EPA/600/R-21/226 | November 2021
www.epa.gov/emergency-response-research
United States
Environmental Protection
Agency
&EPA
Evaluation of Analytical Methods for
Detection of Bacillus anthracis
Surrogate Spores: Compatibility with
Real-World Maritime Environmental
Samples Collected from USCG
Assets and Facilities
Office of Research and Development
Homeland Security Research Program
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EPA/600/R-21/226
November 2021
DRAFT REPORT
for
Evaluation of Analytical Methods for Detection of Bacillus anthracis
Surrogate Spores: Compatibility with Real-World Maritime
Environmental Samples Collected from USCG Assets and Facilities
EPA Contract Number: EP-C-16-014
Task Order: 68HERC20F0237
Sanjiv Shah and Michael Worth Calfee
U.S. Environmental Protection Agency
Kent Hofacre, Scott Nelson, Ryan James, and Patrick Keyes
Battelle Memorial Institute
Columbus, Ohio 43201
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Disclaimer
The U.S. Environmental Protection Agency (EPA) through its Office of Research and Development
(ORD) directed and managed this work. This study was funded through the Analysis for Coastal
Operational Resiliency (AnCOR) Project by the U.S. Department of Homeland Security Science and
Technology Directorate under interagency agreement HSHQPM-17-X-00245. This report was
prepared by Battelle Memorial Institute under EPA Contract Number EP-C-16-014; Task Order
68HERC20F0237. This report has been reviewed and approved for public release in accordance with
the policies of the EPA. Mention of trade names or commercial products does not constitute
endorsement or recommendation for use of a specific product. The contents are the sole
responsibility of the authors and do not necessarily represent the official views of EPA, DHS S&T,
or the United States Government.
Questions concerning this document, or its application should be addressed to:
Dr. Sanjiv Shah
U.S. Environmental Protection Agency Headquarters
1200 Pennsylvania Avenue
Mail Code: E343-06
Washington, DC 20460
Shah. Sanjiv@epa.gov
202-564-9522
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Foreword
The U.S. Environmental Protection Agency (EPA) is charged by Congress with protecting the Nation's
land, air, and water resources. Under a mandate of national environmental laws, the Agency strives to
formulate and implement actions leading to a compatible balance between human activities and the
ability of natural systems to support and nurture life. To meet this mandate, EPA's research program is
providing data and technical support for solving environmental problems today and building a science
knowledge base necessary to manage our ecological resources wisely, understand how pollutants affect
our health, and prevent or reduce environmental risks in the future.
The Center for Environmental Solutions and Emergency Response (CESER) within the Office of
Research and Development (ORD) conducts applied, stakeholder-driven research and provides
responsive technical support to help solve the Nation's environmental challenges. The Center's research
focuses on innovative approaches to address environmental challenges associated with the built
environment. We develop technologies and decision-support tools to help safeguard public water
systems and ground water, guide sustainable materials management, remediate sites from traditional
contamination sources and emerging environmental stressors, and address potential threats from
terrorism and natural disasters. CESER collaborates with both public and private sector partners to foster
technologies that improve the effectiveness and reduce the cost of compliance, while anticipating
emerging problems. We provide technical support to EPA regions and programs, states, tribal nations,
and federal partners, and serve as the interagency liaison for EPA in homeland security research and
technology. The Center is a leader in providing scientific solutions to protect human health and the
environment.
This report focuses on the evaluation of analytical methods for the U.S. Coast Guard (USCG)
preparedness to respond to anthrax contamination incidents. This work was coordinated and managed by
the EPA's Homeland Security Research Program (HSRP) under the Department of Homeland Security
(DHS) funded Analysis for Coastal Operational Resiliency (AnCOR) project.
Gregory Sayles, Director; Center for Environmental Solutions and Emergency Response
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Table of Contents
Page
Disclaimer i
Foreword ii
Acronyms and Abbreviations x
Acknowledgments xii
Executive Summary xiii
1.0 INTRODUCTION 1
1.1 Background 1
1.2 Objective 2
1.3 Scope 2
2.0 MATERIALS AND METHODS 3
2.1 Target Maritime Surface/Material Sampled 3
2.1.1 Aluminum on Response Boats 4
2.1.2 Nonskid Tread 5
2.1.3 Touchscreens (On-board) 6
2.1.4 Concrete Piers 7
2.1.5 Wash Water (Small Vessels) 8
2.1.6 Gravel 9
2.1.7 Soil 10
2.1.8 Vegetation 11
2.1.9 Field Blanks 12
2.2 Sampling Methods 12
2.2.1 Sponge Stick Sampling Method 12
2.2.2 Vacuum Filter Cassette Sampling Method 14
2.2.3 Grab Sampling Method 14
2.3 Sampling Representative Maritime Surfaces/Materials 16
2.3.1 Surfaces Sampled with Sponge Sticks 17
2.3.2 Surfaces Sampled with Vacuum Filter Cassettes 21
2.3.3 Materials Sampled Using a Grab Method 22
2.4 Test Matrix 27
2.5 Overall Method Implementation 28
2.6 Microbiological Methods 29
2.6.1 Spore Stock 29
2.6.2 Spiking Samples 30
2.6.3 Sample Processing for Spore Recovery 31
2.6.4 Culture Method 33
2.6.5 RV-PCR Method 34
2.7 Data Reduction and Analysis 37
2.7.1 Percent Recovery of Presumptive BtkT1B2 Colonies 37
2.7.2 RV-PCR Method 38
2.7.3 Presentation of Results 38
3.0 RESULTS AND DISCUSSION 39
3.1 Sponge Stick Sample Analysis Results 39
3.1.1 Sponge Stick Sample Culture Analysis 39
3.1.2 Colony Confirmation by PCR 45
3.1.3 Sponge Stick Sample RV-PCR Analyses 48
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Table of Contents (Cont,)
Page
3.1.4 Analytical Method Comparison of Sponge Stick Samples 50
3.1.5 Analysis of Controls 56
3.2 Vacuum Filter Cassette Sample Analysis Results 56
3.2.1 Vacuum Filter Cassette Sample Culture Analysis 56
3.2.2 Colony Confirmation by PCR 61
3.2.3 Vacuum Filter Cassette Sample RV-PCR Analysis 63
3.2.4 Analytical Method Comparison of VFC Samples 65
3.2.5 Analysis of Controls 70
3.2.6 Considerations for Culture Analysis False Positive Results for Sponge Sticks
and VFCs 70
3.3 Grab Sample Analysis Results 70
3.3.1 Grab Sample Culture Analysis 71
3.3.2 Colony Confirmation by PCR 77
3.3.3 Grab Sample RV-PCR Analysis 79
3.3.4 Analytical Method Comparison for Grab Samples 83
3.3.5 Analysis of Controls (Grab) 88
4.0 QUALITY ASSURANCE/QUALITY CONTROL 89
4.1 Equipment Calibration 89
4.2 QC Results 89
4.3 Operational Parameters 89
4.4 Audits 89
4.4.1 Performance Evaluation Audit 89
4.4.2 Technical Systems Audit 90
4.4.3 Data Quality Audit 90
4.5 QA/QC Reporting 90
4.6 Data Review 90
5.0 SUMMARY OF METHOD OBSERVATIONS AND EXPERIENCES 91
5.1 Sample Processing Considerations 91
5.2 Method Qualitative Assessment 92
5.2.1 Culture Method 92
5.2.2 RV-PCR Method 92
5.2.3 Time/Cost Estimates 92
5.3 Culture Processing Considerations 93
5.3.1 BHIB Enrichment Culture Analysis 93
5.4 RV-PCR Processing Considerations 94
5.4.1 Biological Safety Level 3 Considerations 94
5.4.2 Suggestions to Improve RV-PCR Throughput 95
5.5 Sponge Stick Sample Analysis 95
5.5.1 Biological Safety Level 3 Considerations 95
5.5.2 Sponge Stick Method Considerations 95
5.6 Vacuum Filter Cassette Sample Analysis 95
5.7 Grab Sample Analysis 96
5.8 Difficult-to-Analyze Sample Types and Recommendations 96
6.0 CONCLUSIONS 98
7.0 REFERENCES 99
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Table of Figures
Page
Figure 1. Representative Area of Aluminum (Cabin Roof) Sampled on an RBS II (White
Sampling Template Shown) 4
Figure 2. Representative Nonskid Tread Adhered to Aluminum Surfaces Sampled on an
RBM 5
Figure 3. Representative Touchscreen Sampled (12 in x 12 in Screen on an RBS) 6
Figure 4. Representative Concrete Pier Surface Sampled 7
Figure 5. Representative Deck Surface (Primarily Nonskid Tread with Some Aluminum) of
an RBM Washed for the Collection of Wash Water Samples 8
Figure 6. Gravel Lot on Base Portsmouth as the Source of the Gravel Samples 9
Figure 7. Representative Sources of the Soil Samples Collected on the Grounds (Left,
Loam) and Shoreline (Right, Sand) at Base Portsmouth 10
Figure 8. Representative Sources of the Vegetation (Left, Grass; Right, Saltmarsh Grass)
Samples Collected on the Grounds and Shoreline at Base Portsmouth 11
Figure 9. Prewetted Sponge Stick from 3M Used for Surface Sampling 13
Figure 10. Vacuum Filter Cassettes (37-mm Diameter), Assembled (Left) and
Disassembled (Right) for Surface Sampling 14
Figure 11. RBS Starboard Stern Scupper 15
Figure 12. Wash Water Runoff Collection Apparatus 15
Figure 13. Sponge Stick Sampling Location of Aluminum Surfaces from the RBS 18
Figure 14. Sponge Stick After Sampling Aluminum Surface of the RBS 18
Figure 15. Sponge Stick Sampling Location of Nonskid Tread from the RBS and RBM 19
Figure 16. Sponge Stick After Sampling Nonskid Tread from the RBS and RBM 19
Figure 17. Sponge Stick Sampling of On-Board Touchscreens from the RBM 20
Figure 18. Sponge Stick After Sampling Touchscreen Surface on the RBS 20
Figure 19. Location of VFC Sampling of Nonskid Tread from the RBM 21
Figure 20. VFC After Sampling Nonskid Tread Surface of the RBM 21
Figure 21. Location of VFC Sampling of Concrete Piers 22
Figure 22. VFCs After Sampling Concrete Pier 22
Figure 23. RBS Washdown that Generated the Wash Water for Collection 23
Figure 24. Vessel Washdown Water Nontraditional Sampling Method Applied to an RBS
Scupper 24
Figure 25. Collection of Gravel Grab Sampling on the Base Grounds and a Close-up of the
Gravel 24
Figure 26. Representative Sample of Gravel in a 1-L Nalgene Bottle 25
Figure 27. Grab Sampling Images Depicting Collection of Soil on the Base Grounds (Left)
and of Sand Along the Shoreline (Right) 25
Figure 28. Collected Soil Sample in a 250-mL or 1-L Bottle (Left, Loamy Soil; Right, Sandy
Soil) 26
Figure 29. Grab Sampling Images Depicting Collection of Grass on the Base Grounds
(Left) and at the Shoreline (Right) 26
Figure 30. Collected Grass Sample in a 1-L Bottle (Left, Base Ground Grass; Right,
Shoreline Grass) 27
Figure 31. Process Flow Chart Depicting Key Process Steps in Chronological Order 28
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Table of Figures (Cont.)
Page
Figure 32. Sponge Stick (A), Vacuum Filter Cassette (B), Gravel (C), Vegetation (D), and
Soil (E) Being Spiked with Bf/cT1B2 Suspension 31
Figure 33. Manifold Containing 16 Filter Vials (A); Capping Tray (B); and Capped Filter
Vials Containing BHIB (C) 35
Figure 34. Presumptive Btk T1B2 Spore Recovery from Sponge Sticks Spiked with Btk
T1B2 Spores After Having Sampled Small Boat Aluminum Surfaces 41
Figure 35. Presumptive Bf/cT1B2 Spore Recovery (%) from Sponge Sticks Spiked with Btk
T1B2 Spores After Having Sampled Nonskid Tread Surfaces 41
Figure 36. Presumptive Btk T1B2 Spore Recovery (%) from Sponge Sticks Spiked with Btk
T1B2 Spores After Having Sampled On-Board Touchscreen Surfaces 42
Figure 37. Culture Images of Spore Recovery from Small Boat 2 Marine Grade Aluminum
Surface Sampled Using Sponge Stick and Plated on TSA 43
Figure 38. Culture Images of Spore Recovery from Nonskid Boat 1 Surface Sampled
Using Sponge Stick and Plated on TSA 44
Figure 39. Culture Images of Spore Recovery from Touch Screen Boat 2 Surface Sampled
Using Sponge Stick and Plated on TSA 45
Figure 40. RV-PCR Analysis of Bf/cT1 B2 Spores Recovered from Sponge Sticks Spiked
with Btk T1B2 Spores After Having Sampled Nonskid Tread Surfaces 49
Figure 41. RV-PCR Analysis of Bf/cT1 B2 Spores Recovered from Sponge Sticks Spiked
with Btk T1B2 Spores After Having Sampled Nonskid Tread Surfaces 49
Figure 42. RV-PCR Analysis of Bf/cT1 B2 Spores Recovered from Sponge Sticks Spiked
with Bf/cT1B2 Spores After Having Sampled On-Board Touchscreen Surfaces;
Positive Response Equals ACt >9 50
Figure 43. Presumptive Btk T1B2 Spore Recovery (%) from VFCs Spiked with Bf/cT1B2
Spores After Having Sampled Nonskid Tread Surfaces 57
Figure 44. Presumptive Btk T1B2 Spore Recovery (%) from VFCs Spiked with Bf/cT1B2
Spores After Having Sampled Concrete Pier Surfaces 58
Figure 45. Culture Images of Spore Recovery from Nonskid 1 Surface Sampled Using
Vacuum Filter Cassette and Plated on TSA 59
Figure 46. Culture Images of Spore Recovery from Concrete Pier 1 Surface Sampled
Using Vacuum Filter Cassette and Plated on TSA 60
Figure 47. Vacuum Filter Cassette Following Sampling of Surfaces 61
Figure 48. RV-PCR Analysis of Bf/cT1 B2 Spores Recovered from VFCs Spiked with Btk
T1B2 Spores After Having Sampled Nonskid Tread Surfaces 64
Figure 49. RV-PCR Analysis of Bf/cT1 B2 Spores Recovered from VFCs Spiked with Btk
T1B2 Spores After Having Sampled Nonskid Tread Surfaces 64
Figure 50. Presumptive Bf/cT1B2 Spore Recovery Percentage (Average ± One Standard
Deviation of N = 3 Replicates) from Wash Water Grab Samples Spiked with Btk
T1B2 Spores 72
Figure 51. Presumptive Bf/cT1B2 Spore Recovery Percentage (Average ± One Standard
Deviation of N = 3 Replicates) from Gravel Grab Samples Spiked with Bf/cT1B2
Spores 72
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Table of Figures (Cont.)
Page
Figure 52. Presumptive Bf/cT1B2 Spore Recovery Percentage (Average ± One Standard
Deviation of N = 3 Replicates) from Soil Grab Samples Spiked with Bf/cT1B2
Spores 73
Figure 53. Presumptive Bf/cT1B2 Spore Recovery Percentage (Average ± One Standard
Deviation of N = 3 Replicates) from Vegetation Grab Samples Spiked with Btk
T1B2 Spores 73
Figure 54. Culture Images of Spore Recovery from Washdown Grab Samples Plated on
TSA 74
Figure 55. Culture Images of Spore Recovery from Gravel Grab Samples Plated on TSA 75
Figure 56. Culture Images of Spore Recovery from Soil Grab Samples Plated on TSA 76
Figure 57. Culture Images of Spore Recovery from Vegetation Grab Samples Plated on
TSA 76
Figure 58. RV-PCR Analysis of Bf/cT1 B2 Spores Recovered from Vessel Wash Water
Grab Samples 81
Figure 59. RV-PCR Analysis of Bf/cT1 B2 Spores Recovered from Gravel Grab Samples 81
Figure 60. RV-PCR Analysis of Bf/cT1 B2 Spores Recovered from Soil Grab Samples 82
Figure 61. RV-PCR Analysis of Bf/cT1 B2 Spores Recovered from Vegetation (Grass) Grab
Samples 82
Figure 62. Autoclaving HDPE Bottles Compromises their Structure 91
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Table of Tables
Page
Table 1. Maritime Sample Type (Surface or Material) and Number of Sample Sets
Collected per Sampling Method 3
Table 2. Maritime Sample Type (Surface or Material) and Number of Samples Collected
per Sampling Method 17
Table 3. Sample Analysis Test Matrix for All Collected Real-World Maritime Samples 28
Table 4. Bf/cT1B2 TaqMan PCR Assay Primers and Probe Sequences 37
Table 5. Presumptive Bf/cT1B2 Spores Recovered from Laboratory-Spiked Sponge Sticks
that Previously Sampled Different Maritime Surfaces 40
Table 6. Summary of the Accuracy of Identification of Presumptive Btk T1B2 Colonies by
PCR Confirmation from Spiked Sponge Sticks Used to Sample Different Maritime
Surfaces 47
Table 7. RV-PCR Analyses of Spiked Sponge Sticks that Were Used to Sample Different
Maritime Surfaces for Detection of Btk T1B2 Spores 48
Table 8. Analytical Method Comparison Displaying Culture Presumptive, Culture ID with
PCR Confirmation, and RV-PCR Replicates Positively Identified (N = 3) for
Surfaces Sampled with Sponge Sticks 52
Table 9. Analytical Method Comparison Displaying Culture ID with PCR Confirmation and
RV-PCR for Surfaces Sampled with Sponge Sticks 55
Table 10. Presumptive Btk T1B2 Spores Recovered and Associated Spore Recovery (%)
from Laboratory-Spiked Vacuum Filter Cassettes that had Previously Sampled
Different Maritime Surfaces 57
Table 11. Summary of the Accuracy of Identification of Presumptive Btk T1B2 Colonies by
PCR Confirmation from Spiked VFCs Used to Sample Different Maritime
Surfaces 62
Table 12. RV-PCR Analyses of Spiked Vacuum Filter Cassette that Were Used to Sample
Different Maritime Surfaces for Detection of Btk T1B2 Spores 63
Table 13. Analytical Method Comparison Displaying Culture Presumptive, Culture ID with
PCR Confirmation, and RV-PCR Replicates Positively Identified (N = 4) for
Surfaces Sampled with Vacuum Cassettes 66
Table 14. Analytical Method Comparison Displaying Culture ID with PCR Confirmation and
RV-PCR for Surfaces Sampled with Vacuum Filter Cassettes 69
Table 15. Volume of Recovered Suspension Concentrated onto MicroFunnel Filter 70
Table 16. Presumptive Bf/cT1B2 Spores Recovered and Associated Spore Recovery (%)
from Laboratory-Spiked Grab Samples 71
Table 17. Summary of the Accuracy of Identification of Presumptive Btk T1B2 Colonies by
PCR Confirmation from Spiked Grab Samples 78
Table 18. PCR Analysis of BHIB Enrichment of Soil Pellet 79
Table 19. RV-PCR Analyses of Bf/cT1B2 Spores Spiked Grab Samples 80
Table 20. Analytical Method Comparison Displaying Culture Presumptive, Culture ID with
PCR Confirmation, and RV-PCR Replicates Positively Identified (N = 4) for Grab
Samples 84
Table 21. Analytical Method Comparison Displaying Culture ID with PCR Confirmation and
RV-PCR Replicates for Grab Samples 87
Table 22. Performance Evaluation Audits 89
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List of Appendices
Page
APPENDIX A: WORK INSTRUCTION FOR SURFACE SAMPLING USING CELLULOSE
SPONGE STICKS A-1
APPENDIX B: WORK INSTRUCTION FOR SURFACE SAMPLING USING VACUUM
CASSETTE FILTERS B-1
APPENDIX C: WORK INSTRUCTION FOR WATER WASHDOWN COLLECTION C-1
APPENDIX D: WORK INSTRUCTION FOR GRAVEL SAMPLING D-1
APPENDIX E: WORK INSTRUCTION FOR SOIL SAMPLE COLLECTION E-1
APPENDIX F: WORK INSTRUCTION FOR VEGETATION SAMPLING F-1
APPENDIX G: WORK INSTRUCTION FOR FORMULATIONS OF RECIPES USED IN
BIOLOGICAL TEST METHODS G-1
APPENDIX H: WORK INSTRUCTION FOR SPIKING WITH BACILLUS THURINGIENSIS
KURSTAKI (Btk) HD-7 T1B2 SPORES H-1
APPENDIX I: WORK INSTRUCTION FOR BACILLUS THURINGIENSIS KURSTAKI (Btk)
T1B2 SPORE RECOVERY FROM MARITIME SAMPLES - SPONGE STICKS, VACUUM
CASSETTES, AND GRAB SAMPLES 1-1
APPENDIX J: WORK INSTRUCTION FOR CULTURE OF BACILLUS THURINGIENSIS
KURSTAKI (Btk) T1B2 SPORES RECOVERED FROM SPONGE STICK WIPES, VACUUM
FILTER CASSETTES, AND GRAB SAMPLES J-1
APPENDIX K: WORK INSTRUCTION FOR MANUAL DNA EXTRACTION AND
PURIFICATION FROM BACILLUS THURINGIENSIS KURSTAKI (Btk) T1B2 SPORES K-1
APPENDIX L: WORK INSTRUCTION FOR REAL-TIME PCR ANALYSIS FOR BACILLUS
THURINGIENSIS KURSTAKI (Btk) T1B2 DNA L-1
APPENDIX M: WORK INSTRUCTION FOR SELECTING PRESUMPTIVE BACILLUS
THURINGIENSIS KURSTAKI (Btk) T1B2 COLONIES FOR QPCR CONFIRMATION M-1
APPENDIX N: WORK INSTRUCTION FOR BHIB ENRICHMENT FOR CULTURE N-1
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Acronyms and Abbreviations
AnCOR Analysis for Coastal Operational Resiliency
ASTM American Society for Testing and Materials, now ASTM International
B. anthracis Bacillus anthracis
Ba Bacillus anthracis
RHIR Brain Heart Infusion Broth
BSC Biological Safety Cabinet
Btk T1B2 Bacillus thuringiensis Sub sp. kurstaki T1B2
°C Degree(s) Celsius
CBR Chemical, Biological, Radiological
CDC Centers for Disease Control and Prevention
CESER Center for Environmental Solutions and Emergency Response
CFU Colony Forming Unit(s)
cm Centimeter(s)
Ct Threshold Cycle
dEhO Distilled Water
DHS S&T U.S. Department of Homeland Security Science and Technology Directorate
DNA Deoxyribonucleic Acid
EPA U.S. Environmental Protection Agency
ERLN Environmental Response Laboratory Network
ft Feet
g Gram(s)
h Hour
HDPE High-Density Polyethylene
HSMMD Homeland Security and Materials Management Division
HSPD Homeland Security Presidential Directive(s)
HSRP Homeland Security Research Program
ID Identification
in Inch(es)
IT Interagency Team
L Liter(s)
|iL Microliter(s)
MCE Methyl Cellulose Ester
MGAL Marine Grade Aluminum
min Minute(s)
mL Milliliter(s)
ModG Modified G
MRST Maritime Response Security Team
MSKTD Marine Grade Aluminum and 50% Nonskid
NSF National Strike Force
NTC No Template Control
ORD Office of Research and Development
PBS Phosphate Buffered Saline
PBST Phosphate Buffered Saline with 0.05% Tween 20
PBSTE Phosphate Buffered Saline with 0.05% Tween 20 and 30% Ethanol
PC Positive Control
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PCR
Polymerase Chain Reaction
PE
Performance Evaluation
Pg
Picogram
PMP
Paramagnetic Particle
QA
Quality Assurance
QAPP
Quality Assurance Project Plan
QC
Quality Control
QMP
Quality Management Plan
qPCR
Quantitative PCR
RBM
Response Boat Medium
RBS
Response Boat Small
rcf
Relative Centrifugal Force
RH
Relative Humidity
rpm
Revolution(s) per Minute
RV-PCR
Rapid Viability-PCR
sec
Second
SOP
Standard Operating Procedure
ss
Sponge Stick
T&E II
Testing and Evaluation II Program
TSA
Trypticase Soy Agar
TSB
Trypticase Soy Broth
USCG
United States Coast Guard
VFC
Vacuum Filter Cassette
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Acknowledgments
This report was prepared for the EPA's Homeland Security Research Program (HSRP) within EPA's
Office of Research and Development (ORD). Dr. Sanjiv Shah was the project lead. Contributions of the
following individuals and organizations to the development of this document are acknowledged.
United States Environmental Protection Agency
Dr. Sanjiv Shah, Center for Environmental Solutions and Emergency Response
Dr. Michael (Worth) Calfee, Center for Environmental Solutions and Emergency Response
Dr. Sara Taft, Center for Environmental Solutions and Emergency Response
Dr. Shannon Serre, Office of Land and Emergency Management
Mr. Leroy Mickelsen, Office of Land and Emergency Management
Ms. Ramona Sherman, Center for Environmental Solutions and Emergency Response
Dr. Kristen Willis, Office of Chemical Safety and Pollution Prevention
Dr. Christine Tomlinson, Office of Land and Emergency Management
United States Department of Homeland Security
Dr. Don Bansleben
Dr. Jane Tang
External Peer-Reviewers
Dr. Heather Moulton-Meissner, Centers for Disease Control and Prevention
Dr. Rebecca Bushon, U.S. Geological Survey
United States Coast Guard
Mr. Emile Benard
LCDR. Clifton Graham
Capt. Kirsten Trego
Battelle Memorial Institute
Mr. Kent Hofacre
Mr. Scott Nelson
Mr. Patrick Keyes
Ms. Hiba Shamma
Mr. Anthony Smith
Ms. Lindsay Catlin
Mr. Nate Poland
Mr. Ken Connelly
Mr. Dave Albertson
Ms. Emily Breech
Dr. Ryan James
Mr. Zachary Willenberg
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Executive Summary
The U.S. Environmental Protection Agency (EPA) is the primary federal agency responsible for the
protection and decontamination of indoor/outdoor structures and water infrastructure vulnerable to
chemical, biological, and radiological (CBR) terrorist attacks. Under the Homeland Security Research
Program (HSRP) of the EPA Office of Research and Development (ORD) conducts research to develop
methods and technologies able to rapidly and cost-effectively remediate areas affected by CBR terrorist
attacks. On the National Response Team, EPA, and the U.S. Coast Guard (USCG), working along with
other federal agencies, provide technical assistance, resources, and coordination of preparedness,
planning, response, and recovery activities for emergencies involving hazardous substances, pollutants
and contaminants, oil, and weapons of mass destruction in natural and technological disasters, and other
environmental incidents of national significance. In such instances, EPA and USCG provide Federal On-
Scene Coordinators and coordinate preparedness for and response to hazard incidents that occur in the
inland zone and the coastal zone, respectively. The USCG installations, facilities, and assets, due to their
unique roles and responsibilities in national security, could also be targets of CBR terrorism attacks.
Therefore, under the Department of Homeland Security (DHS) Science and Technology Directorate
(S&T) funded Analysis for Coastal Operational Resiliency (AnCOR) project, the EPA-HSRP is
providing support to the USCG in its efforts to be better prepared to respond to bioterrorism incidents.
EPA and USCG have formed an Interagency Team (IT) to support research under the AnCOR project of
which this task was a part.
EPA-HSRP has developed extensive protocols for sampling, analysis, and decontamination to respond
to biological contamination incidents; however, the response to any contamination incident is specific to
the affected site and surrounding environment. The coastal zone facilities and assets of the USCG—
including small and large boats and other vehicles in diverse geographical areas and maritime
environmental conditions—can pose complex and unique challenges for adapting existing methods or
developing new ones for sampling, analysis, and decontamination to respond to biological
contamination incidents. The performance of the methods may, in part, depend on the outdoor surfaces
and materials being sampled and analyzed. The USCG bases and ports, by nature of their mission and
location, may have unique surfaces and/or environments that could affect sampling and analysis
methods. The diversity of surfaces at a USCG base that would be impacted during bioagent remediation
necessitate proactive sample collection approaches to define the ongoing extent of contamination, the
effectiveness of completed decontamination, and the need for waste disposal. The purpose of this project
was twofold: 1) to evaluate the microbiological plate culture and EPA's Rapid Viability Polymerase
Chain Reaction (RV-PCR) analytical methods included in the EPA-ORD Protocol for Detection of
Bacillus anthracis (Ba) from Environmental Samples During the Remediation Phase of an Anthrax
Incident for their compatibility with detection of Ba surrogate spores in real-world maritime
environmental samples collected from the USCG coastal zone assets and their immediate surroundings;
and 2) to understand difficulties associated with processing and analyzing those samples and identify
capability gaps in this mission space.
Determination of contamination status of USCG facilities and assets is necessary to make decisions
regarding safety and deploy ability. This report provides data and information that can be used to inform
sampling operations and strategies following an outdoor biological contamination incident impacting a
USCG base. Ultimately, it is desired that these findings will facilitate recovery following a large-scale
biological incident.
Two sampling campaigns were successfully completed at Base Portsmouth, one on 04 November 2020
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and one on 26 March 2021, to collect samples of residual inert and biological deposits on representative
nonporous and porous maritime asset surfaces (e.g., aluminum on boats, nonskid tread on decks of
watercraft, touchscreens, concrete piers) and surrounding grounds and infrastructure and materials
(e.g., soil, vegetation, gravel). Established and commonly used EPA methods for sponge stick wipes
(57 samples), vacuum filter cassettes (VFCs, 48 samples), and grab samples (48 samples) for bulk
material collection were utilized. Samples were then transported to the laboratory and spiked with target
spore loads of Bacillus thuringiensis subsp. kurstaki (Btk) T1B2 barcoded spores. Spores were then
recovered and processed by microbiological plate culture (culture method) and RV-PCR.
Overall, for the samples collected (sponge sticks, VFCs, and grab samples), the culture method resulted
in 10 false positive results, as determined by PCR screening, and the RV-PCR method resulted in 0 false
positives. Overall, there were 19 false negative results for the culture method and 26 false negative
results for RV-PCR. An abundance of background microbial load compared to the spiked target spore
load and particulates within samples contribute to false negative results. Samples with high microbial
background load can mask the identification of target colonies on agar plates and lead to RV-PCR signal
suppression. Particulates within samples can reduce the amount of sample volume processed and
increase sample process times during filtration steps, particularly for vegetation and soil grab samples.
The results of the performance of both the culture and the RV-PCR analytical methods are presented and
discussed. Also, briefly presented are potential mitigation suggestions for sample types that are difficult
to process.
In overall conclusions, both the culture and the RV-PCR methods are valuable methods and can give
similar results for relatively clean samples. The culture method generally takes longer time to provide
sample analysis results. The background microbial flora in complex environmental samples can
overwhelm culture plates and obscure colony morphology of the target biothreat agent, leading to false
negative results with the culture method. Additionally, background microbial flora with a similar or
identical morphology to the target biothreat agent can be present within samples, triggering PCR
screening of colonies and possibly repeated PCR screening (to minimize risk of false negatives) if
presumptive morphology is present in large numbers. The RV-PCR method can provide rapid results,
which is of high significance in a wide-area incident involving multiple cities and environments. It is
akin to a biological indicator, it gives a positive or negative result and there is no iterative or repeat
analysis on sample aliquots, giving the method a clear end of analysis without the need for multiple
follow-up PCR screenings. RV-PCR constitutes a small laboratory footprint and requires less culture
media, resulting in relatively less BSL-3 waste. The method, however, needs to be less labor-intensive
and use of automated liquid handling and DNA extraction is essential. Complex environmental samples
such as soil, grass, and other grab samples, are difficult to analyze using the current sample processing
methods to recover spores. To mitigate this problem, a major emphasis needs to be placed on
development of improved and high-throughput sample processing methods for such complex samples.
xiv
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1.0 INTRODUCTION
1.1 Background
On the National Response Team, the U.S. Environmental Protection Agency (EPA) and the U.S. Coast
Guard (USCG) work along with other federal agencies, providing technical assistance, resources and
coordination on preparedness, planning, response, and recovery activities for emergencies involving
hazardous substances, pollutants and contaminants, oil, and weapons of mass destruction in natural and
technological disasters, and other environmental incidents of national significance. EPA and USCG
provide Federal On-Scene Coordinators and coordinate preparedness for and response to hazard
incidents in the inland zone and coastal zone, respectively.
Since the 2001 terrorist attacks and the Amerithrax (Anthrax) incidents, EPA has also been fulfilling its
homeland security responsibilities, as assigned by various Presidential Directives, by expanding its
original leadership role in environmental protection, decontamination, and cleanup during the
contaminations caused by hazardous chemical, biological, and radiological (CBR) substances to include
CBR terrorism incidents. As a result, EPA has been a focal point for many resources, including research
and development products, to respond to CBR incidents and protect human health and the environment.
USCG was also involved in the responses to the 2001 terrorist attacks and the Amerithrax (Anthrax)
incidents (USCG, 2015). Especially, USCG through its National Strike Force (NSF) was extensively
engaged in the 9/11 terrorist attack response, as well as in supporting the Amerithrax incidents cleanup
at the Capitol Hill and other response locations. In particular, the NSF provided tactical entry teams,
specialized equipment, management support, and a deputy incident commander for the anthrax response
emergency phase. USCG continues to enhance and expand their capabilities to respond to bioterrorism
incidents and effectively protect human health, and coastal zone assets and facilities (Maritime
Environmental Response Mission).
EPA's Homeland Security Research Program (HSRP) provides science and technology-based solutions
needed to effectively respond to and recover from natural or man-made disasters, including bioterrorism
incidents. Under the Department of Homeland Security (DHS) Science & Technology Directorate
(S&T) funded Analysis for Coastal Operational Resiliency (AnCOR) project, the EPA-HSRP is
providing support to the USCG in its efforts to be better prepared to respond to bioterrorism incidents.
EPA-HSRP has developed extensive protocols for sampling, analysis, and decontamination to respond
to biological contamination incidents; however, the response to any contamination incident is specific to
the affected site and surrounding environment. The coastal zone facilities and assets of the USCG—
including small and large boats and other vehicles in diverse geographical areas and maritime
environmental conditions—can pose complex and unique challenges for adapting existing methods or
developing new ones for sampling, analysis, and decontamination to respond to biological
contamination incidents.
Following a biohazard contamination incident such as release of Bacillus anthracis (Ba), accurate
sample analysis results help determine the extent and magnitude of contamination, which informs
responders for selection of decontamination strategies and helps determine the success of
decontamination. Finally, sample analysis helps the responsible authorities make reoccupancy decisions.
The focus of this task order was to evaluate the gold-standard microbiological plate culture and EPA's
Rapid Viability Polymerase Chain Reaction (RV-PCR, Letant et al., 2011) analytical methods and
associated sample processing procedures for their compatibility with detection of Ba surrogate spores in
1
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the real-world maritime environmental samples collected from the USCG coastal zone assets and their
immediate surroundings. Both of these analytical methods are described in detail in the EPA-ORD
Protocol for Detection of Bacillus anthracis from Environmental Samples During the Remediation
Phase of an Anthrax Incident (EPA, 2017) and both methods were evaluated using barcoded Bacillus
thuringiensis subsp. kurstaki (Btk) as the contaminant. Btk is a commonly used surrogate for Ba, and the
genetic barcode insert (termed T1B2) allowed for differentiation of naturally occurring Btk and the
spores applied for this study.
This study also assesses the challenges associated with processing and analyzing samples collected from
USCG facilities and assets, and identifies analytical method capability gaps. Results from this study will
help the USCG to recover rapidly following a biological contamination incident and return assets to duty
after successful decontamination. The outcome of this study will provide data and information to
improve sampling operations and strategies that will facilitate recovery following a large-scale
biological incident.
1.2 Objective
The overall objective of this task was to gather and generate data useful for EPA and USCG decision-
makers and other first responders regarding analytical method performance and impact of interferences
on Btk spore detection sensitivity that can lead to more effective planning and execution for the recovery
of a USCG base following a biological incident.
1.3 Scope
This task had three steps. First, in collaboration with the EPA and USCG, multiple surface types and
bulk (grab) sample types expected to be encountered in a wide-area biological agent contamination
incident involving a USCG base (and that were prevalent and available for sampling), were prioritized
and selected (e.g., vessel surfaces, vessel washdown water, pier surfaces, wide-area base surfaces).
Second, these sample types were collected using various sampling methods (sponge stick wipes, vacuum
filter cassettes (VFC), bulk [grab] samples) from the USCG installation at Portsmouth, VA. Lastly, the
collected samples were returned to Battelle's laboratory, spiked with barcoded Btk T1B2 spores, and
analyzed using existing EPA methods for both culture and RV-PCR to determine the impact of the
sample matrix on the recovery and analysis of spores from various sample matrices.
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2.0 MATERIALS AND METHODS
2.1 Target Maritime Surface/Material Sampled
The maritime sample type (surface or material sampled) and number of samples sets collected for the
applied sampling method are provided in Table 1. A sample set is defined as one sample type and one
associated sampling method, the three sampling methods used were: 1) surface sampling using sponge
stick (SS) wipes, surface sampling using 37-mm VFCs, and grab (bulk sample of material collected)
sampling. At least nine replicates for each sample set were collected for subsequent laboratory analysis,
from which three replicates were spiked with 0 spores, three with 300 spores, and three with 3,000
spores. The total number of sample sets collected (including field blanks) is shown in Table 1.
Table 1. Maritime Sample Type (Surface or Material) and Number of Sample Sets Collected per Sampling
Method.
