&EPA
United States
Environmental Protection
Agency
EEPA/600/R-19/083 | June 2019
www.epa.gov/homeland-security-research
Evaluation of Analytical Methods for
The Detection of Bacillus Anthracis
Spores: Compatibility With Real-World
Samples Collected From Outdoor And
Subway Surfaces
Office of Research and Development
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EPA/600/R-19/083
June 2019
EVALUATION OF ANALYTICAL METHODS FOR
THE DETECTION OF BACILLUS ANTHRACIS
SPORES: COMPATIBILITY WITH REAL-WORLD
SAMPLES COLLECTED FROM OUTDOOR AND
SUBWAY SURFACES
June 2019
U.S. Environmental Protection Agency
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EPA/600/R-19/083
June 2019
Disclaimer
The U.S. Environmental Protection Agency (EPA), through its Office of Research and
Development's National Homeland Security Research Center, funded and managed this
evaluation. The document was prepared by Battelle Memorial Institute under EPA Contract
Number EP-C-15-002; Task Order 0009. This document was reviewed in accordance with EPA
policy prior to publication. Note that approval for publication does not signify that the contents
necessarily reflect the views of the Agency. Mention of trade names or commercial products
does not constitute endorsement or recommendation for use of a specific product.
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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EPA/600/R-19/083
June 2019
TABLE OF CONTENTS
Page
EXECUTIVE SUMMARY xiii
1.0 INTRODUCTION 1
1.1 Background 1
1.2 Objective 2
1.3 Scope 2
2.0 MATERIALS AND METHODS 4
2.1 Sampling Methods 4
2.1.1 Sponge-Sticks 4
2.1.2 Vacuum Filter Cassettes 5
2.2 Sampled Surfaces 5
2.3 Test Matrix 8
2.4 Microbiological Methods 10
2.4.1 Spore Bank 11
2.4.2 Spore Loading (Spiking) 12
2.4.3 Spore Recovery 13
2.4.4 Culture Method 14
2.4.5 RV-PCR Method 16
2.5 Method Implementation 20
2.6 Data Reduction and Analysis 21
2.6.1 Culture - Percent Recovery 21
2.6.2 RV-PCR 22
2.6.3 Presentation of Results 23
3.0 RESULTS AND DISCUSSION 25
3.1 Sponge-Stick Analyses Results 25
3.1.1 Culture Method 25
3.1.2 Sponge-Stick TSB Enrichment 39
3.1.3 RV-PCR Method 40
3.2 Vacuum Filter Cassette Analyses Results 53
3.2.1 Culture Method 53
3.2.2 Vacuum Filter Cassette TSB Enrichment 61
3.2.3 RV-PCR Method 62
3.3 Summary of Detection Accuracy 68
4.0 Quality Assurance/Quality Control 74
4.1 Equipment Calibration 74
4.2 QC Results 74
4.3 Operational Parameters 75
4.4 Audits 75
4.4.1 Performance Evaluation Audit 75
4.4.2 Technical Systems Audit 75
4.4.3 Data Quality Audit 75
4.5 QA/QC Reporting 76
4.6 Data Review 76
5.0 SUMMARY OF METHOD OBSERVATIONS AND EXPERIENCES 77
6.0 CONCLUSIONS AND RECOMMENDATIONS 80
7.0 REFERENCES 82
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LIST OF TABLES
Page
Table 1. Test Matrix for Sponge-Stick Samples 9
Table 2. Test Matrix for Vacuum Filter Cassette Samples 10
Table 3. Target 5. a. Sterne Spore Loading Levels Spiked onto Each Sample Substrate 12
Table 4. Recovery Efficiencies for Presumptive B. a. Sterne Spores from Sponge-Stick
Surface Samples Cultured in SBA Medium 26
Table 5. RV-PCR Analyses of Sponge-Stick Surface Samples for Detection of B. a. Sterne
Spores Using Chromosomal and pXOl Gene Targets (N = 3 Replicates for 0
Nominal Spike; N = 5 for 15, 150, and 1,500 Nominal Spike) 42
Table 6. Recovery Efficiencies for Presumptive B. a. Sterne Spores from Vacuum Filter
Cassette Surface Samples Cultured in SBA Medium 55
Table 7. RV-PCR Analyses of Vacuum Filter Cassette Surface Samples for Detection of
B. a. Sterne Spore Chromosomal and pXOl Gene Targets (N = 3 Replicates for 0
Nominal Spike; N = 5 for 15, 150, and 1,500 Nominal Spike) 63
Table 8. Summary of the Accuracy of the Analytical Method Response to Detect
B. a. Sterne on Sponge-Stick Samples 70
Table 9. Summary of the Accuracy of the Analytical Method Response to Detect
B. a. Sterne on Vacuum Filter Cassette Samples 72
Table 10. Paired Overall Positive and Negative B. a. Sterne Detection Results and
Frequency for Culture and Molecular Analysis Methods, Sponge-Stick and
Vacuum Filter Cassette Sample Results Pooled 73
Table 11. Performance Evaluation Audits 75
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LIST OF FIGURES
Page
Figure 1. Pre-Wetted Sponge-Stick from 3M Used for Surface Sampling 4
Figure 2. Vacuum Filter Cassette (37-mm Diameter) Assembled (Left) and Disassembled
(Right) Used for Surface Sampling 5
Figure 3. Electronic Display Panels (Below Ground) Located in Times Square 42nd Street
Station, Near Track 3 - Sampled with Sponge-Sticks 6
Figure 4. Carpet Surface Located by the Jackie O Entrance to Station, Off 42nd Street -
Sampled with Vacuum Filter Cassette 7
Figure 5. Sponge-Stick (left) and Vacuum Filter Cassette (right) After Spiking with the B.
