EPA/6Q0/R-21/212 | September 2021
www.epa.gov/emergency-response-research
United States
Environmental Protectio
Agency
&EPA
Evaluation and Testing of
Outdoor Surface Sample
Collection and Analysis Methods
Office of Research and Development
Homeland Security Research Program
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September 2021
FINAL REPORT
for
Evaluation and Testing of Outdoor Sample
Collection and Analysis Methods
EPA Contract Number: EP-C-16-014
Task Order 19F0208
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 IA HSHQPM-17-X-00245. This report was
prepared by Battelle Memorial Institute under EPA Contract Number EP-C-16-014; Task Order
68HERC20F0208. 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. Worth Calfee
U.S. Environment Protection Agency
109 T.W. Alexander Drive
Mail Code: E343-06
Research Triangle Park, NC 27711
calfee.worth@epa.gov
919-541-7600
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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 groundwater, 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 performance and limitations of traditional and innovative
sampling and analysis methods, as well as sample collection methods that leverage existing United
States Coast Guard (USCG) maintenance procedures, when applied to USCG assets and bases. The
findings can be used to better plan and execute the recovery of a USCG base, or any large outdoor urban
area, following a biological contamination incident. This work was coordinated with and managed by
the EPA's Homeland Security Research Program (HSRP) under the Department of Homeland Security
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 iii
Foreword iv
Acronyms and Abbreviations xiii
Acknowledgments xv
Executive Summary xvi
1.0 INTRODUCTION 1
1.1 Background 1
1.2 Objective 1
1.3 Scope 2
2.0 MATERIALS AND METHODS 3
2.1 Target Surface/Material Sampled 3
2.1.1 Surface/Material Selection 3
2.1.2 Surface/Material Preparation 5
2.1.3 Btk Contaminated Surface/Material Storage 7
2.2 Sampling Methods 9
2.2.1 Traditional Sampling Methods 9
2.2.2 Nontraditional Sampling Methods 13
2.3 Test Matrix 21
2.4 Microbiological Methods 23
2.4.1 Spore Stock 24
2.4.2 Spiking Positive Controls 24
2.4.3 Sample Processing for Spore Recovery 25
2.4.4 Culture Method 26
2.4.5 RV-PCR Method 27
2.5 Overall Method Implementation 30
2.6 Data Reduction and Analysis 32
2.6.1 Percent Recovery of Presumptive Btk T1B2 Spores 32
2.6.2 RV-PCR Method 33
2.6.3 Presentation of Results 33
3.0 RESULTS AND DISCUSSION 34
3.1 Sponge Stick Sample Analysis Results 34
3.1.1 Sponge Stick Sample Culture Analyses 34
3.1.2 Btk T1B2 Confirmation 39
3.1.3 Sponge Stick Sample RV-PCR Analyses 41
3.1.4 Analytical Method Comparison 44
3.1.5 Analysis of Controls 46
3.2 Vacuum Filter Cassette Sample Analysis Results 46
3.2.1 Vacuum Filter Cassette Sample Culture Analysis 46
3.2.2 BtkT1B2 Confirmation 50
3.2.3 Vacuum Filter Cassette Sample RV-PCR Analysis 52
3.2.4 Analytical Method Comparison 55
3.2.5 Analysis of Controls 56
3.3 Grab Sample Analyses Results (Gravel and Bilge Water) 56
3.3.1 Grab Sample Culture Analyses 56
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Table of Contents (Cont.)
Page
3.3.2 BtkT1B2 Confirmation 63
3.3.3 Grab Sample RV-PCR Analysis 64
3.3.4 Analytical Method Comparison 67
3.3.5 Analysis of Controls (Bilge Water) 68
3.3.6 Analysis of Controls (Gravel) 68
3.4 Traditional Sampling Method Performance (Spore Recovery) Ranking 68
3.5 Nontraditional Sample Analysis Results 69
3.5.1 Nontraditional Sample Culture Analyses 69
3.5.2 BtkT1B2 Confirmation 75
3.5.3 Nontraditional Sample RV-PCR Analyses 75
3.5.4 Analytical Method Comparison 79
3.5.5 Analysis of Controls 79
3.6 Summary of Detection Accuracy of Presumptive Colonies 79
4.0 QUALITY ASSURANCE/QUALITY CONTROL 81
4.1 Equipment Calibration 81
4.2 QC Results 81
4.3 Operational Parameters 81
4.4 Audits 81
4.4.1 Performance Evaluation Audit 81
4.4.2 Technical Systems Audit 82
4.4.3 Data Quality Audit 82
4.5 QA/QC Reporting 82
4.6 Data Review 82
5.0 SUMMARY OF METHOD OBSERVATIONS AND EXPERIENCES 83
5.1 Sample Processing Considerations 83
5.2 Method Qualitative Assessment 83
5.2.1 Culture Method 84
5.2.2 RV-PCR Method 84
5.2.3 Time/Cost Estimates 84
5.3 Culture Processing Considerations 85
5.3.1 BHIB Enrichment Culture Analysis 85
5.4 RV-PCR Processing Considerations 86
5.4.1 Biological Safety Level 3 Considerations 86
5.4.2 Suggestions to Improve RV-PCR Throughput 86
5.5 Sponge Stick Sample Analysis 88
5.5.1 Biological Safety Level 3 Considerations 88
5.5.2 Sponge Stick Method Considerations 88
5.6 Vacuum Filter Cassette Sample Analysis 88
5.7 Washdown and Grab Sample Analysis 88
6.0 CONCLUSIONS AND RECOMMENDATIONS 89
7.0 REFERENCES 92
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List of Appendices
APPENDIX A: SEA SALT COMPOSITION REVIEW
APPENDIX B: SPRAY TABLE DESCRIPTION AND CHARACTERIZATION
APPENDIX C: VESSEL WATER WASHDOWN NONTRADITIONAL SAMPLING METHOD
WORK INSTRUCTION
APPENDIX D: SIMULATED RAINWATER FORMULATION
APPENDIX E: SIMULATED RAINWATER NONTRADITIONAL SAMPLING METHOD WORK
INSTRUCTION
APPENDIX F: SQUEEGEE NONTRADITIONAL SAMPLING METHOD WORK
INSTRUCTION
APPENDIX G: BRISTLE BRUSH NONTRADITIONAL SAMPLING METHOD WORK
INSTRUCTION
APPENDIX H: FORMULATIONS OF RECIPES USED IN BIOLOGICAL TEST
METHODS
APPENDIX I: WORK INSTRUCTION FOR SPIKING WITH BACILLUS THURINGIENSIS
KURSTAKI HD-1 T1B2 SPORES
APPENDIX J: WORK INSTRUCTION FOR BACILLUS THURINGIENSIS KURSTAKI T1B2
SPORE RECOVERY FROM OUTDOOR SAMPLES
APPENDIX K: WORK INSTRUCTION FOR CULTURE OF BACILLUS THURINGIENSIS
KURSTAKI T1B2 SPORES RECOVERED FROM OUTDOOR SURFACES
APPENDIX L: WORK INSTRUCTION FOR MANUAL DNA EXTRACTION AND
PURIFICATION FROM BACILLUS THURINGIENSIS KURSTAKI T1B2 SPORES
APPENDIX M: WORK INSTRUCTION FOR REAL-TIME PCR ANALYSIS FOR BACILLUS
THURINGIENSIS KURSTAKI T1B2 DNA
APPENDIX N: WORK INSTRUCTION FOR SELECTING PRESUMPTIVE BACILLUS
THURINGIENSIS KURSTAKI T1B2 COLONIES FOR QPCR CONFIRMATION
APPENDIX 0: WORK INSTRUCTION FOR ENRICHMENT FOR CULTURE
APPENDIX P: OUTDOOR TEMPERATURE AND RH PLOTS
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Table of Figures
Page
Figure 1. Representative Images of the Seven Surface Types 4
Figure 2. Photograph of Spray Table Used to Apply Sea Salt Spray and Bf/cT1B2 Spores
onto Target Surfaces 6
Figure 3. Illustration (Top View) of Location of Reference Coupons on the Spray Table and
Representative Sea Salt Spray Residue Collected (mg/cm2) at those Locations
Used to Demonstrate Ability to Apply a Target Sea Salt Residue onto Target
Surfaces 6
Figure 4. Illustration of Location of Reference Coupons on the Spray Table and
Representative Btk Spore Load (CFU/cm2) Collected at those Locations Used to
Demonstrate Ability to Apply a Btk T1B2 Spores onto Target Surfaces 7
Figure 5. Ventilated Shed Used to Store Contaminated Surfaces During 180 Days of
Environmental Exposure 8
Figure 6. Photograph of the Contaminated Surfaces (Facing Up) Contained Within the
Ventilated Shed for Up to 180 Days of Environmental Exposure 9
Figure 7. Example of Sample Locations Over a 2 x 8 ft Area of Target Surface and
Schedule for Sponge Stick and VFC Surface Sampling 10
Figure 8. Prewetted Sponge Stick from 3M Used for Surface Sampling (left) and Sampling
a Glass Surface (right) 10
Figure 9. Vacuum Filter Cassette (37-mm Diameter) Assembled (Left) and Disassembled
(Right) Used for Surface Sampling 11
Figure 10. Photograph of the Bilge Water Grab Sample Being Collected (left) from a
Medium Endurance Cutter at Base Portsmouth 12
Figure 11. Photograph of a Sample of Gravel in 1-L Nalgene Bottle Containing 500 mL
PBST 12
Figure 12. Inclined Surface Support Fixture Used to Support the 2 ft x 2 ft Target Surfaces
for Nontraditional Sampling Methods 13
Figure 13. Vessel Washdown Method Spray Pattern 15
Figure 14. Vessel Washdown Nontraditional Sampling Method Applied to Marine Grade
Aluminum 15
Figure 15. Spray Nozzle Producing Simulated Rainfall 16
Figure 16. Simulated Rainfall Landing on Shingles 17
Figure 17. Uniformity of Simulated Rainfall on Target Surface 17
Figure 18. Squeegee Surface Wipe Sampling Pattern 18
Figure 19. Surface Sampling Marine Grade Aluminum Using a Squeegee 19
Figure 20. Bristle Brush Sampling Pattern 20
Figure 21. Surface Sampling Marine Grade Aluminum Using a Bristle Brush (left) and
Rinsing of the Brush by Submerging in Bucket of Water (right) 21
Figure 22. Sponge Stick (A), Vacuum Filter Cassette (B), and Gravel (C) Being Spiked with
Btk T1B2 Suspension for Use as Analytical Controls 25
Figure 23. Manifold Containing 16 Filter Vials (A), Capping Tray (B), and Capped Filter
Vials Containing BHIB (C) 28
Figure 24. Process Flow Chart Depicting Key Process Steps in Chronological Order 31
Figure 25. Presumptive Bf/cT1B2 Spores Recovered from Surfaces with a Low Target
Load of 3,200 Btk Spores and Sampled Using a Sponge Stick 36
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Table of Figures (Cont.)
Page
Figure 26. Presumptive Bf/cT1B2 Spores Recovered from Surfaces with a High Target
Load of 320,000 Btk Spores and Sampled Using a Sponge Stick 37
Figure 27. Culture Images of Day 1 and Day 30 Spore Recovery from Glass Salted
Surface Plated on TSA 38
Figure 28. Culture Images of Day 90 and Day 180 Spore Recovery from Glass Salted
Surface Plated on TSA 38
Figure 29. Sponge Stick Used to Sample GLASS-SA Collected Following 180 Days of
Environmental Exposure Shows Environmental Exposure Led to an Accumulation
of Grime on the Surfaces 39
Figure 30. RV-PCR Analysis of Btk T1B2 Spores Recovered from Surfaces with a Low
Target Load of 3,200 Btk Spores and Sampled Using a Sponge Stick 43
Figure 31. RV-PCR Analysis of Btk T1B2 Spores Recovered from Surfaces with a High
Target Load of 320,000 Btk Spores and Sampled Using a Sponge Stick 43
Figure 32. Presumptive Bf/cT1B2 Spores Recovered from Surfaces with a Low Target
Load of 3,200 Btk Spores and Sampled Using a Vacuum Filter Cassette 48
Figure 33. Presumptive Bf/cT1B2 Spores Recovered from Surfaces with a High Target
Load of 320,000 Btk Spores and Sampled Using a Vacuum Filter Cassette 48
Figure 34. Culture Images of Day 1 through Day 180 Spore Recovery from Nonskid Clean
Surface Plated on TSA 49
Figure 35. Vacuum Filter Cassette Following Sampling of a Nonskid Tread Showing
Collection of Cottonwood Seeds that Deposited on the Surface 50
Figure 36. RV-PCR Analysis of Btk T1B2 Spores Recovered from Surfaces with a Low
Target Load of 3,200 Btk Spores and Sampled Using Vacuum Cassette Filters 54
Figure 37. RV-PCR Analysis of Btk T1B2 Spores Recovered from Surfaces with a High
Target Load of 320,000 Btk Spores and Sampled Using a Vacuum Cassette
Filter 54
Figure 38. Presumptive Bf/cT1B2 Spores Recovered from Surfaces with a Low Target
Load of 3,200 Btk Spores and Sampled Using a Grab Method 58
Figure 39. Presumptive Bf/cT1B2 Spores Recovered from Surfaces with a High Target
Load of 320,000 Btk Spores and Sampled Using a Grab Method 58
Figure 40. Poor Recovery from Filter Membrane for Gravel Samples Collected at Day 30 59
Figure 41. Culture Images of Day 1 and Day 30 Spore Recovery from Gravel Clean Plated
on TSA 60
Figure 42. Culture Images of Day 90 and Day 180 Spore Recovery from Gravel Clean
Plated on TSA 61
Figure 43. Photograph of Bilge Water Unspiked (A, B) and Spiked High (C and E) and Low
(D and F) with Btk T1B2 Spores After 30 Days of Outdoor Ambient Exposure 61
Figure 44. Culture Images of Day 1 and Day 30 Spore Recovery from Bilge Water Plated
on TSA 62
Figure 45. Culture Images of Day 90 and Day 180 Spore Recovery from Bilge Water
Plated on TSA 63
Figure 46. RV-PCR Analysis of Btk T1B2 Spores Recovered from Surfaces with a Low
Target Load of 3,200 Btk Spores and Sampled Using a Grab Method 66
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Table of Figures (Cont.)
Page
Figure 47. RV-PCR Analysis of Btk T1B2 Spores Recovered from Surfaces with a High
Target Load of 320,000 Btk Spores and Sampled Using a Grab Method 66
Figure 48. Presumptive Bf/cT1B2 Spores Recovered from Surfaces with a Low Target
Load of 3,200 Btk Spores and Sampled Using Various Nontraditional Sampling
Methods 74
Figure 49. Presumptive Bf/cT1B2 Spores Recovered from Surfaces with a High Target
Load of 320,000 Btk Spores and Sampled Using Various Nontraditional Sampling
Methods 74
Figure 50. RV-PCR Analysis of Btk T1B2 Spores Recovered from Surfaces with a Low
Target Load of 3,200 Btk Spores and Sampled Using Various Nontraditional
Sampling Methods 78
Figure 51. RV-PCR Analysis of Btk T1B2 Spores Recovered from Surfaces with a High
Target Load of 320,000 Btk Spores and Sampled Using Various Nontraditional
Sampling Methods 78
Figure 52. Autoclaving HDPE Bottles Compromise Structure 83
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Table of Tables
Page
Table 1. Test Matrix for Samples Collected Using Traditional Sampling Methods 22
Table 2. Test Matrix for Samples Collected using Nontraditional Sampling Methods 23
Table 3. Target Btk T1B2 Spore Loading Levels Spiked onto Each Sample Type for
Positive Control 25
Table 4. Bf/cT1B2 TaqMan PCR Assay Primers, Probe, and Amplicon Sequences 30
Table 5. Presumptive Bf/cT1B2 Spore Recovery from Different Surfaces Sampled Using
Sponge Sticks 35
Table 6. Summary of the Accuracy of Identification of Presumptive Btk T1B2 by PCR
Confirmation from Surfaces Sampled with Sponge Sticks Stored in Outdoor
Conditions Over Time 40
Table 7. RV-PCR Analyses of Sponge Stick Surface Samples for Detection of Btk T1B2
Spores 41
Table 8. Analytical Method Comparison Displaying Culture Presumptive, Culture ID with
PCR Confirmation and RV-PCR Replicates Detected (N = 3) for Surfaces
Sampled with Sponge Sticks 45
Table 9. Analytical Method Comparison Displaying Culture ID with PCR Confirmation and
RV-PCR Replicates Detected (N = 3) for Surfaces Sampled with Sponge Sticks 45
Table 10. Presumptive Btk T1B2 Spores Recovered from Different Surfaces Sampled
Using Vacuum Filter Cassettes 47
Table 11. Summary of the Accuracy of Identification of Presumptive Btk T1B2 by PCR
Confirmation from Surfaces Sampled with Vacuum Cassettes Stored in Outdoor
Conditions Over Time 51
Table 12. RV-PCR Analyses of Vacuum Filter Cassette Surface Samples for Detection of
Btk T1B2 Spores Using T1B2 Barcode Target 53
Table 13. Analytical Method Comparison Displaying Culture Presumptive; Culture ID with
PCR Confirmation and RV-PCR Replicates Positively Detected (N = 3) for
Surfaces Sampled with Vacuum Cassettes 55
Table 14. Analytical Method Comparison Displaying Culture ID with PCR Confirmation and
RV-PCR Replicates Detected (N = 3) for Surfaces Sampled with Vacuum
Cassettes 56
Table 15. Recovery Efficiencies for Presumptive Bf/cT1B2 Spores from Gravel and Bilge
Water Grab Samples Cultured on TSA Medium 57
Table 16. Summary of the Accuracy of Identification of Presumptive Btk T1B2 by PCR
Confirmation from Grab Samples Stored in Outdoor Conditions Over Time 64
Table 17. RV-PCR Analyses of Grab Samples for Detection of Bf/cT1B2 Spores Using
T1B2 Barcode Target 65
Table 18. Analytical Method Comparison Displaying Culture Presumptive, Culture ID with
PCR Confirmation and RV-PCR Replicates Detected (N = 3) for Gravel and Bilge
Water Samples 67
Table 19. Analytical Method Comparison Displaying Culture ID with PCR Confirmation and
RV-PCR Replicates Detected (N = 3) for Gravel and Bilge Water Samples 67
Table 20. Percent Recovery Values for Day 1 Traditional Surfaces Inoculated with a High
Load of Btk T1B2 Spores 69
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Table of Tables (Cont.)
Page
Table 21. Recovery Efficiencies for Presumptive Btk T1B2 Spores from Nontraditional
Washdown Samples Cultured on TSA Medium (N = 3) 71
Table 22. Percent Recovery Values for Nontraditional Surfaces Inoculated with a High
Load Target of Btk T1B2 Spores 73
Table 23. Summary of the Accuracy of Identification of Presumptive Btk T1B2 Colonies by
PCR Confirmation from Nontraditional Collection Methods 75
Table 24. RV-PCR Analyses of Nontraditional Washdown Samples for Detection of Btk
T1B2 Spores Using T1B2 Barcode Target (N = 3 Replicates) 76
Table 25. Summary of the Accuracy of Identification of Presumptive Btk T1B2 Colonies by
PCR Confirmation from Surfaces Stored in Outdoor Conditions Over Time 80
Table 26. Performance Evaluation Audits 82
Table 27. Washdown Samples with Highest Btk T1B2 Spore Recovery Available in To
Aliquot and Resulting Ct Values 87
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Acronyms and Abbreviations
AnCOR Analysis for Coastal Operational Resiliency
ASTM American Society for Testing and Materials
B. anthracis Bacillus anthracis
Ba Bacillus anthracis
BHIB Brain Heart Infusion Broth
BILGE Bilge Sample Designation
BSC Biological Safety Cabinet
Btk T1B2 Bacillus thuringiensis kurstaki T1B2
°C Degree(s) Celsius
CDC Centers for Disease Control and Prevention
CESER Center for Environmental Solutions and Emergency Response
CFU Colony Forming Unit(s)
CL Clean Sample Designation
cm Centimeter(s)
ACt Differential Cycle Threshold
Ct Cycle Threshold
dH20 Distilled Water
DNA Deoxyribonucleic Acid
EPA U.S. Environmental Protection Agency
ERLN Environmental Response Laboratory Network
FMRF Florida Materials Research Facility
ft Feet
GLASS Glass Sample Designation
GRAVL Gravel Sample Designation
h Hour(s)
HDPE High Density Polyethylene
HSMMD Homeland Security and Material Management Division
HSPD Homeland Security Presidential Directive
HSRP Homeland Security Research Program
ID Identification (Identifier)
in Inch(es)
IT Interagency Team
L Liter(s)
|iL Microliter(s)
jam Micrometer
m Meter
mg Milligram
MGAL Marine Grade Aluminum Sample Designation
min Minute(s)
mL Milliliter(s)
mm Millimeter
ModG Modified G
MSKID Marine Grade Aluminum and 50% Nonskid Sample Designation
N Normal
NSKID Nonskid Tread Sample Designation
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NTC
No Template Control
ORD
Office of Research and Development
PBS
Phosphate Buffered Saline
PBST
Phosphate Buffered Saline with 0.05% (v/v) Tween 20
PBSTE
Phosphate Buffered Saline with 0.05% (v/v) Tween 20 and 30% (v/v) Ethanol
PCR
Polymerase Chain Reaction
Pg
Picogram
PMP
Paramagnetic Particle
Psig
Pounds Per Square Inch
QA
Quality Assurance
QAPP
Quality Assurance Project Plan
QC
Quality Control
QMP
Quality Management Plan
qPCR
Quantitative Polymerase Chain Reaction
rcf
Relative Centrifugal Force
RH
Relative Humidity
rpm
Revolution(s) per Minute
RV-PCR
Rapid Viability PCR
SA
Sea Salt Spray Sample Designation
sec
Second(s)
SHINGLES
Shingles Sample Designation
SOP
Standard Operating Procedure
ss
Sponge Stick
STREAMS III
Scientific, Testing, Research, Engineering, and Modeling Support III Program
SW
Sea Water Sample Designation
T&E II
Testing and Evaluation II Program
TNTC
Too Numerous To Count
TSA
Trypticase Soy Agar
TW
Tap Water Sample Designation
USCG
United States Coast Guard
VFC
Vacuum Filter Cassette
v/v
Volume to Volume (ratio)
WI
Work Instruction
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Acknowledgments
This document was developed by the U.S. Environmental Protection Agency's (EPA's) Homeland
Security Research Program (HSRP) within EPA's Office of Research and Development. Dr. Worth
Calfee was the project lead for this document. Contributions of the following individuals and
organizations to the development of this document are acknowledged.
United States Environmental Protection Agency
Dr. Michael (Worth) Calfee, Center for Environmental Solutions and Emergency Response
Dr. Sanjiv Shah, Center for Environmental Solutions and Emergency Response
Mr. Leroy Mickelsen, Office of Land and Emergency Management
Dr. Sara Taft, Center for Environmental Solutions and Emergency Response
Dr. Shannon Serre, Office of Land and Emergency Management
Dr. Anne Mikelonis, Center for Environmental Solutions and Emergency Response
Ms. Erin Silvestri, Center for Environmental Solutions and Emergency Response
Mr. John Archer, Center for Environmental Solutions and Emergency Response
Dr. Helen Y. Buse, Center for Environmental Solutions and Emergency Response
Ms. Katrina McConkey, Center for Environmental Solutions and Emergency Response
Mr. Mark Durno, EPA Region 5
Ms. Jessica Duffy, EPA Region 3
Mr. Steven Wolfe, EPA Region 5
Mr. Jason Musante, EPA Region 9
United States Department of Homeland Security
Dr. Don Bansleben
United States Coast Guard
Mr. Emile Benard
LT. Omar Borges
Mr. Edward J. Primeau
CDR. Benjamin Perman
LCDR. Clifton Graham
Battelle Memorial Institute
Mr. Scott Nelson
Mr. Patrick Keyes
Ms. Hiba Shamma
Mr. Anthony Smith
Ms. Lindsay Catlin
Mr. Nate Poland
Ms. Emily Breech
Dr. Ryan James
Mr. Zachary Willenberg
Mr. Kent Hofacre
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Executive Summary
The U.S. Environmental Protection Agency (EPA) is charged by Congress with protecting the Nation's
land, air, and water resources. EPA is designated as a coordinating agency under the National Response
Framework to prepare for, respond to, and recover from a threat to public health, welfare, or the
environment caused by actual or potential oil and hazardous materials incidents. Hazardous materials
include chemical, biological, and radiological substances, whether accidentally or intentionally released.
EPA is expected to respond to outdoor contamination incidents to characterize and remediate the
impacted sites, and thus, determine the performance of available sampling and analysis methods for
outdoor environments is a high priority need. The performance of the methods may, in part, depend on
the outdoor surface being sampled and analyzed. The United States Coast Guard (USCG) bases and
ports are potential targets, and by nature of their mission and location, may have unique surfaces and/or
environments that could affect sampling and analysis methods, or provide an opportunity to collect
novel samples via their maintenance activities. The diversity and magnitude 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. EPA and USCG have formed an Interagency Team (IT) to support research
under the Analysis for Coastal Operational Resiliency (AnCOR) program, of which this study is a part.
This study will help the USCG recover rapidly following a biological contamination incident and return
assets to duty. Determination of asset contamination status is necessary to make decisions regarding
asset safety and deployability. The outcome of the study described in 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.
Surfaces common to USCG bases (marine grade aluminum, nonskid tread, glass, gravel, and bilge
water) were sprayed with barcoded Bacillus thuringiensis subspecies kurstaki spores, designated
Btk T1B2, and exposed to outdoor environmental conditions (but not direct sunlight or precipitation) for
up tol80 days. Traditional sampling techniques were used to collect samples periodically throughout the
outdoor exposure (1, 30, 90, and 180 days). In total, 168 traditional samples collected from the surfaces
were analyzed, comprising 72 sponge sticks, 48 vacuum filter cassettes (VFCs), and 48 grab samples.
Additionally, nontraditional methods incorporating water washdown and equipment readily available at
USCG bases (squeegee and bristle brush) were used to collect samples from surfaces within one week of
Btk T1B2 spore inoculation. In total, 160 nontraditional water washdown samples were collected for
analysis.
Spore recoveries from traditional sampling methods were 37 to 41 % (sponge sticks), 25 to 26 % (gravel
and bilge water), and < 1 % VFCs when sampled prior to exposure to the outdoor environment (Day 1).
Spore recovery from surfaces using nontraditional sampling methods was highest when a physical
wiping of the surface was incorporated with the washdown method; 18 to 19% recovery with squeegee
or bristle brush compared to < 8% for washdown methods without a physical wipe.
The foremost conclusion was that the detection and/or quantification of Btk T1B2 spores via either the
culture or rapid viability polymerase chain reaction (RV-PCR) method and sampled using traditional
EPA sampling methods was extremely dependent on the duration the surface experienced outdoor
ambient conditions - the longer the outdoor exposure duration, the greater the background (interfering)
microorganisms or inert contamination and potential physical loss, resulting in a decrease in the ability
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to identify (detect) and quantify the presence of Btk T1B2 spores. The ability of the EPA methods to
detect and quantify began to be adversely affected between the 30-day and 90-day sample collections.
By the end of 180 days of outdoor exposure, previously detectable and quantifiable amounts of Btk
could not be measured. Also, the nontraditional sampling methods that are associated with typical vessel
maintenance (e.g., vessel water washdown, scrubbing with a wet bristle brush, or wiping excess water
with a squeegee) or collecting rainwater runoff from a shingle roof are all plausible sampling collection
methods that can yield a composite sample over a relatively larger surface area than from a traditional
active sampling method.
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1.0 INTRODUCTION
1.1 Background
The U.S. Environmental Protection Agency (EPA) is designated as a coordinating agency under the
National Response Framework to prepare for, respond to, and recover from a threat to public health,
welfare, or the environment caused by actual or potential oil and hazardous materials incidents.
Hazardous materials include chemical, biological, and radiological substances, whether accidentally or
intentionally released. The anthrax incidents in 2001 highlighted the need to improve the U.S.
Government's response to terrorist attacks. Congress passed the Public Health Security and Bioterrorism
Preparedness and Response Act (Bioterrorism Act) in 2002, and the Office of the President issued a
series of Homeland Security Presidential Directives (HSPDs) to specify the responsibilities of federal
agencies. The EPA's roles and responsibilities related to homeland security are protecting human health
and the environment from bioterrorism.
Following a bioterrorist attack, materials contaminated with biological agent pose significant health
threats. The EPA's Homeland Security and Material Management Division (HSMMD), within the
Center for Environmental Solutions and Emergency Response (CESER), conducts research to develop
methods and technologies to rapidly and cost effectively remediate areas affected by a bioterrorism
attack. Methods for detection and characterization of a biological agent following a bioterrorism incident
include swabs, wipes, vacuum, and grab sampling, as well as bioagent-specific analytical method(s).
These methods have been evaluated for their application in indoor environments; however, their
performance when utilized in an outdoor environment is unknown. EPA is expected to respond to
outdoor contamination incidents to characterize and remediate the impacted sites, and thus, determining
the performance of available sampling and analysis methods for outdoor environments is a high priority
need. The performance of the methods may, in part, depend on the outdoor surface being sampled and
analyzed. Additionally, the amount of time between the contamination incident and the collection of
samples may impact recovery efficiency.
The U.S. Coast Guard (USCG) bases and ports are a potential target, and by nature of their mission and
location, may have unique surfaces and/or environments that could affect sampling and analysis
methods, or provide an opportunity to collect novel samples via their maintenance activities. The
diversity and magnitude 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. EPA
and USCG have formed an Interagency Team (IT) to support research under the Analysis for Coastal
Operational Resiliency (AnCOR) program, for which this study is a part.
This study will help the USCG to recover rapidly following a biological contamination incident and
returning assets to duty. Determination of asset contamination status is necessary to make decisions
regarding asset safety and deployability. The outcome of the study described in this report will provide
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.
1.2 Objective
The objective of this study was to gather and generate data useful for EPA, USCG decision-makers, and
other first responders regarding sampling and analytical method performance, when samples are
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collected in a realistic maritime or urban setting during a bioagent contamination incident. The impact of
contamination aging under outdoor, uncontrolled environmental conditions was also evaluated to inform
expectations of method performance following sample collection operations occurring days, weeks, or
months following an incident. The findings can be used to better plan and execute the recovery of a
USCG base, or any large outdoor urban area, following a biological contamination incident.
Specifically, the study is to evaluate the performance and limitations of traditional and innovative
sampling and analysis methods, as well as sample collection methods that leverage existing USCG
maintenance procedures, when applied to USCG assets and bases.
1.3 Scope
The work described in this report evaluated sampling and analysis methods targeting Bacillus spores
associated with an outdoor setting relevant to a USCG base or port. Multiple outdoor surface types
expected to be encountered in a wide-area contamination incident involving a USCG bases were
identified for use in the evaluation. Representative surface types were prepared to represent the surfaces
of a USCG asset (e.g., marine grade aluminum with sea salt spray deposited onto it to represent a small
or medium boat's exterior surface). Traditional (wipes, vacuum, grab) and nontraditional (e.g., site-
specific maintenance approaches) sampling methods were considered for evaluation to assess their
effectiveness when they are applied to representative surfaces of USCG assets (e.g., boats, ships,
buildings) and USCG base-relevant surface types (e.g., marine grade aluminum). The samples collected
were analyzed using existing EPA methods for both culture and rapid viability polymerase chain
reaction (RV-PCR) to determine sample collection efficiency and confirm the presence of Bacillus
thuringiensis subsp. kurstaki (Btk) spores, used to simulate Bacillus anthracis (Ba) spores.
The surfaces were inoculated with barcoded Bacillus thuringiensis subspecies kurstaki spores,
designated hereafter as Btk T1B2 spores, by spraying a suspension with known spore concentration at a
controlled rate and fixed duration onto the target surface. Experimentally contaminated surfaces were
used to assess traditional sampling methods and were stored in an outdoor ambient air environment (but
under cover shielded from sunlight, precipitation, and thus physical wash-off) to experience daily and
seasonal temperature and relative humidity (RH) cycles and collection of depositing particulate matter.
The outdoor environmental exposure began in winter (January/February through July/August) for a
maximum exposure duration of 180 days in Columbus, Ohio. Surface samples were collected after 1, 30,
90, and 180 days of environmental exposure. In addition, surface types common to USCG facilities and
vessels were experimentally contaminated to evaluate nontraditional, composite sample collection
methods. Nontraditional sampling methods utilized procedures and supplies already available on USCG
bases and stations to increase the likelihood that sampling operations could commence early in the
response with minimal additional training on procedures. For all tests, spore loading (number of spores
inoculated per unit area of surface) was included as a study variable to understand the limits of the
sampling method when combined with its associated analytical method to quantify or identify low
numbers of Btk T1B2 spores. The recovery efficiency of the sample collection method was determined
by quantifying the spores recovered using a current EPA culture method. Whether the presence of viable
Btk can be detected (but not quantified) was assessed using an existing EPA RV-PCR method. Agent
collection efficiency by sampling method and sampled surface was determined.
The information generated from this study can then be used as reliable estimators to quantify and/or
detect and identify the presence of B. anthracis that will be needed for a recovery operation.
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2.0 MATERIALS AND METHODS
2.1 Target Surface/Material Sampled
2.1.1 Surface/Material Selection
Five flat solid nonporous surfaces were selected by the AnCOR Project Team to be representative of
ubiquitous surfaces encountered at a USCGbase or station: 1) marine grade aluminum (designated
[MGAL]); 2) glass (designated [GLASS]); 3) nonskid tread (designated [NSKID]); 4) shingles
(designated [SHINGLES]), and 5) a combination of half marine grade aluminum and half nonskid tread
(designated [MSKID]). Two (2) additional materials were used in the study: gravel (designated
GRAVL) and bilge water (designated BILGE). Representative images of the seven surfaces/materials
are presented in Figure 1.
2.1.1.1 Marine Grade Aluminum
Marine grade aluminum type 5086, 0.0625-inch (in)-thick (Part No. 5865T14, McMaster-Carr Supply
Company, Douglasville, GA) was selected as one target material to represent a common surface
associated with USCG small and medium boat construction. The specific MGAL selected was consistent
with the MGAL used for other ongoing EPA decontamination studies. Sheets of MGAL (2 feet (ft) x
4 ft) were purchased for use. Two (2) sheets were butted end-to-end to prepare for traditional sampling
method assessment for a total of 16 ft2 available for sampling. The sheets were cut into 2 ft x 2 ft squares
for use with the nontraditional sampling method assessments.
2.1.1.2 Glass
Tempered glass (safety glass) was selected as a representative glass that is commonly used in windshield
applications. Tempered glass sheets 1 ft x 2 ft and 3/16-in-thick (Item# E-SHGL1224, Allen Display,
Midlothian, VA) were purchased. Eight (8) sheets were butted end-to-end to prepare for traditional
sampling method assessment for a total of 16 ft2 available for sampling.
2.1.1.3 Shingles
The selected shingles were a fiberglass/asphalt construction (Royal Sovereign® Shingles; GAF Materials
Corporation; Parsippany-Troy Hills, NJ) purchased from Home Depot. The shingles were of a make
similar to shingles used previously for other EPA decontamination studies. The shingles were adhered to
a metal substrate in an overlapping manner as if they were installed on a roof. A square section of
shingles measuring 2 ft x 2 ft constituted one surface sample replicate for use with the nontraditional
sampling method assessments.
2.1.1.4 Nonskid Tread
The nonskid (anti-slip) tread selected was 3M™ Safety-Walk™ Coarse Tapes & Treads, Model #710-
24 x 30, black, custom, purchased from Grainger, Lake Forest, IL in 24-in-wide x 30-ft-long rolls
(Item No. 2JDA8). The tread selected has been used previously for other ongoing EPA decontamination
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 was coated with a pressure-sensitive adhesive covered by a removable protective liner. A
surface measuring 2 ft x 2 ft constituted one surface sample replicate for use with the nontraditional
sampling method assessments.
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2.1.1.5 Half Marine Grade Aluminum and Half Nonskid Tread
The mixed surface comprising half marine grade aluminum and half nonskid tread (MSKID) was
prepared using the starting materials described in Sections 2.1.1.1 and 2.1.1.4 in a manner shown in
Figure 1. A square surface measuring 2 ft x 2 ft constituted one surface sample replicate for the
nontraditional sampling method.
2.1.1.6 Gravel
Medium River Rock (Mr. Mulch; Columbus, Ohio) was selected as the representative gravel. The gravel
was washed by using sterilized water, but the gravel was not further cleaned or sterilized. The equivalent
amount of gravel that would be collected in the field (1 liter (L) Nalgene bottle filled to $ mark) was
spread in a tray measuring ~5 in x 8 in.
2.1.1.7 Bilge Water
Bilge water was obtained from the ballast of a Medium Endurance Cutter (Tampa, FL). The purple
coloration is contamination from "jacket water" that commonly enters the bilge. The bilge water was
used "as-received." It was stored at 2 to 8°C until aliquots were used for spiking with Btk spores.
Figure L Representative Images of the Seven Surface Types.
A) Marine Grade Aluminum; B) Glass; C) Shingles; D) Half Marine Grade Aluminum and Half Nonskid
Tread; E) Nonskid Tread; F) Gravel; and G) Bilge Water.