Sample Types
Number of Sets Collected for each Sampling Method
SS Wipe
37-mm VFC
Grab
Aluminum on response boats
2
-
-
Nonskid tread
2
2
-
Touch screens (on-board)
2
-
-
Concrete on piers
-
2
-
Wash water, small vessels
-
-
1 (+1)
Gravel
-
-
1
Soil
-
-
1 (+1)
Vegetation
-
-
1 (+1)
Field blanks
2
1
2
Total
7
5
5
(a) Sample purposely not collected.
(b) Extra sample set (at least nine replicate samples, three samples for each of three spore spike levels) collected and analyzed.
(c) Extra sample collected as back-up, but not analyzed.
3
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2.1.1 Aluminum on Response Boats
Response Boat Small II (RBS, 29-ft) and Response Boat Medium (RBM, 45-ft) were the primary focus
of the sampling effort when the target surface was exterior aluminum. The aluminum was a marine
grade aluminum (samples designated [MGAL]), type 5086, which is the typical metal used in the
construction of small and medium response boats for the USCG. A representative image of the
aluminum surface sampled is shown in Figure 1.
Figure 1. Representative Area of Aluminum (Cabin Roof) Sampled on an RBS II (White Sampling
Template Shown).
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2.1.2 Nonskid Tread
Nonskid tread (designated NSKD) adhered to surfaces of an RBM is depicted in Figure 2. The same
nonskid tread is also used on RBS vessels. The nonskid (antislip) tread was 3M™ Safety-Walk™
Coarse Tapes and Treads. The tread appears to be similar to the tread used in other EPA
decontamination and surface sampling research. Per the manufacturer product description, the product
consists of large abrasive particles (24-grit aluminum oxide) bonded by a tough, durable polymer to a
dimensionally stable plastic film. The reverse side is coated with a pressure-sensitive adhesive covered
by a removable protective liner.
Figure 2. Representative Nonskid Tread Adhered to Aluminum Surfaces Sampled on an RBM.
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2.1.3 Touchscreens (On-board)
Glass touchscreen displays of various response boats (small and medium) and a Maritime Response
Security Team (MRST) 36-ft Zodiac Hurricane boat were sampled. A typical touchscreen measured
12 x 12 inches (in) and two or three such screens were present on a single boat. Consequently, touch
screens from three or four boats were sampled to collect the minimum of nine replicate samples per
sample set for subsequent analysis. A representative on-board touchscreen in an RBS is shown in
Figure 3.
Figure 3. Representative Touchscreen Sampled (12 in x 12 in Screen on an RBS).
6
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2.1.4 Concrete Piers
Concrete piers along the dock for RBM and RBS vessels and the piers for the large ships were sampled.
There was no known difference in the concrete of the two piers sampled, but they were selected to
purposely collect samples from two distinct locations on Base Portsmouth. Photographs depicting the
piers for the ships and a close-up of the concrete are provided in Figure 4.
Figure 4. Representative Concrete Pier Surface Sampled.
7
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2.1.5 Wash Water (Small Vessels)
Response boats are routinely washed by the USCG with freshwater from a hose at the base pier after sea
excursions. All exterior surfaces are rinsed (no detergent added), and the water runs off and empties into
the sea via drain ports (deck drains and scuppers) located throughout the boat. The wash water
represents a composite grab sample that could be readily and rapidly obtained with little additional
equipment or training. A vessel wash water sample was collected from the bow of an RBS and stern
deck of an RBM. The washdown water would flow over various surfaces, primarily the aluminum and
nonskid tread, but included glass windows in the case of the RBS. An example area of the deck surface
washed on an RBM is depicted in Figure 5.
Figure 5. Representative Deck Surface (Primarily Nonskid Tread with Some Aluminum) of an RBM
Washed for the Collection of Wash Water Samples.
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2.1.6 Gravel
Gravel is common throughout the base for unpaved roads, but highly trafficked areas exist. An example
gravel parking lot is shown in Figure 6.
Figure 6. Gravel Lot on Base Portsmouth as the Source of the Gravel Samples
9
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2.1.7 Soil
Soil type will vary across USCG bases based on geographical location and can also vary within a
specific base. Two soil types were selected for collection at Base Portsmouth: a loam type sample
representative of the grounds that support growth of grass and a sand type collected in close proximity to
the shoreline. The primary sample selected for completion of the test matrix was the loam soil, and the
sand was retained as an extra. Representative sources of the soil samples collected on the grounds and
shoreline at Base Portsmouth are shown in Figure 7.
Figure 7. Representative Sources of the Soil Samples Collected on the Grounds (Left, Loam) and Shoreline
(Right, Sand) at Base Portsmouth.
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2.1.8 Vegetation
Like soil, the vegetation on a USCG base will depend on the geographical location and can vary within a
specific base. For this study, grass was selected as representative vegetation to sample. Similar to the
rationale for soil collection , the grass selected as the primary vegetation sample for collection and
analysis was representative for Base Portsmouth and regularly mowed as part of grounds upkeep
(Figure 8). The grass was beginning to grow for the season but had not yet been mowed. A tall saltmarsh
grass (SparUna alterniflora) growing along the shoreline was sampled as an extra sample set.
Figure 8. Representative Sources of the Vegetation (Left, Grass; Right, Saltmarsh Grass) Samples
Collected on the Grounds and Shoreline at Base Portsmouth.
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2.1.9 Field Blanks
Field blanks were collected as controls by handling the sampling media in the same manner as surface
samples except that the sampling media did not contact a surface or material. For example, the sponge
stick wipes were removed from their original manufacturer's packaging and immediately placed (not
contacting a surface) in the receptacle for packaging, and the bag was sealed, and shipped to the
laboratory. For the washdown water blank, the non-sterile water used to wash the boat was collected
from the end of the freshwater supply hose and collected in a 1-L sterile bottle.
2.2 Sampling Methods
Note that personnel conducting the sampling were not required to wear full personal protective
equipment (nitrile gloves were worn) as the sampling was not performed to collect a Ba target or to
establish a field sampling method.
2.2.1 Sponge Stick Sampling Method
3M sponge sticks™ prewetted with a neutralizing buffer (3M, St. Paul, MN Part number SSL10NB),
shown in Figure 9, were purchased for sample collection per established EPA sampling methods
(Rose et al., 2011, EPA, 2013, and Tufts et al., 2014) and the Center for Disease Control and
Prevention's (CDC's) Anthrax Surface Sampling Guide (CDC, 2021). The sponge sticks were used to
sample a 10 in x 10 in (645 square centimeter (cm2)) area (defined by a template overlaying the target
surface) following the sampling pattern (30 linear passes over the area in a vertical, horizontal, and
diagonal pattern) defined in the EPA sampling method. The Work Instruction to collect sponge stick
samples is provided in Appendix A.
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IM
M
-i
Figure 9. Prewetted Sponge Stick from 3M Used for Surface Sampling.
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2.2.2 Vacuum Filter Cassette Sampling Method
VFCs, 37-millimeter (mm)-diameter, 0.8 micrometer (|am) pore mixed cellulose ester (MCE) membrane
(SKC, Inc. Eighty Four, PA, Part No. SKC 225-3-01), were used for surface sample collection per
established EPA sampling methods (Calfee, 2013). An assembled and disassembled VFC are shown in
Figure 10. The VFCs were used to sample a 12 in x 12 in (929 cm2) area (defined by a template
overlaying the target surface) over a 5-minute (min) (300-second (sec)) sampling duration following the
sampling pattern (50 linear passes over the area in a vertical S-pattern followed by 50 linear passes in a
horizontal S-pattern, with each pass being ~3-sec duration) defined in the EPA sampling method (Calfee
et al., 2013). The EPA-specified >5 liters/minute (L/min) sampling rate was used. The Work Instruction
to collect VFC samples is provided in Appendix B.
VFC Inlet
Figure 10. Vacuum Filter Cassettes (37-mm Diameter), Assembled (Left) and Disassembled (Right) for
Surface Sampling.
2.2.3 Grab Sampling Method
The grab sampling method was specific to the material being sampled or collected. Four (4) grab
sampling methods were employed, one each for: 1) boat wash water runoff, 2) gravel, 3) soil, and
4) vegetation.
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2.2.3.1 Boat Wash Water Runoff Sampling Method
The RBS and REM wash water runoff collection were new methods developed on this project and were
specific to the boat type from which the sample was collected. The primary target for the one required
sample was the RBS, which had stern and bow scuppers or drain ports. The stern drains were too close
to the waterline and thus a collection container could not be placed low enough to catch the runoff The
stern scuppers on the RBS were -30 to 60 centimeters (cm) (~1 to 2 feet (ft)) above the water line and
allowed adequate room to position a collection container. Figure 11 shows a close-up view of the
starboard stern scupper/drain port. The water collection apparatus developed, shown in Figure 12,
comprises a 1-L sterile bottle (like those bottles used for other grab sampling methods) secured with
hose clamps at the end of an extendable pole. (The pole used was one that comes with a bristle brush
from West Marine). The Work Instruction to collect wash water samples is provided in Appendix C.
Figure 12. Wash Water Runoff Collection Apparatus.
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2.2.3.2 Gravel Sampling Method
Gravel sampling was performed per established EPA method for sampling rail ballast (Serre and
Oudejans, 2017). The method entailed one operator donning gloves and randomly grabbing handfuls of
stones from the source and placing them into sterile 1-L bottles to the half-full line. Bottles were capped
with their lids and sealed with parafilm. Each bottle of gravel represented a single sample replicate. The
Work Instruction to collect gravel samples is provided in Appendix D.
2.2.3.3 Soil Sampling Method
The soil sampling method entailed one operator donning gloves and using a small garden hand spade to
remove (by scraping) the top 1 to 2 in of the soil, then scooping the soil into a 1-L sterile bottle. Two (2)
1-L sterile bottles were filled with the soil to collect a composite soil sample. Note, no field
measurements of soil temperature, moisture content, or pH were taken. Bottles were capped with their
lids and sealed with parafilm. The Work Instruction to collect soil samples is provided in Appendix E.
The soil was mixed in the laboratory, and a fixed quantity was used to analyze from each sample
replicate.
2.2.3.4 Vegetation Sampling Method
Vegetation sampling, specifically grass, was performed as described in Mikelonis et al., 2020. The
method entailed one operator donning gloves, grabbing a handful of grass, clipping the grass just above
the soil and then placing the grass into a 1-L sterile bottle. Two (2) 1-L sterile bottles were filled with
the grass to collect a single sample replicate. If the grass length exceeded the height of the bottle, the
grass was folded to fit within the bottle. Bottles were capped with their lids and sealed with parafilm.
The Work Instruction to collect grass samples is provided in Appendix F.
2.3 Sampling Representative Maritime Surfaces/Materials
Samples of representative maritime surfaces and grab materials were collected in two sampling
campaigns conducted at Base Portsmouth in Portsmouth, VA. Campaign #1 occurred on a clear, sunny
day, 04 November 2020, from approximately 0900 to 1800 hours (h). Early morning temperature and
relative humidity (RH) were 20°C and 50% to a mid-day high temperature of 23°C and RH 43% and an
end-of-day condition of 17°C and 65% RH. Sampling Campaign #2 occurred on a mostly cloudy
morning from 0800 to 1200 h on 26 March 2021. Early morning conditions were 22°C and 80% RH
with the temperature rising to 25°C and RH dropping to 66% by noon. Rain occurred on 25 March 2021.
In all sampling events, no dew or unevaporated rain was present and surfaces or materials were dry
when they were sampled. As discussed above, three traditional sampling methods: sponge sticks, VFCs,
and grab were used to collect samples from surfaces and materials commonly found at USCG bases.
EPA has established sampling protocols for these methods, which were summarized in Work
Instructions for the field team to execute. A summary of the samples collected is provided in Table 2.
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Table 2. Maritime Sample Type (Surface or Material) and Number of Samples Collected per Sampling
Method.
Sample Types
Surface/Material
Source Description
Sampling
Campaign
Date
Air
T/RH
(°C) / (%)
Samples Using Each Method
SS Wipe
37-mm
VFC
Grab
Aluminum on
response boats
#1, RBS washed
11/04/2020
21 / 46
12
--
--
#2, RBS not-washed
11/04/2020
20/50
12
--
--
Nonskid tread
RBM
11/04/2020
20/36
12
12
--
RBS
11/04/2020
23/43
12
12
--
Touch screens
(on-board)
RBS, RBM, MRST
11/04/2020
21 / 36
9
--
--
RBS, RBM, MRST
3/26/2021
22/80
11
--
--
Concrete on piers
Base (Large Vessels)
11/04/2020
19/54
--
12
--
Base (Response Boats)
11/04/2020
21 / 51
--
12
--
Wash water, small
vessels
RBS
3/26/2021
25/66
--
--
1
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Figure 13. Sponge Stick Sampling Location of Aluminum Surfaces from the RBS.
» •
• •
't" - ,
Figure 14. Sponge Stick After Sampling Aluminum Surface of the RBS.
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2.3.1.2 Sponge Sticks - Nonskid Tread
Exterior and exposed nonskid tread surfaces of an RBM (Sample Set #1) and an RBS (Sample Set #2)
were sampled during the first sampling campaign in November 2020. The sampling location and sample
collection are shown in Figure 15. Images of the sponge stick after sample collection from the RBS are
provided in Figure 16.
Figure 16. Sponge Stick After Sampling Nonskid Tread from the RBS and RBM.
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2.3.1.3 Sponge Sticks - On-Board Touchscreens
An image depicting the sampling location and sample collection on-board an RBM is provided in
Figure 17. An image of the sponge stick after sample collection from the RBS is provided in Figure 18.
Figure 17. Sponge Stick Sampling of On-Board Touchscreens from the RBM.
Figure 18. Sponge Stick After Sampling Touchscreen Surface on the RBS.
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2.3.2 Surfaces Sampled with Vacuum Filter Cassettes
2.3.2.1 Vacuum Filter Cassettes - Nonskid Tread
An image depicting the sampling location and sample collection is provided in Figure 19. An image of
the VFC after sample collection from the RBM is provided in Figure 20.
Figure 19. Location of VFC Sampling of Nonskid Tread from the RBM.
Figure 20. VFC After Sampling Nonskid Tread Surface of the RBM.
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Figure 21. Location of VFC Sampling of Concrete Piers.
Figure 22. VFCs After Sampling Concrete Pier.
2.3.2.2 Vacuum Filter Cassettes - Concrete Pier
Images depicting the sampling locations and sample collection are provided in Figure 21. An image of
the VFCs after sample collection from the concrete pier are provided in Figure 22.
2.3.3 Materials Sampled Using a Grab Method
The materials sampled and the sampling methods for the four grab samples are described below.
2.3.3.1 Grab - Vessel Wash Water
Boats are commonly washed with a fresh water source (no detergent added) available at the dock. The
washdown was performed by a USCG staff member and thus considered representative of an actual
washdown using the freshwater supply at the dock and associated 5/8-in-diameter garden hose with an
adjustable brass spray nozzle. The specifications of the nozzle were not known, but it appeared to be
similar to a heavy-duty adjustable brass spray nozzle (Dramm Model # 14033591; Home Depot) that has
been used to apply the water for washdown by EPA during other decontamination applications and
sampling method development projects. For those studies, a target flow rate of 4 ± 1 l./min operating at
a source pressure of 30 pounds per square in gauge (psig) was used; by observation, a similar volumetric
flow rate was used for the vessel washdown during sample collection. The spray nozzle setting was
arbitrary and adjusted to produce a small (estimated as <30-cm-diameter) cone at 1-m distance. Nozzle-
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to-surface distance was also variable, but was estimated to have typically ranged from 0.5 to 1.5 m. The
fill rate of the 1-L bottles was somewhat variable, but the bottles typically filled at an estimated rate of at
least 2 L/min at peak flow (a 1-L bottle was filled within 30 sec.). The washdown did not follow a
scripted pattern or established method/protocol. The washdown was performed in a manner so that the
water preferentially flowed to the drain from which the sample was collected. The flow and force of the
water was such that the wash water flow was directed toward the drain. The washdown was focused on
the collection side of the boat. An estimated area of 4 square meters (m2) was washed in a <5 min
period. The exterior surface area covered by the washdown of the RBS was glass windows, aluminum
roof and deck, and nonskid tread on the deck. The exterior surface area covered by washdown of the
RBM was comprised mostly of nonskid tread with some exposed aluminum. This approach was
adequate to create a water stream flowing from the scupper of the RBS and side drains of the RBM that
allowed for collection (the flow was high enough to prevent rinse water from adhering to the outer
surface of the boat and flowing into the sea). A photograph depicting water washdown of the RBS is
provided in Figure 23, and the washdown water sampling method is shown in Figure 24.
Figure 23. RBS Washdown that Generated the Wash Water for Collection.
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Figure 24. Vessel Washdown Water Nontraditional Sampling Method Applied to an RBS Scupper.
2.3.3.2 Grab-Gravel
Images depicting grab sample collection of gravel and a close-up of the source gravel are provided in
Figure 25. A representative sample of gravel in a 1-L Nalgene bottle is shown in Figure 26. Gravel was
sampled by collection into a 1-L Nalgene bottle to the lA full mark, ~900 g.
Figure 25. Collection of Gravel Grab Sampling on the Base Grounds and a Close-up of the Gravel.
24
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Figure 26. Representative Sample of Gravel in a 1-L Nalgene Bottle.
2.3.3.3 Grab - Soil
Images of soil sample collection from the grounds of Base Portsmouth are provided in Figure 27. The
completed bulk sample of soil collected in a filled 1-L bottle is shown in Figure 28.
Figure 27. Grab Sampling Images Depicting Collection of Soil on the Base Grounds (Left) and of Sand
Along the Shoreline (Right).
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Figure 28. Collected Soil Sample in a 250-niL or 1-L Bottle (Left, Loamy Soil; Right, Sandy Soil).
2.3.3.4 Grab - Vegetation (Grass)
An image of a grass sample being collected from the grounds of Base Portsmouth is provided in
Figure 29. The completed bulk sample of grass collected in a 1-L bottle is shown in Figure 30.
IW&43
Figure 29. Grab Sampling Images Depicting Collection of Grass on the Base Grounds (Left) and at the
Shoreline (Right).
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Figure 30. Collected Grass Sample in a 1-L Bottle (Left, Base Ground Grass; Right, Shoreline Grass).
2.4 Test Matrix
Each of the collected surface samples described in Sections 2.3.1 and 2.3.2 was processed to recover
spiked Btk T1B2 spores, and the recovered spore suspension was analyzed to quantify and identify
recovered Btk T1B2 spores to assess the EPA-provided culture and RV-PCR methods.
The completed test matrices for traditional sampling methods: sponge sticks, VFCs, and grab samples
are provided in Table 3. In total. 135 collected samples were analyzed, comprising 54 sponge sticks,
36 VFCs, and 45 grab samples. Nominally, triplicate samples for each of two Btk T1B2 spore loading
levels (300 and 3,000 colony forming units |CFU|) and triplicate samples of unspiked (0 CFU load)
samples were analyzed for each sample type. Additional spikes and blanks were included as controls and
are discussed in the results section.
Following sample processing, the recovered sample volume was split nominally in half, and therefore
the total target spores available listed in Table 3 were divided by two to represent the number of
Btk T1B2 spores available for each of the two analytical methods (culture and RV-PCR), as described in
the "U.S. EPA Protocol for Detection of Bacillus anthracis in Environmental Samples During the
Remediation Phase of an Anthrax Incident, Second Edition" (EPA, 2017). The method details are
discussed in further detail in Sections 2.6.3 to 2.6.5. Negative controls that were handled only within the
analytical laboratory were included to assess the potential for sample cross-contamination. Field blank
samples were collected to serve as a baseline to represent the expected best-case performance of the
method because of the absence of potentially competing or interfering grime or flora.
Culture and RV-PCR analytical methods were used to detect and/or quantify recovered Btk T1B2 spores
from spiked samples and subsequently recovered in the sample extracts. Trypticase Soy Agar (TSA) was
the primary medium used for all culture analyses.
27
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Table 3. Sample Analysis Test Matrix for All Collected Real-World Maritime Samples.
Sample Type
Sample ID
Sampling
Method
Replicate
Samples per
Btk T1B2
Spore Spike
Target Spore
Load
Analytical Method
(CFU)'"
Culture
Molecular
Small Boat Marine
Grade Aluminum
SBMGAL-1
SS
3, 3, 3
0, 300, 3,000
TSA
RV-PCR
Small Boat Marine
Grade Aluminum
SBMGAL-2
ss
3, 3, 3
0, 300, 3,000
TSA
RV-PCR
Touchscreen
TCHSCRN-1
SS
3, 3, 3
0, 300, 3,000
TSA
RV-PCR
Touchscreen
TCHSCRN-2
ss
3, 3, 3
0, 300, 3,000
TSA
RV-PCR
Nonskid Tread
NSKID-1
ss
3, 3, 3
0, 300, 3,000
TSA
RV-PCR
Nonskid Tread
NSKID-2
ss
4, 4, 4
0, 300, 3,000
TSA
RV-PCR
Nonskid Tread
NSKID-1
VFC
4, 4, 4
0, 300, 3,000
TSA
RV-PCR
Nonskid Tread
NSKID-2
VFC
4, 4, 4
0, 300, 3,000
TSA
RV-PCR
Concrete Pier
CONPIER-1
VFC
4, 4, 4
0, 300, 3,000
TSA
RV-PCR
Concrete Pier
CONPIER-2
VFC
4, 4, 4
0, 300, 3,000
TSA
RV-PCR
Boat Washdown
Water
SBWASH-1
Grab
3, 3, 3, 3
0, 300, 3,000,
30,000
TSA
RV-PCR
Boat Washdown
Water
SBWASH-2
Grab
3
30,000
TSA
RV-PCR
Gravel
GRAVEL-1
Grab
3, 3, 3
0, 300, 3,000
TSA
RV-PCR
Soil
SOIL-1
Grab
3, 3, 3, 3
0, 3,000, 30,000,
300,000
TSA
RV-PCR
Vegetation (Grass)
GRASS-1
Grab
3, 3, 3, 3
0, 300, 3,000,
30,000
TSA
RV-PCR
(a) Nominally half of the target quantity of spores loaded were available for each of the two analytical methods.
2.5 Overall Method Implementation
The traditional procedures used to spike/recover/analyze the sponge sticks, VFCs, grab and
nontraditional methods are shown as they occur in chronological order, as depicted graphically in the
process flow diagram of Figure 31.
Spore Spike
and Spore
Recovery
Extracted
SSW, VFC or
Filter
TSA,
Incubate
Overnight
BHIB
Enrichment
BHIB.
Incubate
Overnight
BHIB.
Incubate 2
Days
Day 1
(Monday)
Enumerate
and
~ Select
Colonies for
PCR Screen
DNA
Extraction
{t, and t,)
Day 2
(Tuesday)
Colony PCR
PCR
Analysis
Streak for
Isolation if
Colony PCR
Negative
f
i
Day 3
(Wednesday)
Select Presumptive Btk
Colonies and Prepare
BHIB Broth for PCR
Day 4
(Thursday)
Colony PCR
~ and BHIB
Broth PCR
Day 5
(Wednesday of
Following Week)
Figure 31. Process Flow Chart Depicting Key Process Steps in Chronological Order.
28
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The methods implemented, in the form of Work Instructions followed by the analytical staff, are
provided in Appendices H through N. These Work Instructions also complement the microbiological
methods described in Section 2.6, and emphasize glove-changing schedules that were implemented to
minimize cross-contamination. Work Instructions were reviewed, as needed, with the EPA Project Team
to ensure consistency with published methods.
The above process workflow was used to analyze a batch of 16 samples per trial, with 1 trial conducted
per week. For each weekly trial, the test samples (e.g., sponge sticks or VFCs) were spiked using
Btk T1B2 spores suspended in water or PBST per "Work Instruction for Spiking with Bacillus
thuringiensis kurstaki {Btk) HD-1 T1B2 Spores" in Appendix H. The spores spiked onto test samples
were recovered following the "Work Instruction for Bacillus thuringiensis kurstaki {Btk) T1B2 Spore
Recovery from Maritime Samples - Sponge Sticks, Vacuum Cassettes, and Grab Samples," process
described in Appendix I. The recovered suspension volume was then split equally between the culture
method and RV-PCR. The culture aliquot was plated onto TSA media and incubated overnight as
described in the "Work Instruction for Culture of Bacillus thuringiensis kurstaki {Btk) T1B2 Spores
Recovered from Sponge Stick Wipes, Vacuum Filter Cassettes, and Grab Samples" process in
Appendix J. The To RV-PCR aliquot was stored frozen while the recovered spores enriched overnight,
then the 7/aliquot was removed, and the DNA was extracted from both To and 7/aliquots per the "Work
Instruction for Manual DNA Extraction and Purification from Bacillus thuringiensis kurstaki {Btk) T1B2
Spores" process described in Appendix K. The extracted DNA was then analyzed using the real-time
PCR assay described in Section 2.6.5.4 and per the "Work Instruction for Real Time PCR Analysis for
Bacillus thuringiensis kurstaki {Btk) T1B2 DNA" process described in Appendix L. The real-time PCR
assay was also used to confirm or refute presumptive Btk T1B2 colonies selected from the culture
analysis per the "Work Instruction for Selecting Presumptive Bacillus thuringiensis kurstaki {Btk) T1B2
Colonies for qPCR Confirmation" process described in Appendix M. Selected samples for which the
culture was a nondetect were further analyzed using an enrichment per the "Work Instruction for BfflB
Enrichment for Culture" process described in Appendix N.
2.6 Microbiological Methods
Bacillus thuringiensis subsp. kurstaki {Btk) with the T1B2 genetic barcode (Buckley et al., 2012) was
selected as the surrogate for Ba in the current study because it is physically and genetically similar to Ba
(Tufts et al., 2014 and Greenberg et al., 2010) and has been used previously for outdoor testing research
conducted by EPA, and is planned to be used in future outdoor release testing by EPA. Use of Btk with
the T1B2 barcode makes it distinguishable from wild-type/naturally occurring Btk at the molecular level
and provides a level of resolution for the study so that naturally occurring Btk did not confound the PCR
results.
Traditional sample processing and analytical methods (both a culture and RV-PCR analytical method)
were conducted as described in the EPA Protocol (EPA, 2017), with modification to incubation
temperature, culture media and real-time PCR assay to optimize detection of Btk T1B2.
Following are sections that summarize specific procedures and steps applied to conduct the study.
2.6.1 Spore Stock
A single spore stock of Btk with T1B2 barcode was used as the biological test agent for the entire study.
Btk is commonly used as a biopesticide, the T1B2 barcoded version was produced to allow for
differentiating environmental Btk spores from those used in a test event (Buckley et al., 2012). The Btk
29
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T1B2 strain was handled as a Risk Group I agent following the Biosafety in Microbiological and
Biomedical Laboratories guidelines and Battelle biosafety work practices for such agents and was
reviewed by the Institutional Biosafety Committee for compliance with recombinant organisms. A spore
bank was produced using sporulation broth as follows and used as needed for the duration of the study.
An isolate of Btk T1B2 was provided by EPA and streaked for isolation on TSA, then incubated
overnight at 30 ± 2 degrees Celsius (°C). An isolated colony was then used to inoculate 50 milliliter
(mL) aliquots of nutrient broth and incubated overnight at 30 ± 2°C with shaking at 200 revolutions per
minute (rpm). Modified G (ModG) (500 mL) of sporulation broth (see Appendix G, Table 1 for
formulation details) was inoculated with 50 mL of the overnight Btk T1B2 culture, and then incubated in
a 3-L Fernbach flask at 30 ± 2°C with shaking at 200 rpm. The culture was observed via wet mount
microscopy every 1 to 3 days for sporulation. Following 5 days of incubation, the ModG culture reached
> 99% sporulation.
The sporulated culture was centrifuged at 10,000 relative centrifugal force (rcf) for 12 min in multiple
250-mL bottles. After removing and discarding the supernatant, the resulting pellets were resuspended
to a total volume of approximately 100 mL with sterile distilled water (dFhO), transferred into a sterile
glass vessel, and heat shocked at 60 to 65°C for 1 h in a water bath with gentle agitation. (Note: a
control flask with a thermometer was used to ensure the desired temperature was achieved and
maintained during the heat shock step). The spores were then washed twice by repeated centrifugations
at 10,000 rcf for 12 min using 100-mL dFhO per wash. After the final centrifugation, the spores were
resuspended to a total volume of 100 mL in sterile dFhO. The spore bank was assigned a unique lot
number and stored refrigerated at 2 to 8°C. Spore bank concentration was determined by spread plating
serial dilutions onto TSA, followed by 30 ± 2°C overnight incubation and enumeration of CFU.
2.6.2 Spiking Samples
On the day of sample processing for spore recovery, Btk T1B2 spore stock was vortex-mixed and diluted
using sterile dFhO or phosphate buffered saline with 0.05% Tween 20 (PBST) and used to directly spike
the samples at two spore spike levels (300 CFU or 3,000 CFU). For washdown and vegetation samples,
a third spore spike level of 30,000 CFU was incorporated. For soil samples, the 300 CFU spore level
was omitted, and samples were processed with 3,000 CFU, 30,000 CFU and 300,000 CFU. Three
replicates of each sample were used for each spore spike level, including zero spore spike level. Each
spiking stock was spread plated onto TSA on the day of testing to calculate the actual concentration of
spores spiked in CFU/mL.
Each sponge stick was positioned in a specimen cup so that the dirty side was facing up and Btk T1B2
spores were directly spiked onto the surface of each sponge stick (the sides of the sponge that could
contact the specimen cup wall were not spiked; see Figure 32). For VFCs, the final spiking stock
concentrations were directly applied over the surfaces of collected particulates and filter. Gravel was
spiked on the top layer of -900 grams (g) of gravel. Sponge sticks, VFCs, gravel, and soil were spiked
with 100-|iL volume total in a dropwise fashion; washdown samples were spiked with 100-|iL volume;
and vegetation samples were spiked with 500-|iL volume and larger droplet size to enable downward
movement of the spiking stock into the grass grab sample. Spiking procedure is further outlined in
Appendix H.
30
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A
• / J
% *}<¦
•
kf
D
Figure 32. Sponge Stick (A), Vacuum Filter Cassette (B), Gravel (C), Vegetation (D), and Soil (E) Being
Spiked with Btk T1B2 Suspension
2.6.3 Sample Processing for Spore Recovery
Throughout the spore recovery procedure, gloves were changed between handling samples to limit the
likelihood of cross-contamination. Spore recovery methods were summarized as work instructions for
the laboratory staff to execute (Appendix I).
2.6.3.1 Sponge Sticks
Following sample collection, samples were stored at 2 to 8°C until sample spiking and spore recovery.
The remaining sponge stick handle was removed, and the sponge stick was unfolded, transferred
aseptically to a Stomacher bag (Seward, Bohemia, NY) containing 90 mL cold (2 to 8°C) Phosphate
Buffered Saline with 0.05% Tween 20 and 30% ethanol (PBSTE), then homogenized for 1 min at
260 rpm in a Stomacher 400. Each sample then sat for 10 min to allow foam to settle before removing
the sponge. Absorbed liquid was expelled from the sponge into the Stomacher bag and the sponge was
removed. The suspension (-90 mL) was gently mixed by pipetting up and down three times with a
sterile 50-mL pipet, then the suspension was split into two (2) 50-mL sterile conical tubes and
centrifuged at 3,500 rcf for 15 min in a swinging bucket rotor at 4°C with the brake off. To concentrate
the sample, -65 mL of supernatant was removed and the remaining -25 mL of supernatant was used to
suspend the pellets. The suspension was split in half and used for culture-based analysis as described in
Section 2.6.4 and RV-PCR analysis as described in Section 2.6.5.
31
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2.6.3.2 Vacuum Filter Cassettes
Following sample collection, samples were stored at 2 to 8°C until sample spiking and spore recovery.
Spore recovery using 5 mL of PBSTE was added to the conical tube containing the nozzle and tubing
and set aside. Six (6) mL total of PBSTE was used to rinse and recover particulates collected within the
VFC by adding 2 mL of PBSTE in three successive rinse steps. Following the second rinse step, the
filter was transferred to the 2-ounce (oz.) cup containing rinsate. The nozzle and tubing containing 5 mL
PBSTE was sonicated in a sonicating bath for 1 min, then vortexed for 2 min and combined with filter
rinsate in the 2-oz. cup. The 2-oz. cup containing filter and 11 mL of PBSTE was sonicated in a
sonicating bath for 3 min. As much suspension as possible, typically ~ 8 mL, was transferred to a 15-mL
conical tube, and the suspension was split in half and used for culture-based analysis as described in
Section 2.6.4 and RV-PCR analysis as described in Section 2.6.5.
2.6.3.3 Grab Samples (Wash Water, Gravel, and Vegetation)
Following sample collection, samples were stored at 2 to 8°C until they were spiked. Spores were
recovered by adding 500 mL of sterile PBST to the 1-L bottles containing gravel or vegetation and
shaking the bottle vigorously with one hand on the bottom and the other on the top using an over the
shoulder back-and-forth motion for 2 min. The sample was allowed to settle for 30 sec and then the
eluent was poured into a clean sterile 500 mL container. The washdown water or eluent from gravel and
vegetation was mixed vigorously by hand for 30 sec; then, the liquid was poured into a 0.45-|im filter
funnel (MicroFunnel™ Filter, Pall Corporation, Washington, NY, Cat. 4804) to the 100 mL gradation
line. If the volume passed through the filter without becoming clogged, an additional 100-mL aliquot
and 50-mL aliquot was added for a total of 250 mL for gravel and a total of 500 mL for washdown or
vegetation eluate. If a 100-mL or 50-mL aliquot took longer than 10 min to pass through the filter, no
further volume was added. At 30-min post-sample addition, if the sample did not completely pass
through, the remaining volume in the filter unit was carefully removed. The total volume vacuum
filtered was documented. The filter membrane was then removed using sterile forceps and transferred to
a 50-mL conical tube so that it was positioned in the bottom half of the tube with the inlet side of the
membrane facing the center of the tube. Then, 10 mL of PBSTE was added and vortex-mixed at
maximum speed on a platform vortex for 10-sec bursts for 2 min to dislodge spores. The suspension in
tubes was allowed to settle for 2 min, then transferred to a 50-mL conical tube. An additional 10 mL of
PBSTE was added to the 50-mL tube containing the membrane and vortexed as described for the first 10
mL, then pooled with the first 10 mL for each sample. This 20-mL pooled volume was vortex-mixed,
allowed 30 sec of settling time, and then split in half for culture-based analysis as described in
Section 2.6.4 and RV-PCR analysis as described in Section 2.6.5.
2.6.3.4 Grab Samples (Soil)
Soil collected from the field was homogenized by manual shaking and then parsed into 50-mL conical
tubes, each sample containing 10 g of soil. Following spiking of soil with target spore load of Btk T1B2,
40 mL of PBST was added to each soil sample and vortex-mixed for 30 sec, followed by bath sonication
for 10 min. The samples were then manually mixed for 2 min. Each sample was then spun at 1,000 rcf
for 5 min in a swinging bucket centrifuge and the supernatant was transferred to a clean 50 mL tube,
leaving -2.5 mL of supernatant in the pelleted soil. The supernatant and pellet were heat shocked at
70 ± 2°C for 1 h. The supernatant was split in half, with -18 mL available for culture analysis and
-18 mL available for RV-PCR.
32
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Sterile soil was processed alongside the field samples as a control. The sterilization process was
completed by spreading soil onto a Pyrex glass dish and autoclaving twice at 121°C for 45 min with soil
cooling to room temperature in between.
Moisture analysis (based on ASTM International (ASTM) D 2216) and pH analysis (based on EPA
Method 9045D) were performed on the unsterilized and sterilized soil.
Moisture analysis: Soil (~5 g) was placed in pre-weighed aluminum dish using Sartorius Balance
(R200D, Sartorius Instruments, McGrawPark, Illinois). Heated in oven at 105°C overnight
(Thermodyne Furnace, 30400, Barnstead Thermolyne Corporation, Ramsey, MN). Transferred heated
samples into a desiccator to cool to room temperature, then weighed the soil again.
W /(Mcws-Mcs)/(Mcs-Mc)JxlOO
where: W= water content (%); Mcws = mass of container and wet sample (g); Mcs = mass of container
and dry sample (g); and Mc = mass of container (g).
pH analysis: Soil (-14 g) was transferred to 50-mL tube and 15 mL of reagent grade water was added
and stirred for 10 min. The samples were centrifuged at 9,000 rpm for 20 min, then supernatant was
decanted and pH was measured using a Thermo Scientific Orion Dual Star pH/ISE Benchtop instrument.
2.6.4 Culture Method
Culture-based microbiological analysis was performed on each sample by filtering the recovered spore
suspension through 0 .45 - (am MicroFunnel filters (Pall Corporation, Washington, NY, Cat. 4804), then
placing the filters onto solid bacterial growth media (TSA) or spread plating 0.1 mL of the recovered
extract onto TSA. A modification to the EPA Protocol (EPA, 2017) used in the current study was that
sample analysis proceeded directly to filter-plate or 0.1 mL spread plating of undiluted samples without
dilutions, since spike levels were at or near the method detection limit. Work instructions for the culture
method are detailed in Appendix J.
For MicroFunnel filter analysis, each filter was prewetted with 5 mL of PBST, 10 mL of PBST was
added to each MicroFunnel filter to suspend aliquots, and then 1-mL to 8-mL aliquots of the extract
were applied and vacuum filtered. The walls of each MicroFunnel filter were rinsed with 10 mL of
PBST and filtered through the MicroFunnel filter, then the filter membrane was removed and placed
onto TSA media.
Colonies with a typical Btk T1B2 morphology following overnight incubation at 30 ± 2°C were counted
to determine percent spore recovery. Typical Btk T1B2 morphology on TSA is 2 to 5 mm in diameter,
flat or slightly convex with edges that are irregular, and has a ground-glass appearance.