a. Sterne Suspension 13
Figure 6. Manifold Containing 16 Filter Vials (Top); Capping Tray (Middle); Capped Filter
Vials Containing BHIB (Bottom) 17
Figure 7. Process Flow Chart Depicting Key Method Process Steps in Chronological Order 20
Figure 8. Percent Recovery Efficiencies (Average ± One Standard Deviation of N = 5
Replicates) of Presumptive B. a. Sterne Spores from Floor Tile Sponge-Stick
Samples Using SBA Medium 29
Figure 9. Percent Recovery Efficiencies (Average ± One Standard Deviation of N = 5
Replicates) of Presumptive B. a. Sterne Spores from Concrete Floor Sponge-Stick
Samples Using SBA Medium 29
Figure 10. Percent Recovery Efficiencies (Average ± One Standard Deviation of N = 5
Replicates) of Presumptive B. a. Sterne Spores from Steps (Metal) Sponge-Stick
Samples Using SBA Medium 30
Figure 11. Percent Recovery Efficiencies (Average ± One Standard Deviation of N = 5
Replicates) of Presumptive B. a. Sterne Spores from Wall Tile Sponge-Stick
Samples Using SBA Medium 30
Figure 12. Percent Recovery Efficiencies (Average ± One Standard Deviation of N = 5
Replicates) of Presumptive B. a. Sterne Spores from Glass Window Sponge-Stick
Samples Using SBA Medium 31
Figure 13. Percent Recovery Efficiencies (Average ± One Standard Deviation of N = 5
Replicates) of Presumptive B. a. Sterne Spores from Electronic Display Panel
(Below Ground) Sponge-Stick Samples Using SBA Medium 31
Figure 14. Percent Recovery Efficiencies (Average ± One Standard Deviation of N = 5
Replicates) of Presumptive B. a. Sterne Spores from Glass Panel Sponge-Stick
Samples Using SBA Medium 32
Figure 15. Percent Recovery Efficiencies (Average ± One Standard Deviation of N = 5
Replicates) of Presumptive B. a. Sterne Spores from Fluorescent Light Fixture
Sponge-Stick Samples Using SBA Medium 32
Figure 16. Percent Recovery Efficiencies (Average ± One Standard Deviation of N = 5
Replicates) of Presumptive B. a. Sterne Spores from Overhead Sign Sponge-Stick
Samples Using SBA Medium 33
Figure 17. Percent Recovery Efficiencies (Average ± One Standard Deviation of N = 5
Replicates) of Presumptive B. a. Sterne Spores from Metro Card Machine Sponge-
Stick Samples Using SBA Medium 33
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LIST OF FIGURES (CONT.)
Page
Figure 18. Percent Recovery Efficiencies (Average ± One Standard Deviation of N = 5
Replicates) of Presumptive B. a. Sterne Spores from Subway Car Filter Grille
Sponge-Stick Samples Using SBA Medium 34
Figure 19. Percent Recovery Efficiencies (Average ± One Standard Deviation of N = 5
Replicates) of Presumptive B. a. Sterne Spores from Field Blank Sponge-Stick
Samples Using SBA Medium 34
Figure 20. Percent Recovery Efficiencies (Average ± One Standard Deviation of N = 5
Replicates) of Presumptive B. a. Sterne Spores from Concrete Sidewalk Sponge-
Stick Samples Using SBA Medium 35
Figure 21. Percent Recovery Efficiencies (Average ± One Standard Deviation of N = 5
Replicates) of Presumptive B. a. Sterne Spores from Electronic Display Panel
(Above Ground) Sponge-Stick Samples Using SBA Medium 35
Figure 22. Percent Recovery Efficiencies (Average ± One Standard Deviation of N = 5
Replicates) of Presumptive B. a. Sterne Spores from Crosswalk Signal Sponge-
Stick Samples Using SBA Medium 36
Figure 23. Percent Recovery Efficiencies (Average ± One Standard Deviation of N = 5
Replicates) of Presumptive B. a. Sterne Spores from Telephone Booth Sponge-
Stick Samples Using SBA Medium 36
Figure 24. Percent Recovery Efficiencies (Average ± One Standard Deviation of N = 5
Replicates) of Presumptive B. a. Sterne Spores from Painted Crosswalk Sponge-
Stick Samples Using SBA Medium 37
Figure 25. Percent Recovery Efficiencies (Average ± One Standard Deviation of N = 5
Replicates) of Presumptive B. a. Sterne Spores from Granite Bench Sponge-Stick
Samples Using SBA Medium 37
Figure 26. Sponge-Stick Samples from Street Grating Contained Background Flora that
Interfered with Identification of B. a. Sterne Morphology to a Greater Degree
Compared to Other Surfaces (both images were inoculated with 2 mL of extract) 38
Figure 27. Sponge-Stick Samples: Subway Car Filter Grille (Top Left); Steps (Top Right);
Crosswalk Signal (Bottom Left); Telephone Booth (Bottom Right) 39
Figure 28. RV-PCR Analysis of B. a. Sterne Spores Recovered from Floor Tile Sponge-
Stick Samples Using Chromosomal and pXOl Gene Targets (Average ± One
Standard Deviation for N > 3 Replicates); Positive Response Equals ACt >9 44
Figure 29. RV-PCR Analysis of B. a. Sterne Spores Recovered from Concrete Floor
Sponge-Stick Samples Using Chromosomal and pXOl Gene Targets (Average ±
One Standard Deviation for N > 3 Replicates); Positive Response Equals ACt >9 44
Figure 30. RV-PCR Analysis of B. a. Sterne Spores Recovered from Steps (Metal) Sponge-
Stick Samples Using Chromosomal and pXOl Gene Targets (Average ± One
Standard Deviation for N > 3 Replicates); Positive Response Equals ACt >9 45
Figure 31. RV-PCR Analysis of B. a. Sterne Spores Recovered from Wall Tile Sponge-Stick
Samples Using Chromosomal and pXOl Gene Targets (Average ± One Standard
Deviation for N > 3 Replicates); Positive Response Equals ACt >9 45
Figure 32. RV-PCR Analysis of B. a. Sterne Spores Recovered from Glass Window Sponge-
Stick Samples Using Chromosomal and pXOl Gene Targets (Average ± One
Standard Deviation for N > 3 Replicates); Positive Response Equals ACt >9 46
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LIST OF FIGURES (CONT.)