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2.1.2 Surface/Material Preparation
2.1.2.1 Cleaning
All smooth, nonporous surfaces of marine grade aluminum and glass were first cleaned by wiping with
70% isopropyl alcohol to remove surface grime and allowed to air dry. Compressed house air was used
to blow off loose surface dust and debris from all nonskid tread and shingles. When surfaces were used
with no other pretreatment before contaminating with Btk spores, the surface was termed clean (meaning
it represented a clean ambient environment) and designated [CL],
2.1.2.2 Sea Salt Spray Application
A subset of the cleaned surfaces was subsequently treated with a sea salt spray, designated [SA], to mimic
their presence in a marine environment. The amount of salt deposited on a surface that may be
encountered when sampling could vary by orders of magnitude, depending, in part, on factors such as
location, season, and time for salt to accumulate. The salt loading level to target in the EPA outdoor
sampling study of USCG-relevant surfaces is recommended based on a Florida Materials Research
Facility (FMRF) data set, as these data represent continuous measurements on a monthly basis for over
15 years at a location considered one of the most corrosive environments in the United States. The salt
loading level to apply to USCG-relevant surfaces was recommended based on a chloride deposition of
200 milligrams (mg)/square meter (m2)/day, equal to the 75th percentile of observation at the FMRF. This
level is also above or near 11 of the 12 monthly median values and is thus expected to represent a
relatively high salt loading flux. The amount of sodium chloride as the primary salt source of chloride ion
measurement corresponds to a total salt deposition of 330 mg/m2/day (milligrams/square meter/day).
Using a 5-day exposure as a basis, the total salt deposition would be 1,650 mg/m2 or 0.17 mg/square
centimeters (cm2). Considering the spread and uncertainty of deposited salt, a dry weight salt residue
loading of 0.15 ± 0.05 mg/cm2 was targeted to apply to the surfaces before application of Btk. A brief
literature review was conducted to determine a reasonable salt residue loading to target and is summarized
in Appendix A.
A commercially available sea salt dry formulation (item "ASTMD1141 Substitute Ocean Water Salts,"
G2MT Labs; Houston, TX) was purchased and mixed with sterile deionized water to achieve a
concentration of 41.95 mg/milliliter (mL). The sea salt spray dry residue was achieved by spraying the
simulated sea salt solution described above onto the target surface using the spray table depicted in
Figure 2. A hydraulic spray nozzle (Model XR 8001 VS, Agro-Chem Inc.; Wabash, IN) was supported on
a translating beam at a fixed distance of 33 cm above the target surface. The spray was initiated at one end
of the table, before the leading edge of the target surface to be sprayed, then translated over the
243-centimeter (cm)-long target surface at a rate of-100 cm/s. A belt-driven carriage supporting the spray
nozzle was controlled by a variable speed drive motor to translate the spray over the surface. The spray
nozzle spray rate was 330 milliliters (mL)/min when operated at 30 pounds per square inch gauge (psig).
The target dry salt residue loading was achieved by controlling the rate of spray nozzle traverse over the
target surface and number of passes. When a surface was treated with sea salt spray, at least 24 hours (h)
was permitted for the water to evaporate before the surface was sampled or before spraying with the Btk
T1B2 suspension. A Work Instruction (WI) for the operation of the spray table is provided in Appendix B.
Eight (8) aluminum foil coupons of 47-millimeter (mm) diameter were spatially distributed on the spray
table to characterize the sea salt deposition. After a spray event, the coupons were dried, and the mass of
salt residue was determined gravimetrically. A representative spatial distribution of the measured dry salt
residue in units of mg/cm2 is shown in the illustration in Figure 3. In the representative example shown,
the average deposition was 0.14 mg/cm2, compared to a target of 0.15 mg/cm2. The actual sea salt residue
was determined for each surface contamination trial by placing the eight coupons on the table, recovering,
and analyzing as described or by mass balance.
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Figure 2. Photograph of Spray Table Used to Apply Sea Salt Spray and Btk T1B2 Spores onto Target
Surfaces.
Note: Two 2 ft x 4 ft sections of glass on table-top shown.
Figure 3. Illustration (Top View) of Location of Reference Coupons on the Spray Table and Representative
Sea Salt Spray Residue Collected (mg/cm2) at those Locations Used to Demonstrate Ability to Apply a
Target Sea Salt Residue onto Target Surfaces.
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2.1.2.3 Barcoded Btk T1B2 Spores Spray Application
Btk T1B2 spores were applied to the target surfaces using the spray table and method described above
for application of sea salt spray at two nominal Btk T1B2 spore loadings of 10 colony forming units
(CFU)/cm2 (denoted "low") and 1,000 CFU/cm2 (denoted "high"). Characterization tests were
performed in a manner similar to the sea salt spray, except that spores were recovered from reference
coupons, then enumerated to quantify the spore loading levels, as described in Section 2.4.3.1. The two
Btk T1B2 spore loading levels were achieved by spraying two different Btk spore suspension
concentrations, differing by a nominal two orders of magnitude (2 x 103 and 1 x 105 CFU/mL suspended
in water), prepared from Btk T1B2 stock described in Section 2.4.1.
A representative spatial distribution of the measured Btk spore load deposited onto a target surface (high
loading level) in units of CFU/cm2 is shown in the illustration in Figure 4. The outline of the two 2 ft x
4 ft target surfaces is shown relative to the reference coupon placement. The average Btk spore load
measured in this representative example was 760 CFU/cm2, which was deemed acceptable to
demonstrate an ability to achieve the nominal Btk T1B2 loading level of 1,000 CFU/cm2. The actual
Btk T1B2 spore loading was determined for each surface contamination trial by placing the eight clean
coupons on the table, recovering, and analyzing as described. A total of 16 ft2 of surface was inoculated
with Btk spores for each surface type in the test matrix for traditional sampling method assessment. This
size allowed collection of all 12 replicate sponge stick (SS) or vacuum filter cassette (VFC) samples
from a surface contaminated in a single event (eliminating surface-to-surface Btk loading variability),
since the sponge stick method requires a 10-in x 10-in area per sample and the VFC requires a 12-in x
12-in area per sample.
Y
Axis
i
T
1
i
A'
:
1 530 1
860 J
;
| 1200 |
6501
^ Axis
¦ 2
'
690
1 660
i
1 910 1
1 550 1
Figure 4. Illustration of Location of Reference Coupons on the Spray Table and Representative Btk Spore
Load (CFU/cm2) Collected at those Locations Used to Demonstrate Ability to Apply a Btk T1B2 Spores
onto Target Surfaces.
2.1.3 Btk Contaminated Surface/Material Storage
2.1.3.1 Surfaces for Traditional Sampling Methods
Following spray application of barcoded Btk T1B2 spores, the surfaces were stored overnight in an
indoor laboratory environment to allow the surfaces to dry. The day following Btk spore application, the
surface was sampled using traditional sampling methods (Section 2.2.1), and the collected sample was
designated as the Day 1 sample. Subsequently, within 24 h, the surfaces were moved to an outdoor
ventilated shed for 180 days. Following nominally 30, 90, and 180 days of storage within the outdoor
shed, replicate coupons were sampled. Sufficient replicate coupons were included so that each coupon
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was sampled only once during the study. The shed was required to ensure proper containment of the
genetically modified Btk T1B2 spores to meet regulatory compliance requirements, yet achieve outdoor
environmental conditions needed to meet the project objectives.
On the Battelle Memorial Institute Campus, at 505 King Avenue, Columbus Ohio; an 8 ft x 13 ft
ventilated shed (Tremont Model; SKU 19302658; Menards, Eau Claire, WI) was erected outdoors on a
concrete pad, but under a hip-roof overhang as depicted in the photograph of Figure 5. The shed was
modified to equip an exhaust blower with in-line high efficiency particulate air filter to provide
containment of the Btk T1B2 organism, protect the surfaces from being disturbed by rodents and
animals, yet maintain temperature and humidity conditions of the outdoor environment. The blower
pulled ambient air into the shed through the screened windows of the two front shed doors and any other
unit. The air temperature and RH in the shed was monitored, but not controlled; thus, the sprayed
surfaces experienced the ambient conditions. The shed did provide cover and protection from
precipitation, but the continuous airflow through the shed allowed for deposition of ambient particulate
matter and condensation to form/evaporate with the diurnal cycle. Contaminated surfaces contained
within the shed are shown in the two photographs in Figure 6.
Figure 5. Ventilated Shed Used to Store Contaminated Surfaces During 180 Days of Environmental
Exposure.
Shed under an overhang shown in the left image. Middle image shows the back side (opposite of the front
doors) with a HEPA filter in housing and blower to pull ambient air through the shed. Inside of shed (right
image) showing support racks to place test surfaces.
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Figure 6. Photograph of the Contaminated Surfaces (Facing Up) Contained Within the Ventilated Shed for
Up to 180 Days of Environmental Exposure.
2.1.3,2 Materials for Nontraditional Sampling Methods
Following spray application of Btk T1B2 spores, the surfaces were stored at a minimum of overnight,
but not more than two weeks in an indoor laboratory environment to allow the surfaces to dry before
sampling. There was no exposure duration or prescribed environmental conditions for coupon surfaces
in the assessment of the nontraditional sampling methods.
2.2 Sampling Methods
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 the
methods, which were then summarized in Wis for the field team to execute.
Nontraditional washdown methods using tap water, simulated seawater, and simulated rainwater were
assessed for collection from solid surfaces commonly found at USCG bases. Squeegee brush and bristle
brushes were used in combination with washdown methods.
2.2.1 Traditional Sampling Methods
When sponge sticks or VTCs were used to sample the surface, the three areas sampled at each of the
four time points were randomized and defined before sampling started to help minimize any sample bias
due to variations of applied Btk T1B2 spores and/or ambient exposure impacts. An example of one of
the random sampling schemes is shown in Figure 7. Note that often four replicate samples were
collected per time point to provide an extra sample if there was a processing issue.
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tl
*2
tl
tl
t3
t4
t2
t3
t4
*2
*3
t4
tl
t4
t2
t3
Figure 7. Example of Sample Locations Over a 2 x 8 ft Area of Target Surface and Schedule for Sponge
Stick and VFC Surface Sampling.
Sample Time Points: tt = 1 Day, t2 = 30 Days, % = 90 Days, t4 = 180 Days.
2.2,1.1 Sponge Sticks
3M Sponge Sticks™ prewetted with a neutralizing buffer (3M St. Paul, MN Part number SSL1GNB) -
shown in Figure 8 - were purchased for sample collection per established EPA sampling methods
(EPA, 2013 and Tufts et al., 2014) and Centers for Disease Control and Prevention's (CDC's) Surface
Sampling Procedures for Bacillus anthracis Spores from Smooth, Nonporous Surfaces (CDC, 2012).
The sponge sticks were used to sample a 10 in x 10-in (645 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, using all six sides of the sponge) defined in the EPA sampling method.
Figure 8. Prewetted Sponge Stick from 3M Used for Surface Sampling (left) and Sampling a Glass Surface
(right).
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2.2.1.2 Vacuum Filter Cassettes
VFCs, 37-mm-diameter, 0.8 micrometer (|am) pore mixed cellulose ester membrane (Part No. SKC 225-
3-01, SKC, Inc., Eighty Four, PA) were purchased for surface sample collection per established EPA
sampling methods (Calfee, 2013). An assembled and disassembled VFC are shown in Figure 9. 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, 2013).
The EPA specified 5 L/min sampling rate was used.
Figure 9. Vacuum Filter Cassette (37-mm Diameter) Assembled (Left) and Disassembled (Right) Used for
Surface Sampling.
2.2.1.3 Grab - Bilge Water
Bilge water was sampled from a Medium Endurance Cutter ballast located at the Portsmouth, Virginia
USCG base in three, sterilized 10-L carboy containers. The bilge water had a noticeable purple tint
caused by "jacket water" (water that circulates to cool the main diesel engines), per USCG personnel. A
picture of the bilge water sample is provided in Figure 10. The carboys of bilge water samples were
shipped in chests with cold packs to Battelle's laboratories via Federal Express. The bilge water was
stored in 10-L carboys at 4°C until samples were obtained for analysis. When an aliquot was retrieved
for analysis, the 10-L carboy was rolled back and forth on its side to mix the contents, then 0.5 L was
collected using a 100-mL serological pipette and stored in a clean sterile bottle.
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Figure 10. Photograph of the Bilge Water Grab Sample Being Collected (left) from a Medium Endurance
Cutter at Base Portsmouth.
2.2.1.4 Grab-Gravel
A photograph of a representative grab sample of gravel is shown in Figure 11. Gravel was stored in
260-cm2 sample trays and sampled by collection into a 1-L Nalgene bottle to the % full mark, -900 g.
Then, 500 mL of sterile phosphate buffered saline with 0.05% (v/v) Tween20 (PBST) was added to each
1-L bottle containing gravel sample, and each bottle was shaken 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 settled
for 30 sec, and then the eluent was poured into a clean, sterile 500-mL container.
Figure 11. Photograph of a Sample of Gravel in 1-L Nalgene Bottle Containing 500 mL PBST.
Before Shaking (Left) and After Shaking to Extract (Right)
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2.2.2 Nontraditional Sampling Methods
Four (4) nontraditional sampling methods (wash water, water runoff, wash water with squeegee, and
wash water with bristle brush) were defined and implemented, and each yielded a composite sample
collected from a surface area larger than the surface area sampled by traditional methods. The
nontraditional sampling methods were intended to mimic cleaning and maintenance procedures that are
performed on USCG bases and stations. Utilizing procedures and supplies already available on USCG
bases and stations increases the likelihood that sampling operations could commence early in the
response with minimal additional training on procedures. Requests for cleaning and maintenance
procedures were made, but responses did not include detailed procedures that were readily adaptable to
define a sampling method. Alternatively, information concerning the types of water (fresh versus
seawater) and representative equipment (e.g., squeegees or bristle brushes) used for vessel washdown
procedures were obtained from USCG personnel for adaptation to sampling methods.
In all instances, nontraditional sampling methods were applied to 2 ft x 2 ft areas of the target surface to
generate a single sample. The 2 ft x 2 ft target surface was placed on an inclined support (-30°) so that
the applied water would mn off and collect in a trough that then flowed to a drain port and into an
autoclaved 2-L Nalgene sample collection beaker to determine the volume of water collected and then
subsequently transferred into a clean (autoclaved) 1-L polypropylene bottle for storge under refrigerated
conditions (2 to 8°C) until analyzed. The word "water" is used generically in the method descriptions;
the actual water used was either simulated seawater, tap (fresh) water, or rainwater as per the test matrix.
A photograph of the surface support fixture is shown in Figure 12.
Figure 12. Inclined Surface Support Fixture Used to Support the 2 ft x 2 ft Target Surfaces for
Nontraditional Sampling Methods.
The sampling order was always to collect from a blank (not purposely contaminated with Btk) surface
followed by surfaces with a low Btk T1B2 spore load and ending with surfaces with the high Btk T1B2
spore load. This approach was taken to minimize Btk carryover from one sample replicate to another.
The same surface supporting structure and collection trough were used for all four sampling methods,
and the structure was decontaminated with bleach and rinsed with deionized water between uses with
the different methods.
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2.2.2.1 Vessel Water Washdown
Boats are commonly washed down with fresh or seawater source (no detergent added) after returning
from deployment. Both tap water (designated TW) and seawater (designated SW) were used as water
sources. The hard tap water (type Organization for Economic Co-operation and Development hard
water) was prepared per a recipe standard used by EPA (EPA, 2019). The starting water was deionized
and hardened to a 375 parts per million calcium cation concentration using a calcium carbonate solution.
The pH was adjusted to 7.0 ± 0.2 using 1 normal (N) sodium hydroxide or 1 N hydrochloric acid, as
appropriate. The prepared hard tap water was filter-sterilized before use. The seawater used was the
same as that used to spray to deposit seawater salt onto the surface before application of Btk spores
(see Section 2.2.2.2).
A heavy duty adjustable brass spray nozzle (Dramm Model # 14033591; Home Depot, Atlanta, GA) was
used to apply the water for washdown; EPA has used this nozzle for ongoing decontamination
applications. Similarly, the operating conditions used here were based on previous use of the spray
nozzle. The nozzle had no graduations to control delivered flowrate of water, and thus was characterized
before use with a target flow rate of 4 ± 1 L/min operating at a source pressure of 30 psig. The nozzle
was positioned at a right angle -12 in above the target surface, and the resulting cone covered a 12- to
14-in-diameter area. The nozzle was kept at this height and swept across the test surface according to the
pattern shown in Figure 13. A total of five passes (labeled P1-P5 in the Figure 13) was performed at
~3 sec per pass for a total of-15 sec of spray. The passes were positioned so that the spray cone slightly
overlapped the edges of the test surface by 2 to 3 in. A photograph of the washdown method being
performed is shown in Figure 14. At the conclusion of the spray event, 15 to 20 sec was allowed for
water to drain off and into the collection beaker. After collection of the runoff, a clean, dry, disposable
cloth or wipe was used to remove any standing water or droplets from the basin surface and collection
trough. A WI for the procedure is provided in Appendix C.
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Spray Basin
(30" x 30")
PI
P2
P3
P4
P5
Spray Width
(12" -14")
Spray Path
Water Flow
Collection/Drainage
Trough
Figure 13. Vessel Washdown Method Spray Pattern.
Figure 14. Vessel Washdown Nontraditional Sampling Method Applied to Marine Grade Aluminum.
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2.2.2.2 Simulated Rainfall
A spray nozzle (Spray Systems, Pomona, CA Model # GG-2.8W) was selected and characterized for use
to achieve a target rainfall rate of 1 in/h. The spray nozzle and operating conditions were selected based
on previous EPA research (Mikelonis et al., 2021) requiring simulated rainfall events. A simulated
rainwater formulation was prepared based on a brief literature review. In summary, the rainfall
composition is highly variable depending, in part, on season and geographic location, as well as decade.
It was decided to use a formulation as reported in a study by Schoettle et al. (1992), as it was within the
range of ion concentration and acidity considered reasonable for use in the timeframe of this study. A
summary of the rationale for the formulation is provided in Appendix D. The nozzle was positioned
-7 ft above the target surface, the maximum distance of fall that could be achieved and maintain proper
containment of organisms within a controlled space. The spray nozzle was operated at 5 psig pressure
resulting in an output flow of 1.15 ± 0.15 L/min and is shown in operation in Figure 15. The simulated
rainfall impinging on the shingle surface is shown in Figure 16. Most of the generated droplets (-85%)
fell outside the target 2 ft x 2 ft surface, but rainfall rate and distribution measurements made to
characterize the rainfall simulator resulted in a measured rainfall rate of 0.92 in/h on target. A plot of
measured rainfall rate (average of 0.92 in/'h) by location using 64 collection bottles covering the 2 ft x
2 ft target surface in an 8 x 8 grid is shown in the heat map in Figure 17. The rainfall event was 5 min in
duration, resulting in -0.8 L of rainwater runoff per sample collected into a collection beaker. At the
conclusion of the rain event, 1 min was allowed for water to drain off and into the collection beaker.
After collection, a clean, dry, disposable cloth or wipe was used to remove any standing water or
droplets from the basin surface and collection trough. A WI for the procedure in provided in
Appendix E.
Figure 15. Spray Nozzle Producing Simulated Rainfall.
16
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Figure 16. Simulated Rainfall Landing on Shingles.
Average Rainfall (in/h)
0.8
0.9
0.9
1.0
1.0
1.0
1.0
0.9
0.9
0.9
0.9
0.9
1.0
1.0
1.0
1.0
0.9
0.9
0.9
0.8
0.9
1.0
1.0
1.0
0.9
0.9
O.E
0.9
0.8
0.9
1.0
1.0
1.0
0.9
0.8
0.8
0.8
0.9
0.9
0.9
1.0
0.9
0.8
0.8
0.9
0.9
0.9
0.8
1.0
1.0
0.9
0.9
0.9
0.9
0.8
0.8
1.0
1.0
1.0
0.9
0.9
0.9
0.8
0.8
Figure 17. Uniformity of Simulated Rainfall on Target Surface.
17
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2.2.2.3 Squeegee
In consultation with USCG representatives, a 12-in-wide Shurhold, quick-connect stainless steel
squeegee (West Marine, Watsonville, C A Model # 2673184) was selected as a representative squeegee.
A new squeegee was used for each sample collected. A maintenance procedure was not provided as a
guide to specify details of use, so the following procedure was agreed upon for use:
Rinse the surface with tap water using the vessel washdown procedure described in Section 2.2.2.1
and illustrated in Figure 14. Collect this pre-wet rinsate sample as described in Section 2.2.2.1.
Immediately following the spray, pass the squeegee over the surface five times in an overlapping
pattern as shown in Figure 18. The entire surface area is covered by the squeegee at least twice and
some areas a third time by following the pattern depicted in Figure 18.
Each pass of the squeegee overlaps the previous pass by ~3 in.
Pull the squeegee from the top of the surface to the bottom so that the flow of water is also in the
direction of gravity flow, toward the catch trough. The squeegee surface wipe sampling being
performed is shown in Figure 19.
Flush the trough with a ~2-sec water spray to further rinse the trough into the collection beaker.
Wipe the surface support and collection trough with a clean, dry, disposable cloth or wipe to
remove any standing water or droplets before use with the next surface.
The procedure resulted in ~1 L of water being collected per sample. A WI for the procedure is provided
in Appendix F.
12" Squeegee Coverage
Pass#l
T
^ \ ^ Sample Surface
3" to Basin Edge (24" x 24")
Pass #2
Pass #3
Sample Pass
lx Pass 2x Pass 3x Pass
Pass 04
Pass#5
Figure 18. Squeegee Surface Wipe Sampling Pattern.
18
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Figure 19. Surface Sampling Marine Grade Aluminum Using a Squeegee.
2.2.2.4 Bristle Brush
In consultation with USCG representatives, an 8-in-wide, Stowaway 3.0 Deck Brush (West Marine,
Watsonville, CA Model # 12830840) was used as the bristle brush to scrub 2 ft x 2 ft surfaces of marine
grade aluminum with nonskid surface (50/50) salted surfaces. A maintenance procedure was not
provided as a guide to specify details of use, so the following procedure was agreed upon for use:
Prewet the surface with water using the vessel washdown procedure described in Section 2.2.2.1
and illustrated in Figure 14, except complete each pass in ~1 sec for a total of a 5-sec spray. Any
runoff from pre-wetting is collected as part of the sample.
Submerge the brush in a clean 2-gallon bucket containing 5 L of the desired washdown water
(either tap water or seawater as prepared and as used in other sampling approaches of this study).
Use a new brush and a new bucket and water for each surface sampled.
Pass the brush over the surface seven times in an overlapping pattern as shown in Figure 20; images
of the brushing and submerging of the brush are shown in Figure 21.
The overlap is half of the brush width (~4 in). As shown, the brushing pattern results in each
surface being brushed two times.
Rinse the brushed surface with a second water spray following the same procedure described in
Section 2.2.2.1 and illustrated in Figure 14, using the five passes at 3 sec per pass for a total rinse
time of 15 sec.
The second washdown rinsate is combined with the initial prewet rinsate of the surface. A total of
~1 L of washdown liquid was collected and stored in a clean (autoclaved) 1-L Nalgene bottle.
Submerge and lift the bristle brush into the bucket with 5 L of water three times. Leave the bru sh
submerged in the water and pour 1 L of the brush rinse water into a clean (autoclaved)
19
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polypropylene bottle for subsequent analysis as a bristle brush rinse sample.
Wipe the surface support and collection trough with a clean, dry, disposable cloth or wipe to
remove any standing water or droplets before use with the next surface.
The procedure resulted in ~1 L of surface rinse water being collected per sample, and 1 L of the bristle
brush rinse from the 5 L of water used to rinse the brush collected per sample. A WI for the procedure is
provided in Appendix G.
8" Brush Coverage
Brush Coverage
lx Pass
2x Pass
Figure 20. Bristle Brush Sampling Pattern.
20
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Figure 21. Surface Sampling Marine Grade Aluminum Using a Bristle Brush (left) and Rinsing of the
Brush by Submerging in Bucket of Water (right).
2.3 Test Matrix
Each of the collected surface samples described in Sections 2.2.1 and 2.2.2 was processed to recover
spores, and the resultant suspension was analyzed to quantify and identify recovered Btk T1B2 spores to
assess the EPA-provided culture and RV-PCR methods (EPA, 2017).
The completed test matrices for traditional sampling methods (sponge sticks, VFCs, and grab samples)
are provided in Table 1 and nontraditional sampling methods in Table 2. In total, 168 traditional samples
collected from the surfaces were analyzed, comprising 72 sponge sticks, 48 VFCs, and 48 grab samples.
Nominally, triplicate samples for each of two Btk spore loading levels and four exposure durati ons
(totaling 12 samples per surface) were analyzed. In total, 160 nontraditional water washdown samples
were collected for analysis. Nominally, triplicate samples for each of two Btk spore loading levels plus
two blank surface samples (totaling eight samples per surface) were analyzed. Additional spikes and
blanks were included as controls and are discussed in the results section.
The surface sample target and associated identifier (ID) used to uniquely name the samples collected are
provided in the first two columns. In the surface ID, the suffix of "-CL" or "-SA" indicated whether the
surface was clean (no salt spray) or sprayed with simulated seawater, respectively. The target spray
coverage was 10 or 1,000 CFU/cm2 for solid surfaces and 1 CFU/mL or 100 CFU/mL for bilge water.
The surface area sampled varied by sample collection type, as defined in Table 1 and Table 2. Following
physical extraction, the recovered sample volume was split nominally in half, and the total Btk T1B2
spores available listed in Table 1 and Table 2 was divided by two to represent the number of Btk TLB2
spores available for each of the two detection methods: culture and RV-PCR (EPA, 2017). The method
details are discussed further in Sections 2.4.3 to 2.4.5. Each surface type was sampled in quadruplicate
at 1 day, 30 days, 90 days, and 180 days post-spray; three of the sample replicates were analyzed and
one was stored as a backup sample. Negative controls that were handled only within the analytical
laboratory were included to assess the potential for sample cross-contamination. Field blank and method
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 microorganisms.
21
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Table 1. Test Matrix for Samples Collected Using Traditional Sampling Methods.
Surface1"
(Surface ID)
Sample Type
Surface
Area
Sampled
(cm2)
Replicate
Samples per 1,
30. 90. and 180
Days Exposure
Duration
Target Spore Load
(CFU/cm2)'" :l
Total Spores Available
per Analytical Method
(CFU)ldl
Low
High
Low
High
Glass
(GLASS-SA)
Sponge Stick
(SS)
645
3, 3, 3, 3
10
1,000
3,200
3.2 x 105
Marine Grade
Aluminum
(MGAL-CL)
Sponge Stick
(SS)
645
3, 3, 3, 3
10
1,000
3,200
3.2 x 105
Marine Grade
Aluminum
(MGAL-SA)
Sponge Stick
(SS)
645
3, 3, 3, 3
10
1,000
3,200
3.2 x 105
Nonskid
(NSKID-CL)
Vacuum Filter
Cassette
(VFC)
929
3, 3, 3, 3
10
1,000
4,600
4.6 x 105
Nonskid
(NSKID-SA)
Vacuum Filter
Cassette
(VFC)
929
3, 3, 3, 3
10
1,000
4,600
4.6 x 105
Gravel
(GRAVL-CL)
Grab
258(e)
3, 3, 3, 3
10
1,000
1,300
1.3 x 105
Bilge Water
(BILGE-CL)
Grab
250 mL®
3, 3, 3, 3
1
CFU/mL
100
CFU/mL
130
1.3 x 104
(a) Surfaces were pretreated with simulated seawater (salt water) spray (SA) or left clean (CL) prior to inoculating (loading)
with Btk T1B2 spores.
(b)Low target number of Btk T1B2 spores was 10 CFU/cm2 bilge water directly inoculated with 1 CFU/mL Btk T1B2 spores.
See Section 2.4.2 for discussion.
(c)High target number of Btk T1B2 spores was 1,000 CFU/cm2 bilge water directly inoculated with 100 CFU/mL Btk T1B2
spores.
(d) Nominally half of the target quantity of spores loaded onto the surface were available (samples split) for analysis using two
analytical methods: culture (trypticase soy agar [TSA] media) and molecular (RV-PCR). See Sections 2.4.3 to 2.4.5 for
discussion.
(e) Projected surface area is the area of the container that was covered with gravel.
(f) Spiked 250 mL of liquid with Btk T1B2 spore suspension.
22
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Table 2. Test Matrix for Samples Collected using Nontraditional Sampling Methods.
Surface'3'
(Surface ID)
Sample
Type
Water
Type
Replicates""
Target Spore Load
(CFU/cm2)(c)
Total Spores
Available on Surface
(CFU)(d)
Low
High
Low
High
Marine Grade
Aluminum/Nonskid
(MSKID-CL)
Vessel
Washdown
Tap
2, 3, 3
10
1,000
1.9 x104
1.9x10®
Marine Grade
Aluminum/Nonskid
(MSKID-SA)
Vessel
Washdown
Tap
2, 3, 3
10
1,000
1.9 x104
1.9x10®
Marine Grade
Aluminum/Nonskid
Vessel
Washdown
Sea
2, 3, 3
10
1,000
1.9 x104
1.9x10®
(MSKID-CL)
Marine Grade
Aluminum/Nonskid
Vessel
Washdown
Sea
2, 3, 3
10
1,000
1.9 x104
1.9x10®
(MSKID-SA)
Roofing Shingles
(SHING-CL)
Water
Runoff
Tap
2, 3, 3
10
1,000
1.9 x104
1.9x10®
Roofing Shingles
(SHING-CL)
Water
Runoff
Sea
2, 3, 3
10
1,000
1.9 x104
1.9x10®
Roofing Shingles
(SHING-CL)
Water
Runoff
Rain
2, 3, 3
10
1,000
1.9 x104
1.9x10®
Marine Grade
Aluminum
(MGAL-SA)
Squeegee
Tap
2, 3, 3
10
1,000
1.9 x 104
1.9x10®
Marine Grade
Aluminum/Nonskid
(MSKID-SA)
Bristle
Brush
Tap
2, 3, 3
10
1,000
1.9 x 104
1.9x10®
(a) Surfaces were pretreated with simulated seawater (salt water) spray (SA) or left clean (CL) prior to inoculating (loading)
with Btk T1B2 spores. Each surface replicate sampled was 2 x 2 ft (3,720 cm2).
(b) Two blanks (not inoculated with Btk spores) and three replicates for the low and high Btk spore inoculation.
(c)Low target number of Btk T1B2 spores was 10 CFU/cm2; high target number of Btk T1B2 spores was 1,000 CFU/cm2.
(d) Nominally half of the target quantity of Btk T1B2 spores loaded onto the surface were available (samples split) for analysis
using two analytical methods: culture (TSA media) and molecular (RV-PCR). See Sections 2.4.3 to 2.4.5 for discussion.
2.4 Microbiological Methods
Bacillus thuringiensis subsp. kurstaki {Btk) with T1B2 genetic barcode (Buckley et al., 2012) was
selected as the surrogate for Bacillus anthracis (Ba) in the current study because it is physically and
genetically similar to Ba (Tufts et al., 2014 and Greenberg et al., 2010), 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 naturally occurring Btk
species by PCR analysis and provided 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 for Detection of Bacillus anthracis in Environmental
Samples During the Remediation Phase of an Anthrax Incident (EPA, 2017), with modification to spore
recovery to allow for sample splitting, 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.
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2.4.1 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, and 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 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 a 50-mL aliquot
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 H, 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
in 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 stock was assigned a unique lot
number and stored refrigerated at 2 to 8°C. Spore stock concentration was determined by spread plating
serial dilutions onto TSA, followed by 30 ± 2 °C overnight incubation and enumeration of CFU.
2.4.2 Spiking Positive Controls
The matrix used to sample the surface (sponge stick and VFC) or the material being sampled (gravel or
washdown liquid) was spiked and processed alongside collected samples to serve as positive controls for
the spore recovery and analytical methods.
On the day of sample spore recovery, Btk T1B2 spore stock was vortex-mixed and diluted using sterile
dFhO and used to directly spike the sample type at the two target Btk T1B2 spore load levels shown in
Table 3. Each spiking suspension was spread plated onto TSA on the day of testing to calculate the
actual concentration of spores spiked in CFU/mL. The loading levels in Table 3 represent the maximum
target loading of CFU that could have been collected at Day 1 for (low) 10 CFU/cm2 and (high)
1,000 CFU/cm2. Spores were applied over test surfaces and the actual spike level applied to positive
controls throughout testing.
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Table 3. Target Btk T1B2 Spore Loading Levels Spiked onto Each Sample Type for Positive Control.
Sample Type
Loading Level
Target Spike Level (CFU)
Sponge Stick
Low
6.5 x103
High
6.5 x105
Vacuum Filter Cassette
Low
9.3 x103
High
9.3 x105
Grab (Gravel)
Low
2.6 x103
High
2.6 x105
Grab (Bilge)
Low
2.5 x102
High
2.5 x104
Tap Water Washdown or Runoff
Low
3.7 x104
High
3.7 x106
Seawater Washdown or Runoff
Low
3.7 x104
High
3.7 x106
Rainwater Runoff
Low
3.7 x104
High
3.7x10®
Each sponge stick to be spiked with Btk T1B2 spores was positioned in a specimen cup so that the dirty
side was facing up and 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 22). For VFCs, the spore load levels
were directly applied over the surfaces of collected particulates and filter. Gravel was spiked on the top
layer of-900 g of gravel using microliter volumes.
& j
Figure 22. Sponge Stick (A), Vacuum Filter Cassette (B), and Gravel (C) Being Spiked with Btk T1B2
Suspension for Use as Analytical Controls.
2.4.3 Sample Processing for Spore Recovery
Throughout the recovery procedure, gloves were changed between handling samples to limit the
likelihood of cross-contamination between samples.
2.4.3.1 Metal Reference Coupons
Following Btk T1B2 spore spray application, metal reference coupons were collected from the spray
table and placed into 50-mL tubes containing 10 mL of PBST, vortexed for 30 sec and plated (0.1 mL
spread plate or milliliter volumes on MicroFunnel filters) on TSA. The CFU/mL recovered from the
reference coupons were divided by the size of the reference coupon (6.25 cm2); the average CFU/cnr is
described as the determined load for each surface type.
6
25
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2.4.3.2 Sponge Sticks
Following sample collection, samples were stored at 2 to 8°C until spore recovery. Spore recovery using
90 mL cold (2 to 8°C) phosphate buffered saline with 0.05% (v/v) Tween 20 and 30% (v/v) ethanol
(PBSTE) was added into a Stomacher bag (Seward, Bohemia, NY). The remaining handle was removed,
and the sponge stick was unfolded and aseptically added to the Stomacher bag and homogenized for 1
min at 260 rpm. 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 in half in two 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. The pellets were suspended in -25
mL of the supernatant to concentrate the sample. This pooled suspension was split in half and used for
culture-based analysis described in Section 2.4.4 and RV-PCR analysis as described in Section 2.4.5.
2.4.3.3 Vacuum Filter Cassettes
Following sample collection, samples were stored at 2 to 8°C until spore recovery. Spore recovery using
5 mL of PBSTE was added to the conical tube containing the nozzle and tubing and was set aside.
Six (6) mL 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 described in Section 2.4.4 and RV-PCR
analysis as described in Section 2.4.5.
2.4.3.4 Nontraditional (Washdown) and Grab Samples (Bilge Water and Gravel)
Following sample collection, samples were stored at 2 to 8°C until spore recovery. Washdown, bilge
water, or gravel suspension was mixed vigorously by hand for 30 sec. Then, the liquid was poured into a
0 .45 - (am filter funnel (MicroFunnel filter; Pall Corporation, Washington, NY) 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 were added for a total of 250 mL. 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 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 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 tubes settled
for 2 min, then the volume was 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, then
allowed 30 sec of settle time and then split in half for culture-based analysis described in Section 2.4.4
and RV-PCR analysis as described in Section 2.4.5.
2.4.4 Culture Method
Culture-based analysis was performed on each sample by filtering the recovered spore suspension
through MicroFunnel filter funnels (Pall Corporation, Washington, NY Cat. 4804) then placing the
26
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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 for Detection of Bacillus anthracis in Environmental Samples
During the Remediation Phase of an Anthrax Incident, Second Edition (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, rather than inclusion of spread plate analysis of 10"1 and 10"2 dilutions for all samples, since
spore load levels were at or near the method detection limit.
For MicroFunnel filter analysis, initially, each filter was prewetted with 5 mL of PBST, then 10 mL of
PBST was added to each MicroFunnel filter, then milliliter volumes of the recovered spore suspensions
were applied to the 10 mL PBST and vacuum filtered. The walls of each MicroFunnel filter were rinsed
with 10 mL of PBST and filtered through the filter, then the filter membrane was removed and placed
onto TSA media.
Following overnight incubation at 30 ± 2°C, colonies with typical Btk T1B2 morphology were counted
to determine percent spore recovery. Typical Btk T1B2 colony morphology on TSA are 2 to 5 mm in
diameter, flat or slightly convex with edges that are irregular, and have a ground-glass appearance.
Two (2) different microbiologists enumerated colonies over the course of the project, all of whom were
trained by the lead microbiologist on the project to identify presumptive Btk T1B2 more consistently
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 observed during culture analysis are reported as presumptive Btk T1B2.
A small subset of presumptive Btk T1B2 colonies was screened using a PCR assay targeting the T1B2
barcode. A portion of a single colony was suspended in 100 microliter (|iL) of PCR-grade water, heated
for 5 min at 95 ± 2°C, centrifuged at 14,000 rpm (18,407 rcf) for 2 min and the supernatant was
analyzed in triplicate. An average cycle threshold (Ct) value of < 40 was recorded as a positive result.