Two different microbiologists enumerated colonies over the course of the project, all of whom were
trained by the project's lead microbiologist to consistently identify presumptive Btk T1B2 based on
colony morphology. The lead microbiologist periodically reviewed the enumeration results to help
ensure consistency and integrity, which is an important consideration and factor in the application of the
method because the culture analysis was subjective to the assessment of colony morphology. Colonies
identified during culture analysis are reported as presumptive Btk T1B2.
33
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A small subset of presumptive Btk T1B2 were screened using a real-time PCR assay targeting the T1B2
barcode. A portion of a single colony or up to 10 colonies were pooled and suspended in 100 |iL of
PCR-grade water, heated for 5 min at 95 ± 2°C, centrifuged at 14,000 rpm for 2 min and the supernatant
was analyzed in triplicate. An average threshold cycle (Ct) value of < 40 was recorded as a positive
result. The work instruction for colony PCR is located in Appendix M.
For Brain Heart Infusion Broth (BHIB) enrichment, the spores that remained on the filter or sponge
were enriched following spore recovery within the 50 mL conical tube or specimen cup by adding
25 mL of BHIB, then incubated at 30 ± 2°C for 24 to 48 h. If Btk T1B2 colony morphology was not
observed on TSA plates from culture analysis (spread plate or MicroFunnel Filters), turbid BHIB was
then streaked onto three TSA plates for isolation and incubated overnight at 30 ± 2°C. Colonies with Btk
T1B2 morphology that were isolated on these streak plates were screened using a real-time PCR assay
targeting the T1B2 barcode. If colonies with Btk T1B2 morphology were not isolated on streak plates, an
aliquot of the BHIB suspension (50 |iL) was pelleted by centrifugation at 12,000 rcf for 2 min,
supernatant was discarded, and the pellet was suspended in 100 |iL of PCR-grade water. The suspended
pellet was lysed at 95 ± 2°C for 5 min, then screened using real-time PCR assays. An average Ct value
of < 40 was recorded as a positive result. The work instruction for BHIB enrichment is located in
Appendix N.
2.6.5 RV-PCR Method
2.6.5.1 Further Sample Processing for RV-PCR
Following filtration of half (12.5 mL for sponge stick; ~5 mL for VFC; 10 mL for wash water, gravel,
and vegetation; or -17 mL for soil samples) of recovered extract through the Whatman™ Autovial™
filter vials (with polyvinylidene difluoride membrane, Whatman, Marlborough, MA Cat.
AV125NPUAQU), two buffer washes were performed according to the EPA Protocol (EPA, 2017). The
first wash was 12.5 mL of cold (4°C) high salt buffer (10X PBS) followed by 12.5 mL of cold (4°C) low
salt wash buffer (IX PBS). The top portion of the manifold was then removed and placed into a capping
tray with pre-filled luer lock caps to seal the filter vials. Five (5) mL of cold (4°C) BHIB was then added
to each filter vial, the vials were capped, and then vortex-mixed for 10 min on a setting of 7. Images of
the manifold and capping tray are provided in Figure 33. Following the vortex step, the broth culture
was mixed by pipetting up and down -10 times and before incubating, a 1-mL aliquot was transferred to
a screw cap tube and stored at -20°C as the time zero (To) aliquot. The capped filter vials were then
incubated overnight for -16 h (time final, Tf) in an incubator shaker set to 30 ± 2°C at 230 rpm. Note
that the EPA Protocol specified 9 h or longer (EPA, 2017); the 16-h incubation allowed for a standard
work schedule to be maintained rather than the overnight shift that would have been required by a 9-h
incubation.
34
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B
.\ S ' M \* Ix »1( •((' •' £
v j/ tff rW *
C
Figure 33. Manifold Containing 16 Filter Vials (A); Capping Tray (B); and Capped Filter Vials Containing
BHIB (C).
Following overnight incubation (-16 h) of the filter vials, the vials were mixed on the platform vortex
for 10 min with speed set to 7. Following the vortex step, the culture suspension was mixed by pipetting
up and down -10 times, and a 1-mL aliquot was transferred to screw cap tubes and labeled as the 7/
aliquot. These processing steps are described in Appendix I.
2.6.5.2 DNA Extraction and Purification
Prior to extraction of deoxyribonucleic acid (DNA), the lysis buffer with antifoam reagent and the
alcohol wash was added according to the manufacturer's instructions in the Magnesil Blood Genomic,
Max Yield System Kit (Promega, Madison, WI, Cat. MD1360) and a heat block was preheated to 80°C.
All screw-capped, 1-mL aliquots were thawed and centrifuged at 14,000 rpm (18,188 rcf) for 10 min
(4°C), and 800 j.iL of the supernatant from each tube was removed and discarded. To extract the DNA,
800 |iL of lysis buffer was added to each tube and the samples were mixed by vortexing on high
(-1,800 rpm) in 10-sec pulses for a total of 60 sec. Each tube was then vortex-mixed for 10 sec at low
speed directly before the lysate was transferred to a 2-mL labeled Eppendorf tube. The lysate tube was
then incubated at room temperature for 5 min. Uniformly resuspended paramagnetic particles (PMPs)
(600 |iL) were added to each lysate tube and the samples were mixed by vortexing. After vortexing each
To and I < tube for 10 sec (high, -1,800 rpm), the samples were incubated at room temperature for 5 min.
35
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The samples were then placed on the magnetic stand with the hinged side of the tube facing toward the
magnet after briefly resuspending the particles by vortexing. The magnetic rack was then inverted to
ensure all PMPs were contacting the magnet. After 10 sec, the tubes were opened, and the liquid
removed without disturbing the PMPs. Lysis buffer (360 |iL) was then added to each To and 7/tube, the
tube was capped and vortexed for 10 sec. The tubes were then placed on the magnetic stand and inverted
again. The supernatant was then removed, and 360 |iL of salt wash solution was added to each tube. The
tubes were capped and vortexed for 10 sec, placed on the magnetic stand, and inverted. The supernatant
was removed without disturbing the PMP pellet. The pelleted PMPs were washed a second time with
360 |iL of salt wash solution.
After removal of the second salt wash supernatant, 500 |iL of alcohol wash (70% ethanol) was added to
each tube. The tubes were vortexed for 10 sec, placed on the magnetic stand, and inverted. The
supernatant was then removed, and two more alcohol washes were conducted for a total of three 500-|iL
alcohol washes. A fourth alcohol wash was then conducted using 500 |iL of 70% ethanol. After the
supernatant from the wash was removed, all tubes were opened and allowed to air-dry for 2 min. The
open tubes were then heated at 80°C in a heat block inside a Biological Safety Cabinet (BSC) until the
PMPs were dry (-20 min). DNA was then eluted from the PMPs by the addition of 200 |iL of elution
buffer to each To and 7/tube. The tubes were then closed, vortexed for 10 sec, and incubated in the heat
block for 80 sec. The tubes were then vortexed another 10 sec and incubated in the heating block for 1
min. The vortexing and heating was repeated four times for a total of five times. The tubes were then
removed from the heating block and incubated at room temperature for at least 5 min. Each tube was
briefly vortexed and then centrifuged at 2,000 rpm (371 rcf) at 4°C for 1 min. The tubes were then
vortexed and placed on the magnetic stand for at least 30 sec. The eluate was collected (-80 to 90 |iL)
and transferred to clean labeled 1.5-mL tubes on a cold block. The tubes were centrifuged at 14,000 rpm
(18,188 rcf) at 4°C for 5 min to pellet any particles remaining with the eluted DNA. The supernatant was
carefully removed and transferred to a new 1.5-mL tube using a new tip for each tube. The To and 7/
DNA extracts were stored at 4°C until RV-PCR analysis or at -20°C if the analysis could not be
performed within 24 h. The work instruction for DNA purification is Appendix K.
2.6.5.3 Btk T1B2 DNA Preparation
Genomic DNA of Btk T1B2 was extracted for use as a positive control for RV-PCR based analysis. The
Btk T1B2 vegetative cell culture that DNA was extracted from originated from the spore stock used for
spike/recovery tests. DNeasy Blood & Tissue Kit (Part No. 69504, Qiagen, Germantown, MD) was used
following the manufacturer provided Gram-positive bacteria protocol to extract Btk DNA. The resulting
DNA was quantified by Quant-iT™ PicoGreen™ dsDNA Assay Kit (Invitrogen, Waltham, MA, Cat.
PI 1496). The purified DNA was assigned a unique lot number, dispensed as multiple aliquots, stored
frozen at below -20°C, and used as needed as the positive control for PCR analysis.
2.6.5.4 Real-Time PCR Assay
The specific tag 2 primer sequences from Buckley et al. (2012) were paired with a TaqMan probe
designed using the PrimerQuest Tool (Integrated DNA Technologies, Coralville, IA). When comparing
amplification of Btk T1B2 control DNA using the specific tag 2 primers described in Buckley et al.
(2012)—coupled with SYBR Green chemistry with the TaqMan PCR assay combining the specific tag 2
primers and the TaqMan probe designed using the PrimerQuest Tool—the sensitivity was similar;
however, the SYBR Green assay had amplification in no template control wells, therefore analysis was
performed using the TaqMan assay (Table 4).
36
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Table 4. Btk T1B2 TaqMan PCR Assay Primers and Probe Sequences.
Btk T1B2 Oligo
Sequence
Length
Tm(°C)
GC (%)
Forward Primer
GGT ACA AGC AAC GAT CTC CAG AAT
24
64.5
45.8
Probe
6FAM-CGC CGA CGC TTT ACA TAC TAT GAG AGG-
MGBNFQ
27
67.5
51.9
Reverse Primer
TGA AGG TTA ATT AGC GCA TTT GAA
24
62.0
33.3
Amplicon
GGT ACA AGC AAC GAT CTC CAG AAT TCG CCG
ACG CTT TAC ATA CTA TGA GAG GCA CCT TAA
GGT GTC TTT TCT TTT TGG ACA TTA CAT CCA TTT
TGT TTT TCC ACC TTA TTT CAA ATG CGC TAA TTA
ACC TTC A
133
71.3
39.1
The PCR assay Master Mix was prepared using the recipe provided in the formulations and recipes
appendix (Appendix G, Table 2). Each sample DNA extract was assayed in triplicate reactions. Controls
consisted of four positive control wells containing 50 picograms (pg) of DNA extracted from Btk T1B2
and four no template controls (NTCs) were also included with each assay. An Applied Biosystems 7500
Fast Real-Time PCR Instrument (Waltham, MA) was used for PCR assay development and testing.
Thermocycler conditions with a fast ramp rate were:
Stage 1: 1 cycle at 50°C for 2 min
Stage 2: 1 cycle at 95°C for 2 min
Stage 3: 45 cycles at 95°C for 3 sec followed by 60°C for 30 sec
The work instruction for real-time PCR is Appendix L.
2.7 Data Reduction and Analysis
2.7.1 Percent Recovery of Presumptive Btk T1B2 Colonies
The percent recovery (Erecovery) of Btk T1B2 spores from each spiked sample was calculated by dividing
the number of presumptive Btk T1B2 CFU recovered (Nrecover) from the sample by the actual number of
Btk T1B2 spores spiked (Nspike), as determined by stock suspension titer for each test, then multiplied by
100. Nrecover is a product of the presumptive Btk T1B2 spore concentration (Crecover) (CFU/mL) and the
total volume of extract used to recover the spores (V'extract) (mL). Mathematically, the percent recovery is
expressed as follows:
r * V
j-i sn/\ recover v extract
^recovery (%) = T, * 100%
^ spike
Further, the number of presumptive Btk T1B2 spores present in the volume of recovered suspension
plated onto spread plates or via MicroFunnel filter membrane was divided by the suspension volume
analyzed to yield a presumptive Btk T1B2 spore concentration (Crecover) (CFU/mL). The recovered
suspension volume (Vextract) was used to determine Btk T1B2 CFU recovered from the sample. The
percent recovery was calculated for all volumes plated. The reported percent recovery was determined
using the below rules:
37
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1) Report the percent recovery from the aliquot that has between 20 to 80 CFU on MicroFunnel
Filter membranes.
2) Report the higher-volume aliquot percent recovery if the CFU counted from both aliquots is
less than 20.
3) Report the higher-volume aliquot percent recovery if the CFU counted from both aliquots is
between 20 to 80.
4) Report the lower-volume aliquot percent recovery if the background microbial flora on the
high-volume aliquot produces numerous colonies or a lawn of growth, thus complicating the
identification of Btk T1B2 colonies.
5) Report the percent recovery from the spread plate that has between 25 and 250 CFU. Note,
since spike levels were at or near the method detection limit for samples processed in this study,
10"1 and 10"2 dilutions were not spread plated as described in EPA Protocol (EPA, 2017).
The number of CFU is reported as presumptive BtkTYBl colonies. PCR analysis of presumptive
colonies is required to positively confirm the presence of Btk T1B2. To perform this task, a portion of
the presumptive colony was collected into 100 |iL of PCR-grade water in microcentrifuge tubes. The
colony suspension was then heated for 5 min on a heat block at 95°C. The lysate was cooled and then
centrifuged at 14,000 rpm (18,188 rcf) for 2 min, and the supernatant was analyzed using the real-time
PCR assay targeting the Btk T1B2 target. The work instruction for colony PCR is located in
Appendix M.
2.7.2 RV-PCR Method
The Ct values for the To and 7/timepoints as well as the delta Ct value (ACt) were reported. The ACt is
generated by subtracting the average Ct (from triplicate reactions) value generated by the 7/aliquot from
the average Ct (triplicate reactions) value generated by the To aliquot. A ACt > 9 value indicates that
viable Btk T1B2 spores were detected in the sample if the following criterion was met:
The ACt must be greater than or equal to 9 for the Btk T1B2 target: (ACt = Ct (To) - Ct (Tfi„ai) > 9)
Additional criteria exist for the positive confirmation of a sample if analyzing samples obtained from an
actual incident, but for this study the above criterion was used.
2.7.3 Presentation of Results
The method employed to recover Btk T1B2 spores was consistent with current EPA methods, as
described in Section 2.6.4. In the instance of an actual biological agent release, the entire suspension of
spores recovered from samples would be analyzed using either a culture method or a RV-PCR method.
In the study performed and reported here, however, the recovered suspension was split as described in
Sections 2.6.4 and 2.6.5, so that approximately half of the suspension was used for culture analysis and
the other half for RV-PCR analysis. Consequently, neither the culture nor the RV-PCR method
processed the total quantity of spores available in the suspension for analysis. Rather, each split sample
suspension had a maximum of nominally half the actual spiked spore quantity available for their
respective analyses. Therefore, in the presentation of results in tables and figures, unless explicitly noted
otherwise, column headers or axis labels denote the nominal maximum number of recovered spores
available in the sample for its respective analysis, which was half of the target spore load.
38
-------
3.0 RESULTS AND DISCUSSION
As described in the previous section, all results presented in plots have an x-axis title and labels of 0,
150, and 1,500 CFU representing the nominal number of spores available for analysis. Similarly, the
summary results in the tables contain the same nominal quantity of spores available, and the determined
quantity of spores applied to the surface sampler substrate being assessed. This convention of presenting
the results was considered the most accurate and consistent representation and allowed for the most
unambiguous discussion and interpretation of results across all sample types and analytical methods,
recognizing that the samples were originally spiked with target quantities of Btk T1B2 spores of 0, 300,
and 3,000 CFU, but recovered suspensions from processed samples were split into approximately equal
volume for the two analyses.
Note that the spores available for analysis represent the maximum number of spores available (assumes
100% spore recovery from the filter and no physical losses associated with processing of samples); it is
not an absolute indication of the analytical method limit of identification. Rather, it is a measure of the
method's end-to-end performance to detect Btk T1B2.
3.1 Sponge Stick Sample Analysis Results
3.1.1 Sponge Stick Sample Culture Analysis
A summary of the average and standard deviation values of the measured recovery of presumptive
Btk T1B2 spores from sponge sticks that were used to wipe maritime surfaces and then spiked
(inoculated) in the laboratory with a target of 300 or 3,000 CFU is presented in Table 5. The nominal
quantity of spores available for analysis of 150 and 1,500 CFU represents one-half the target spore load
applied to the surfaces and the determined number of spores available represents one-half the measured
number of Btk T1B2 spores based on the Btk suspension titer and volume applied on the day of spiking.
The spore recovery percentage of presumptive Btk colonies recovered as determined by culture analysis
using TSA plates are plotted in Figure 34 through Figure 36. The quantity of presumptive Btk T1B2
colonies for each sponge stick sample used in the percent recovery calculations is reported in Table 5.
39
-------
Table 5. Presumptive Btk T1B2 Spores Recovered from Laboratory-Spiked Sponge Sticks that Previously
Sampled Different Maritime Surfaces.
Surface Type
(Sample ID)
Sample
Replicates
Spores Available for Analysis
(CFU)
Spore
Recovery
(CFU)
(X ± CT)l :l
Spore Recovery
(%)
Nominal1"
Determined"51
(X±c)
(X ± CT),dl
Small Boat Aluminum
(SBMGAL-1)
3
0
0
12 ± 1.0
N/A
3
150
87 ± 34
33 ± 8.6
40 ± 5.3
3
1,500
870 ± 340
440±110
52 ± 7.3
Small Boat Aluminum
(SBMGAL-2)
3
0
0
17 ± 13
N/A
3
150
87 ± 34
37 ± 21
50 ±42
3
1,500
870 ± 340
410 ± 79
50 ± 8.9
Nonskid Tread
(NSKID-1)
3
0
0
18 ± 6.8
N/A
3
150
87 ± 34
94 ± 68
99 ±40
3
1,500
870 ± 340
330 ± 40
41 ± 12
Nonskid Tread
(NSKID-2)
4
0
0
130±180
N/A
4
150
130 ± 55
100 ± 92
120± 150
4
1,500
1,300 ± 550
500±170
39 ± 7.8
On-Board Touchscreen
(TCHSCRN-1)
3
0
0
5.4 ± 5.0
N/A
3
150
140 ± 55
280± 310
170± 170
3
1,500
1,400 ± 550
390 ± 360
32 ±27
3
0
0
0
N/A
On-Board Touchscreen
(TCHSCRN-2)
3
150
150 ±130
39 ± 42
25 ± 9.0
3
1,500
1,400 ± 550
610± 930
26 ±27
(a) Nominally one-half of the target spore load on the surface and assuming 100 % recovery of spores.
(b) Based on the spiking suspension titer measured per trial, 100 % recovery, and one-half of extract used for culture analysis.
(c) Presumptive Btk T1B2 colonies based on morphology, and one-half of extract used for culture analysis.
(d) Calculated using the actual spore loading applied during spiking and total presumptive Btk T1B2 spores recovered on each
sponge stick sample.
40
-------
Small Boat Marine Grade Aluminum {Sponge Stick Wipe)
100
90
80
70
\P
O
Q.
to
•2 60
> 50
Q.
E
3
cn
20
10
~ SBMGAL 1 ~ SBMGAL 2
Nominal BtkTlB2 Spore Available for Analysis (CFU)
Figure 34. Presumptive Btk T1B2 Spore Recovery from Sponge Sticks Spiked with Btk T1B2 Spores After
Having Sampled Small Boat Aluminum Surfaces
Average ± One Standard Deviation of N = 3 Replicates. Sponge Stick Spiked with Nominal 300 or 3,000 CFU
of Btk T1B2 Spores.
Nonskid (Sponge Stick Wipe)
140
120
80
INSKID1
~ NSKID2
a
E
3
ul
OJ
£
M—
O
>¦
-------
For nonskid 2, 150 CFU nominal spore load percent recovery values are not plotted in Figure 35 because
percent recovery of 120 ±150 % is not an accurate reflection of target spores recovered. The percent
recovery values, particularly for low spore level samples, are inflated from the presence of background
microorganisms with Btk morphology.
Small Boat Touchscreen (Sponge Stick Wipe)
100
\P
O
Q.
to
90
80
70
60
> 50
4-»
Q.
E
3
l/l
w
£
40
30
20
10
~ TCHSCRN 1
~ TCHSCRN 2
TCHSCRN 1
170 ±170
% recovery
^ %
Nominal Btk T1B2 Spore Available for Analysis (CFU)
Figure 36. Presumptive Btk T1B2 Spore Recovery (%) from Sponge Sticks Spiked with Btk T1B2 Spores
After Having Sampled On-Board Touchscreen Surfaces
Average ± One Standard Deviation ofN = 3 Replicates. Sponge Stick Spiked with Nominal 300 or 3,000 CFU
of Btk T1B2 Spores.
For touch screen, 150 CFU nominal spore load percent recovery recovery values are not plotted in
Figure 36 because percent recovery of 170 ± 170 % is not an accurate reflection of target spores
recovered. The percent recovery values, particularly for low spore level samples, are inflated from the
presence of background microorganisms with Btk morphology.
The images in Figure 37 through Figure 39 show examples of culture plates with 2-mL or 8-mL
volumes on TSA at all three spike levels: 0 CFU spike, 300 CFU spike, and 3,000 CFU spike.
Presumptive colonies were present on zero spike samples, although the number of presumptive colonies
increased as the spike level was increased.
42
-------
'¦I
r
>
%
-
\ ™ -A. 7k.- % ¦¦ t
y-^%XX? i
1
. ¦• :• %.•••
IE:
^ • ,
II
Figure 37. Culture Images of Spore Recovery from Small Boat 2 Marine Grade Aluminum Surface
Sampled Using Sponge Stick and Plated on TSA.
Presumptive Btk colonies were observed in zero spore spike samples (Average <1 CFU/mL).
Image Descriptions: (A) Zero Spore Spike, 2 mL; (B) Zero Spore Spike, 8 mL; (C) 300 Spore Spike, 2 mL; (D)
300 Spore Spike, 8 mL; (E) 3,000 Spore Spike, 2 mL; (F) 3,000 Spore Spike, 8 mL.
43
-------
Figure 38. Culture Images of Spore Recovery from Nonskid Boat 1 Surface Sampled Using Sponge Stick
and Plated on TSA.
Presumptive Btk colonies were observed in zero spore spike samples (Average < 3 CFU/mL when plating 2 mL
volume for Boat 1, < 15 CFU/mL when plating 2 mL volume for Boat 2).
Image Descriptions: (A) Zero Spore Spike, 2 mL; (B) Zero Spore Spike, 8 mL; (C) 300 Spore Spike, 2 mL; (D)
300 Spore Spike, 8 mL; (E) 3,000 Spore Spike, 2 mL; (F) 3,000 Spore Spike, 8 mL
44
-------
Figure 39. Culture Images of Spore Recovery from Touch Screen Boat 2 Surface Sampled Using Sponge
Stick and Plated on TSA.
Presumptive Btk colonies were not observed in zero spore spike samples.
Image Descriptions: (A) Zero Spore Spike, 2 mL; (B) Zero Spore Spike, 8 mL; (C) 300 Spore Spike, 2 mL; (D)
300 Spore Spike, 8 mL; (E) 3,000 Spore Spike, 2 mL; (F) 3,000 Spore Spike, 8 mL
~ \
3.1.2 Colony Confirmation by PCR
Based on the colony morphology, presumptive Btk T1B2 colonies were identified for all surfaces (52 of
57 samples) sampled using sponge sticks with the following exceptions: 1) on-board touchscreen from
Boat 1 (one replicate); 2) on-board touchscreen from Boat 2 (three replicates); and 3) nonskid tread from
Boat 2 (one replicate). The presence of presumptive Btk T1B2 morphology did not mean that the Btk
T1B2 spores were recovered from the surfaces. Nineteen (19) samples with inert and biological deposits
were 0-spike samples, meaning they were not inoculated with Blk T i B2 spores, yet presumptive Btk
colonies were still isolated from the spore recovery. Colonies with morphology indistinguishable from
Btk T1B2 were present on the culture plates, as indicated by a negative PCR result for presumptive Btk
T1B2 colonies. The confirmation of target Btk T1B2 was assessed by colony PCR from the initial
45
-------
culture plates, colony PCR from BHIB enrichment culture from the sponge stick samples, or PCR of an
aliquot of the BHIB enrichment culture from the sponge stick samples. Results from PCR confirmatory
testing are shown in Table 6. A total of six sample replicates were false positive samples; however, the
colony PCR Ct values were 37, 37, 35, 32, and 38. By comparison, the eight spiked field blank or
laboratory spike samples had a Ct value of 21.2 ± 0.6. The two other false positive samples had Ct
values of 37 and 39 for the BHIB enrichment culture from sponge samples. The cause of these high Ct
value false positive samples is either low level contamination between samples or potentially
nonspecific PCR amplification.
46
-------
Table 6. Summary of the Accuracy of Identification of Presumptive Btk T1B2 Colonies by PCR Confirmation from Spiked Sponge Sticks Used to
Sample Different Maritime Surfaces.
Surface Type
(Sample ID)
Nominal Spore
Load
(CFU)
Culture Replicates
Presumptive
Positive''"
Colonies from
Initial Culture
Plates PCR-
Screened
(# PCR+)1151
Colony PCR Ct
(X±CT)l:l
Colonies from
BHIB Streak Plates
PCR-Screened
(# PCR +)ldl
BHIB PCR-
Screened
(# PCR+)101
BHIB PCR Ct
(X±CT)'"
Small Boat
Aluminum
(SBMGAL-1)
0
3 of 3
9(0)
N/A
0
2(1)
37.2
150
3 of 3
7(2)
22.2 ± 0.4
1 (0)
0
N/A
1,500
3 of 3
12(3)
22.8 ± 0.8
0
0
N/A
Small Boat
Aluminum
(SBMGAL-2)
0
3 of 3
4(1)
37.2
0
0
N/A
150
3 of 3
12(3)
22.7 ± 1.7
0
0
N/A
1,500
3 of 3
12(3)
22.5 ± 1.0
0
0
N/A
Nonskid Tread
(NSKID-1)
0
3 of 3
6(2)
36.0 ± 1.0
0
2(0)
N/A
150
3 of 3
12 (2)
22.9 ± 1.0
0
1 (0)
N/A
1,500
3 of 3
12(3)
23.3 ± 1.4
0
0
N/A
Nonskid Tread
(NSKID-2)
0
3 of 4
21 (1)
32.1
0
1 (0)
N/A
150
4 of 4
27(3)
25.7 ± 4.6
1 (0)
1 (0)
N/A
1,500
4 of 4
31 (4)
22.6 ± 1.3
1 (0)
0
N/A
On-Board
Touchscreen
(TCHSCRN-1)
0
2 of 3
11 (1)
38.1
0
1 (1)
39.1
150
3 of 3
21 (3)
23.6 ± 3.2
0
0
N/A
1,500
3 of 3
21 (3)
20.9 ± 0.6
0
0
N/A
On-Board
Touchscreen
(TCHSCRN-2)
0
0 of 3
0
N/A
0
0
N/A
150
3 of 3
28 (3)
20.8 ± 3.1
0
0
N/A
1,500
3 of 3
30 (3)
20.5 ± 0.8
0
0
N/A
(a) Presumptive Btk T1B2 was present on initial culture plates.
(b) Number of colonies PCR-screened from initial plating, with number of PCR positive replicates in parentheses.
(c) Colony PCR Ct values for positive samples (Ct value of < 40).
(d) Number of colonies PCR-screened from BHIB streak plates, with number of PCR positive replicates in parentheses.
(e) Number of samples with PCR screening of BHIB enrichment culture, with number of PCR positive replicates in parentheses.
(f) BHIB enrichment culture PCR Ct values for positive samples (Ct value of < 40).
47
-------
3.1.3 Sponge Stick Sample RV-PCR Analyses
A summary of the average and standard deviation of the RV-PCR ACt values for the detection of Btk
T1B2 spores recovered from sponge sticks that were used to wipe maritime surfaces and then spiked
(inoculated) with Btk T1B2 spores in the laboratory with a target of 300 or 3,000 CFU is presented in
Table 7. The nominal quantity of spores available for analysis represents one-half the target spore load
applied to the surfaces, and the determined number of spores available represents one-half the measured
number of Btk T1B2 spores applied based on the Btk suspension titer and volume applied on the day of
spiking. Sample replicates with an RV-PCR ACt value > 9 are RV-PCR positive, indicating that viable
Btk T1B2 spores were recovered. The RV-PCR ACt results are plotted in Figure 40 through Figure 42.
The plots all depict the ACt threshold value of 9 as a dashed line with an area shaded in red representing
a negative detection result, and an area of green representing a positive detection result.
Table 7. RV-PCR Analyses of Spiked Sponge Sticks that Were Used to Sample Different Maritime Surfaces
for Detection of Btk T1B2 Spores.
Surface Type
(Sample ID)
Number of
Replicates
Spores Available for
Analysis
(CFU)
ACt11:1 (X ± o)
RV-PCR
Replicates
Positive11"
Nominal'-1'
Determined"51
Small Boat Aluminum
(SBMGAL-1)
3
0
0
0.4 ±2.5
0
3
150
87 ± 34
16.9 ± 5.7
3
3
1,500
870 ± 340
21.1 ± 5.1
3
Small Boat Aluminum
(SBMGAL-2)
3
0
0
0± 0
0
3
150
87 ± 34
15.6 ± 2.2
3
3
1,500
870 ± 340
22.2 ± 0.9
3
Nonskid Tread
(NSKID-1)
3
0
0
1.4 ± 1.3
0
3
150
87 ± 34
15.3 ± 8.0
3
3
1,500
870 ± 340
14.9 ± 4.6
3
Nonskid Tread
(NSKID-2)
4
0
0
0.6 ±2.6
0
4
150
130 ± 55
11.3 ± 2.6
3
4
1,500
1,300 ± 550
17.2 ± 2.0
4
On-Board Touchscreen
(TCHSCRN-1)
3
0
0
4.6 ±4.1
0
3
150
140 ± 55
21.1 ± 3.2
3
3
1,500
1,400 ± 550
23.1 ± 2.4
3
On-Board Touchscreen
(TCHSCRN-2)
3
0
0
0± 0
0
3
150
150±130
20.1 ± 3.5
3
3
1,500
1,400 ± 550
23.0 ± 2.1
3
(a) Nominally one-half of the target spore load on the surface and assuming 100% recovery of spores.
(b) Based on the spiking suspension titer measured per trial, 100 % recovery, and one-half of extract used for culture analysis.
(c) PCR assay for T1B2 Barcode Gene Target.
(d) Number of replicates with a RV-PCR ACt value > 9.
48
-------
35
Small Boat Marine Grade Aluminum (Sponge Stick Wipe)
30
~ SBMGAL1 I 1 SRMCnAI 7
¦Threshold
25 ¦
4 20 ¦
15
10
5 ¦
Nominal BtkTlB2 Spore Available for Analysis (CPU)
\
Figure 40. RV-PCR Analysis of Btk T1B2 Spores Recovered from Sponge Sticks Spiked with Btk T1B2
Spores After Having Sampled Nonskid Tread Surfaces.
Average ± One Standard Deviation ofN>3 Replicates. Sponge Stick Spiked with Nominal 300 or 3,000 CFU
of Btk T1B2 Spores. Positive Result Equals ACt>9.
35
30 -
25 -
4 20
5
10 ¦
Nonskid (Sponge Stick Wipe)
-t-n _L
1 iNKKini
~ NSKID 2
¦ Threshold
JA
so
Nominal BtkTlB2 Spore Available for Analysis (CFU)
Figure 41. RV-PCR Analysis of Btk T1B2 Spores Recovered from Sponge Sticks Spiked with Btk T1B2
Spores After Having Sampled Nonskid Tread Surfaces.
Average ± One Standard Deviation ofN>3 Replicates. Sponge Stick Spiked with Nominal 300 or 3,000 CFU
of Btk T1B2 Spores. Positive Result Equals ACt>9.
49
-------
35
Touch Screen (Sponge Stick Wipe)
30
I 1TCHSCRN 1 I 1TCHSCRN 2
¦ Threshold
25 ¦
4 20 ¦
5 15
<
10
5 ¦
\
Nominal BtkTlB2 Spore Available for Analysis (CPU)
Figure 42. RV-PCR Analysis of Btk T1B2 Spores Recovered from Sponge Sticks Spiked with Btk T1B2
Spores After Having Sampled On-Board Touchscreen Surfaces; Positive Response Equals ACt > 9.
Average ± One Standard Deviation ofN = 3 Replicates. Sponge Stick Spiked with Nominal 300 or 3,000 CFU
of Btk T1B2 Spores. Positive Result Equals ACt >9.
3.1.4 Analytical Method Comparison of Sponge Stick Samples
Culture analysis identified presumptive Btk T1B2 colonies for most samples, even zero spike samples,
indicating that background microbial flora included wild-type Btk or another organism that had a
morphology indistinguishable from Btk T1B2 on TSA plates. Three exceptions occurred where
presumptive Btk T1B2 colonies were not present: 1) on-board touchscreen from Boat 1 (one replicate);
2) on-board touchscreen from Boat 2 (three replicates); and 3) nonskid tread from Boat 2 (one replicate).
Colony PCR from initial culture plates; colony PCR of colonies isolated from BHIB enrichment culture
of the sponge stick samples; and/or PCR analysis of the BHIB enrichment culture was therefore required
to confirm or refute the presence of Btk T1B2. To compare the two methods, culture with PCR
confirmation and RV-PCR results were assessed to determine which method may be more likely to
detect viable spores that have been spiked onto sponge sticks that contain outdoor interferents.
PCR screening of presumptive Btk T1B2 colonies was negative in some cases, indicating that
background microbial flora with colony morphology indistinguishable from the morphology of Btk
T1B2 were present on TSA culture plates; and hence, present in the samples collected in the field. It is
possible that wild-type/naturally occurring Btk and their presence led to an inflation in presumptive
spore recovery values by the culture method. Presumptive culture identification by colony morphology,
colony identification confirmed by PCR, and RV-PCR results are shown in Table 8.
For culture analysis of sponge stick samples, 48 of 57 (84 %) that had been used to collect inert and
biological deposits were determined to be true positive or true negative. A true positive is defined as a
sample spiked with Btk T1B2 spores that was confirmed positive by PCR. A true negative is defined as a
sample that was not spiked with Btk T1B2 spores and tested negative in PCR confirmatory screening.
Six samples were false positive and three samples were false negative using the culture method. For the
50
-------
six false positive samples, colony PCR Ct values were < 40, each measuring between 32 to 38, or a
BHIB culture PCR Ct value of 37. For comparison, colony PCR Ct values of Btk T1B2 isolated from
field blanks or laboratory blanks (new sponge spiked with Btk T1B2 spores) was 21.2 ± 0.6. For the
three false negative samples, a minimum of 26 presumptive Btk colonies were available for colony PCR;
however, only one colony from each of these samples was screened. If more colonies were screened for
these samples, it is possible the sample would have been confirmed positive. The EPA Protocol specifies
that one to three colonies from MicroFunnel filters and a minimum of three colonies from spread plates
should be PCR-screened for target confirmation (EPA, 2017). All field blank and laboratory blank
controls were true negatives (19 of 19).
For RV-PCR analysis of sponge stick samples, 56 of 57 (98 %) that had been used to collect inert and
biological deposits were determined to be true positives or true negatives. A true positive is defined as a
sample spiked with Btk T1B2 spores that had a ACt of > 9. A true negative is defined as a sample that
was not spiked with Btk T1B2 spores and had a ACt of < 9. The one false negative sample had been
spiked with 300 CFU target spore load and had a ACt of 8.7. One laboratory blank sample was a false
positive with a ACt of 9.8.
51
-------
Table 8. Analytical Method Comparison Displaying Culture Presumptive, Culture ID with PCR
Confirmation, and RV-PCR Replicates Positively Identified (N = 3) for Surfaces Sampled with Sponge
Sticks.
Sample
Surface
Actual Spike
Level (CFU)
Presumptive
Culture
Result
Culture PCR
Confirmation
Culture %
Recovery
RV-PCR
Result
RV-PCR ACt
Marine
Grade
Aluminum,
Boat 1
0
Positive
Negative
N/A
Negative
-1.92
0
Positive
Positive
N/A
Negative
0
0
Positive
Negative
N/A
Negative
3.1
250
Positive
Negative
33.8
Positive
22.8
150
Positive
Positive
44
Positive
16.5
120
Positive
Positive
41.7
Positive
11.5
2,500
Positive
Positive
45.1
Positive
26.9
1,500
Positive
Positive
52
Positive
18.9
1,200
Positive
Positive
59.6
Positive
17.5
Marine
Grade
Aluminum,
Boat 2
0
Positive
Negative
N/A
Negative
0
0
Positive
Negative
N/A
Negative
0
0
Positive
Positive
N/A
Negative
0
250
Positive
Positive
27.6
Positive
14.6
150
Positive
Positive
24
Positive
14.1
120
Positive
Positive
99
Positive
18.1
2,500
Positive
Positive
39.5
Positive
21.3
1,500
Positive
Positive
53.6
Positive
23.1
1,200
Positive
Positive
56
Positive
22.3
Touch
Screen, Boat
1
0
Positive
Positive
N/A
Negative
0
0
Positive
Negative
N/A
Negative
7.9
0
Negative
Negative
N/A
Negative
5.8
150
Positive
Positive
42.2
Positive
24.8
340
Positive
Positive
111.6
Positive
19.9
340
Positive
Positive
367.1
Positive
18.7
1,500
Positive
Positive
51.2
Positive
23.9
3,400
Positive
Positive
44.1
Positive
20.4
3,400
Positive
Positive
1.43
Positive
25.1
Touch
Screen, Boat
2
0
Negative
Negative
N/A
Negative
0
0
Negative
Negative
N/A
Negative
0
0
Negative
Negative
N/A
Negative
0
110
Positive
Positive
30.6
Positive
22.2
180(a)
Positive
Positive
86.7
Positive
21.9
600
Positive
Positive
29.2
Positive
16
1,100
Positive
Positive
18.6
Positive
21.2
1,800
Positive
Positive
27.6
Positive
22.6
6,000
Positive
Positive
56.1
Positive
25.3
(a'Stock enumeration plate outside 25 - 250 CFU range.