Page
Figure 33. RV-PCR Analysis of B. a. Sterne Spores Recovered from Electronic Display
Panel (Below Ground) Sponge-Stick Samples Using Chromosomal and pXOl
Gene Targets (Average ± One Standard Deviation for N> 3 Replicates); Positive
Response Equals ACt >9 46
Figure 34. RV-PCR Analysis of B. a. Sterne Spores Recovered from Glass Panel Sponge-
Stick Samples Using Chromosomal and pXOl Gene Targets (Average ± One
Standard Deviation for N > 3 Replicates); Positive Response Equals ACt >9 47
Figure 35. RV-PCR Analysis of B. a. Sterne Spores Recovered from Fluorescent Light
Fixture Sponge-Stick Samples Using Chromosomal and pXOl Gene Targets
(Average ± One Standard Deviation for N > 3 Replicates); Positive Response
Equals ACt >9 47
Figure 36. RV-PCR Analysis of B. a. Sterne Spores Recovered from Overhead Sign
Sponge-Stick Samples Using Chromosomal and pXOl Gene Targets (Average ±
One Standard Deviation for N > 3 Replicates); Positive Response Equals ACt >9 48
Figure 37. RV-PCR Analysis of B. a. Sterne Spores Recovered from Metro Card Machine
Sponge-Stick Samples Using Chromosomal and pXOl Gene Targets (Average ±
One Standard Deviation for N > 3 Replicates); Positive Response Equals ACt >9 48
Figure 38. RV-PCR Analysis of B. a. Sterne Spores Recovered from Subway Car Filter
Grille Sponge-Stick Samples Using Chromosomal and pXOl Gene Targets
(Average ± One Standard Deviation for N > 3 Replicates); Positive Response
Equals ACt >9 49
Figure 39. RV-PCR Analysis of B. a. Sterne Spores Recovered from Field Blank Sponge-
Stick Samples Using Chromosomal and pXOl Gene Targets (Average ± One
Standard Deviation for N > 3 Replicates); Positive Response Equals ACt >9 49
Figure 40. RV-PCR Analysis of B. a. Sterne Spores Recovered from Concrete Sidewalk
Sponge-Stick Samples Using Chromosomal and pXOl Gene Targets (Average ±
One Standard Deviation for N > 3 Replicates); Positive Response Equals ACt >9 50
Figure 41. RV-PCR Analysis of B. a. Sterne Spores Recovered from Electronic Display
Panel (Above Ground) Sponge-Stick Samples Using Chromosomal and pXOl
Gene Targets (Average ± One Standard Deviation for N> 3 Replicates); Positive
Response Equals ACt >9 50
Figure 42. RV-PCR Analysis of B. a. Sterne Spores Recovered from Crosswalk Signal
Sponge-Stick Samples Using Chromosomal and pXOl Gene Targets (Average ±
One Standard Deviation for N > 3 Replicates); Positive Response Equals ACt >9 51
Figure 43. RV-PCR Analysis of B. a. Sterne Spores Recovered from Telephone Booth
Sponge-Stick Samples Using Chromosomal and pXOl Gene Targets (Average ±
One Standard Deviation for N> 3 Replicates); Positive Response Equals ACt >9 51
Figure 44. RV-PCR Analysis of B. a. Sterne Spores Recovered from Street Grate Sponge-
Stick Samples Using Chromosomal and pXOl Gene Targets (Average ± One
Standard Deviation for N > 3 Replicates); Positive Response Equals ACt >9 52
Figure 45. RV-PCR Analysis of B. a. Sterne Spores Recovered from Painted Crosswalk
Sponge-Stick Samples Using Chromosomal and pXOl Gene Targets (Average ±
One Standard Deviation for N > 3 Replicates); Positive Response Equals ACt >9 52
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LIST OF FIGURES (CONT.)
Page
Figure 46. RV-PCR Analysis of B. a. Sterne Spores Recovered from Granite Bench Sponge-
Stick Samples Using Chromosomal and pXOl Gene Targets (Average ± One
Standard Deviation for N > 3 Replicates); Positive Response Equals ACt >9 53
Figure 47. Subway Car Filter (SCFILT) Representative Images, Sample Receipt, Addition
of Extraction Buffer, and Culture Growth on SB A Filter Spiked with 30, 300, or
3,000 Spores (from left to right, respectively) 56
Figure 48. Percent Recovery Efficiencies (Average ± One Standard Deviation of N =5
Replicates) of Presumptive B. a. Sterne Spores from Concrete Floor Vacuum Filter
Cassette Samples Using SBA Medium 57
Figure 49. Percent Recovery Efficiencies (Average ± One Standard Deviation of N = 5
Replicates) of Presumptive B. a. Sterne Spores from Steps (Metal) Vacuum Filter
Cassette Samples Using SBA Medium 57
Figure 50. Percent Recovery Efficiencies (Average ± One Standard Deviation of N = 5
Replicates) of Presumptive B. a. Sterne Spores from Carpet Vacuum Filter Cassette
Samples Using SBA Medium 58
Figure 51. Percent Recovery Efficiencies (Average ± One Standard Deviation of N = 5
Replicates) of Presumptive B. a. Sterne Spores from Subway Car Filter Vacuum
Filter Cassette Samples Using SBA Medium 58
Figure 52. Percent Recovery Efficiencies (Average ± One Standard Deviation of N = 5
Replicates) of Presumptive B. a. Sterne Spores from Concrete Sidewalk Vacuum
Filter Cassette Samples Using SBA Medium 59
Figure 53. Percent Recovery Efficiencies (Average ± One Standard Deviation of N = 5
Replicates) of Presumptive B. a. Sterne Spores from Pavement Vacuum Filter
Cassette Samples Using SBA Medium 59
Figure 54. Percent Recovery Efficiencies (Average ± One Standard Deviation of N = 5
Replicates) of Presumptive B. a. Sterne Spores from Field Blank Vacuum Filter
Cassette Samples Using SBA Medium 60
Figure 55. Vacuum Filter Cassettes (Top Left to Right: Subway Car Filter, Pavement, Floor
Concrete; Bottom Left to Right: Carpet, Steps, Sidewalk Concrete) 61
Figure 56. RV-PCR Analysis of B. a. Sterne Spores Recovered from Concrete Floor
Vacuum Filter Cassette Samples Using Chromosomal and pXOl Gene Targets
(Average ± One Standard Deviation for N > 3 Replicates); Positive Response
Equals ACt >9 64
Figure 57. RV-PCR Analysis of B. a. Sterne Spores Recovered from Steps (Metal) Vacuum
Filter Cassette Samples Using Chromosomal and pXOl Gene Targets (Average ±
One Standard Deviation for N > 3 Replicates); Positive Response Equals ACt >9 65
Figure 58. RV-PCR Analysis of B. a. Sterne Spores Recovered from Carpet Vacuum Filter
Cassette Samples Using Chromosomal and pXOl Gene Targets (Average ± One
Standard Deviation for N > 3 Replicates); Positive Response Equals ACt >9 65
Figure 59. RV-PCR Analysis of B. a. Sterne Spores Recovered from Subway Car Filter
Vacuum Filter Cassette Samples Using Chromosomal and pXOl Gene Targets
(Average ± One Standard Deviation for N > 3 Replicates); Positive Response
Equals ACt >9 66
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LIST OF FIGURES (CONT.)