For brain heart infusion broth (BHIB) enrichment, the filter or sponge was enriched following spore
recovery within the 50-mL conical tube or specimen cup by adding 25 mL of BHIB, then incubating 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 broth was 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 broth 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.
2.4.5 RV-PCR Method
2.4.5.1 Further Sample Processing for RV-PCR
Following filtration of half (12.5 mL for sponge stick, ~5 mL for VFC, or 10 mL for gravel, bilge, and
washdown samples) of recovered spore suspension through the Whatman™ Autovial™ filter vials (with
polyvinylidene difluoride membrane; Whatman, Marlborough, MA Cat. AV125NPUAQU) set in a
manifold, two buffer washes were performed according to EPA Protocol 2017, Second Edition
(EPA, 2017). The first wash was with 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
27
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removed and placed into a capping tray with prefilled 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 shown in Figure 23. Following the
vortex step, the BHIB broth was mixed by pipetting up and down ~10 times and 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 (-16 h, /)) instead of 9 h as described in the EPA Protocol for Detection
of Bacillus anthracis in Environmental Samples During the Remediation Phase of an Anthrax Incident,
Second Edition (EPA, 2017) in an incubator shaker set to 30 ± 2°C at 230 rpm.
A ^ . \z ill -(c ! V
c
Figure 23. Manifold Containing 16 Filter Vials (A), Capping Tray (B), and Capped Filter Vials Containing
BHIB (C).
Following overnight incubation (~16 h), the filter vials were mixed on the platform vortex for 10 min
with speed set to 7. (Note, the 16-h incubation was longer than the 9-h incubation specified in the EPA
Protocol for Detection of Bacillus anthracis in Environmental Samples During Remediation Phase of an
Anthrax Incident in the 2012 version (EPA, 2012). The 2017, Second Edition of the protocol specified
9 h or longer for complex and post-decontamination samples. The 16-h incubation allowed for a
standard work schedule to be maintained rather than require an overnight shift that would have been
required by a 9-h incubation.) 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 final time (fj) aliquot and
deoxyribonucleic acid (DNA) was immediately extracted or stored at -20°C.
28
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2.4.5.2 DNA Extraction and Purification
Prior to extraction of 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 pre-heated to 80°C. All screw-capped,
1-mL aliquot tubes of To and 7/were thawed and centrifuged at 14,000 rpm (18,407 rcf) for 10 min
(4°C), and 800 |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 (Eppendorf, Enfield,
CT). 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 Tfmai tube for 10 sec (high, -1,800 rpm), the samples were
incubated at room temperature for 5 min.
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 Tfmai tube,
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 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
70% ethanol 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 Tfmai 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 for 1 min were repeated four more 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 (18,407 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 Tfmai DNA extracts were stored at 4°C until RV-PCR analysis or at -20°C if RV-PCR could
not be performed within 24 h.
2.4.5.3 6f/cT1B2 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. The Wizard® Genomic DNA Kit (Promega, Madison, WI) was used following an
internal Battelle method specific for extracting B. anthracis. The resulting DNA was quantified by
29
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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 < -20°C, and used as
needed as the positive control for PCR analysis.
2.4.5.4 Real-Time PCR Assay
The specific tag 2 primer sequences from Buckley et al. (2012) were paired with a TaqMan probe
designed using 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 assay chemistry and the TaqMan PCR assay combining the specific
tag 2 primers and the TaqMan probe designed using PrimerQuest Tool, the sensitivity was similar,
however, the SYBR Green assay had nonspecific amplification in all negative control wells (no template
controls [NTCs]), therefore analysis was performed using the TaqMan assay after discussing these
results with the client. The inclusion of a third oligonucleotide (probe) in the PCR assay also imparts
more specificity of detection (Table 4).
Table 4. Btk T1B2 TaqMan PCR Assay Primers, Probe, and Amplicon Sequences.
Btk
T1B2
Oligo
Sequence
Length
Tm
GC
Percent
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 I I I 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 conditions provided in Appendix H. Each sample
DNA extract was assayed in triplicate reactions. Controls consisted of four positive control wells
containing 50 picogram (pg) of DNA extracted from Btk T1B2 and four NTCs were also included with
each assay. An Applied Biosystems 7500 Fast Real-Time PCR Instrument 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
2.5 Overall Method Implementation
The procedures used to spike/recover/analyze the sponge sticks, VFCs, grab, and nontraditional samples
as they occur in chronological order, are depicted graphically in the process flow diagram in Figure 24.
The process flow chart describes the workflow followed for this project. Calendar time could be reduced
for both culture and RV-PCR methods. For culture, colonies from agar plates and BHIB enrichment
culture could be PCR-screened following overnight incubation for confirmation of Btk T1B2. There is a
note in the EPA Protocol for a culture method that allows for faster analysis of samples by combining
and concentrating the remaining spore suspension and remainder of all dilutions from plating onto a
MicroFunnel membrane filter and plate, instead of BHIB enrichment (EPA 2017). Additional analysis of
30
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BHIB enrichment of the sponge or filter by streaking turbid BHIB onto solid media would, as is usually
done by established laboratory networks, require an extra day or more of incubation followed by PCR;
however, there is not an equivalent analysis for RV-PCR. RV-PCR only analyzes the whole spore
recovery suspension. For RV-PCR, if the enrichment time is reduced from 16 h (overnight) to 9 h, as
described in the EPA Protocol (EPA, 2017) the DNA extraction could be performed by a third-shift staff
or automated method and PCR analysis could potentially be completed within 24 h.
Spore Spike
and Spore
Recovery
Extracted
SSW, VFC or
Filter
TSA,
Incubate
Overnight
BHIB
Enrichment
BHIB,
Incubate
Overnight
BHIB,
Incubate 2
Days
Enumerate
&
~ Select
Colonies for
PCR Screen
-+ Colony PCR
DNA
Extraction
and t,)
Day 1
(Monday)
Day 2
(Tuesday)
PCR
Analysis
Streak for
Isolation if
Colony PCR
Negative
. J
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 24. Process Flow Chart Depicting Key Process Steps in Chronological Order.
The methods implemented, in the form of Wis followed by the analytical staff, are provided in
Appendices I through O. These Wis also complement those microbiological methods described in
Section 2.4, and emphasize glove-changing schedules that were implemented to minimize cross-
contamination. Wis were reviewed, as needed, with the EPA Project Team to ensure consistency with
published methods.
The above method was used to analyze a batch of 16 samples, with one batch conducted per week. Each
batch consisted of samples collected from surfaces and positive controls spiked with a maximum Btk
T1B2 spore load that could be collected for the low (10 CFU/cm2) or high (1,000 CFU/cm2) that was
available on the surface area sampled per "Work Instruction for Spiking with Btk T1B2 Spores" in
Appendix I. The collected samples were recovered following the "Work Instruction for Btk T1B2 Spore
Recovery from Outdoor Surfaces," as described in Appendix J. The recovered spore suspension volume
was then split equally between the culture method and RV-PCR. The aliquot for the culture method was
plated onto TSA media and incubated overnight as described in the "Work Instruction for Culture of Btk
T1B2 Spores Recovered from Outdoor Surfaces" in Appendix K. The To RV-PCR aliquot was stored
frozen while the recovered spores enriched overnight, then the Tfmai aliquot was removed, and the DNA
was extracted from both To and Tfmai aliquots per "Work Instruction for Manual DNA Extraction and
Purification from Btk T1B2 Spores " in Appendix L. The extracted DNA was then analyzed using the
PCR assay described in Section 2.4.5.4 and per "Work Instruction for Real-Time PCR for Btk T1B2
Spores DNA" in Appendix M. PCR was also used to confirm or refute presumptive Btk T1B2 spores
selected from the culture analysis per "Work Instruction for Selecting Presumptive Btk T1B2 Colonies
for qPCR Confirmation" in Appendix N. Selected samples for which the culture was a nondetect were
further analyzed using an enrichment procedure per "Work Instruction for Enrichment for Culture," in
Appendix O.
31
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2.6 Data Reduction and Analysis
2.6.1 Percent Recovery of Presumptive Btk T1B2 Spores
The percent recovery efficiency (Erecovery) of Btk T1B2 spores from each sprayed surface 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 sprayed over the surface area sampled (Nspray), as determined
by test coupon extraction, 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
(Vextract) (mL). Mathematically, the percent recovery is expressed as follows:
r * V
j-i sn/\ recover vextract . nnn,
^recovery (%) = 77 * 100%
iv cnrm;
Further, the number of presumptive Btk T1B2 spores present in the volume of recovered suspension
collected onto spread plates or MicroFunnel filter membrane was divided by the suspension volume
analyzed to yield a presumptive Btk T1B2 spore concentration (Crecover) (CFU/mL). The 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 rules
below:
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 microorganisms on the
high-volume aliquot produce 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, 10"1 and 10"2 dilutions were not plated in all cases as described in "U.S. EPA Protocol for
Detection of Bacillus anthracis in Environmental Samples During the Remediation Phase of an
Anthrax Incident, Second Edition" (EPA, 2017). Rather, the volume plated was included or
excluded depending on spore load level and expected recovery.
The number of CFU is reported as presumptive Btk T1B2 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 (18,407 rcf) for 2 min and the supernatant was analyzed using the real-time
PCR assays targeting Btk T1B2.
32
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2.6.2 RV-PCR Method
The Ct values for the To and Tfmai timepoints as well as the delta Ct value (ACt) were reported. The ACt
is generated by subtracting the average Ct (from triplicate reactions) generated by the Tfinal 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 below criterion was met:
The ACt must be greater than or equal to 9 for the Btk T1B2 target
(ACt = Ct (To) - Ct (Tfinal) > 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.
2.6.3 Presentation of Results
The method employed to recover Btk T1B2 spores was consistent with current EPA methods, as
described in Section 2.4.3. In the instance of an actual biological release, the entire suspension of spores
recovered from samples would be analyzed either using a culture method or an RV-PCR method. In the
study performed and reported here, however, the spore suspension was split as described in Sections
2.4.4 and 2.4.5, 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 part of the suspension of
recovered spores 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 Btk T1B2 spore load.
As described in Section 2.4.2, the surfaces were sprayed with a target quantity of Btk T1B2 spores at a
low (10 CFU/cm2) and high (1,000 CFU/cm2) level. The reported spore load for each surface analyzed
was based on the Btk T1B2 spore load applied and measured from the eight metal test coupons
distributed across the spray table during surface spray. As expected, there was variability in the
measured spore titer for each trial. Consequently, the summary tables of results also contain a
determined spore level, which provides the reader with information on spore load level actually applied
to each surface.
33
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3.0 RESULTS AND DISCUSSION
Because recovered spore suspension from each collected sample was split equally for the two analysis
methods (culture and RV-PCR), summary results tables indicate one-half the nominal target Btk T1B2
spore load and one-half the determined spore load that was applied to the surfaces sampled, assuming
100% recovery efficiency. 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 the surface types and analytical methods, recognizing that the surfaces were originally
sprayed with target quantities of Btk T1B2 spores double the quantity that is represented in the tables
and plots.
Note, the determined number of spores available for analysis represents the maximum number of spores
available and is half of the average quantity of spores recovered from eight reference metal coupons
placed on the spray table with the target surface (see Section 2.1.2.3). The number assumes 100%
recovery from the surface and no physical losses associated with processing of samples. It is not an
absolute indication of the limit of detection of the analytical method, rather, it is a measure of the end-to-
end performance of the method to detect and identify Btk T1B2 spores.
Plots and data summary tables of the outdoor temperature and RH experienced by the surfaces over the
duration of the study are presented in Appendix P. The surfaces experienced outdoor conditions from
mid-winter (February 2020) through late summer (September 2020); they were not exposed to direct
sunlight, precipitation, or wind from the ambient.
3.1 Sponge Stick Sample Analysis Results
3.1.1 Sponge Stick Sample Culture Analyses
A summary of the average and standard deviation of the measured recovery efficiencies of presumptive
Btk T1B2 spores recovered from surfaces using sponge sticks are presented in Table 5. The nominal
quantity represents one-half the target Btk T1B2 spore load applied to the surfaces and the determined
number of spores available represents one-half the number of presumptive Btk T1B2 spores recovered
from the metal reference coupon enumeration (see Section 2.4.3.1) on the day of the spray application.
The quantity of presumptive Btk colonies recovered as determined by culture analysis using TSA plates
is plotted in Figure 25 (Low Btk T1B2 Spore Load) and Figure 26 (High Btk T1B2 Spore Load).
The clean MGAL surface with a low Btk T1B2 spore load of 3,200 CFU applied had measurable
quantities of presumptive Btk spores recovered after 1 day and 30 days of environmental exposure. After
90 days of environmental exposure, an overwhelming number of presumptive Btk colonies were too
numerous to count (TNTC), but the colony PCR screens were negative. This TNTC growth may have
been due to the inadvertent storage of the collected sponge stick samples at room temperature for 1 week
post sampling, rather than at 2 to 8°C as all other samples were stored, potentially allowing background
microorganisms to grow. At 180 days of environmental exposure, no presumptive Btk were observed, in
part due to background growth overwhelming culture plates. Both sea salt sprayed GLASS and MGAL
surfaces had measurable presumptive Btk spores recovered through 180 days of environmental exposure;
however, none of the presumptive Btk were confirmed PCR positive for target Btk T1B2 spores beyond
90 days for GLASS and 30 days for MGAL for the salted surfaces.
As shown in Table 5, surfaces with a high Btk T1B2 spore load of 320,000 CFU applied had measurable
quantities of presumptive Btk recovered throughout the 180 days of environmental exposure for all
34
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surface types and followed a similar trend in percent recovery, starting at approximately 40% at 1 day,
25% at 30 days, 5% at 90 days, and <1% at 180 days. The clean MGAL surface after 90 days of
environmental exposure had an overwhelming number of presumptive Btk colonies that were TNTC, but
the colony PCR screens were negative. This TNTC growth may have been due to the inadvertent storage
of the collected sponge stick samples at room temperature for 1 week post sampling, rather than at 2 to
8°C as all other samples were stored, potentially allowing background microorganisms to grow. The sea
salt sprayed glass surface was the only surface with presumptive Btk T1B2 colonies confirmed by PCR
analysis after 180 days of environmental exposure, although all surfaces did have the same relative
number (<100 CFU) of total presumptive Btk.
The spore recovery was fairly consistent (within a factor of two to three) across the surface material
types for surfaces that started clean or with a sea salt residue when comparing within the same target Btk
T1B2 spore loading level.
Table 5. Presumptive Btk T1B2 Spore Recovery from Different Surfaces Sampled Using Sponge Sticks.
Surface ID1"
Spores Available for Analysis
(CFU)
Environmental
Exposure
Duration
(Days)
Spore Recovery (CFU)
(X ± CT),dl
Spore
Recovery
Efficiency
(%)
(X ± o)101
Nominal"51
Determined,:l
GLASS-SA-LOW
3.2 x 103
1.8 x 103
1
3.9 x 102 ± 1.2 x 102
22 ± 5.5
30
3.4 x 102± 4.0 x 101
19 ± 1.8
90
5.9 x 101 ± 1.2 x 101
3.3 ± 0.6
180
1.9x102±2.5x 102®
11 ± 11.50
GLASS-SA-HIGH
3.2 x 105
2.7 x 105
1
1.1 x 105± 3.9x 103
41.4 ± 1.2
30
6.6 x 104 ± 1.1 x 104
24.4 ± 3.4
90
1.3 x 104 ± 2.6 x 103
4.9 ± 0.8
180
3.6 x 101 ± 1.2 x 101
0.01 ± 0.0
MGAL-CL-LOW
3.2 x 103
7.0 x 103
1
9.0 x 102± 9.2 x 101
12.6 ± 1.1
30
4.8 x 102 ± 1.4 x 102
6.8 ± 1.6
90
BKGD(fg)
BKGD(fg)
180
0 ± 0®
0 ± 0®
MGAL-CL-HIGH
3.2 x 105
2.3 x 105
1
8.5 x 104 ± 1.5 x 104
36.6 ± 5.2
30
5.4 x 104 ± 1.1 x 104
23.3 ± 3.9
90
BKGDM
BKGD(fg)
180
2.9 x 101 ± 8.2 x 101(f>
0.01 ± 0.01
MGAL-SA-LOW
3.2 x 103
5.5 x 102
1
1.5 x 102 ± 1.7 x 101
28.0 ±2.6
30
2.9 x 102 ± 1.5 x 102
52.2 ± 22.6
90
4.4 x 102 ± 5.3 x 102®
79.7 ± 78.60
180
7.5 x101 ± 7.4 x101(f)
13.7 ± 11.00
MGAL-SA-HIGH
3.2 x 105
2.4 x 105
1
9.3 x 104 ± 1.1 x 104
38.6 ± 3.6
30
7.0 x 104 ± 1.3 x 103
29.0 ± 0.5
90
7.8 x 103 ± 2.8 x 103
3.2 ± 0.9
180
1.4 x 101 ± 1.3 x 101(f)
0.01 ± 0.0(f)
(a) (N = 3) GLASS-SA = glass with sea salt spray; MGAL-CL = marine grade aluminum clean; MGAL-SA = marine grade
aluminum with sea salt spray.
(b) Nominally one-half of the target Btk T1B2 spore load on the surface and assuming 100% recovery of spores.
(c) Based on the metal reference coupon enumeration measured with each spray application, 100% recovery efficiency, and
one-half of the extract used for culture analysis.
(d) Presumptive Btk T1B2 colonies based on morphology and one-half of extract used for culture analysis.
(e) Calculated using the actual spore loading on each surface and total presumptive Btk T1B2 spores recovered on each sponge
stick sample.
(f) Presumptive Btk T1B2 colonies were PCR negative or not screened by PCR.
(g) BKGD = Culture plates were overwhelmed with background growth.
35
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The presumptive Btk T1B2 spores recovered for all surfaces sampled using the sponge stick method are
shown in Figure 25 (Low Btk T1B2 Spore Load) and Figure 26 (High Btk T1B2 Spore Load) with data
represented in total logio CFU recovered. Since background microorganisms interfered with the
identification of presumptive Btk on culture plates from the low spore load samples more than the high
spore load samples due to sample dilutions plated, trends for spore stability using the high spore load
surfaces are considered more reliable. Using the high spore load surfaces as an indicator, the spore load
recovered was stable through 30 days of environmental exposure on all surface types. Then, there was
an approximate 1 logio decline in the quantity of presumptive Btk spores recovered between 30 days and
90 days of environmental exposure. An additional 2 to 3 logio decline was observed between 90 days
and 180 days of environmental exposure. The glass surface was the only surface that had presumptive
Btk T1B2 spores confirmed by PCR at 90 days of environmental exposure for the low load surfaces and
180 days of environmental exposure for the high load surfaces (Figure 26). As the number of
presumptive Btk colonies declined, the background microorganisms and grime collected increased,
interfering with the identification of presumptive Btk on TSA plates. As a result, a higher standard
deviation was observed when lower numbers of target Btk T1B2 spores were collected and as time of
environmental exposure increased. The decline in recovered Btk T1B2 spores may have been reduced
with environmental exposure because spores were less likely to be collected onto the sponge stick as a
layer of microorganisms and grime accumulated on top of the surface and spores, or because the spores
were more tightly attached to the surface as time increased, or the viability of the spores decreased with
time.
I I SS-G LASS-SA-Low
I I SS-MGAL-CL-Low
I I SS-MGAL-SA-Low
Initial Load SS-GLASS-SA
Initial Load SS-MGAL-CL
Initial Load SS-MGAL-SA
LL
180
(Days)
Figure 25. Presumptive Btk T1B2 Spores Recovered from Surfaces with a Low Target Load of 3,200 Btk
Spores and Sampled Using a Sponge Stick.
Average ± One Standard Deviation ofN = 3. GLASS-SA, Glass with Sea Salt Spray; MGAL-CL: Marine Grade
Aluminum Clean; MGAL-SA: Marine Grade Aluminum with Sea Salt Spray.
30
90
Duration of Environmental Exposur
36
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w>
£-4
oo 2
E 1
Initial Load SS-GLASS-SA
Initial Load SS-MGAL-CL
Initial Load SS-MGAL-SA
30 90
Duration of Environmental Exposure (Days)
180
Figure 26. Presumptive Btk T1B2 Spores Recovered from Surfaces with a High Target Load of 320,000 Btk
Spores and Sampled Using a Sponge Stick.
Average ± One Standard Deviation ofN = 3. GLASS-SA, Glass with Sea Salt Spray; MGAL-CL: Marine Grade
Aluminum Clean; MGAL-SA: Marine Grade Aluminum with Sea Salt Spray.
The images of culture plates in Figure 27 and Figure 28 show the increasing levels of background
microorganisms and grime recovered from sponge stick wipe samples as storage time increased on glass
surfaces with a pretreatment of salt spray. At Day 1 and Day 30, background microorganisms did not
interfere with identifying Btk T1B2 morphology on TSA culture plates. Surfaces accumulated
environmental microorganisms and grime as storage time increased while the number of presumptive
Btk T1B2 spores being recovered from the surfaces decreased, leading to an increase in background
interference, making differentiation of presumptive Btk T1B2 colonies from background
microorganisms and grime at Day 90 and Day 180 more challenging.
37
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Figure 27. Culture Images of Day 1 and Day 30 Spore Recovery from Glass Salted Surface Plated on TSA.
No Interfering Background Growth or Grime Observed: (A) Day 1, 0.01 mL from high Bth T1B2 spore load
surface; (B) Day 1, 2 mL from high Btk T1B2 spore load surface; (C) Day 30, 0.1 mL from low Btk T1B2 spore
load surface; (D) Day 30, 0.01 mL from high Btk T1B2 spore load surface.
Figure 28. Culture Images of Day 90 and Day 180 Spore Recovery from Glass Salted Surface Plated on TSA.
Background Growth and Grime Interfered with Btk T1B2 Morphology Identification: (A) Day 90, 0.1 mL from
low Btk T1B2 spore load surface; (B) Day 90, 2 mL from low Btk T1B2 spore load surface; (C) Day 180, 0.1
mL from low Btk T1B2 spore load surface; (D) Day 180, 2 mL from high Btk T1B2 spore load surface; (E)
Day 180, 8 mL from high Btk T1B2 spore load surface.
38
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The presence of material interfering with the analysis is not surprising considering that the sponge sticks
used to sample the surfaces were noticeably dirtier as environmental exposure time increased, as shown
in Figure 29 (Day 1 sponge sticks appeared clean).
Figure 29. Sponge Stick Used to Sample GLASS-SA Collected Following 180 Days of Environmental
Exposure Shows Environmental Exposure Led to an Accumulation of Grime on the Surfaces.
3.1.2 Btk T1B2 Confirmation
Presumptive Btk TJB2 colonies were observed for all surfaces sampled using sponge sticks from Day 1
through Day 180 with the lone exception of MGAL-CL-LOW surface sampled on Day 180; for this
condition and timepoint, presumptive Btk T1B2 colonies were not present on culture plates. The
presence of presumptive Btk T1B2 based only on the colony morphology did not mean that the Btk
T1B2 spores persisted and were recovered from the surfaces. 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 plates streaked from BHIB enrichment culture of
the sponge stick, or PCR of an aliquot of the BITIB enrichment culture from the sponge stick. Results
from PCR confirmatory testing are shown in Table 6.
For surfaces inoculated with a low Btk T1B2 spore load of 3.5 logio (3.2 x 103 CFU), presumptive
Btk T1B2 colonies were confirmed to be present on GLASS-SA, MGAL-CL, and MGAL-SA surfaces
by PCR analysis at 30 days of outdoor storage. The only low Btk T1B2 spore load surface with
Btk T1B2 confirmed beyond 30 days was GLASS-SA with target Btk T1B2 confirmed by PCR at
Day 90 for all three replicates. Btk T1B2 was not confirmed to be present at Day 180 for any of the low
Btk T1B2 spore load surfaces.
For surfaces inoculated with a high Btk T1B2 spore load of 5.5 logio (3.2 x 105 CFU), presumptive
Btk T1B2 colonies were also confirmed to be present on GLASS-SA, MGAL-CL, and MGAL-SA at
30 days of outdoor storage. Presumptive Btk T1B2 was confirmed to be present by PCR analysis at
Day 180 for GLASS-SA for one of the three replicates sampled. For the MGAL surfaces, all three
sample replicates were confirmed positive by PCR analysis at Day 90 from the salted surface, but none
were confirmed to be positive at Day 90 for the clean surfaces, likely because the plates were
overwhelmed by background growth. The high amount of background growth on the MGAL-CL surface
at Day 90 may have been due to inadvertent storage of the collected sponge sticks at room temperature
for 1 week prior to their being analyzed, rather than 2 to 8°C as all other samples were stored,
potentially allowing background microorganisms to grow.
39
-------
It is possible that more replicates could have been confirmed positive for the culture method if more
presumptive Btk T1B2 colonies were PCR-screened and/or more BHIB enrichment of the sponge stick
samples were PCR-screened. However, none of the six colonies PCR-screened from the MGAL surfaces
with a low Btk T1B2 spore load were PCR positive at Day 90, and the PCR screen of the BHIB
enrichment culture from MGAL surfaces was also PCR negative for all three replicates, indicating that
target Btk T1B2 spores may not have been present or could not be detected at Day 90. At Day 180, the
high Btk T1B2 spore load surfaces consistently show a four-log reduction in presumptive Btk T1B2
recovered, and if that trend holds true for the low Btk T1B2 spore load surfaces with an initial load of
3.5 logio, it is reasonable that zero or very few spores would have been collected from the low Btk T1B2
load surfaces at Day 180, as the data indicate.
Table 6. Summary of the Accuracy of Identification of Presumptive Btk T1B2 by PCR Confirmation from
Surfaces Sampled with Sponge Sticks Stored in Outdoor Conditions Over Time.
Surface ID1"
Sample Type
Days of
Environmental
Exposure
Culture
Replicates
Presumptive
Positive"51
Colonies from
Initial Culture
Plates PCR-
screened1'^
Colonies from
BHIB Streak
Plates PCR-
Screened"11
BHIB Broth
PCR-
Screened101
1
3 of 3 (100%)
1 of 1 (100%)
Not Tested
Not Tested
GLASS-SA-LOW
Sponge Stick
30
3 of 3 (100%)
3 of 3 (100%)
Not Tested
Not Tested
(SS)
90
3 of 3 (100%)
3 of 3 (100%)
Not Tested
Not Tested
180
3 of 3 (100%)
0 of 3 (0%)
Not Tested
0 of 3 (0%)
1
3 of 3 (100%)
1 of 1 (100%)
Not Tested
Not Tested
GLASS-SA-HIGH
Sponge Stick
30
3 of 3 (100%)
1 of 1 (100%)
Not Tested
Not Tested
(SS)
90
3 of 3 (100%)
1 of 1 (100%)
Not Tested
Not Tested
180
3 of 3 (100%)
1 of 2 (50%)
Not Tested
1 of 1 (100%)
1
3 of 3 (100%)
1 of 1 (100%)
Not Tested
Not Tested
MGAL-CL-LOW
Sponge Stick
30
3 of 3 (100%)
1 of 1 (100%)
Not Tested
Not Tested
(SS)
90
3 of 3 (100%)
0 of 3 (0%)
Not Tested
0 of 3 (0%)
180
0 of 3 (0%)
Not Tested
Not Tested
0 of 3 (0%)
1
3 of 3 (100%)
0 of 1 (0%)
1 of 1 (100%)
Not Tested
MGAL-CL-HIGH
sponge stick
30
3 of 3 (100%)
1 of 1 (100%)
Not Tested
Not Tested
(SS)
90
3 of 3 (100%)
0 of 3 (0%)
Not Tested
Not Tested
180
3 of 3 (100%)
0 of 2 (0%)
Not Tested
0 of 1 (0%)
1
3 of 3 (100%)
2 of 2 (100%)
Not Tested
Not Tested
MGAL-SA-LOW
Sponge Stick
30
3 of 3 (100%)
4 of 4 (100%)
Not Tested
Not Tested
(SS)
90
3 of 3 (100%)
0 of 3 (0%)
Not Tested
0 of 3 (0%)
180
3 of 3 (100%)
0 of 3 (0%)
Not Tested
0 of 3 (0%)
1
3 of 3 (100%)
Not Tested
3 of 3 (100%)
Not Tested
MGAL-SA-HIGH
Sponge Stick
30
3 of 3 (100%)
Not Tested
Not Tested
Not Tested
(SS)
90
3 of 3 (100%)
3 of 3 (100%)
Not Tested
Not Tested
180
3 of 3 (100%)
0 of 2 (0%)
Not Tested
0 of 1 (0%)
(a) GLASS-SA = Glass with Sea Salt Spray; MGAL-CL = Marine Grade Aluminum Clean; MGAL-SA = Marine Grade
Aluminum with Sea Salt Spray.
(b) Presumptive Btk T1B2 was present on initial culture plates.
(c) Number of colonies PCR-screened from initial plating, with percent PCR positive.
(d) Number of colonies PCR-screened from BHIB streak plates, with percent PCR positive.
(e) Number of samples with PCR screening of BHIB enrichment broth, with percent PCR positive.
40
-------
3.1.3 Sponge Stick Sample RV-PCR Analyses
A summary of the average and sample standard deviation of the RV-PCR ACt values for the detection of
Btk T1B2 spores recovered from sponge stick surface samples is presented in Table 7. The nominal
quantity represents one-half the target Btk T1B2 spore load applied to the surfaces, and the determined
number of spores available represents one-half the number of presumptive Btk T1B2 spores recovered
from metal reference coupon enumeration (see Section 2.4.3.1) during the day of the spray application.
Sample replicates with a RV-PCR ACt value > 9 are RV-PCR positive, indicating that viable Btk T1B2
spores were recovered.
Table 7. RV-PCR Analyses of Sponge Stick Surface Samples for Detection of Btk T1B2 Spores.
Spores Available for Analysis
(CFU)
Environmental
Exposure
Duration
(Days)
ACt (X ± a)
RV-PCR
Surface ID1"
Nominal"5'
Determined'0'
T1B2 Barcode
Target
Replicates
Positive""
1
25.2 ±0.1
3
GLASS-SA-LOW
3.2 x 103
1.8 x 103
30
13.6 ±0.5
3
90
8.0 ±0.9
1
180
1.1 ±0.8
0
1
21.0 ±2.1
3
GLASS-SA-HIGH
3.2 x 105
2.7 x 105
30
20.6 ±2.4
3
90
13.9 ±0.7
3
180
7.3 ±0.5
0
1
25.3 ±0.2
3
MGAL-CL-LOW
3.2 x 103
7.0 x 103
30
15.5 ±1.4
3
90
2.1 ±1.6
0
180
0±0
0
1
22.4 ±1.5
3
MGAL-CL-HIGH
3.2 x 105
2.3 x 105
30
16.2 ±2.8
3
90
9.7 ± 1.4
2
180
8.0 ±4.2
2
1
25.1 ±0.8
3
MGAL-SA-LOW
3.2 x 103
5.5 x 102
30
11.2 ±0.4
3
90
5.0 ±4.1
1
180
-0.8 ±0.6
0
1
20.9 ±2.6
3
MGAL-SA-HIGH
3.2 x 105
2.4 x 105
30
17.8 ±3.0
3
90
15.0 ±3.6
3
180
2.5 ± 1.5
0
(a) GLASS-SA = Glass with Sea Salt Spray; MGAL-CL = Marine Grade Aluminum Clean; MGAL-SA = Marine Grade
Aluminum with Sea Salt Spray.
(b) Nominally one-half of the target Btk T1B2 spore load on the surface and assuming 100% recovery of spores.
(c) Based on the metal reference coupon enumeration measured each spray application, 100% recovery efficiency, and one-
half of extract used for RV-PCR analysis.
(d) Number of replicates (N = 3) with a RV-PCR ACt value > 9.
41
-------
Samples collected from the low Btk T1B2 spore load (3,200 CFU) were RV-PCR positive for all
replicates through 30 days of environmental exposure. At 90 days of environmental exposure, only one
sample replicate from the GLASS-SA and MGAL-SA were RV-PCR positive, and zero sample
replicates were positive for MGAL-CL, possibly due to the increased amount of background
microorganisms observed on the culture plates, as described in Sections 3.1.1 and 3.1.2. At 180 days of
environmental exposure, zero sample replicates were RV-PCR positive for surfaces with low Btk T1B2
spore load.
Samples collected from the high Btk T1B2 spore load (320,000 CFU) were RV-PCR positive for all
sample replicates through 90 days of environmental exposure except for one sample replicate from the
MGAL-CL surface at the 90-day timepoint. At 180 days of environmental exposure, there were only
two sample replicates RV-PCR positive, both from the MGAL-CL surface at the high Btk T1B2 spore
load.
The RV-PCR ACt results are plotted in Figure 30 (Low Btk T1B2 Spore Load) and Figure 31 (High Btk
T1B2 Spore Load). 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. The RV-PCR response was consistent across the surface material types and whether they started
clean or had a sea salt residue.
For the low Btk T1B2 spore load, RV-PCR ACt signal was suppressed from a value of -25 at Day 1 to a
value of -13 at 30 days of environmental exposure. There was no reduction of presumptive Btk colonies
from culture plates from 1 day to 30 days, and 100% (7 of 7) of the colonies PCR-screened from
GLASS-SA, MGAL-CL, and MGAL-SA at the 30-day timepoint were PCR positive, indicating that a
high percentage of the presumptive Btk colonies observed and counted were Btk T1B2, suggesting that
RV-PCR signal suppression was due to growth competition of background organisms that accumulated
from 30-day environmental exposure, not a reduction in number of Btk T1B2 spores collected and
recovered. Despite the -12 ACt reduction from 1 day to 30 days, all replicates were still RV-PCR
positive.
For the high Btk T1B2 spore load, RV-PCR ACt signal suppression was observed between 1 day and
30 days of environmental exposure, but at lower levels compared to the low Btk T1B2 spore load
surfaces, with a ACt reduction of -6 for MGAL-CLh and -3 for MGAL-SA. There was no reduction in
ACt for GLASS-SA surfaces between Day 1 and Day 30, indicating that a higher level of Btk T1B2
spores applied to filter vials at pre-enrichment, compared to background microorganisms, reduces the
impact of growth competition from background microorganisms and as a result, yields a higher ACt
value
42
-------
30
25 ¦
20 ¦
u
<
O)
2? 15
a>
>
<
10 -
5 ¦
¦ SS-GLASS-SA-Low
3 SS-MGAL-CL-Low
' ' SS-MGAL-SA-Low
— — Threshold
1 30 90 180
Duration of Environmental Exposure (Days)
Figure 30. RV-PCR Analysis of Btk T1B2 Spores Recovered from Surfaces with a Low Target Load of
3,200 Btk Spores and Sampled Using a Sponge Stick.
Average ± One Standard Deviation for N = 3 Replicates. GLASS-SA, Glass with Sea Salt Spray; MGAL-CL:
Marine Grade Aluminum Clean; MGAL-SA: Marine Grade Aluminum with Sea Salt Spray. Positive Response
Equals ACt > 9.
30
25 ¦
20
u
<
v
M 15
fTJ
w
0)
>
<
10 ¦
¦ SS-GLASS-SA-High
3 SS-MGAL-CL-High
~ SS-MGAL-SA-High
- Threshold
F=h
1 30 90 180
Duration of Environmental Exposure (Days)
Figure 31. RV-PCR Analysis of Btk T1B2 Spores Recovered from Surfaces with a High Target Load of
320,000 Btk Spores and Sampled Using a Sponge Stick.
Average ± One Standard Deviation for N = 3 Replicates. GLASS-SA, Glass with Sea Salt Spray; MGAL-CL:
Marine Grade Aluminum Clean; MGAL-SA: Marine Grade Aluminum with Sea Salt Spray. Positive Response
Equals ACt >9.
43
-------
3.1.4 Analytical Method Comparison
Overall, culture and RV-PCR analytical methods performed similarly for the detection of Btk T1B2
spores collected from glass and marine grade aluminum surfaces with or without a pretreatment of sea
salt. As discussed below for results of each individual surface, the ability to collect and detect Btk
decreased with duration of environmental exposure. Because the surfaces were protected from direct
precipitation, sunlight, and wind, the results reported here are expected to be higher than would be
experienced had the surfaces been exposed directly to all elements.
Presumptive Btk T1B2 colonies were observed for the entire 180-day duration of environmental
exposure for all surface types except for MGAL-CL with low Btk T1B2 spore load, which had
presumptive Btk T1B2 colonies observed following 90 days of environmental exposure but not at
180 days. A subset of presumptive T1B2 colonies were PCR-screened from the initial culture plates;
colony PCR of BHIB enrichment culture of the sponge stick; and/or PCR analysis of 50-|iL BHIB
enrichment culture. PCR screening of presumptive Btk T1B2 colonies was negative in some cases,
indicating that background microorganisms with morphology indistinguishable from Btk T1B2 were
present on TSA culture plates, perhaps from wild-type/naturally occurring spores. The presence of
background morphologies indistinguishable from target Btk T1B2 led to an inflation in presumptive
culture recovery values, particularly for the 90- and 180-day environmental exposure samples, as well as
uncertainty about the culture-generated results and how many presumptive colonies should be PCR-
screened. 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 collected from surfaces
stored in outdoor conditions. Presumptive culture identification, colony identification confirmed by
PCR, and RV-PCR results are shown in Table 8 for each day samples were collected from surfaces.