52
-------
Table 8. Analytical Method Comparison Displaying Culture Presumptive, Culture ID with PCR
Confirmation, and RV-PCR Replicates Positively Identified (N = 3) for Surfaces Sampled with Sponge
Sticks (Cont.)
Sample
Surface
Actual Spike
Level (CFU)
Presumptive
Culture
Result
Culture PCR
Confirmation
Culture %
Recovery
RV-PCR
Result
RV-PCR ACt
0
Positive
Negative
N/A
Negative
0
0
Positive
Positive
N/A
Negative
1.7
0
Positive
Positive
N/A
Negative
2.4
Nonskid,
Boat 1
250
Positive
Negative
134.4
Positive
9.3
150
Positive
Positive
107.3
Positive
12.3
120
Positive
Positive
55.8
Positive
24.42
2,500
Positive
Positive
27.3
Positive
12
1,500
Positive
Positive
48.8
Positive
20.2
1,200
Positive
Positive
47.9
Positive
12.4
0
Positive
Negative
N/A
Negative
-2.5
0
Positive
Positive
N/A
Negative
3.8
0
Positive
Negative
N/A
Negative
1.1
0
Negative
Negative
N/A
Negative
0
250
Positive
Negative
135.2
Positive
9.5
Nonskid,
120
Positive
Positive
322.9
Positive
13.1
Boat 2
340
Positive
Positive
17.2
Negative
8.7
360
Positive
Positive
9.6
Positive
14
2,500
Positive
Positive
38.7
Positive
18.7
1,200
Positive
Positive
48
Positive
15.9
3,400
Positive
Positive
41.3
Positive
19
3,600
Positive
Positive
29.3
Positive
15
Results for all samples processed, including positive and negative controls, are shown in Table 9.
Culture method correctly determined 67 of 76 samples (88%) with six false positive samples; however,
all six of these samples had colony PCR Ct values between 32 to 38 or BHIB enrichment culture PCR of
37. Colony PCR of Btk T1B2 from the eight spiked field blank or laboratory spike (new sponge stick
spiked with Btk T1B2 spores) controls had a Ct value of 21.2 ± 0.6, suggesting that Ct values between
32 to 38 might be caused by low level contamination or potentially nonspecific PCR amplification. The
RV-PCR method correctly determined 74 of 76 samples (97%), with only one false positive laboratory
blank with a ACt value of 9.78 and one false negative sample from a 300-target spore load sample that
had a ACt value of 8.7.
For culture analysis, the marine grade aluminum surfaces sampled with sponge sticks had two false
positive samples with Ct values of 37 when PCR analysis was performed on colonies and BHIB
enrichment culture of the processed sponge stick samples and one false negative sample. The false
negative sample had 33 presumptive colonies and only one was PCR-screened, so it is possible that the
sample would have been confirmed positive if more colonies were screened. RV-PCR results matched
the expected outcomes in all cases.
53
-------
For culture analysis, the touch screen surfaces sampled with sponge sticks had one false positive sample
with a Ct value of 39 when PCR analysis was performed on BHIB enrichment culture of the processed
sponge stick samples. The same sample replicate had a Ct value of 38 for colony PCR. For all eight of
the spiked field blank or laboratory spike (new sponge stick spiked with Btk T1B2) controls, colony
PCR of Btk T1B2 colonies resulted in a Ct value of 21.2 ± 0.6. Therefore, a Ct value of 38 for a colony
PCR reaction may have been caused by low level contamination or potentially nonspecific PCR
amplification. RV-PCR results matched the expected outcome in all cases.
For culture analysis, the nonskid tread surfaces sampled with sponge sticks had three false positive
samples. The first had a colony PCR Ct value of 37, and the BHIB enrichment of the extracted sponge
stick was PCR negative. The second had a colony PCR Ct value of 35 with four presumptive Btk
colonies pooled, and the third had a colony PCR Ct value of 32 for a pool of 10 presumptive Btk
colonies. Colony PCR of the eight spiked field blank or laboratory spike samples had a Ct value of 21.2
± 0.6, therefore Ct values between 32 to 37 may have been caused by low level contamination or
potentially nonspecific PCR amplification. There were two false negative samples for culture analysis,
although only one presumptive colony each was PCR-screened with 26 or 28 well-isolated colonies
available for PCR screen. However, the BHIB enrichment cultures of the processed sponge stick
samples were also negative for both replicates.
54
-------
Table 9. Analytical Method Comparison Displaying Culture ID with PCR Confirmation and RV-PCR for Surfaces Sampled with Sponge Sticks.
Culture Method (PCR Confirmation)
RV-PCR Method
Surface Type
True Positive or
False Positive
False Negative
True Positive or
False Positive
False Negative
True Negative
Sample
Sample
True Negative
Sample
Sample
Small Boat Aluminum 1
7
1
-| (a)
9
0
0
Small Boat Aluminum 2
8
1
0
9
0
0
Nonskid 1
6
2
-| (b)
9
0
0
Nonskid 2
10
1
-| (b)
11
0
-| (d)
Touchscreen 1
8
1
0
9
0
0
Touchscreen 2
9
0
0
9
0
0
Controls
19
0
0
18
-| (C)
0
Totals
67
6
3
74
1
1
Positive PCR threshold of 40 for colony PCR and BHIB enrichment culture PCR. The definition for Positive Ct threshold per the EPA Protocol (EPA, 2017) is < 40.
(a)Only PCR-screened 1 colony from membrane plate, 33 were presumptive.
(b) Only PCR-screened 1 colony from membrane plate, 28 and 26 were presumptive on 2-mL plate.
(c) ACt value of 9.78 of laboratory blank, may be caused by cross-contamination that occurred during DNA extraction or nonspecific amplification.
(d) ACt value of 8.67.
55
-------
3.1.5 Analysis of Controls
For sponge sticks, there were a total of eight zero spike sponge sticks that served as reagent control
laboratory blanks, three zero spike sponge sticks that were opened in the field that served as field blanks,
three (3) 300-CFU spiked field blanks, three (3) 3,000-CFU spiked field blanks, one (1) 300-CFU spiked
sponge stick, and one (1) 3,000-CFU spiked sponge stick that was handled in the laboratory only
(laboratory spike).
All controls performed as expected except for one zero spore spike sponge stick that was RV-PCR
positive with a ACt of 9.8 (laboratory blank). For all eight of the spiked field blank or laboratory spike
(new sponge stick spiked with Btk T1B2) samples where colony PCR was performed, the average Ct
value was 21.2 ± 0.6.
3.2 Vacuum Filter Cassette Sample Analysis Results
3.2.1 Vacuum Filter Cassette Sample Culture Analysis
A summary of the average and standard deviation values of the measured recovery of presumptive Btk
T1B2 spores from VFCs that were used to sample maritime surfaces and then spiked (inoculated) in the
laboratory with a target of 300 or 3,000 CFU is presented in Table 10. The nominal quantity of spores
available for analysis of 150 and 1,500 CFU represents one-half the target spore load applied to the
surfaces, and the determined number of spores available represents one-half the measured number of Btk
T1B2 spores applied based on the Btk suspension titer and volume applied on the day of spiking. The
spore recovery percentage of presumptive Btk colonies recovered was determined by culture analysis
using TSA plates. The spore recovery percentages of presumptive Btk colonies recovered as determined
by culture analysis using TSA plates is plotted in Figure 43 and Figure 44.
56
-------
Table 10. Presumptive Btk T1B2 Spores Recovered and Associated Spore Recovery (%) from Laboratory-
Spiked Vacuum Filter Cassettes that had Previously Sampled Different Maritime Surfaces.
Surface Type
(Sample ID)
Sample
Replicates
Spores Available for Analysis
(CFU)
Spore
Recovery
(CFU)
(X ± o)'c'
Spore Recovery
(%)
Nominal'3'
Determined'13'
(X±o)
(X ± o)'d»
Nonskid Tread
(NSKID-1)
4
0
0
140 ± 94
N/A
4
150
110 ± 72
180 ± 56(e)
200 ± 92
4
1,500
1,100 ± 720
330±190
47 ± 52
Nonskid Tread
(NSKID-2)
4
0
0
11 ± 19
N/A
4
150
110 ± 72
46 ± 42
71 ± 97
4
1,500
1,100 ± 720
150 ± 70
16 ± 4.7
Concrete Pier
(CONPIER-1)
4
0
0
60 ± 39
N/A
4
150
110 ± 72
110 ±110
100 ± 88
4
1,500
1,100 ± 720
220±120
22 ± 8.1
Concrete Pier
(CONPIER-2)
4
0
0
75 ± 62
N/A
4
150
110 ± 72
54 ± 31
62 ±47
4
1,500
1,100 ± 720
120 ± 76
11 ± 3.2
(E" Nominally one-half of the target spore load on the surface and assuming 100% recovery of spores.
(bl Based on the spiking suspension titer measured per trial, 100 % recovery, and one-half of extract used for culture analysis.
(cl Presumptive Btk T1B2 colonies based on morphology, and one-half of extract used for culture analysis.
(dl Calculated using the actual spore loading applied during spiking and total presumptive Btk T1B2 spores recovered on each
VFC sample.
Nonskid (Vacuum Filter Cassette)
100
90
80
70
60
50
40
30
20
10
4
£L
E
o
2-
-------
For nonskid 1 and 2, 150 CFU nominal spore load percent recovery values were not plotted in Figure 43
because percent recovery of 200 ± 92 % and 71 ± 97 % is not an accurate reflection of target spores
recovered. The percent recovery values, particularly for 150 CFU nominal spore level samples are
inflated from the presence of background microorganisms with Btk morphology.
For Concrete Pier 1, 150 CFU nominal spore load percent recovery values were not plotted in Figure 44
because percent recovery of 100 ± 88 % is not an accurate reflection of target spores recovered. The
percent recovery values, particularly for 150 CFU nominal spore level samples, are inflated from the
presence of background microorganisms with Btk morphology.
120
E
t/1
CD
o 100
a.
80
60
Q.
E
ai 40
o
2"
w
>
o
20
Concrete Pier (Vacuum Filter Cassette)
CONPIER 1
100 ± 88 %
recovery
~ CONPIER 1
~ CONPIER 2
Nominal BtkTlB2 Spore Available for Analysis (CFU)
Figure 44. Presumptive Btk T1B2 Spore Recovery (%) from VFCs Spiked with Btk T1B2 Spores After
Having Sampled Concrete Pier Surfaces.
Average ± One Standard Deviation ofN = 4 Replicates. VFCs Spiked with Nominal 300 or 3,000 CFU of Btk
T1B2 Spores.
The images in Figure 45 and Figure 46 show examples of culture plates with 1 mL or 3 mL volumes on
TSA at all three spike levels: 0 CFU spike, 300 CFU spike, and 3,000 CFU spike. Presumptive colonies
were present on zero spike samples, although the number of presumptive colonies increased as spike
level increased.
58
-------
r--'v7 SHE
.
.v> -yy :¦
¦¦
;pi
' : ' . i
¦¦¦.¦IB
'
¦IB
r*V; ;
f ?
j
I Hi
LB
ISS&il
tmr M
-v.". •
WiPl
t
i -^h;, :
[¦' -
1 1
* J
'' '*- '
Ut; 4 vi
\ J0i 4 - ^
Figure 45. Culture Images of Spore Recovery from Nonskid 1 Surface Sampled Using Vacuum Filter
Cassette and Plated on TSA.
Presumptive Btk colonies were observed in zero spore spike samples (Average ~5S CFU/mL when plating 1 mL
volume for Boat 1, < 3 CFU/mL when plating 1 mL volume for Boat 2).
Image Descriptions: (A) Zero Spore Spike, 1 mL; (B) Zero Spore Spike, 3 mL; (C) 300 Spore Spike, 1 mL; (D)
300 Spore Spike, 3 mL; (E) 3,000 Spore Spike, 1 mL; (F) 3,000 Spore Spike, 3 mL
59
-------
Figure 46. Culture Images of Spore Recovery from Concrete Pier 1 Surface Sampled Using Vacuum Filter
Cassette and Plated on TSA.
Presumptive Btk colonies were observed in zero spore spike samples (Average ~10 CFU/mL when plating 1 ml.
volume for Pier 1,-13 CFU/mL when plating 1 mL volume for Pier 2).
Image Descriptions: (A) Zero Spore Spike, 1 mL; (B) Zero Spore Spike, 3 mL; (C) 300 Spore Spike, 1 mL; (D)
300 Spore Spike, 3 mL; (E) 3,000 Spore Spike, 1 mL; (F) 3,000 Spore Spike, 3 mL
The VFCs were not grossly loaded with particulates when viewed prior to spore recovery, as shown in
Figure 47.
60
-------
Figure 47. Vacuum Filter Cassette Following Sampling of Surfaces.
Image Descriptions: Top Row: VFCs collected from concrete pier; Bottom Row: VFCs collected from nonskid
tread.
3.2.2 Colony Confirmation by PCR
Based on the colony morphology, presumptive Btk T1B2 colonies were identified for all surfaces (46 of
48 samples) sampled using VFCs except for two replicates sampled from nonskid tread on Boat 2. The
presence of presumptive Btk T1B2 morphology did not mean that Btk T1B2 spores were recovered from
the surfaces. Sixteen (16) of these samples with inert and biological deposits were 0-spike samples,
meaning they were not inoculated with Btk T1B2 spores, yet presumptive Btk colonies were still isolated
from the spore recovery. Colonies with morphology indistinguishable from Btk T1B2 were present on
the culture plates, as indicated by a negative PCR result for presumptive Btk T1B2 colonies. The
confirmation of target Btk T1B2 was assessed by colony PCR from the initial culture plates, colony PCR
from BHIB enrichment of the vacuum membrane, or PCR of an aliquot of the BHIB enrichment culture
from the vacuum membrane sample. Results from PCR confirmatory testing are shown in
Table 11. There was a total of five sample replicates that were false positive samples; however, the
colony PCR Ct values were 32, 34 and 37. By comparison, the 11 spiked field blank or laboratory spike
samples had a Ct value of 21.2 ± 1.4. The other false positive samples had Ct values of 38.3 (laboratory
blank) and 40 for BHIB enrichment culture PCR. Ct values between 32 and 37 for colony PCR and Ct
values between 38 and 40 for BHIB enrichment culture PCR may have been caused by low level
contamination or potentially nonspecific PCR amplification.
61
-------
Table 11. Summary of the Accuracy of Identification of Presumptive Btk T1B2 Colonies by PCR Confirmation from Spiked VFCs Used to
Sample Different Maritime Surfaces.
Culture
Replicates
Presumptive
Positive'3'
Colonies from
Colonies from
Surface Type
(Sample ID)
Nominal Spore
Load
(CFU)
Initial Culture
Plates PCR-
Screened
(# PCR +)(b)
Colony PCR Ct
(X ± CT) ,cl
BHIB Streak
Plates PCR-
Screened
(# PCR+)(t"
BHIB PCR-
Screened
(# PCR +)
Nonskid Tread
(NSKID-1)
0
4 of 4
31 (0)
N/A
3(0)
4(1)
40.0
150
4 of 4
31 (0)
N/A
3(0)
4(1)
37.6
1,500
4 of 4
31 (3 of 4)
25.6 ±2.6
0
1 (1)
31.1
Nonskid Tread
(NSKID-2)
0
2 of 4
11 (0)
N/A
1 (0)
3(0)
N/A
150
4 of 4
25 (2 of 4)
24.3 ± 1.2
1 (0)
2(2)
34.7 ±0.2
1,500
4 of 4
31 (4 of 4)
23.0 ± 1.9
0
0
N/A
Concrete Pier
(CONPIER-1)
0
4 of 4
32 (2 of 4)
33.6 ± 1.4
3(0)
3(0)
N/A
150
4 of 4
25 (2 of 4)
24.2 ±0.8
1 (0)
1 (1)
34.3
1,500
4 of 4
31 (3 of 4)
25.2 ±2.3
1 (1)
1 (1)
23.3
Concrete Pier
(CONPIER-2)
0
4 of 4
31 (1 of 4)
37.3
2(0)
3(0)
N/A
150
4 of 4
31 (3 of 4)
24.5 ±2.0
0
1 (1)
31.7
1,500
4 of 4
31 (4 of 4)
22.5 ± 1.2
0
0
N/A
(a) Presumptive Btk T1B2 was present on initial culture plates.
(b) Number of colonies PCR-screened from initial plating, with number of PCR positive replicates in parentheses.
(c) Colony PCR Ct values for positive samples (Ct value of < 40).
(d) Number of colonies PCR-screened from BHIB streak plates, with number of PCR positive replicates in parentheses.
(e) Number of samples with PCR screening of BHIB enrichment culture, with number of PCR positive replicates in parentheses.
(f) BHIB enrichment culture PCR Ct values for positive samples (Ct value of < 40).
62
-------
3.2.3 Vacuum Filter Cassette Sample RV-PCR Analysis
A summary of the average and standard deviation values of the RV-PCR ACt values for the detection of
Btk T1B2 spores recovered from VFCs that were used to sample maritime surfaces and then spiked
(inoculated) with Btk T1B2 spores in the laboratory with a target of 300 or 3,000 CFU are presented in
Table 12. The nominal quantity of spores available for analysis represents one-half the target spore load
applied to the surfaces and the determined number of spores available represents one-half the measured
number of Btk T1B2 spores applied based on the Btk suspension titer and volume applied on the day of
spiking. Sample replicates with a RV-PCR ACt value > 9 are RV-PCR positive, indicating that viable
Btk T1B2 spores were recovered. The plots depict an area shaded in red that is the region of a negative
detection result and an area of green that is a positive detection result, delineated by the Btk T1B2
barcode target ACt value > 9. The RV-PCR ACt results are plotted in Figure 48 and Figure 49. The plots
all depict the ACt threshold value of 9 as a dashed line.
Table 12. RV-PCR Analyses of Spiked Vacuum Filter Cassette that Were Used to Sample Different
Maritime Surfaces for Detection of Btk T1B2 Spores.
Surface Type
(Sample ID)
Number of
Replicates
Spores Available for
Analysis
(CFU)
ACfcl (X ± o)
RV-PCR
Replicates
Positive11"
Nominal'-1'
Determined"31
Nonskid Tread
(NSKID-1)
4
0
0
1.0 ± 1.2
0
4
150
110 ± 72
4.3 ±4.3
1
4
1,500
1,100 ± 720
10.5 ± 2.0
3
Nonskid Tread
(NSKID-2)
4
0
0
0.0 ± 0.0
0
4
150
110 ± 72
12.5 ± 1.3
4
4
1,500
1,100 ± 720
16.7 ± 3.9
4
Concrete Pier
(CONPIER-1)
4
0
0
0.0 ± 0.0
0
4
150
110 ± 72
7.4 ± 8.4
2
4
1,500
1,100 ± 720
15.9 ± 5.0
3
Concrete Pier
(CONPIER-2)
4
0
0
0.0 ± 0.0
0
4
150
110 ± 72
15.0 ± 4.4
4
4
1,500
1,100 ± 720
16.6 ± 4.8
4
(a) Nominally one-half of the target spore load on the surface and assuming 100% recovery of spores.
(b) Based on the spiking titer measured each test trial, 100% recovery efficiency, and one-half of extraction used for RV-PCR
analysis.
(c) PCR assay forTlB2 Barcode Gene Target.
(d) Number of replicates with a RV-PCR ACt value > 9.
63
-------
35
Nonskid (Vacuum Filter Cassette)
30
~ NSKID1
I INSKID?
¦ Threshold
25 ¦
< 20 ¦
15
10
5 ¦
\
Nominal BtkTlB2 Spore Available for Analysis (CPU)
Figure 48. RV-PCR Analysis of Btk T1B2 Spores Recovered from VFCs Spiked with Btk T1B2 Spores
After Having Sampled Nonskid Tread Surfaces.
Average ± One Standard Deviation ofN = 4 Replicates. VFCs Spiked with Nominal 300 or 3,000 CFU of Btk
T1B2 Spores. Positive Result Equals ACt > 9.
35
Concrete Pier (Vacuum Filter Cassette)
30
~ CONPIER 1 I 1 fflNPIFR 7
¦ Threshold
25 ¦
<-> -ir,
<1 20 ¦
15
10 -
Nominal Btk T1B2 Spore Available for Analysis (CFU)
Figure 49. RV-PCR Analysis of Btk T1B2 Spores Recovered from VFCs Spiked with Btk T1B2 Spores
After Having Sampled Nonskid Tread Surfaces.
Average ± One Standard Deviation ofN = 4 Replicates. VFCs Spiked with Nominal 300 or 3,000 CFU of Btk
T1B2 Spores. Positive Result Equals ACt > 9.
64
-------
3.2.4 Analytical Method Comparison of VFC Samples
Culture analysis identified presumptive Btk T1B2 colonies for all samples, except for two replicates
sampled from nonskid tread on Boat 2, indicating that background microbial flora included wild-type
Btk or another organism that had a morphology indistinguishable from Btk T1B2 on TSA plates. Colony
PCR from initial culture plates, the colony PCR of colonies isolated from BHIB enrichment culture of
the VFC samples, and/or PCR analysis of the BHIB enrichment culture was therefore required to
confirm or refute the presence of Btk T1B2. To compare the two methods, culture with PCR
confirmation and RV-PCR results were assessed to determine which method may be more likely to
detect viable spores that have been spiked onto VFCs that contain outdoor interferents.
PCR screening of presumptive Btk T1B2 colonies was negative in some cases, indicating that
background microbial flora with colony morphology indistinguishable from the morphology of
Btk T1B2 were present on TSA culture plates; and hence, present in the samples collected in the field. It
is possible that wild-type/naturally occurring Btk and their presence led to an inflation in presumptive
spore recovery values by the culture method. Presumptive culture identification by colony morphology,
colony identification confirmed by PCR, and RV-PCR results are shown in Table 13.
For culture analysis of VFC samples, 40 of 48 (83%) that had been used to collect inert and biological
deposits were true positives or true negatives. A true positive is defined as a sample spiked with Btk
T1B2 spores that was confirmed positive by PCR. A true negative is defined as a sample that was not
spiked with Btk T1B2 spores and was negative for PCR confirmatory screening. Four (4) samples were
false positive and four samples were false negative using the culture method. For the four false positive
samples, colony PCR Ct values were < 40, each measuring between 32 to 37, or a BHIB culture PCR Ct
value of 40. For comparison, colony PCR Ct values of Btk T1B2 isolated from field blanks or laboratory
blanks (new VFC spiked with BtkTYQl spores) was 21.2 ± 1.4. Twenty-two (22) of 23 field blank and
laboratory blank controls were true negatives, with one false positive with a BHIB culture Ct value of
38.
For RV-PCR, 41 of 48 samples (87%) that had been used to collect inert and biological deposits were
determined as true positives or true negatives. A true positive is defined as a sample spiked with
Btk T1B2 spores that had a ACt of > 9. A true negative is defined as a sample that was not spiked with
Btk T1B2 spores and had a ACt of < 9. All seven nontrue sample results were false negative samples;
five were spiked with a 300-CFU target spore load and two were spiked with a 3,000-CFU target spore
load. ACt values for the false negative samples ranged from 0 to 8.8.
65
-------
Table 13. Analytical Method Comparison Displaying Culture Presumptive, Culture ID with PCR
Confirmation, and RV-PCR Replicates Positively Identified (N = 4) for Surfaces Sampled with Vacuum
Cassettes.
Surface
Type
Actual Spike
Level (CFU)
Presumptive
Culture
Result
Culture PCR
Confirmation
Culture %
Recovery
RV-PCR
Result
RV-PCR ACt
0
Positive
Positive
N/A
Negative
1.9
0
Positive
Negative
N/A
Negative
2.1
0
Positive
Negative
N/A
Negative
0
0
Positive
Negative
N/A
Negative
0
410
Positive
Negative
126.1
Negative
1.5
Nonskid,
120
Positive
Negative
275
Negative
6.6
Boat 1
250
Positive
Positive
114.4
Positive
9.1
97(a)
Positive
Negative
348.1
Negative
0
4,100
Positive
Positive
11.5
Positive
10.4
1,200
Positive
Positive
29.3
Positive
12.8
2,500
Positive
Positive
23.3
Positive
10.8
970
Positive
Positive
153.2
Negative
8
0
Positive
Negative
N/A
Negative
0
0
Negative
Negative
N/A
Negative
0
0
Negative
Negative
N/A
Negative
0
0
Positive
Negative
N/A
Negative
0
410
Positive
Positive
10.2
Positive
11.4
Nonskid,
120
Positive
Positive
18.3
Positive
13.8
Boat 2
250
Positive
Positive
39.6
Positive
11.3
97(a)
Positive
Positive
264.6
Positive
13.3
4,100
Positive
Positive
8.6
Positive
19.6
1,200
Positive
Positive
16.5
Positive
19.6
2,500
Positive
Positive
18.9
Positive
16.2
970
Positive
Positive
22.3
Positive
11.4
0
Positive
Negative
N/A
Negative
0
0
Positive
Positive
N/A
Negative
0
0
Positive
Positive
N/A
Negative
0
0
Positive
Negative
N/A
Negative
0
410
Positive
Positive
126.1
Negative
7.3
Concrete
120
Positive
Positive
24.4
Positive
20.4
Pier 1
250
Positive
Positive
48.4
Negative
0
97(a)
Positive
Negative
269.2
Positive
10
4,100
Positive
Positive
12.3
Positive
18.6
1,200
Positive
Positive
19.3
Positive
20.1
2,500
Positive
Positive
30.4
Negative
8.8
970
Positive
Positive
33.4
Positive
15.9
(E" Stock enumeration plate outside 25 - 250 CFU range.
66
-------
Table 13. Analytical Method Comparison Displaying Culture Presumptive, Culture ID with PCR
Confirmation, and RV-PCR Replicates Positively Identified (N = 4) for Surfaces Sampled with Vacuum
Cassettes (Cont.)
Surface
Type
Actual Spike
Level (CFU)
Presumptive
Culture
Result
Culture PCR
Confirmation
Culture %
Recovery
RV-PCR
Result
RV-PCR ACt
0
Positive
Negative
N/A
Negative
0
0
Positive
Positive
N/A
Negative
0
0
Positive
Negative
N/A
Negative
0
0
Positive
Negative
N/A
Negative
0
410
Positive
Positive
20.6
Positive
11.6
Concrete
120
Positive
Positive
33.6
Positive
21.5
Pier 2
250
Positive
Positive
74.8
Positive
13.4
97(a)
Positive
Positive
153.2
Positive
13.7
4,100
Positive
Positive
10.7
Positive
14.3
1,200
Positive
Positive
7.9
Positive
23.4
2,500
Positive
Positive
9.8
Positive
16.2
970
Positive
Positive
19
Positive
12.6
(E" Stock enumeration plate outside 25 - 250 CFU range.
Results for all samples processed, including positive and negative controls, are shown in Table 14. The
culture method correctly determined 62 of 71 samples (87%) with five false positives; however, all five
of these samples had colony PCR Ct values between 32 to 37, or a Ct of 38 to 40 for BHIB PCR. Colony
PCR of Btk T1B2 from the 11 spiked field blank or laboratory spike (new vacuum cassette spiked with
Btk T1B2 spores) controls had a Ct value of 21.2 ± 1.4, suggesting that Ct values between 32 and 37
might be caused by low level contamination or potentially nonspecific PCR amplification. The culture
method also had four false negative replicates. One of the four false negative sample replicates had only
one presumptive Btk colony screened of 47 that were available on the 1-mL MicroFunnel membrane
plate. The EPA Protocol specifies that one to three colonies from MicroFunnel filters and a minimum of
three colonies from spread plates should be PCR-screened for target confirmation (EPA, 2017). For the
other four false negative replicates, 8 to 10 colonies were PCR-screened in addition to PCR screening of
BHIB enrichment culture PCR; therefore, if additional colony PCR screenings were performed, the false
negative results may not have changed.
The RV-PCR method correctly determined 64 of 71 samples (90%) with zero false positive samples and
seven false negative samples, five of which were at the 300-target spore load.
For culture analysis, nonskid tread surfaces sampled with VFCs had one false positive sample that had a
Ct value of 40 when PCR analysis was performed on a BHIB enrichment culture of the processed VFC
sample, and the colony PCR was negative. There were three false negative samples at the 300 CFU
spike level with only the fourth replicate confirmed positive, although the BHIB culture PCR Ct was
high (37.6). RV-PCR results had zero false positives and three false negatives at the 300 CFU level and
one false negative at the 3,000 CFU level.
For culture analysis, concrete pier surfaces sampled with VFCs had three false positive samples with Ct
values of 32, 34, and 37 for colony PCR. For all 11 spiked field blanks or laboratory spikes (new
vacuum cassette spike with Btk T1B2 spores), controls resulted in a Ct value of 21.2 ± 1.4. Therefore, a
Ct value between 32 and 37 may have been caused by low level contamination or potentially nonspecific
67
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PCR amplification. Additionally, analysis of BHIB enrichment culture PCR of the processed vacuum
membrane was negative for all three of these false positive samples. There was one false negative
sample at the 300 CFU spike level for culture analysis. RV-PCR results had two false negative samples
at the 300 CFU level and one at the 3,000 CFU level.
68
-------
Table 14. Analytical Method Comparison Displaying Culture ID with PCR Confirmation and RV-PCR for Surfaces Sampled with Vacuum Filter
Cassettes.
Culture Method
RV-PCR Method
Surface Type
True Positive or
False Positive
False Negative
True Positive or
False Positive
False Negative
True Negative
Sample
Sample
True Negative
Sample
Sample
Nonskid 1
8
1
3(a)
8
0
4
Nonskid 2
12
0
0
12
0
0
Concrete Pier 1
9
2
1(b)
9
0
3
Concrete Pier 2
11
1
0
12
0
0
Controls
22
1
0
23
0
0
Totals
62
5
4
64
0
7
Positive PCR threshold of 40 for colony PCR and BHIB enrichment culture PCR. The definition for Positive Ct threshold per the EPA Protocol (EPA, 2017) is < 40.
(a) One replicate spiked with 97 CFU, which is the low end of the acceptance range for a sample spiked with Btk T1B2 spores.
(b) One replicate spiked with 97 CFU, which is the low end of the acceptance range for a sample spiked with Btk T1B2 spores.
69
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3.2.5 Analysis of Controls
For VFCs, there were a total of seven zero spike VFCs that served as reagent control laboratory blanks,
four zero spike VFCs that were opened in the field that served as field blanks, four (4) 300-CFU spiked
field blanks, four (4) 3,000-CFU spiked field blanks, two (2) 300-CFU spiked VFCs, and two (2) 3,000-
CFU spiked VFCs that were handled in laboratory only (laboratory spike).
All controls performed as expected except for one zero spike vacuum cassette that was culture positive
with a BHIB culture PCR Ct value of 38. For all 11 of the spiked field blank or laboratory spike (new
VFC spiked with Btk T1B2) samples on which colony PCR was performed, the average Ct value was
21.2 ± 1.4.
3.2.6 Considerations for Culture Analysis False Positive Results for Sponge Sticks and
VFCs
Regarding false positive results for colony PCR and BHIB enrichment PCR, it is important to establish
limit of detection thresholds for distinguishing a positive PCR response from a negative PCR response.
These thresholds will vary depending on the PCR assay used for detection and should be established
experimentally, although a Ct value threshold of 40 is the norm for any PCR assay used for biothreat
detection and was therefore used for this study. For culture PCR analysis, a Ct value threshold of < 40
was used to establish positive results for colony PCR and BHIB enrichment PCR. Comparatively, for
RV-PCR, a ACt value threshold of > 9 was used to establish positive results. For example, if the To
aliquot was undetected, a value of 45 (total number of PCR cycles run) was assigned; and if the 7/
aliquot resulted in a Ct value of < 36, the sample was positive for RV-PCR (To - Tf = ACt). A baseline
equivalent to the To used for RV-PCR is not included in BHIB enrichment PCR analysis, and its absence
may have led to more false positive results for culture method compared to RV-PCR method.
3.3 Grab Sample Analysis Results
For grab samples, 500 mL of PBST was added to sample. However, particulates such as soil and other
debris limit filtration onto MicroFunnel filter membranes (0.45 |im), slowing processing speed and
reducing the total volume that can be analyzed, were present as shown in Table 15.
Table 15. Volume of Recovered Suspension Concentrated onto MicroFunnel Filter.
Grab Sample Type
PBST Volume (mL)
Volume of Suspension Processed
through MicroFunnel Filter, 0.45 pm
(X±CT)
SBWASH 1 and 2
500
500 ±0
Gravel
500
250 ±0
Soil
40
N/A
Vegetation
500
131.5 ± 42.1
The water content of the soil was 26.18 ± 0.01% with a pH of 6.615 ± 0.002, and the sterilized soil water
content was 0.97 ± 0.00%, with a pH of 5.621 ± 0.002.
70
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3.3.1 Grab Sample Culture Analysis
A summary of the average and standard deviation values of the measured recovery of presumptive Btk
T1B2 spores from spiked wash water, gravel, soil, and vegetation (grass) samples is presented in
Table 16. The nominal quantity represents one-half the target spore load applied to the grab sample, and
the determined number of spores available represents one-half the number of presumptive Btk T1B2
spores spiked, assuming the full sample volume was processed.
The spore recovery percentage of presumptive Btk colonies recovered as determined by culture analysis
using TSA plates is plotted in Figure 50 through Figure 53.
The percent recovery of presumptive Btk T1B2 spores for grab samples was highly variable and
unreliable with percent recoveries > 100% for all surfaces analyzed with less than 15,000 spore load
with few exceptions (gravel with 1,500 CFU, and soil samples) due to the presence of high background
microbial growth.
Table 16. Presumptive Btk T1B2 Spores Recovered and Associated Spore Recovery (%) from Laboratory-
Spiked Grab Samples.
Grab Sample
Type
Sample
Replicates
Spores Available for Analysis
(CFU)
Spore
Recovery
(CFU)
(X ± CT)ICI
Spore Recovery
(%)
Nominal'-'1
Determined"51
(X±o)
(X ± CT)ldl
3
0
0
850 ± 490
N/A
Wash Water-1
3
150
90 ± 0
1,200 ± 330
1,400 ± 370
3
1,500
900 ± 0
1,300 ±670
150 ± 74
3
15,000
30,000 ± 0
4,700 ± 770
16 ± 2.6
Wash Water-2
0
N/A
N/A
N/A
N/A
3
15,000
30,000 ± 0
7,100 ±420
24 ± 1.4
Gravel
0
0
1,400 ± 1,900
N/A
3
150
180 ± 0
370 ± 270
200±150
3
1,500
1,800 ± 0
1,600 ± 910
88 ± 51
3
0
0
760 ± 340
N/A
Soil
3
1,500
2,200 ± 0
840±100
38 ± 4.6
3
15,000
22,000 ± 0
1,800 ± 350
8.2 ± 1.6
3
150,000
220,000 ± 0
11,000 ± 7,000
5.1 ± 3.2
1
0
0
0
0
Sterile Soil
1
1,500
2,200 ± 0
930
42
1
15,000
22,000 ± 0
7,600
35
1
150,000
220,000 ± 0
46,000
21
3
0
0
6,700 ±4,100
N/A
Vegetation (Grass)
3
150
55 ± 0
9,900 ±2,700
18,000 ± 4,900
3
1,500
550 ± 0
12,000 ±4,400
2,200 ± 810
3
15,000
30,000 ± 0
12,000 ±2,600
40 ± 8.7
(a) Nominally one-half of the target spore load applied to the grab sample type and assuming 100% recovery of spores.
(b) Based on the spiking suspension titer measured per trial, 100 % recovery, and one-half of extract used for culture analysis.
(c) Presumptive Btk T1B2 colonies based on morphology and one-half of suspension used for culture analysis.
(d) Calculated using the actual spore loading applied during spiking and total presumptive Btk T1B2 spores recovered from
each sample.
71
-------
100
£ 90
Q)
O
Q.
LD
«N
CO
80
70
60
•B 50
Q.
E
3
0)
i_
Q_
M—
O
2r
o
40
30
20
10
Wash Water 1 (Grab)
Wash Water 1 recoveries for 150
CFIJ and 1,500 CFIJ of 1,400 %
and 150 %, respectively
^ % \
Nominal Spores Available for Analysis (CFU)
Figure 50. Presumptive Btk T1B2 Spore Recovery Percentage (Average ± One Standard Deviation of N :
Replicates) from Wash Water Grab Samples Spiked with Btk T1B2 Spores.
Wash Water 1 percent recovery values for 150 and 1,500 CFU nominal spore load are not plotted in
Figure 50 because percent recovery values of 1,400 ±370 % and 150 ± 74 % are not an accurate
reflection of target spores recovered. The percent recovery values are inflated from the presence of
background microorganisms with Btk morphology.
140
SjP
ss
o
Q.
rvj 100
CO
CO
0)
t>
Q.
E
D
-------
Gravel percent recovery values for 150 CFU nominal spore load are not plotted in Figure 51 because
percent recovery of 200 ± 150 % is not an accurate reflection of target spores recovered. The percent
recovery values are inflated from the presence of background microorganisms with Btk morphology.
100
SP
e. 90
0)
o
Q.
to
PM
CO
CL
£
o
>*
aj
o
u
50
40
30
20
10
Soil (Grab)
is
¦%
%
Nominal Spores Available for Analysis (CFU)
Figure 52. Presumptive Btk T1B2 Spore Recovery Percentage (Average ± One Standard Deviation of N
Replicates) from Soil Grab Samples Spiked with Btk T1B2 Spores.
100
£ 90
o
u
Q)
a:
80 ¦
70 ¦
5 60
CD
0)
50
4->
Q.
