determination of Chlorine Dioxide andChlorite using the HACH Test Kit

Prepared by: __________________________________________ Date: 11/15/2012

Stella McDonald, ARCADIS Work Assignment Leader

Reviewed by: __________________________________________ Date: 11/15/2012


Approved by: __________________________________________ Date: 11/15/2012


Prepared for

National Homeland Security Research CenterOffice of Research and Development



Prepared by

ARCADIS U.S., Inc.4915 Prospectus Drive, Suite F

Durham, NC 27713

A19

MOP-3148Revision 1April 2011Page 2 of 4

MOP-3148

TITLE: IODOMETRIC METHOD FOR THE DETERMINATION OF CHLORINEDIOXIDE AND CHLORITE USING THE HACH TEST KIT

SCOPE: This MOP is intended for measurement of bleach (FAC) or chlorine dioxide (ClO2)in the DTRL.

PURPOSE: This document provides the standard procedure for sample titration using theHACH Test Kit.

Equipment

HACH digital titrator

Magnetic stir bar

Buret

Reagents

HACH digital titrator cartridge (2.26N stabilized sodium thiosulfate (STS), cat. No.26869-01)

HACH starch indicator solution (cat. No. 349-32)

6N Hydrochloric Acid (HCl)

Phosphate buffer concentrate

Potassium Iodide

Deionized water

1. PROCEDURE

1.1 Preparation of Potassium Iodide Phosphate Buffer (KIPB) Solution

Add 5 mL of phosphate buffer concentrate and 50 g KI to a 1.0 L volumetric flask. Bring up to1.0L with deionized water.

1.2 Preparation of Sample

1. Insert a clean delivery tube into the 2.26N STS titrant solution cartridge. Attach the cartridgeto the titrator body (see Figure 1).

A20

MOP-3148Revision 2


Figure 1. Digital Titrator body, delivery tube, and reagent cartridge

2. Flush the delivery tube by turning the delivery knob to eject a few drops of titrant. Reset thecounter to zero and wipe off the tip.

3. In a 250 ml beaker, add 20 mL of KIPB and 5 mL of sample

4. Fill beaker to about the 200 mL mark with deionized water.

5. Add a stir bar and place beaker on a stir plate

1.3 Titration: ClO2 and Chlorite

For FAC measurements of bleach, proceed directly to Step 3

1. Place the delivery tube tip into the solution and titrate with 2.26N STS until the solution is paleyellow. From the Digital Titrator, record the number of digits required (A).

2. Calculate the volume of titrant delivered (VA):

VA (ml) = A/800

3. Reset the counter to zero and add ~5 mL of 6N HCL to beaker.

4. Titrate with 2.26 N STS until the solution is pale yellow, add 1 dropper of starch indicatorand continue titration until the solution becomes colorless. Record the number of digitsrequired (B).

5. Calculate the volume of titrant delivered (VB):VB (ml) = B/ 800

A21

MOP-3148Revision 1April 2011Page 4 of 4

Calculations

In the following equations, 5 represents the sample size in mL, 2.26 represents the normality of theSTS, the other constants are the equivalent weights (mg/eq) per electron, and VA and VB are asdefined previously.

Bleach (ppm FAC) = VB * 2.26 *35453 / 5

Chlorine dioxide (ppm) = VA * 2.26 * 67452 / 5

Chlorite (ppm) = (VB – 4 * VA) *2.26 * 16863 / 5

If VB is not greater than 4 * VA, then the solution contains chlorine and must be reformulated.

A22

MOP-3165

Revision 3

July 2013

Page 1 of 8


Sponge Sample Collection Protocol

Prepared by: __________________________________________ Date: 7/17/2013

Stella McDonald, ARCADIS Work Assignment Leader

Reviewed by: __________________________________________ Date: 7/17/2013


Approved by: __________________________________________ Date: 7/17/2013


Prepared for





Prepared by

ARCADIS U.S., Inc.

4915 Prospectus Drive, Suite F Durham, NC 27713

A23

MOP-3165

Revision 3

July 2013

Page 2 of 8

MOP 3165

TITLE: SPONGE SAMPLE COLLECTION PROTOCOL

SCOPE: This MOP outlines the procedure for collecting spores using a 3M Sponge-Stick™.

PURPOSE: To provide a procedure for the collection of spore samples using a Sponge-Stick™

in a consistent and repeatable manner.

MATERIALS

3M Sponge-Sticks™ (P/N SSL10NB), hereafter referred to as ‘sponge’

One Seward stomacher bag (P/N BL6041/CLR) per kit

Disposable gloves

Sterilized sampling templates

One Fisher Sterile sampling bag with flat wire enclosure (7” x 12”, P/N 14-955-194) per kit

One Fisher Sterile sampling bag with flat wire enclosure (10” x 14”, P/N 01-002-53) per kit

for overpack

Dispatch wipes

1.0 PREPARATION

All materials needed for collection of each sample will be prepared in advance using aseptic

technique. A sample kit for a single sponge sample will be prepared as follows:

1.1 One stomacher bag will be uniquely labeled as specified in the project QAPP.

1.2 A 10” x 14” bag will be labeled with the same ID as the stomacher bag.

1.3 One stomacher bag, and one 9.5” x 12” unlabeled bag will be placed in the overpack bag.

1.4 A sterile Sponge-Stick will be added to the overpack bag.

1.5 Each prepared bag is one sampling kit.

A24

MOP-3165

Revision 3

July 2013

Page 3 of 8

2.0 PROCEDURE FOR 12” X 12” SAMPLING AREAS

NOTE: For sampling surface dimensions not outlined directly in this MOP, follow the

12" x 12" sampling procedure for areas larger than 3” x 3” and the 2” x 2”

procedure for areas smaller than 3” x 3”. The area must be at least 1.5” x 1.5” to

accommodate the dimensions of the sponge stick itself. Number of passes will

vary with the dimensions of the surface being sampled. It is important that each

pass overlaps the previous pass, and that each direction (horizontal/vertical/

diagonal) is sampled as described in this MOP.

A two person team will be used, employing aseptic technique throughout. The team will consist of

a sampler and a sample handler. In some cases, a third person may be needed to move samples.

Throughout the procedure, the support person will log anything they deem to be significant into the

laboratory notebook.

In general, the team works from the least contaminated sample set (i.e., control blanks) towards the

most contaminated sample set (i.e., positive controls).