Table 9 compares culture identification confirmed by PCR and RV-PCR results, because presumptive
results were not used to confirm the presence of target Btk T1B2 spores. Neither culture nor RV-PCR
consistently had more confirmed positive sample replicates at longer environmental exposure durations
as there was variability in the results from the two methods. Similarly, neither method consistently
demonstrated more confirmed positive sample replicates at the same environmental durations.
For GLASS-SA with low Btk T1B2 spore load, more sample replicates were confirmed positive for
culture (3) than RV-PCR (1) at 90 days. For GLASS-SA with high Btk T1B2 spore load, more replicates
were confirmed positive for culture (1) than RV-PCR (0) at 180 days.
For MGAL-CL low Btk T1B2 spore load, more replicates were confirmed positive for RV-PCR (3) than
culture (1) at 30 days, although PCR confirmation screening was performed on only one sample replicate
(only one colony screened, and that colony was PCR positive) and all three replicates had a uniform
concentration of recovered presumptive Btk colonies. Therefore, we assumed that if multiple colonies
were screened from each replicate, more colonies would have been confirmed positive. The "U.S. EPA
Protocol for Detection of Bacillus anthracis in Environmental Samples During the Remediation Phase of
an Anthrax Incident, Second Edition EPA," protocol specifies that 1 to 3 colonies from MicroFunnel
filters and a minimum of three colonies from spread plates should be PCR-screened for target Btk T1B2
confirmation, screening only one colony was not adequate and deviates from the protocol (EPA, 2017).
For MGAL-CL high Btk T1B2 load, two of three replicates were RV-PCR positive at 180 days of
environmental exposure, and zero replicates were confirmed positive by culture analysis at 180 days after
screening two colonies from the initial culture plates and PCR screening the BHIB broth by PCR for one
replicate; three colonies screened at 90 days of environmental storage were all PCR negative.
For MGAL-SA low Btk T1B2 load, one sample replicate was positive for RV-PCR at 90 days of
environmental exposure and zero replicates were confirmed PCR positive at 90 days after PCR screening
44
-------
of three colonies, one per sample replicate and PCR screening of BHIB enrichment culture for each of the
three replicates. For MGAL-SA high Btk T1B2 spore load, all replicates were confirmed positive for both
methods at 90 days and all sample replicates were negative for culture and RV-PCR methods at 180 days.
Table 8. Analytical Method Comparison Displaying Culture Presumptive, Culture ID with PCR
Confirmation and RV-PCR Replicates Detected (N = 3) for Surfaces Sampled with Sponge Sticks.
Surface ID(a)
Analytical Method
Replicates Detected
Day 1
Day 30
Day 90
Day 180
Presumptive Culture ID
3
3
3
3
GLASS-SA-LOW
Culture ID with PCR Confirmation
1
3
3
0
RV-PCR
3
3
1
0
Presumptive Culture ID
3
3
3
3
GLASS-SA-HIGH
Culture ID with PCR Confirmation
1
1
1
1
RV-PCR
3
3
3
0
Presumptive Culture ID
3
3
3
0
MGAL-CL-LOW
Culture ID with PCR Confirmation
1
1
0
0
RV-PCR
3
3
0
0
Presumptive Culture ID
3
3
3
3
MGAL-CL-HIGH
Culture ID with PCR Confirmation
1
1
0
0
RV-PCR
3
3
2
2
Presumptive Culture ID
3
3
3
3
MGAL-SA-LOW
Culture ID with PCR Confirmation
2
3
0
0
RV-PCR
3
3
1
0
Presumptive Culture ID
3
3
3
3
MGAL-SA-HIGH
Culture ID with PCR Confirmation
3
0
3
0
RV-PCR
3
3
3
0
(a) GLASS-SA = Glass with Sea Salt Spray; MGAL-CL = Marine Grade Aluminum Clean; MGAL-SA = Marine Grade
Aluminum with Sea Salt Spray.
Table 9. Analytical Method Comparison Displaying Culture ID with PCR Confirmation and RV-PCR
Replicates Detected (N = 3) for Surfaces Sampled with Sponge Sticks.
Surface ID(a)
Analytical Method
Last Day of Detection
(# Replicates Confirmed)
GLASS-SA-LOW
Culture w/PCR Confirmation
Day 90 (3)
RV-PCR
Day 90 (1)
GLASS-SA-HIGH
Culture w/PCR Confirmation
Day 180 (1)
RV-PCR
Day 90 (3)
MGAL-CL-LOW
Culture w/PCR Confirmation
Day 30 (1)
RV-PCR
Day 30 (3)
MGAL-CL-HIGH
Culture w/PCR Confirmation
Day 30 (1)
RV-PCR
Day 180 (2)
MGAL-SA-LOW
Culture w/PCR Confirmation
Day 30 (3)
RV-PCR
Day 90 (1)
MGAL-SA-HIGH
Culture w/PCR Confirmation
Day 90 (3)
RV-PCR
Day 90 (3)
(a) GLASS-SA = Glass with Sea Salt Spray; MGAL-CL = Marine Grade Aluminum Clean; MGAL-SA = Marine Grade
Aluminum with Sea Salt Spray.
45
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3.1.5 Analysis of Controls
All reagent, laboratory blank, and field blank controls from sponge sticks were negative for culture and
RV-PCR. Three (3) method blank samples from traditional sampling methods were positive, salted
marine grade aluminum (2) and glass (1) had 1.3, 0.8 and 0.1 CFU/mL recovered from the culture
aliquot with corresponding RV-PCR ACt values of 23.1, 25.1, and 25.3, respectively, indicating that
very low levels of spores, perhaps as few as one, could lead to a positive ACt value, particularly for
samples with low background microorganisms. These method blank samples were most likely
contaminated by low levels of Btk T1B2 from being within the spray laboratory for salt application,
which is the same laboratory/spray table where Btk T1B2 was applied. Method blanks from all other
surface types were negative for both culture and RV-PCR.
Sponge sticks directly spiked with Btk T1B2 at 6.5 x 103 or 6.5 x 105 CFU target Btk T1B2 spore load
were positive for culture and RV-PCR when processed alongside samples collected from surfaces. The
average percent recovery for the 6.5 x 103 CFU positive control samples was 77 ± 69% and had a RV-
PCR ACt value for of 24.6 ± 1.3. The average percent recovery for the 6.5 x 105 CFU positive control
samples was 75 ± 42% and had a RV-PCR ACt value for of 24.6 ± 0.8.
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 of the measured recovery efficiencies of presumptive
Btk T1B2 spores recovered from surfaces that were sprayed with Btk T1B2 spores, then sampled using
VFCs, are presented in Table 10. The nominal quantity represents one-half the target Btk T1B2 spore
load applied to the surfaces and the determined number of spores available represents one-half the
number of presumptive Btk T1B2 spore recovered from metal reference coupon enumeration
(see Section 2.4.3.1) during the day of the spray application. The quantity of presumptive Btk colonies
recovered as determined by culture analysis using TSA plates is plotted in Figure 32 (Low Btk T1B2
Spore Load) and Figure 33 (High Btk T1B2 Spore Load).
The percent recovery of presumptive Btk T1B2 were generally low (< 3%) for both clean and salted
nonskid surfaces. The surfaces with a low Btk T1B2 spore load of 4,700 CFU and high Btk spore load of
470,000 applied had quantifiable recoveries of presumptive Btk colonies through 90 days. At 180 days
of environmental exposure, only NSKID-CL low Btk T1B2 spore load had presumptive Btk colonies,
which had two presumptive colonies on a 3.5-mL volume aliquot plated on a membrane filter that had
grime present, making colony morphology difficult to identify clearly, and one of those two colonies
was PCR negative, the second colony was not PCR-screened.
The low Btk T1B2 spore load level (4,700 CFU) initially recovered zero colonies from the clean surface
and an average of seven colonies from the salted surface. Even the high Btk T1B2 spore load level
(470,000 CFU) surfaces had an average of only 40 to 120 colonies recovered at 1 day post spore
application, an approximate 3 logio difference between Btk load and initial recovery (Figure 32).
46
-------
Table 10. Presumptive Btk T1B2 Spores Recovered from Different Surfaces Sampled Using Vacuum Filter
Cassettes.
Surface ID1"
Spores Available for Analysis
(CFU)
Days of
Environmental
Exposure
Spore Recovery
(CFU)
(X ± CT)ldl
Spore
Recovery
Efficiency (%)
(X ± CT)101
Nominal"51
Determined'^1
NSKID-CL-LOW
4.7 x 103
3.5 x 103
1
0± 0
0± 0
30
3.2 x 101 ± 2.1 x 10°
0.9 ± 0.1
90
7.7 x 101 ± 2.2 x 101®
2.2 ± 0.5®
180
1.1 x 10° ± 1.8x10°®
0.03 ± 0.040
NSKID-CL-HIGH
4.7 x 105
3.5 x 105
1
4.1 x 101 ± 2.0 x 101
0.01 ± 0
30
1.2 x 102 ± 1.8 x 101
0.03 ± 0
90
1.8 x 102 ± 1.7 x 101
0.05 ± 0
180
0 ± 0®
0 ± 0®
NSKID-SA-LOW
4.7 x 103
3.1 x 103
1
7.3 x 10° ± 8.4 x 10°
0.2 ± 0.2
30
2.8x10° ±0^
0.1 ± 0®
90
2.7 x 101 ± 1.6 x 101
0.9 ± 0.4
180
0 ± 0®
0 ± 0®
NSKID-SA-HIGH
4.7 x 105
3.5 x 105
1
1.2 x 102 ± 1.0 x 101
0.03 ± 0
30
9.6 x 101 ± 4.0 x 101
0.03 ± 0
90
1.2 x 102 ± 2.9 x 101
0.03 ± 0.01
180
0 ± 0®
0 ± 0®
(a) NSKID-CL = Nonskid Tread, Clean; NSKID-SA = Nonskid Tread with Sea Salt Spray.
(b) Nominally one-half of the target Btk T1B2 spore load on the surface and assuming 100% recovery of spores.
(c) Based on the metal reference coupon enumeration measured with each spray application, 100% recovery efficiency, and
one-half of extract used for culture analysis.
(d) Presumptive Btk T1B2 colonies based on morphology and one-half of extract used for culture analysis.
(e) Calculated using the actual spore loading on each surface and total presumptive Btk T1B2 spores recovered on each VFC
sample.
(f) Presumptive Btk T1B2 colonies were PCR negative or not screened by PCR.
47
-------
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The images of culture plates in Figure 34 show the increasing levels of background microorganisms and
grime recovered as storage time increased on nonskid surfaces. At Day 1 and Day 30, background
microorganisms did not interfere with identifying Btk T1B2 morphology on TSA culture plates. Surfaces
accumulated environmental microorganisms and grime as storage time increased while the number of
Btk T1B2 spores being recovered from the surfaces decreased, leading to an increase in interference of
identification of presumptive Btk T1B2 colonies at Day 90 and Day 180.
Figure 34. Culture Images of Day 1 through Day 180 Spore Recovery from Nonskid Clean Surface Plated
on TSA.
(A) Day 1, 0.1 mL from high Btk T1B2 spore load surface; (B) Day 30, 4 mL from low Btk T1B2 spore load
surface; (C) Day 90, 4 mL from low Btk T1B2 spore load surface; (D) Day 180, 3 mL from high Btk T1B2
spore load surface.
The presence of material interfering with the analysis is not surprising considering that the VFCs used to
sample the surfaces were noticeably dirtier as environmental exposure time increased, as shown in
Figure 35.
49
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Figure 35. Vacuum Filter Cassette Following Sampling of a Nonskid Tread Showing Collection of
Cottonwood Seeds that Deposited on the Surface.
3.2.2 Btk T1B2 Confirmation
Presumptive Btk T1B2 colonies were observed for all surfaces sampled from Day 1 through Day 90,
except for NSKID-CL-LOW surface sampled on Day 1; for this condition and timepoint, presumptive
Btk T1B2 colonies were not present on culture plates. Recovery of presumptive Btk T1B2 colonies using
VFCs was very low from the onset, with Day 1 recovery approximately 3 logio CFU lower than the
ini tial load sprayed onto the high Btk T1B2 spore load surfaces. The presence of presumptive Btk T1B2
morphology did not mean that the Btk T1B2 spores persisted and were recovered from the surfaces.
Colonies with morphology indistinguishable from Btk T1B2 were present on the culture plates, as
indicated by a negative PCR result for presumptive Btk T1B2 coloni es. The confirmation of target Btk
T1B2 was assessed by colony PCR from the initial culture plates, colony PCR from BHIB enrichment
culture, or PCR of an aliquot of the BHIB enrichment culture from the vacuum cassette filter. Resul ts
from PCR confirmatory testing are shown in Table 11.
For surfaces inoculated with a low Btk T1B2 spore load of 3.7 logio (4.7 x 103 CFU), presumptive Btk
T1B2 colonies were not observed on initial culture plates for the NSKID-CL surface at Day I. However,
coloni es with presumptive Btk T1B2 morphology were isolated from streak plates of the BHIB culture,
and one of those colonies was confirmed to be Btk T1B2 target. At Day 30, an average of 32 CFU
(6 CFU/mL) and 3 CFU (0.5 CFU/mL) presumptive Btk T1B2 colonies per sample replicate were
observed on initial culture plates for NSKID-CL and NSKID-SA, respectively. Only one of the six
(17%) colonies screened from the initial culture plates were PCR positive, and none of the five colonies
isolated from BHIB enrichment streak plates were PCR positive at Day 30. At Day 90, the number of
presumptive Btk T1B2 colonies isolated from initial culture plates increased slightly from Day 30, but
50
-------
0 of 10 (0%) were PCR positive and 1 of 4 colonies isolated from BHIB enrichment streak plates was
positive. At Day 180, only one replicate had presumptive Btk on initial culture plates (0.5 CFU/mL), but
the colony screened was PCR negative and PCR analysis of the BHIB enrichment broth from all
replicates was negative.
For surfaces inoculated with a high Btk T1B2 spore load of 5.7 logio (4.7 x 105 CFU), presumptive
Btk T1B2 colonies were observed on initial culture plates for all replicates at a steady level of 100 CFU
(25 CFU/mL) through 90 days, and all four colonies (100%) screened were PCR positive. At Day 180,
colonies with Btk T1B2 morphology were not isolated from any of the sample replicates.
The consistent culture recovery of presumptive Btk across three aliquot volumes (0.1, 1, and 3 mL) of
spore recovery on culture plates for the high Btk T1B2 spore load NSKID surfaces collected with
vacuum cassettes suggests that the presumptive Btk were target Btk T1B2 spores isolated through
Day 90 despite only screening four presumptive Btk colonies, all of which were confirmed positive by
PCR.
Table 11. Summary of the Accuracy of Identification of Presumptive Btk T1B2 by PCR Confirmation from
Surfaces Sampled with Vacuum Cassettes Stored in Outdoor Conditions Over Time.
Surface ID(a)
Sample
Type
Days of
Environmental
Exposure
Culture
Replicates
Presumptive
Positive""
Colonies from
Initial Culture
Plates PCR-
Screened(c)
Colonies
from BHIB
Streak Plates
PCR-
Screened(tl)
BHIB Broth
PCR-
Screened(e)
1
0 of 3 (0%)
Not Tested
1 of 1 (100%)
1 of 1 (100%)
NSKID-CL-LOW
Vacuum
30
3 of 3 (100%)
1 of 3 (33%)
0 of 2 (0%)
Not Tested
Cassette
90
3 of 3 (100%)
0 of 6 (0%)
Oof 2(0%)
Not Tested
180
1 of 3 (33%)
0 of 1 (0%)
Not Tested
0 of 3 (0%)
1
3 of 3 (100%)
1 of 1 (100%)
Not Tested
Not Tested
NSKID-CL-HIGH
Vacuum
30
3 of 3 (100%)
Not Tested
Not Tested
Not Tested
Cassette
90
3 of 3 (100%)
1 of 1 (100%)
Not Tested
Not Tested
180
0 of 3 (0%)
Not Tested
Not Tested
Not Tested
1
2 of 3 (100%)
1 of 1 (100%)
1 of 1 (100%)
Not Tested
NSKID-SA-LOW
Vacuum
30
3 of 3 (100%)
0 of 3 (0%)
0 of 3 (0%)
Not Tested
Cassette
90
3 of 3 (100%)
0 of 4 (0%)
1 of 2 (50%)
Not Tested
180
0 of 3 (0%)
Not Tested
Not Tested
0 of 3 (0%)
1
3 of 3 (100%)
Not Tested
Not Tested
Not Tested
NSKID-SA-HIGH
Vacuum
30
3 of 3 (100%)
1 of 1 (100%)
Not Tested
Not Tested
Cassette
90
3 of 3 (100%)
1 of 1 (100%)
Not Tested
Not Tested
180
0 of 3 (0%)
Not Tested
Not Tested
Not Tested
(a) NSKID-CL = Nonskid Tread Clean; NSKID-SA = Nonskid Tread with Sea Salt Spray.
(b) Presumptive Btk T1B2 was present on initial culture plates.
(c) Number of colonies PCR-screened from initial plating, with percent PCR positive.
(d) Number of colonies PCR-screened from BHIB streak plates, with percent PCR positive.
(e) Number of samples with PCR screening of BHIB enrichment broth, with percent PCR positive.
51
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3.2.3 Vacuum Filter Cassette Sample RV-PCR Analysis
A summary of the average and sample standard deviation of the RV-PCR ACt values for the detection of
Btk T1B2 spores recovered from VFC surface samples is presented in Table 12. The nominal quantity
represents one-half the target Btk T1B2 spore load applied to the surfaces, and the determined number of
spores available represents one-half the number of presumptive Btk T1B2 spores recovered from metal
reference coupon enumeration (see Section 2.4.3.1) during the day of the spray application. Sample
replicates with a RV-PCR ACt value > 9 are RV-PCR positive, indicating that viable Btk T1B2 spores
were recovered.
Samples collected from the low Btk T1B2 spore load of 4,700 CFU were RV-PCR positive for 1 of 3
replicates through 90 days of environmental exposure. At 180 days of environmental exposure, zero
replicates were positive, due to low recovery and an increased amount of background microorganisms
on those plates, as described in Sections 3.2.1 and 3.2.2. Samples collected from the high Btk T1B2
spore load of 470,000 CFU were RV-PCR positive for all replicates through 90 days of environmental
exposure. At 180 days of environmental exposure, only 1 of 3 replicates was RV-PCR positive.
The RV-PCR ACt results are plotted in Figure 36 (Low Btk T1B2 Spore Load) and Figure 37 (High Btk
T1B2 Spore Load). The plots all 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 to be a positive result.
For the low Btk T1B2 spore load, RV-PCR ACt signal begins at the threshold value of 9 and maintains
that level through 30 days before being cut in half at 90 days and was absent by 180 days. Signal was
consistent between the clean and salted surfaces. For the high Btk T1B2 spore load, RV-PCR ACt signal
was level at -22 through 30-days then fell to -17 at 90 days and was consistent between clean and salted
surfaces. At 180 days, the signal tapered off to 7.8 for the clean surface and 3.4 for the salted surface.
52
-------
Table 12. RV-PCR Analyses of Vacuum Filter Cassette Surface Samples for Detection of Btk T1B2 Spores
Using T1B2 Barcode Target.
Surface ID(a)
Spores Available for Analysis
(CFU)
Days of
Environmental
Exposure
ACt (X ± a)
RV-PCR
Replicates
Positive""
Nominal"5'
Determined'0'
1
8.7 ±9.8
1
NSKID-CL-LOW
4.7 x103
3.5 x103
30
8.5 ±4.5
1
90
3.8 ±5.4
1
180
0 ±0
0
1
25.3 ±0.1
3
NSKID-CL-HIGH
4.7 x105
3.5 x105
30
23.5 ±0.6
3
90
16.8 ± 1.8
3
180
7.8 ±1.8
1
1
12.2 ±8.9
1
NSKID-SA-LOW
4.7 x103
3.1 x 103
30
8.2 ±9.3
1
90
5.2 ±6.2
1
180
-0.5 ±0.8
0
1
19.1 ±5.5
3
NSKID-SA-HIGH
4.7 x105
3.5 x105
30
21.5 ±2.0
3
90
16.1 ±2.9
3
180
3.4 ±4.8
1
(a)NSKID-CL = Nonskid Tread Clean; NSKID-SA = Nonskid Tread, with Sea Salt Spray.
(b) Nominally one-half of the target Btk T1B2 spore load on the surface and assuming 100% recovery of spores.
(c) Based on the metal reference coupon enumeration measured with each spray application, 100% recovery efficiency, and
one-half of extract used for culture analysis.
(d) Number of replicates (N = 3) with a RV-PCR with ACt value > 9.
53
-------
30
25 ¦
20 ¦
u
<
01
2? 15
to
i_
01
>
<
10 ¦
¦ VCF-NSKID-CL-Low
~ VCF-NSKID-SA-Low
- Threshold
T
1 30 90 180
Duration of Environmental Exposure (Days)
Figure 36. RV-PCR Analysis of Btk T1B2 Spores Recovered from Surfaces with a Low Target Load of
3,200 Btk Spores and Sampled Using Vacuum Cassette Filters.
Average ± One Standard Deviation for N> 3 Replicates. NSKID-CL: Nonskid Tread Clean; NSKID-SA:
Nonskid Tread with Sea Salt Spray.; Positive Response Equals ACt>9.
30
25 ¦
20 ¦
u
<
0)
2? 15
CD
k-
3 Replicates. NSKID-CL: Nonskid Tread Clean; NSKID-SA:
Nonskid Tread with Sea Salt Spray.; Positive Response Equals ACt>9.
54
-------
3.2.4 Analytical Method Comparison
Overall, culture and RV-PCR analytical methods performed similarly for the detection of Btk T1B2
spores collected from nonskid surfaces with or without a pretreatment of sea salt. Presumptive Btk T1B2
recovery efficiencies were low, with < 1% recovery for all surfaces at Day 1 of sampling. The highest
recovery value of presumptive Btk T1B2 was < 3%, at Day 90 on the nonskid tread clean, low Btk T1B2
spore load surface, but zero of the six colonies screened from this timepoint were colony PCR positive,
indicating that background microorganisms with morphology indistinguishable from Btk T1B2 were
present on TSA culture plates, perhaps from wild-type/naturally occurring Btk, and their presence led to
an inflation in culture recovery values. Inefficient recovery resulted in 1 of 3 RV-PCR replicates from
each timepoint from Day 1 to Day 90 and zero positive replicates at Day 180 for samples collected from
the low Btk T1B2 spore load surfaces. Samples collected from the high Btk T1B2 spore load surfaces
were all positive through 90 days and 1 of 3 replicates was positive at 180 days. Presumptive culture
identification, culture identification confirmed by PCR, and RV-PCR results are shown in Table 13.
Table 14 compares culture identification confirmed by PCR and RV-PCR results as presumptive results
do not confirm the presence of target Btk T1B2 spores. Comparing confirmed culture results with RV-
PCR results, the methods performed similarly for the detection of target Btk T1B2 spores, with a slight
edge to the RV-PCR method because one replicate from the clean and sea salt sprayed surface with high
Btk T1B2 spore load was RV-PCR positive at 180 days and zero were confirmed positive for culture at
180 days.
Table 13. Analytical Method Comparison Displaying Culture Presumptive; Culture ID with PCR
Confirmation and RV-PCR Replicates Positively Detected (N = 3) for Surfaces Sampled with Vacuum
Cassettes.
Surface ID(a)
Analytical Method
Replicates Detected
Day 1
Day 30
Day 90
Day 180
Presumptive Culture ID
0
3
3
1
NSKID-CL-LOW
Culture ID with PCR Confirmation
1
1
0
0
RV-PCR
1
1
1
0
Presumptive Culture ID
3
3
3
0
NSKID-CL-HIGH
Culture ID with PCR Confirmation
1
0
1
0
RV-PCR
3
3
3
1
Presumptive Culture ID
2
3
3
0
NSKID-SA-LOW
Culture ID with PCR Confirmation
2
0
1
0
RV-PCR
1
1
1
0
Presumptive Culture ID
3
3
3
0
NSKID-SA-HIGH
Culture ID with PCR Confirmation
0
1
1
0
RV-PCR
3
3
3
1
(a) NSKID-CL = Nonskid Tread Clean; NSKID-SA = Nonskid Tread with Sea Salt Spray.
55
-------
Table 14. Analytical Method Comparison Displaying Culture ID with PCR Confirmation and RV-PCR
Replicates Detected (N = 3) for Surfaces Sampled with Vacuum Cassettes.
Surface ID(a)
Analytical Method
Last Day of Analytical Method
Detection (# Replicates Confirmed)
NSKID-CL-LOW
Culture w/PCR Confirmation
Day 30 (1)
RV-PCR
Day 90 (1)
NSKID-CL-HIGH
Culture w/PCR Confirmation
Day 90 (1)
RV-PCR
Day 180 (1)
NSKID-SA-LOW
Culture w/PCR Confirmation
Day 90 (1)
RV-PCR
Day 90 (1)
NSKID-SA-HIGH
Culture w/PCR Confirmation
Day 90 (1)
RV-PCR
Day 180 (1)
(a) NSKID-CL = Nonskid Tread Clean; NSKID-SA = Nonskid Tread with Sea Salt Spray.
3.2.5 Analysis of Controls
VFCs directly spiked with Btk T1B2 at 9.3 x 103 or 9.3 x 105 CFU spore load were positive for culture
and RV-PCR when processed alongside samples collected from surfaces. The average percent recovery
for the 9.3 x 103 CFU positive control samples was 12 ± 12% and had an RV-PCR ACt value of
24.1 ± 1.2. The average percent recovery for the 9.3 x 105 CFU positive control samples was 41 ± 35%
(one replicate with 939% recovery value was excluded) and had a RV-PCR ACt value of 23.1 ± 2.7.
3.3 Grab Sample Analyses Results (Gravel and Bilge Water)
Of the 31 gravel samples that were filter-concentrated, 1 gravel sample at Day 1 clogged the filter at
135 mL, preventing the target volume of 250 mL from being analyzed. This sample was culture and RV-
PCR positive. All other samples, 30 of 31, did not clog the filter prior to 250 mL being concentrated.
Of the 32 bilge water samples that were filter-concentrated, the target volume of 250 mL was never met,
and all filters clogged before reaching this volume. The average volume concentrated before filter clog
for all samples was 109 mL. The 180-day samples clogged the filter at an average volume of 50 mL,
compared to the average of 125 mL for Days 1 through 90. The 180-day samples likely had more
sediment than Day 1 through 90 samples due to the collection method, which was by withdrawing
sample volume using a serological pipet. At Day 180, less volume was available for collection in the
storage carboys compared to the previous timepoints, and the serological pipet was therefore closer to
the settled sediment, which may have led to the collection of more sediment, and as a result, less volume
filtered before becoming clogged.
3.3.1 Grab Sample Culture Analyses
A summary of the average and standard deviation of the measured recovery efficiencies of presumptive
T1B2 spores recovered from gravel and bilge water loaded with Btk T1B2 spores, then sampled using
the grab method are presented in Table 15. The nominal quantity represents one-half the target Btk T1B2
spore load applied to the gravel surface, as determined by the amount of presumptive Btk recovered
from metal reference coupons (see Section 2.4.3.1), or directly spiked into the bilge water. The quantity
of presumptive Btk colonies recovered as determined by culture analysis using TSA plates are plotted in
Figure 38 (LowBtk T1B2 Spore Load) and Figure 39 (High Btk T1B2 Spore Load).
The percent recovery of presumptive Btk T1B2 for gravel was variable, ranging from 0 to >100% for
low Btk T1B2 spore load of 1,300 CFU applied, in part from the presence of high background growth.
The 0% recovery value for 30-day samples was due to incomplete recovery of bacteria and particulates
56
-------
from the filter membrane, as shown in (Figure 40). For this timepoint, all replicates were confirmed
PCR positive from BHIB enrichment of the filter membrane. The high Btk T1B2 spore load of 1.3 x 105
CFU applied also had variability in percent recovery, ranging from an average of 0.1 to 26%. The
presence of Btk T1B2 spores was confirmed by PCR for low and high Btk T1B2 spore load gravel
samples through 180 days of environmental exposure by PCR of colonies from initial culture plates,
BHIB culture streak plates, or 50 |iL of BHIB culture.
Table 15. Recovery Efficiencies for Presumptive Btk T1B2 Spores from Gravel and Bilge Water Grab
Samples Cultured on TSA Medium.
Surface ID1"
Sample
Replicates
Spores Available for
Analysis (CFU)
Environmental
Exposure
(Days)
Spore Recovery
(CFU)
(X ± CT)ldl
Spore
Recovery
Efficiency
(%)
(X ± CT)I0»"
Nominal"51
Determined'1^
GRAVL-CL-
LOW
3
1.3 x 103
7.0 x 102
1
1.5 x 103 ± 4.8 x 102
210 ± 54.3
30
0 ± 0(9'
0± 0
90
8.0 x 101 ± 7.2 x 101
11.1 ± 8.2
180
5.3 x 101 ± 6.8 x 101
7.4 ± 7.7
GRAVL-CL-
HIGH
3
1.3 x 105
1.1 x 105
1
2.9 x 104 ± 2.3 x 104
26.0 ± 18.4
30
1.1 x 102± 3.9 x 101O)
0.1 ± 0.03
90
9.5 x 103 ± 6.2 x 103
9.2 ±4.9
180
1.6 x 104 ± 5.2 x 103
15.7 ± 4.1
BILGE-CL-LOW
3
5.0 x 101
1.5 x 102
1
5.2 x 101 ± 7.3 x 10°
40.5 ± 11.2
30
5.3 x 101 ± 2.0 x 101
32.6 ± 15.0
90
2.3 x 101 ± 4.1 x 10°
12.5 ± 1.8
180
2.9 x 101 ± 1.1 x 101
38.1 ± 11.7
BILGE-CL-
HIGH
3
5.6 x 103
2.0 x 104
1
5.9 x 103 ± 4.5 x 102
25.1 ± 2.8
30
3.6 x 103 ± 1.0 x 103
15.6 ± 2.8
90
4.2 x 103 ± 1.2 x 103
17.4 ± 3.8
180
2.6 x 103± 3.1 x 102
31.4 ± 4.4
(a) GRAVL-CL = Gravel Clean; BILGE-CL = Bilge Water Clean.
(b) Nominally one-half of the target Btk T1B2 spore load on the surface and assuming 100% recovery of spores.
(c) Based on the metal reference coupon enumeration measured with each spray application, 100% recovery efficiency, and
one-half of extract used for culture analysis.
(d) Presumptive Btk T1B2 colonies based on morphology, and one-half of extract used for culture analysis.
(e) Calculated using the actual spore loading on each surface and total presumptive Btk T1B2 spores recovered from each
sample.
(f) Presumptive Btk T1B2 colonies were confirmed by PCR for all conditions.
(g) Incomplete recovery of bacteria from the filter membrane.
The percent recovery from bilge water was consistent throughout the 180-day environmental exposure,
the low Btk T1B2 spore load samples averaged 40.5% at Day 1 and 38.1% at Day 180. The high
BtkTYQl spore load samples averaged 25.1% at Day 1 and 31.4% at Day 180.
57
-------
3
Li.
u
a
qo
o
£"4
0)
>
o
u
HI
cc
o
Q.
1/1
0)
>
Q.
E
3
I GRB-GRAVL-CL-Low
I GRB-BILGE-CL-Low
Initial Load GRB-GRAVL-CL
Initial Load GRB-BILGE-CL
30 90
Duration of Environmental Exposure (Days)
180
Figure 38. Presumptive Btk T1B2 Spores Recovered from Surfaces with a Low Target Load of 3,200 Btk
Spores and Sampled Using a Grab Method.
Average ± One Standard Deviation ofN = 3 Replicates. GRA VL-CL: Gravel Clean; BILGE-CL: Bilge Water,
Clean.
u
35
60
I GRB-GRAVL-CL-High
] GRB-BILGE-CL-High
Initial Load GRB-GRAVL-CL
Initial Load GRB-BILGE-CL
m 2
E 1
30
90
180
Duration of Environmental Exposure (Days)
Figure 39. Presumptive Btk T1B2 Spores Recovered from Surfaces with a High Target Load of 320,000 Btk
Spores and Sampled Using a Grab Method.
Average ± One Standard Deviation ofN = 3 Replicates. GRA VL-CL: Gravel Clean; BILGE-CL: Bilge Water,
Clean.
58
-------
Figure 40. Poor Recovery from Filter Membrane for Gravel Samples Collected at Day 30.
Note: The vortex step typically dislodged most of the sediment observed on the filter membrane. However,
Day 30 samples had visible sediment that remained on the filter membrane post-vortex mixing, leading to poor
recovery values.
For gravel samples, the images of culture plates in Figure 41 show that initially (Day 1), background
microorganisms did not interfere with detection of presumptive Btk morphology on 0.1-mL spread
plates. Thus, Btk T1B2 was easily observed and confirmed by PCR analysis. However, when a 2-mL
aliquot was collected onto a membrane and placed onto a TSA plate, the soil particulates on the washed
gravel interfered with morphology identification, which makes the detection of low levels of spores
challenging (Figure 41B). At Day 30, the recovery of presumptive Btk was significantly reduced (Figure
41C). The cause was an inadequate removal of particulates from the membrane, as observed in Figure
40. Even though particulate removal from the filter membrane for the 30-day timepoint was poor, all
high Btk T1B2 spore load samples were confirmed positive by colony PCR from 0.1-mL spread plates,
and all low Btk T1B2 spore load samples were confirmed positive by PCR analysis of the BHIB broth.
Figure 42 shows images of culture plates from Day 90 and Day 180. Background microorganisms did
not interfere with identification of presumpti ve Btk when 0.01 mL was spread plated for high Btk T1B2
spore load surfaces, leading to confirmation of Btk T1B2 by colony PCR from initial culture plates. The
background microorganisms did complicate the confirmation of Btk T1B2 from 1-mL membrane
plating. For this condition, two of three colonies that were PCR-screened from the initial culture plates
were PCR negative. However, all three samples were confirmed positive by PCR of the BHIB culture
for the low Btk T1B2 spore load surfaces sampled at 90 days. At 180 days of environmental storage,
more background microorganisms were present, although when 0.1 mL was spread plated for high
Btk T1B2 spore load surfaces, colonies were confirmed positive for all three replicates from initial
culture plates. For low Btk spore load surfaces, milliliter volumes were plated onto membranes and
background growth and soil obscured the detection of presumptive Btk colonies. However, all three low
Btk T1B2 spore load samples were confirmed positive by PCR of the BHIB culture at 180 days.
59
-------
Figure 41. Culture Images of Day 1 and Day 30 Spore Recovery from Gravel Clean Plated on TSA.
Soil on gravel interferes with colony morphology identification when 2-mL aliquot is plated: (A) Day 1, 0.1 mL
from high Btk T1B2 spore load surface; (B) Day 1, 2 mL from low Btk T1B2 spore load surface; (C) Day 30,
0.1 mL from low Btk T1B2 spore load surface.
60
-------
Figure 42. Culture Images of Day 90 and Day 180 Spore Recovery from Gravel Clean Plated on TSA.
Soil on gravel interferes with colony morphology identification when 1- or 2-mL aliquot is plated: (A) Day 90,
0.1 mL from high Btk T1B2 spore load surface; (B) Day 1, 1 mL from low Btk T1B2 spore load surface
(circled colony was presumptive Btk positive but PCR negative); (C) Day 180, 0.1 mL from high Btk T1B2
spore load surface; (D) 1 mL (low Btk T1B2 spore load sample); (E) 2 mL (low Btk T1B2 spore load sample).
The bilge water spiked with Btk T1B2 spores changed color from the original purple/pink to a tan while
stored in the outdoor environment. The color of the unspiked control bilge water did not change under
the same storage conditions. Photographs of the spiked and unspiked bilge water are shown in Figure 43.
Figure 43. Photograph of Bilge Water Unspiked (A, B) and Spiked High (C and E) and Low (D and F) with
Btk T1B2 Spores After 30 Days of Outdoor Ambient Exposure.
61
-------
For bilge water samples, the images of culture plates in Figure 44 show that initially (Day 1),
background microorganisms did not interfere with identification of presumptive Btk morphology on
0.1-mL spread plates. Thus, Btk T1B2 was easily observed and confirmed by PCR analysis. However,
when three colonies from 2-mL aliquots of the low Bik T1B2 spore load sample were PCR-screened,
two of three were PCR negative. The problem of presumpti ve Btk coloni es being PCR negative persisted
beyond Day 1 but at a lower rate, with 2 of 9 presumptive Btk colonies that were screened between
Days 30 and 180 PCR negative from 2-mL or 8-rnL membrane plates. Figure 45 shows images of
culture plates from Day 90 and Day 180. Of the colonies screened using PCR from low Btk T1B2 spore
load bilge water, two of three colonies from 90 days and three of three colonies from 180 days were
confirmed positive. Background microorganisms did not interfere with identification of presumptive Btk
when 0.1 mL was spread plated for high Btk T1B2 spore load samples, leading to confirmation of Btk
T1B2 by colony PCR from initial culture plates through Day 180. The level of presumptive Btk was
stable throughout 180 days of environmental storage.
Figure 44. Culture Images of Day 1 and Day 30 Spore Recovery from Bilge Water Plated on TSA.
Background microorganisms did not interfere when < 1 mL was plated. When 2 mL or 8 mL was plated, some
presumptive Btk colonies screened were PCR negative: (A) Day 1, 0.1 mL from high Btk T1B2 spore load
carboy (PCR positive); (B) Day 1, 2 mL from low Btk T1B2 spore load carboy (PCR negative); (C) Day 1, 8 mL
from field blank carboy (PCR negative); (D) Day 30, 0.1 mL from high Btk T1B2 spore load carboy
(PCR positive); (E) 2 mL from low Btk T1B2 load carboy (PCR positive); (F) 8 mL field blank carboy
(PCR negative).