E
3
40
30 :
20
10
Vegetation (Grab)
Vegetation recoveries for 150
CFU and 1,500 CFU of 18,000 %
and 2,200 %, respectively
Nominal Spores Available for Analysis (CFU)
Figure 53. Presumptive Btk T1B2 Spore Recovery Percentage (Average ± One Standard Deviation of N
Replicates) from Vegetation Grab Samples Spiked with Btk T1B2 Spores.
73
-------
Vegetation percent recovery values for 150 and 1,500 CFU nominal spore loads are not plotted in
Figure 53 because percent recoveries of 18,000 % and 2,200 % are not an accurate reflection of target
spores recovered. The percent recovery values are inflated from the presence of background
microorganisms with Btk morphology.
For grab samples, representative images of the culture plates are shown in Figure 54 through Figure 57.
Presumptive Btk colonies were isolated from all nonsterile sample types, including the zero spore spike
samples, resulting in unreliable spore recovery values.
Figure 54. Culture Images of Spore Recovery from Wash down Grab Samples Plated on TSA.
Presumptive Btk colonies were observed in zero spike samples and 2 mL volumes plated were overwhelmed
with background growth.
Image Descriptions: (A) Zero Spore Spike, ft 1 mL; (B) Zero Spore Spike, 2 mL; (C) 300 Spore Spike, 0.1 mL;
(D) 300 Spore Spike, 2 mL; (E) 3,000 Spore Spike, 0.1 mL; (F) 3,000 Spore Spike, 2 mL
74
-------
Figure 55. Culture Images of Spore Recovery from Gravel Grab Samples Plated on TSA.
Image Descriptions: (A) Zero Spore Spike, 1 mL; (B) Zero Spore Spike, 4 mL; (C) 300 Spore Spike, 1 mL; (D)
300 Spore Spike, 4 mL; (E) 3,000 Spore Spike, 1 mL; (F) 3,000 Spore Spike, 4 mL
75
-------
Figure 56. Culture Images of Spore Recovery from Soil Grab Samples Plated on TSA.
Presumptive Btk colonies were observed in zero spike samples. Image Descriptions: (A) Zero Spike Sterile Soil,
0.1 mL; (B) 3,000 Spike Sterile Soil, 0.1 mL; (C) 30,000 Spike Sterile Soil, 0.1 mL; (D) 300,000 Spike Sterile
Soil, 0.1 mL; (E) Zero Spike Nonsterile Soil, 0.1 mL; (F) 3,000 Spike Nonsterile Soil, 0.1 mL; (G) 30,000 Spike
Nonsterile Soil, 0.1 mL; (H) 300,000 Spike Nonsterile Soil, 0.1 mL.
Figure 57. Culture Images of Spore Recovery from Vegetation Grab Samples Plated on TSA.
Presumptive Btk colonies overwhelmed zero spike samples. Image Descriptions: (A) Zero Spike, 2 mL; (B) 300
Spike, 2 mL; (C) 3,000 Spike, 2 mL.
76
-------
3.3.2 Colony Confirmation by PCR
Based on the colony morphology, presumptive Btk T1B2 colonies were identified for all grab samples
(48 of 48 samples). Background microbial flora levels were high, even more so than observed for
sponge stick and vacuum filter cassette samples due to the larger surface area sampled and subsequent
concentration alongside target organism onto the filter membrane. Twelve of these samples with inert
and biological deposits were 0-spike samples, meaning they were not inoculated with Btk T1B2 spores,
yet presumptive Btk colonies were still isolated from the spore recovery. Colonies with morphology
indistinguishable from the morphology of Btk T1B2 were present on the culture plates, as indicated by a
negative PCR result for presumptive Btk T1B2 colonies. The confirmation of target Btk T1B2 was
assessed by colony PCR from the initial culture plates, colony PCR from the BHIB enrichment culture,
or PCR of an aliquot of the BHIB enrichment culture. Results from PCR confirmatory testing are shown
in Table 17.
For all grab samples (wash water, gravel, soil, and vegetation) spiked at the 300 Btk T1B2 spore level,
only one replicate of the gravel samples was positive by culture analysis as confirmed by colony PCR.
At the 3,000 CFU target spore load level, all three wash water sample replicates, two gravel sample
replicates, one soil replicate, and one vegetation sample replicate were confirmed positive by colony
PCR. Additionally, one 3,000 CFU replicate was confirmed positive by PCR of the BHIB culture
enriched soil pellet, although the PCR Ct value was 38.7.
Since detection limits were relatively high for grab samples compared to sponge stick and vacuum filter
cassette samples, a 30,000 CFU target spore load was added to the test matrix for wash water and
vegetation. Additionally, a 300,000 CFU target spore load was added to the test matrix for soil. At the
30,000 CFU target spore load level, all replicates were confirmed positive by colony PCR for sample
types tested (wash water, vegetation, and soil) with Ct values <21. Soil samples were processed with
target spore loads of 300,000 CFU and were confirmed culture positive by PCR with Ct values < 21.
77
-------
Table 17. Summary of the Accuracy of Identification of Presumptive Btk T1B2 Colonies by PCR Confirmation from Spiked Grab Samples.
Grab Samples
Nominal Spore
Load
(CFU)
Culture
Replicates
Presumptive
Positive1'"0
Colonies from
Initial Culture
Plates PCR-
Screened
(# PCR +)(b)
Colony PCR Ct
(X±CT)l:l
Colonies from
BHIB Streak
Plates PCR-
Screened
(# PCR +)(tl)
BHIB PCR-
Screened
(# PCR +)(e)
BHIB PCR Ct
(X±CT)'"
Wash Water-1
0
3 of 3
30 (0 of 3)
N/A
30 (0)
3(0)
N/A
150
3 of 3
30 (0 of 3)
N/A
20 (0)
3(0)
N/A
1,500
3 of 3
30 (3 of 3)
23.3 ± 1.2
10 (0)
3(2)
38.6 ±0.7
15,000
3 of 3
30 (3 of 3)
18.2 ±0.7
0
0
N/A
Wash Water-2
15,000
3 of 3
30 (3 of 3)
18.9 ±0.5
0
0
N/A
Gravel
0
3 of 3
30 (0 of 3)
N/A
3(0)
0
N/A
150
3 of 3
28 (1 of 3)
22.4
2(0)
0
N/A
1,500
3 of 3
30 (2 of 3)
23.1 ±0.6
1 (0)
0
N/A
Soil
0
3 of 3
27 (0 of 3)
N/A
0
3(0)
N/A
1,500
3 of 3
33 (1 of 3)
22.1
2(0)
3(2)
38.1 ±0.6
15,000
3 of 3
33 (3 of 3)
20.4 ±0.5
3(0)
3(3)
37.0 ± 0.6
150,000
3 of 3
33 (3 of 3)
20.4 ± 1.0
0
3(3)
34.6 ±1.9
Sterile Soil
0
Oof 1
0
N/A
0
1 (0)
N/A
1,500
1 of 1
11 (1 of 1)
19.7
0
1 (1)
20.5
15,000
1 of 1
11 (1 of 1)
21.8
0
1 (1)
21.2
150,000
1 of 1
10 (1 of 1)
19.8
1 (1)
1 (1)
20.2
Vegetation
0
3 of 3
30 (0)
N/A
0
3(0)
N/A
150
3 of 3
30 (0)
N/A
2(0)
3(0)
N/A
1,500
3 of 3
30 (1 of 3)
29.0
2(0)
2(0)
N/A
15,000
3 of 3
30 (3 of 3)
19.7 ±0.8
0
0
N/A
(a) Presumptive Btk T1B2 was present on initial culture plates.
(b) Number of colonies PCR-screened from initial plating, with number of PCR positive replicates in parentheses.
(c) Colony PCR Ct values for positive samples (Ct value of < 40).
(d) Number of colonies PCR-screened from BHIB streak plates, with number of PCR positive replicates in parentheses.
(e) Number of samples with PCR screening of BHIB enrichment culture, with number of PCR positive replicates in parentheses.
(f) BHIB enrichment culture PCR Ct values for positive samples (Ct value of < 40).
78
-------
Data suggest the limit of detection is lower for the culture method when PCR is performed on BHIB
enrichment culture of the soil pellet compared to colony PCR screen of isolated colonies for the culture
method. However, the Ct values generated from BHIB enrichment are > 36 for 3,000 and 30,000 target
spore spike levels. The BHIB enrichment culture PCR data in Table 18 suggest that soil chemical
components (inhibitors) are not interfering with PCR analysis of the enriched soil pellet, with Ct values
of 20.2 to 21.2 in sterile soil and suppressed Ct values of 33.7 to 38.9 in nonsterile soil. Therefore, the Ct
value suppression seen in nonsterile soil is likely due to growth competition from the background
microbial flora present in the soil.
Table 18. PCR Analysis of BHIB Enrichment of Soil Pellet.
Sample Type
PCR (Ct Values)
0 Spores
(X±CT)
3,000 Spores
(X±CT)
30,000 Spores
(X±CT)
300,000 Spores
(X±CT)
Nonsterile Soil
Not Detected
38.9 ± 1.6
37.0 ±0.6
33.7 ± 0.6
Sterile Soil
Not Detected
20.5
21.2
20.2
3.3.3 Grab Sample RV-PCR Analysis
A summary of the average and standard deviation of the RV-PCR ACt values for the detection of Btk
T1B2 spores recovered from grab samples is presented in Table 19. Sample replicates with a RV-PCR
ACt value > 9 are considered positive, indicating that viable Btk T1B2 spores were recovered.
The RV-PCR ACt results are plotted in Figure 58 through Figure 61. The plots all depict the ACt
threshold value of 9 as a dashed line, with an area shaded in red representing a negative detection result,
and an area of green representing a positive detection result. Wash water grab samples were RV-PCR
negative at the 300 CFU and 3,000 CFU target spore load levels. The 30,000 CFU target spore load
level was added since detection limits were high for this grab sample type. All replicates processed at
the 30,000 CFU target spore load were RV-PCR positive, with an average ACt value of 12.2. Gravel
grab samples were RV-PCR negative for two of three replicates at the 300 CFU and 3,000 CFU target
spore load levels. The positive replicate ACt values were 10.4 (300 CFU) and 9.8 (3,000 CFU).
Soil grab samples were RV-PCR negative for two of three replicates at the 3,000 CFU target spore load
level. The ACt for the RV-PCR positive replicate at the 3,000 CFU target spore load was 10.4. All
replicates at the 30,000 CFU and 300,000 CFU target spore load were RV-PCR positive. The average
ACt value of the 30,000 CFU target spore load was 13.3, and the average ACt value of the 300,000 CFU
target spore load was 16.6.
Vegetation grab samples were RV-PCR negative at the 300 CFU and 3,000 CFU target spore load
levels. The 30,000 CFU target spore load level was added since detection limits were high for this grab
sample type. All replicates processed at the 30,000 CFU target spore load were RV-PCR positive with
an average ACt value of 12.6.
79
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Table 19. RV-PCR Analyses of Btk T1B2 Spores Spiked Grab Samples.
Grab Sample Type
Number of
Replicates
Spores Available for
Analysis
(CFU)
ACt|,:l (X ± ct)
RV-PCR
Replicates
Positive11"
Nominal'"
Determined"51
Wash Water-1
3
0
0
0.0 ± 0.0
0
3
150
90 ± 0
0.0 ± 0.0
0
3
1,500
900 ± 0
7.0 ± 0.8
0
3
15,000
30,000 ± 0
12.2 ± 0.6
3
Wash Water-2
3
0
0
Not Tested
Not Tested
3
15,000
30,000 ± 0
14.7 ± 0.8
3
Gravel
3
0
0
0.7 ± 1.3
0
3
150
180 ± 0
7.5 ±2.6
1
3
1,500
1,800 ± 0
6.9 ± 3.1
1
Soil
3
0
0
0.6 ± 1.0
0
3
1,500
2,200 ± 0
8.7 ± 1.5
1
3
15,000
22,000 ± 0
13.3 ± 1.2
3
3
150,000
220,000 ± 0
16.6 ± 0.6
3
Sterile Soil
1
0
0
0
0
1
1,500
2,200 ± 0
25.0
1
1
15,000
22,000 ± 0
24.8
1
1
150,000
220,000 ± 0
24.5
1
Vegetation (Grass)
3
0
0
0.0 ± 0.0
0
3
150
55 ± 0
0.0 ± 0.0
0
3
1,500
550 ± 0
1.6 ± 1.5
0
3
15,000
30,000 ± 0
12.6 ± 1.3
3
(a) Nominally one-half of the target spore load on the surface and assuming 100% recovery of spores.
(b) Based on the spiking titer measured each test trial, 100% recovery efficiency, and one-half of extraction used for RV-PCR
analysis.
(c)PCR assay for T1B2 Barcode Gene Target.
(d) Number of replicates with a RV-PCR ACt value > 9.
80
-------
35
Wash Water (Grab)
30
Wash Water 1 I iWach Watpr ?
¦Threshold
25 ¦
<-> ir,
<1 20
10
9.
35
Gravel (Grab)
30 ¦
I 1 GRAVEL 1
¦ Threshold
25
->n
<3 20
15 ¦
10
5 ¦
i
so
Nominal Btk T1B2 Spore Available for Analysis (CFU)
Figure 59. RV-PCR Analysis of Btk T1B2 Spores Recovered from Gravel Grab Samples.
Average ± One Standard Deviation ofN = 3 Replicates. Gravel Spiked with Nominal 300 or 3,000 CFU of Btk
T1B2 Spores. Positive Response Equals ACt > 9.
81
-------
35
30
25
u
<1 20
01
M
2
o>
Soil (Grab)
15
10 ¦
5 :
0
; ^
~ Non-Sterile Soil
~ Sterile Soil
¦ Threshold
Nominal Btk T1B2 Spore Available for Analysis (CFU)
Figure 60. RV-PCR Analysis of Btk T1B2 Spores Recovered from Soil Grab Samples.
Average ± One Standard Deviation ofN = 3 Replicates. Soil Spiked with Nominal 3,000, 30,000, or 300,000
CFU of Btk T1B2 Spores. Positive Response Equals ACt> 9.
35
30
Vegetation (Grab)
¦ Vegetation
¦Threshold
25
u
•a 20
g 15 ¦
<
10 ¦
5
0
Nominal Btk T1B2 Spore Available for Analysis (CFU)
%
Figure 61. RV-PCR Analysis of Btk T1B2 Spores Recovered from Vegetation (Grass) Grab Samples.
Average ± One Standard Deviation ofN = 3 Replicates. Vegetation (Grass) Spiked with Nominal 300 or 3,000
CFU of Btk T1B2 Spores. Positive Response Equals ACt>9.
82
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3.3.4 Analytical Method Comparison for Grab Samples
Culture analysis identified presumptive Btk T1B2 colonies for all grab field samples, indicating that
background microbial flora included wild-type Btk or another organism that had a morphology
indistinguishable from Btk T1B2 on TSA plates. PCR confirmation was performed on either colonies
from initial culture plates, colonies isolated from BHIB enrichment culture of the grab samples, and/or
the BHIB enrichment culture to confirm or refute the presence of Btk T1B2. To compare the two
methods, culture with PCR confirmation and RV-PCR results were assessed to determine which method
may be more likely to detect viable spores that have been deposited onto wash water, gravel, soil, or
vegetation containing outdoor interferents.
PCR screening of presumptive Btk T1B2 colonies were negative in some cases, indicating that
background microbial flora with morphology indistinguishable from that of Btk T1B2 were present on
TSA culture plates, therefore present in the samples collected in the field. It is possible that wild-
type/naturally occurring Btk led to an inflation in presumptive spore recovery values by the culture
method. Presumptive culture identification by colony morphology, colony identification confirmed by
PCR, and RV-PCR results are shown in Table 20.
For culture analysis of field grab samples, 36 of 48 (75%) were true positives or true negatives. A true
positive is defined as a sample spiked with Btk T1B2 spores that was confirmed positive by PCR. A true
negative is defined as a sample that was not spiked with Btk T1B2 spores and was negative for PCR
confirmatory screening. All 12 nontrue sample results were false negative using the culture method,
meaning they were not confirmed positive by PCR following colony PCR or PCR of BHIB culture. All
16 field blank and laboratory blank controls were true negatives.
For RV-PCR analysis of field grab samples, 30 of 48 (63%) were true positives or true negatives. A true
positive is defined as a sample spiked with Btk T1B2 spores that had a ACt of > 9. A true negative is
defined as a sample that was not spiked with Btk T1B2 spores and had a ACt of < 9. All 18 nontrue
sample results were false negative samples, eight were spiked with 300 CFU target spore load and ten
were spiked with 3,000 CFU target spore load. ACt values for the false negative samples ranged from 0
to 8.3.
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Table 20. Analytical Method Comparison Displaying Culture Presumptive, Culture ID with PCR
Confirmation, and RV-PCR Replicates Positively Identified (N = 4) for Grab Samples.
Sample
Actual Spike
Level (CFU)
Presumptive
Culture
Result
Culture PCR
Confirmation
Culture %
Recovery
RV-PCR
Result
RV-PCR ACt
Washdown 1
0
Pos
tive
Negative
N/A
Negative
0
0
Pos
tive
Negative
N/A
Negative
0
0
Pos
tive
Negative
N/A
Negative
0
180(a)
Pos
tive
Negative
1,611.00
Negative
0
180(a)
Pos
tive
Negative
1,555.00
Negative
0
180(a)
Pos
tive
Negative
944.00
Negative
0
1,800
Pos
tive
Positive
96.3
Negative
7.29
1,800
Pos
tive
Positive
233.33
Negative
7.57
1,800
Pos
tive
Positive
114.81
Negative
6.07
60,000
Pos
tive
Positive
18.3
Positive
11.54
60,000
Pos
tive
Positive
13.2
Positive
12.72
60,000
Pos
tive
Positive
15.6
Positive
12.44
Washdown 2
60,000
Pos
tive
Positive
25.1
Positive
14.74
60,000
Pos
tive
Positive
22.3
Positive
13.84
60,000
Pos
tive
Positive
23.9
Positive
15.42
Washdown
Field Blanks
0
Pos
tive
Negative
N/A
Negative
0
0
Pos
tive
Negative
N/A
Negative
4.42
180(a)
Pos
tive
Positive
19.44
Positive
24.85
600
Pos
tive
Positive
90
Positive
13.15
1,800
Pos
tive
Positive
31.67
Positive
24.83
6,000
Pos
tive
Positive
26.5
Positive
25.06
Gravel
0
Pos
tive
Negative
N/A
Negative
2.2
0
Pos
tive
Negative
N/A
Negative
0
0
Pos
tive
Negative
N/A
Negative
0
360
Pos
tive
Negative
97.2
Negative
6.7
360
Pos
tive
Negative
133.3
Negative
5.4
360
Pos
tive
Positive
377.8
Positive
10.4
3,600
Pos
tive
Positive
30
Positive
9.8
3,600
Pos
tive
Negative
113.3
Negative
3.6
3,600
Pos
tive
Positive
121.1
Negative
7.2
Gravel Lab
Blank
0
Pos
tive
Negative
N/A
Negative
0
360
Pos
tive
Positive
11.1
Positive
12.2
3,600
Pos
tive
Positive
24.4
Positive
18.2
(E" Stock enumeration plate outside 25 - 250 CFU range.
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Table 20. Analytical Method Comparison Displaying Culture Presumptive, Culture ID with PCR
Confirmation, and RV-PCR Replicates Positively Identified (N = 4) for Grab Samples (Cont.)
Sample
Actual Spike
Level (CFU)
Presumptive
Culture
Result
Culture PCR
Confirmation
Culture %
Recovery
RV-PCR
Result
RV-PCR ACt
0
Positive
Negative
N/A
Negative
0
0
Positive
Negative
N/A
Negative
0
0
Positive
Negative
N/A
Negative
1.7
4,400
Positive
Positive
42.4
Negative
8.3
4,400
Positive
Positive
39.4
Positive
10.4
Soil
4,400
Positive
Negative
33.3
Negative
7.4
44,000
Positive
Positive
7.6
Positive
14.2
44,000
Positive
Positive
10
Positive
13.7
44,000
Positive
Positive
7
Positive
11.9
440,000
Positive
Positive
1.4
Positive
16.9
440,000
Positive
Positive
6.8
Positive
15.9
440,000
Positive
Positive
7.1
Positive
16.9
0
Negative
Negative
N/A
Negative
0
Sterile Soil
4,400
Positive
Positive
42.4
Positive
25
44,000
Positive
Positive
34.6
Positive
24.8
440,000
Positive
Positive
20.7
Positive
24.5
0
Positive
Negative
N/A
Negative
0
0
Positive
Negative
N/A
Negative
0
0
Positive
Negative
N/A
Negative
0
110
Positive
Negative
16,094.30
Negative
0
110
Positive
Negative
23,579.60
Negative
0
Vegetation
110
Positive
Negative
14,424.20
Negative
0
1,100
Positive
Negative
2,335.10
Negative
0
1,100
Positive
Negative
1,393.90
Negative
3.1
1,100
Positive
Positive
2,998.10
Negative
1.7
60,000
Positive
Positive
50.3
Positive
12.6
60,000
Positive
Positive
36.7
Positive
13.9
60,000
Positive
Positive
34.2
Positive
11.2
Vegetation
Control
0
Negative
Negative
N/A
Negative
0
110
Positive
Positive
6.9
Positive
22.1
(PBST)
1,100
Positive
Positive
15.7
Positive
22.3
Results for all samples processed, including positive and negative controls, are shown in Table 21. For
culture, 52 of 64 samples (81%) were determined as true positives or true negatives using the culture
method. All 12 nontrue sample results were false negative using the culture method, meaning they were
not confirmed positive by PCR following colony PCR or PCR of BHIB culture. All 16 field blank and
laboratory blank controls were true negatives. The RV-PCR method correctly determined 46 of 64
samples (72%), with zero false positives and 18 false negatives.
Many of the grab samples had a high load of presumptive Btk colonies, including the zero spore spike
samples. Therefore, an important consideration for the culture method is establishing a maximum
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number of presumptive colonies that should be screened using PCR. The method currently lists that a
minimum of three colonies be screened from spread plates or 1 - 3 colonies from membrane filter plates.
For samples with a high number of background organisms with presumptive Btk morphology, a
maximum number of colonies or a method for pooling multiple colonies or pooling and mixing growth
from culture plates would need to be established.
For wash water surface culture analysis, all three 300 CFU target spore load level samples were false
negatives. For RV-PCR, all 300 CFU and 3,000 CFU target spore load level samples were false
negatives.
For gravel samples, two of three 300 CFU target spore load level samples were false negative for both
culture and RV-PCR. At the 3,000 CFU target spore load level, one of three replicates was false
negative for culture and two of three replicates were false negative for RV-PCR.
For soil samples, one of three replicates was false negative for culture and two of three replicates were
false negative for RV-PCR at the 3,000 CFU spike level. All replicates were positive at the 30,000 CFU
and 300,000 CFU target spore load levels for both culture and RV-PCR.
For vegetation culture analysis, all three 300 CFU target spore load level and two of three 3,000 CFU
target spore load level samples were false negatives. For RV-PCR, all 300 and 3,000 CFU target spike
level samples were false negatives.
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Table 21. Analytical Method Comparison Displaying Culture ID with PCR Confirmation and RV-PCR Replicates for Grab Samples.
Sample Type
Culture Method
RV-PCR Method
True Positive or
True Negative
False Positive
Sample
False Negative
Sample
True Positive or
True Negative
False Positive
Sample
False Negative
Sample
SBWASH 1
9
0
3
6
0
6
SBWASH 2
3
0
0
3
0
0
GRAVEL
6
0
3
5
0
4
SOIL
11
0
1
10
0
2
VEGETATION
7
0
5
6
0
6
CONTROLS
16
0
0
16
0
0
Totals
52
0
12
46
0
18
Positive PCR threshold of 40 for colony PCR and BHIB culture PCR. The definition for Positive Ct threshold per the EPA Protocol (EPA, 2017) is < 40.
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3.3.5 Analysis of Controls (Grab)
Water that was used for washdown of the small boat was analyzed as a field blank control at the zero
spike, 300 CFU target spike and 3,000 CFU target spike levels. Sample replicates for each condition
above were determined as true positives or negatives for both culture and RV-PCR analysis methods. A
true positive or true negative indicates that the result matches the expected outcome, for example, if a
sample spiked with Btk T1B2 was positive, it was a true positive. Gravel purchased from a home
improvement store was included as a laboratory blank control at the zero spike, 300 CFU target spike
and 3,000 CFU target spike levels. Each condition was determined as a true positive or negative (for
both culture and RV-PCR analysis methods). Soil that was sterilized by autoclave treatment was
included as a control at the zero spike, 3,000, 30,000, and 300,000 CFU target spike levels. Each
condition was determined as a true positive or negative (for both culture and RV-PCR analysis
methods). As a vegetation control, 500 mL of PBST was included at the zero spike, 300 CFU target
spike and 3,000 CFU target spike levels and processed alongside vegetation samples. Each condition
was determined as a true positive or negative for both culture and RV-PCR analysis methods.
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4.0 QUALITY ASSURANCE/QUALITY
CONTROL
Quality assurance (QA)/quality control (QC) procedures were performed in accordance with the
Scientific, Testing, Research, and Modeling, Support (STREAMS III) Program Quality Management
Plan (QMP). The QA/QC procedures and results are summarized below.
4.1 Equipment Calibration
All equipment (e.g., pipettes, incubators, water baths, refrigerators/freezers) used at the time of the
evaluation was verified as being certified, calibrated, or validated.
4.2 QC Results
QC efforts conducted during testing included positive and negative controls for both spread plate
samples and qPCR. In addition, Btk spike suspensions were quantified to verify either CFU/mL titer or
target spike concentrations.
Positive and negative control results were within the target requirements for the qPCR. Applied
Biosystems 7500 Fast system performance was assessed according to internal standard operating
procedures (SOPs) and maintained at regular intervals—monthly (optical and background calibration),
every 6 months (dye calibration), and annually (RNase P calibration). For culture analysis, the PC spore
stock maintained a single morphological appearance consistent with Btk T1B2 throughout the study, as
determined at the beginning of each trial. Media and reagents used for culture analysis were screened
(negative controls) and had no growth, showing that reagents used were not the source of contamination.
4.3 Operational Parameters
Micropipettes, thermometers, and timers used were calibrated against a traceable standard at regular
intervals (every 6 months or annually) and used only within the acceptable calibration interval
established by internal SOPs.
4.4 Audits
4.4.1 Performance Evaluation Audit
Performance evaluation audits were conducted to assess the quality of the results obtained during these
experiments. Table 22 summarizes the PE audits that were performed; the equipment was verified to be
within an acceptable tolerance range.
Table 22. Performance Evaluation Audits
Measurement
Audit
Allowable
Actual
Procedure
Tolerance
Tolerance
Volume of liquid from
micropipettes
Gravimetric evaluation
± 10%
Passed calibration as
found/as returned
Time
Compared to independent
clock
± 2 sec/h
Passed calibration as
found/as returned
Temperature
Compared to independent
calibrated thermometer
±2°C
Passed calibration as
found/as returned
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4.4.2 Technical Systems Audit
A technical system audit was conducted on laboratory procedures under STREAMS Task Orders in
January 2021 and July 2021 to ensure that tests were being conducted in accordance with the appropriate
QAPP and QMP.
4.4.3 Data Quality Audit
At least 10% of data acquired during the evaluation were audited. Data were reviewed from November
9, 2020 through May 17, 2021. A QA auditor traced the data from the initial acquisition, biologic plate
counts, PCR ACt calculation, data reduction and statistical analysis, to final reporting to ensure the
integrity of reported results. All calculations performed on the data undergoing the audit were verified.
No issues were noted with the data collection and reporting process, and all calculations were performed
accordingly.
4.5 QA/QC Reporting
Each assessment and audit was documented in accordance with the QAPP and QMP. For these tests, no
findings were noted during the TSA or in the data quality audit, and no follow-up corrective action was
necessary. QA/QC procedures were performed in accordance with the QAPP.
4.6 Data Review
Records and data generated in the evaluation received a QC/technical review before they were utilized
in calculating or evaluating results and prior to incorporation in this report.
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5.0 SUMMARY OF METHOD
OBSERVATIONS AND EXPERIENCES
While implementing the method, key observations and experiences were noted that will be useful to
understand and/or take into consideration for future iterations or versions.
5.1 Sample Processing Considerations
Sponge sticks, VFCs, wash water, and grab samples each have unique processing procedures,
complicating the analyses by requiring different protocols and equipment. Samples will need to be
batched according to sample type and transported to a laboratory with the necessary equipment. Sponge
sticks require the use of a Stomacher 400 and swinging bucket centrifuge, VFCs require a bath
sonicator, and wash water and grab samples require a filter manifold for concentrating the sample
volume onto a filter membrane.
For gravel ballast sample processing, the 500 mL bottles (Daigger Item # EF2247C) specified in the
extraction protocol for gravel ballast (Serre and Oudejans, 2017) are high-density polyethylene (HDPE)
and are not autoclavable. The bottles become misshapen when autoclaved at 121°C gravity cycle for 15
min, Figure 62 shows rounding of the bottom of a bottle postautoclaving that could lead to sample spills
during processing. These bottles should be sterilized by irradiation or another method, or a replacement
product that can be autoclaved should be considered.
Figure 62. Autoclaving HDPE Bottles Compromises their Structure
Grab sample suspension/rinsate can lead to clogged MicroFunnel membranes, limiting the total volume
that can be analyzed per sample.
For soil samples with a 0.25 g/mL ratio of soil to extraction buffer, limit of detection may not improve
even if more volume were available for analysis for either method (culture or RV-PCR) because the high
background microbial flora that survive the heat shock and are recovered alongside target spores and the
particulates lead to clogging of the filter. Data suggest for the soil sample, plating >0.1 mL suspension
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will produce an overwhelming level of background microbial growth. The sterile soil sample spiked
with 3,000 CFU target spore level had < 10 colonies per 0.1 mL plate, so plating dilution series would
make detection of spores less likely. Filtering 20 mL through the filter vial for the RV-PCR method
averaged 41 min, with an additional -15 min for each of the two subsequent washes with 10X PBS and
IX PBS, making this a time-consuming process.
5.2 Method Qualitative Assessment
Given our experiences running analytical methods, there are pros and cons for both methods.
5.2.1 Culture Method
The strengths of the culture method are that it allows for a quantifiable measure of target; isolation of
target organism; and confirmatory PCR screening of colonies that provides a definitive result.
Weaknesses of the culture method are that background microbial flora can overwhelm culture plates and
obscure colony morphology, leading to false negative results. Additionally, background microbial flora
with a similar or identical morphology can be present within samples, triggering PCR screening of
colonies and possibly repeated PCR screening (to minimize risk of false negatives) if presumptive
morphology is present in large numbers.
5.2.2 RV-PCR Method
The strengths of the RV-PCR method are that it is akin to a biological indicator, it gives a positive or
negative result and there is no iterative or repeat analysis on sample aliquots, giving the method a clear
end of analysis without the need for multiple follow-up PCR screenings. The method can provide rapid
results, which is of high significance in a wide-area incident involving multiple cities and environments.
RV-PCR constitutes a small laboratory footprint and requires less culture media, resulting in relatively
less BSL-3 waste. The weaknesses of the RV-PCR method are that it does not allow for quantification of
target; target organism is not isolated for banking (unless additional streak plating is performed using the
7/aliquot); DNA purification steps are time-consuming; each sample is split into To and 7/aliquots
resulting in two DNA purification extractions per sample and six PCR reactions per assay (may be
improved if automated DNA extraction is performed, and multiplex assays are available and validated);
and the presence of background microbial flora could compete with target organism growth during
enrichment, suppressing signal due to interferent and potentially leading to false negative results.
5.2.3 Time/Cost Estimates
The sample analyses were performed in laboratory analysis batches of 16 samples using a single
manifold system for RV-PCR. The 16 samples were the maximum that was deemed reasonable to
process considering a normal 8:00 AM to 5:00 PM workday, without overtime and/or a night shift that
may be used by the EPA's Environmental Response Laboratory Network (ERLN) if actual emergency
response samples were being processed. A single batch was completed over four to five consecutive
days of operation, starting with sample control spiking and spore recovery and culturing on Day 1 (refer
to Figure 31 in Section 2.7). (Note: had these been actual samples collected postbiological release, the
spiking activity would, obviously, not be performed by the ERLN). Day 2 consisted of culture colony
counting from agar plates, presumptive colony selection for PCR screening, and nucleic acid extraction
for RV-PCR. Day 3 involved PCR analysis of colonies from culture plates and To and 7/aliquots for
RV-PCR. For samples that were not confirmed positive by colony PCR, additional streak plates of the
enriched filter or sponge stick were performed on Day 4 and Day 5 for the culture method. If incubation
time for RV-PCR was reduced to 9 h and a night shift performed nucleic acid extraction, results could
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be completed in the next day after spore recovery. If additional enrichment of the sponge stick or filter is
not performed for the culture method, indicated as optional in the EPA Protocol for initial and clearance
stages, results could be completed the next day after spore recovery. The text below is quoted from page
53 (EPA, 2017).
Note: "For faster sample analysis results during the initial stages of an incident (e.g., incident
characterization) and during post-decontamination/clearance phase, it is recommended that the
remainder of all suspensions (e.g., undiluted, 10'1 and 10~2 dilutions) be filtered using an
additional MicroFunnel™ and plated as described above, instead ofproceeding with enrichment
in TSB."
Estimated staff time to spike 16 samples with Btk spores and then process and analyze them was
approximately 56 h of labor and $1,500 of consumables. The estimation of 56 h of staff time was
distributed as follows:
8 h for activities related specifically to the spiking of the materials being assessed, which was a
requirement of the study, but not an activity that would be performed had these been actual field
samples. This task included time to prepare the stock suspensions, enumerate stock suspensions,
spike the samples, and prepare associated documentation.
• 10 h for spore recovery.
14 h for culture analysis.
• 24 h for RV-PCR analysis.
If the EPA method had been followed without any changes (most notably the samples would not be split
for analysis and either the culture only or the RV-PCR method only would have been used), a batch of
16 samples would take an estimated 34 h of labor and $1,000 of consumables to perform culture analysis
(with PCR confirmation of at least three colonies per sample). To process the same number of samples,
an estimated 40 h of labor and $1,200 of consumables would be required using RV-PCR analysis. Each
of these methods can generate results within two days for analysis of the recovered spore suspension;
additional time would be required for enrichment of the sponge stick or filter. The labor required for
nucleic acid extraction for 16 samples is -14 h; exploring options to reduce this labor or using an
automated sample processing or/and automated nucleic acid extraction procedure may increase
throughput and reduce sample cost. Thus, there is no significant time or cost advantage for one method
over the other as per the methods with modifications used in this effort.
5.3 Culture Processing Considerations
Background growth and grime interfere with target Btk T1B2 morphology identification on culture
plates. Presumptive Btk T1B2 colonies need to be PCR-screened to confirm or refute the presence of the
target organism. The method defines that a minimum of three presumptive colonies (for this project,
Btk T1B2) are screened to confirm the presence of the target organism. The method should define a
maximum number of colonies that should be screened; otherwise, all presumptive colonies would need
to be screened.
5.3.1 BHIB Enrichment Culture Analysis
The culture method detailed in the EPA Protocol (EPA, 2017) instructs users to streak turbid BHIB
enrichment culture in triplicate on solid media plates; then, if any colonies with target morphology are
isolated, to PCR screen those target colonies. A similar protocol is also used by the CDC-LRN. PCR on
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a 50-|iL aliquot of the BHIB culture is performed only if a colony with target morphology is not
observed on streak plates. In previous evaluation of BHIB enrichment culture, data showed that the
target organism was not isolated from sponge sticks and VFCs when streaked from turbid enrichment
broth for nonfield blank samples due to the significant presence of the competing background organisms
in those samples; however, the target organism was present in enrichment broth for many VFC samples,
as determined by PCR analysis of an aliquot of broth (Calfee et al., 2019). Given these results, it would
be preferred to perform PCR on enrichment broth, and only streak additional plates from the enrichment
broth if attempting to isolate the target organism from BHIB enrichment following a positive PCR
identification.
BHIB enrichment for sponge sticks samples is not as effective as enrichment for 47-mm filter membrane
samples, which may be because a 25-mL volume of BHIB does not completely cover the sponge within
a specimen cup and/or because the recovery of spores from VFCs is not as efficient as from sponge
sticks; thus, more spores remain on the VFC membrane than on the sponge.
5.4 RV-PCR Processing Considerations
The RV-PCR method requires great care and diligence to implement effectively. Most notably, the
method requires changing gloves between samples for each step, which is onerous and time-consuming.
However, this added measure is critical when analyzing samples from the field collected after an
incident, as the samples and associated results are high-value and high-impact—they will support key
decisions in the response and impact response timelines, credibility, and cost. During the RV-PCR
method, when applying vacuum to the filter vial manifold, the filtrate is pooled in the manifold reservoir
and contacting the bottom of the filter vials near the vacuum source. It is recommended to increase the
depth of the bottom section of the manifold so that the filtrate does not pool and contact the bottom of
filter vials.
Recovered sample suspensions with high particulate loads can clog the RV-PCR filter vials. As a result,
the below two rules were applied to expedite sample processing and the inclusion of buffer washes:
• At 15-min, post-sample addition to the filter vial. If the sample did not pass through the filter
vial, a reduced volume of high and low salt wash buffer (5 mL) was added, rather than omitting
one or both entirely.
• At 1-h, post-sample addition to the filter vial. If the sample did not pass through the filter vial,
the high and low salt wash steps were omitted.
The RV-PCR manifold, capping tray, and manifold incubator rack for holding manifold/capping trays in
the shaker incubator are custom-manufactured equipment. Scaling up sample processing would need to
consider supply chain and time required to manufacture custom parts.