All members shall wear dust masks to minimize potential contamination of the samples.

Depending on the situation, respiratory protection beyond a dust mask may be required to protect

the sampling team (e.g., SAR; this will be specified in the project QAPP). New disposable lab

coats are required for the sample handler when changing between different types of materials or

when direct contact between the coupon and lab coat occurs.

2.1 The sampler will don sterile gloves and place the disposable template over the area to be

sampled.

2.2 The support person will remove a sample kit from the sampling bin and record the sample

tube number on the sampling log sheet next to the corresponding coupon code just

recorded.

2.3 The sampler and support person will verify the sample code and ensure that the correct

coupon and location are being sampled.

2.4 The support person will:

a) Open the outer sampling bag touching the outside of the bag.

b) Touching only the outside of the (10” x 14”) bag, remove the sponge, and hand it to

the sampler.

c) Remove the stomacher bag, being careful to not touch the inside of the outer sampling

bag, and open it touching only the outside.

2.5 The sampler will remove the sterile sponge from its package. Grasp the sponge near the top

of the handle. Do not handle below the thumb stop.

2.6 The sampler will wipe the surface to be sampled using the moistened sterile sponge by

A25

MOP-3165

Revision 3

July 2013

Page 4 of 8

laying the widest part of the sponge on the surface, leaving the leading edge slightly lifted.

Apply gentle but firm pressure and use an overlapping ‘S’ pattern to cover the entire

surface with horizontal strokes (Figure 1). Use the other hand to hold the template during

sampling, being careful not to touch the surface.

Figure 1. First pass with sponge – horizontal strokes using one side of the sponge

2.7 The sampler will turn the sponge over and wipe the same area again using vertical ‘S’-

strokes (Figure 2).

Figure 2. Second pass with sponge – vertical strokes using the other side of the sponge

A26

MOP-3165

Revision 3

July 2013

Page 5 of 8

2.8 The sampler will the use the edges of the sponge (narrow sides) to wipe the same area using

diagonal ‘S’-strokes (Figure 3). The sponge will be flipped to use the opposite side

immediately after the longest stroke at opposite corners.

Figure 3. Third pass with sponge – diagonal strokes using the edges of the sponge

2.9 The sampler will use the tip of the sponge to wipe the perimeter of the sampling area

(Figure 4).

Figure 4. Final (fourth) pass with sponge – perimeter wipe using the tip of the sponge

2.10 The sample handler will open the stomacher bag, careful not to touch the inside of the bag.

2.11 The sampler will place the end of the sponge in the bag, holding the handle outside the

opening of the bag.

2.12 The sample handler will grasp the sponge from outside of the bag, and help the sample break

off the handle of the sponge. The handle below the thumbstop should not touch the inside of

the stomacher bag.

A27

MOP-3165

Revision 3

July 2013

Page 6 of 8

2.13 The sample handler will securely seal the stomacher bag and wipe the outside with a

disinfecting wipe.

2.14 The sample handler will then place the stomacher bag inside the unlabeled sterile bag.

2.15 The sample handler will place this in the overpack bag and wipe the overpack bag with

disinfecting wipes.

2.16 The sample handler will place the overpack bag in the sample bin.

NOTE: Remove excessive air from the re-sealable plastic bags to increase the number of

samples that can be shipped in one container.

2.17 The sampler will dispose of the template if present. The coupon handler will remove the

coupon, if present.

2.18 Both members will remove outer gloves and discard. Clean gloves should be worn for each

new sample.

3.0 PROCEDURE FOR 2” X 2” COUPONS

A two person team will be used, employing aseptic technique throughout. The team will consist of

a sampler and a sample handler. In cases where coupons are mobile, a third person will be needed

to move coupons. Only the coupon handler will handle coupons.

Throughout the procedure, the support person will log anything they deem to be significant into the

laboratory notebook.

In general, the team works from the least contaminated sample set (i.e., control blanks) towards the

most contaminated sample set (i.e., positive controls).

All members shall wear dust masks to minimize potential contamination of the samples.

Depending on the situation, respiratory protection beyond a dust mask may be required to protect

the sampling team (e.g., SAR; this will be specified in the project QAPP). New disposable lab

coats are required for the sample handler when changing between different types of materials or

when direct contact between the coupon and lab coat occurs.

3.1 The support person will remove a sample kit from the sampling bin and record the sample

tube number on the sampling log sheet next to the corresponding coupon code just

recorded.

3.2 The sampler and support person will verify the sample code and ensure that the correct

coupon and location are being sampled.

3.3 The support person will:

d) Open the outer sampling bag touching the outside of the bag.

e) Touching only the outside of the (10” x 14”) bag, remove the sponge, and hand it to

the sampler.

A28

MOP-3165

Revision 3

July 2013

Page 7 of 8

f) Remove the stomacher bag, being careful to not touch the inside of the outer sampling

bag, and open it touching only the outside.

3.4 The sampler will remove the sterile sponge from its package. Grasp the sponge near the top

of the handle. Do not handle below the thumb stop.

3.5 Align the sponge with the widest part in contact with the surface to be sampled in the upper

left corner, as seen in Figure 5. Sample by moving the sponge down to the bottom edge of

the sampled area along the left edge applying even pressure to the sponge tip.

3.6 Align the sponge with the widest part in contact with the surface on the same side as in Step

3.5 in the upper right corner. Sample by moving the sponge down to the bottom edge of the

sampled area along the right edge applying even pressure to the sponge tip.

3.7 Rotate the coupon 90 degrees.

3.8 Flip the sponge over and repeat Steps 3.5 and 3.6.

3.9 Flip the sponge on one side (the flat part of the handle will be in the vertical orientation) and

sample horizontally across the coupon from upper left corner to lower right, overlapping

30% each stroke.

3.10 Rotate the coupon 90 degrees.

3.11 Flip the sponge to the other side and repeat Step 3.9.

3.12 The sample handler will open the stomacher bag, careful not to touch the inside of the bag.

3.13 The sampler will place the end of the sponge in the bag, holding the handle outside the

opening of the bag.

Figure 5 - Sponge on 2x2 coupon Figure 5 - Sponge on 2x2 coupon Figure 5. Sponge on 2” x 2” coupon (Side A)

A29

MOP-3165

Revision 3

July 2013

Page 8 of 8

3.14 The sample handler will grasp the sponge from outside of the bag, and help the sample break

off the handle of the sponge. The handle below the thumbstop should not touch the inside of

the stomacher bag.