62
-------
Figure 45. Culture Images of Day 90 and Day 180 Spore Recovery from Bilge Water Plated on TSA.
(A) Day 90, 0.1 mL from high Btk T1B2 spore load carboy; (B) Day 90, 1 mL from high Btk T1B2 spore load
carboy; (C) Day 90, 8 mL from low Btk T1B2 spore load carboy; (D) Day 180, 0.1 mL from high Btk T1B2
spore load carboy; (E) 1 mL from high Btk T1B2 spore load carboy; (F) 8 mL field blank carboy.
3.3.2 Btk T1B2 Confirmation
Presumptive Btk T1B2 colonies were observed for all gravel and bilge water grab samples from Day 1
through Day 180, with the lone exception of Day 30 for gravel with low Btk T1B2 spore load, due to
incomplete recovery as described in Section 3.3.1
The presence of Btk T1B2 was confirmed by PCR for low and high Btk T1B2 spore load gravel and
bilge water samples through 180 days of environmental exposure by colony PCR from the initial culture
plates, PCR of BHIB culture, or PCR of 50 uL of BHIB culture.
For gravel and bilge samples with low Btk T1B2 spore load, 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. At the low Btk T1B2 spore load level, 25% (2 of 8) presumptive colonies were PCR
positive for gravel and 67% (8 of 12) presumptive colonies were PCR positive for bilge water. The
confirmation of Btk T1B2 spores was assessed by colony PCR from the initial culture plates, PCR of
BHIB culture, or PCR of 50 j.iL of BHIB culture. Results from PCR confirmatory testing are shown in
Table 16.
63
-------
Table 16. Summary of the Accuracy of Identification of Presumptive Btk T1B2 by PCR Confirmation from
Grab Samples Stored in Outdoor Conditions Over Time.
Surface ID(a)
Sample
Type
Days of
Environmental
Exposure
Culture
Replicates
Presumptive
Positive"5'
Colonies from
Initial Culture
Plates PCR-
Screened(c)
Colonies
from BHIB
Streak Plates
PCR-
Screened(tl)
BHIB Broth
PCR-
Screened(e)
1
3 of 3 (100%)
1 of 3 (33%)
Not Tested
Not Tested
GRAVL-CL-LOW
Grab
30
0 of 3 (0%)
Not Tested
Not Tested
3 of 3 (100%)
90
3 of 3 (100%)
1 of 3 (33%)
Not Tested
3 of 3 (100%)
180
3 of 3 (100%)
0 of 2 (0%)
Not Tested
3 of 3 (100%)
1
3 of 3 (100%)
3 of 3 (100%)
Not Tested
Not Tested
GRAVL-CL-HIGH
Grab
30
3 of 3 (100%)
3 of 3 (100%)
Not Tested
Not Tested
90
3 of 3 (100%)
3 of 3 (100%)
Not Tested
Not Tested
180
3 of 3 (100%)
3 of 3 (100%)
Not Tested
Not Tested
1
3 of 3 (100%)
1 of 3 (33%)
Not Tested
Not Tested
BILGE-CL-LOW
Grab
30
3 of 3 (100%)
2 of 3 (66%)
2 of 2 (100%)
Not Tested
90
3 of 3 (100%)
2 of 3 (66%)
Not Tested
Not Tested
180
3 of 3 (100%)
3 of 3 (100%)
Not Tested
Not Tested
1
3 of 3 (100%)
3 of 3 (100%)
Not Tested
Not Tested
BILGE-CL-HIGH
Grab
30
3 of 3 (100%)
3 of 3 (100%)
Not Tested
Not Tested
90
3 of 3 (100%)
3 of 3 (100%)
Not Tested
Not Tested
180
3 of 3 (100%)
3 of 3 (100%)
Not Tested
Not Tested
(a) GRAVL-CL = Gravel Clean; BILGE-CL = Bilge Water, Clean.
(b)Presumptive Btk T1B2 was present on initial culture plates.
(c) Number of colonies PCR-screened from initial plating, with percent PCR positive.
(d) Number of colonies PCR-screened from BHIB streak plates, with percent PCR positive.
(e) Number of samples with PCR screening of BHIB enrichment broth, with percent PCR positive.
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 gravel surface samples and bilge water are presented in Table 17.
Sample replicates with a RV-PCR ACt value > 9 are RV-PCR positive, indicating that viable Btk T1B2
spores were recovered.
Gravel and bilge water samples were RV-PCR positive for low and high Btk T1B2 spore loads for all
replicates except for the gravel samples collected following 180 days of environmental exposure, two of
three replicates were positive for low and high Btk T1B2 spore loads.
The RV-PCR ACt results are plotted in Figure 46 (Low Btk T1B2 Spore Load) and Figure 47 (High
Btk T1B2 Spore Load). The plots all 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 to be a positive result.
64
-------
For gravel low Btk T1B2 spore load, RV-PCR ACt signal is level, starting at 16 and dropping slightly to
14 at 90 days, then reducing to just above the threshold (ACt of 9.9) at 180 days. For bilge water, the
ACt signal is begins at 21.8 and ends at 19.9 at 180 days for the low Btk T1B2 spore load.
The high Btk T1B2 spore load for gravel follows a trend similar to the low Btk T1B2 spore load, with
signal level through 90 days and then decreased by 180 days (ACt of 14.6). For bilge water, the ACt
signal begins at 21.9 and ends at 22.3 at 180 days for the high i?^TlB2 spore load.
Table 17. RV-PCR Analyses of Grab Samples for Detection of Btk T1B2 Spores Using T1B2 Barcode
Target.
Surface ID(a)
Spores Available for
Analysis (CFU)
Days of
Environmental
Exposure
ACt (X ± a)
RV-PCR
Replicates
Positive""
Nominal"5'
Determined'0'
1
16.1 ±5.4
3
GRAVL-CL-LOW
1.3 x 103
7.0 x102
30
14.4 ±0.5
3
90
13.9 ±0.2
3
180
9.9 ±1.2
2
1
21.7 ±1.8
3
GRAVL-CL-HIGH
1.3 x 105
1.1 x 105
30
24.7 ±1.1
3
90
19.7 ±3.1
3
180
14.6 ±6.3
2
1
21.8 ±3.0
3
BILGE-CL-LOW
5.0 x 101
1.5 x 102
30
19.1 ±2.7
3
90
17.8 ±4.2
3
180
19.9 ±1.4
3
1
21.9 ±2.2
3
BILGE-CL-HIGH
5.6 x103
2.0 x104
30
22.8 ±1.2
3
90
21.9 ±2.2
3
180
22.3 ±0.2
3
(a) GRAVL-CL = Gravel Clean; BILGE-CL = Bilge Water, Clean.
(b) Nominally one-half of the target Btk T1B2 spore load on the surface and assuming 100% recovery of spores.
(c) Based on the metal reference coupon enumeration measured with each spray application, 100% recovery efficiency, and
one-half of extract used for culture analysis.
(d) Number of replicates analyzed (N = 3) with an RV-PCR ACt value > 9.
65
-------
30
25
20
4-i
u
<3
<
10
I GRB-GRAVL-CL-Low
I GRB-BILGE-CL-Low
Threshold
1 30 90 180
Duration of Environmental Exposure (Days)
Figure 46. RV-PCR Analysis of Btk T1B2 Spores Recovered from Surfaces with a Low Target Load of
3,200 Btk Spores and Sampled Using a Grab Method.
Average ± One Standard Deviation for N = 3 Replicates. GRAVL-CL: Gravel Clean; BILGE-CL: Bilge Water,
Clean; Positive Response Equals ACt>9.
30
25
20
+j
u
<1
<
10
5
0
Duration of Environmental Exposure (Days)
30
90
I GRB-GRAVL-CL-High
I GRB-BILGE-CL-High
¦ Threshold
180
Figure 47. RV-PCR Analysis of Btk T1B2 Spores Recovered from Surfaces with a High Target Load of
320,000 Btk Spores and Sampled Using a Grab Method.
Average ± One Standard Deviation for N = 3 Replicates. GRAVL-CL: Gravel Clean; BILGE-CL: Bilge Water,
Clean; Positive Response Equals ACt>9.
66
-------
3.3.4 Analytical Method Comparison
Culture analysis performed slightly better than RV-PCR analysis for gravel samples because all
replicates were confirmed PCR positive through 180 days for culture, compared to two of three RV-PCR
samples positive at 180 days. For bilge water, both culture and RV-PCR were positive for all replicates
throughout the 180 days of environmental exposure.
Presumptive culture identification, culture identification confirmed by PCR, and RV-PCR results are
shown in Table 18 for each day of analysis. Table 19 compares culture identification confirmed by PCR
and RV-PCR results, because presumptive results are not used to confirm the presence of Btk T1B2
spores.
Table 18. Analytical Method Comparison Displaying Culture Presumptive, Culture ID with PCR
Confirmation and RV-PCR Replicates Detected (N = 3) for Gravel and Bilge Water Samples.
Surface ID(a)
Analytical Method
Replicates Detected
Day 1
Day 30
Day 90
Day 180
Presumptive Culture ID
3
0
3
3
GRAVL-CL-LOW
Culture ID with PCR Confirmation
1
3
3
3
RV-PCR
3
3
3
2
Presumptive Culture ID
3
3
3
3
GRAVL-CL-HIGH
Culture ID with PCR Confirmation
3
3
3
3
RV-PCR
3
3
3
2
Presumptive Culture ID
3
3
3
3
BILGE-CL-LOW
Culture ID with PCR Confirmation
1
3
2
3
RV-PCR
3
3
3
3
Presumptive Culture ID
3
3
3
3
BILGE-CL-HIGH
Culture ID with PCR Confirmation
3
3
3
3
RV-PCR
3
3
3
3
(a) GRAVL-CL = Gravel Clean; BILGE-CL = Bilge Water, Clean.
Table 19. Analytical Method Comparison Displaying Culture ID with PCR Confirmation and RV-PCR
Replicates Detected (N = 3) for Gravel and Bilge Water Samples.
Surface ID(a)
Analytical Method
Last Day of Analytical Method
Detection (# Replicates Confirmed)
GRAVL-CL-LOW
Culture w/PCR Confirmation
Day 180 (3)
RV-PCR
Day 180 (2)
GRAVL-CL-HIGH
Culture w/PCR Confirmation
Day 180 (3)
RV-PCR
Day 180 (2)
BILGE-CL-LOW
Culture w/PCR Confirmation
Day 180 (3)
RV-PCR
Day 180 (3)
BILGE-CL-LOW
Culture w/PCR Confirmation
Day 180 (3)
RV-PCR
Day 180 (3)
(a) GRAVL-CL = Gravel Clean; BILGE-CL = Bilge Water, Clean.
67
-------
3.3.5 Analysis of Controls (Bilge Water)
All reagent, laboratory blank, and field blank samples were negative for RV-PCR. These samples were
all bilge water samples. Laboratory blank bilge water was stored at 2 to 8°C for the duration of the
study, and field blank bilge water was stored under outdoor conditions. Of the eight laboratory blank and
field blank samples analyzed, four had low level (0.6 CFU/mL) presumptive Btk colonies present.
However, colony PCR analysis of those colonies was negative. One colony with a background
morphology was screened from a laboratory blank sample and generated an average Ct value of 39.5,
and a colony isolated from the BHIB culture streak plate also generated an average Ct value of 39.3,
indicating that a background organism may have been present that cross-reacted at low levels with the
T1B2 real-time PCR assay. A colony was isolated from BHIB culture from another field blank that did
not have presumptive Btk colonies present that was subsequently analyzed by PCR and generated an
average Ct value of 39.2.
For positive control bilge water samples, sterile water was directly spiked with 2.5 x 102 or 2.6 x 104
CFU Btk T1B2 spores and was positive for culture and RV-PCR when processed alongside bilge water
samples. The average percent recovery for the 2.5 x 102 CFU positive control samples was 65 ± 23% and
had a RV-PCR ACt value for of 20.9 ± 3.6. The average percent recovery for the 2.5 x 104 CFU positive
control samples was 132 ± 44% and had a RV-PCR ACt value for of 23.6 ± 2.5.
3.3.6 Analysis of Controls (Gravel)
All reagent, laboratory blank, and field blank samples were negative for RV-PCR. One sample had
presumptive Btk present on culture plates, but the single colony screened was PCR negative.
Gravel that was directly spiked with 2.6 x 103 or 2.6 x 105 CFU Btk T1B2 spores was positive for culture
and RV-PCR when processed alongside outdoor samples. The average percent recovery for the 2.6 x 103
CFU positive control samples was 68 ± 68 % and had a RV-PCR ACt value of 19 ± 0.7. The average
percent recovery for the 2.6 x 105 CFU positive control samples was 120 ± 39% and had a RV-PCR ACt
value for of 21.5 ± 2.2.
3.4 Traditional Sampling Method Performance (Spore Recovery) Ranking
Using the spore percent recovery values for Day 1 sampling of surfaces with a high Btk T1B2 spore load
of 1,000 CFU/cm2 for surfaces and 100 CFU/mL for bilge water as the condition for comparison, the
rank order for traditional sampling methods from best to worst is shown in Table 20. The recovery
efficiencies were very comparable for the grab and sponge stick sampling methods, but the VFC spore
percent recoveries were at least two orders of magnitude lower when applied to the nonporous solid
surfaces. The VFC recovery efficiencies were so low that VFC sampling of such surfaces is not
recommended.
68
-------
Table 20. Percent Recovery Values for Day 1 Traditional Surfaces Inoculated with a High Load of Btk
T1B2 Spores.
Sample
Surface
Surface
Spore Recovery T1 (Day 1)
Ranking
Type
Treatment
Actual (%)
Avg (%)
STDEV
Sponge
Stick
42.7
Glass
Salt
39.8
41.4
1.2
1
41.7
Sponge
Stick
Marine Grade
37.7
Aluminum
Salt
34.6
38.6
3.6
2
(MGAL)
43.3
Sponge
Stick
Marine Grade
42.2
Aluminum
Clean
37.9
36.6
5.2
3
(MGAL)
29.6
0.1
Grab
Gravel
Clean
40.6
26.0
18.4
4
37.4
24.7
Grab
Bilge Water
Clean
28.7
25.1
2.8
5
22.0
Vacuum
0.04
Filter
Nonskid (NSKID)
Salt
0.03
0.03
0.00
6
Cassette
0.04
Vacuum
0.02
Filter
Nonskid (NSKID)
Clean
0.01
0.01
0.00
7
Cassette
0.01
Note: High load target was 1,000 CFU/cnr litk T1B2 spores applied to surfaces and 100 CFU/mL spores for bilge water.
3.5 Nontraditional Sample Analysis Results
None of the 94 nontraditional samples clogged the 47-mm membrane filter used for sample
concentration. Therefore, 250 mL was processed for each sample. If nontraditional surfaces had been
exposed to outdoor environmental conditions for up to 180 days, filter clogging may have been an issue
as observed in bilge water samples and to a lesser degree, the gravel samples. Additionally, the surfaces
had relatively low levels of background microorganisms, which is known to compete with enrichment of
Btk T1B2 organisms, compared to traditional surfaces that accumulated background organisms while
being stored outdoors. If the surfaces of the nontraditional samples had been exposed to the environment
for up to 180 days, it is possible that high microorganism background would have been sampled.
Therefore, the nontraditional samples could have the same limitations of process as the traditional
samples.
3.5.1 Nontraditional Sample Culture Analyses
A summary of the average and standard deviation of the measured recovery efficiencies of presumptive
Btk T1B2 spores recovered from surfaces that were sprayed with Btk T1B2 spores, then sampled using
nontraditional washdown methods is presented in Table 21. The nominal quantity represents one-half
the target Btk T1B2 spore load applied to the surfaces and the determined number of spores available
represents one-half the number of presumptive Btk T1B2 spore recovered from metal reference coupon
enumeration during the day of the spray application (see Section 2.4.3.1).
69
-------
The quantity of presumptive Btk colonies recovered as determined by culture analysis using TSA plates
is plotted in Figure 48 (Low Btk T1B2 Spore Load) and Figure 49 (High Btk T1B2 Spore Load).
Surfaces with low Btk T1B2 spore load of 19,000 CFU applied and high Btk T1B2 spore load of
1,900,000 CFU applied were easily observed on spread plates and membrane plating in the absence of
background microorganisms and excess dirt and grime since all these surfaces were clean and not
exposed to outdoor environmental conditions. Nontraditional methods involved washing down surfaces
commonly found on boats, 50% marine grade aluminum and 50% nonskid (MSKID), with either
simulated tap water or simulated sea water that had a pretreatment of seawater spray or no seawater
spray (clean). For these MSKID surfaces with pretreatment of seawater spray, the simulated seawater
washdown had better percent recoveries at 8.38% ± 2.32% (LowBtk T1B2 Spore Load) and 7.62% ±
2.09% (High Btk T1B2 Spore Load) compared to tap water washdown 4.64% ± 1.44% (Low Btk T1B2
Spore Load) and 2.98% ± 0.68% (High Btk T1B2 Spore Load). For clean MSKID surfaces, the seawater
washdown percentage was better for low Btk T1B2 spore load surfaces, 4.62% ± 1.19% compared to
2.82% ± 0.26%, not for high Btk T1B2 spore load surfaces 5.35% ± 1.69% and 6.58% ± 0.66%.
Water runoff from roofing shingles was evaluated using simulated seawater, tap water, or simulated
rainwater. Precent recoveries were lowest for rainwater runoff with 1.37% ± 1.10% (Low Btk T1B2
Spore Load) and 0.43 ± 0.26 (High Btk T1B2 Spore Load), with <1% recovery following a second
rainwater rinse of the same surfaces; followed by simulated seawater runoff 1.04 ± 0.21 (Low Btk T1B2
Spore Load) and 1.20 ± 0.19 (High Btk T1B2 Spore Load); and simulated tap water had the best percent
recoveries of 2.77 ± 1.57 (Low i?^TlB2 Spore Load) and 2.44 ± 1.25 (High i?^TlB2 Spore Load).
Bristle brush scrubbing with a tap water washdown of a salt spray MSKID surface had the best overall
recovery at 22.2% ± 1.5% (Low Btk T1B2 Spore Load) and 18.2% ± 2.5% (High Btk T1B2 Spore
Load). Analysis of the brush rinse water recovered <1%.
Squeegee wiping with a tap water washdown of a salt spray marine grade aluminum (MGAL) surface
had the second-best overall recovery at 14.4% ± 1.7% (Low Btk T1B2 Spore Load) and 19.2% ± 4.0%
(High Btk T1B2 Spore Load).
A physical wiping of the surface with a bristle brush or squeegee wipe improves Btk T1B2 spore
recovery and would be reasonable to implement in the field. For marine grade aluminum with 50%
cover of nonskid material, incorporation of the bristle brush scrub improved recovery (18.2% recovery
compared to < 8%) and washdown of marine grade aluminum with a squeegee wipe resulted in the
highest recovery for nontraditional sampling methods (19.2%).
70
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Table 21. Recovery Efficiencies for Presumptive Btk T1B2 Spores from Nontraditional Washdown Samples Cultured on TSA Medium (N = 3).
Sample Type
(Spore Load)
Surface
Surface
T reatment
Washdown
Water Type
Spores Available for Analysis
(CFU)
Spore Recovery
(CFU)
(X ± CT)l:l
Spore
Recovery
Efficiency (%)
(X ± CT)ldl
Nominal1"
Determined"31
Squeegee
(LOW)
Marine Grade Aluminum
(MGAL)
Salt Spray
Tap Water
1.9 x 104
1.8 x 104
2.5 x 103± 3.6 x 102
14.4 ± 1.7
Squeegee
(HIGH)
Marine Grade Aluminum
(MGAL)
Salt Spray
Tap Water
1.9 x 10s
1.5 x 10s
2.9 x 105± 7.3 x 104
19.2 ±4.0
Bristle Brush
(LOW)
50% Marine Grade
Aluminum/50% Nonskid
(MSKID)
Salt Spray
Tap Water
1.9 x 104
1.8 x 104
3.9 x 103± 3.1 x 102
22.2 ± 1.5
Bristle Brush Rinse(e)
(LOW)
50% Marine Grade
Aluminum/50% Nonskid
(MSKID)
Salt Spray
Tap Water
1.9 x 104
1.8 x 104
5.6 x 101 ± 1.9 x 101
0.32 ± 0.09
Bristle Brush
(HIGH)
50% Marine Grade
Aluminum/50% Nonskid
(MSKID)
Salt Spray
Tap Water
1.9 x 10s
1.8 x 10s
3.3 x 105± 5.4 x 104
18.2 ±2.5
Bristle Brush Rinse(e)
(HIGH)
50% Marine Grade
Aluminum/50% Nonskid
(MSKID)
Salt Spray
Tap Water
1.9 x 10s
1.8 x 10s
5.8 x 103± 6.8 x 103
0.32 ± 0.31
Vessel Washdown
(LOW)
50% Marine Grade
Aluminum/50% Nonskid
(MSKID)
Salt Spray
Seawater
1.9 x 104
1.2 x 104
1.0 x 103± 3.5 x 102
8.38 ±2.32
Vessel Washdown
(HIGH)
50% Marine Grade
Aluminum/50% Nonskid
(MSKID)
Salt Spray
Seawater
1.9 x 10s
1.4 x 10s
1.1 x 105± 3.6x 104
7.62 ±2.09
Vessel Washdown
(LOW)
50% Marine Grade
Aluminum/50% Nonskid
(MSKID)
Salt Spray
Tap Water
1.9 x 104
5.6 x 104
2.6 x 103± 9.8 x 102
4.64 ± 1.44
Vessel Washdown
(HIGH)
50% Marine Grade
Aluminum/50% Nonskid
(MSKID)
Salt Spray
Tap Water
1.9 x 10s
2.4 x 10®
7.2 x 104 ± 2.0 x 104
2.98 ± 0.68
Vessel Washdown
(LOW)
50% Marine Grade
Aluminum/50% Nonskid
(MSKID)
Clean
Seawater
1.9 x 104
1.5 x 104
7.0 x 102 ± 2.2 x 102
4.62 ± 1.19
Vessel Washdown
(HIGH)
50% Marine Grade
Aluminum/50% Nonskid
(MSKID)
Clean
Seawater
1.9 x 10s
1.6 x 10s
8.8 x 104± 3.4 x 104
5.35 ± 1.69
Vessel Washdown
(LOW)
50% Marine Grade
Aluminum/50% Nonskid
(MSKID)
Clean
Tap Water
1.9 x 104
4.1 x 104
1.2 x 103± 1.3 x 102
2.82 ± 0.26
Vessel Washdown
(HIGH)
50% Marine Grade
Aluminum/50% Nonskid
(MSKID)
Clean
Tap Water
1.9 x 10s
2.2 x 10®
1.5 x 105± 1.8 x 102
6.58 ± 0.66
71
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Table 21. Recovery Efficiencies for Presumptive Btk T1B2 Spores from Nontraditional Washdown Samples Cultured on TSA Medium (N = 3).
(Cont.)
Sample Type
(Spore Load)
Surface
Surface
T reatment
Washdown
Water Type
Spores Available for Analysis
(CFU)
Spore Recovery
(CFU)
(X ± CT)ICI
Spore
Recovery
Efficiency (%)
(X ± CT)ldl
Nominal'"
Determined"51
Water Runoff
(LOW)
Shingles (SHING)
Clean
Seawater
1.9 x 104
3.2 x 104
3.3 x 102± 8.0 x 101
1.04 ± 0.21
Water Runoff
(HIGH)
Shingles (SHING)
Clean
Seawater
1.9 x 10s
2.0 x 10®
2.5 x 104 ± 4.7 x 103
1.20 ± 0.19
Water Runoff
(LOW)
Shingles (SHING)
Clean
Tap Water
1.9 x 104
3.0 x 104
8.2 x 102± 5.7 x 102
2.77 ± 1.57
Water Runoff
(HIGH)
Shingles (SHING)
Clean
Tap Water
1.9 x 10s
1.9 x 10s
4.5 x 104 ± 2.9 x 104
2.44 ± 1.25
Water Runoff
(LOW)
Shingles (SHING)
Clean
Rainwater
1.9 x 104
1.5 x 104
2.0 x 102 ± 2.1 x 102
1.37 ± 1.34
Water Runoff; 2nd
Rinse®
(LOW)
Shingles (SHING)
Clean
Rainwater
1.9 x 104
1.5 x 104
5.8 x 101
0.40
Water Runoff
(HIGH)
Shingles (SHING)
Clean
Rainwater
1.9 x 10s
1.2 x 10s
5.3 x 103± 4.2 x 103
0.43 ± 0.26
Water Runoff; 2nd
Rinse®
(HIGH)
Shingles (SHING)
Clean
Rainwater
1.9 x 10s
1.2 x 10s
2.2 x 103
0.20
(a) Nominally one-half of the target I ilk T1B2 spore load on the surface and assuming 100% recovery of spores.
(b) Based on the metal reference coupon enumeration measured with each spray application, 100% recovery efficiency, 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 on each surface and total presumptive Btk T1B2 spores recovered from each sample.
(e) Bristle brush was rinsed with tap water after being used to brush the MSKID surface. Rinsed to assess whether spores were retained on the brush.
(f) Second rainwater application to shingles immediately after the first rainwater application to collect the primary sample analyzed.
72
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Using the spore recovery efficiency results for the high spore load condition as a basis for comparison,
the rank order for nontraditional sampling method from best to worst is below and detailed in Table 22.
Table 22. Percent Recovery Values for Nontraditional Surfaces Inoculated with a High Load Target of Btk
T1B2 Spores.
Sample Type
Surface
Surface
Treatment
Washdown Water
Type
Spore Recovery Efficiency
(%)
Ranking
Actual
Avg
STDEV
Marine Grade
18.2
Squeegee
Aluminum
Salt Spray
Tap Water
24.6
19.2
4.0
1
(MGAL)
14.9
50% Marine
18.5
Bristle Brush
Grade
Salt Spray
Tap Water
15.0
18.2
2.5
2
Wash down
Aluminum/50%
21.0
Nonskid (MSKID)
50% Marine
10.3
Vessel
Grade Aluminum
Salt Spray
Seawater
7.4
7.6
2.1
3
Wash down
(MGAL)/50%
Nonskid (NSKID)
5.2
50% Marine
7.3
Vessel
Grade
Clean
Tap Water
6.6
6.6
0.7
4
Wash down
Aluminum/50%
5.7
Nonskid (MSKID)
50% Marine
7.7
Vessel
Grade Aluminum/
Clean
Seawater
4.0
5.4
1.7
5
Wash down
50% Nonskid
4.3
(MSKID)
50% Marine
2.6
Vessel
Grade Aluminum/
Salt Spray
Tap Water
3.9
3.0
0.7
6
Wash down
50% Nonskid
2.4
(MSKID)
4.2
Water runoff
Shingles
Clean
Tap Water
1.9
2.4
1.3
7
1.3
1.0
Water runoff
Shingles
Clean
Seawater
1.1
1.2
0.2
8
1.4
0.2
Water Runoff
Shingles
Clean
Rainwater
0.3
0.4
0.3
9
0.8
50% Marine
0.1
Bristle Brush
Grade
Salt Spray
Tap Water
0.8
0.3
0.3
10
Rinse
Aluminum/50%
0.1
Nonskid (MSKID)
Water Runoff,
2nd Rinse
Shingles
Clean
Rainwater
0.2
0.2
N/A
11
Note: High load target was 1,000 CFU/cm2 Btk T1B2 spores applied to surfaces.
73
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2 6
U
3
tLO
2 5
a>
>
o 4
u ^
a>
OC
O 3
Q.
i/l
a>
>
s. 2
E
~
l/i
2 1
a.
[=]SW-MSKID-CL
l=ISW-SHNG-CL
) SW-MSKID-SA
~ TWBB-MSKID-SA
UTW-SHNG-CL
DTWBBR-MSKID-SA
IIW-MSKID-CL
I RW-SHMG-CL
• Initial Loading
ITW-MSKID-SA
ITWSQ-MGAL-SA
-Target Loading
Vessel Washdown Water Water Runoff Squeegee
Non-Traditional Sampling Method
Bristle Brush
Figure 48. Presumptive Btk T1B2 Spores Recovered from Surfaces with a Low Target Load of 3,200 Btk
Spores and Sampled Using Various Nontraditional Sampling Methods.
Average ± One Standard Deviation of N = 3 Replicates Sample ID Key: AABBB-CCCC-DD, where AA =
washdown water type; BBB = brush or squeegee scrub (if used); CCCCC = Surface Type; DD = Surface
pretreatment (Clean or Salt Sprayed).
3
LL
u
S
o
o
Cd
0>
u.
o
Q.
cn
a
>
Q.
E
~
W A
J? 1
~ SW-MSKIO-CL 1 I SW-MSKID-SA > 1 TW-MSKin-ri
~ SW-SHNG-CL I ITW-SHNG-CL > ' RW-SHNG-CL
3 TWBB-MSKID-SA I 1TWBBR-MSKID-SA Initial loading
TW-MSKID-SA
1 TWSQ-MGAl-SA
— —Target Loading
Vessel Washdown Water Water Runoff Squeegee
Non-Traditional Sampling Method
Bristle Brush
Figure 49. Presumptive Btk T1B2 Spores Recovered from Surfaces with a High Target Load of 320,000 Btk
Spores and Sampled Using Various Nontraditional Sampling Methods.
Average ± One Standard Deviation of N = 3 Replicates Sample ID Key: AABBB-CCCC-DD, where AA =
washdown water type; BBB = brush or squeegee scrub (if used); CCCCC = Surface Type; DD = Surface
pretreatment (Clean or Salt Sprayed).
74
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3.5.2 Btk T1B2 Confirmation
For nontraditional washdown methods, background microorganisms were low, and there was not a
reduction in Btk T1B2 over time. Samples were collected following a dry time but stability over time
was not determined. Therefore, colonies were observed and counted on spread plates in most cases, not
membrane plates with mL volumes. As a result, 73 of 74 (99%) of the presumptive colonies screened
from the initial culture plates and 4 of 4 (100%) of the colonies isolated from BHIB enrichment streak
plates were confirmed positive by PCR (Table 23). The only presumptive Btk T1B2 colony that was
PCR negative was isolated from a method blank sample.
Table 23. Summary of the Accuracy of Identification of Presumptive Btk T1B2 Colonies by PCR
Confirmation from Nontraditional Collection Methods.
PCR Input
Number (%) of Presumptive Btk T1B2 Confirmed Positive by PCR
Presumptive Btk T1B2 from
Initial Plates
73 of 74 (99%)
Presumptive Btk T1B2 from
BHIB Streak Plates
4 of 4 (100%)
3.5.3 Nontraditional Sample RV-PCR Analyses
A summary of the average and sample standard deviation of the RV-PCR ACt values for the detection of
Btk T1B2 spores recovered from nontraditional samples are presented in Table 24. Sample replicates
with a 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 50 (Low Btk T1B2 Spore Load) and Figure 51 (High
Btk T1B2 Spore Load). The plots all 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 to be a positive result. All samples analyzed were RV-PCR positive.
75
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Table 24. RV-PCR Analyses of Nontraditional Washdown Samples for Detection of Btk T1B2 Spores Using T1B2 Barcode Target (N = 3
Replicates).
Sample Type
Surface
Surface
Washdown
Spores Available for Analysis
(CFU)
ACt
RV-PCR
Replicates
Positive'0'
(Spore Load)
Treatment
Water Type
Nomina l(a)
Determined"5'
(X±o)
Squeegee
(LOW)
Marine Grade
Aluminum (MGAL)
Salt Spray
Tap Water
1.9 x104
1.8 x104
21.9 ± 1.7
3
Squeegee
(HIGH)
Marine Grade
Aluminum (MGAL)
Salt Spray
Tap Water
1.9x10®
1.5x10®
23.3 ±0.7
3
Bristle Brush
(LOW)
50% Marine Grade
Aluminum/50%
Nonskid (MSKID)
Salt Spray
Tap Water
1.9 x104
1.8 x104
23.8 ±0.1
3
Bristle Brush
Rinsed (LOW)
50% Marine Grade
Aluminum/50%
Nonskid (MSKID)
Salt Spray
Tap Water
1.9 x104
1.8 x104
24.0 ±0.2
3
Bristle Brush
(HIGH)
50% Marine Grade
Aluminum/50%
Nonskid (MSKID)
Salt Spray
Tap Water
1.9x10®
1.8x10®
22.2 ± 0.2
3
Bristle Brush
Rinse(d)
(HIGH)
50% Marine Grade
Aluminum/50%
Nonskid (MSKID)
Salt Spray
Tap Water
1.9x10®
1.8x10®
24.2 ± 0.2
3
Vessel Washdown
(LOW)
50% Marine Grade
Aluminum/50%
Nonskid (MSKID)
Salt Spray
Seawater
1.9 x104
1.2 x104
21.5 ± 1.0
3
Vessel Washdown
(HIGH)
50% Marine Grade
Aluminum/50%
Nonskid (MSKID)
Salt Spray
Seawater
1.9x10®
1.4x10®
21.4 ± 1.9
3
Vessel Washdown
(LOW)
50% Marine Grade
Aluminum/50%
Nonskid (MSKID)
Salt Spray
Tap Water
1.9 x104
5.6 x104
23.3 ± 1.1
3
Vessel Washdown
(HIGH)
50% Marine Grade
Aluminum/50%
Nonskid (MSKID)
Salt Spray
Tap Water
1.9x10®
2.4x10®
21.8 ±2.4
3
Vessel Washdown
(LOW)
50% Marine Grade
Aluminum/50%
Nonskid (MSKID)
Clean
Seawater
1.9 x104
1.5 x104
25.9 ±0.1
3
Vessel Washdown
(HIGH)
50% Marine Grade
Aluminum/50%
Nonskid (MSKID)
Clean
Seawater
1.9x10®
1.6x10®
25.2 ±0.8
3
76
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Table 24. RV-PCR Analyses of Nontraditional Washdown Samples for Detection of Btk T1B2 Spores Using T1B2 Barcode Target (N = 3
Replicates). (Cont.)
Sample Type
(Spore Load)
Surface
Surface
Treatment
Washdown
Water Type
Spores Available for Analysis
(CFU)
ACt
(X±o)
RV-PCR
Replicates
Positive'0'
Nomina l 9.
(d)Bristle brush was rinsed with tap water after being used to brush the MSKID surface. Rinsed to assess whether spores were retained on the brush.
(e)Second rainwater application to shingles immediately after the first rainwater application to collect the primary sample analyzed.
77
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35
30
25
u
«a
& 20
< 15
10
DSW-MSKIO-CL
ZtSW-SHNG-Cl
~ TW8B-MSKID-SA
~ SW-MSKID-SA ¦
DTW-SHWG-CL IB
~ TWBBR-MSKID-SA -
=*TW-MSKID-CL
j RW-SHNG-Cl
Threshold
ITW-MSKID-SA
I TWSQ-MGAl-SA
Vessel Washdown Water Water Runoff Squeegee Bristle Brush
Nontraditional Sampling Method
Figure 50. RV-PCR Analysis of Btk T1B2 Spores Recovered from Surfaces with a Low Target Load of
3,200 Btk Spores and Sampled Using Various Nontraditional Sampling Methods.
Average ± One Standard Deviation for N = 3 Replicates. ID Key: AABBB-CCCC-DD, where AA = wash down
water type; BBB = brush or squeegee scrub (if used); CCCCC = Surface Type; DD = Surface pretreatment
(Clean or Salt Sprayed). Positive Response Equals ACt> 9.
35
30
25
y 20
a>
M
n
| 15
<
E=JSW-MSKID-CL
] SW-MSKID-SA i i Tuij-Mtuin-ri
I I RW-SHNG-Cl
1 1 twb r-msk I rt-SA I ITWB6R-MSKID-SA Threshold
I !T«i. 9.
78
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3.5.4 Analytical Method Comparison
All samples that were sprayed with target Btk T1B2 spores were positive for both culture and RV-PCR
analyses. The nontraditional sample surfaces were sprayed with the same target Btk T1B2 spore loads as
surfaces sampled using traditional methods (10 CFU/cm2 and 1,000 CFU/cm2); however, the surface
area sampled was greater for the nontraditional collection methods, thus 1 x 104to 1 x 106 CFU were
available for collection, and relatively low background growth and grime were present on these surfaces.
A higher ratio of Btk T1B2 spores to background microorganisms and limited grime allows for easier
identification of Btk morphology for culture analysis and quantification. Likewise, the higher ratio of
Btk T1B2 spores to background microorganisms and limited grime allows for a more favorable
condition for the target organism to grow exponentially in BHIB during the enrichment step for RV-
PCR.
3.5.5 Analysis of Controls
Reagent and laboratory blank controls for nontraditional samples were negative for culture and RV-
PCR. Method blank samples, which were handled within the washdown laboratory, had some
contamination from being near the washdown procedure.
Two (2) method blank samples from the squeegee nontraditional sampling method with 0.3 and
0.4 CFU/mL recovered from the culture aliquot with corresponding ACt values of 19.5 and 24.2.
Three method blank samples from the marine grade aluminum with nonskid coating were also positive,
two were positive for both culture and RV-PCR with 0.3 CFU/mL spores and corresponding RV-PCR
ACt values of 13.9, 9.5 and 15.0. The ACt values for these samples were not as high as the others
because the To samples had an average ACt of 36.0, decreasing the ACt.