5.4.1 Biological Safety Level 3 Considerations
Transfer of the RV-PCR manifold/capping tray method into a BSL-3 laboratory may present sample
handling challenges, but they are expected to be manageable with proper training of experienced staff.
The filter vials are sealed via a capping tray with a compression luer cap that does leak on occasion and
are arranged in a tray with little space between vials, making physical wiping of the vials with
decontaminant a challenge. Direct contact between the metal capping tray and plastic bags during
shaking incubation and platform vortex mixing can lead to bag tears. Therefore, packaging of manifold
within durable (8-mil thickness recommended) bags or use of a biocontainment box with absorbent is
recommended for incubation and platform vortex mixing to avoid select agent release during these steps.
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Considerations for proper containment and effective implementation of containment by properly trained
and experienced staff are expected to work in such an environment.
5.4.2 Suggestions to Improve RV-PCR Throughput
5.4.2.1 Nucleic Acid Extraction
For a set of 16 samples, the nucleic acid extraction procedure takes -14 h of labor and consumes 624
1-mL micropipette tips, 96 200-|iL micropipette tips, and 80 2-mL microcentrifuge tubes in addition to
the nucleic acid kit consumables. PCR analysis of a 50-|iL aliquot of the 7/BHIB from filter vials using
thermolysis instead of nucleic acid extraction procedure may produce results similar to the To and 7/
aliquots that were extracted using the nucleic acid extraction procedure, with less labor, consumables
and biohazardous waste generated. As per the EPA Technical Lead for this effort, however, considering
the post-2001 Amerithrax response queries from the U.S. Government Accountability Office, stringent
DNA extraction procedures were warranted to minimize PCR inhibition and were included in the EPA
Protocol (EPA, 2017). The RV-PCR process of washing filter vials with 10X and IX PBS may decrease
PCR inhibitors to a level that reduces the risk of a false negative sample. Alternatively, a different DNA
purification technique could be utilized. Bushon et al. 2021 found that a Qiagen DNA purification kit
reduced processing times compared to the Promega purification procedure utilized in this study.
5.5 Sponge Stick Sample Analysis
5.5.1 Biological Safety Level 3 Considerations
Transfer of the sponge stick method into a BSL-3 laboratory will present sample handling challenges.
Stomacher 400 equipment footprint fills the depth of a Class II BSC and is a high energy
homogenization process in a nonrigid container (Stomacher bag) that is subject to puncture from
particulates recovered from heavily soiled surfaces, and leakage may occur.
Stomacher bag stands are available to hold bags upright to prevent tipping and spillage, but the
stand fills the depth of a Class II BSC.
Transferring volume from the Stomacher bag to and from tubes is subject to dripping and
spillage.
5.5.2 Sponge Stick Method Considerations
The sponge stick method uses 90 mL of buffer to extract a sponge. The next step following stomaching
is to reduce the volume by centrifugation. Percent recovery could potentially be gained by reducing the
volume used for stomaching.
5.6 Vacuum Filter Cassette Sample Analysis
Recovery efficiencies are low for this sampling method, possibly due to poor removal of spores from
surfaces or poor recovery from the vacuum cassette. A vortex mix rather than bath sonication may
improve recovery of spores from the VFC filter. The bubble and cavitation energy of a bath sonicator
may not transfer through plastic tubes/2 oz. cups, and the signal may be damped by racks or distributed
nonuniformly. A previous program assessing sonication in the recovery of spores from soil samples did
not improve spore recovery (Silvestri, 2016).
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5.7 Grab Sample Analysis
The approach used for this project for analysis of washdown or grab samples was to concentrate the
collected eluate (250 mL) onto a 47-mm mixed ester cellulose filter membrane (0.45 |im), then recover
the spores from the filter membrane using 20 mL of extraction buffer (PBSTE) with agitation for culture
and RV-PCR analysis methods. The concentration of suspension onto this 47-mm filter and subsequent
removal of the spores may reduce recovery of spores for both culture and RV-PCR analysis. Culturing
the 47-mm filter directly onto solid media for the culture approach or broth enrichment of this filter may
improve detection limits.
Additionally, grab samples can contain particulates that clog the filter membrane, limiting the total
volume of sample that can be processed. A centrifugation step, rather than membrane filtration, for
concentration should be considered to allow more sample volume to be processed. For vegetation
samples, the membrane filter clogged following filtration of 132 ± 42 mL of the 500 mL available.
Bushon et al. 2021 demonstrated that results obtained using a centrifugation step were comparable to
membrane filtration and took less time.
5.8 Difficult-to-Analyze Sample Types and Recommendations
Grab samples had fewer true positive/true negative (true results) samples for both culture (75 %) and
RV-PCR (63 %) methods. All of the nontrue grab sample results were false negative, meaning the
samples were spiked with Btk T1B2 spores, and the samples were not confirmed positive by PCR. For
each of the grab sample types, background microorganisms recovered alongside target spores are the
main contributor to false negative samples, either masking the presence of target colonies or
outcompeting the target organism for nutrients. To reduce the impact of background microorganisms,
30% ethanol was a component in the recovery buffer for wash water, gravel, and vegetation to inhibit
growth or reduce viability of vegetative organisms without affecting the Btk spores. For soil samples, a
heat shock (70°C) was incorporated to reduce the viability of vegetative organisms. Background
microorganisms with presumptive Btk morphology were still recovered at levels of 30 to 100 CFU/mL
(wash water 1), 8.8 to 180 CFU/mL (gravel), 105 to 167 CFU/mL (vegetation) and 23 to 57 CFU/mL
(soil) for zero spike samples. Total background microorganisms for these samples were 1,490 to 1,800
CFU/mL (wash water 1), 80-262 CFU/mL (gravel), 334-750 CFU/mL (vegetation), and 8,040 to 23,160
CFU/mL (soil) for zero spike samples. By comparison, the 300 CFU spike level samples would have a
maximum of 15 CFU/mL following concentration of the 500 mL spore recovery volume to 20 mL for
wash water, gravel, and vegetation or 7.5 CFU/mL for the soil samples.
Particulates in grab samples can reduce the maximum total volume concentrated by filtration,
particularly for vegetation and soil samples. However, particulates will vary in field samples, so it is
expected to be a problem to overcome for all grab samples. For vegetation samples, 132 ± 42 mL of
500 mL total volume was processed through the MicroFunnel filter (47 mm, with 0.45 |im pore size).
The RV-PCR filter vials are also susceptible to clogging or reduced flow rates from particulates within
grab samples. The soil samples averaged 41 min of filtration time for 20 mL of spore recovery to pass
through the filter vial, and additional time was required for each of the two filter vial wash steps.
Reduction of background microorganisms prior to culture could be explored using chemical treatment,
perhaps a higher concentration of ethanol, and/or heat shock for all grab sample types. Another
possibility is to evaluate selective broth media for enrichment rather than BHIB, which is a nutrient rich
nonselective medium.
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Removal of particulates by centrifugation would be a method to explore for reducing the impact of filter
clogging. Post-centrifugation, the supernatant could be concentrated onto a filter, and the particulates
could be resuspended in a buffer or media for detection of target spores separately or recombined with
the supernatant following the filtration of the supernatant to maximize sample capture onto filter
membranes and shorten filtration times.
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6.0 CONCLUSIONS
Samples of residual inert and biological deposits on representative maritime asset surfaces
(e.g., aluminum on boats, nonskid tread on decks of boats, touchscreens, and concrete piers) and
surrounding grounds and infrastructure and materials (e.g., soil, vegetation, and gravel) were collected
using established EPA methods for sponge stick wipes, VFCs, and grab samples for bulk material
collection. The ambient background of inert and biological material would also be present if sampled
following a biological contamination incident such as anthrax. Two sampling campaigns were
successfully completed at the USCG Base Portsmouth, VA (one on 04 November 2020 and one on
26 March 2021).
Overall, for samples with maritime inert and biological deposits (sponge sticks, VFCs, and grab), the
culture method resulted in 10 false positive results, and the RV-PCR method resulted in 0. Overall, there
were 19 false negative results for the culture method and 26 false negative results for RV-PCR for
samples with maritime inert biological deposits (sponge sticks, VFCs, and grab).
Samples with high microbial background load can mask the identification of target colonies on agar
plates for the culture method and lead to RV-PCR signal suppression. Particulates within samples can
reduce the amount of sample volume processed and increase sample processing times during filtration
steps, particularly for vegetation and soil grab samples. Isolation of background microorganisms with
target morphology from environmental samples complicates culture analysis, and requires PCR
confirmation, which can delay sample analysis results.
Biological decontamination response scenarios will be widely varied and as demonstrated by this
project, require multiple different sample types to determine extent of contamination, decontamination
efficacy, and clearance phase monitoring. Given all these variables, there may be instances where both
the culture methods and RV-PCR methods described here are applicable for use. In general, our
summary of the culture method is that it is labor- and reagent/material-intensive, but very
straightforward for laboratory staff to accomplish. Our observation of the RV-PCR method is that it has
the potential to be much more streamlined, with less labor and fewer laboratory consumables. However,
to deal with the low contamination levels and relatively high background in the samples included in this
project, the RV-PCR method required labor intensive steps—such as sequential mixing and wash steps,
DNA purification, and custom manifolds and equipment—which may negate some of the
advantages. Our data demonstrated that one example of a scenario where the culture method may be
preferable would be for grab samples (75% correct results for culture compared to 63% for RV-PCR)
and one example of a scenario when the RV-PCR methods would be preferable would be sponge sticks
(98%) correct results for RV-PCR compared to 84% for culture). The bottom line is that each response
will have to be considered on its own merits, and both methods will likely be used in various situations.
It would be valuable to assess ways to simplify both analytical methods to improve turnaround time and
reduce the amount of biohazardous waste generated. To reduce iterative analysis with the culture
method, a maximum number of colonies screened from a sample should be defined. Otherwise, field
samples with high background microbial loads with presumptive target morphology would need an
indefinite number of colonies screened. In addition, spread plating multiple dilutions in triplicate is
useful in establishing a quantitative measure of the presumptive colonies, but for detection purposes,
triplicate plating may not be necessary and reducing to single or duplicate replicates could reduce the
total number of plates (media) consumed. For the RV-PCR method, a reduction in processing steps
could be evaluated in a sample complexity-dependent manner to determine which steps are crucial for
detection. Additionally, use of automated sample processing and nucleic extraction can further expedite
sample analysis using the RV-PCR method with more accuracy.
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7.0 REFERENCES
Buckley, P., Rivers, B., Katoski, S., Kim, M.H., Kragi, F.J., Broomall, S., Krepps, M., Skowronski,
E.W., Rosenzweig, C.N., Palkoff, S., Emanuel, P., Gibbons, H.S. (2012). Genetic Barcodes for
Improved Environmental Tracking of an Anthrax Simulant. Applied and Environmental
Microbiology 78(23): 8272-8280.
Bushon, R.N., Brady, A.M.G., Kephart, C.M., Gallardo, V. (2021). Evaluation of a Modified Rapid
Viability-Polymerase Chain Reaction Method for Bacillus atrophaeus Spores in Water Matrices.
Journal of Microbiological Methods 188: 106293.
Calfee, M. W., Rose, L.J., Morse, S., Mattorano, D., Clayton, M., Touati, A., Griffin-Gatchalian, N.,
Stone, C., McSweeney, N. (2013). Comparative evaluation of vacuum-based surface sampling
methods for collection of Bacillus spores. Journal of Microbiological Methods 95(3): 389-396.
Calfee, M. W., Shah, S., Lee, S., Mickelsen, R., Cruz, F., Karim, K., Ackelsberg, J., Gemelli, M.,
Hofacre, K. Evaluation of Analytical Methods for the Detection of Bacillus Anthracis Spores:
Compatibility with Real-World Samples Collected from Outdoor and Subway Surfaces. U.S.
Environmental Protection Agency, Washington, DC, EPA/600/R-19/083, 2019
Centers for Disease Control and Prevention (CDC). Anthrax Surface Sampling Guide, Available at
https://www.cdc.gov/niosh/topics/anthrax/sampling.html Accessed 9/27/2021.
Greenberg DL, Busch JD, Keim P, Wagner DM. Identifying experimental surrogates for Bacillus
anthracis spores: a review. Investig Genet. 2010 Sep 1;1(1):4. doi: 10.1 186/2041 -2223-1 -4. PMTD:
21092338; PMC1D: PMC2988482
Letant, S.E., Murphy, G.A., Alfaro, T.M., Avila, J.R., Kane, S.H., Raber, E., Bunt, T.M., Shah, S.R.
(2011) Rapid- Viability PCR Method for Detection of Live, Virulent Bacillus anthracis in
Environmental Samples. Applied and Environmental Microbiology 77(18): 6570-8.
Mikelonis, A.M., et al., "Comparison of surface sampling methods for an extended duration outdoor
biological contamination study." Environ Monit Assess, 2020. 192(7): p. 455.
Rose, L.J., Hodges, L., O'Connell, H., Noble-Wang, J. (2011). National Validation Study of a Cellulose
Sponge Wipe-Processing Method for Use after Sampling Bacillus anthracis Spores from Surfaces.
Applied and Environmental Microbiology 77(23): 8355-8359.
Serre, S. and Oudejans, L. Underground Transport Restoration (UTR) Operational Technology
Demonstration (OTD). U.S. Environmental Protection Agency, Washington, DC, EPA/600/R-
17/272, 2017.
Silvestri, E. E., Feldhake, D., Griffin, D., Lisle, J., Nichols, T.zl., Shah, SN., Pemberton, A., Schaefer
III, F.W. (2016). Optimization of a Sample Processing Protocol for Recovery of Bacillus anthracis
Spores from Soil. Journal of Microbiological Methods 130: 6-13.
Tufts, J. A., Meyer, K.M., Calfee, M.W., Lee, S.D. (2014). Composite Sampling of a Bacillus anthracis
Surrogate with Cellulose Sponge Surface Samplers from aNonporous Surface PloS ONE 9(12):
el 14082.
Tufts, J. A.M., Calfee, M.W., Lee, S.D., Ryan, S.P. (2014) Bacillus thuringiensis as a Surrogate for
Bacillus anthracis in Aerosol Research. World Journal of Microbiology and Biotechnology 30:
1453 1461. Available at: https://doi.org/10.1007/sll274-013-1576-x: Accessed 9/24/21.
USCG 2015. Proceedings of the Marine Safety & Security Council, Spring 2015, The Coast Guard
Journal of Safety Security at Sea, 72(1), 2015.
U.S. EPA2013. Bio-Response Operational Testing and Evaluation Project-Phase 1: Decontamination
Assessment. U.S. Environmental Protection Agency, Washington, D.C., EPA/600/R-13/168.
U.S. EPA 2017. EPA Protocol for Detection of Bacillus anthracis in Environmental Samples During the
Remediation Phase of an Anthrax Incident, 2nd Edition. U.S. Environmental Protection Agency,
Washington, DC.
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Evaluation of Analytical Methods for
Detection of Bacillus anthracls Surrogate
Spores: Compatibility with Real-World
Maritime Environmental Samples
Collected from USCG Assets and Facilities
Appendices A-N
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List of Appendices
Page
APPENDIX A: WORK INSTRUCTION FOR SURFACE SAMPLING USING CELLULOSE
SPONGE STICKS A-1
APPENDIX B: WORK INSTRUCTION FOR SURFACE SAMPLING USING VACUUM
CASSETTE FILTERS B-1
APPENDIX C: WORK INSTRUCTION FOR WATER WASHDOWN COLLECTION C-1
APPENDIX D: WORK INSTRUCTION FOR GRAVEL SAMPLING D-1
APPENDIX E: WORK INSTRUCTION FOR SOIL SAMPLE COLLECTION E-1
APPENDIX F: WORK INSTRUCTION FOR VEGETATION SAMPLING F-1
APPENDIX G: WORK INSTRUCTION FOR FORMULATIONS OF RECIPES USED IN
BIOLOGICAL TEST METHODS G-1
APPENDIX H: WORK INSTRUCTION FOR SPIKING WITH BACILLUS THURINGIENSIS
KURSTAKI (Btk) HD-7 T1B2 SPORES H-1
APPENDIX I: WORK INSTRUCTION FOR BACILLUS THURINGIENSIS KURSTAKI (Btk)
T1B2 SPORE RECOVERY FROM MARITIME SAMPLES - SPONGE STICKS, VACUUM
CASSETTES, AND GRAB SAMPLES 1-1
APPENDIX J: WORK INSTRUCTION FOR CULTURE OF BACILLUS THURINGIENSIS
KURSTAKI (Btk) T1B2 SPORES RECOVERED FROM SPONGE STICK WIPES, VACUUM
FILTER CASSETTES, AND GRAB SAMPLES J-1
APPENDIX K: WORK INSTRUCTION FOR MANUAL DNA EXTRACTION AND
PURIFICATION FROM BACILLUS THURINGIENSIS KURSTAKI (Btk) T1B2 SPORES K-1
APPENDIX L: WORK INSTRUCTION FOR REAL-TIME PCR ANALYSIS FOR BACILLUS
THURINGIENSIS KURSTAKI (Btk) T1B2 DNA L-1
APPENDIX M: WORK INSTRUCTION FOR SELECTING PRESUMPTIVE BACILLUS
THURINGIENSIS KURSTAKI (Btk) T1B2 COLONIES FOR QPCR CONFIRMATION M-1
APPENDIX N: WORK INSTRUCTION FOR BHIB ENRICHMENT FOR CULTURE N-1
ix
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APPENDIX A: WORK INSTRUCTION FOR SURFACE SAMPLING
USING CELLULOSE SPONGE STICKS
1.0 References
1.1 Miscellaneous Operation Procedure (MOP) 6583 "Assembly of 3MSponge Stick™
Kits. " Prepared by Arcadis, Inc. for the National Homeland Security Research Center
1.2 CDC NIOSH: Surface sampling procedures for Bacillus anthracis spores from
smooth, non-porous surfaces
1.3 CDC NIOSH: Instructor Guide for Anthrax Surface Sampling
2.0 Surface Preparation
Note: This procedure utilizes pre-prepared 3M sponge stick sampling kits, which are
assembled in accordance with Reference 1.1. For information on assembly, refer to this
document.
All preparation and sampling will be done in two-person teams consisting of a sampler
and an assistant.
2.1 Before beginning, the sampler and assistant will don a new pair of gloves overtop
of their primary personal protective equipment (PPE). The assistant need only don a
new pair at the start of the first sample. A glove change for the assistant is not
necessary provided that the gloves remain unsoiled throughout their use.
2.2 Next, the sampler will obtain a clean 10" x 10" Teflon® sampling template from the
assistant and place it over the desired area, if necessary, using pieces of tape on the
outside edges to secure the template in place.
2.2.1 If the surface is unable to accommodate a 10" x 10" sampling area,
measure out an area equivalent to 100 in2. The same sampling procedure
will remain unchanged for the alternate test area.
3.0 Sampling
3.1 When ready to sample, the assistant will retrieve one pre-made sponge stick kit and
open the outer overpack bag, as well as the bag containing the sponge stick. Care will
be taken to ensure that the assistant does not touch the sponge or its handle. The
assistant will then present the open sponge stick bag to the sampler.
3.2 The sampler will remove the sponge stick by grasping the handle above the thumb
stop, making sure not to touch below the stop.
3.3 In total, the sampler will make four passes over the area inside of the template.
3.3.1 Pass 1: Starting in a corner of the template, place the sponge flat on the
surface against one of its widest faces and apply gentle, but firm pressure.
Ensure that the entire face of the sponge is in contact with the surface.
While keeping the sponge pressed down, move the sponge horizontally, in
an overlapping S-pattern over the entire area of the template. Covering the
entire area will take approximately eight passes of the sponge.
A-1
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3.3.2 Pass 2: Turn the sponge over (to the opposite wide face) and in a similar
fashion, use a vertical, overlapping S-pattern to cover the entire surface.
Covering the entire area will again take approximately eight passes of the
sponge.
3.3.3 Pass 3: Turn the sponge so that one of the narrow faces of the sponge will
be flat against the surface. Starting in a corner of the template area, use a
similar overlapping S-pattern to cover the surface, diagonally toward the
opposite corner. Strokes will be 45° to the previous two patterns. Upon
reaching the center of the surface, flip the sponge over (to the opposite
narrow face) and continue sampling the remaining half of the surface using
the same technique. Covering the entire area will take approximately 14
passes of the sponge.
A-2
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3.3.4 Pass 4: Finally, using the full width of the sponge tip, wipe the perimeter
of the sampling area once, in a circular pattern.
3.4 While the sampler is working, the assistant will document the layout of the
sampling location, which can be done using pictures, drawings, comments, or a
combination of the three.
4.0 Sample Recovery
4.1 When the sampler has finished, the assistant will open a specimen container and
hold it out for the sampler.
4.2 Without handling or taking the cup from the assistant, the sampler will press the
sponge into the bottom of the cup and break off the head by moving the handle back
and forth until it separates.
4.2.1 The sampler should take care to not touch below the thumb stop on the
sponge stick while attempting to break off the head.
4.3 Once the head has been separated into the cup, the assistant will then secure the lid,
label the container accordingly, and seal the lid with a strip of parafilm.
4.4 The assistant will place the cup into a resealable secondary container (one secondary
container will hold all the samples from one location).
A-3
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4.5 The overpack bag and stomacher bag (if included) from each kit will be retained and
packed back into their resealable secondary containment (bag, bin, etc.).
4.5.1 Attempt to remove excess air from the samples and secondary containers to
aid in repackaging.
4.6 While the assistant is packing, the sampler will move the template to the next
sampling location and then discard their outer glove gloves.
4.7 Steps 2.1 through 4.6 will be repeated for each sample taken.
5.0 Storage and Shipping
Note: The following procedure will be performed prior to storing samples, moving
samples offsite, or shipping samples to the laboratory.
5.1 Wipe the internal surfaces of any sample shipping or storage container with a bleach
or disinfectant wipe.
5.2 When all samples from a location have been collected, sealed, and placed in
secondary containment, wipe down the outside of the secondary containment with
bleach or a disinfectant wipes and then move the secondary containers into storage,
along with any corresponding overpack and stomacher bags.
5.2.1 Attempt to remove as much excess air from within containers as possible.
5.3 If shipping the samples, place enough cold packs or water ice among the secondary
containers to ensure that samples remain cold for the duration of the trip to the
processing laboratory.
5.4 Any samples that will not be shipped/moved the day of collection, will be retained in
a refrigerator or freezer until they can be shipped.
5.5 Samples will be processed within 48 hours of collection.
A-4
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APPENDIX B: WORK INSTRUCTION FOR SURFACE SAMPLING
USING VACUUM CASSETTE FILTERS
1.0 References
1.1 Miscellaneous Operating Procedure (MOP) 3164: "Procedure for 37MM Cassette
and Trace Evidence Filter Vacuum Sampling of Large and Small Coupons
Prepared by Arcadis, Inc. for the National Homeland Security Research Center
2.0 Flow Check
Note: This procedure utilizes pre-prepared 37-mm vacuum cassette sampling kits, which
were assembled in accordance with Reference 1.1 above. For information on assembly of
these kits refer to this document.
All preparation and sampling will be done in two-person teams consisting of a sampler
and an assistant.
2.1 Start the sampling pump.
2.2 Using a length of Tygon® tubing (approximately 3 ft long), attach a 37-mm cassette,
denoted "Flow Check" to the pump.
2.3 Attach a rotameter or equivalent flow measuring device upstream from the cassette
and adjust the pump until a flow of 5 ± 0.5 liters per minute is achieved.
2.4 Record the rotameter value as well as the setpoint/flow readout value on the pump.
2.4.1 This value will be considered representative of the flow through cassettes
being used for sampling.
2.5 When pump adjustment is complete, remove the vacuum cassette and leave the
length of Tygon tubing attached for later sampling.
3.0 Sampling Preparation
3.1 Before beginning, the sampler and assistant will don a new pair of gloves over top
of their primary personal protective equipment (PPE). At a minimum, the sampler
will always don a new pair of gloves before beginning a new sample; however, the
assistant need not change gloves provided they remain unsoiled.
3.2 Next, the sampler will obtain a clean 12" x 12" template from the assistant and
place it over the desired area, if necessary, using pieces of tape on the outside edges
to secure the template in place.
3.2.1 If the surface is unable to accommodate a 12" x 12" sampling area,
measure an alternate area equivalent to 144 in2. The same sampling
procedure will be used for the alternate test area.
3.3 When ready to proceed, the assistant will open a pre-made cassette kit and remove
the sealed cassette bag from within.
3.4 When able, the assistant will record the bag collection ID on the sampling log sheet,
ensuring that the sample ID matches the current location.
B-1
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3.5 The assistant will then open the cassette bag, and hold it so that the sampler can
remove the cassette and any components within.
3.5.1 The sampler may need to assemble the cassette filters by removing the red
plugs, attaching the front nozzle, and attaching a back-PVC adaptor. Ensure
that the red plugs are kept clean and saved for later use.
3.6 When the cassette is ready, the assistant will ensure that the length of Tygon tubing
used in Step 2.2 is attached to the sampling pump and will remove any previously
used PVC adaptors from the free end. The free end will then be handed to the
sampler.
3.7 The sampler will connect this end to the vacuum cassette filter using the
downstream PVC adaptor on the cassette.
3.7.1 After attaching the tubing, the sampler will ensure that the orientation of
the cassette is correct, and all fittings are appropriately attached.
3.8 At this time, the assistant will obtain a stopwatch in order to time the sampling
procedure.
4.0 Sampling
4.1 Each cassette sample will consist of two portions, each lasting 150 seconds for a total
of 300 seconds of sampling. For the first portion, the sampler will perform
horizontal S-strokes across the width of the template at an approximate rate of
3 seconds per pass for a total of approximately 50 passes covering the entire area.
During the second portion, the sampler will perform vertical S-strokes across the
area at a similar rate, for an additional 50 passes covering the entire area. Figure B-l
illustrates the sampling pattern for each portion.
Figure B-l. Mock illustration of the first (left) and second (right) portion vacuum
cassette sampling patterns.
4.2 When ready to begin sampling, the sampler will position the cassette nozzle in the
corner of the template and turn on the pump. Upon initiating flow, the sampler will
begin vacuuming over the area, using horizontal S-strokes.
4.2.1 The Tygon nozzle should be kept angled such that the tapered end of the
nozzle is flush with the surface.
4.3 As soon as the sampler begins sampling, the assistant will start the stopwatch and
monitor the progress of the sampler.
B-2
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4.4 While the sampler is working, the assistant will document the layout of the
sampling location, which can be done using pictures, drawings, comments, or a
combination of the three.
4.5 When the first portion (150 seconds) has elapsed, the assistant will prompt the
sampler to change direction of sampling and will continue to monitor progress.
4.5.1 It may be necessary for the assistant to call out intermediate times during
the sample portions (i.e., halfway, 75 seconds, 30 seconds left) in order to
ensure that the sampler is keeping a good pace and covering the entire area
in the allotted time. At conclusion of the first portion, the sampler should
return to a corner of the template before continuing with the vertical passes.
4.6 At the conclusion of the second portion (300 seconds), the assistant will prompt the
sampler to stop the pump and conclude the sample.
5.0 Sample Recovery
5.1 After completion of the sample, the sampler will remove the nozzle from the
cassette and retain it in their hand, while holding the cassette with the opposite hand.
5.1.1 The sampler will keep the nozzle only in one hand, designated the
"nozzle" hand. To avoid cross contamination, the sampler will not swap
the nozzle between hands.
5.2 The assistant will retrieve a conical tube from the cassette kit, remove its lid, and
present the open end to the sampler.
5.3 The sampler will place the nozzle (adaptor end down) into the tube, while
continuing to hold the cassette with their opposite hand.
5.4 The assistant will secure the cap on the tube and place it inside the additional,
labeled bag inside the cassette kit.
5.5 Next, the sampler will use their "nozzle" hand to remove the cassette from the
tubing connecting it to the pump. When removed, the sampler will hold out the
cassette to the assistant.
5.5.1 The Tygon tubing will be retained for the next sample in the set; however,
the upstream PVC adaptor will be discarded. A new section of Tygon
tubing will be used with each new sample set (i.e., new sample location).
5.6 The assistant will retrieve the two cassette end plugs, take the cassette from the
sampler, and use the plugs to seal the ends of the cassette. The cassette will then be
placed in the same labeled bag as the conical tube.
5.7 The assistant will immediately seal the bag, wipe the outside with a bleach or
disinfectant wipe, and place it back into its original secondary containment (i.e. the
cassette kit bag).
5.7.1 Attempt to remove as much air as possible before sealing.
5.8 The secondary containment will be resealed by the assistant and wiped down with a
bleach or disinfectant wipes.
5.8.1 Attempt to remove as much air as possible before sealing.
5.9 The final, double contained sample will be placed into a resealable storage bin/bag.
5.10 The sampler will discard their outer gloves.
B-3
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5.11 Steps 3.1 through 5.9 will be repeated for each sample collected, until the sample
matrix is finished.
5.12 If the sample collection is finished for the location, perform a post-set flow check of
the pump, following the procedure in Steps 2.1 through 2.5.
5.13 Record the post-set flow in the appropriate data sheet, as well as the set point/readout
flow from the pump.
6.0 Storage and Shipping
Note: The following procedure will be performed prior to storing samples, moving
samples offsite, or shipping samples to the laboratory.
6.1 Wipe the internal surfaces of any shipping or storage container with a bleach or
disinfectant wipe.
6.2 When all samples from a location have been collected, sealed, and placed in
secondary containment, wipe down the outside of each secondary containment with
bleach or disinfectant wipes and then move the containers into the storage/shipping
container.
6.2.1 Attempt to remove as much excess air from within containers as possible.
6.3 If shipping the samples, place enough cold packs or water ice among the secondary
containers to ensure that samples remain cold for the duration of the trip to the
processing laboratory.
6.4 Any samples that are not shipped the day of collection, will be retained in a
refrigerator or freezer until they can be shipped.
6.5 Samples will be processed within 48 hours of collection.
B-4
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APPENDIX C: WORK INSTRUCTION FOR WATER WASHDOWN
COLLECTION
1. Don clean nitrile gloves.
2. Remove bottles from Ziploc bag (retain bag) and then the lids from the five sterile
bottles.
3. Start tap water flow used for boat washdown and allow to run for at least 15 sec to flush
the hose before sample collection.
4. Don a second pair of clean nitrile gloves (over the first pair).
5. Target flow rate of 4 ± 1 L/min operating at a source pressure of 30 psig should be
determined by filling a 4-L vessel and adjusting flow accordingly.
6. Set spray nozzle setting to produce a small (estimated to < 30-cm-diamter) cone at 1-m
distance. Nozzle-to-surface distance will be variable, but attempt to maintain distance of
0.5 to 1.5 m.
7. Note: The washdown does not need to follow a scripted pattern or establish
method/protocol, but should be performed in a manner so that the water preferentially
flows to the drain from which the sample was collected. The washdown focuses on the
collection side of the boat. An estimated area of 4 m2 should be washed in a < 5-min
period. The exterior surface area covered by the washdown of the RBS should cover
glass windows, aluminum roof and deck, and non-skid tread on the deck.
8. Fill the 1-L bottles with the washdown water using the pole/bottle holder for sampling
from the drains/scuppers on the side of the watercraft. Fill all five bottles consecutively.
9. Turn off water.
10. Doff outer nitrile gloves and don a clean second pair.
11. Secure lids on the bottles and apply parafilm around the lid/bottle interface to help seal
and secure the lid.
12. Place each bottle in a Ziploc bag (retain bag).
13. Place bottles in cooler and pack the cold packs around them.
14. Doff all gloves.
15. Fill remaining void volume in cooler with bubble wrap. Seal cooler by wrapping with
supplied duct tape.
16. Record the following information on this form:
a. Date:
b. Start/Stop Time:
c. Operator Name:
d. Air Temperature/Relative Humidity (using supplied logger):
Notes/Comments:
C-1
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APPENDIX D: WORK INSTRUCTION FOR GRAVEL SAMPLING
Materials:
1-L Nalgene bottle (Thermo Scientific Item# 2187-0032)
14" x 10" secondary containment bag
- Nitrile gloves
Field Collection (Hot Zone):
1. At the sampling location, sampler and support person each don a new pair of gloves.
2. Support person obtains the appropriate sample kit, containing one wide-mouth, 1-L,
Nalgene bottle; check and record sample number.
3. Support person opens the overpack bag, maneuvers Nalgene bottle to the bag opening,
and opens Nalgene lid using one hand to hold the bottle through the bag, and the other to
twist and remove the lid.
Note: Support person should maintain the lid in one hand and bottle in the other
hand throughout the collection. Do not place lid or bottle down and do not remove
bottle from overpack bag.
4. While support person holds bottle and lid, sampler person collects gravel at a depth of
0 to 10 cm using gloved hands.
5. Sampler person drops each gravel piece into the bottle without touching the bottle.
6. Samper person collects enough gravel to fill the 1-L bottle to the 1/2 full mark.
7. Support person places the cap back on the bottle, allows the bottle to drop to the bottom
of the overpack bag, and secures the overpack bag opening.
8. Support person stores the bottle/bag containing the sample.
9. Support person and sampler person doff outer pair of gloves.
Field Sampling Kit Preparation:
All materials needed for collection of each sample will be prepared in advance using aseptic
technique. A sample kit for a single ballast sample will be prepared as follows:
1. Using a permanent marker, mark the 1/2 full level (83 mm (3.25") from the bottom) on
each 1-L Nalgene bottle.
2. One 1-L, sterile, Nalgene bottle and one 14" x 10" overpack bag will be uniquely labeled
as specified in the sample analysis plan.
3. Two additional labels will be added to the overpack bag (these labels will be affixed to
the laboratory extraction sample bottle and containment bag upon sample processing).
4. The sterile, labeled, 1-L Nalgene bottle will be added to the overpack bag.
5. Each prepared bag is one sampling kit.
Reference:
Boehm, A. B., J. Griffith, C. McGee, T. A. Edge, H. M. Solo-Gabriele, R. Whitman, Y. Cao, M.
Getrich, J. A. Jay, D. Ferguson, K. D. Goodwin, C. M. Lee, M. Madison and S. B. Weisberg
(2009). "Faecal indicator bacteria enumeration in beach sand: a comparison study of extraction
methods in medium to coarse sands." J Appl Microbiol 107(5): 1740-1750.
D-1
-------
APPENDIX E: WORK INSTRUCTION FOR SOIL SAMPLE
COLLECTION
1. Don fresh gloves over base pair. (If multiple samples are to be taken per site, don multiple
gloves over base pair.)
3. Open the zip-top bag containing a pre-sterilized, 1-L sterile bottle.
4. Open the bottle, placing the cap open side down on a sterile surface or holding it carefully in
your hand. Avoid touching the inside of the cap.
5. Using a small garden hand spade, remove (by scraping) the top 1 to 2 inches of the soil then
scoop the soil into the 1-L sterile bottle. Two, 1-L sterile bottles need to be filled with the soil to
collect a composite soil sample. Cap bottles with lid and seal with parafilm.
6. Upon return to the laboratory, mix the soil and use a fixed quantity for each sample replicate
to analyze.
Figure E-l. Soil collection using hand spade and bottle (and completed soil sample - far
right).
7. Wipe down tube with an alcohol wipe, taking care to avoid the label.
8. Place sample bottle into the prepared oveipack bag and seal.
9. Wipe down the large zip-top bag with a bleach wipe.
10. Place the zip-top bag into a second zip-top bag for transport.
E-1
-------
APPENDIX F: WORK INSTRUCTION FOR VEGETATION SAMPLING
Materials:
1-L Nalgene bottle (Thermo Scientific Item# 2187-0032)
14" x 10" secondary containment bag
- Nitrile gloves
Shears
Field Collection (Hot Zone):
1. At the sampling location, sampler and support person each don a new pair of gloves.
2. Support person obtains the appropriate sample kit containing one wide-mouth, 1-L,
Nalgene bottle; check and record sample number.
3. Support person opens the overpack bag, maneuvers Nalgene bottle to the bag opening,
and opens Nalgene lid using one hand to hold the bottle through the bag, and the other to
twist and remove the lid.
Note: Support person should maintain the lid in one hand and bottle in the other
hand throughout the collection. Do not place lid or bottle down and do not remove
bottle from overpack bag.
4. While support person holds bottle and lid, the sampler person collects vegetation by
grabbing a handful of grass and clipping the grass just above the soil using gloved hands.
5. Sampler person then places the grass into a 1-L sterile bottle, without touching the bottle.
6. Samper person collects enough vegetation to fill two 1-L bottles for each replicate
sample.
7. If the grass length exceeds the height of the bottle, the grass should be folded to fit within
the bottle.
8. Support person places the cap back on the bottle, allows the bottle to drop to the bottom
of the overpack bag, and secures the overpack bag opening.
9. Support person stores the bottle/bag containing the sample.
10. Support person and sampler person doff outer pair of gloves.
Field Sampling Kit Preparation:
All materials needed for collection of each sample will be prepared in advance using aseptic
technique. A sample kit for a single ballast sample will be prepared as follows:
1. One (1) 1-L, sterile, Nalgene bottle and one 14" x 10" overpack bag will be uniquely
labeled as specified in the sample analysis plan.
2. Two (2) additional labels will be added to the overpack bag (these labels will be affixed
to the laboratory extraction sample bottle and containment bag upon sample processing).