Page
Figure 60. RV-PCR Analysis of B. a. Sterne Spores Recovered from Concrete Sidewalk
Filter Vacuum Filter Cassette Samples Using Chromosomal and pXOl Gene
Targets (Average ± One Standard Deviation for N > 3 Replicates); Positive
Response Equals ACt >9 66
Figure 61. RV-PCR Analysis of B. a. Sterne Spores Recovered from Pavement Vacuum
Filter Cassette Samples Using Chromosomal and pXOl Gene Targets (Average ±
One Standard Deviation for N > 3 Replicates); Positive Response Equals ACt >9 67
Figure 62. RV-PCR Analysis of B. a. Sterne Spores Recovered from Field Blank Vacuum
Filter Cassette Samples Using Chromosomal and pXOl Gene Targets (Average ±
One Standard Deviation for N > 3 Replicates); Positive Response Equals ACt >9 67
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LIST OF APPENDICES
Page
APPENDIX A: TARGET SURFACES A-l
APPENDIX B: FORMULATIONS OF RECIPES USED IN BIOLOGICAL TEST
METHODS B-l
APPENDIX C: WORK INSTRUCTION FOR SPIKING WITH BACILLUS
ANTHRACIS STERNE SPORES-SPG STICKS C-l
APPENDIX D: WORK INSTRUCTION FOR SPIKING WITH BACILLUS
ANTHRACIS STERNE SPORES-VCF D-l
APPENDIX E: WORK INSTRUCTION TOK BACILLUS ANTHRACIS STERNE
SPORE RECOVERY-SPG STICKS E-l
APPENDIX F: WORK INSTRUCTION TOK BACILLUS ANTHRACIS STERNE
SPORE RECOVERY-VCF F-l
APPENDIX G: WORK INSTRUCTION FOR CULTURE OF BACILLUS
ANTHRACIS SPORES RECOVERED FROM SPG STICKS G-l
APPENDIX H: WORK INSTRUCTION FOR CULTURE OF BACILLUS
ANTHRACIS SPORES RECOVERED FROM VCF H-l
APPENDIX I: WORK INSTRUCTION FOR MANUAL DNA EXTRACTION AND
PURIFICATION FROM BACILLUS ANTHRACIS SPORES 1-1
APPENDIX J: WORK INSTRUCTION RV-PCR TOK BACILLUS ANTHRACIS
SPORES-SPG STICKS J-l
APPENDIX K: WORK INSTRUCTION RV-PCR IO R HA CILL US AMI IRA CIS
SPORES-VCF K-l
APPENDIX L: WORK INSTRUCTION FOR SELECTING PRESUMPTIVE
BACILLUS ANTHRACIS STERNE COLONIES FOR QPCR
CONFIRMATION L-l
APPENDIX M: WORK INSTRUCTION FOR ENRICHMENT FOR CULTURE
NON-DETECTS-SPG STICKS M-l
APPENDIX N: WORK INSTRUCTION TSB ENRICHMENT FOR CULTURE-VCF . N-l
APPENDIX O: CULTURE RESULTS FOR SPONGE-STICK SAMPLES USING
SHEEP BLOOD AGAR MEDIUM O-l
APPENDIX P: RV-PCR RESULTS FOR SPONGE-STICK SAMPLES USING
CHROMOSOMAL AND pXOl GENE TARGETS P-l
APPENDIX Q: CULTURE RESULTS FOR VCF SAMPLES USING SHEEP BLOOD
AGAR MEDIUM Q-l
APPENDIX R: RV-PCR RESULTS FOR VCF SAMPLES USING CHROMOSOMAL
AND pXOl GENE TARGETS R-l
APPENDIX S: TSB ENRICHMENT PCR RESULTS FOR SPONGE-STICK
SAMPLES S-l
APPENDIX T: TSB ENRICHMENT PCR RESULTS FOR VACUUM FILTER
CASSETTES T-l
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Abbreviations and Acronyms
Acronym
Definition
B. a. Sterne
Bacillus anthracis Sterne
B. anthracis
Bacillus anthracis
BHIB
Brain Heart Infusion Broth
BMBL
Biosafety in Microbiological and Biomedical Laboratories
BSC
Biological Safety Cabinet
°C
Degree(s) Celsius
CDC
Centers for Disease Control and Prevention
CFU
Colony Forming Unit(s)
Ct
Threshold Cycle
dH20
Distilled Water
DNA
Deoxyribonucleic Acid
DOHMH
Department of Health and Mental Hygiene
EPA
U.S. Environmental Protection Agency
ERLN
Environmental Response Laboratory Network
FAM
Fluorescent reporter dye on 5' end of PCR probe (6-carboxyfluorescein); emits at
-517 nm
HVAC
Heating, Ventilation, and Air Conditioning
ID
identification
L
Liter(s)
jiL
Microliter(s)
MCE
Methyl Cellulose Ester
min
minute
mL
Milliliter(s)
ModG
Modified G
NRF
National Response Framework
NTC
No Template Control
NYC
New York City
PBS
Phosphate Buffered Saline
PBST
Phosphate Buffered Saline plus 0.05% Tween
PC
Positive Control
PCR
Polymerase Chain Reaction
PE
Performance Evaluation
Pg
picogram
PMP
Paramagnetic Particle
PVDF
Polyvinyldiene Difluoride
QA
Quality Assurance
QAPP
Quality Assurance Project Plan
QC
Quality Control
QMP
Quality Management Plan
qPCR
quantitative PCR
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Abbreviations and Acronyms (Cont.)