The method blank samples from shingles contained 1.9 CFU/mL of presumptive Btk However, the two
colonies screened were PCR negative. The shingles surfaces were not sterile, so it is possible that
background organisms with presumptive Btk morphology were isolated.
For nontraditional spikes, water (tap, simulated sea water, simulated rainwater) directly spiked with
3.7 x 104 or 3.7 x 106 CFU target Btk T1B2 spore load were positive for culture and RV-PCR when
processed alongside washdown samples. The average percent recovery for the 3.7 x 104 CFU positive
control samples was 56 ± 16 % and had a RV-PCR ACt value for of 24.6 ± 0.9. The average percent
recovery for the 3.7 x 106 CFU positive control samples was 40 ± 20% and had a RV-PCR ACt value for
of 25.0 ± 1.8.
3.6 Summary of Detection Accuracy of Presumptive Colonies
Storage of surfaces in outdoor conditions led to an accumulation of background microorganisms and
grime that had a negative impact on identification of presumptive Btk T1B2 colonies. At Day 1 and
Day 30, presumptive Btk colonies isolated from initial plating of the spore recovery on TSA were
confirmed positive by PCR at a rate of 81% and 82%, respectively. For Day 90 and Day 180, this rate of
confirmation dropped to 48% and 52%, respectively. Not only were there fewer presumptive Btk T1B2
colonies being collected from surfaces at 90 and 180 days, but more of the presumptive colonies were
PCR negative, meaning that an environmental microorganism that was indistinguishable from Btk T1B2,
perhaps wild-type/naturally occurring Btk, was being deposited onto the surfaces. Table 25 summarizes
the total number of colonies screened by PCR from the initial plate as well as those isolated from BHIB
enrichment streak plates for surfaces that were sampled using traditional methods.
79
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For nontraditional washdown methods, background microorganisms were low and the Btk T1B2 spore
load was high, negating the impact of background microorganisms on identification of presumptive
Btk T1B2 colonies, as demonstrated with 73 of 74 (99%) of the presumptive colonies screened from the
initial culture plates and 4 of 4 (100%) of the colonies isolated from BHIB enrichment streak plates
being confirmed positive by PCR.
Table 25. Summary of the Accuracy of Identification of Presumptive Btk T1B2 Colonies by PCR
Confirmation from Surfaces Stored in Outdoor Conditions Over Time.
PCR Input
Number (%) of Presumptive Btk T1B2 Confirmed Positive by PCR
Day 1
Day 30
Day 90
Day 180
Presumptive Btk T1B2 from
Initial Plates
29 of 36 (81%)
31 of 38 (82%)
22 of 46 (48%)
15 of 29 (52%)
Presumptive Btk T1B2 from
BHIB Streak Plates
14 of 15 (93%)
4 of 10 (40%)
0 of 0 (0%)
0 of 0 (0%)
80
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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 were 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 PCR. In addition, quantification of the Btk spray and spike suspensions were performed
each day of spraying surfaces and spiking control samples to verify CFU/mL titer concentrations.
Positive and negative control results were within the target requirements for the PCR. 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, 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 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 26 summarizes the PE audits that were performed; equipment was within acceptable
tolerance range.
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Table 26. 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
4.4.2 Technical Systems Audit
Observations and findings from the technical system audit were documented and submitted to the
laboratory technical lead for response. The technical system audit was conducted on January 13, 2021,
to ensure that tests were being conducted in accordance with the appropriate QAPP and QMP. As part of
the audit, test procedures were compared to those specified in the QAPP and Wis, and data acquisition
and handling procedures were reviewed. All procedures were according to documentation and no
findings were noted.
4.4.3 Data Quality Audit
At least 10% of data acquired during the evaluation were audited. Data were reviewed from February 9
through April 8, 2021. A QA auditor traced the data from the initial acquisition, biologic plate counts,
PCR delta CT calculation, data reduction and statistical analysis, to final reporting to ensure the integrity
of the 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 technical system audit or in the data quality audit, and no follow-up
corrective action was necessary. QA/QC procedures were performed in accordance with the QAPP.
There was one QAPP deviation: uncontaminated surfaces were not included in the 180-day test of
traditional sampling methods.
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.
82
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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 of the method.
5.1 Sample Processing Considerations
Sponge sticks, VFCs, wash water, and grab samples each have unique extraction 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 necessaiy 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.
For gravel ballast sample processing, the 500-mL bottles (Daigger, Buffalo Grove, IL Item # EF2247C)
specified in the extraction protocol for gravel ballast (Serre and Oudejans, 2017) are made of high
density polyethylene (HOPE) and are not autoclavable. The bottles become misshapen when autoclaved
at 121°C gravity cycle for 15 min, Figure 52 shows rounding of the bottom of a bottle post-autoclaving
that could lead to sample spills during processing. These bottles should be sterilized by irradiation or a
replacement product that can be autoclaved should be considered.
1
Figure 52. Autoclaving HDPE Bottles Compromise Structure.
5.2 Method Qualitative Assessment
Given experiences running analytical methods, there are pros and cons for both methods, discussed in
the sections below.
83
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5.2.1 Culture Method
The strengths of the culture method are that it allows for a quantifiable measure of target organisms,
isolation of target organism, and confirmatory PCR screening of colonies that gives a definitive
confirmatory result. Weaknesses of the culture method are that background microorganisms can be
present that overwhelm culture plates and obscure colony morphology that could lead to false negative
results. Additionally, background microorganisms with a similar or identical morphology can be present
within samples, requiring PCR screening of colonies and possibly repeated PCR screening (to minimize
risk of false positives) if presumptive morphology is present in large numbers. Such background bacteria
can have serious consequences if the traditional culture method is used without PCR screening,
especially for analysis of complex and dirty environmental samples.
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 iterative or repeated PCR screening. The method can give rapid results which is
of high significance in a wide-area incident involving multiple cities. It can have a small laboratory
footprint, and less culture media is required compared to the culture method. The weakness of the RV-
PCR method are that it does not allow for quantification of target organisms, no isolation of target
organism for banking, 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
unless multiplex assays are available and validated, and the presence of background microorganisms
competes with target organism growth during enrichment, suppressing signal and potentially leading to
false negative results.
5.2.3 Time/Cost Estimates
The sample analyses were performed in 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. If these had been actual samples
collected post-biological release, the spiking activity would, obviously, not be performed by the ERLN.
Day 2 consisted of culture plate colony counting and presumptive colony selection and nucleic acid
extraction for RV-PCR. Day 3 for 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 BHIB culture were performed on Day 4 and Day 5 for the culture method. As described in
Section 2.5, if incubation time for RV-PCR were reduced to 9 h as per the EPA Protocol (EPA 2017)
and a night shift performs nucleic acid extraction, results could be completed in the next day after spore
recovery. For culture analysis, BHIB enrichment is described as optional to expedite results, allowing
culture results to be completed the next day after spore recovery. Text for this option is quoted below
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' and 10~2 dilutions) be filtered using an
additional MicroFunnel™ and plated as described above, instead ofproceeding with
enrichment."
84
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Estimated staff time to process 16 samples was approximately 64 h of labor and $1,500 of consumables.
The 64 h of staff time budget was approximately distributed by:
8 h for activities related specifically to the spiking of the filters being assessed, which was a
requirement of this study, but not an activity that would be performed had these been actual field
samples. This task included time to prepare the spore spiking suspensions, enumerate spore
suspension, spike the samples, and generate the associated documentation.
• 10 h for spore recovery.
10 h for culture analysis.
• 24 h for RV-PCR analysis.
Additionally, 4 h was needed for PCR confirmation analysis of samples suing the culture method,
when performed.
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 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
the analytical methods can generate results within 2 days for analysis of the recovered spore suspension.
However, for the culture method, additional time would be required for BHIB enrichment culture and
PCR confirmation (as per the CDC-LRN protocol). The labor required for nucleic acid extraction for
16 samples is -14 h. Exploring options to reduce this labor or using an automated extraction procedure
would increase throughput and reduce sample cost.
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
target organism. The method defines that a minimum of three presumptive colonies from spread plates
or 1 to 3 colonies from MicroFunnel filter plates (for this project, Btk T1B2) be screened to confirm the
presence of 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 "U.S. EPA Protocol for Detection of Bacillus anthracis in Environmental Samples
During the Remediation Phase of an Anthrax Incident, Second Edition" (EPA, 2017) currently 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 a 50-|iL aliquot of the BHIB culture is performed only if colony with target
morphology is not observed on streak plates. In previous evaluation of BHIB enrichment culture, data
showed that the target was not isolated from sponge sticks and VFCs when streaked from turbid
enrichment broth for nonfield blank samples due to the high number of competing background
organisms present in those samples. However, target was present in enrichment broth for many VFC
samples, as determined by PCR analysis of an aliquot of broth (EPA/600/R-19/083, June 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
culture following a positive PCR identification.
85
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BHIB enrichment of sponge stick samples is not as effective as enrichment of 47-mm filter membrane
samples. A couple possibilities for this are because the 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 the sponge.
5.4 RV-PCR Processing Considerations
The RV-PCR method requires great care and diligence to implement effectively. Most notable, the
method requires changing gloves between samples for each step, which is onerous and time-consuming,
is critical when analyzing samples from the field collected after an incident because the samples and
associated results are high-value and high-impact - the results will support key decisions in the response
and thus impact response timelines, credibility, and cost. During the RV-PCR method, when applying
vacuum to the filter vial manifold, the filtrate pooled in the manifold reservoir and contacted 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.
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.
RV-PCR manifold, capping tray, and manifold incubator racks 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 is 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 biocontainment box with absorbent is
recommended for incubation and platform vortex mixing to avoid select agent release during these steps.
Considerations for proper containment and effective implementation of containment are expected to be
effectively implemented with properly trained and experienced staff.
5.4.2 Suggestions to Improve RV-PCR Throughput
5.4.2.1 RV-PCR To Baseline
Nucleic acid extraction and analysis of the To samples may not be necessary for all samples. To aliquots
with spore loads of 2.9 x 105 CFU or more result in minimal Ct values, as seen in the two nontraditional
surfaces with the highest target spore load. The two surfaces with the highest Btk T1B2 recovery were:
1) marine grade aluminum/nonskid salted surface that was washed down with tap water and scrubbed
86
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with a bristle brush, and 2) marine grade aluminum washdown with tap water and squeegee wipe, each
having 5.5 logio CFU (3.3 x 105 CFU and 2.9 x 105 CFU) available per analytical method. The RV-PCR
To Ct values averaged 43.2, 43.5, and 43.4 for the bristle brush method and 43.2, 45.0, and 45.0 for the
squeegee method, indicating that up to 3.3 x 105 spores loaded into the RV-PCR filter vial generates a
PCR signal of > 43. Minimal PCR Ct value signal from these samples may be due to spores binding to
the filter vial membrane or ineffective spore lysis during DNA extraction procedure. Table 27 shows the
washdown samples with the highest Btk T1B2 spore recovery in the To aliquot and Ct values generated.
These results may indicate that To analysis only needs to be included if a 7/aliquot is positive, to
exclude the possibility that target signal was from nonviable target (free DNA and DNA coated on
nonviable spores). Another consideration is that the method for agitating filter vials could be simplified.
The current method for agitating filter vials involves a platform vortex step and mixing by pipetting up
and down manually 10 times. Elimination of the platform vortex step should be considered because the
filter vial/capping tray does not fit within the platform vortex (VWR, Radnor, PA VX-2500 Cat. 58816-
115) without overhanging which has led to secondary bag tearing. The platform vortex step following
overnight incubation of the filter vials before removal of the Tfinal aliquot should also be considered for
removal and rely on mixing by pipetting up and down alone.
Table 27. Washdown Samples with Highest Btk T1B2 Spore Recovery Available in To Aliquot and
Resulting Ct Values.
Sample ID
Spores
Recovered
(CFU)
Ct
Average Ct
Ct STDEV
Marine Grade Aluminum/Nonskid
(Salted) with Bristle Brush and
Washdown To, Replicate 1
3.3 x105
45.00
43.22
3.09
45.00
39.65
Marine Grade Aluminum/Nonskid
(Salted) with Bristle Brush and
Washdown To, Replicate 2
45.00
43.49
2.61
45.00
40.47
Marine Grade Aluminum/Nonskid
(Salted) with Bristle Brush &
Washdown To, Replicate 3
45.00
43.44
2.70
45.00
40.33
Marine Grade Aluminum (Salted)
with Squeegee and Washdown
To, Replicate 1
2.9 x105
45.00
43.21
3.09
39.64
45.00
Marine Grade Aluminum (Salted)
with Squeegee and Washdown
To, Replicate 2
45.00
45.00
0.00
45.00
45.00
Marine Grade Aluminum (Salted)
with Squeegee and Washdown
To, Replicate 3
45.00
45.00
0.00
45.00
45.00
5.4.2.2 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 50 |iL of the BHIB enriched filter vial using
thermolysis instead of nucleic acid extraction procedure may produce results similar to the To and 7/
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aliquots that were extracted using the nucleic acid extraction procedure, with less labor, fewer
consumables and less biohazardous waste generated. The RV-PCR process of washing filter vials with
10X and IX PBS may reduce PCR inhibitors to a level that reduces the risk of a false negative sample.
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,
including:
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 drippage.
5.5.2 Sponge Stick Method Considerations
The sponge stick method uses 90 mL of buffer to extract a sponge, and the next step following
stomaching is to reduce the volume by centrifugation. Efficiency could potentially be gained by
reducing the volume used for stomaching.
5.6 Vacuum Filter Cassette Sample Analysis
Recovery efficiencies are low for this extraction 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 efficiency 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 dampened by racks or distributed
nonuniformly. A previous program assessing sonication in the recovery of spores from soil samples did
not improve spore recovery (Silvestri, et al. 2016).
5.7 Washdown and Grab Sample Analysis
The approach used for this project for analysis of washdown or grab samples was to concentrate the
collected suspension (250 mL) onto a 47-mm mixed ester cellulose filter membrane (0.45 |im), then
extract 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 efficiency 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.
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6.0 CONCLUSIONS AND
RECOMMENDATIONS
Btk spores with T1B2 barcode were successfully applied, via controlled spray deposition, to various
ubiquitous maritime surfaces (e.g., marine grade aluminum, glass, nonskid tread, roof shingles; both
clean and sea salt spray-contaminated) associated with a USCG base. Traditional (established EPA
sampling methods: e.g., sponge stick wipes, VFCs, and grab samples) were employed to recover Btk
spores from the surfaces. Composite samples collected using nontraditional sampling methods
(e.g., collection of water used in washdown of a vessel or runoff from a shingle roof) were also collected
in simulated laboratory conditions. All samples were analyzed using established EPA culture and
molecular methods.
Although a direct comparison cannot be made with other research because of differences in outdoor
environmental exposure conditions and surface/sampling differences, the results for the traditional
sampling of solid surfaces using sponge sticks and VFCs were generally consistent with the results from
another EPA study (Mikelonis, et. al., 2021). In the Mikelonis study, initial (within lday) spore
recoveries of solid surfaces using sponge stick wipes were as high as 56% for a smooth stainless surface
to 1.8% for a rough asphalt surface. Recoveries were < 0.01% after 37 days and no detectable spores
were recovered in samples collected after 210 days of exposure. Spores persisted longer in the study
reported here compared to the Mikelonis research, most likely due to the surfaces in the study reported
here not being directly exposed to sunlight and precipitation like the surfaces in the Mikelonis study.
The order of magnitude recovery of spores using VFCs was comparably low (generally <1 %) for both
studies. The quantity of spores recovered in the Mikelonis study decreased with exposure duration,
while they were fairly steady for the study reported here, most likely attributed to greater physical losses
occurring in the Mikelonis study.
The foremost conclusion was that the detection and/or quantification of T1B2 barcoded Btk via either
the culture or molecular method and sampled using traditional EPA sampling methods was very
dependent on the duration the surface experienced outdoor ambient conditions - the longer the outdoor
exposure duration, the greater the background (interfering) microorganisms or inert contamination and
potentially physical loss, resulting in a decrease in the ability to identify (detect) and quantify the
presence of T1B2 barcoded Btk. In most instances, the ability of the EPA methods to detect and quantify
began to be adversely affected between the 30-day and 90-day sample collections. By the end of
180 days of outdoor exposure, previously detectable and quantifiable amounts of Btk could not be
measured. Additionally,:
The presence of deposited sea salt spray residue on the surface did not hinder the EPA methods.
The VFC did not effectively collect Btk spores from the nonskid tread and this method is not
recommended as a preferred sampling method.
The effects of the environmental exposure measured in this study are expected to be the least severe
as the surfaces were not exposed to direct sunlight, precipitation, and wind.
• Btk T1B2 could be detected within the bilge water with no loss in detection or quantification by
culture or RV-PCR over the 180 days of outdoor environment exposure.
• Btk T1B2 could be detected within gravel samples throughout the 180 days of outdoor
environmental exposure by culture and RV-PCR analysis for all sample replicates except for one
replicate each at the low and high Btk T1B2 spore load for RV-PCR, indicating that the gravel
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recovery method is a good candidate for detection of target spores following prolonged
environmental exposure. All three 180-day environmental exposure replicates with low Btk T1B2
spore load level were confirmed positive by enrichment of the 47-mm mixed ester cellulose filter,
with Ct values of 36.3, 36.0, and 37.0. If a cycle threshold of 36 were set for BHIB enrichment of
the filter, only one replicate would be confirmed positive at 180 days. For comparative purposes,
the ACt values generated from the RV-PCR aliquot of these samples were 8.4, 10.2, and 11.2.
• Neither culture nor RV-PCR consistently had more confirmed positive sample replicates at a longer
environmental exposure durations as there was variability in the results from the two methods.
Similarly, neither method consistently demonstrated more confirmed positive sample replicates at
the same environmental durations.
An RV-PCR analysis method improvement consideration is to assess whether current DNA
extraction and purification procedures could be replaced with automated nucleic acid extraction.
For a batch of 16 samples, the DNA extraction procedure takes approximately 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.
A culture analysis method improvement suggestion is to consider evaluation of BHIB enrichment
culture by PCR analysis prior to streaking triplicate agar plates to isolate the target organism.
Previous data (EPA/600/R-19/083, June 2019) showed that target was not isolated when streaked
from turbid broth with background organisms present, while PCR analysis of 50 |iL of the BHIB
culture was PCR positive.
An RV-PCR analysis method improvement suggestion is to consider processing samples without
the use of custom manifold, custom capping tray and filter vials. Concentrating samples by
centrifugation or other filter membrane may provide more flexibility in supply chain and allow for
safer processing within BSL-3 laboratories by incorporating incubation and mixing steps in
standard sealed tubes.
The nontraditional sampling methods that are associated with typical vessel maintenance (e.g., vessel
water washdown, scrubbing with a wet bristle brush, or wiping excess water with a squeegee) or
collecting rainwater runoff from a shingle roof are all plausible sampling collection methods that can
yield a composite sample over a relatively larger surface area than sampling using a traditional active
sampling method. Comparing the 1-day sponge stick traditional sampling methods with the squeegee or
bristle brush nontraditional sampling methods, the squeegee and bristle brush sampling methods yielded
comparable (within a factor of 2.2) spore recoveries for sponge stick (-19% compared to 41%). All
nontraditional sampling methods generated a sample that was positive when analyzed with RV-PCR. It
is therefore recommended that nontraditional sampling methods be considered as viable sample
collection methods in a sampling plan:
The water type (freshwater versus seawater) did not appear to impact spore recovery or the
analytical method.
Addition of physical wiping of surfaces with a bristle brush scrub or squeegee wipe improves target
spore recovery. For marine grade aluminum with 50% cover of nonskid material, incorporation of
the bristle brush scrub improved recovery (18.2% recovery compared to < 8%) and washdown of
marine grade aluminum with a squeegee wipe resulted in the highest recovery for nontraditional
sampling methods (19.2%).
The usefulness of collecting rainwater runoff from a roof should be assessed considering the
collection efficiency was relatively low (< 3 %). Collecting rainwater runoff from a roof may only
provide a worthwhile sample if the initial water runoff can be collected before the source is
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depleted and the sample diluted. If rainwater runoff is to be further considered, it is recommended
that the spore recovery be assessed over prolonged rain events to better understand and support the
collection of initial runoff collection.
These results, along with outcomes from numerous other AnCOR projects, are useful in advancing the
state of the science and building capabilities for remediation and recovery at a USCG base in the event
of a bioterrorism incident. Additionally, the results from this study will be used in planning a field-scale
demonstration of response and recovery capabilities following a simulated large outdoor biological
aerial release.
The conclusions are based on the results obtained for the Btk T1B2 spore loading applied in this study
and may be different if different spore loadings were used. For example, if the spore loadings were
orders of magnitude higher, then it is possible that the adverse effect of accumulating interferents or any
loss of spores from the surfaces may not have been detectable over the 180-day outdoor ambient
exposure. This study purposely sought to apply relatively low Btk T1B2 spore loadings to better
understand the limits of the methods as applied to representative USCG surfaces.
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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.
Calfee, Michael, S. Shah, S. Lee, Ronald Mickelsen, F. Cruz, K. Karim, J. Ackelsberg, M. Gemelli, and
K. Hofacre. 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.
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.
Centers for Disease Control and Prevention, Surface Sampling Procedures for Bacillus anthracis Spores
from Smooth, Non-Porous Surfaces (2012), Available at:
https://www.cdc.gov/niosh/topics/anthrax/sampling.html Accessed 9/13/21.
Greenberg, D.L., Busch, J.D., Keim, P., Wagner, D.M. Identifying Experimental Surrogates for Bacillus
anthracis Spores: A Review. (2010) Investigative Genetics 1(1):4. doi: 10.1186/2041-2223-1-4.
Mikelonis, A.M., Calfee, M.W., Lee, S.D., Touati, A., and Ratliff, K. (2021) Rainfall Washoff of Spores
From Concrete and Asphalt Surfaces. Water Resources Research 57(3): e2020WR028533.
Schoettle, A. and Hubbard, R. A Rain Simulator for Greenhouse Use (1992). USDA Forest Service.
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.
U.S. Environmental Protection Agency, Standard Operating Procedure for Preparation of Hard Water
and Other Diluents for Preparation of Antimicrobial Products (2019). SOP Number: MB-30-02.
U.S. Environmental Protection Agency Protocol for Detection of Bacillus anthracis in Environmental
Samples During the Remediation Phase of an Anthrax Incident, 2017, Second Edition. U.S.
Environmental Protection Agency, Washington, DC, EPA/600/R-17/213 | July 2017.
https://cfpub.epa.gov/si/si public record report.cfm?Lab=NHSRC&dirEntryId=338673. Accessed
9/6/21.
U.S. Environmental Protection Agency (2013). Bio-Response Operational Testing and Evaluation
Project - Phase 1: Decontamination Assessment. EPA/600/R-13/168 Washington, D.C., U.S.
Environmental Protection Agency.
U.S. Environmental Protection Agency, Protocol for Detection of Bacillus anthracis in Environmental
Samples During the Remediation Phase of an Anthrax Event (2012). U.S. Environmental Protection
Agency. Washington, D.C. EPA/600/R-12/577.
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Evaluation and Testing of Outdoor Sample
Collection and Analysis Methods
Appendices A-P
-------
List of Appendices
APPENDIX A: SEA SALT COMPOSITION REVIEW A-1
APPENDIX B: SPRAY TABLE DESCRIPTION AND CHARACTERIZATION B-1
APPENDIX C: VESSEL WATER WASHDOWN NONTRADITIONAL SAMPLING
METHOD WORK INSTRUCTION C-1
APPENDIX D: SIMULATED RAINWATER FORMULATION D-1
APPENDIX E: SIMULATED RAINWATER NONTRADITIONAL SAMPLING METHOD
WORK INSTRUCTION E-1
APPENDIX F: SQUEEGEE NONTRADITIONAL SAMPLING METHOD WORK
INSTRUCTION F-1
APPENDIX G: BRISTLE BRUSH NONTRADITIONAL SAMPLING METHOD WORK
INSTRUCTION G-1
APPENDIX H: FORMULATIONS OF RECIPES USED IN BIOLOGICAL TEST
METHODS H-1
APPENDIX I: WORK INSTRUCTION FOR SPIKING WITH
BTKHD-1 T1B2 SPORES 1-1
APPENDIX J: WORK INSTRUCTION FOR BACILLUS THURINGIENSIS KURSTAKI
T1B2 SPORE RECOVERY FROM OUTDOOR SAMPLES J-1
APPENDIX K: WORK INSTRUCTION FOR CULTURE OF BACILLUS
THURINGIENSIS KURSTAKI T1B2 SPORES RECOVERED FROM OUTDOOR
SURFACES K-1
APPENDIX L: WORK INSTRUCTION FOR MANUAL DNA EXTRACTION AND
PURIFICATION FROM BACILLUS THURINGIENSIS KURSTAKI T1B2 SPORES L-1
APPENDIX M: WORK INSTRUCTION FOR REAL-TIME PCR ANALYSIS FOR
BACILLUS THURINGIENSIS KURSTAKI T1B2 DNA M-1
APPENDIX N: WORK INSTRUCTION FOR SELECTING PRESUMPTIVE BACILLUS
THURINGIENSIS KURSTAKI T1B2 COLONIES FOR QPCR CONFIRMATION N-1
APPENDIX 0: WORK INSTRUCTION FOR ENRICHMENT FOR CULTURE 0-1
APPENDIX P: OUTDOOR TEMPERATURE AND RH PLOTS P-1
NOTE: Work Instructions are templates that were completed for each test, at the time of testing.
These templates are included here. Completed work instruction templates for each test are not
included.
-------
Appendix A
A-1
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Salt Aerosol Deposition Levels in a Marine Environment
(Kent Hofacre and Aaron Richardson, 12 Feb 2020)
Salt aerosols are produced during the breaking of waves and ocean white caps. Aerosols produced in
coastal regions due to breaking of waves tend to produce larger salt particles than those produced in the
ocean. Salt aerosols in the atmosphere exist as liquid droplets and solid particles and are transported to
surfaces by wind and gravity. The aerosol concentration is a function of many parameters including wind
speed and distance inland from the shore. The objective of this review was to identify data regarding salt
surface deposition to provide a basis for the loading to be used in testing to evaluate sample collection
methods from surfaces in a marine environment.
A literature search was completed using the ScienceDirect database and Google searches to identify
sources of data. Key search terms included chloride, salt, marine, steel, surface, and deposition. There is a
wealth of references related quantifying or modeling the effects of salt deposition on metal or concrete in
a marine environment. Studies of corrosion rates tended to measure weight loss of a sample over time. No
studies were identified that directly measured the deposition loading on a surface by swabbing or
otherwise collecting a sample for analysis. Rather, the wet candle method is a common reference method
used to quantify chloride deposition (ISO 9225, 2012; ASTM G140-02, 2019).
The wet candle method includes a wetted gauze wrapped around a cylinder that passively collects the salt
aerosol. The collected salt is extracted, and the mass of chloride collected is determined by chemical
analysis. The mass of chloride deposited is normalized based on the mass collected divided by the
sampler surface area and the collection duration to provide the flux (mg/m2/day). The ASTM method
recommends a 30-day exposure. Deposition will vary depending on orientation of the surface, but the wet
candle method has been used to classify the severity of exposure in a variety of marine locations (ASTM
G140-02, 2019).
A summary of measured chloride deposition values from the literature are summarized in Table 1. The
measured deposition is a strong function of inland distance. This review focused on measured deposition
at or near the coast. The data represent a sampling of measurements made from a variety of locations
around the world and meteorological conditions. To represent worst case, the table primarily summarizes
the highest reported values which is typically less than 800 mg/m2/day. The exception is Alcantara et al.
(2015) who note that the measured salinity is higher than that found normally in the literature.
Table 1. Summary of Chloride Deposition Values from Literature
Reference
Location
Method
Chloride Deposition
(mg/m2/day)
Morcillo et al. (2000)
Spain
Wet Candle
7.3 to 114.5
Corvo et al. (1995)
Cuba
Dry Plate
Up to 525
Mustafa and Yusof (1994)(a)
Malaysia
Wet Candle
Up to 400
Meira et al. (2006)
Brazil
Wet Candle
Up to 750
Cook et al. (1998)(b)
Mexico
Not reported
170
Cook et al. (1998)(b)
U.S.
Not reported
311
Alcantara et al. (2015)
Spain
Wet Candle
393 to 1,905
(a) As reported in Meira et al. (2006).
(b) As reported in Guedes Sores et al. (2009).
Data was also requested from Battelle's Florida Materials Research Facility (FMRF), an outdoor facility
for subtropical exposure studies located along the ocean front in Ponce Inlet, FL. The site provides two
A-2
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acres of exposure area within 70 meters of the ocean and its location is rated among the most corrosive
environments in the United States (Battelle, 2019). ISO defines six corrosivity categories ranging from
CI (very low) to C5 (very high) and CX (extreme) (ISO 9223:2012). The FMRF is categorized as a C5.
Since 2000, chloride deposition has been measured on a monthly basis using the wet candle method (ISO
9225). The chloride deposition by month over the period from 2000 to 2016 is shown in Figure 1. The
blue points represent measured monthly values. Monthly deposition values ranged from <10 to about
1,500 mg/m2/day. The red bars indicate the median value for each month. Salt aerosol deposition varied
by month presumably due to the differences in the wind conditions. The highest mean deposition and
mean wind speeds (data not shown) were both observed in October. Conversely, the lowest mean
deposition and mean wind speed was in July. An increase in salinity with increased wind speed is
consistent with the literature (Meira et aL 2007; Meira et al, 2008; Morcillo et al., 2000). The red, blue,
and green lines in Figure 1 represent the 50th, 75th, and 90th percentile measurements from the entire data
set. The 50th, 75th, and 90th percentile depositions were 107, 202, and 439 mg/nr/dav, respectively.
Figure 1. Chloride Deposition Data Collected between 2000 and 2016 from the Florida Materials
Research Facility
As discussed above, the amount of salt deposited on a surface that may be encountered when sampling
could van by orders of magnitude, depending on factors of location, season, and time for salt to
accumulate. The salt loading level to target in the EPA Outdoor sampling study of USCG-relevant
surfaces is recommended based on Battelie's FMRF data set as this represents continuous measurements
on a monthly basis for over 15 years at a location considered one of the most corrosive environments in
the U.S. The studies identified in the literature are typically shorter studies, generally less than a year.
Specifically, the salt loading level to apply to USCG-relevant surfaces is recommended to be based on a
A-3
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chloride deposition of 200 mg/m2/day, equal to the 75th percentile of observation at the FMRF. This level
is also above or near 11 of the 12 monthly median values and is thus expected to represent a relatively
high salt loading flux. Using sodium chloride (NaCl) as the primary salt source of chloride ion measured,
this corresponds to a total salt deposition of 330 mg/m2/day. Using a 5-day exposure as a basis, the total
salt deposition would be 1,650 mg/m2 or 0.165 mg/cm2. Considering the spread and uncertainty of
deposited salt, a salt loading of 0.15±0.05 mg/cm2 will be targeted to apply to the surfaces before
application of Btk.
References
ASTM G 140-02(2019), "Standard test method for determining atmospheric chloride deposition rate by
wet candle method," ASTM International, 2019.
ISO 9223:2012, "Corrosion of metals and alloys - corrosivity of atmospheres - classification,
determination, and estimation," International Organization for Standardization, 2012.
ISO 9225:2012, "Corrosion of metals and alloys - corrosivity of atmospheres - measurement of
environmental parameters affecting corrosivity of atmospheres," International Organization for
Standardization, 2012.
Alcantara, J., B. Chico, I. Diaz, D. de la Fuentes, and M. Morcillo, "Airborne chloride deposit and its
effect on marine atmospheric corrosion of mild steel," Corrosion Science, Vol. 97, pp. 74-88, 2015.
Battelle, "Battelle Florida Materials Research Facility," Brochure, 2019.
Cook, D.C., A.C. Van Orden, J.J. Carpio, S.J. Oh, "Atmospheric corrosion in the Gulf of Mexico,"
Hyperfine Interactions, Vol. 113, pp. 319-329, 1998.
Corvo, F. N. Betancourt, and A. Mendoza, "The influence of airborne salinity on the atmospheric
corrosion of steel," Corrosion Science, Vol. 37, No. 12, pp. 1889-1901, 1995.
Guedes Sores. C., Y. Garbatov, A. Zayed, and G. Wang," Influence of environmental factors on corrosion
of ship structures in marine environments," Corrosion Science, Vol. 51, pp. 2014-2026, 2009.
Meira, G.R., C. Andrade, I.J. Padaratz, C. Alonso, and J.C. Borba, "Measurements and modelling of
marine salt transportation and deposition in a tropical region in Brazil," Atmospheric Environment, Vol.
40, pp. 5596-5607, 2006.
Meira, G.R., C. Andrade, I.J. Padaratz, C. Alonso, and J.C. Borba, "Salinity of marine aerosols in a
Brazilian coastal area - influence of wind regime," Atmospheric Environment, Vol. 41, pp. 8431-8441,
2007.
Meira, G.R., C. Andrade, C. Alonso, I.J. Padaratz, and J.C. Borba, "Modelling sea-salt transport and
deposition in marine atmosphere zone - a tool for corrosion studies," Corrosion Science, Vol. 50, pp.
2724-2731,2008.
Morcillo, M., B. Chico, L. Mariaca, and E. Otero, "Salinity in marine atmospheric corrosion: its
dependence on the wind regime existing in the site," Corrosion Science, Vol. 42, pp. 91-104, 2000.
Mustafa, M.A. and K.M. Yusof, "Atmospheric chloride penetration into concrete in semi-tropical marine
environment," Cement and Concrete Research, Vol. 24, pp. 661-670, 1994.
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Appendix B
B-1
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Work Instructions for Operation of the Test Surface Spray Table
1.0 Loading Sample
Note: Loading of sample solution should be done before placement of samples to avoid
contamination caused by potential drips or splashes. Consult Figure 2 for location of
valves on the spray trolley.
1.1 Ensure that nitrogen pressure to the reservoir has been shut off and that residual
pressure has been vented through the relief valve (VI).
1.2 Remove the top plug and ensure that the shut-off valve (V3) is closed.
1.3 Obtain a 60 mL syringe and a 3-4" piece of 1/8" ID TvgonR tubing. Attach the tubing
to the end of the syringe and begin filling with test solution.
1.4 Once full, feed the tubing into the reservoir until the end is ~2-3" inside.
1.5 Begin filling the reservoir. Refill the syringe and repeat the process until the reservoir
has been filled with -275 mL of solution or until a desired volume.
1.5.1 Note: The maximum fill volume of the reservoir is 300 mL; however, it is
recommended to use less than this volume to avoid dripping/spilling from
the reservoir.
1.6 Once full, replace the stainless-steel plug and retighten. Ensure all valves (VI, V2,
V3) are closed.
Figure 1: Image of the test surface spray table.
B-2
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Figure 2: Image of the spray trolley and valve location.
2.0 Spraying Test Surfaces
Note: Two personnel are required for the following portion of the procedure. The two
personnel shall be referred to as the operator and assistant. If instructions do not specify a
single person, then the action may be performed by either. Consult Figure 3 for layout the
layout of the control box.
2.1 Begin laying down test surfaces/sampling targets onto the spray table. Material type
and location will be determined by the test plan.
After laying out all test surfaces and targets, ensure that the spray trolley is located at
the start position (opposite end as the control box).
When ready to begin spraying, the operator will position themselves at the control
box of the spray table and the assistant at the opposite end of the table.
The assistant will pressurize the reservoir by opening valve V2 and check to ensure
that the system is reaching target pressure (as determined by the test plan). If
necessary, the source nitrogen regulator may be adjusted until the desired pressure is
observed.
Once pressurized, the operator will ensure that the control box is set to the proper
speed setting (as determined by the test plan), the RUN/BRAKE toggle is set to
BRAKE, and that FORWARD/REVERSE toggle is set to FORWARD. If necessary,
adjust the speed setting/toggle positions.
The operator will now power the unit by flipping up the POWER toggle. The front
green indicator light should turn on to confirm the unit is now being powered.
The operator will indicate to the assistant when he/she is ready to begin spraying.
2.2
2.3
2.4
2.5
2.6
2.7
B-3
-------
2.8 When instructed by the operator, the assistant will open the spray valve (V3),
initiating the spray. The assistant will verify that the spray fan looks consistent
before signaling the operator to traverse the trolley.
2.9 At this time, the operator will flip the RUN/BRAKE toggle to RUN and watch the
trolley traverse the test surfaces/targets.
2.10 As the trolley reaches the marked stop position, the operator will flip the
RUN/BRAKE Toggle back to BRAKE and immediately shut off the spray valve
(V3) on the reservoir.
2.11 Return the trolley to the start position and repeat steps 2.7 -2.10 if additional passes
are necessary. If solution volume in the reservoir becomes low, depressurize the
reservoir and reload with solution according to steps 1.1 - 1.6.
3.0 Shutdown
3.1 When ready to stop testing, power down the control until by switching off the
POWER toggle and return the trolley to the start position.
3.2 Empty the reservoir of any residual liquid by placing a bottle/high walled container
over the nozzle tip and opening the spray valve (V3).
3.3 Once empty, close the spray valve (V3) and depressurize the reservoir by closing the
nitrogen source (V2) venting residual pressure through the relief valve (VI).
B-4
-------
Appendix C
C-1
-------
Work Instruction for Pressurized Washdown of Test Surfaces
Note: Sample collection will be performed from levels of low analyte concentration to high
analyte concentration. For example, blank samples will be sampled first, followed by low
concentration samples, and finally high concentration samples. The test system will be
decontaminated and rinsed before returning to a low analyte concentration from a higher one as
well as at the end of each testing period.