3.15 The sample handler will securely seal the stomacher bag and wipe the outside with a

disinfecting wipe.

3.16 The sample handler will then place the stomacher bag inside the unlabeled sterile bag.

3.17 The sample handler will place this in the overpack bag and wipe the overpack bag with

disinfecting wipes.

3.18 The sample handler will place the overpack bag in the sample bin.

NOTE: Remove excessive air from the re-sealable plastic bags to increase the number of

samples that can be shipped in one container.

3.19 Both members will remove outer gloves and discard. Clean gloves should be worn for each

new sample.

4.0 REFERENCES

Sponge sample collection protocol adapted from:

National Validation Study of a Cellulose Sponge Wipe-Processing Method for Use after Sampling

Bacillus anthracis Spores from Surfaces. Rose, Laura J.; Hodges, Lisa; O’Connell, Heather;

Noble-Wang, Judith. Appl. Environ. Microbiol. 2011, 77(23):8355.

A30

MOP 3194

Revision 0

August 2013

Page 1 of 5


Procedure for Fabricating 18” X 18” Upholstery Coupons

for Liquid Inoculation

Prepared by: __________________________________________ Date: 8/9/2013 Stella McDonald, ARCADIS Work Assignment Leader

Reviewed by: __________________________________________ Date: 8/9/2013 Dahman Touati, ARCADIS Project Manager Approved by: __________________________________________ Date: 8/9/2013 Worth Calfee, EPA Work Assignment Manager

Prepared for





Prepared by

ARCADIS U.S., Inc.


Durham, NC 27713

A31

MOP 3194

Revision 0

August 2013

Page 2 of 5

MOP 3194

TITLE: PROCEDURE FOR FABRICATING 18” X 18” UPHOLSTERY COUPONS FOR

LIQUID INOCULATION

SCOPE: This MOP describes the procedure for constructing 18” x 18” upholstery coupons

with the foam and fabric layers adhered together.

PURPOSE: The purpose of this MOP is to ensure consistent manufacturing (materials and

procedure) of these coupons.

1.0 INTRODUCTION

Section 2.0 details the fabrication procedure for the material coupons. Section 3.0 describes how

the 18 mm coupon punches (for liquid inoculation) are created.

2.0 FABRICATION OF 18” X 18” UPHOLSTERY COUPONS

The materials to be used for the fabrication of the upholstery coupons are detailed in the table

below.

Material Description Vendor Part Number

Upholstery Fabric Bryant

Indoor/Outdoor Pine

www.fabric.com 0298925

Upholstery Foam 1” x 24” x 108” High

Density Upholstery

Foam

OnlineFabricStore 124108-2645

Upholstery Adhesive 3MTM Foam Fast 74

Spray Adhesive Clear

3MTM 62495049504

Plywood ¾” Pine plywood Lowe’s 35677

Prepare the upholstery coupons as follows:

1. Cut an 18 in x 18 in piece of foam padding, an 18 in x 18 in x 3/4 in piece of plywood (not

pressure treated), and a 24 in x 24 in piece of upholstery fabric.

A32

MOP 3194

Revision 0

August 2013

Page 3 of 5

2. Place the 18 in x 18 in piece of foam padding in the center of an 18 in x 18 in x 3/4 in piece

of plywood.

3. Spray two layers of 3MTM

FoamFast 74 to the surface of the foam then, quickly cover with a

24 in x 24 in piece of upholstery fabric to cover the foam.

4. Fold excess fabric underneath and staple to the back side of the plywood backing as shown in

Figures 1a and b.

a. b.

Figure 1. Front (a) and back (b) of assembled upholstery cushion

3.0 FABRICATION OF 18 MM PUNCHES

1. Place a 17.5” x 17.5” grid with 3.5” x 3.5” sections on the surface of the coupon (Figure 2).

A33

MOP 3194

Revision 0

August 2013

Page 4 of 5

Figure 2. 17.5” x 17.5” Punch Grid

2. Place an 18 mm punch at the center of a 3.5” x 3.5” section of the grid and punch through the

upholstery and foam, stopping at the plywood (DO NOT PUNCH THROUGH THE

PLYWOOD).

3. Retain the 18 mm punch

4. Continue until 18mm punches have been removed from each section of the grid (Figure 3).

17.5”

17.5”

3.5”

3.5”

A34

MOP 3194

Revision 0

August 2013

Page 5 of 5

Figure 3. Punch Grid on Upholstery Coupon with 18mm Punches

18 mm coupon

(removed)

18 mm coupon (installed)

Material section

Punch grid

A35

MOP 3195

Revision 0

September 2013

Page 1 of 5


General Procedure for Immersion Decontamination

Prepared by: __________________________________________ Date: 9/6/2013 Stella McDonald, ARCADIS Work Assignment Leader

Reviewed by: __________________________________________ Date: 9/6/2013 Dahman Touati, ARCADIS Project Manager Approved by: __________________________________________ Date: 9/6/2013 Worth Calfee, EPA Work Assignment Manager

Prepared for





Prepared by

ARCADIS U.S., Inc.


Durham, NC 27713

A36

MOP 3195

Revision 0

September 2013

Page 2 of 5

MOP 3195

TITLE: GENERAL PROCEDURE FOR IMMERSION DECONTAMINATION

SCOPE: This MOP details the procedure for immersion decontamination of carpet,

upholstery, personal protective equipment (PPE; nitrile gloves), and books.

PURPOSE: The purpose of this procedure is to ensure all immersion decontaminations are

performed in a consistent manor.

Equipment and Supplies:

75-Gallon immersion tank (10 cu ft Poly Trough, EZ Grout Corporation, p/n HTP10)

Polypropylene mesh (McMaster Carr, p/n 30145T51)

Stir rod

Oakton pH 5 meter, Acorn Series

Ventilating polypropylene mesh bag, white, 21" wide x 31 ½" high (McMaster Carr, p/n

9883T63)

Decontaminant solution (see Table 1)

1.0 INTRODUCTION

Prior to beginning any decontamination procedure, all test materials, equipment, and supplies

should be prepared as described in the work assignment’s QAPP. While containing

decontaminant solution, the immersion tank should be placed on a spill deck.

Table 1 lists several decontaminants, their active ingredient(s), and the appropriate preparation

and analysis method.