3. The sterile, labeled 1-L Nalgene bottle will be added to the overpack bag.
4. Each prepared bag is one sampling kit.
F-1
-------
APPENDIX G: WORK INSTRUCTION FOR FORMULATIONS OF
RECIPES USED IN BIOLOGICAL TEST METHODS
Spore Production
Table 1. Components of Modified G Sporulation Medium
Ingredient
Amount/L
Yeast Extract
2.0 g
(NH4)2S04
2.0 g
CaCl2 • 2H20
0.03 g
CuS04 • 5H20
0.005 g
FeS04 • 7H20
0.0005 g
MgS04 • 7H20
0.2 g
MnS04 • H20*
0.06 g
ZnS04 • 7H20
0.005 g
k2hpo4
0.5 g
dH20
1000 mL
*MnS04 • H20 substituted for MnS04 • 4H20. If MnS04 • 4H20 is used, add 0.05 g.
Table 2. Real-Time PCR Assay Conditions
Component
Volume for One Reaction (|aL)
TaqMan Fast Advanced Master Mix
(Applied Biosystems, Cat. 4444556)
12.5
Platinum Taq Polymerase
(Invitrogen, Cat. 10-966-034)
0.1
Btk T1B2 Forward Primer (25 jiM)
1.0
Btk T1B2 Reverse Primer (25 |iM)
1.0
Btk T1B2 Probe (2 |iM)
1.0
PCR Grade Water
4.4
Template
5.0
Total Volume
25
G-1
-------
APPENDIX H: WORK INSTRUCTION FOR SPIKING WITH BACILLUS
THURINGIENSISKURSTAKI(Btk) HD-7 T1B2 SPORES
I. PURPOSE/SCOPE
To spike Sponge Stick Wipes (SSW), Vacuum Filter Cassettes (VFC), and Grab (GRB) samples
for spore recovery testing.
II. MATERIALS/EQUIPMENT
Materials
lleni
Ma n ii I'acl ii rer
Lot Nilmher
Kxp.
Dale
Storage
Temp.
Bacillus thuringiensis
kurstaki {Btk) HD-1
T1B2 Stock
(2 x 108 CFU/mL)
In house
HD1.T1B2.120919
TBD
2-8 °C
Sterile Deionized
(DI) Water
RT
TSA
2-8 °C
1.5- or 2-mL Tubes
Eppendorf
N/A
RT
Sterile Forceps
N/A
N/A
N/A
RT
VFC
SKC
18109-7E1-219
N/A
RT
Sponge Stick
3M
RT
Specimen Cup
N/A
RT
Equipment
Item
Manufacturer
Serial
Nil in her
Thermomeler/Uees #
Calibration
Due
Biosafety
Cabinet
(BSC)
The Baker Company
N/A
Micropipette
Type:L1000
Rainin
N/A
Micropipette
Type:L200
Rainin
N/A
Micropipette
Type:L10 or
L20
Rainin
N/A
Refrigerator
Fisher
ST/A = Not Applicable
Other Supplies and Equipment
• Micropipette filter tips
• Biohazard bags
Performed by: Date:
Page 1 of 7
WI1 (Appendix H)-SPIKE
H-1
-------
III. PROCEDURE
A. Decontaminate the BSC with DNA Erase; bleach and isopropanol prior to use.
B. Name SSW, VFC, and GRB samples as follows:
1. Label each sample with sample ID per the following:
i. AAA-BBB-CCC-DDD
1. AAA = Sample #
2. BBB = Sample Type
3. CCC = Location
4. DDD = Spore Spike Level
ii. Electronically populate table below with sample names to be prepared on
each day from the Sample Log.
Ssimple
#
Ssimple
Type
Locution
liller \ iill
"I'vpc
Spore
Spike
1 .e\el
(dl)
Siimple II)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
C. Spike Samples
1. Prepare Spiking Stocks
i. Fill in information from stock tube.
Orgsinism
Lot
Prep (Isile
Concenlrsilion
BtkHD-1 T1B2
HD1.T1B2.120919
12/09/2019
2 X 108 cfu/mL
Performed by: Date:
Page 2 of 7
WI1 (Appendix H)-SPIKE
H-2
-------
ii. Target stock concentrations:
(oiKTiilmlion
Total Spores per KM) ill.
3.0 X 106 cfu/mL
300,000
3.0 X 105 cfu/mL
30,000
3.0 X 104cfu/mL
3,000
3.0 X 103 cfu/mL
300
iii. Prepare dilutions of stock in sterile DI water. Vortex stock on high for
30 seconds prior to preparing dilutions.
Show calculations:
Dilution 1: (2.0 X 108 cfu/mL)*(X)=(3.0 X 107cfu/mL)(l mL) 150 |iL of stock into
850 |iL H20
Dilution 2: (3.0 X 107 cfu/mL)*(X)=(3.0 X 106cfu/mL)(l mL) -> 100 |iL of Dilution 1 into
900 |iL H20
Dilution 3: (3.0 X 106 cfu/mL)*(X)=(3.0 X 105 cfu/mL)(l mL) -> 100 |iL of Dilution 2 into
900 |iL H20
Dilution 4: (3.0 X 105 cfu/mL)*(X)=(3.0 X 104cfu/mL)(1.5 mL) -> 150 |iL of Dilution 3 into
1,350 |iLH20
Dilution 5: (3.0 X 104 cfu/mL)*(X)=(3.0 X 103 cfu/mL)(1.5 mL) -> 150 |iL of Dilution 4 into
1,350 |iL H20
Dilution 6: (3.0 X 103 cfu/mL)*(X)=(3.0 X 102cfu/mL)(1.5 mL) -> 150 |iL of Dilution 5 into
1,350 |iLH20
2. Spike Sponge Sticks
i. Position sponge in specimen cup so that the dirty side is facing up. Change
forceps between samples.
ii. Prior to spiking sponges, vortex the stock for 30 seconds.
iii. Per sponge, transfer a 120-|iL aliquot of the appropriate Stock tube
(Dilution 4 for 3,000 CFU and Dilution 5 for 300 CFU) into a 1.5-ml tube.
iv. Place ten (10) 5-|iL droplets onto each side of the sponge stick (20 5-|iL
droplets total), being as careful as possible to avoid having spiked surfaces
contact the specimen cup wall. Position sponge as shown in Figure H-l.
The same pipet tip can be used to place all 20 droplets; dispose of the
120 |iL aliquot once each sponge has been spiked.
v. Seal the specimen cup and process immediately using spore recovery
Work Instruction (WI) or store @ 2 to 8 °C.
2-8 °C Start time: Date/Initials:
Performed by:
WI 1 (Appendix H)-SPIKE
Date:
Page 3 of 7
H-3
-------
3. Spike VFCs
i. Wipe each cassette with 10% bleach solution or bleach wipes followed by
a clean Kimwipe and discard wipes into an autoclavable biohazard bag.
ii. Prior to spiking filters, vortex the stock for 30 seconds.
iii. Per VFC, transfer a 120-|iL aliquot of the appropriate Stock tube
(Dilution 4 for 3,000 CFU and Dilution 5 for 300 CFU) into a 1.5-ml tube.
iv. Remove the red plug and apply 20 5-|iL droplets onto each filter as shown
in Figure H-2. The same pipet tip can be used to place all 20 droplets;
dispose of the 120-|iL aliquot once each VFC has been spiked.
v. Seal the VFC and process immediately using spore recovery WI or store
@ 2 to 8 °C.
2-8 °C Start time: Date/Initials:
4. Spike Wash Water Grab Samples
i. Wipe 1-L container with 10% bleach solution or bleach wipes.
ii. Prior to spiking samples, vortex the stock for 30 seconds.
iii. Per wash water grab sample, transfer a 100-|iL aliquot of the appropriate
Stock tube (Dilution 3 for 30,000 CFU, Dilution 4 for 3,000 CFU and
Dilution 5 for 300 CFU) into a 1.5-ml tube containing 400 |iL sterile FhO,
for 500 |iL volume total. Mix by vortex.
iv. Apply twenty (20) 25-|iL droplets into the wash water liquid and pipette
tip submerged slightly into wash water. The same pipet tip can be used to
add all 20 droplets.
v. Seal the container and process within 1 hour of spiking or store @ 2 to
8 °C.
2-8 °C Start time: Date/Initials:
5. Spike Gravel Grab Samples
i. Wipe a 1-L container with 10% bleach solution or bleach wipes.
ii. Prior to spiking samples, vortex the stock for 30 seconds.
iii. Per gravel grab sample, transfer a 120-|iL aliquot of the appropriate Stock
tube (Dilution 4 for 3,000 CFU and Dilution 5 for 300 CFU) into a 1.5-ml
tube.
iv. Apply twenty (20) 5-|iL droplets onto the surface of the gravel (top layer)
as shown in Figure H-3. The same pipet tip can be used to place all 20
droplets; dispose of the 120-|iL aliquot once each gravel grab sample has
been spiked.
v. Seal the 1-L container and process immediately using spore recovery WI
or store @ 2 to 8 °C.
2-8 °C Start time: Date/Initials:
Performed by: Date:
Page 4 of 7
WI 1 (Appendix H)-SPIKE
H-4
-------
6. Spike Grass Grab Samples
i. Wipe a 1-L container with 10% bleach solution or bleach wipes.
ii. Prior to spiking filters, vortex the stock for 30 seconds.
iii. Per grass grab sample, transfer a 100-|iL aliquot of the appropriate Stock
tube (Dilution 3 for 30,000 CFU, Dilution 4 for 3,000 CFU and Dilution 5
for 300 CFU) into a 1.5-ml tube containing 400 |iL sterile FhO, for
500 |iL volume total. Mix by vortex.
iv. Apply twenty (20) 25-|iL droplets onto the surface of the grass to cover as
many grass surface areas as possible as shown in Figure H-4. The same
pipet tip can be used to place all 20 droplets.
v. Seal the 1-L container and process within 1 hour of spiking or store @ 2 to
8 °C.
2-8 °C Start time: Date/Initials:
7. Spike Soil Grab Samples
i. Wipe a 50-mL container with 10% bleach solution or bleach wipes.
ii. Prior to spiking samples, vortex the stock for 30 seconds.
iii. Per soil sample, transfer a 100-|iL aliquot of the appropriate Stock tube
(Dilution 2 for 300,000, Dilution 3 for 30,000, or Dilution 4 for 3,000
CFU) onto the 10-g soil aliquot in a dropwise fashion to distribute the
spike throughout the soil sample. Mix by vortex.
iv. Seal the 50-mL container and process within 1 hour of spiking or store @
2 to 8 °C.
2-8 °C Start time: Date/Initials:
8. Enumerate stock
i. Spread 100-|iL aliquots of Dilutions 5 and 6 onto TSA in triplicate.
ii. Incubate plates
1. Invert the plates and incubate them at 30°C ± 2°C for 18 to 24
hours. Btk produces flat or slightly convex, 2 to 5 mm colonies,
with edges that are slightly irregular and have a "ground glass"
appearance.
Incubation start Date/Time: Initials:
Incubation end Date/Time: Initials:
Performed by: Date:
Page 5 of 7
WI1 (Appendix H)-SPIKE
H-5
-------
iii Plate counts
1. Record counts in the table below.
Dilution #
Media Type
Volume/
(Dilution on
Plate)
Plate Counts
Average
Counts
CFU/mL
Plate
1
Plate
2
Plate
3
5 (3.0 X 103 cfu/mL)
TSA
100 jiL/
(10-1)
6 (3.0 X 102 cfu/mL)
TSA
100 |j.L/
(10-1)
1 |Hnc- 'i *
Position with folded side up or stick side up. Do not spike the sides of the sponge that could contact
the specimen cup wall.
Place ten (10) 5-juL evenly dispersed droplets on each side for a total of twenty (20) 5-|llI. droplets.
Figure H-l. Spiking Diagram for Sponge Sticks
Figure H-2. Spiking Diagram for VFC
Performed by:
WI1 (Appendix H)-SPIKH
Date:
Page 6 of 7
H-6
-------
Figure H-3. Spiking of Gravel Grab Samples
i
Figure H-4. Spiking of Grass Grab Samples
Reviewed by:
Date:
Performed by:
WI1 (Appendix H)-SPIKE
Date:
Page 7 of 7
H-7
-------
APPENDIX I: WORK INSTRUCTION I OR BACILLI S THURINGIENSIS
KURSTAKI (Btk) T1B2 SPORE RECOVERY FROM MARITIME SAMPLES
- SPONGE STICKS, VACUUM CASSETTES, AND GRAB SAMPLES
I. PURPOSE/SCOPE
To recover Bacillus thuringiensis kurstaki (.Btk) T1B2 spores from Sponge-Stick Wipes (SSW),
Vacuum Cassette Filters (VFC), and Grab (GRB) samples.
II. MATERIALS/EQUIPMENT
Materials
llciii
Man ii I'acl ii rcr
Lot Nil in her
Kxp.
Storage
Initials and
Dale
Temp.
Dale
Extraction Buffer
with Tween 20 +
In-house
2-8 °C
30% Ethanol
Phosphate Buffered
Saline with 0.05%
Teknova
RT
Tween 20 (PBST)
Stomacher Lab
Blender Bags
Seward
N/A
RT
Stomacher Bag
Racks
Seward
BA6096
N/A
RT
10X PBS
Teknova
2-8 °C
IX PBS (pH 7.4)
Teknova
2-8 °C
Brain Heart
Infusion Broth
BD
2-8 °C
(BHIB)
Conical Tubes
N/A
RT
15-mL
Conical Tube
50-mL
Falcon
N/A
RT
Screw Top Flask
250 mL
Corning
N/A
RT
0.45-|im Filter Vials
Whatman
N/A
RT
2-mL Tubes
RT
Sterile Forceps
Unomedical
N/A
RT
N/A = Not Applicable
Performed by:
Wl 2 (Appendix l)-SPG VFC AND GRAB SPORE RECOVERY
Date:
Page 1 of 19
1-1
-------
Equipment
lll'ill
M;i n ii fact ii ivr
Sorisil Number
Thcrnioinclcr /
Rees #
C';ilibr:ilioii
Duo
luitiiils/
Dsile
Biosafety
Cabinet (BSC)
The Baker
Company
57553
N/A
57544
Micropipette
Type: L1000
Rainin
N/A
Incubator
Shaker
New Brunswick
590644988
Refrigerator
Swinging
Bucket
Centrifuge
Beckman
Coulter
X59221
N/A
N/A
Stomacher
Seward
40142
N/A
N/A
Sonicator Bath
Bransonic
RNC010140514E
N/A
N/A
Water Bath
N/A = Not Applicable
Other Supplies and Equipment
• Forceps
• Biohazard bags
• Bleach
• 5-mL, 25-mL, and 100-mL serological pipets
• Pipette aid
• Ziplock bags
• Stainless Steel SureSeal Cassette Opener, SKC Cat. 225-13-5A
Performed by:
Wl 2 (Appendix l)-SPG VFC AND GRAB SPORE RECOVERY
Date:
Page 2 of 19
1-2
-------
Filters - Electronically update this table with sample names from the Sample Log
Sample #
Sample
Type
Location
liher Vial
Type
Spore Spike
Level (CI l )
Sample II)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
III. PROCEDURE
A. Sample Processing: Spore Recovery for Sponge-Stick Samples
Note: Process samples from negative control to high inoculation level. Change gloves when
working from an inoculated sample to a sample containing a lower inoculation level, or if
contamination of gloves is suspected. Pre-aliquot reagents from the kit to prevent
contamination of reagents between runs.
1. Prior to sample processing, prepare the following items:
• Fill sample tube rack with 50-mL screw cap conical tubes and label as appropriate; two
50-mL conical tubes are required per sample.
• In a BSC, attach the vacuum manifold to the vacuum trap, waste container (with 400 mL
of bleach), and vacuum source. Attach the filter vials to the manifold, using outer rows
first. Verify that all filter vials are completely pushed down. Place a red pull tab tapered
plug in each filter vial.
• Document filter vial and sample tube labels.
• Extraction Buffer with Tween 20 + Ethanol (1,500 mL) will be needed per set of 16
samples (90 mL per sample).
• High salt wash buffer (lOx PBS) (225-mL aliquot) in a 250-mL screw capped bottle per
set of 16 samples (12.5 mL per sample).
Performed by:
Wl 2 (Appendix l)-SPG VFC AND GRAB SPORE RECOVERY
Date:
Page 3 of 19
1-3
-------
• Low salt wash buffer (lx PBS) (225-mL aliquot) in a 250-niL screw capped bottle per
set of 16 samples (12.5 mL per sample).
2. Add 90 mL cold (4°C) Extraction Buffer with I w een 20 + Ethanol to each Stomacher bag.
3. Using sterile forceps, remove the remaining portion of the sponge-stick handle and unfold the
sponge.
4. Aseptically add a sponge-stick to a Stomacher bag. Open one bag at a time; close and seal
bag prior to moving to the next sample. Note: Save specimen cup for broth enrichment of
sponge.
5. Place an unsealed bag containing a sample into the Stomacher so the sponge rests evenly
between the homogenizer paddles and stomach each sample for 1 min at 260 rpm
(Figure 1-1). Open the door of the Stomacher and remove the bag. Reseal bag.
Figure 1-1. Sponge is opened and centered between paddle positions.
6. Stomach all sponges; removal of bag from Stomacher begins the settle time. Allow bags to
sit for 10 min to allow elution suspension foam to settle.
7. Grab the sponge from the outside of the bag with hands. With the bag closed, move the
sponge to the top of the bag while using hands to expel liquid from the sponge.
8. Open the bag, remove sponge, and place into a labeled specimen cup using sterile forceps.
Store sponge at 2 to 8°C until enrichment in BFTIB (See WI #7 Appendix N: Work
Instruction for BH1B Enrichment for Culture).
9. Follow steps described above for each sample, changing forceps between samples.
10. Gently mix the suspension in the Stomacher bag up and down three times with a sterile
50-mL pipet. Remove half of the suspension volume (-45-46 mL) and place it in a 50-mL
screw cap centrifuge tube (Aliquot 1). Place the remaining suspension (-45-46 mL) into a
second 50-mL tube (Aliquot 2). Adjust the suspension volumes so that volume is equal in
both tubes.
11. Process the suspension for each sample, as described above.
12. Place 50-mL tubes into sealing centrifuge buckets and decontaminate centrifuge buckets
before removing them from the BSC.
13. Centrifuge tubes at 3,500 X g with the brake off for 15 min in a swinging bucket rotor at 4°C.
Performed by:
WI 2 (Appendix Q-SPG VFC AND GRAB SPORE RECOVERY
Date:
Page 4 of 19
1-4
-------
14. Each sample has two pelleted aliquots (Aliquot 1 and Aliquot 2). Using a sterile 50-mL pipet,
remove the supernatant from Aliquot 1 and discard it in an autoclavable leak-proof biohazard
container. The pellet may be easily disturbed and not visible, so keep the pipet tip away from
the bottom of the tube. Stop pipetting when meniscus reaches the 5-mL gradation level of on
50-mL Falcon tube, leaving -2 to 3 mL in each tube. Next, using the same pipet, remove
20 mL of supernatant from Aliquot 2 and add it to Aliquot 1 pellet. Discard the remaining
supernatant from Aliquot 2 into an autoclavable leak-proof biohazard container.
15. Vortex Aliquot 1 (containing -22 mL of supernatant) for 30 sec to resuspend the pellet, then
transfer entire volume to Aliquot 2.
16. Vortex Aliquot 2 for 30 sec to resuspend the pellet. This pooled suspension of -25 mL will
be used for culture and RV-PCR analytical methods. Record total volume for each sample in
Table 1-1.
Table 1-1. Volume of Sample Recovered from Sponge Sticks.
Sam pie
Nil m her
Sample II)
Total Volume
Recovered from
Sponge-Slick
Recorded In :
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17. Transfer 11 mL of the pooled extract and store on ice or in refrigerator until processed on
same day using WI #4 Appendix J: Work Instruction for Culture of Recovered Spores.
18. Place manifold and Whatman Autovial filter vials with red caps in BSC. Label all filter vials.
Record filter vial labels.
19. Vortex each RV-PCR aliquot and allow 3 to 5 min of settle time to avoid loading large
particulates into filter vial. Transfer 12.5 mL of the pooled suspension volume from each
Page 5 of 19
Performed by: Date:
WI 2 (Appendix l)-SPG VFC AND GRAB SPORE RECOVERY
1-5
-------
tube to the corresponding labeled filter vial by lifting red cap slightly. Change serological
pipets and gloves between samples.
20. Complete filtration of liquid through filter vials. Turn off vacuum pump.
Note 1: At 15 min post-sample addition, if sample has not completely passed through the
filter vial, a reduced volume of high salt wash buffer will be added (5 mL) to avoid
prolonged filtering delays. It is desired to add a lower volume of each salt wash buffer (10X
and IX) than to omit one or both entirely.
Note 2: At 1 hour post-sample addition, if sample has not completely passed through the
filter vial, the high salt and low wash steps will be omitted.
Sample #
Sample II)
Sample
\(lrihion
Volume ol'
W ash IJulTers
Recorded
Start
lime1
Kml
lime2
I0X
l\
hv:
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
1 Record the time of adding the final sample to filter vial.
2 Record end time for samples that have clogging and meet the criteria in Notes 1 and 2 above.
21. Proceed to RV-PCR processing section (Section E) below, with filter vial manifold.
B. Sample Processing: Spore Recovery for VFC Samples
Note: Process samples from negative control to high inoculation level. Change gloves when
working from an inoculated sample to a sample containing a lower inoculation level, or if
contamination of gloves is suspected. Pre-aliquot reagents to prevent contamination of
reagents between runs.
1. Prior to sample processing, prepare the following items:
• Fill sample tube rack with 15-mL screw cap conical tubes and label as appropriate, each
containing 11 mL sterile Extraction Buffer with Tween 20 + Ethanol.
Performed by:
Wl 2 (Appendix l)-SPG VFC AND GRAB SPORE RECOVERY
Date:
Page 6 of 19
1-6
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• One labeled 2-oz sterile cup with lid per sample, sterilized by autoclave (gravity cycle,
121°C for 15 min).
• In a BSC, attach the vacuum manifold to the vacuum trap, waste container (with 250 ml
of bleach), and vacuum source. Attach the filter vials to the manifold, using outer rows
first. Verify that all filter vials are completely pushed down. Place a red pull tab tapered
plug in each filter vial.
• Document filter vial and sample tube labels.
2. For each 37-mm filter cassette, prepare one 15-mL conical tube containing 11 mL of sterile
Extraction Buffer with Tween 20 + Ethanol and label one 2-oz sterile cup.
3. In the BSC, remove the conical tube containing the nozzle and the cassette from the
containment bags and wipe the outside of the conical tube with a disinfectant and place it into
a rack. Aseptically add 5 mL of Extraction Buffer with Tween 20 + Ethanol (from the 11 mL
of a pre-measured aliquot of PBST + Ethanol [PBSTE]) to the conical tube containing the
nozzle and tubing and set aside.
4. Remove the band from around the cassette using sterile scissors. Wipe each cassette with
10% bleach solution or bleach wipes followed by a clean Kimwipe and discard wipes into an
autoclavable biohazard bag.
5. Remove the red plug from the inlet of the cassette; the plug on the back side should be kept
in place. Using a pipette dispense 2 mL of Extraction Buffer with Tween 20 + Ethanol from
the tube now containing the 6 mL into the cassette and replace plug. Roll the cassette around
to allow the liquid to touch all surfaces of the inside of the cassette. If there is a large quantity
of parti culate matter, more Extraction Buffer with Tween 20 + Ethanol may be required.
Particulate matter should be dampened enough to prevent aerosolization.
6. Using the cassette tool, pry open the top section of the cassette, using care not to spill the
Extraction Buffer with Tween 20 ± Ethanol inside the cassette and set aside, plug side down
as shown in Figure 1-2. Set the bottom portion containing the filter aside carefully (filter side
up), and using a pipette rinse the walls of the cassette with 2 mL of Extraction Buffer with
Tween 20 + Ethanol. Transfer the rinsate using the same pipette to the appropriately labeled
2-oz sterile cup.
Figure 1-2. Vacuum Cassette with Top Section Removed
Performed by:
Wl 2 (Appendix Q-SPG VFC AND GRAB SPORE RECOVERY
Date:
Page 7 of 19
1-7
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7. Using the cassette tool, remove the middle section of the cassette (this piece is holding the
filter in place) and place on top of top section as shown in Figure 1-3. Using sterile forceps
aseptically remove the filter without picking up the support filter underneath. Place the filter
in the 2-oz cup with the rinsates.
Figure 1-3. Vacuum Cassette with Top and Middle Sections Removed
8. Use the remainder of the 6 mL Extraction Buffer with Tween 20 + Ethanol to rinse walls of
the middle and top sections (configuration shown in Figure 1-3, image on left) of the cassette
and transfer rinsate to 2-oz cup. Discard the cassette sections, support filter, plugs, and
transfer pipette in an autoclavable biohazard bag.
9. Disinfect the outside of the 2-oz cup with 10% bleach solution and place in tray.
Decontaminate the BSC with 10% bleach solution and don a fresh pair of gloves in between
samples. Repeat procedure described above for each 37-mm filter cassette.
10. Seal the conical tubes containing 5 mL Extraction Buffer with Tween 20 + Ethanol. tubing,
and nozzle with Parafilm. Place the rack of conical tubes into the sonicating bath to a level
that allows at least 1 inch (-2.5 cm) of tube to be above the water line. Place a weight on top
of the tubes to prevent them from floating or tipping over. Sonicate for 1 min and remove
tubes from the sonicating bath. Dry and disinfect each tube with a 10% bleach solution.
11. Vortex the conical tubes 2 min using platform vortex at Setting 10 (high setting), then
transfer the 5 mL. Extraction Buffer with Tween 20 + Ethanol to the appropriate 2-oz cup. To
transfer volume, use 1-mL micropipette to remove volume collected in the tubing nozzle,
then use pipette tip to remove nozzle from the 15-mL conical tube. Before disposing of
nozzle, depress pipette piston to expel any remaining extract volume from the nozzle into the
15-mL conical tube (See Figure 1-4).
Figure 1-4. Nozzle Removal Using 1-mL Pipette
Performed by:
Wl 2 (Appendix Q-SPG VFC AND GRAB SPORE RECOVERY
Date:
Page 8 of 19
1-8
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12. Seal all of the 2-oz cups with Parafilm. Place the rack of 2-oz cups in the sonicating bath and
cover with a weight on top of the cups to prevent them from floating or tipping over. There
should be 1 inch (-2.5 cm) between the level of the water and the cup lids. Sonicate for
3 min without heat. Remove rack from the bath and dry each cup with a Kimwipe and place
in the BSC. Place cups in a sealable plastic lidded box.
13. Using a 10-mL serological pipet, transfer as much suspension as possible from each 2-oz cup
to a 15-mL conical tube. Record total volume for each sample in Table 1-2. Note: Save 2-oz
cups containing filter. Store at 2 to 8 °C until enrichment in Trypticase Soy Broth on same
day (See WI #7 Appendix N: Work Instruction for BHIB Enrichment for Culture).
Table 1-2. Volume of Sample Recovered from VFC
Sam pie
Nil m her
Sample II)
Total Volume
Recovered from
VI (
Volume Available
per Analytical
Method
( Total Volume -h 2)
Recorded by:
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
14. Vortex each sample, then allow 3 to 5 min settle time to avoid transferring large particulates
into filter vial and cause clogging. Transfer half (~5 mL) volume of each sample to
corresponding labeled filter vial. Change serological pipets between each sample.
15. Store the remaining half (~5 mL) of the pooled extract for microbiological analysis (WI #4
Appendix J: Culture of Recovered Spores). Store aliquot on ice or in refrigerator until
processed on same day. Change serological pipets between each sample.
16. Complete filtration of liquid through filter vials. Turn off vacuum pump.
Performed by:
WI 2 (Appendix l)-SPG VFC AND GRAB SPORE RECOVERY
Date:
Page 9 of 19
1-9
-------
Note 1: At 15 min post-sample addition, if sample has not completely passed through the
filter vial, a reduced volume of high salt wash buffer will be added (5 mL) to avoid
prolonged filtering delays. It is desired to add a lower volume of each salt wash buffer (10X
and IX) than to omit one or both entirely.
Note 2: At 1 hour post-sample addition, if sample has not completely passed through the
filter vial, the high salt and low wash steps will be omitted.
Ssimple #
Snmple II)
Ssimple
\(lrihion
Volume of
\\ sisli liullci s
Recorded
Slsirt
lime1
Kml
lime2
I0X
l\
hv:
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
1 Record the time of adding the final sample to filter vial.
2 Record end time for samples that have clogging and meet the criteria in Notes 1 and 2 above.
Performed by:
Wl 2 (Appendix l)-SPG VFC AND GRAB SPORE RECOVERY
Date:
Page 10 of 19
1-10
-------
C. Spore Recovery for Grab Samples (Wash Water, Gravel Vegetation - See Section D for
Soil Samples)
Note: Process samples from negative control to high inoculation level. Change gloves when
working from an inoculated sample to a sample containing a lower inoculation level, or if
contamination of gloves is suspected. Pre-aliquot reagents to prevent contamination of
reagents between runs.
1. Prior to sample processing, prepare the following items:
• In a BSC, attach vacuum manifold to waste container containing appropriate amount of
bleach for a final concentration of 1% NaOCl after collecting all waste fluids.
• In a BSC, attach the RV-PCR vacuum manifold to the vacuum trap, waste container
(with 500 mL of bleach), and vacuum source. Attach the filter vials to the manifold,
using outer rows first. Verify that all filter vials are completely pushed down. Place a red
pull tab tapered plug in each filter vial.
• Document filter vial and sample tube labels.
2. Add 20 mL of Extraction Buffer with PBSTE to a MicroFunnel unit with 0.45-|im GN-6
Metricel membrane (Pall ID: 4800 or equivalent); this filtration unit will be referred to as the
filter unit. Apply vacuum after PBSTE completely passes through membrane, turn off
vacuum, and apply the membrane to a Trypticase Soy Agar plate using sterile forceps. This
sample will serve as a negative control. Incubate this control at 30°C overnight and check for
sterility.
Start time End time Sterility (Yes/No)
3. Weigh grab samples before and after extraction and record weights below.
Siim plo
Nil m her
Siimple 11)
I're- Kxlmction
Weight ((1 nuns)
I'osl-Kxlmclion
Weight ((inuns)
Recorded hv:
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Performed by:
Wl 2 (Appendix l)-SPG VFC AND GRAB SPORE RECOVERY
Page 11 of 19
Date:
1-11
-------
4. Carefully add 500 mL of sterile Phosphate Buffered Saline with 0.05% Tween 20 (PBST) to
each 1-L sample bottle. Parafilm seal lid and place into secondary bag with absorbent.
5. Shake vigorously for 2 min by hand.
a. Grasp 1-L sample bottle with one hand on bottom of bottle, the other hand around
the bottle near the top. Hold bottle over shoulder and shake vigorously back and
forth.
6. Allow sample to settle for 30 sec.
7. Pour off eluent into clean, labeled, 500-mL container. Ensure sample labels for each
collection bottle match their respective eluent bottle.
8. Vigorously mix 0.5 L grab eluate aliquots by hand for 30 sec. Allow 30 sec of settle time.
9. Pour mixed grab eluate into filter unit to the 100-mL gradation line.
10. Apply vacuum until entire 100 mL passes through membrane. Once complete, break vacuum
pressure, then close valve.
11. Repeat Steps 8 through 10 five times with an additional 100 mL of grab eluate, for a total of
500 mL of grab eluate (250 mL for gravel) onto a single 47-mm filter.
Note: If filter becomes clogged, less than 500 mL of sample (250 mL for gravel) will be
processed. Record volume filtered in the table below. At 30 min post-sample addition, if
sample has not completely passed through the filter, the remaining volume in the filter unit
will be removed.
Sum pic #
S:i in plo 11)
lillrnlion
Slnrl Time
lilliitlion
Knd l ime
Tot si 1 Volume
Tillered
Recorded IK:
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Performed by:
Wl 2 (Appendix l)-SPG VFC AND GRAB SPORE RECOVERY
Date:
Page 12 of 19
1-12
-------
12. Remove the filter membrane using sterile forceps and transfer to a 50-mL conical tube.
Position the membrane in the bottom half of the conical tube with the inlet side of the
membrane facing the center of the tube. Avoid placing the filter into the conical portion of
the tube.
13. Repeat Steps 5 through 12 for all samples.
14. Add 10 mL of PBSTE to 50-mL conical tubes containing membrane filters.
15. Vortex at maximum speed on platform vortex in 10-sec bursts for 2 min to dislodge spores.
16. Let tubes settle for 2 min, then transfer volume to a clean 50-mL conical tube.
17. Repeat extraction of each membrane filter by adding another 10 mL of PBSTE to the 50-mL
conical tube with membrane.
18. Repeat Steps 15 and 16, transferring volume to the same 50-mL conical tube per sample for a
total recovered pooled spore recovery volume of 20 mL.
Note: Save filter membrane. Store at 2 to 8°C until enrichment in BHIB on same day (See
WI #7 Appendix N: Work Instruction for BHIB Enrichment for Culture).
19. Vortex each 20-mL sample, then allow 30 sec of settle time to avoid transferring large
particulates into RV-PCR filter vial. Transfer half (10 mL) volume of each sample to
corresponding labeled RV-PCR filter vial.
20. Store the remaining half (10 mL) of the pooled spore recovery volume for microbiological
analysis (WI #4 Appendix J: Work Instruction for Culture of Recovered Spores). Store
aliquot on ice or in refrigerator until processed on same day.
21. Complete filtration of liquid through RV-PCR filter vials. Turn off vacuum pump.
Note 1: At 15 min post-sample addition, if sample has not completely passed through the
filter vial, a reduced volume of high salt wash buffer will be added (5 mL) to avoid
prolonged filtering delays, It is desired to add a lower volume of each salt wash buffer (10X
and IX) than to omit one or both entirely.
Note 2: At 1 hour post-sample addition, if sample has not completely passed through the
filter vial, the high salt and low wash steps will be omitted.
Performed by:
WI 2 (Appendix l)-SPG VFC AND GRAB SPORE RECOVERY
Date:
Page 13 of 19
1-13
-------
Sample #
Sample II)
Sample
\(hlhion
Volume of'
W ash liullct s
Recorded
Slaii
lime1
Iml
lime2
I0X
l\
hv:
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
1 Record the time of adding the final sample to filter vial.
2 Record end time for samples that have clogging and meet the criteria in Notes 1 and 2 above.
D. Spore Recovery for Soil Samples
Note: Process samples from negative control to high inoculation level. Change gloves when
working from an inoculated sample to a sample containing a lower inoculation level, or if
contamination of gloves is suspected. Pre-aliquot reagents to prevent contamination of
reagents between runs.
1. Prior to sample processing, prepare the following items:
• Set one water bath to 75 to 80 C, and a second to 70 to 75°C (optional).
• In a BSC, attach the RV-PCR vacuum manifold to the vacuum trap, waste container
(with 500 mL of bleach), and vacuum source. Attach the filter vials to the manifold,
using outer rows first. Verify that all filter vials are completely pushed down. Place a red
pull tab tapered plug in each filter vial.
• Document filter vial and sample tube labels.
• Weigh 10 ± 0.1 g soil aliquots in 50-mL tubes.
Performed by:
Wl 2 (Appendix l)-SPG VFC AND GRAB SPORE RECOVERY
Date:
Page 14 of 19
1-14
-------
S;nii|)lc #
Ssimplc II)
Ssimplc \Yeiglil («)
Wciglu'il/Uccordi'il hv:
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
2. Add 40 mL of PBST to each 10-g soil sample. Parafilm tubes.
3. Vortex samples for 30 sec.
4. Sonicate samples for 10 min in bath sonicator.
5. Manually mix each sample for 2 min.
6. Spin at 1,000 x g for 5 min.
7. Transfer supernatant to a clean 50-mL tube (leaving -2.5 mL of supernatant with each
pellet). Save pellet aliquots for heat shock.
8. Measure pH of supernatant using pH strips. Parafilm tubes.
Performed by:
Wl 2 (Appendix l)-SPG VFC AND GRAB SPORE RECOVERY
Date:
Page 15 of 19
1-15
-------
Ssimple #
Ssimplc II)
Volume Recovered
Ssimplc pll
Recorded In :
per S:impie (ml.)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
9. Heat shock supernatant and pellets at 70 ± 2°C for 1 hour with intermittent mixing of tubes.
One-hour heat time starts when pilot tube containing 40 mL of PBST reaches 70 ± 2°C. Once
pilot tube reaches temperature, turn down water bath to avoid heating above 70 ± 2°C, or
transfer to second water bath set to 70°C. Post-heat shock and allow samples to cool to
ambient temperature. Measure temperature of pilot tube to assess return to ambient
temperature.
Batch 1: Heat Start Time Heat End Time
Time Ambient Temperature Reached
Batch 2: Heat Start Time Heat End Time
Time Ambient Temperature Reached
10. Split supernatant in half, -20 mL for culture and -20 mL for RV-PCR analysis.
11. Vortex each ~20-mL RV-PCR aliquot, then allow 30 sec of settle time to avoid transferring
large particulates into RV-PCR filter vial. Transfer 12.5 mL volume of each sample to
corresponding labeled RV-PCR filter vial. Add additional volume to filter vials if clogging
does not occur, up to full 20 mL.
12. Store culture aliquot (-20 mL) and pellet at 2 to 8°C for microbiological analysis (WI #4
Appendix J: Culture of Recovered Spores).
13. Complete filtration of liquid through RV-PCR filter vials. Turn off vacuum pump.
Note 1: At 15 min post-sample addition, if sample has not completely passed through the
filter vial, a reduced volume of high salt wash buffer will be added (5 mL) to avoid
Performed by:
WI 2 (Appendix l)-SPG VFC AND GRAB SPORE RECOVERY
Date:
Page 16 of 19
1-16
-------
prolonged filtering delays. It is desired to add a lower volume of each salt wash buffer (10X
and IX) than to omit one or both entirely.
Note 2: At 1 hour post-sample addition, if sample has not completely passed through the
filter vial, the high salt and low wash steps will be omitted.