Acronym
Definition
rcf
relative centrifugal force
rpm
revolution(s) per minute
RV-PCR
Rapid Viability PCR
RWI
Real-World Interferent
SBA
Sheep Blood Agar
SOP
Standard Operating Procedure
T&E II
Testing and Evaluation II Program
TOCOR
Task Order Contracting Officer's Representative
TSA
Technical Systems Audit
TSB
Trypticase Soy Broth
VFC
Vacuum Filter Cassette
VIC
Fluorescent reporter dye on 5' end of PCR probe (emits at -551 nm)
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Acknowledgements
This document was developed by the 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. Worth Calfee, National Homeland Security Research Center
Dr. Sanjiv Shah, National Homeland Security Research Center
Mr. Leroy Mickelsen, Office of Land and Emergency Management
Dr. Sang Don Lee, National Homeland Security Research Center
Mr. Francisco Cruz, Office of Compliance and Enforcement Assurance
New York City Department of Health and Mental Hygiene
Ms. Kobria Karim
Dr. Joel Ackelsberg
New York City Transit
Mr. Mike Gemelli
Battelle Memorial Institute
Mr. Scott Nelson
Mr. Anthony Smith
Ms. Hiba Shamma
Dr. Ryan James
Mr. Zachary Willenberg
Dr. Rachel Spurbeck
Dr. Aaron Wenzel
Ms. Delaney Pfister
Ms. Jennifer Beare
Mr. Nate Russart
Mr. Kent Hofacre
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EXECUTIVE SUMMARY
Under Emergency Support Function #10 of the National Response Framework (NRF), the
United States Environmental Protection Agency (EPA) is responsible for the remediation of land
and public infrastructure following a biological contamination incident such as an act of
bioterrorism involving the release of Bacillus anthracis (B. anthracis) in an urban area. (NRF:
https://www.fema.gOv/media-library/assets/dociiments/l 17791). EPA, in coordination with other
Government agencies, National Laboratories, and Stakeholders have conducted studies to
support preparation for that role, which have included release of surrogates for B. anthracis
spores in outdoor environments and subway stations to better understand the transport of aerosol
releases and to assess models to predict spread of the particles. Further, EPA has developed
analytical methods including microbiological culture, polymerase chain reaction (PCR), and
Rapid Viability (RV) PCR quantification and identification protocols for B. anthracis species
that are used by EPA's Office of Emergency Management Environmental Response Laboratory
Network (ERLN) (Shah, 2017). EPA and the Centers for Disease Control and Prevention (CDC)
have also developed and established surface sampling methods using a wetted Sponge-Stick™
(Rose et al., 2011) and a vacuum filter cassette (VFC) (Calfee, 2013), and associated organism
recovery methods from those sampling media to be used with the developed culture and RV-
PCR analytical methods.
Following a biological contamination incident, the spatial extent of the contamination should be
determined using established sampling and analytical methods such as those noted above. Both
surface sampling methods will also collect ambient particulate matter that has accumulated on
surfaces to be sampled, along with the target organism when surface samples are taken. That
ambient particulate matter can then be present in the sample extract used to identify the presence
of B. anthracis. The collected and recovered ambient particulate matter represent potential real-
world interferents (RWIs) to the EPA culture and molecular analysis methods and may adversely
impact their ability to identify the presence of B. anthracis spores. EPA therefore seeks to assess
the impact of potential RWIs (present in Sponge-Stick and VFC samples) on current culture and
molecular analysis methods. Having an assessment of the impact, if any, will help EPA
understand limitations of the current methods for contaminant spread and extent mapping and
identify possible opportunities or needs for method improvement.
A surface sampling campaign was conducted in the mid-town Manhattan (Times Square and
Grand Central Station areas) in November 2017 using Sponge-Sticks and VFCs to sample target
surfaces representative of those of relevance to EPA for site remediation. The target surfaces
were selected in coordination with EPA, New York City (NYC) Transit personnel, NYC
Department of Health and Mental Hygiene (DOHMH), and local law enforcement. The surface
samples collected contained material that would be representative of such matter that would also
be collected if sampling for B. anthracis, post-bioterrorism incident. The surface sampling was
not being conducted to characterize background organisms or to baseline whether a target
organism was present. No such analyses were performed on the collected samples.
The surface samples collected from the field were sent to the analytical laboratory and stored in
refrigerated conditions (2 to 8°C) until spiked with a known quantity of Bacillus anthracis Sterne
(B. a. Sterne) spores (as a surrogate for other virulent B. anthracis strains) to apply a target of 30,
300, or 3,000 spores per surface sample. The spores were then recovered from the samples, along
with any physically removed material previously collected from the surface sample and
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subsequently analyzed to identify the presence of B. a. Sterne using EPA's culture and RV-PCR
methods. The culture method used Sheep Blood Agar (SBA) as the growth medium. The RV-
PCR method including PCR assays targeting chromosomal and a pXOl plasmid genes was used
with independent analyses of samples run for each assay.