Table 1: Active Ingredients and Titration Methods for Select Decontaminants

Decontaminant Active Ingredient Preparation

Method

Analysis Method

Diluted Bleach Hypochlorite MOP 3181 Iodometric Method

(MOP 3128-A)

pH-Adjusted Bleach

(pAB)

Hypochlorite MOP 3128-A Iodometric Method

(MOP 3128-A)

Spore-Klenz® Hydrogen peroxide (H2O2)

and peracetic acid (PAA)

Per product label Ceric Sulfate Titration

(MOP 3196)

A37

http://www.mcmaster.com/#9883T63

MOP 3195

Revision 0

September 2013

Page 3 of 5

2.0 CARPET AND UPHOLSTERY

1. Prepare approximately 35 gallons of decontaminant solution (see Table 1) in the immersion

tank. Perform the necessary data quality indicator (DQI) checks to verify the solution is

within the quality specifications stated in the QAPP.

2. Lower the sterilized mesh (ethylene oxide or vapor H2O2 sterilization) into the immersion

tank containing the decontaminant solution so that it covers the bottom and walls of the tank.

3. Analyze the solution for active ingredient(s) (see Table 1), pH, and temperature then discard

the sample.

4. Place the entire batch of material sections into the immersion tank (see QAPP for batch

information).

5. Allow to soak for the predetermined immersion time (nominally 15 minutes).

6. After soaking for the required amount of time, remove the material sections from the

immersion tank by lifting the mesh and allow to drain over the immersion tank for 5 minutes.

7. Place the inoculated coupons in the established sampling area for sample collection. Place the

uninoculated material sections (do not contain 18 mm inoculated coupons) directly into the

waste storage bag oriented horizontally.

8. Collect samples from inoculated material sections per the QAPP. Then place horizontally in

the same waste storage bag used in Step 7 in the following order:

1 inoculated coupon

2 uninoculated coupons

1 inoculated coupon

2 uninoculated coupons

1 inoculated coupon

9. Collect a sample of the residual decontaminant solution.

10. Analyze the residual decontaminant solution for the active ingredient(s) (see Table 1), pH,

and temperature.

A38

MOP 3195

Revision 0

September 2013

Page 4 of 5

3.0 NITRILE GLOVES (PPE)

1. Prepare approximately 35 gallons of decontaminant solution (see Table 1) in the immersion

tank. Perform the necessary DQI checks to verify the solution is within the quality

specifications stated in the QAPP.

2. Place the entire batch of gloves (see QAPP for batch information) into a sterilized

polypropylene mesh bag (ethylene oxide or vapor H2O2 sterilization).

3. Lower the mesh bag containing the gloves into the immersion tank containing the

decontaminant solution so that it covers the gloves. If necessary, add weights to the bag to

keep it submersed.


5. After soaking for the required amount of time, remove the gloves from the immersion tank

by lifting the mesh bag and allow to drain over the immersion tank for 5 minutes.

6. Place the inoculated (white) gloves in the established sampling area for sample collection.

Place the uninoculated (blue) gloves directly into the waste storage bag.

7. Collect samples from the inoculated gloves per the QAPP. Then place in the same waste

storage bag used in Step 6.


9. Analyze the residual decontaminant solution for the active ingredient(s), pH, and

temperature.

4.0 BOOKS

1. Prepare 35 gallons of decontaminant solution in the immersion tank (see Table 1). Perform

the necessary DQI checks to verify the solution is within the quality specifications stated in

the QAPP.

2. Place the entire batch of books (see the QAPP for batch information) into a sterilized

polypropylene mesh bag (ethylene oxide or vapor H2O2 sterilization).

3. Lower the mesh bag containing the books into the immersion tank containing the

decontaminant solution so that it covers the books.


A39

MOP 3195

Revision 0

September 2013

Page 5 of 5

5. After soaking for the required amount of time, remove the books from the tank by lifting the

mesh bag and allow to drain over the immersion tank for 5 minutes.

6. Place the inoculated books in the established sampling area for sample collection. Place the

uninoculated books directly into the waste storage bag.

7. Collect samples from the inoculated books per the QAPP. Then place into the same waste

storage bag as the uninoculated books (Step 6).


9. Analyze the residual decontaminant solution for the active ingredient(s) (see Table 1), pH,

and temperature.

A40

MOP 6535a Revision 4

January 2013 Page 1 of 8

Miscellaneous Operating Procedure (MOP) 6535a:

Serial Dilution: Spread Plate Procedure to Quantify Viable Bacterial Spores

Prepared by: __________________________________________ Date: 2/11/2013 Nicole Griffin Gatchalian, ARCADIS Work Assignment Leader

Reviewed by: __________________________________________ Date: 2/11/2013 Dahman Touati, ARCADIS Project Manager

Approved by: __________________________________________ Date: 2/11/2013 Worth Calfee, EPA Work Assignment Manager

Prepared for

National Homeland Security Research Center Office of Research and Development

U.S. Environmental Protection Agency Research Triangle Park, NC 27711

Prepared by

ARCADIS U.S., Inc.


A41



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 as specified in QAPP or Test Plan (e.g., sterile water, Phosphate Buffered Saline with Tween 20 (PBST))

Media plates as specified in QAPP or Test Plan (e.g., Trypticase Soy Agar (TSA) plates)

Microliter pipettes with sterile tips

Sterile beads placed inside a test tube (used for spreading samples on the media surface according to MOP 6555 (Petri Dish Media Inoculation Using Beads) or cell spreaders

Vortex mixer

1.0 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 uL of sterile diluent to each of the tubes.

2. Label three media plates for each dilution that will be plated. These dilutions will be plated in triplicate.

3. Mix original spore suspension by vortexing thoroughly for 30 seconds. Immediately after the

cessation of vortexing, transfer 100 uL of the stock suspension to the 10-1 tube. Mix the 10-1 tube by vortexing for 10 seconds, and immediately pipette 100 uL to the 10-2 tube. Repeat this process until the final dilution is made. It is imperative that used pipette tips be exchanged for a sterile tip each time a new dilution is started.

4. To plate the dilutions, vortex the dilution to be plated 10 seconds, immediately pipette 100

uL of the dilution onto the surface of a media plate, taking care to dispense all of the liquid

A42



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 and aseptically spread the aliquotted dilution on the surface of the media either by

use of glass beads (MOP 6555) or cell spreader (the method used may be directed in the QAPP or Test Plan) until the entire sample is distributed on the surface of the agar plate. Repeat for all plates.