Sample #
Sample II)
Sample
\(ldhion
Volume of
Wash Buffers
Recorded
Slaii
Time1
Iml
lime2
I0X
l\
bv:
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
1 Record the time of adding the final sample to filter vial.
2 Record end time for samples that have clogging and meet the criteria in Notes 1 and 2 above.
E. RV-PCR Sample Processing: Buffer Washes and Broth Culture
1. Place into BSC: a Ziplock bag with orange caps (one per filter vial), 10-mL serological pipets
and cold (4°C) 10X PBS in 250-mL screw cap bottle.
2. Transfer 12.5 mL of cold (4°C) high salt wash buffer (lOx PBS) to each filter vial using a
10-mL serological pipet. Change pipet and gloves between each sample.
3. Complete filtration of liquid through the filter vials.
4. Place into the BSC: 10-mL serological pipets and cold (4°C) IX low salt wash buffer in
250-mL screw cap bottle.
5. Transfer 12.5 mL cold (4°C) low salt wash buffer ( lx PBS) to each filter vial using a
10-mL serological pipet. Change pipet and gloves between each sample.
6. Complete filtration of liquid through filter vials. Turn off vacuum pump.
7. Using an Allen wrench, unscrew the top of the manifold and break the seal on manifold using
a plate sealer to separate the top of the manifold.
Performed by:
Wl 2 (Appendix l)-SPG VFC AND GRAB SPORE RECOVERY
Date:
Page 17 of 19
1-17
-------
8. Using a tray preloaded with caps, move the top of the manifold with the filters still in place
and firmly press down, capping the bottoms of the filters. Repeat pressing down on each
filter vial to ensure a good seal.
9. Place bleach soaked wipes onto the manifold to soak up the filtered waste and disinfect for
20 min.
10. Place into the BSC: 5-mL serological pipets, l,000-[xL pipet, l,000-[xL tips, cold (2-8°C)
BHIB aliquoted in 50-mL conical tubes, sharps container, and orange caps.
11. Pipet 5 mL of cold BHIB into each filter vial using a 5-mL serological pipet. Use a new
pipet for each filter vial. Dispose of the red cap and place the orange cap firmly into the top
of the filter. Change gloves between each sample.
12. Record the time of the BHIB addition; this represents To. Bleach wipe the filter vial.
Time of BHIB addition:
13. Place the rack of capped filter vials in a plastic bag, seal, double bag, and bleach the bag.
14. Vortex the filter vials for 10 min on the platform vortexer, Setting 7.
Start Time: End Time: Speed:
15. Place 2-mL screw cap tubes for To aliquots onto ice in the BSC.
16. After vortexing, transfer filter vials to the BSC. Remove bag.
17. Uncap one filter vial at a time and open the corresponding 2-mL tube. Using a 1-mL pipette
or serological pipet (if filter deteriorated), gently pipet up and down ~10X to mix. Transfer
1 mL from each vial to the corresponding pre-chilled (on ice) 2-mL screw cap tube for To.
Cap the tube and place it back onto ice. Wipe the filter vial with a bleach soaked laboratory
wipe. Change gloves between each sample. After transferring the To aliquots for all samples,
place the filter vial rack in a transfer container, seal, and bleach the container. Store the To
aliquot at -20 °C overnight.
To -20°C Storage Start Time: End time:
Initial/Date:
18. Transfer the filter vial rack to the shaker incubator. Secure the rack. Incubate at 30 ± 2°C at
230 rpm, overnight (i.e., 16 hours from the addition of BHIB to the filter vials). These
samples are referred to as the Tf samples. Following incubation record turbidity observation
and volume remaining in the table below.
Start Time: End Time: Speed:
Temperature:
Performed by:
Wl 2 (Appendix l)-SPG VFC AND GRAB SPORE RECOVERY
Date:
Page 18 of 19
1-18
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Sample #
Sample II)
'I'll rhid
(Yes/No)
Volume
Remaining (ml.)
Recorded by:
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
19. Proceed to WI #3 Appendix K: Work Instruction for DNA Purification to process To and Tf
samples
IV. Technical Review
Performed by: Date:
Performed by:
WI 2 (Appendix l)-SPG VFC AND GRAB SPORE RECOVERY
Date:
Page 19 of 19
1-19
-------
APPENDIX J: WORK INSTRUCTION FOR CULTURE OF BACILLUS
THURINGIENSIS KURSTAKI (Btk) T1B2 SPORES
RECOVERED FROM SPONGE STICK WIPES, VACUUM FILTER
CASSETTES, AND GRAB SAMPLES
I. PURPOSE/SCOPE
Culture of Bacillus thuringiensis kurstaki (Btk) T1B2 spores recovered from sponge stick wipes
(SSW), vacuum filter cassettes (VFC), and grab (GRB) samples following the EPA/600/R-
17/213 published by the EPA July 2017.
II. MATERIALS/EQUIPMENT
Materials
hem
.Manuracliirer
Lot N il in her
Kxp.
Dale
Storage
Temp.
Phosphate Buffered
Saline with Tween
(0.05%) (PBST)
Teknova
2-8°C
Microfunnel filters
PALL
RT
Trypticase Soy Agar
2-8°C
N/A = Not Applicable
Equipment
Item
Manuracliirer
Serial Number
Thcrmomclcr/Uccs #
Calibration
Due
Biosafety
Cabinet
(BSC)
The Baker Company
N/A
Stationary
Incubator
N/A
N/A
Vacuum
Manifold
Gelman Sciences
N/A
N/A
N/A
N/A = Not Applicable
Other Supplies and Equipment
• Forceps
• Bleach
• 5-mL, 10-mL, and 25-mL Serological Pipettes
• Pipette Aid
Performed by:
Wl 4 (Appendix J)-Culture SSW VFC and Grab
Date:
Page 1 of 4
J-1
-------
Filters - Electronically update this table with sample names from the Sample Log
Sam pie
#
Sample
Type
Location
Filler
Villi
Type
Spore
Spike Level
«TT )
Sample II)
1
SSW
2
VFC
3
GRB
4
5
6
7
8
9
10
11
12
13
14
15
16
Performed by:
Wl 4 (Appendix J)-Culture SSW VFC and Grab
Date:
Page 2 of 4
J-2
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III. PROCEDURE
Note: The following procedure is to be carried out with the extract taken from WI #2
(Appendix I — Work Instruction for Btk Spore Recovery). Process 2-3 PBST-only negative
control filter funnels alongside samples.
A. Culture Method
1. Label filter funnels per sample as indicated below. For some samples, the neat sample will be
spread in triplicate, as indicated below.
Sample #
Sample II)
Volume lo Plate
N/A
PBST Negative Control
8 mL
1
SSW
2 mL and 8 mL
2
VFC
1 mL and 3 mL
3
GRB
0.1 mL, 1 mL, and 4 mL
4
5
6
7
8
9
10
11
12
13
14
15
16
2. Place the filter funnels onto the vacuum manifold in a Class II BSC.
3. Add 5 mL of PBST to each filter funnel. Apply vacuum.
4. With the vacuum valve closed and the vacuum pressure released, place 10 mL of PBST into
each filter cup.
5. Vortex each sample, then allow 3 to 5 minutes of settle time to avoid loading large
particulates into filter funnel. For each SSW, VFC, and GRB sample, add the volume
indicated in Step 1. Save any remaining volume of culture aliquot and store at 2 to 8°C.
6. Close the vacuum valve and release the vacuum pressure. Rinse the walls of each filter
funnel using 10 mL of PBST. Apply vacuum.
7. With the vacuum valve closed and the vacuum pressure released, remove the membrane from
the filter funnel and place onto TSA. Dispose of filter bases and then change glove.
Performed by:
WI 4 (Appendix J)-Culture SSW VFC and Grab
Date:
Page 3 of 4
J-3
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8. Incubate plates inverted overnight at 30°C ± 2°C. Following incubation, save culture plates
to PCR screen presumptive Btk colonies.
a. Btk produces flat or slightly convex colonies, with edges that are slightly irregular and
have a "ground glass" appearance.
Incubation Start Date/Time: Initials:
Incubation End Date/Time: Initials:
9. Enter results into the tables below.
Sample II)
Btk TIB.
(11/2 ml.
' Colonies
( IT/8 ml.
Tolal (
(All Mori
(1 1/2 ml.
olonies
)holo«ics)
( 1 l /S ml.
I'BST \cgali\ c 1
\ A
\ A
PBST Negative #2
N/A
N/A
Sample II)
Btk
0.1 ml.
11B2 ( oh
0.1 ml.
uiies
0.1 ml.
I<
(All
0.1 ml.
rial Coloni
Morpholo1
0.1 ml.
es
zies)
0.1 ml.
IV. Technical Review
Reviewed by: Date:
Performed by:
Wl 4 (Appendix J)-Culture SSW VFC and Grab
Date:
Page 4 of 4
J-4
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APPENDIX K: WORK INSTRUCTION FOR MANUAL DNA
EXTRACTION AND PURIFICATION FROM BACILLUS
THURINGIENSIS KURSTAKI (Btk) T1B2 SPORES
I. PURPOSE/SCOPE
Manual DNA extraction and purification of Bacillus thuringiensis kurstaki (Btk) T1B2 spores
from recovered surfaces.
II. MATERIALS/EQUIPMENT
Materials
llein
Maniilai'lurer
l.ot Nilmher
Kxp.
Dale
Storage
Temp.
Initials/Dale
Lysis Buffer
Promega
RT
PMP
Promega
RT
Salt Wash
Solution
Promega
RT
Alcohol Wash
Promega
RT
70% Ethanol
Inhouse
RT
Elution Buffer
Promega
RT
N/A = Not Applicable
Equipment
Item
MiHiMl'iK-lurcr
Serisil Number
Thermometer/
Rees #
C'iilibriition
Due
Initials/
Ditto
Biosafety
Cabinet (BSC)
The Baker
Company
57544
N/A
Micropipette
Type: L200
Rainin
N/A
Micropipette
Type: L200
Rainin
N/A
Micropipette
Type: L1000
Rainin
N/A
Micropipette
Type: L1000
Rainin
N/A
Ultra-Low
Freezer
Woods
X34664
Refrigerator
Thermo Fisher
35840
Centrifuge
Eppendorf
X58983
N/A
N/A
Heat Block
VWR
949039
N/A
N/A
Thermometer
N/A
N/A = Not Applicable
Performed by: Date:
Wl 3 (Appendix K)-Manual DNA Extraction and Purification
Page 1 of 4
K-1
-------
Other Supplies and Equipment
• Micropipette tips
• Biohazard bags
• Bleach
• Prepare tubes
III. PROCEDURE
A. Manual DNA Extraction and Purification
Prepare lysis buffer with anti-foam according to manufacturer's instructions in the Magnesil
Blood Genomic, Max Yield System, Kit. Prepare the alcohol wash solution by adding ethanol and
isopropyl alcohol according to manufacturer's instructions. Prepare 70% ethanol by adding 6 mL
sterile water to 14 mL EtOH. Transfer sufficient volume of buffer to sterile, 100-mL reservoir
immediately before use. Pre-heat heat block to 80°Cprior to Section 10.4.8.
NOTE: Process samples from negative control to high inoculation level. Change gloves when
moving from an inoculated sample to a sample containing a lower inoculation level, or if
contamination of gloves is suspected. Pre-aliquot reagents from the kit to prevent
contamination of reagents between runs.
1. After the overnight (16 hours) incubation, remove the filter vial manifold from the shaker
incubator. Thaw To aliquots if they were stored at -20°C.
2. Vortex filter vials for 10 minutes on platform vortexer with speed set to 7.
Start: End: Speed:
3. Transfer the filter vial manifold to the BSC, remove and discard bags.
4. Set up 2-mL screw cap tubes for Tf aliquots in a tube. Do not use 1,5-mL tubes. Transfer Tf
aliquot screw cap tubes to the BSC.
5. Transfer the filter vial rack to the BSC. Uncap one filter vial at a time and transfer 1 mL to
corresponding 2-mL tube after gently pipetting up and down -10 to mix.
6. Centrifuge 2-mL screw cap tubes (both To and Tf) at 14,000 rpm for 10 minutes (4°C).
Start: End: Speed:
7. Remove 800 [xL of the supernatant from each tube, using a l,000-[xL pipet and dispose to
waste. Do not disturb the pellet.
8. Add 800 [xL of lysis buffer using a l,000-[xL pipet, using a new tip for each sample. Cap the
tubes and mix by vortexing on high (-1,800 rpm) in 10 second pulses for a total of 60 seconds.
9. Vortex each screw cap tube briefly (low speed, 5 to 10 seconds) and transfer the entire sample
volume to a 2-mL Eppendorf tube (ensure the tubes are labeled correctly during transfer).
Incubate the To and Tf lysate tubes at room temperature for 5 minutes.
10. Vortex the PMPs on high (-1,800 rpm) for 30 to 60 seconds, or until they are uniformly
resuspended. Keep PMPs in suspension by briefly vortexing (3 to 5 seconds) before adding to
each To and Tf lysate tube.
11. Uncap one tube at a time and add 600 [xL of PMPs to each To and Tf tube (containing 1 mL
sample).
Page 2 of 4
Performed by: Date:
Wl 3 (Appendix K)-Manual DNA Extraction and Purification
K-2
-------
12. Vortex each To and Tf tube for 5 to 10 seconds at high speed. Incubate at room temperature for
5 minutes, briefly vortex, and then place on the magnetic stand with hinged-side of the tube
facing toward the magnet.
13. Invert tubes 180 degrees (upside down) turning away from you, then right side-up, then upside
down toward you, then right side-up (caps up) position, allowing all PMPs to contact the
magnet.
14. Check to see if any beads are in the caps and if so, repeat the tube inversion cycle again. Let
the tubes sit for 5 to 10 seconds before opening. Maintain the tube layout when transferring
tubes between the magnetic stand and tube rack.
15. Uncapping one tube at a time, withdraw all liquid using a l,000-[xL pipet, placing the pipet tip
in the bottom of the 2-mL tube. Be sure to remove all liquid without disturbing PMPs. Use a
new pipet tip to remove any residual liquid, if necessary. If liquid remains in the tube cap,
remove by pipetting.
16. Uncap each tube one at a time and add 360 [xL of lysis buffer using a l,000-[xL pipet. Vortex
on low setting for 5 to 10 seconds, and transfer to tube rack.
17. Vortex each tube for 5 to 10 seconds (low) and place back on the magnetic stand. After all
tubes are in the stand, follow tube inversion cycle, as described in Step 13.
18. Remove all the liquid as described in Step 15. Use a new tip for each To and Tf tube.
Wash Steps:
19. Uncap each tube one at a time and add 360 [xL of Salt Wash Solution. Remove tube rack off
of magnetic stand. Vortex on low setting for 5 to 10 seconds, and transfer to tube rack. Place
tube rack back on magnetic stand. Invert as described in Step 13. Remove all the liquid as
described in Step 15. Use a new tip for each To and Tf tube. This is 1st Salt Wash.
20. Uncap each tube one at a time and add 360 [xL of Salt Wash Solution. Remove tube rack off
of magnetic stand. Vortex on low setting for 5 to 10 seconds, and transfer to tube rack. Place
tube rack back on magnetic stand. Invert as described in Step 13. Remove all the liquid as
described in Step 15. Use a new tip for each To and Tf tube. This is 2nd Salt Wash.
21. Uncap each tube one at a time and add 500 [xL of Alcohol Wash Solution. Remove tube rack
off of magnetic stand. Vortex on low setting for 5 to 10 seconds, and transfer to tube rack.
Place tube rack back on magnetic stand. Invert as described in Step 13. Remove all the liquid
as described in Step 15. Use a new tip for each To and Tf tube. This is 1st Alcohol Wash.
22. Uncap each tube one at a time and add 500 [xL of Alcohol Wash Solution. Remove tube rack
off of magnetic stand. Vortex on low setting for 5 to 10 seconds, and transfer to tube rack.
Place tube rack back on magnetic stand. Invert as described in Step 13. Remove all the liquid
as described in Step 15. Use a new tip for each To and Tf tube. This is 2nd Alcohol Wash.
23. Uncap each tube one at a time and add 500 [xL of Alcohol Wash Solution. Remove tube rack
off of magnetic stand. Vortex on low setting for 5 to 10 seconds, and transfer to tube rack.
Place tube rack back on magnetic stand. Invert as described in Step 13. Remove all the liquid
as described in Step 15. Use a new tip for each To and Tf tube. This is 3rd Alcohol Wash.
24. Uncap each tube one at a time and add 500 [xL of 70% Ethanol. Remove tube rack off of
magnetic stand. Vortex on low setting for 5 to 10 seconds, and transfer to tube rack. Place tube
Performed by: Date:
Wl 3 (Appendix K)-Manual DNA Extraction and Purification
Page 3 of 4
K-3
-------
rack back on magnetic stand. Invert as described in Step 13. Remove all the liquid as described
in Step 15. Use a new tip for each To and Tf tube. This is 4th Alcohol Wash.
25. If necessary, use a 200-|iL pipet to remove remaining 70% ethanol, being careful to not
disturb PMPs.
26. Open all To and Tf tubes and air dry for 2 minutes.
27. Close tubes and transfer to heat block. Re-open tubes once placed on the heat block at 80°C
until the PMPs are dry (-20 minutes, or until dry). Allow all the alcohol solution to evaporate
since alcohol may interfere with analysis. If residual condensation is present, do not remove,
leave it in place.
Start: End: Temperature:
28. DNA elution: While they are in the heating block add 200 [xL of elution buffer to each To and
Tf tube, and close tube. Vortex for 10 seconds and place back on heating block for 80 seconds.
29. Briefly vortex the tubes (5 to 10 seconds) taking care to prevent the liquid from entering the
tube cap and let the tube sit in the heating block for 1 minute. Reduce vortex speed if liquid
appears to enter the tube cap lid.
30. Repeat Step 29 four more times.
31. Remove the tubes from the heating block, place them in a tube rack in the BSC, and incubate at
room temperature for at least 5 minutes.
32. Briefly vortex each tube (5 to 10 seconds) on low speed and centrifuge at 2,000 rpm, 4°C for
1 minute.
33. Briefly vortex each tube and place on the magnetic stand for at least 30 seconds.
34. Collect liquid from each To and Tf tube and transfer -80-90 uL to a clean, labeled, 1.5-mL tube
on ice (check tube labels to ensure the correct order). Use a new tip for each tube. Visually
verify absence of PMP carryover during final transfer. If magnetic bead carryover occurred,
place 1.5-mL tube on magnet, collect liquid, and transfer to a clean, labeled, 1.5-mL tube.
35. Centrifuge tubes at 14,000 rpm at 4°C for 5 minutes to pellet any particles remaining with the
eluted DNA; carefully remove supernatant from all samples and transfer to a new 1.5-mL tube
using a new tip for each tube.
Start: End:
36. Store To and T, DNA extract tubes at 4°C until PCR analysis. Continue to PCR analysis.
Note: If PCR cannot be performed within 24 hours, freeze DNA extracts at -20°C.
Labeled:
Start:
End:
Date/Time:
Storage Location:
Storage Temperature:
IV. Technical Review
Performed by:
Date:
Page 4 of 4
Performed by:
Wl 3 (Appendix K)-Manual DNA Extraction and Purification
Date:
K-4
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APPENDIX L: WORK INSTRUCTION FOR REAL-TIME PCR ANALYSIS
FOR BACILLUS THURINGIENSIS KURSTAKI (Btk) T1B2 DNA
I. PURPOSE/SCOPE
Real-time PCR analysis for Bacillus thuringiensis kurstaki (Btk) T1B2 DNA.
II. MATERIALS/EQUIPMENT
Enter material lot and expiration dates used into FORM A:
Materials
hem
Manufacturer
Product Nilmher
TaqMan Fast Advanced PCR Mix (2x)
Applied Biosystems
4444556
Platinum Taq DNA Polymerase
Invitrogen
10966-034
Custom Primers and Probes w/ 6-FAM
Reporter Dye
Applied Biosystems
Custom
PCR Grade Water
Fisher Scientific
BP2484100
Optical Plate Seal
ThermoFisher
4311971
Equipment
llem
Manufacturer
Serial Number
Thcrmomclcr/Uccs #
Calibration
Due
Biosafety
Cabinet (BSC)
Baker
Thermo Forma
N/A
Micropipette
Type: 10
N/A
Micropipette
Type: 20
N/A
Micropipette
Type: 200
N/A
Micropipette
Type: 1000
N/A
Freezer
Centrifuge
LabNet
K4070898
N/A
N/A
7500 Fast
Applied
Biosystems
275017115
N/A
N/A = Not Applicable
Other Supplies and Equipment
• Micropipette tips, 96-well 0.1 mL FAST plates, optical caps, bleach, DNA erase, 70%
isopropanol
Attach FORM A: Date:
Performed by:
Wl 5 (Appendix L) Real-Time PCR, Btk T1B2
Date:
Page 1 of 7
L-1
-------
III. PROCEDURE
A. Prepare samples for qPCR
Note: This step must be performed in the BSC outside the PCR clean room set-up area.
Prepare a fresh aliquot of PCR-grade water per sample batch to use for 1:10 dilutions and
NTCs.
1. To and T/DNA extracts: Label 1.5-mL tubes with the sample identifier and "10-fold
dilution." Add 90 [xL of PCR-grade water to the tubes.
2. Mix To and Tf DNA extracts by vortexing (3 to 5 seconds), spin at 14,000 rpm for 2 minutes,
and transfer 10 [xL of supernatant to 1.5-mL Eppendorf tubes with 90 [xL of PCR-grade
water, maintaining the plate layout.
Note: No centrifugation is required if PCR analysis is conducted immediately after DNA
elution.
B. Real-time PCR Analysis of DNA Extracts
1. Decontaminate the PCR workstation by treating all work surfaces with bleach solution,
followed by 70% isopropanol. After decontamination, discard gloves and replace with a new
clean pair.
Note: If gloves become contaminated, they should be disposed of and fresh gloves donned.
Only open one tube at a time throughout the process. At no point, should more than one tube
be open. Do not allow hands (gloved or otherwise) to pass over an open tube, PCR plate, or
any reagent container. All usedpipet tips, gloves and tubes must be discarded in a biohazard
autoclave bag.
2. Determine the number of reactions that are to be run. Prepare a sufficient volume of Master
Mix to allow for one extra reaction for every 10 reactions, so that there is enough Master Mix
regardless of pipetting variations. For each batch of samples, PCR Master Mix should be
made for four positive controls (PCs), four NTCs, and six DNA extracts per sample (three for
To and three for TfDNA extracts). Record sample names and reaction numbers on FORM A.
3. In a clean PCR-preparation hood, pipet 20 [xL of Master Mix into the wells of the PCR plate.
Label four wells as NTC.
4. Add 5 [xL of PCR-grade water into each of the NTC wells.
5. Lightly seal the NTC wells with optical caps and cover all other wells of the plate using
optical caps.
6. Vortex each sample briefly, then add 5 |xL to each sample well. Lightly seal the sample wells
with optical caps.
7. Vortex the PC, B. thuringiensis kurstaki T1B2 DNA [10 pg/1 [xL or 50 pg/5 jliL] by adding
5 |xL to each PC well. Tightly seal the wells using optical caps.
C. Within the Post-Amplification Laboratory, load 96-well plates onto 7500 Fast.
1. Set up 7500 Fast (TaqMan)
a. Open the 7500 Fast Software and select New Experiment
Performed by:
Wl 5 (Appendix L) Real-Time PCR, Btk T1B2
Date:
Page 2 of 7
L-2
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i. Set Experiment Properties:
1. Enter an Experiment Name
2. Select 7500 Fast (96 wells)
3. Select Quantitation - Standard Curve
4. Select TaqMan Reagents
5. Select Fast (-40 minutes to complete a run)
ii. Plate Step
1. Define the Target and Samples
a. Define a target with designated reporter (6-FAM) and None as
the quencher. Multiple targets can be selected if more than one
target will be run on the plate.
b. Define samples by selecting Add New Sample for all samples,
include NTCs and standard curve concentrations as sample
names.
2. Assign Targets and Samples
a. Highlight the wells that will be used for this assay, then check
the assign box to assign the target. Check appropriate task
(Unknown, Standard, or Negative Control).
b. Highlight the sample wells, then check the assign box to assign
the sample.
c. Highlight the standard curve wells, to enter the sample name,
then enter a quantity for each standard under the assign target
pane.
d. Select ROX as the passive reference from the Passive Reference
drop down box.
iii. Run Method
1. Under Graphical View, enter 25 |iL as the reaction volume.
2. Set thermocycling conditions to match the below settings:
Temperature PC)
Time
Cycles
50.0
2:00
Hold
95.0
2:00
Hold
0
<):<>*
45
(id 0
11:30
25 |iL Total Volume
3. Select Save As and assign unique plate file name and save in project
folder.
Performed by:
Wl 5 (Appendix L) Real-Time PCR, Btk T1B2
Date:
Page 3 of 7
L-3
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iv. Start Run
1. Centrifuge the plate at 300 x g for 1 to 2 minutes at room temperature or
in Labnet's MPS-1000 Mini Plate Spinner briefly. Check that the
samples are at the bottom of the wells and no bubbles are at the bottom
of the wells.
2. Select Start Run.
3. When run is complete, burn the file to a CD.
4. Remove 96-well plate from the 7500 Fast and dispose.
D. Analysis
1. Open the assay with the most current version of 7500 Fast software
a. Select the Analysis Tab
b. Select Plot Settings:
i. Plot Type: ARn vs Cycle
ii. Graph Type: Log
iii. Plot Color: Well
c. Select Options:
i. Target: Select target that was assigned to wells
ii. Threshold: Uncheck Auto and Auto Baseline
iii. Show: Check Threshold, Baseline Start
d. In Amplification Plot, set the Threshold to 0.1.
e. In View Well Table, view Ct values for all samples. Adjust the baseline manually in the
Amplification Plot so that the Baseline End is two Ct values below the lowest Ct value
whole number, ignoring values to the right of the decimal. For example, if the lowest Ct
value is 22.610105, the Baseline End cursor should be set to 20.
f. After moving Baseline End, recheck the Ct values and adjust again if necessary.
2. Save file with the file extension "_Analyzed"
3. Export Results
a. Select Export
b. Check the Results option, one file
c. Enter a unique plate file name with run date and initials
d. Select file type, .xls (Excel)
e. Browse File Location to save in project-specific location
f. Select Start Export, then Close Export Tool
Performed by:
Wl 5 (Appendix L) Real-Time PCR, Btk T1B2
Date:
Page 4 of 7
L-4
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4. Print Report
a. Select Print Report
b. Check the below selections and then Print Report:
i. Experiment Summary
ii. Results Summary
iii. Amplification Plot
iv. Standard Curves
v. Results Table (By Well)
c. Under Analysis Setting, Select Multicomponent Plot
i. Highlight all NTC wells, then select Print from the icon on the Multicomponent
Plot
ii. Highlight all Standard wells, then select Print from the icon on the
Multicomponent Plot
iii. Highlight all Sample wells, then select Print from the icon on the
Multicomponent Plot
d. Annotate printouts
i. Initial and date every page
ii. Initial, date, and error or otherwise annotate all errors and comments
iii. Indicate which, if any, wells of the standard curve were omitted
iv. Indicate multicomponent results for each well on the Results Table
5. Quality Control Acceptance Criteria
a. Verify the below acceptance criteria are met
• Amplification in PC wells
• NTC wells have no amplification
IV. Data Calculations
Calculate the average Ct value from the replicate reactions for To and Tf DNA extracts of
each sample. Subtract the average Ct value of the Tf DNA extract from the average Ct value
of the To DNA extract to generate delta Ct value (ACt). If there is no Ct value for the To
DNA extract (i.e., the To is non-detect), use 45 (total number of PCR cycles used) as the Ct
value.
Performed by: Date:
V. Technical Review
All data will receive technical review and QC review in accordance with QA. 1-005.
Technical Review Initials/Date:
QC Review Initials/Date:
Performed by:
Wl 5 (Appendix L) Real-Time PCR, Btk T1B2
Date:
Page 5 of 7
L-5
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DNA ASSAY: 96-Well Plate Setup for Fast 7500 (FORM A)
Project: Barcode:
Target: Btk T1B2
1. Calculate the total number of reactions per plate:
Sample wells + 4 NTC wells + 4 PCs + extras = total rxns/plate (Y)
2. Prepare the Master Mix by combining the following reagents in an appropriate tube
according to the following calculation:
Reagent volume (X) x total rxns/plate (Y) = total volume of reagent needed
Reagent
Manufacturer
Lot No.
Exp.
Date
X
Y
Total
Volume
(jiL)
TaqMan Fast
Advanced Master
Mix (Cat. 4444556)
Applied
Biosystems
12.5 |iL
Platinum Taq
Polymerase
Invitrogen
0.1 |iL
Btk T1B2 For.
Primer (25 |iM)
In-House
TBD
1 |iL
Btk T1B2 Rev.
Primer (25 |iM)
In-House
TBD
1 |iL
Btk T1B2 Probe
(2 nM)
In-House
TBD
1 |xL
PCR Grade Water
4.4 |xL
Total
20 |iL
3. Distribute 20 |iL of Master Mix into each reaction well, as indicated in the plate layout,
below. Loosely cover all wells containing Master Mix with caps.
4. Add 5 |iL of PCR-grade water to each of the NTC Wells. Cap wells tightly.
5. Add 5 |iL of PNC (Method Blank) to the corresponding wells and secure the caps.
6. Add 5 |iL of Sample to the corresponding wells and secure the caps.
7. Add 5 |iL of PC to the corresponding wells and secure the caps.
Positive Control Prep Date
Positive Control Lot
8. Centrifuge the plate using Labnet's MPS-1000 Mini Plate Spinner at room temperature,
and then load the plate onto the 7500 Fast.
Performed by:
Wl 5 (Appendix L) Real-Time PCR, Btk T1B2
Date:
Page 6 of 7
L-6
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1
2
3
4
5
6
7
8
9
10
11
12
A
B
C
D
E
F
G
H
PC
50
PS
PC
50
PS
PC
50 pg
PC
50
PS
NTC
NTC
NTC
NTC
Technicians
Signature
Date
Master Mix, NTC
Samples
Standards
Analyst
Reviewed By: Date:
Performed by:
Wl 5 (Appendix L) Real-Time PCR, Btk T1B2
Date:
Page 7 of 7
L-7
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APPENDIX M: WORK INSTRUCTION FOR SELECTING
PRESUMPTIVE BACILLUS THURINGIENSIS KURSTAKI (Btk) T1B2
COLONIES FOR QPCR CONFIRMATION
I. PURPOSE/SCOPE
Select and screen Bacillus thuringiensis kurstaki (Btk) T1B2 colonies recovered on culture plates
using qPCR.
II. MATERIALS/EQUIPMENT
Materials
Item
Ma n ii fact ii rer
l.ol Nilmher
Kxp.
Dale
Storage
Temp.
PCR-grade water
Teknova
RT
l-|iL loop, 10-|iL
loop or inoculating
needles
N/A
RT
1.5- or 2-mL tubes
N/A
N/A
RT
N/A = Not Applicable
Equipment
llem
.Maniilai'lurer
Serial Number
Calibration
Due
Biosafety
Cabinet (BSC)
The Baker Company
Heat Block
VWR
N/A
Thermometer
Camera
N/A
N/A
N/A
N/A = Not Applicable
Other Supplies and Equipment
• Bleach
• 5-mL, 10-mL, and 25-mL serological pipettes
III. PROCEDURE
A. Selecting Colonies
1. Pipette 100 |iL of PCR-grade water into 1.5- or 2-mL tubes.
2. Select colonies. Take pictures of colonies that are selected.
3. Use l-|iL loop, 10-|iL loop, or inoculating needle to select the colony.
4. Immerse needle into PCR-grade water and rotate to dislodge cellular material.
Page 1 of 2
Performed by: Date:
Wl 6 (Appendix M)-Colony Screen
M-1
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5. Colonies from a single sample can be pooled to increase the number of presumptive colonies
screened. Up to 10 colonies can be pooled within a 100-|iL volume of PCR-grade water.
Repeat Steps 3 and 4 to pool multiple colonies from a single sample and record the number
of colonies pooled in the table below.
6. Lyse the colony suspension for 5 minutes on a heat block at 95 ± 2°C.
Incubation Start Date/Time: Initials:
Incubation End Date/Time: Initials:
7. Store lysed suspension at - 20°C for qPCR analysis.
8. Prior to qPCR analysis, thaw tubes, centrifuge at 14,000 rpm for 2 minutes. Use supernatant
for qPCR.
Filters - Record Filter ID and Morphology for Selected Colonies
I'u he
#
liller II)
Volume (ml.)
Morphology
(IJlk or
Background)
# of Colonics
Pooled
PC U Result
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
IV. Technical Review
Reviewed by: Date:
Performed by:
Wl 6 (Appendix M)-Colony Screen
Date:
Page 2 of 2
M-2
-------
APPENDIX N: WORK INSTRUCTION FOR
BHIB ENRICHMENT FOR CULTURE
I. PURPOSE/SCOPE
Enrich extracted sponge or filter in Brain Heart Infusion Broth (BHIB).
II. MATERIALS/EQUIPMENT
Materials
llem
Maniilai'lurer
l.ol Nilmher
Kxp.
Dale
Storage
Temp.
Initials/Dale
PCR-grade water
Teknova
RT
10-|iL loop or
inoculating
needles
RT
1.5- or 2-mL
tubes
RT
Trypticase Soy
Agar (TSA)
plates
2 to 8°C
BHIB
RT
N/A = Not Applicable
Equipment
llem
Manuracliirer
Serial Number
Thermomele
r/Uees #
Calibration
Due
Initials/
Dale
Biosafety
Cabinet (BSC)
The Baker
Company
N/A
Incubator
Precision
Thermometer
Traceable
N/A
N/A
N/A
Heat Block
VWR
Refrigerator
Fisher
C3274822
115
8/2020
N/A = Not Applicable
Other Supplies and Equipment
• 25-mL serological pipettes
III. PROCEDURE
A. Enrichment of Sponges and Filters
1. Add 25 mL of BHIB to each specimen cup containing the extracted sponge or filter.
2. Incubate cups at 30°C ± 2°C for 24-48 hours.
Page 1 of 4
Performed by: Date:
Wl 7 (Appendix N) BHIB Enrichment
N-1
-------
Incubation Start Date/Time: Initials:
Incubation End Date/Time: Initials:
3. Evaluate the BHIB enrichment for samples.
I. If broth is not turbid, record as no growth (NG) and incubate for an additional
24 hours.
II. If broth is turbid, record as positive growth (G+) and proceed to Step 4.
Ssiin pic
N il in her
liher II)
(¦rowlli (C+
r
24 hours
or No (i row 111
48 hours
Recorded hv:
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
4. For samples that have not been confirmed positive by culture membrane plating, streak
turbid samples onto TSA. Cap tightly and mix BHIB with growth for 30 seconds.
Remove a loopful of broth with a 10-[xL loop and streak triplicate TSA plates for
isolation. Store enriched samples at 2 to 8°C.
5. Incubate the isolation plates and BHIB with growth at 30°C ± 2°C for a maximum of
three days.
Incubation Start Date/Time: Initials:
Incubation End Date/Time: Initials:
6. Examine plates for Btk colonies.
I. If presumptive Btk colonies are isolated and positive identification has not
already been confirmed by PCR from a representative sample, record the sample
Page 2 of 4
Performed by: Date:
Wl 7 (Appendix N) BHIB Enrichment
N-2
-------
in the table below as a colony selection sample and proceed to PCR confirmation
from BHIB streak plates (Section B).
II. If NO presumptive Btk colonies are isolated and positive identification has not
already been confirmed by PCR from a representative sample, record the sample
in the table below as a BHIB Analysis sample and proceed to PCR confirmation
of BHIB Enriched Samples (Section C).
Sample #
1 iller II)
Colony Selection
or IS 111 IS Analysis
N il in her of
Colonies
Screened
PCU Ucsiill
Recorded
hy:
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
B. Selecting Colonies
1. Pipette 100 |iL of PCR-grade water into 1.5- or 2-mL tubes.
2. Select colonies.
3. Use l-|iL loop, 10-|iL loop or inoculating needle to select the colony.
4. Immerse needle into PCR-grade water and rotate to dislodge cellular material.
5. Colonies from a single sample can be pooled to increase the number of presumptive
colonies screened. Up to 10 colonies can be pooled within a 100-|iL volume of PCR-
grade water. Repeat Steps 3 and 4 to pool multiple colonies from a single sample and
record the number of colonies pooled in the above table.
6. Proceed to Lysis and Storage (Section D)
C. PCR Confirmation of BHIB Enriched Samples
Performed by:
Wl 7 (Appendix N) BHIB Enrichment
Date:
Page 3 of 4
N-3
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1. Transfer 50 [xL of broth with growth to a microcentrifuge tube.
2. Centrifuge at 12,000 x g for 2 minutes.
3. Remove and discard the supernatant in an autoclavable biohazard container. Add 100 [xL
of PCR-grade water to the tube containing the bacterial pellet.
4. Resuspend the pellet by flicking the tube.
5. Proceed to Lysis and Storage (Section D)
D. Lysis and Storage
1. Lyse colony screen and BHIB enrichment samples for 5 minutes on a heat block at 95 ±
2. Store lysed suspension at -20°C for qPCR analysis or refrigerator if processed same day.
3. Prior to qPCR analysis, thaw tubes, centrifuge @ 14,000 rpm for 2 minutes. Use
supernatant for PCR analysis.
IV. Technical Review
2°C.
Incubation Start Date/Time:
Initials:
Incubation End Date/Time:
Initials:
Reviewed by:
Date:
Performed by:
Wl 7 (Appendix N) BHIB Enrichment
Date:
Page 4 of 4
N-4
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