The performance of the culture method was assessed by determining percent recovery efficiency
of presumptive B. a. Sterne spores spiked onto the samples, which was also used to define
frequency of false positives and false negatives. The performance of the molecular method was
assessed by whether a positive identification was made, which was then used to determine a
frequency of accurate identification, false positives, and false negatives.
The foremost conclusion is that the B. anthracis RV-PCR analysis method was very accurate (>
97%) in correctly identifying the presence or absence of B. a. Sterne in Sponge-Stick samples that
had previously collected background material from real-world surface sampling. The culture
method was less accurate (77%) in correctly identifying the presence or absence of B. a. Sterne in
the same Sponge-Stick samples, meaning the presence of real-world material collected during
surface sampling can hinder the culture method performance. The 18 field blank samples
analyzed - 15 samples spiked with B. a. Sterne and 3 samples not spiked - were 100% accurately
identified by the culture method.
Neither the culture nor molecular analytical methods performed as well with surface samples
collected using the VFC compared to the Sponge-Sticks sampling method. The decrease in
performance as measured by the accuracy of properly identifying the presence or absence of
B. a. Sterne spiked onto the samples was attributed primarily to poor physical recovery of
B. a. Sterne from the VFC methyl cellulose ester (MCE) collection substrate and, also, possibly,
to the collected ambient particulate matter. It is possible that the spiking method (drops of a
B. a. Sterne suspension applied directly onto the VFC collection substrate) affected the physical
recovery of organisms for subsequent analysis. Collection of the B. a. Sterne as an aerosol while
simultaneously collecting the ambient particulate matter (as would be the case in an actual
sampling campaign following a bioterrorism incident) may yield improved recovery efficiencies.
This effect, however, may be most apparent when there is an opportunity to collect much ambient
particulate matter.
RV-PCR can be used to positively identify viable B. a. Sterne in presence of complex, dirty
sample matrices from Sponge-Stick surface samples. The background flora and grime collected
on the Sponge-Stick can impact the lower limit of detection and/or suppress the sensitivity of the
B. a. Sterne signal, yet samples with as few as a nominal quantity of 15 B. a. Sterne spores could
be positively identified in the presence of real-world background matter.
The RV-PCR method requires great care and diligence to implement effectively. Most notable,
the method required changing gloves between procedural samples for each step, which is time-
consuming. Glove changing is critical to avoid cross-contamination samples, which would
negatively impact key decisions in the response, response timelines, credibility, and cost.
The results from this study will be useful to those analyzing samples collected following a
bioterrorism incident. The study demonstrates that results from traditional culture-based methods
may be confounded by an overwhelming presence of background flora, obscuring the presence of
B. anthracis spores.
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1.0 INTRODUCTION
1.1 Background
Under Emergency Support Function #10 of the National Response Framework (NRF), the
United States Environmental Protection Agency (EPA) is responsible for the remediation of land
and public infrastructure following a biological contamination incident such as a bioterrorism
incident involving release of Bacillus anthracis (B. anthracis) in an urban area. (NRF:
https://www.fema.gOv/media-library/assets/dociiments/l 17791). EPA, in coordination with other
Government agencies, National Laboratories, and Stakeholders have conducted studies to
support preparation for that role, which have included releases of surrogates in outdoor
environments and subway stations to better understand the transport of aerosol releases and to
assess models to predict spread of the particles. EPA has developed culture, polymerase chain
reaction (PCR), and Rapid Viability (RV) PCR quantification and identification protocols for
B. anthracis for use by EPA's Office of Emergency Management Environmental Response
Laboratory Network (ERLN) (Shah, 2017). EPA and the Centers for Disease Control and
Prevention (CDC) have developed and established surface sampling methods using a wetted
Sponge-Stick (Rose et al., 2011) and a vacuum filter cassette (VFC) (Calfee, 2013), and
associated organism recovery methods from those sampling media to be used with the developed
culture and RV-PCR methods.
Following a biological contamination incident, the spatial extent of the contamination should be
determined using established sampling and analytical methods such as those noted above. Both
surface sampling methods will collect ambient particulate matter that has accumulated on
surfaces, along with the target organism when surface samples are taken. That collected ambient
particulate matter can then be present in the sample extract used to identify the presence of
B. anthracis. The collected and recovered ambient particulate matter represent potential real-
world interferents (RWIs) to the EPA culture and molecular analysis methods and may adversely
impact their ability to identify the presence of B. anthracis spores. It is therefore critical to assess
the impact of potential RWIs (present in Sponge-Stick and VFC samples) on current culture and
molecular analysis methods. Having an assessment of the impact, if any, will help determine
limitations of the current methods for contaminant spread and extent mapping and identify
possible opportunities or needs for method improvement.
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1.2 Objective
The objective of this study was to assess the impact of RWIs collected on Sponge-Stick and VFC
samples on the current EPA-recommended culture and molecular methods for identification of
viable B. anthracis spores in environmental samples.
1.3 Scope
A surface sampling campaign was conducted in mid-town Manhattan (Times Square and Grand
Central Station) in November 2017 using Sponge-Sticks and VFCs to collect particulate matter
that had accumulated on selected surfaces and be representative of such matter that would also
be collected if sampling for a target organism, post-bioterrorism incident. The surface sampling
was not being conducted to characterize background organisms or to baseline whether a target
organism was present. No such analyses were performed on the collected samples.
The surface samples collected from the field were sent to the analytical laboratory and stored in
refrigerated conditions until spiked with Bacillus anthracis Sterne (B. a. Sterne) spores. The
spores were then recovered from the samples, along with any physically removed material
previously collected from the surface sample and analyzed to identify the presence of
B. a. Sterne using EPA culture and RV-PCR methods. (Initially, samples were analyzed using
the 2012 version of the analytical methods, which did not include an enrichment step, but then
the enrichment culture step was added per the 2017 version (2nd Edition) of the EPA's Protocol
for Detection of B. anthracis Spores from Environmental Samples During the Remediation
Phase of an Anthrax Incident so that the analytical methods used here were as consistent as
possible with the current EPA methods). The performance of the culture method was assessed by
determining percent recovery efficiency of presumptive B. a. Sterne spores spiked onto the
samples, which was also used to define frequency of false positives and false negatives. The
performance of the molecular method was assessed by whether a positive identification was
made, and that was then used to determine a frequency of accurate identification, false positives,
and false negatives.