6. Incubate the plates for the optimum time period at the optimum growth temperature for the

target organism (incubation conditions will vary depending on the organism’s optimum growth temperature and generation time. This information can be found in Bergey’s Manual of Determinative Bacteriology or it will be provided with the ATCC certification.

7. Manually enumerate the colony forming units (CFU) on the media 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). A hand held tally counter or an electronic counting pen may be used to assist the person counting, but may not be used as the primary source for the count. Quality control (QC) requirements for bacterial enumeration will be addressed per QAPP or test plan. However, in general, the following QC practices should always be adhered to: a. The arrangement of plates and tubes, and the procedure for preparing dilutions and

enumerating CFU should be done the exact same way each time. This helps prevent systematic errors and often helps determine the cause of problems when a discrepancy is found.

b. A visual check of the graduated pipette tip should be made during each use to ensure the pipette is pulling properly.

c. Samples should acclimate to room temperature for 1 hour prior to plating.

d. Samples should be processed (extracted and plated) from the least contaminated to the most contaminated.

e. When a target range of CFU is known, three dilution factors are plated to bracket the expected results (0, -1, and -2, if the -1 dilution factor was the target).

f. Enumerated colonies and results should be verified that the results are the target organism, and that second counts have been performed. Second counts must be completed on 25% of significant data, and must be within 10% of the first count. If CFUs are found to have more than a 10% difference between first and second counts, then a third count is to be completed.

A43



g. Pictures should be taken of any plates that are contaminated or have results out of the normal

8. Record all quantitative data in the “Serial Dilution/Plating Results Sheet”. Target range for statistically significant counts is 30-300 CFU. Data that fall out of the 30-300 CFU range are addressed in MOP 6584 (Procedure for Replating Bacteria Spore Extract Samples) and MOP 6565 (Filtration and Plating of Bacteria from Liquid Extracts).

2.0 CALCULATIONS Total abundance of spores (CFU) within extract:

(Avg CFU / volume (mL) plated) × (1 / tube dilution factor) × 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 mL =

(2000) × (1000) × 20 = 4.0 × 107 CFU

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

A44



Serial Dilution/Plating Results Sheet Page 1 of ______

TEST INFORMATION EPA Project No. PI Technician Name Test Date Technician Signature Test No.

RESULTS Date: Volume Plated:

Tube Dilution

Sample ID Plate Repl. 100 10-1

10-2 10-3

10-4 10-5 10-6

A B C

A B C

A B C

A B C

A B C

A B C

NOTES:

A45



Page 2 of ______


10-2 10-3

10-4 10-5 10-6

A B C

A B C

A B C

A B C

A B C

A B C

A B C

A B C

A B C

A B C

NOTES:

A46



Page _____ of ______


10-2 10-3

10-4 10-5 10-6

A B C

A B C

A B C

A B C

A B C

A B C

A B C

A B C

A B C

A B C

NOTES:

A47



Page _____ of ______


10-2 10-3

10-4 10-5 10-6

A B C

A B C

A B C

A B C

A B C

A B C

A B C

A B C

A B C

A B C

NOTES:

A48

MOP 6562 Revision 1

February 2013 Page 1 of 6


Preparing Pre-Measured Tubes with Aliquoted Amounts of Phosphate Buffered Saline with Tween 20 (PBST)

Prepared by: __________________________________________ Date: 2/12/2013 Nicole Griffin Gatchalian, ARCADIS Work Assignment Leader Reviewed by: __________________________________________ Date: 2/12/2013 Dahman Touati, ARCADIS Project Manager Approved by: __________________________________________ Date: 2/12/2013 Worth Calfee, EPA Work Assignment Manager

Prepared for



Prepared by

ARCADIS U.S., Inc.


A49

MOP 6562 Revision 1


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 “non-sterile 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 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 ml bottle.

5. Change label to reflect that the PBST is now sterile. Include initials and date of sterilization. The label should now include information on when the PBST was initially made and when it was sterilized and by whom.

6. Each batch of PBST should be used within 90 days.

2.0 PREPARING 20 ML/5 ML PBST TUBES FOR USE DURING

EXPERIMENTATION Twenty (20) ml or five (5) ml of the prepared PBST will be added to each sterile 50-ml conical tube as detailed below. Each flat of conical tubes contains 25 tubes, so one 500 ml sterile bottle of PBST should fill approximately one flat when 20 ml tubes are needed and four flats when 5 ml tubes are needed.

A50

MOP 6562 Revision 1


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: - 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 to not touch the tip to any surface.

5. Hold the pipetter with the first three fingers of your right (or dominant) hand. With your left hand (or non-dominant hand), pick up a bottle of the PBST and use the bottom of your right hand to unscrew the lid. Place the lid upside down on the benchtop and quickly flame the lip of the bottle. Turn the bottle and repeat, taking care to thoroughly flame the lip without getting the glass so hot that it shatters.

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

7. Quickly pick up one of the tubes that you have loosened the cap on, and use the bottom

of your right hand to remove the cap. Completely discharge the entire pipette into the tube, taking care 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.

A51

MOP 6562 Revision 1


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 labeled 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:

A52

MOP 6562 Revision 1


- A sterile beaker of microcentrifuge tubes. - Sufficient tubes of sterile PBST to fill these tubes (PBST may be aseptically transferred to 50 ml conical tubes for an easier aseptic transfer to the microcentrifuge tubes- it is easier than working from a 500 ml reagent bottle. Make certain that these 50 ml conical tubes are labeled to when the PBST was made, sterilized, etc.). - 1000 µL micropipette. - 1000 µL sterile pipette tips - Microcentrifuge tube racks. - Labeled beaker or waste container used to hold non-regulated waste, such as tips, under the hood.

2. Carefully remove the microcentrifuge tubes one at a time from the beaker and close the top on each one before placing it in the tube rack. Place the tubes in the rack skipping every other row. Fill up two racks doing this.

3. Add 900 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 micropitte and tips. Change tips whenever after 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 900µL PBST TUBES 1. Dispose of the waste that was put in the labeled beaker or waste container (micropipette

tips and tubes) in the nonregulated waste. Then, place this beaker in the “To be decontaminated via sterilization- contaminated glassware” bin or if it is a disposable container, then it can be put in the non-regulated waste container.