Obtain a test surface and place it inside the sampling basin. Ensure that the surface is
contacting the support pins at the bottom of the basin and is centered between the side
walls.
If necessary, vent any residual pressure from the pressurized spray kit and close off the
overpressure nitrogen cylinder.
Uncap the spray kit reservoir and fill it with the appropriate washdown liquid (tap water
or saltwater). Recap the reservoir when finished.
o Note: The Rinse Kit reservoir can hold ~8 L. Reloading of liquid may be
necessary throughout the testing depending on number of samples and usage rate.
Attach the Dramm heavy-duty adjustable brass nozzle (Home Depot Model # 14033591)
to the outlet of the spray kit and open the nozzle to the desired setting.
o Note: The appropriate setting will be marked on the nozzle itself. This setting
corresponds to a nozzle opening which will achieve the desired flow rate of 3 - 5
L/min at 30 PSIG, and which will produce a spray cone with a diameter of 12" -
14" when positioned -12" above the test surface.
Ensure that the spray valve (on/off valve) on the spray kit is shut-off and attach the
overpressure line to the reservoir.
Set the nitrogen overpressure cylinder to 30 PSIG. When ready to pressurize the
reservoir, open the cylinder secondary valve.
Using a large (e.g. 4 L) graduated cylinder (or equivalent containment) and calibrated
stopwatch, ensure that the flowrate from the nozzle is within 3 to 5 L/min.
o To do this, position the nozzle above the graduated cylinder and open the spray
valve to initiate the spray. Simultaneously, start the stopwatch and allow the
nozzle to spray for a predetermined amount of time (15 seconds, 30 seconds, etc.).
Shut the nozzle off at the conclusion of the time and measure the volume of liquid
dispensed. The flow rate of the nozzle is the amount of liquid dispensed divided
by the spray time, in minutes.
If the nozzle is outside of the acceptable range, adjust the opening as necessary and
reattempt the measurement. Once within range, repeat the measurement two additional
times for three total measurements.
When ready to begin sample collection, place the collection beaker underneath the trough
outlet.
With the spray valve off, hold the nozzle -12" above (normal to) the test surface. At this
height, the nozzle should generate a cone that is -12" - 14" in diameter.
The nozzle will be kept at this height and swept across the test surface according to the
pattern shown in Figure 1. A total of 5 passes (labeled PI - P5 in the figure) will be
C-2
-------
performed at ~3 seconds per pass for a total of-15 seconds of spray. The passes will be
positioned so that the spray cone slightly overlaps the edges of the test surface by 2-3".
When ready to sample, turn on the nozzle and execute the spray event as shown in
Figure 1.
Spray Basin
(30" x 30")
Figure 1: Washdown method test surface spray pattern.
Runoff from the test surface will accumulate in the collection beaker placed at the trough
outlet. At conclusion of spray event, allow the surface an additional 15-20 seconds to
drain off any additional liquid.
Once drained, removed the test surface and set it aside in the staging area. Remove the
collection beaker and record the volume of runoff collected. Transfer the runoff to an
autoclaved, sealable vessel for storage.
After collection of the runoff, obtain a clean, dry, disposable cloth or wipe and remove
any standing water or droplets from the basin surface and collection trough. Dispose of
the cloth when finished.
C-3
-------
Figure 2: Example of surface washdown
C-4
-------
Appendix D
D-1
-------
Rainwater Formulation
Simulated rain formulation given in Table 2 of Schoettle and Hubbard, 1992 (see below).
The rationale for the recommendation follows:
1. Stormwater recipes (absent the metals) were considered, but the ion concentrations in the
simulated stormwater recipes were one to two orders of magnitude higher than that in the
Scheottle paper and/or per the National Atmospheric Deposition Program
(http://nadp.slh.wisc.edu/data/animaps.aspx).
2. The stormwater recipe didn't have prevalent ions of ammonium and sulfate found in rainwater
a. Scheottle paper formulation had both ions, among others.
b. The website http://nadp.slh.wisc.edu/data/animaps.aspx reports presence of
ammonium and sulfate.
c. Sigma Aldrich sells a simulated rainwater that also has ammonium and sulfate. Based on
European Union research. See sites: Simulated rain water ERM8 certified Reference
Material I Sigma-Aldrich and SIMULATED RAINWATER (major compounds) I LGC
Standards
i. Both contain the sulfate and ammonium ions.
3. There certainly is no one "right" formulation, and the formulation provided in the Scheottle
paper seems reasonable (and if anything has ion concentrations higher than in the NADP data).
Table 2. Average tonic composition of ambient rain from Rocky Mountain National Park. Colorado
for June through September (1984 and 1985). and the recipe for preparation of the rain solution
(simulant) of the same ionic composition.
AMBIENT RAIN SIMULATED RAIN
Ion Concentration Compound 1:1,000 concentrate
(mg/L) added (g/L)
Ca* 4
0 508
CaC03
0.619
CI*
0.23
CaClj 2H?0
0.724
Mg* *
0081
MgS04 7HaO
0822
so;
1.66
k2so4
0 183
K*
0.083
NaNQj
0.447
Na*
0.121
(NH4)2S04
1.064
nh;
029
h3po4
0.005
po;
trace
hno3*
139 ml
no3
1 68
H2S04b
266 ml
*1:100 dilution of concentrated HNO
b 1:1000 dilution of concentrated HJ>
c
D-2
-------
Appendix E
E-1
-------
Work Instruction for Rainwater Washdown of Test Surfaces
Note: Sample collection will be performed from levels of low analyte concentration to high
analyte concentration. For example, blank samples will be sampled first, followed by low
concentration samples, and finally high concentration samples. The test system will be
decontaminated and rinsed before returning to a low analyte concentration from a higher one as
well as at the end of each testing period.
Obtain a test surface and place it inside the sampling basin. Ensure that the surface is
contacting the support pins at the bottom of the basin and is centered inside of the acrylic
side shields.
o Note: The basin has side shields installed which are designed to divert droplets
which would not directly contact the test surface away from the collection area.
If necessary, vent any residual pressure from the pressurized spray kit and close off the
overpressure nitrogen cylinder.
Uncap the spray kit reservoir and fill it with the appropriate washdown liquid (rainwater).
Recap the reservoir when finished.
o Note: The Rinse Kit reservoir can hold ~8 L. Reloading of liquid may be
necessary throughout the testing depending on number of samples and usage rate.
Ensure that spray both has the correct nozzle installed (Spray Systems Model: GG-2.8W)
and shut off the spray valve (on/off valve).
Attach the overpressure line to the reservoir.
Set the nitrogen overpressure cylinder so that the gauge located directly next to the nozzle
reads 5 PSIG. When ready to pressurize the reservoir, open the cylinder secondary valve,
o Note: It may be necessary to let the system purge with water to establish the
correct nozzle pressure. If necessary, remove the test surface from the basin (to
avoid accidental wetting) and troubleshoot the nozzle pressure.
Using a large (e.g. 4 L) graduated cylinder (or equivalent containment) and calibrated
stopwatch, ensure that the flowrate from the nozzle is 1.0 to 1.3 L/min.
o To do this, position graduated cylinder below the nozzle and open the spray valve
to initiate the spray. Simultaneously, start the stopwatch and allow the nozzle to
spray for a predetermined amount of time (60 seconds, 120 seconds, etc.). Shut
the nozzle off at the conclusion of the time and measure the volume of liquid
dispensed. The flow rate of the nozzle is the amount of liquid dispensed divided
by the spray time, in minutes.
If the flowrate is outside of the acceptable range, check to ensure that the regulator on the
overpressure cylinder is set to the correct pressure and/or that there are no clogs in the
nozzle tip or tubing. If necessary, obtain a new nozzle and replace.
When ready to begin sample collection, place the collection beaker underneath the trough
outlet and ensure that the spray booth side shields are drawn shut.
Initiate the spray by opening the spray valve. Simultaneously, start a calibrated stopwatch
to track the duration of the spray event.
Ensure that the nozzle pressure is reading 5 PSIG and that the spray pattern appears even.
Allow the rain event to continue for 5 minutes, at which point shut off the spray valve.
E-2
-------
At conclusion of the spray, wait an additional 60 seconds to allow the test surface time to
drain.
Remove the test surface and store it in the staging area.
o Note: If performing a second spray event on the same surface, the sheet may be
left on the basin.
Remove the collection beaker and record the volume of runoff collected. Transfer the
runoff to an autoclaved, seal able vessel for storage.
After collection of the runoff, obtain a clean, dry, disposable cloth or wipe and remove
any standing water or droplets from the basin surface and collection trough. Dispose of
the cloth when finished.
Figure 1: Rainwater collection basin with test surface.
E-3
-------
Figure 2: Rain spray nozzle during a rain event.
E-4
-------
Appendix F
F-1
-------
Work Instruction for Squeegee Sampling of Test Surfaces
Note: Sample collection will be performed from levels of low analyte concentration to high
analyte concentration. For example, blank samples will be sampled first, followed by low
concentration samples, and finally high concentration samples. The test system will be
decontaminated and rinsed before returning to a low analyte concentration from a higher one as
well as at the end of each testing period.
Obtain a test surface and place it inside the sampling basin. Ensure that the surface is
contacting the support pins at the bottom of the basin and is centered between the side
walls.
If necessary, vent any residual pressure from the pressurized spray kit and close off the
overpressure nitrogen cylinder.
Uncap the spray kit reservoir and fill it with the appropriate washdown liquid (tap water
or saltwater). Recap the reservoir when finished.
o Note: The Rinse Kit reservoir can hold ~8 L. Reloading of liquid may be
necessary throughout the testing depending on number of samples and usage rate.
Attach the Dramm heavy-duty adjustable brass nozzle (Home Depot Model # 14033591)
to the outlet of the spray kit and open the nozzle to the desired setting.
o Note: The appropriate setting will be marked on the nozzle itself. This setting
corresponds to a nozzle opening which will achieve the desired flow rate of 3 - 5
L/min at 30 PSIG and which will produce a spray cone with a diameter of 12" -
14" when positioned -12" above the test surface.
Ensure that the spray valve (on/off valve) on the spray kit is shut-off and attach the
overpressure line to the reservoir.
Set the nitrogen overpressure cylinder to 30 PSIG. When ready to pressurize the
reservoir, open the cylinder secondary valve.
Using a large (e.g. 4 L) graduated cylinder (or equivalent containment) and calibrated
stopwatch, ensure that the flowrate from the nozzle is within 3 to 5 L/min.
o To do this, position the nozzle above the graduated cylinder and open the spray
valve to initiate the spray. Simultaneously, start the stopwatch and allow the
nozzle to spray for a predetermined amount of time (15 seconds, 30 seconds, etc.).
Shut the nozzle off at the conclusion of the time and measure the volume of liquid
dispensed. The flow rate of the nozzle is the amount of liquid dispensed divided
by the spray time, in minutes.
If the nozzle is outside of the acceptable range, adjust the opening as necessary and
reattempt the measurement. Once within range, repeat the measurement two additional
times for three total measurements.
When ready to begin sample collection, place the collection beaker underneath the trough
outlet.
With the spray valve off, hold the nozzle -12" above (normal to) the test surface. At this
height, the nozzle should generate a cone that is -12" - 14" in diameter.
F-2
-------
The nozzle will be kept at this height and swept across the test surface according to the
pattern shown in Figure 1. A total of 5 passes (labeled P I - P5 in the figure) will be
performed at ~3 seconds per pass for a total of-15 seconds of spray.
Immediately following the spray, a clean Shurhold 12" squeegee (West Marine Model #:
2673184) will be used to squeegee off the surface. The squeegee will be passed along the
surface 5 times in an overlapping pattern as shown in Figure 2.
o Note: A new squeegee will be used for each individual surface
Spray Width
(12" -14")
Spray Path
Water Flow
Collection/Drainage
Trough
Figure 1: Squeegee pre-rinse spray pattern
12" Squeegee Coverage
Pass#l
T
Pass #2
Pass #3
Pass #4
i
i
i
i
i
i
i
Pas
s#5
i
i
i
i
i
i
i
i
~
i
>
Sample Surface
3" to Basin Edge (24" x 24")
Sample Pass
lx Pass 2x Pass 3x Pass
Figure 2: Squeegee surface sampling pattern
F-3
-------
When ready to sample, turn on the nozzle and execute the spray event as shown in
Figure 1. Immediately following the spray event, obtain a squeegee and perform the
surface sampling as shown in Figure 2.
At conclusion of the sampling (squeegeeing). Remove the test surface and set it aside in
the staging area.
Use a quick 1-2 seconds pulse from the spray kit to clear out any stagnant droplets in
the trough into the collection beaker.
Remove the collection beaker and record the volume of runoff collected. Transfer the
runoff to an autoclaved, sealable vessel for storage.
After collection of the runoff, obtain a clean, dry, disposable cloth or wipe and remove
any standing water or droplets from the basin surface and collection trough. Dispose of
the cloth when finished.
F-4
-------
Appendix G
G-1
-------
Work Instruction for Bristle Brush Sampling of Test Surfaces
Note: Sample collection will be performed from levels of low analyte concentration to high
analyte concentration. For example, blank samples will be sampled first, followed by low
concentration samples, and finally high concentration samples. The test system will be
decontaminated and rinsed before returning to a low analyte concentration from a higher one as
well as at the end of each testing period.
Obtain a test surface and place it inside the sampling basin. Ensure that the surface is
contacting the support pins at the bottom of the basin and is centered between the side
walls.
Obtain a clean 2-gallon bucket (or equivalent) and fill the bucket with 5 L of washdown
liquid (tap water or salt water).
o Note: A new bucket and water will be used for each sample surface.
If necessary, vent any residual pressure from the pressurized spray kit and close off the
overpressure nitrogen cylinder.
Uncap the spray kit reservoir and fill it with the appropriate washdown liquid (tap water
or saltwater). Recap the reservoir when finished.
o Note: The Rinse Kit reservoir can hold ~8 L. Reloading of liquid may be
necessary throughout the testing depending on number of samples and usage rate.
Attach the Dramm heavy-duty adjustable brass nozzle (Home Depot Model # 14033591)
to the outlet of the spray kit and open the nozzle to the desired setting.
o Note: The appropriate setting will be marked on the nozzle itself. This setting
corresponds to a nozzle opening which will achieve the desired flow rate of 3 - 5
L/min at 30 PSIG and which will produce a spray cone with a diameter of 12" -
14" when positioned -12" above the test surface.
Ensure that the spray valve (on/off valve) on the spray kit is shut-off and attach the
overpressure line to the reservoir.
Set the nitrogen overpressure cylinder to 30 PSIG. When ready to pressurize the
reservoir, open the cylinder secondary valve.
Using a large (e.g. 4 L) graduated cylinder (or equivalent containment) and calibrated
stopwatch, ensure that the flowrate from the nozzle is within 3 to 5 L/min.
o To do this, position the nozzle above the graduated cylinder and open the spray
valve to initiate the spray. Simultaneously, start the stopwatch and allow the
nozzle to spray for a predetermined amount of time (15 seconds, 30 seconds, etc.).
Shut the nozzle off at the conclusion of the time and measure the volume of liquid
dispensed. The flow rate of the nozzle is the amount of liquid dispensed divided
by the spray time, in minutes.
If the nozzle is outside of the acceptable range, adjust the opening as necessary and
reattempt the measurement. Once within range, repeat the measurement two additional
times for three total measurements.
When ready to begin sample collection, place the collection beaker underneath the trough
outlet.
G-2
-------
With the spray valve off, hold the nozzle -12" above (normal to) the test surface. At this
height, the nozzle should generate a cone that is -12" - 14" in diameter
The nozzle will be kept at this height and swept across the test surface according to the
pattern shown in Figure 1. A total of 5 passes (labeled PI - P5 in the figure) will be
performed at -1 second per pass for a total of 5 seconds of spray. This quick spray is
intended to pre-wet the surface before it is brushed.
Following the surface wetting, a clean 8.5" West Marine deck brush (West Marine Model
#: 12830840) will be used to scrub the surface. The brush will be submerged in the clean
wash liquid before being passed along the surface 7 times in an overlapping pattern as
shown in Figure 2.
o Note: A new brush will be used for each surface.
After the surface is brushed, a second spray event will be used to wash off the surface.
This event will follow the same pattern shown in Figure 1, but instead will use 5 -3
second passes.
When ready to sample, turn on the nozzle and execute the first pre-wetting spray event as
shown in Figure 1.
Immediately following the pre-wet, obtain a bristle brush and wet the head of the brush
by completely submerging it in the bucket of wash liquid.
Perform the surface scrubbing as shown in Figure 2.
When finished with the scrub, transfer the bri stle brush back to the water bucket and dunk
the head into the water 3 times (physical agitation). After the third dunk, leave the brush
set in the water.
Reobtain the spray kit nozzle and perform the wash down spray event using 5 -3 second
passes according to Figure 1.
Spfay Basin
(30"x 30")
Figure 1: Bristle brush pre-wet and washdown spray pattern
G-3
-------
8" Brush Coverage
4" Overhang
Pass#l
I
i
Pass #2
~
Pass #3
Pass #5
Pass #6
Pass #7
Brush Coverage
lx Pass 2x Pass
Figure 2: Bristle brush sampling pattern
Allow the surface to drain and return to the bristle brush.
Perform three additional dunks of the bristle brush head in the bucket of water before
pulling the brush head out and shaking/twisting it gently to remove any excess liquid. Set
the brush aside in the staging area.
Obtain an autoclaved, sealable vessel and transfer ~1L of the bucket liquid into the
vessel.
Remove the test surface from the basin and set it aside in the staging area.
Remove the collection beaker and record the volume of runoff collected. Transfer the
runoff to an autoclaved, sealable vessel for storage
After collection of the runoff, obtain a clean, dry, disposable cloth or wipe and remove
any standing water or droplets from the basin surface and collection trough. Dispose of
the cloth when finished.
G-4
-------
Figure 3: Example of bristle brush sampling and dunking
-------
Appendix H
H-1
-------
Spore Production
Table 1. Components of Modified G Sporulation Medium
Ingredient
Amount/L
Yeast Extract
2-0 g
(NH4)2fi()4
2.0 g
CaCh • 2H20
0.03 g
CuSOi • 5II;0
0.005 g
FcS04 • 7II30
0.0005 g
MgS04 • 71I2O
0.2 g
M11SO4 • H2O*
0.06 g
ZnS04 • 7II20
0.005 g
K2IIPO1
0.5 g
dll20
1000 mL
*MnSG4 • IhO substituted for M11SO4 • 4H:G. If MnS04 • 411 iO is used, add 0.05 g.
Table 2. ( ompoiuiils of Leighton Doi Sporulation Medium
Component
Amount/L
KCl
188 g
CaCb
0.29 g
FeS04 x 7 H2O
0.003 g
MnSOt s IbO
0.0017 g
MgSOt x 7 H20
0.025 g
Dextrose
0.9 g
Nutrient Broth
16.0 g
H-2
-------
Table 2. Real-Time PCR Assay Conditions
Component Volume for one reaction (jiL)
TaqMan Fast Advanced Master Mix 12.5
(Applied Biosystems, Cat. 4444556)
Platinum Taq Polymerase 0.1
(Invitrogen, Cat. 10-966-034)
Btk T1B2 Forward Primer (25 (.iM) 1
Btk T1B2 Reverse Primer (25 (.iM) 1
Btk T1B2 Probe (2 jiM) 1
PCR Grade Water 4.4
Template 5
Total volume 25
H-3
-------
Appendix I
1-1
-------
Purpose: Generate Stock suspensions for spiking test articles as positive controls.
Materials:
Item
Manufacturer
Product Number/Prep. Date/Lot
Sterile Milli-Q Water
In house
Btk HD-1T1B2 stock (2x
10s CFU/mL)
In house
Lot: HD1.T1B2.120919
TSA
Becton Dickinson
PN: B21283X / Lot:
PBS, 0.05% Tween-20, pH
7.4 (PBST)
Teknova
PN:P0201/
Sponge-Sticks
3M
Specimen cup
Vacuum Filter Cassette
Gravel
Equipment:
Item
Manufacturer
Serial Number
Thermometer/
Rees #
Calibration
Due
Initials & Date
Biosafety
Cabinet (BSC)
Baker
Thermo Forma
N/A
Micropipette
Type: 200
N/A
Micropipette
Type: 1000
N/A
Refrigerator
Incubator
Stock Prep:
1. Prepare four serial 1:10 dilutions of 2 x 10s CFU/mL Btk T1B2 stock by adding 1 mL of stock into
9 mL sterile Milli-Q. water. Label as 2 x 107 CFU/mL, 2 x 10s CFU/mL, 2 x 10s CFU/mL and 2 x 104
CFU/mL.
Spike Sponge-Sticks:
1. Sponge-Sticks will be directly inoculated with either 6,500 CFU or 650,000 CFU. This represents
the number of CFU available for collection following the sampling of a 10" X 10" area that has
been sprayed with 10 CFU/cm2 (6,500 CFU [Low]) and 1,000 CFU/cm2 (650,000 CFU [High]),
a. Calculations:
i. For High Spike, 2 x 10sCFU/mL (X) = 6.5 x 10sCFU
1-2
-------
1. X = 0.325 mL
a. For High Spike, place ten 16.3 (J.L evenly dispersed droplets on
each side for a total of twenty 16.3 jiL droplets, 326 jiL total,
ii. For Low Spike, 2 x 104 CFU/mL (X) = 6.5 x 103 CFU
1. X = 0.325 mL
a. For Low Spike, place ten 16.3 |iL evenly dispersed droplets on
each side for a total of twenty 16.3 j^L droplets, 326 jiL total
Position with folded side up or stick side up. Do not spike the sides of the sponge that could contact
the specimen cup wall.
1. Two VCF per surface spray batch will be directly inoculated with 9,300 CFU or 930,000 CFU. This
represents the number of CFU available for collection following the sampling of a 12" X 12" area
that has been sprayed with 10 CFU/cm2 (9,300 CFU [Low]) and 1,000 CFU/cm2 (930,000 CFU
[High]).
a. Calculations:
i. For High Spike, 2 x 107CFU/mL (X) = 9.3 x 10sCFU
1. X = 0.0465 mL
a. For High Spike, place ten 4.65 |iL evenly dispersed droplets on
VCF for 46.5 (iL total.
ii. For Low Spike, 2 x 10s CFU/mL (X) = 9.3 x 103 CFU
1. X = 0.0465 mL
a. For Low Spike, place ten 4.65 |iL evenly dispersed droplets on
VCF for 46.5 [iL total.
VCF Spike:
1-3
-------
Place ten 4.65 jiL evenly dispersed droplets onto filter.
Gravel Spike:
1. Two 1 L bottles containing gravel filled to the Zi full level per surface spray batch will be directly
inoculated with 2,600 CFU or 260,000 CFU. This represents the number of CFU available for
collection following the sampling of a 5" X 8" area that has been sprayed with 10 CFU/cm2
(2,600 CFU [Low]) and 1,000 CFU/cm2 (260,000 CFU [High]),
a. Calculations:
i. For High Spike, 2 x 105CFU/'mL (X) = 2.6 x 105CFU
1. X = 0.130 mL
a. For High Spike, place ten 13 [iL evenly dispersed droplets onto
surface of gravel in 1 L bottle for a total of 130 (iL.
ii. For Low Spike, 2 x 10' CFU/mL (X) = 2.6 x 103 CFU
1. X = 0.130 mL
a. For Low Spike, place ten 13 |aL evenly dispersed droplets onto
surface of gravel in 1 L bottle for a total of 130 |^L,
Spike Sterile Milli-Q Water (for Bilge water control);
1. Spike 249.875 mL of sterile Milii-Q water with 0.125 mL of 2 x 103 CFU/mL Btk T1B2, mix well.
This is the 1 CFU/mL (250 CFU) Spike water sample.
2. Spike 249.875 mL of sterile Milli-Q water with 0.125 mL of 2 x 105 CFU/mL Btk T1B2, mix well.
This is the 100 CFU/mL (25,000 CFU) Spike water sample.
Spike Water for non-traditional samples:
1. Spike 249.687 mL of rainwater or tap water with 0.313 mL of 2 x 104 CFU/mL Btk T1B2, mix well.
This is the 25 CFU/mL (6,250 CFU) Spike water sample.
2. Spike 249.687 mL of rainwater or tap water with 0.313 mL of 2 x 106CFU/mL Btk T1B2, mix well.
This is the 2,500 CFU/mL (625,000 CFU) Spike water sample.
Stock Enumeration:
1. 2 x 10s CFU/mL stock
a. Prepare three serial 1:10 dilutions of 2 x 10s CFU/mL Btk stock by adding 0.1 mL of stock
into 0.9 mL PBST.
b. Spread 0.1 mL onto TSA plates for 1:100 and 1:1,000 dilutions made for the following
final dilution factor onto plates: 1 x 10"3 and 1 x 10".
c. Incubate at 30 ± 2 °C overnight
1-4
-------
2. 2 x 104CFU/mL stock
a. Prepare two serial 1:10 dilution of 2 x 104 CFU/mL Btk stock by adding 0.1 mL of stock
into 0.9 mL PBST.
b. Spread 0.1 mL onto TSA plates for 1:10 and 1:100 dilutions made for the following final
dilution factor onto plates: 1 x 10"2 and 1 x 10"3.
c. Incubate at 30 ± 2 °C overnight
3. 2 xlO3 CFU/mL stock
a. Prepare a 1:10 dilution of 2 x 103 CFU/mL Btk stock by adding 0.1 mL of stock into 0.9
mL PBST.
b. Spread 0.1 mL onto TSA plates for Neat and 1:10 dilutions made for the following final
dilution factor onto plates: 1 x 10"1, 1 x 10"2.
c. Incubate at 30 ± 2 °C overnight
4. Include negative controls
a. 0.1 mL PBST on TSA
b. 0.1 mL H20 on TSA
c. TSA plate
Incubation Start Time/Date:
Enumeration Results:
Sample ID
Final
Dilution Factor
on Plate
Plate Counts
Average
Counts
CFU/mL
Plate 1
Plate 2
Plate 3
Stock 2 x 10s CFU/mL
1 x 10"3
1 x 10"4
Stock 2 x 104 CFU/mL
1 x 10"2
1 x 10"3
Stock 2x 103 CFU/mL
1 x 101
1 x 10"2
1-5
-------
Appendix J
J-1
-------
I. PURPOSE/SCOPE
To recover Btk T1B2 spores from samples collected from outdoor surfaces.
II. MATERIALS/EQUIPMENT
Materials
Item
Manufacturer
Lot Number
Exp.
Date
Storage
Temp.
Initials & Date
Extraction Buffer with
Tween® 20 + 30%
Ethanol
Inhouse
2-8 °C
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
BHIB
BD
2-8 °C
Conical tubes, 15 mL
N/A
R.T.
Falcon Conical Tube,
50m L
N/A
R.T.
Screw top flask, 250 mL
Corning
N/A
R.T.
0.45 nm filter vials
Whatman
N/A
R.T.
2mL screw cap tubes
N/A
R.T.
Sterile disposable
forceps
Unomedical
N/A
R.T.
N/A = Not Applicable
Equipment
Item
Manufacturer
Serial Number
Thermometer/
Rees #
Calibration
Due
Initials & Date
Biosafety Cabinet
(BSC)
The Baker Company
57553
N/A
57544
Micropipette
Type:L1000
Rainin
N/A
Incubator Shaker
New Brunswick
590644988
Refrigerator
Fisher
C3274822
115
Swinging Bucket
Centrifuge
Beckman Coulter
X59221
N/A
N/A
Stomacher
Seward
40142
N/A
N/A
N/A = Not Applicable
J-2
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Filters - Electronically update this table with samples names from the Sample Log
Sample
#
Sample
type
Surface Type
Filter Vial
Type
Spore level
Sample ID
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Other Supplies and Equipment
• Forceps
• Biohazard bags
• Bleach
• 5 mL, 25 mL and 100 mL Serological Pipets
• Pipette aid
• Ziplock bags
III. PROCEDURE
A. RV-PCR 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
J-3
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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.
• 1,500 ML of Extraction Buffer with Tweens 20 ± Ethanol will be needed per set of 16 samples
(90 mL per sample)
• 225 mL aliquot of High salt wash buffer (lOx PBS) in a 250mL screw capped bottle per set of 16
samples (12.5 mL per sample).
• 225 mL aliquot of low salt wash buffer (Ix PBS) in a 250mL screw capped bottle per set of 16
samples (12.5 mL per sample).
2. Add 90 mL cold (4°C) extraction buffer with Tween® 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 minute at 260 rpm (Figure 1). Open the
door of the Stomacher and remove the bag. Reseal bag.
Figure 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
minutes 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 - 8 °C until enrichment in BHIB (See Wl #7: BHIB 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
J-4
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(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 3500 x g with the brake off, for 15 minutes in a swinging bucket rotor at 4°C.
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 - 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 seconds to resuspend the pellet, then
transfer entire volume to Aliquot 2.
16. Vortex Aliquot 2 for 30 seconds 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.
• Table 1. Volume of sample recovered from Sponge Sticks.
Sample
Number
Sample ID
Total volume recovered
from Sponge-Stick
Recorded by:
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
J-5
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17. Transfer 11 mL of the pooled extract and store on ice or in refrigerator until processed on same day
using Wl #4: 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 3-5 minutes of settle time to avoid loading large particulates
into filter vial. Transfer 12.5 mL of the pooled suspension volume from each 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 minutes 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 ID
Sample Addition
Volume of Wash
Recorded
#
Buffers
by:
Start
End
10X
IX
Time
Time
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Record the time of adding the final sample to filter vial.
2Record 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 D) below, with filter vial manifold.
J-6
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B. RV-PCR Sample Processing: Spore Recovery for VCF 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 Tween9 20 + Ethanol.
• One labeled 2 oz. sterile cup with lid per sample, sterilized by autoclave (Gravity cycle, 121 °C
for 15 minutes).
• 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'1' 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'1' 20 + Ethanol (from the 11 ml of a pre-measured aliquot
of PBST + Ethanol) 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 Tween9 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 particulate
matter, more Extraction Buffer with Tween9 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 Tween9 20 + Ethanol inside the cassette and set aside, plug side down as shown in
Figure 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 Tween9 20 + Ethanol.
Transfer the rinsate using the same pipette to the appropriately labelled 2 oz. sterile cup.
J-7
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Figure 2. Vacuum Cassette with Top Section Removed
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 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 3. Vacuum Cassette with Top and Middle Sections Removed
8, Use the remainder of the 6 rnL Extraction Buffer with Tween® 20 + Ethanol to rinse walls of the
middle and top sections (configuration shown in Figure 2, 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 minute and remove tubes from the
sonicating bath. Dry and disinfect each tube with a 10% bleach solution.
11. Vortex the conical tubes 2 minutes using platform vortex at Setting 10 (high setting), then transfer
the 5 mL Extraction Buffer with Tween'1' 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 4).
J-8
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Figure 4. Nozzle Removal Using 1 mL Pipette
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 minutes 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 2. Note: Save 2 oz. cups containing
filter. Store at 2 - 8 °C until enrichment in TSB on same day (See Wl #7: VCF TSB Enrichment for
Culture).
Table 2. Volume of sample recovered from VCF.
Sample
Number
Sample ID
Total volume
recovered from
VCF
Volume available
per analytical
method (Total
Volume -r 2)
Recorded by:
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
J-9
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14. Vortex each sample, then allow 3-5 minutes 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 (Wl #4: 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.
Note 1: At 15 minutes 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 Addition
Volume of Wash
Recorded
#
Buffers
by:
Start
End
10X
IX
Time
Time
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Record the time of adding the final sample to filter vial.
2Record end time for samples that have clogging and meet the criteria in Notes 1 and 2 above.
17. Proceed to RV-PCR processing section (Section D) below, with filter vial manifold
J-10
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C. Spore Recovery for Gravel, Bilge or Washdown 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% NaOCI after collecting all waste fluids.
• In a BSC, attach the RV-PCR 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. Add 20 mL of Extraction Buffer with Tween 20 + 30% Ethanol (PBSTE) to a MicroFunnel unit with
0.45 nm 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 TSA plate using sterile forceps. This 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 extracted gravel for each sample and record weights below.
Sample
Number
Sample ID
Weight (Grams)
Recorded by:
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
J-11
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Sample
Number
Sample ID
Weight (Grams)
Recorded by:
16
4. Vigorously mix 0.5 L Gravel eluate, Bilge water or 1 L Washdown aliquots by hand for 30 seconds.
5. Pour mixed Gravel eluate, Bilge water or Washdown eluate into filter unit to the 100 mL gradation
line. Allow 30 seconds of settle time.
6. Apply vacuum until entire 100 mL passes through membrane. Once complete, break vacuum
pressure, then close valve.
7. Repeat steps 43 - 44 once with an additional 100 mL of Gravel eluate, Bilge water or Washdown.
8. Repeat steps 43 -44, this time with an additional 50 mL of Gravel eluate, Bilge water or Washdown,
for a total of 250 mL of sample eluate onto a single 47 mm filter.
Note: If filter becomes clogged, less than 250 mL of sample will be processed. Record volume
filtered in the below table. If a 100 mL or 50 mL aliquot of Gravel eluate, Bilge water or Washdown
eluate takes longer than 10 minutes to filter, no further volume will be added to filter. At 30 minutes
post-sample addition, if sample has not completely passed through the filter, the remaining volume
in the filter unit will be removed.
Sample
#
Sample ID
Filtration Start
Time
Filtration End
Time
Total Volume
Filtered
Recorded By:
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
9. 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.
J-12
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10. Repeat steps 42 - 47 for all samples.
11. Add 10 mL of PBSTE to 50 mL conical tubes containing membrane filters.
12. Vortex at maximum speed on platform vortex in 10 second bursts for 2 minutes to dislodge spores.
13. Let tubes settle for 2 minutes, then transfer volume to a clean 50 mL conical tube.
14. Repeat extraction of each membrane filter by adding another 10 mL of PBSTE to the 50 mL conical
tube with membrane.
15. Repeat steps 50 and 51, transferring volume to the same 50 mL conical tube per sample for a total
recovered pooled spore recovery volume of 20 mL. Observe membrane, and record if particulates
were suspended in solution or remain adhered to filter.
Membrane Observations:
Note: Save filter membrane. Store at 2 - 8 °C until enrichment in BHIB on same day (See Wl #7:
BHIB Enrichment for Culture).
16. Vortex each 20 mL sample, then allow 30 seconds 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.
17. Store the remaining half (10 mL) of the pooled spore recovery volume for microbiological analysis
(Wl #4: Culture of Recovered Spores). Store aliquot on ice or in refrigerator until processed on same
day.
18. Complete filtration of liquid through RV-PCR filter vials. Turn off vacuum pump.
Note 1: At 15 minutes 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 Addition
Volume of Wash
Recorded
#
Buffers
by:
Start
End
10X
IX
Time
Time
1
2
3
4
J-13
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Sample
Sample II)
Sample Addition
Volume of Wash
Recorded
#
Buffers
by:
Start
End
10X
IX
Time
Time
5
6
7
8
9
10
11
12
13
14
15
16
Record the time of adding the final sample to filter vial.
2Record end time for samples that have clogging and meet the criteria in Notes 1 and 2 above.
D. 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.
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
minutes.
J-14
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10. Place into the BSC: 5 mL serological pipets, 1000 piL pipet, 1000 piL 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 T0. 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 minutes on the platform vortexer, setting 7.
Start time: End Time: Speed:
15. Place 2 mL screw cap tubes for T0 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 mLtube. Using a 1 mL pipette or
serological pipet (if filter deteriorated), gently pipet up and down ~10 to mix. Transfer 1 mLfrom
each vial to the corresponding pre-chilled (on ice) 2 mL screw cap tube for T0. Cap the tube and
place it back onto ice. Wipe the filter vial with a bleach soaked lab wipe. Change gloves between
each sample.
After transferring the T0 aliquots for all samples, place the filter vial rack in a transfer container,
seal, and bleach the container. Store the T0 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:
Sample
Number
Sample ID
Turbid (Yes/No)
Volume remaining
(mL)
Recorded by:
1
2
3
4
5
6
J-15
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7
8
9
10
11
12
13
14
15
16
19. Proceed to Wl #3: DNA Purification to process T0 and Tf samples
IV. Technical Review
Performed by: Date:
J-16
-------
Appendix K
K-1
-------
I. PURPOSE/SCOPE
Culture of B. thuringiensis T1B2 spores recovered from outdoor surfaces.
II. MATERIALS/EQUIPMENT
Materials
Item
Manufacturer
Lot Number
Exp.
Date
Storage
Temp.
Initials & Date
PBS with Tween
(0.05%)
Teknova
2-8 °C
Microfunnel filters
PALL
R.T.
TSA
BD
2-8 °C
N/A = Not Applicable
Equipment
Item
Manufacturer
Serial Number
Thermometer/
Rees #
Calibration
Due
Initials & Date
Biosafety
Cabinet (BSC)
The Baker Company
N/A
Stationary
Incubator
Precision
9509-003
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 25mL Serological Pipettes
• Pipette aid
K-2
-------
Filters - Electronically update this table with samples names from the Sample Log
Sample
#
Sample
type
Surface Type
Filter
Vial
Type
Spore level
Sample ID
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
III. PROCEDURE
Note: The following procedure is to be carried out with the 10 mL pooled extract taken from step 16
(refer to Wl #2 for Bacillus thuringiensis kurstaki T1B2 spore recovery). Process 2 PBST only negative
control filter funnels alongside samples, one with first set and one with last set
A. Filter funnel plating for low spore load samples or blanks (negative control samples)
1. Label filter funnels per sample and label with volume - 1 mL, 2 mL, 4 mL or 8 mL aliquot to be
plated.