It is important to note that this study was not solely an assessment of the analytical method to
identify B. a. Sterne in the presence of potential RWIs, but rather, the entire method end-to-end
to include physical recovery from the filter media (and other grime or flora associated with the
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filter's operation in its intended use) followed by the B. a. Sterne analytical method. It is that
end-to-end analysis that was the key element to assess method performance. Consequently, the
study provided information on the limitations and opportunity for improvement of the methods
as well as providing a baseline of processing and analyzing samples that may be encountered in
an actual incident response.
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2.0 MATERIALS AND METHODS
2.1 Sampling Methods
Two sampling tools were used to collect samples form target surfaces: Sponge-Sticks and VFCs.
EPA has established sampling protocols for both the methods, which were then summarized in
work instructions for the field team to execute.
2.1.1 Sponge-Sticks
3M Sponge-Sticks™ pre-wetted with a neutralizing buffer (3M Part number SSLIONB) - shown
in Figure 1 - were purchased for sample collection per established EPA sampling methods
(EPA 2013 and Tufts et al., 2014) and CDC's Anthrax Surface Sampling Guide. The Sponge-
Sticks were used to sample a 10 x 10-inch area (defined by a template overlaying the target
surface) following the sampling pattern (30 linear passes over the area in a vertical, horizontal,
and diagonal pattern) defined in the EPA sampling method.
Figure 1. Pre-Wetted Sponge-Stick from 3M Used for Surface Sampling
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2.1.2 Vacuum Filter Cassettes
VFCs, 37-mm-diameter, 0.8 um pore MCE membrane (Part No. SKC 225-3-01) were purchased
for surface sample collection per established EPA sampling methods (Calfee, 2013). An
assembled and disassembled VFC are shown in Figure 2. The VFCs were used to sample a
12 x 12-inch area (defined by a template overlaying the target surface) over a 5-min (300-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 method. A battery-operated personal sampling pump (Leland
Legacy, SKC International, Eighty Four, PA), set to 10 liter/minute (L/min) sampling rate was
used
Figure 2. Vacuum Filter Cassette (37-nim Diameter)
Assembled (Left) and Disassembled (Right) Used for Surface Sampling
2.2 Sampled Surfaces
Both the Sponge-Sticks and VFCs were used to sample target surfaces in the NYC mid-town
Manhattan area (Times Square and Grand Central Station). Representative images of the field
team collecting surface samples with Sponge-Sticks and VFCs are shown in Figure 3 and
Figure 4, respectively. The samples were collected following established EPA sampling
procedures by a sampling team the week of 12 November 2017. Prior to the sampling campaign,
planning, and coordinating meetings with the EPA Task Order Contracting Officer's
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Representative (TOCOR), New York City (NYC) Transit personnel, NYC Department of Health
and Mental Hygiene (DOHMH), and local law enforcement were held in NYC to define priority
target surfaces and coordinate access to locations throughout the Times Square and Grand
Central Station area. The primary rationale and guidelines used to target for sampling included:
• Location of relevance to EPA sites considered for site remediation.
• Having adequate surface area available to collect at least 18 samples (could also include
multiple of the same surface to attain required surface area).
• At least 25% of sampling locations were to be sampled with VFCs.
• At least 50%, but not more than 75%, were to be below ground.
• Locations were dispersed within the Times Square and Grand Central Station vicinity.
• At least two surfaces were to be conducive to sampling with both methods.
Figure 3. Electronic Display Panels (Below Ground) Located in Times Square 42nd Street
Station, Near Track 3 - Sampled with Sponge-Sticks
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Figure 4. Carpet Surface Located by the Jackie O Entrance to Station,
Off 42nd Street - Sampled with Vacuum Filter Cassette
In summary, 23 distinct surfaces were targeted consistent with the above characteristics that also
were consistent with surfaces used during previous EPA sampling campaigns. Three (3) of those
surfaces were sampled using both the Sponge-Stick and YFC methods for a total of 26 distinct
surface/sampling method sets of samples. Field blanks were collected for each the Sponge-Stick
and VFC samples, accounting for 2 of the 26 sample sets collected. The field blanks were
samplers handled in the exact same manner as those samplers used to collect from surfaces,
except that the field blanks never contacted a surface to actively collect a sample (i.e., opened
and exposed to the collection environment, then packaged). They were packaged, shipped, and
stored in the same manner as samples that had been used to sample a surface. Fourteen (14) of
the surfaces were located below ground in areas associated with the subway system, with the
remaining 12 surface locations above ground. Twenty (20) discreet samples were collected for
each surface set comprising 18 samples for subsequent analysis (see Section 2.3) and 2 spare
samples. A total of 520 samples were collected.
A brief summary description, including a photograph, of each target surface and location is
provided in Appendix A.
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2.3 Test Matrix
Each of the collected surface samples described in Sections 2.1 and 2.2 was spiked with
B. a. Sterne spores, extracted, and the extract analyzed to quantify and identify recovered
B. a. Sterne to assess the EPA-provided culture and RV-PCR methods.
The completed test matrices for the Sponge-Stick and VFC samples are given in Table 1 and
Table 2, respectively. In total, 468 surface samples were analyzed, comprising 342 Sponge-Stick
samples and 126 VFC samples.
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 "-A" or "-B"
indicate whether the sample was collected above ground or below ground, respectively. The
target spore load of 0, 30, 300, or 3,000 was the number of spores intended to be spiked onto the
filter. Following physical extraction, the sample volume was split nominally in half to result in 0,
15, 150, and 1,500 B. a. Sterne spores available for each of the two detection methods (culture
and RV-PCR). The method details are discussed in further detail in Section 2.4. Each sample set
of 18 had 5 samples spiked with the target 30, 300, and 3,000 B. a. Sterne spores; the remaining
3 samples that were not purposely spiked (0-load condition) with B. a. Sterne served as negative
controls. The field blanks served as a baseline to represent the expected best-case performance of
the method because of the absence of potentially competing or interfering grime or flora.