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

A53

MOP 6562 Revision 1


3. Replace any unused 50 ml conicals of PBST in the liquid containment on the shelf. Make sure that the tube is labeled as having been opened (date opened and initials of whomever used it). If the tube could possibly be contaminated in any way, dispose of it in non-regulated waste.

4. Wipe down the hood benchtop with ethanol, followed by bleach, followed by DI water and a clean Kimwipe or TechWipe.

A54

MOP 6565 Revision 4


Miscellaneous Operating Procedure (MOP) 6565: Filtration and Plating of Bacteria from Liquid Extracts

Prepared by: __________________________________________ Date: 2/15/2013 Nicole Griffin Gatchalian, ARCADIS Work Assignment Leader

Reviewed by: __________________________________________ Date: 2/15/2013 Dahman Touati, ARCADIS Project Manager

Approved by: __________________________________________ Date: 1/8/2014 Worth Calfee, EPA Work Assignment Manager

Prepared for



Prepared by

ARCADIS U.S., Inc.


A55

MOP 6565 Revision 4


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. This method can also be used to analyze liquid samples such as decon rinsates.

Materials: Petri dishes with appropriate agar

0.2 μm to 0.45 μm Pore-size disposable analytical filter units

P1000 pipette and sterile 1000 μL tips

Serological pipette

Serological pipette tips

Sterile forceps

Sterile deionized water (in ~10 mL aliquots)

1.0 PROCEDURE 1. For each liquid sample to be analyzed, gather the required number of disposable analytical

filter units and Petri dishes containing the desired sterilized/quality control checked (QC’d) media.

NOTE #1: For analysis of 5 to 30 mL extracts, 1 mL and remainder should be filtered; for 31 to 200 mL samples, 1 mL, 10mL, and remainder should be filtered; for samples over 200 mL, additional filter samples may be needed and will be determined on an individual basis.

NOTE #2: For previously plated samples where 10 – 19 CFU were observed, replate

using a 400 μL inoculums: for plates where 20 – 29 CFU were observed, replating using a 200 μL inoculum can be executed rather than filter plating (see MOP 6584).

2. Label plates.

A56

MOP 6565 Revision 4


3. Vortex liquid extract vigorously for 2 minutes, using 10 second bursts (for larger volume

samples, a vigorous mixing by shaking of the sample container can be substituted for vortex mixing). Directly prior to removing an aliquot, again vortex or agitate for 10 seconds.

4. For 1 mL to 10 mL filters (using a pipettor of appropriate size, sterile tip, and aseptic techniques), immediately following vortexing, pipette the pre-determined amount of extract into a 50 mL conical tube containing ~10 mL of sterile deionized water. Vortex the aliquotted solution and the water together and then aseptically pour into one of the filter units.

5. Apply vacuum to the filter unit to pull the liquid through the filter and collect the spores on

the surface of the filter. Immediately following, rinse the filter unit by aseptically adding ~10 mL of sterile deionized water along the inner sides of the unit while it is under vacuum.

For aliquots greater than 10 mL that need filtering, pipette the extract directly into the filter unit using a sterile serological pipette. Immediately following, rinse the filter unit by aseptically adding ~10 mL of sterile deionized water along the inner sides of the unit while it is under vacuum.

NOTE #3: Be sure to note and record the volume of the sample.

6. Aseptically remove the filter from the filter apparatus using sterile forceps, and roll the filter onto the agar surface within the Petri dish (spore side up).

7. Incubate all plates at the optimal growth temperature and time period for the specific organism.

8. Following incubation, enumerate and record the number of CFU on each plate. Be certain to record the volumes of the amount filtered on the data sheets.

2.0 DATA CALCULATIONS

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

filtered

Extract

V

VCFUN

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), and VFiltered is the volume of the extract filtered.

A57

MOP 6580 Revision 2


Miscellaneous Operating Procedure (MOP) 6580: Recovery of Bacillus Spores from 3M Sponge-Stick™ Samples

Prepared by: __________________________________________ Date: 2/12/2013 Nicole Griffin Gatchalian, ARCADIS Work Assignment Leader Reviewed by: __________________________________________ Date: 2/12/2013 Dahman Touati, ARCADIS Project Manager Approved by: __________________________________________ Date: 2/12/2013 Worth Calfee, EPA Work Assignment Manager

Prepared for



Prepared by

ARCADIS U.S., Inc.


A58

MOP 6580 Revision 2


MOP 6580 TITLE: RECOVERY OF BACILLUS SPORES FROM 3M SPONGE-STICK™

SAMPLES SCOPE: This MOP provides the procedure for recovering spores from 3M Sponge-

Stick™ samples.

PURPOSE: To extract and quantify bacterial spores from 3M Sponge-Stick™ samples using a highly repeatable procedure.

MATERIALS

pH-amended bleach

70-90 % Solution of denatured ethanol

Deionized (DI) water

Kimwipes

3M Sponge-Stick™ samples (P/N SSL10NB), hereafter referred to as ‘sponge’

Seward Stomacher® bags (P/N BA6041/CLR)

Phosphate buffered saline with 0.05% TWEEN®20 (PBST) (SIGMA-ALDRICH, Co,

P/N P3563-10PAK)

MicroFunnel Disposable Filter Funnels, Pall Life Sciences (VWR P/N 55095-060) or

Nalgene Sterile Analytical Filter Unit (Fisher P/N 130-4020)

Disposable polystyrene serological pipettes (5mL and 10mL)

Tryptic Soy Agar (TSA) plates

Vortex mixer

Disposable sterile 10µl loops

Disposable sterile forceps

Disposable gloves

Cell spreaders or glass beads for spreading

Racks for 15 mL and 50 mL centrifuge tubes

Sterile, plastic, screw-cap 50 mL centrifuge tubes (e.g. Fisher Cat# 14-959-49A)

A59

MOP 6580 Revision 2


Sterile, plastic, screw-cap 15 mL centrifuge tubes (e.g. Fisher Cat# 14-959-49D)

Pipette tips with aerosol filter for 1 mL and 200 μL

1.0 PREPARATION

Personnel must be familiar with all procedures prior to start.

1.1 Equipment Preparation

a) Begin by donning personal protective equipment (PPE) such as gloves, lab coat, and protective eyewear.

b) Clean the workspace (Biological Safety Cabinet; BSC) by wiping surfaces with pH-amended bleach, next with DI water, and lastly with a 70-90% solution of denatured ethanol. Allow any excess liquid to dry prior to beginning procedure. Make sure the workspace is clean and free of debris.

c) Assemble equipment in the BSC as needed: vortex mixer, filtration manifold,

automatic pipettors, tips, racks, etc.

d) Assemble extra supplies, such as stomacher and reagents, near BSC.