2. Place the filter funnels onto the vacuum manifold in a Class II BSC.
3. Add 5 mL of PBS with 0.05% Tween (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. For each sample, add appropriate volume of pooled extract to one filter funnel.
Apply vacuum.
6. Close the vacuum valve and release the vacuum pressure. Rinse the walls of each filter funnel using
10 mL of PBST. Apply vacuum.
K-3
-------
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.
B. Spread plating for high load samples
1. Prepare a 1:10 dilution of each sample by adding 1 mL of Neat sample into 9 mL of PBST and mix
well by vortex. Neat or serial dilutions may be plated.
2. Spread 0.1 mL of Neat or appropriate serial dilution onto TSA in triplicate.
C. Plate incubation
1. Incubate plates inverted overnight at 30 ± 2°C.
Incubation start Date/Time: Initials:
Incubation end Date/Time: Initials:
2. Enter results enumeration table.
Background
Background
Growth
Interferes
Sample
Number
Sample ID
Volume
plated (mL)
Plate Counts - Btk Morphology
(CFU)
Present
(Yes/No)
with
Morphology
Identification
(Yes/No)
N/A
PBST Neg. (First)
8.00
N/A
N/A
N/A
PBST Neg. (Last)
8.00
N/A
N/A
N/A
TSA Neg.
N/A
N/A
N/A
1E-3 (0.001)
1E-2 (0.01)
1E-1 (0.10)
1
1.00
N/A
N/A
2.00
N/A
N/A
4.00
N/A
N/A
8.00
N/A
N/A
IV. Technical Review
Reviewed by: Date:
K-4
-------
Appendix L
L-1
-------
I. PURPOSE/SCOPE
Manual DNA extraction and purification Btk T1B2 spores from recovered surfaces.
II. MATERIALS/EQUIPMENT
Materials
Item
Manufacturer
Lot Number
Exp.
Date
Storage
Temp.
Initials & Date
Lysis Buffer
Promega
RT
PMPs
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
Manufacturer
Serial Number
Thermometer
/Rees #
Calibration
Due
Initials &
Date
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
Other Supplies and Equipment
• Micropipette tips
• Biohazard bags
• Bleach
• Prepare tubes
L-2
-------
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 h) 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 mLtube after gently pipetting up and down ~10 to mix.
6. Centrifuge 2 mL screw cap tubes (both T0 and Tf) at 14,000 rpm for 10 minutes (4°C).
Start: End: Speed:
7. Remove 800 piL of the supernatant from each tube, using a 1000 piL pipet and dispose to waste. Do not
disturb the pellet.
8. Add 800 piL of lysis buffer using a 1000 piL pipet, using a new tip for each sample. Cap the tubes and
mix by vortexing on high (~1800 rpm) in 10 second pulses for a total of 60 seconds.
9. Vortex each screw-cap tube briefly (low speed, 5-10 seconds) and transfer the entire sample volume
to a 2 mL Eppendorf tube (ensure the tubes are labeled correctly during transfer). Incubate the T0 and
Tf lysate tubes at room temperature for 5 minutes.
10. Vortex the PMPs on high (~1800 rpm) for 30 - 60 seconds, or until they are uniformly resuspended.
Keep PMPs in suspension by briefly vortexing (3-5 seconds) before adding to each T0 and Tf lysate
tube.
11. Uncap one tube at a time and add 600 piL of PMPs to each T0 and Tf tube (containing 1 mL sample).
L-3
-------
12. Vortex each T0 and Tf tube for 5-10 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 5-10 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 1000 piL 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 piL of lysis buffer using a 1000 piL pipet. Vortex on low
setting for 5-10 seconds, and transfer to tube rack.
17. Vortex each tube for 5-10 seconds (low) and place back on the magnetic stand. After all tubes are in
the stand, follow tube inversion cycle, as described in Step A. 13.
18. Remove all the liquid as described in Step A.15. Use a new tip for each T0 and Tf tube.
Wash Steps:
19. Uncap each tube one at a time and add 360 piL of Salt Wash Solution. Remove tube rack off of
magnetic stand. Vortex on low setting for 5-10 seconds, and transfer to tube rack. Place tube rack
back on magnetic. Invert as described in step A.13. Remove all the liquid as described in Step A.15.
Use a new tip for each T0 and Tf tube. This is 1st Salt Wash.
20. Uncap each tube one at a time and add 360 piL of Salt Wash Solution. Remove tube rack off of
magnetic stand. Vortex on low setting for 5-10 seconds, and transfer to tube rack. Place tube rack
back on magnetic. Invert as described in step A.13. Remove all the liquid as described in Step A.15.
Use a new tip for each T0 and Tf tube. This is 2nd Salt Wash.
21. Uncap each tube one at a time and add 500 piL of Alcohol Wash Solution. Remove tube rack off of
magnetic stand. Vortex on low setting for 5-10 seconds, and transfer to tube rack. Place tube rack
back on magnetic. Invert as described in step A.13. Remove all the liquid as described in Step A.15.
Use a new tip for each T0 and Tf tube. This is 1st Alcohol Wash.
22. Uncap each tube one at a time and add 500 piL of Alcohol Wash Solution. Remove tube rack off of
magnetic stand. Vortex on low setting for 5-10 seconds, and transfer to tube rack. Place tube rack
back on magnetic. Invert as described in step A.13. Remove all the liquid as described in Step A.15.
Use a new tip for each T0 and Tf tube. This is 2nd Alcohol Wash.
23. Uncap each tube one at a time and add 500 piL of Alcohol Wash Solution. Remove tube rack off of
magnetic stand. Vortex on low setting for 5-10 seconds, and transfer to tube rack. Place tube rack
L-4
-------
back on magnetic. Invert as described in step A.13. Remove all the liquid as described in Step A. 15.
Use a new tip for each T0 and Tf tube. This is 3rd Alcohol Wash.
24. Uncap each tube one at a time and add 500 piL of 70% Ethanol. Remove tube rack off of magnetic
stand. Vortex on low setting for 5-10 seconds, and transfer to tube rack. Place tube rack back on
magnetic. Invert as described in step A.13. Remove all the liquid as described in Step A. 15. Use a new
tip for each T0 and Tf tube. This is 4th Alcohol Wash.
25. If necessary, use a 200 uL pipet to remove remaining 70% ethanol, being careful to not disturb PMPs.
26. Open all T0 and Tftubes 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 piL of elution buffer to each T0 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 - 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.
Start: End:
32. Briefly vortex each tube (5 - 10 seconds) on low speed and centrifuge at 2000 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 T0 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 mLtube.
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 mLtube using a new tip
for each tube.
L-5
-------
Start: End:
36. Store T0 and T, DNA extract tubes at 4°C until PCR analysis. Continue to WI-RV-PCR-STREAMS.
Note: If PCR cannot be performed within 24 hours, freeze DNA extracts at -20°C.
Labeled:
Date/Time: Storage Temperature:
Storage Location:
IV. Technical Review
Performed by: Date:
Comments:
L-6
-------
Appendix M
M-1
-------
I. PURPOSE/SCOPE
Real-time PCR analysis for B. thuringiensis kurstaki T1B2 DNA.
II. MATERIALS/EQUIPMENT
Enter material lot and expiration dates used into STREAMS Task 6 Wl, Real-Time PCR - FORM A
Materials
Item
Manufacturer
Product Number
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
Item
Manufacturer
Serial Number
Thermometer/
Rees #
Calibration
Due
Initials & Date
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 STREAMS Task 6 Wl, Real-Time PCR - FORM A, Date:
M-2
-------
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 afresh
aliquot of PCR-grade water per sample batch to use for 1:10 dilutions and NTCs.
1. To and TjDNA extracts: Label 1.5 mL tubes with the sample identifier and "10-fold dilution". Add 90 piL
of PCR-grade water to the tubes.
2. Mix TO and Ti DNA extracts by vortexing (3-5 seconds), spin at 14,000 rpm for 2 minutes, and
transfer 10 piL of supernatant to 1.5-mL Eppendorf tubes with 90 piL 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 used pipet 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 ten reactions, so that there is enough Master Mix regardless of
pipetting variations. For each batch of samples, PCR Master Mix should be made for 4 PCs, 4 NTCs, and
6 DNA extracts per sample (3 for TO and 3 for TfDNA extracts). Record sample names and reaction
numbers on STREAMS Task 6 Wl, RV-PCR - FORM A.
3. In a clean PCR-preparation hood, pipet 20 piL of Master Mix into the wells of the PCR plate. Label four
wells as NTC.
4. Add 5 piL of PCR-grade water into each of the NTC wells.
5. Lightly seal the NTC wells with optical caps, cover all other wells of the plate using optical caps.
6. Vortex each sample briefly, then add 5 piLto each sample well. Lightly seal the sample wells with
optical caps.
7. Vortex the Positive Control (PC), B. thuringiensis kurstaki T1B2 DNA [10 pg/1 piL or 50 pg/5 piL and 5
piL] to each PC well. Tightly seal the wells with an optical plate seal, using optical caps.
Performed by: Date:
M-3
-------
C. Within the Post-Amplification Lab, Load 96-well plates onto 7500 Fast.
1. Set up 7500 Fast (TaqMan) - See Section 2 for SYBR settings
a. Open the 7500 Fast Software and select New Experiment
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 piL as the reaction volume.
2. Set thermocycling conditions to match the below settings:
Temperature (°C)
Time
Cvcles
50.0
2:00
Hold
95.0
2:00
Hold
95.0
0:03
45
60.0
0:30
25 piL Total Volume
3. Select Save As, assign unique plate file name and save in project folder.
Performed by: Date:
iv. Start Run
1. Centrifuge the plate at 300 x g for 1-2 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.
M-4
-------
D. Analysis
3. When run is complete, burn the file to a CD.
4. Remove 96-weel plate from the 7500 Fast and dispose.
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"
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: Date:
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
M-5
-------
iii. Indicate which, if any, wells of the standard curve were omitted
iv. Indicate multicomponent results for each well on the Results Table
5. QC Acceptance Criteria
a. Verify the below acceptance criteria are met
• Amplification in PC wells
• NTC wells have no amplification
Data Calculations
Calculate the average Ct value from the replicate reactions for T0 and Tf DNA extracts of each
sample. Subtract the average Ct value of the Tf DNA extract from the average Ct value of the T0 DNA
extract to generate delta Ct value (ACt). If there is no Ct value for the T0 DNA extract (i.e., the T0 is
non-detect), use 45 (total number of PCR cycles used) as the Ct value.
Performed by: Date:
Technical Review
All data will receive technical review and QC review in accordance to QA. 1-005.
Technical Review Initials/Date:
QC Review Initials/Date:
M-6
-------
Appendix N
N-1
-------
I. PURPOSE/SCOPE
Select and screen B. thuringiensis kurstaki T1B2 colonies recovered on culture plates using qPCR.
II. MATERIALS/EQUIPMENT
Materials
Item
Manufacturer
Lot Number
Exp. Date
Storage
Temp.
PCR-Grade Water
Teknova
R.T.
1 nL loop, 10 nL loop or
inoculating needles
N/A
R.T.
1.5 or 2 mL tubes
N/A
N/A
R.T.
N/A = Not Applicable
Equipment
Item
Manufacturer
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 25mL Serological Pipettes
Mil. PROCEDURE
A. Selecting colonies
1. Pipette 100 piL of PCR-grade water into 1.5 or 2 mL tubes.
2. Select colonies. Take pictures of colonies that are selected.
3. Use 1 piL loop, 10 piL 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 piL volume of PCR-grade water. Repeat steps
N-2
-------
3 and 4 to pool multiple colonies from a single sample and record the number of colonies pooled in
the below table.
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 @ 14,000 rpm for 2 minutes. Use supernatant for
qPCR.
Filters - Record Filter ID and Morphology for Selected Colonies
Tube
#
Filter ID
Volume
(mL)
Morphology (Btk
or Background)
# of Colonies
Pooled
PCR Result
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
V. Technical Review
Reviewed by: Date:
N-3
-------
Appendix O
0-1
-------
I. PURPOSE/SCOPE
Enrich extracted sponge or filter in BHIB.
II. MATERIALS/EQUIPMENT
Materials
Item
Manufacturer
Lot Number
Exp.
Date
Storage
Temp.
Initials & Date
PCR-Grade Water
Teknova
R.T.
10 nL loop or
inoculating needles
R.T.
1.5 or 2 mL tubes
R.T.
TSA Plates
2-8 °C
BHIB
R.T.
N/A = Not Applicable
Equipment
Item
Manufacturer
Serial Number
Thermometer/
Rees #
Calibration
Due
Initials & Date
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
• 25mL Serological Pipettes
Mill. PROCEDURE
A. Selecting colonies
9. Add 25 mL of BHIB to each specimen cup containing the extracted sponge or filter.
10. Incubate cups at 30 °C ± 2 °C for 24-48 hours.
Incubation start Date/Time: Initials:
Incubation end Date/Time: Initials:
11. Evaluate the BHIB Enrichment for samples.
a. If broth is not turbid, record as no growth (NG) and incubate for an additional 24 hours.
b. If broth is turbid, record as positive growth (G+) and proceed to Step 4.
0-2
-------
Sample
Number
Filter ID
Growth (G+) or Nc
24 hours
) Growth (NG)
48 hours
Recorded by:
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
12. 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 piL loop and streak triplicate TSA plates for isolation. Store enriched samples at
2 - 8 °C.
13. Incubate the isolation plates and BHIB with growth at 30 °C ± 2 °Cfor a maximum of three days.
Incubation start Date/Time: Initials:
Incubation end Date/Time: Initials:
14. Examine plates for B. thuringiensis colonies.
a. If 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 colony selection sample and proceed to PCR confirmation from BHIB streak plates
(Section B).
b. 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)
0-3
-------
Sample
#
Filter ID
Colony
Selection or
BHIB Analysis
Number of
colonies
screened
PCR Result
Recorded by:
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
B. Selecting Colonies
1. Pipette 100 piL of PCR-grade water into 1.5 or 2 mL tubes.
2. Select colonies.
3. Use 1 piL loop, 10 piL 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 piL 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
1. Transfer 50 piL of broth with growth to a microcentrifuge tube.
2. Centrifuge at 12,000 x g for 2 minutes.
0-4
-------
3. Remove and discard the supernatant in an autoclavable biohazard container. Add 100 piL 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 °C.
Incubation start Date/Time: Initials:
Incubation end Date/Time: Initials:
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
qPCR.
Performed by: Date:
VI. Technical Review
Reviewed by: Date:
0-5
-------
Appendix P
P-1
-------
MGAL (Clean)
Date
Figure P-l. Outdoor Temperature Daily Median, Maximum, and Minimum to Which the Btk T1B2
Contaminated (High and Low Loading) Marine Grade Aluminum (Clean) Surfaces were Exposed
MGAL (Clean)
Date
Figure P-2. Outdoor Relative Humidity Daily Median, Maximum, and Minimum to Which the Btk
T1B2 Contaminated (High and Low Loading) Marine Grade Aluminum (Clean) Surfaces were
Exposed
P-2
-------
MGAL (Salted)
P 20
o>
= 15
I
II
a
E
V# #""" o?"" <£"" ^ rf>"~ (Vs""" o?"" rf>"" (Vs"" rt?"" <0*" a?
,\fev ^ ^ ^ ^ /
,oa
Date
Figure P-3. Outdoor Temperature Daily Median, Maximum, and Minimum to Which the Btk T1B2
Contaminated (High and Low Loading) Marine Grade Aluminum (Salted) Surfaces were Exposed
MGAL (Salted)
Date
Figure P-4. Outdoor Relative Humidity Daily Median, Maximum, and Minimum to Which the Btk
T1B2 Contaminated (High and Low Loading) Marine Grade Aluminum (Salted) Surfaces were
Exposed
P-3
-------
NSKID (Clean)
35
30
it
i s
a
E
* '
*
-I
/«
,
II
fl
AA
L-t. uiii|mi}u
v*
ft
v
V*
A A
11 I I I I IL t til IjI J 11i t I I I I I I I | I I
JL_
Daily Median
/ft oO *|C>
.n
-------
NSKID (Salted)
35
30
If
a.
E
AA
*
A
i.
v*
#
*
%
A A
i>|nnimjijjj.i|,ii
i i|HLitm»mijiiiiiLLim»H|iimiuiiii i|i i
JL_
Daily Median
oO iO »vO
.nc>v 0cJv ,0ov
# tAa ^ <#'
nO rfo
V#0 >1^
i-yO nO
-------
GLASS (Salted)
Date
Figure P-9. Outdoor Temperature Daily Median, Maximum, and Minimum to Which the Btk T1B2
Contaminated (High and Low Loading) Glass (Salted) Surfaces were Exposed
GLASS (Salted)
Date
Figure P-10. Outdoor Relative Humidity Daily Median, Maximum, and Minimum to Which the Btk
T1B2 Contaminated (High and Low Loading) Glass (Salted) Surfaces were Exposed
P-6
-------
GRAVL (Clean)
Date
Figure P-ll. Outdoor Temperature Daily Median, Maximum, and Minimum to Which the Btk
T1B2 Contaminated (High and Low Loading) Gravel (Clean) Surfaces were Exposed
GRAVl (Clean)
Date
Figure P-12. Outdoor Relative Humidity Daily Median, Maximum, and Minimum to Which the Btk
T1B2 Contaminated (High and Low Loading) Gravel (Clean) Surfaces were Exposed
P-7
-------
BILGE (Clean)
Date
Figure P-13. Outdoor Temperature Daily Median, Maximum, and Minimum to Which the Btk
T1B2 Contaminated (High, Low, and No Loading) Bilge Water (Clean) Surfaces were Exposed
20
10
Date
BILGE (Clean)
50 +
30
Figure P-14. Outdoor Relative Humidity Daily Median, Maximum, and Minimum to Which the Btk
T1B2 Contaminated (High, Low, and No Loading) Bilge Water (Clean) Surfaces were Exposed
P-8
-------
Table P-l. Outdoor Temperature and Relative Humidity Daily Median, Minimum, and Maximum
Values over Entire Surface Exposure Period
Date
Daily T
Median ( C)
Daily T Max
(C)
Daily T Min
(C)
Daily RH
Median (%)
Daily RH
Max (%)
Daily RH
Min (%)
2/4/2020
21.9
22.6
21.3
51.4
54.3
49.8
2/4/2020
21.9
22.6
21.3
51.4
54.3
49.8
2/5/2020
4.6
23.1
1.3
54.0
82.7
26.6
2/6/2020
1.3
2.2
-0.6
86.4
90.0
81.6
2/7/2020
-0.9
1.6
-1.9
81.8
87.5
64.8
2/8/2020
0.3
1.5
-1.2
83.2
86.2
74.1
2/9/2020
1.9
4.5
-0.9
81.8
85.4
67.2
2/10/2020
5.4
8.6
3.9
89.4
93.3
82.9
2/11/2020
3.3
4.7
2.1
76.8
92.2
60.5
2/12/2020
1.6
3.6
-0.5
76.8
91.6
58.0
2/13/2020
1.4
2.8
-3.7
82.5
92.1
70.5
2/14/2020
-6.0
-2.7
-7.1
56.8
78.5
43.7
2/15/2020
-0.6
3.8
-7.2
58.4
69.9
45.2
2/16/2020
2.4
8.4
-0.4
64.5
81.6
53.4
2/17/2020
6.7
9.3
-2.0
57.7
85.3
41.8
2/18/2020
7.5
11.2
1.4
72.1
91.0
57.7
2/19/2020
0.6
5.7
-1.8
59.4
77.8
41.7
2/20/2020
-2.1
0.4
-5.1
53.3
69.0
43.0
2/21/2020
-2.0
4.5
-7.1
54.4
70.0
33.0
2/22/2020
1.2
10.0
-4.3
51.5
68.9
30.2
2/23/2020
5.1
11.5
-0.9
49.8
59.2
32.9
2/24/2020
6.2
8.7
2.0
69.6
89.2
58.5
2/25/2020
6.1
7.9
4.1
90.0
92.4
86.1
2/26/2020
2.7
4.1
-2.2
91.1
92.6
81.7
2/27/2020
-1.7
1.0
-3.7
70.7
86.4
50.3
2/28/2020
-2.1
0.7
-5.0
65.2
86.0
50.6
2/29/2020
-2.2
3.0
-6.0
60.4
74.3
37.7
3/1/2020
5.2
11.9
-3.8
52.8
72.6
37.6
3/2/2020
9.3
10.4
7.0
88.4
94.6
55.3
3/3/2020
9.9
14.9
6.6
85.6
96.4
43.9
3/4/2020
6.4
13.9
3.0
62.2
78.0
44.1
3/5/2020
6.7
14.6
-0.4
57.2
80.6
32.1
3/6/2020
3.9
8.5
1.3
70.0
82.3
56.2
3/7/2020
2.8
11.0
-0.2
58.4
66.3
33.3
3/8/2020
9.7
18.6
0.3
44.6
68.9
25.0
3/9/2020
15.4
18.4
8.2
45.4
57.7
34.3
3/10/2020
12.9
15.2
7.0
79.2
90.3
58.3
P-9
-------
Date
Daily T
Median ( C)
Daily T Max
(C)
Daily T Min
(C)
Daily RH
Median (%)
Daily RH
Max (%)
Daily RH
Min (%)
3/12/2020
13.5
17.7
8.8
77.4
91.8
58.3
3/13/2020
10.0
13.9
5.9
54.7
93.6
31.7
3/14/2020
3.5
6.5
1.9
74.3
89.0
38.9
3/15/2020
4.3
9.0
1.6
69.2
84.5
52.9
3/16/2020
6.7
9.5
1.3
61.9
84.8
53.8
3/17/2020
6.7
8.4
2.9
79.5
89.2
63.2
3/18/2020
8.7
11.0
1.6
83.4
96.4
72.8
3/19/2020
10.5
17.7
7.6
94.6
97.0
87.8
3/20/2020
16.3
20.9
4.9
79.4
96.2
68.5
3/21/2020
2.2
4.9
0.4
67.6
72.9
57.7
3/22/2020
6.0
8.8
-1.1
68.9
79.8
47.6
3/23/2020
6.9
9.1
6.1
79.3
92.6
70.4
3/24/2020
7.7
10.2
5.8
70.7
77.3
61.7
3/25/2020
9.6
19.2
4.5
75.0
85.4
41.1
3/26/2020
14.0
21.0
8.1
75.4
88.2
49.7
3/27/2020
13.0
15.2
11.6
84.3
90.1
72.7
3/28/2020
17.0
22.8
12.7
90.9
95.7
77.6
3/29/2020
18.1
20.2
13.9
50.4
95.6
40.2
3/30/2020
9.1
14.0
7.4
59.5
65.3
39.4
3/31/2020
7.1
8.7
5.6
71.5
83.7
59.8
4/1/2020
8.1
10.3
5.5
76.1
86.0
62.4
4/2/2020
9.4
16.7
4.3
65.6
86.0
40.8
4/3/2020
13.3
20.4
5.0
39.9
79.7
25.1
4/4/2020
14.2
22.4
6.2
48.4
68.4
34.6
4/5/2020
10.9
13.5
6.4
81.4
88.6
68.0
4/6/2020
14.4
21.6
3.6
66.7
83.1
28.9
4/7/2020
18.4
25.6
12.4
74.5
92.0
49.9
4/8/2020
17.8
25.8
14.2
75.8
91.1
33.5
4/9/2020
10.2
14.3
4.1
60.8
87.6
31.6
4/10/2020
5.0
7.5
2.5
58.5
73.5
45.3
4/11/2020
9.7
14.8
2.2
54.2
80.8
32.0
4/12/2020
12.7
17.2
8.8
65.0
93.5
44.7
4/13/2020
7.9
14.8
6.2
80.3
94.8
56.8
4/14/2020
5.5
8.2
2.7
44.2
63.4
33.1
4/15/2020
3.5
11.1
0.7
59.3
83.4
33.9
4/16/2020
6.1
10.8
-0.4
43.9
70.8
32.4
4/17/2020
6.5
9.5
4.0
69.2
90.8
41.4
4/18/2020
7.3
12.7
2.5
62.7
87.7
44.8
P-10
-------
Date
Daily T
Median ( C)
Daily T Max
(C)
Daily T Min
(C)
Daily RH
Median (%)
Daily RH
Max (%)
Daily RH
Min (%)
4/20/2020
12.2
18.1
5.4
45.3
76.5
27.9
4/21/2020
9.6
13.4
4.3
43.8
67.3
26.2
4/22/2020
9.7
17.0
1.8
39.8
61.4
31.9
4/23/2020
12.3
13.8
9.3
79.2
91.5
43.6
4/24/2020
11.9
19.9
9.4
78.2
92.5
50.8
4/25/2020
14.5
21.1
10.7
70.6
89.6
42.3
4/26/2020
9.2
13.6
7.5
89.5
91.9
72.8
4/27/2020
11.3
18.0
4.2
59.4
87.8
38.1
4/28/2020
15.9
23.8
10.7
63.5
85.9
46.4
4/29/2020
17.0
21.0
12.1
65.1
87.7
44.6
4/30/2020
10.6
12.1
8.1
83.9
88.7
66.0
5/1/2020
10.0
18.0
8.1
81.5
91.9
54.5
5/2/2020
17.4
27.0
11.0
63.1
80.5
42.7
5/3/2020
20.3
22.1
15.4
67.9
89.4
40.8
5/4/2020
13.3
17.9
10.4
56.8
70.6
41.3
5/5/2020
9.9
13.0
6.5
48.4
82.1
40.6
5/6/2020
8.7
16.2
5.1
57.6
85.1
27.1
5/7/2020
15.0
19.3
5.9
45.8
69.6
29.9
5/8/2020
10.4
14.7
4.8
59.7
83.4
49.3
5/9/2020
5.5
12.3
0.9
43.5
71.5
30.5
5/10/2020
9.3
17.9
5.2
58.8
82.2
40.2
5/11/2020
7.0
8.1
4.1
67.6
84.3
58.5
5/12/2020
10.0
15.7
2.9
51.6
79.4
35.4
5/13/2020
14.3
21.3
4.8
43.2
78.4
28.6
5/14/2020
16.6
24.0
13.4
80.2
93.4
49.5
5/15/2020
18.6
23.5
16.0
86.4
94.0
61.4
5/16/2020
19.2
25.2
14.9
83.3
96.4
57.1
5/17/2020
22.9
27.8
17.8
64.6
81.8
50.0
5/18/2020
20.6
23.0
17.4
81.5
94.9
68.8
5/19/2020
16.1
17.6
12.3
90.6
95.2
84.7
5/20/2020
11.9
13.5
9.7
82.5
86.9
73.4
5/21/2020
14.2
17.3
12.2
88.3
94.4
76.8
5/22/2020
18.1
23.2
15.1
84.8
94.5
61.1
5/23/2020
22.3
28.0
14.2
71.6
93.3
49.4
5/24/2020
24.5
31.4
19.1
73.4
90.6
46.7
5/25/2020
24.6
32.0
20.4
70.5
87.3
42.3
5/26/2020
25.9
32.4
20.6
68.6
85.5
37.6
5/27/2020
25.4
29.9
22.4
61.6
68.4
45.3
p-11
-------
Date
Daily T
Median ( C)
Daily T Max
(C)
Daily T Min
(C)
Daily RH
Median (%)
Daily RH
Max (%)
Daily RH
Min (%)
5/29/2020
21.7
25.9
17.2
75.3
85.3
59.2
5/30/2020
18.4
24.0
16.8
57.8
86.9
39.5
5/31/2020
16.3
21.2
12.7
48.7
77.8
34.2
6/1/2020
18.2
23.8
9.5
43.6
75.5
26.0
6/2/2020
25.7
31.7
17.1
45.7
61.1
36.4
6/3/2020
26.0
31.2
22.3
51.9
70.1
42.4
6/4/2020
22.7
27.2
21.6
78.3
90.2
65.6
6/5/2020
25.0
30.7
20.7
77.8
91.1
54.3
6/6/2020
25.3
31.6
22.2
52.4
87.2
28.9
6/7/2020
22.5
28.4
17.1
49.0
68.8
33.3
6/8/2020
23.8
30.8
17.6
43.9
57.5
28.3
6/9/2020
28.0
34.6
19.6
56.3
66.9
42.0
6/10/2020
25.9
32.3
22.8
73.7
83.0
54.1
6/11/2020
22.3
28.1
18.4
55.0
76.1
31.8
6/12/2020
22.9
28.1
17.1
48.9
68.1
32.6
6/13/2020
19.7
22.3
16.0
62.0
87.0
50.6
6/14/2020
17.8
21.9
13.3
52.3
58.6
43.2
6/15/2020
20.7
26.2
15.1
59.8
76.0
43.7
6/16/2020
22.8
27.9
16.2
53.6
82.8
39.9
6/17/2020
22.5
26.6
18.2
55.3
70.4
39.9
6/18/2020
21.1
25.8
18.9
71.6
89.7
60.7
6/19/2020
23.1
29.6
17.9
71.4
90.7
46.5
6/20/2020
27.2
33.2
19.7
54.6
86.5
31.3
6/21/2020
25.7
33.5
23.0
60.6
70.3
36.8
6/22/2020
23.6
29.5
20.8
73.1
91.5
49.6
6/23/2020
23.3
26.6
20.3
78.3
91.0
52.9
6/24/2020
20.9
26.3
18.7
69.3
78.4
50.0
6/25/2020
23.2
29.2
17.6
58.5
84.8
36.8
6/26/2020
25.5
29.3
19.7
66.2
76.6
49.6
6/27/2020
24.9
27.4
24.0
74.1
85.0
62.7
6/28/2020
25.8
30.6
22.2
72.2
88.5
55.8
6/29/2020
27.2
31.8
22.0
55.7
86.2
43.7
6/30/2020
26.7
31.8
22.9
63.6
77.1
44.2
7/1/2020
27.2
33.0
22.8
56.0
74.7
36.7
7/2/2020
27.1
33.3
21.1
56.8
74.7
35.6
7/3/2020
28.5
34.3
21.7
53.8
78.1
34.3
7/4/2020
27.8
32.9
23.0
60.8
78.1
43.5
7/5/2020
27.8
34.8
22.3
47.9
71.9
30.9
P-12
-------
Date
Daily T
Median ( C)
Daily T Max
(C)
Daily T Min
(C)
Daily RH
Median (%)
Daily RH
Max (%)
Daily RH
Min (%)
7/7/2020
27.0
33.8
23.2
68.6
88.3
44.6
7/8/2020
27.7
34.5
22.2
67.7
89.6
45.3
7/9/2020
29.2
35.1
24.2
65.9
81.6
42.4
7/10/2020
26.9
31.2
25.2
73.7
81.8
59.0
7/11/2020
26.2
31.5
21.5
56.6
78.2
41.0
7/12/2020
23.7
27.6
21.0
70.7
81.2
53.1
7/13/2020
24.4
29.6
19.7
60.1
83.8
45.0
7/14/2020
25.8
31.9
20.3
52.5
71.5
36.8
7/15/2020
28.1
34.0
22.1
54.0
62.3
37.8
7/16/2020
27.2
31.2
25.4
63.7
75.5
55.2
7/17/2020
25.9
33.9
22.6
62.9
85.7
29.3
7/18/2020
28.7
34.6
21.8
65.0
78.6
42.8
7/19/2020
26.6
34.0
24.0
74.4
85.5
48.0
7/20/2020
26.9
32.7
23.4
62.9
86.1
45.6
7/21/2020
25.7
32.7
24.1
60.5
84.4
47.3
7/22/2020
24.8
29.0
22.3
80.7
89.0
62.3
7/23/2020
25.1
29.3
22.7
84.9
88.2
63.0
7/24/2020
26.6
31.3
22.2
68.5
89.7
50.6
7/25/2020
27.2
33.1
21.5
55.1
77.4
40.1
7/26/2020
28.1
34.4
22.2
57.5
82.1
40.4
7/27/2020
26.6
33.5
23.7
75.1
88.6
51.5
7/28/2020
25.4
31.8
22.9
58.5
88.5
38.0
7/29/2020
27.2
33.5
21.2
58.0
79.7
38.2
7/30/2020
25.2
27.6
22.3
73.8
87.9
63.1
7/31/2020
25.1
30.2
21.3
63.0
90.1
45.8
8/1/2020
24.3
29.7
21.9
73.2
89.8
64.5
8/2/2020
22.6
26.1
21.4
81.4
92.2
61.0
8/3/2020
21.3
26.9
19.7
82.8
92.0
64.0
8/4/2020
20.8
24.8
19.0
87.2
93.6
61.8
8/5/2020
20.2
26.5
15.7
62.5
82.7
42.4
8/6/2020
22.2
27.1
17.1
62.1
80.0
45.6
8/7/2020
22.9
29.3
18.4
65.5
77.9
44.5
8/8/2020
23.8
31.8
18.4
62.6
80.1
40.3
8/9/2020
26.2
33.6
20.1
65.2
84.3
41.4
8/10/2020
27.1
32.8
22.2
69.4
82.7
49.7
8/11/2020
24.7
30.3
22.3
73.9
85.4
54.0
8/12/2020
25.5
30.9
22.4
66.6
83.8
48.5
8/13/2020
26.7
33.8
20.2
59.8
78.4
37.5
P-13
-------
Date
Daily T
Median ( C)
Daily T Max
(C)
Daily T Min
(C)
Daily RH
Median (%)
Daily RH
Max (%)
Daily RH
Min (%)
8/15/2020
22.8
28.9
21.4
79.7
87.7
62.0
8/16/2020
22.7
28.8
18.7
79.7
88.4
52.0
8/17/2020
24.1
30.3
18.8
64.2
87.9
40.3
8/18/2020
21.8
26.7
19.5
71.0
81.2
43.7
8/19/2020
21.4
29.0
15.3
54.3
79.7
34.5
8/20/2020
22.2
31.2
15.2
53.6
78.4
30.9
8/21/2020
24.8
29.5
18.6
60.1
75.7
44.4
8/22/2020
24.1
30.3
21.0
72.7
82.8
46.0
8/23/2020
25.0
30.9
20.9
76.1
93.6
48.5
8/24/2020
25.3
31.9
21.6
77.2
86.9
51.4
8/25/2020
27.2
33.8
24.2
67.3
85.1
39.3
8/26/2020
27.1
32.7
22.6
70.0
86.6
47.8
8/27/2020
26.3
30.8
23.8
76.5
85.5
61.2
8/28/2020
25.6
29.0
22.6
84.6
91.2
72.7
8/29/2020
24.0
29.1
22.2
71.2
91.8
45.2
8/30/2020
21.4
26.0
16.2
61.7
81.6
40.7
8/31/2020
22.8
26.6
16.9
69.2
85.3
53.1
9/1/2020
24.1
28.9
21.3
84.9
91.8
63.0
9/2/2020
23.9
27.8
22.8
84.5
95.3
65.5
9/3/2020
23.1
29.6
21.7
85.1
93.9
59.0
9/4/2020
22.5
28.0
18.2
55.1
76.5
30.9
9/5/2020
22.0
29.3
15.1
53.4
76.8
33.8
9/6/2020
22.0
27.6
16.6
63.8
82.9
41.3
9/7/2020
21.0
30.3
18.8
78.6
92.9
53.1
9/8/2020
24.3
31.6
20.2
79.9
95.8
49.5
9/9/2020
24.5
32.9
20.5
76.3
90.3
42.7
9/10/2020
23.6
29.8
20.8
80.4
89.5
57.8
9/11/2020
20.3
22.0
19.0
79.4
83.6
72.9
9/12/2020
23.8
30.2
16.5
80.1
88.6
62.9
9/13/2020
22.9
26.3
20.5
83.1
95.3
54.2
9/14/2020
19.6
23.7
16.3
68.0
88.2
51.5
9/15/2020
17.3
24.1
12.8
63.0
76.5
39.3
9/16/2020
19.0
27.3
12.4
74.5
85.9
43.8
9/17/2020
18.2
24.5
14.4
74.0
88.7
59.8
9/18/2020
15.7
20.9
12.0
57.8
76.2
33.3
9/19/2020
14.5
22.6
8.6
47.3
79.8
27.3
9/20/2020
15.7
24.4
9.4
46.5
74.8
23.8
9/21/2020
16.0
24.1
11.2
51.6
65.3
27.8
P-14
-------
Date
Daily T
Median ( C)
Daily T Max
(C)
Daily T Min
(C)
Daily RH
Median (%)
Daily RH
Max (%)
Daily RH
Min (%)
9/23/2020
19.3
25.4
12.2
66.1
83.5
44.1
9/24/2020
19.6
27.0
16.7
67.1
83.5
42.2
9/25/2020
21.2
26.8
14.6
67.7
87.3
43.5
9/26/2020
21.8
30.0
18.7
71.6
84.7
43.5
9/27/2020
20.9
29.8
18.2
71.3
83.2
42.9
9/28/2020
19.2
24.9
15.2
78.8
83.8
60.0
9/29/2020
15.2
19.0
13.1
70.3
83.7
57.8
9/30/2020
16.0
18.5
11.9
65.4
76.8
52.0
10/1/2020
13.5
19.2
11.6
62.4
68.5
37.0
P-15
-------
vvEPA
United States
Environmental Protection
Agency
PRESORTED STANDARD
POSTAGE & FEES PAID
EPA
PERMIT NO. G-35
Office of Research and Development (8101R)
Washington, DC 20460
Official Business
Penalty for Private Use
$300
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