1.2 Supply Preparation a) Unpack shipping containers directly into a BSC.

b) If sponges are not in Stomacher® bags, label one Stomacher® bag for each sponge

and place in a bag rack.

c) Label two sterile 50 mL centrifuge tubes for each sponge sample and place in tube rack.

d) For each sample, label TSA plates on the agar side of the plate with the sample

number and the appropriate dilution factors, as per MOP 6535a (Serial Dilution: Spread Plate Procedure to Quantify Viable Bacterial Spores).

e) Label two additional plates for filter-plate analysis.

A60

MOP 6580 Revision 2


2.0 PERFORM SPORE EXTRACTION, ELUTION, AND CULTURE PROCEDURE

2.1 Dislodge Spores from the Sample Sponges

a) Begin by donning a new pair of gloves. All subsequent procedures involving manipulation of sponges or spore suspensions must be carried out in a BSC. (Stomaching may occur outside the BSC when samples are double-contained inside the indicated bags.)

b) If the sponges are not in Stomacher® bags, aseptically transfer each sponge to a Stomacher® bag (labeled during step 1.2b) using sterile disposable forceps. Change forceps between samples.

c) Aseptically add 90 mL of PBST to each bag that contains a sponge.

d) Stomach sponges in the PBST by completing the following:

Make certain the Stomacher® is set to MANUAL. Program the Stomacher®

speed to 260 RPM and the timer to 1 minute.

Open the Stomacher® door by raising the lid fully upward and back. The DOOR OPEN icon will be displayed.

Place the stomacher bag containing the sponge sample into a second stomacher bag to contain any leakage in the event the primary containment is compromised. Place the combined bags such that 50 to 60 mm of the top portions protrude above the bag clamp, while making certain that the sponge sample rests evenly between the homogenizer paddles.

Close the door to the Stomacher®. The DOOR OPEN icon will no longer be illuminated.

Stomach each sponge for 1 min by pressing the START button.

When the cycle ends, the Stomacher® will stop. If there is an emergent reason

to stop the stomacher during the 1 minute stomaching period, press the red button or the power button to do so prior to opening the Stomacher®. Stopping the Stomacher® by opening the door can damage the equipment.

Open the door of the Stomacher® and remove the bags containing the sponge.

Grab the sponge from the outside of the bag with your hands. Move the sponge

A61

MOP 6580 Revision 2


to the top of the bag while using your hands to squeeze excess liquid from the sponge.

Remove and discard the sponge using sterile forceps.

e) Repeat steps (b) through (d) for all samples.

f) Allow bags to sit for 10 min to allow elution suspension foam to settle before beginning the concentration step.

2.2 Remove Sponge Elution Suspension

a) Gently mix elution suspension up and down with a 50 mL pipette three times.

b) Split elution suspension volume equally.

Remove half of the suspension volume (~45 mL) with a sterile 50 mL pipette and place it in a 50 mL screw capped centrifuge tube.

Place remaining suspension (~45 mL) into a second 50 mL tube.

c) Record suspension volumes on tubes and data sheet.

d) Repeat steps (a) through (c) for all samples.

2.3 Concentrate Sponge Elution Suspension (Optional)

a) Centrifuge 50 mL centrifuge tubes

Prior to daily use and before placing tubes into centrifuge, follow MOP 6558 (Centrifuge Cleaning Procedure) for cleaning this equipment.

Add centrifuge tubes to rotor, evenly distributing weight.

Centrifuge tubes at 3500 x g for 15 min. Do not use the brake option on the centrifuge to slow the rotor, as re-suspension of pellet may occur.

b) Carefully remove about 42mL of supernatant with a 50 mL pipette and discard to leave approximately 3 mL in each tube. The pellet may be easily disturbed and not visible, so place pipette tip away from the tube bottom or side.

A62

MOP 6580 Revision 2


c) Vortex and sonicate tubes as follows: Set vortex mixer to level 10 and touch activation.

Turn on sonicator water bath.

Vortex tubes for 30 sec.

Transfer tubes to sonicator bath and sonicate for 30 sec.

Repeat vortex and sonication cycles two additional times.

d) Remove suspension from one tube with a sterile 5 mL pipette and place it in the

other tube of the same sample. The combined result is the final sponge elution suspension.

e) Measure final volume of the final sponge elution suspension with 5 mL pipette and record on tube and data sheet.

f) Repeat steps (e) through (i) for all samples.

2.4 Serially Dilute and Plate the Final Spore Elution Suspension

a) Use MOP 6535a to serially dilute and plate samples.

NOTE: If the samples are turbid, wide-orifice pipette tips may be used to prevent clogging of pipette tips.

b) Place all plates in an incubator set at 35 ± 2 ºC for a maximum of 3 days. Plates

should be examined within 18-24 hours after start of incubation. Manually enumerate CFU of target organism and record data.

If the CFU is <300/plate, record actual number.

If the CFU is >300/plate, record as “too numerous to count” (TNTC)

2.5 Capture Spores on Filter Membranes and Culture on TSA

Choose one of the following to methods to filter the final spore elution suspension:

a) Complete filter plating using MOP 6565 (Filtration and Plating of Bacteria from Liquid Extracts).

A63

MOP 6580 Revision 2


b) Complete filter plating using the following method:

1) Place two 0.45 μm (pore-size) Microfunnels on a Pall vacuum manifold (Pall Cat# 15403).

2) Moisten Microfunnel membranes with 5 ml PBST, open vacuum, and vacuum through the filter. All filtering should be done with a vacuum pressure <20 cm Hg.

3) Make certain that the manifold vacuum valve is closed. Turn on the vacuum.

4) With the vacuum valve closed, place 10 mL of PBST into each filter cup.

5) Add 1.0 mL of the final sponge elution suspension to each filter cup.

6) Open valves and allow the suspension to flow through the filter, close the valve.

7) Rinse the walls of each Microfunnel cup with 10 mL of PBST. Reopen the valve to

allow the suspension to flow through the filter.

8) Close the valve, turn off the vacuum pump. Slowly reopen the valve to equalize the pressures.

9) Squeeze the walls of the Microfunnel cup gently and separate the walls from the

base holding the filter. Remove each filter membrane with sterile disposable forceps and place grid-side up on a TSA plate. Make sure that the filter is in good contact with the surface of the agar. If an air pocket occurs under the filter, use the sterile forceps to lift the edge of the filter to release the air pocket for better contact with the agar.

10) Record exact volume of the sponge elution suspension filtered on each plate. It

should be 1 mL. (Greater sample volumes may be used to lower detection limits)

11) Repeat steps (1) through (8) for all each sample.

12) Incubate TSA plates with filter membranes at 35 ± 2 ºC for a maximum of 3 days. Plates should be examined within 18-24 hours after start of incubation. Manually enumerate CFU of target organism and record data.

If the CFU is <300/plate, record actual number.

If the CFU is >300/plate, record as “too numerous to count” (TNTC)

A64

MOP 6584Revision 1



Procedure for Replating Bacteria Spore Extract Samples

Prepared by: __________________________________________ Date: 11/15/2012Nicole Griffin Gatchalian, ARCADIS Work Assignment Leader

Reviewed by: __________________________________________ Date: 11/15/2012Dahman Touati, ARCADIS Project Manager

Approved by: __________________________________________ Date: 11/15/2012Worth Calfee, EPA Work Assignment Manager

Prepared for

National Homeland Security Research CenterOffice of Research and Development

U.S. Environmental Protection AgencyResearch Triangle Park, NC 27711

Prepared by

ARCADIS U.S., Inc.4915 Prospectus Drive, Suite F

Durham, NC 27713

A65

MOP 6584Revision 1


MOP 6584

TITLE: PROCEDURE FOR REPLATING BACTERIA SPORE EXTRACTSAMPLES

SCOPE: Determine the abundance of bacterial spores in a liquid extract that haspreviously been plated.

PRUPOSE: This method is deployed when results from spread-plate methods yield arelative standard deviation (RSD) value greater than 50 or colonies outsideof the acceptable range (30-300 CFU).

Materials:

Liquid suspension of bacterial spores

5.0 mL sterile centrifuge tubes (e.g., USA Scientific 3882-7600) containing 2700 µLdiluent (sterile deionized water, buffered peptone water or phosphate buffered saline)

Trypticase Soy Agar plates

Pipettes with sterile tips

Sterile beads placed inside a test tube (will be used for spreading samples on the agarsurface), or sterile disposable cell spreaders

Vortex mixer

1.0 DETERMING THE TARGET DILUTION

1. Using the original data (most likely obtained initially using MOP 6535a), locate thedilution set where the mean number of colony forming units (CFU) was nearest the30 to 300 range. This is the target dilution.

2. The target dilution will be noted on the tracking sheet, along with the sample ID andtest number. Each work assignment will have its own tracking sheet.

3. For each sample, 100 µL, 200 µL and 400 µL aliquots of the dilutions indicated inTable 1 shall be plated in triplicate.

A66

MOP 6584Revision 1


Table 1.

Average CFU inTarget Dilution

Volumes to Replate per Dilution10x less dilute than

Target (10x-1)Target Dilution (10x)

10x more dilutethan Target (10x+1)

1 – 50 100µl 200µl, 400µl51 – 150 100µl, 200µl, 400µl151 – 300 100µl 200µl, 400µl

4. Upon collection of results, at least one set of triplicate plates must contain all threedata points within the acceptable (30-300 CFU) range to consider the replatesuccessful. If none of the three sets of triplicate plates contain all data within therage, repeat replate procedure.

2.0 PROCEDURE

1. For each bacterial spore suspension to be tested label 2700 µL diluentmicrocentrifuge tubes as follows: 10-1, 10-2, 10-3, 10-4, 10-5, 10-6... (The number ofdilution tubes will vary depending on the concentration of spores in thesuspension.

2. For each liquid sample to be replated, gather the required number of 2700 µLdiluent tubes and Petri dishes containing the desired sterilized/QC’d media.

3. Label three Trypticase Soy agar plates for each dilution that will be plated withthe sample ID and volume to be plated. These dilutions will be plated intriplicate.

4. Mix original spore suspension by vortexing thoroughly for 30 seconds.Immediately after the cessation of vortexing, transfer 300 µL of the stocksuspension to the 10-1 tube. Mix the 10-1 tube by vortexing for 10 seconds, andimmediately pipette 300 µL to the 10-2 tube. Repeat this process until the finaldilution is made. It is imperative that used pipette tips be exchanged for a steriletip each time a new dilution is started.

5. To plate the dilutions, vortex the dilution to be plated for 10 seconds, thenimmediately pipette the desired volume (100, 200, or 400 µL) of the dilution ontothe surface of a TSA plate, taking care to dispense all of the liquid from thepipette tip. If less than 10 seconds elapses between inoculation of all replicateplates, then the initial vortex mixing before the first replicate is sufficient for allreplicates of the sample. Use a new pipette tip for each set of replicate dilutions.

A67

MOP 6584Revision 1


6. Carefully pour the sterile glass beads onto the surface of the TSA plate with thesample and shake until the entire sample is distributed on the surface of the agarplate. Aseptically remove the glass beads. Repeat for all plates.

7. Incubate the plates overnight at 32◦C - 37◦C (incubation conditions will varydepending on the organism’s optimum growth temperature and generation time.)

8. Enumerate the CFU on the agar plates by manually counting with the aid of aplate counting lamp, and a marker (place a mark on the surface of the Petri dishover each CFU when counting, so that no CFU is counted twice).

9. Since each dilution was tested in triplicate, determine the average of the triplicateplate abundances. Only those data between 30 - 300 colonies are suitable for usein data calculation formulas below. High variability below 30 CFU, and highprobability of co-located CFU above 300 are the reasons that only data within thisrange are acceptable for further reduction.

3.0 DATA 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 CFU10-3 100 µL (0.1 mL) 1 15010-3 100 µL (0.1 mL) 2 25010-3 100 µL (0.1 mL) 3 200

Extract total volume = 20 mL

(200 CFU / 0.1 mL) X (1/10-3) X 20 mL =

(2000) X (1000) X 20 = 4.0 X 107 CFU

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

Spore Abundance per mL = (Avg CFU) X (1 / DF) X extract volume

A68

Office of Research and Development (8101R) Washington, DC 20460

Official Business Penalty for Private Use $300

PRESORTED STANDARDPOSTAGE & FEES PAID

EPAPERMIT NO. G-35

Documents

Expedient Approaches for the Management of Wastes