Culture and RV-PCR analytical methods were used to detect and/or quantify recovered
B. a. Sterne spores spiked and subsequently recovered in the sample extracts. Sheep Blood Agar
(SBA) was the primary medium used for all culture analyses. Details regarding the analytical
methods are discussed in Section 2.4.
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Table 1. Test Matrix for Sponge-Stick Samples
Sii rl'sice
S;i in pie
Sii rl';iee 11)
T.irsyM Spore
l.oiids onto
liller'"
Niiiiiiiiiil Spores
A\;iil;il)le per
Mel liocl'1"
Keplie;iles
An;il\lic;il Melhori'"
( ill In re
Mnleeuhir
Floor (Tile)
FLTILE-B
0/30/300/3,000
0/15/150/1,500
3/5/5/5
SBA
RV-PCR
Floor
(Concrete)
FLCON-B
0/30/300/3,000
0/15/150/1,500
3/5/5/5
SBA
RV-PCR
Steps
(w/Metal
Grid)
STEPS-B
0/30/300/3,000
0/15/150/1,500
3/5/5/5
SBA
RV-PCR
Wall Tile
WLTILE-B
0/30/300/3,000
0/15/150/1,500
3/5/5/5
SBA
RV-PCR
Glass
Window
GLSWIN-B
0/30/300/3,000
0/15/150/1,500
3/5/5/5
SBA
RV-PCR
Electrical
Display Panel
EDPAN(B)-B
0/30/300/3,000
0/15/150/1,500
3/5/5/5
SBA
RV-PCR
Glass Panel
GLSPAN-B
0/30/300/3,000
0/15/150/1,500
3/5/5/5
SBA
RV-PCR
Fluor Light
Fixture
FLLFIX-B
0/30/300/3,000
0/15/150/1,500
3/5/5/5
SBA
RV-PCR
Overhead
Sign
OHSIGN-B
0/30/300/3,000
0/15/150/1,500
3/5/5/5
SBA
RV-PCR
Metro Card
Machine
MCMACH-B
0/30/300/3,000
0/15/150/1,500
3/5/5/5
SBA
RV-PCR
Subway Car
Filter Grille
SCGRIL-B
0/30/300/3,000
0/15/150/1,500
3/5/5/5
SBA
RV-PCR
Control
(Field Blank)
FLDBLK-A
0/30/300/3,000
0/15/150/1,500
3/5/5/5
SBA
RV-PCR
Sidewalk
Concrete
SWCON-A
0/30/300/3,000
0/15/150/1,500
3/5/5/5
SBA
RV-PCR
Electrical
Display Panel
EDPAN(A)-A
0/30/300/3,000
0/15/150/1,500
3/5/5/5
SBA
RV-PCR
Crosswalk
Signal
CWSIGN-A
0/30/300/3,000
0/15/150/1,500
3/5/5/5
SBA
RV-PCR
Telephone
Booth
TELEBO-A
0/30/300/3,000
0/15/150/1,500
3/5/5/5
SBA
RV-PCR
Street Grating
STGRAT-A
0/30/300/3,000
0/15/150/1,500
3/5/5/5
SBA
RV-PCR
Crosswalk
Painted
CWPNTD-A
0/30/300/3,000
0/15/150/1,500
3/5/5/5
SBA
RV-PCR
Granite
Bench
GRNBEN-A
0/30/300/3,000
0/15/150/1,500
3/5/5/5
SBA
RV-PCR
(a) Target number of spores spiked onto filter - See Section 2.4.2 for discussion.
(b) Nominally half of the target quantity of spores loaded onto the filter were available for each of the two
analytical methods - See Section 2.4.3 for discussion.
(c) SB A medium; RV-PCR assay, chromosomal and pXOl gene targets.
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Table 2. Test Matrix for Vacuum Filter Cassette Samples
Sii rl';iee
Siimplo
Sii rl';iee 11)
Tsirgel Spore
l.oiids onto
Killer1'"
Nuiiiiiiiil Spores
A\:iil
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2.4.1 Spore Bank
B. a. Sterne spores were used as the biological test agent for the entire study. This organism is a
vaccine strain produced by Colorado Serum Company and is frequently used as surrogate to fully
virulent B. anthracis strains such as Ames. The B. a. Sterne strain was handled as a Risk Group II
agent following the Biosafety in Microbiological and Biomedical Laboratories (BMBL)
guidelines and Battelle biosafety work practices for such agents. A spore bank was produced
using sporulation broth as follows and used as needed for the duration of the study.
A cell bank of B. a. Sterne 34F2 prepared previously at Battelle from BEI Resources (BEI
NR-1400) was used to grow an overnight culture on TSB. Isolated colonies were then used to
inoculate 50 milliliter (mL) aliquots of nutrient broth and incubated overnight at 35 to 37 degrees
Celsius (°C) with shaking at 200 revolutions per minute (rpm). Modified G (ModG) (500 mL) of
sporulation broth (see Appendix B, Table 1 for formulation details) was inoculated with 50 mL of
the overnight B. a. Sterne culture, and then incubated in a 3-liter (L) Fernbach flask at 35 to 37°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.
(Note, a spore bank was also prepared using Leighton-Doi medium [see Appendix B, Table 2 for
formulation details], but spores from the ModG medium were used because > 99% sporulation
was not achieved with Leighton-Doi and there was more cellular debris compared to spores
prepared in the ModG medium.)
The sporulated culture was centrifuged at 10,000 relative centrifugal force (rcf) for 12 minutes in
multiple 250-mL bottles. After removing and discarding the supernatant, the resulting pellets
were resuspended to a total volume of approximately 100 mL with sterile distilled water (dFhO),
transferred into a sterile glass vessel, and heat shocked at 60 to 65°C for 1 hour 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 minutes using 100 mL dFhO per
wash. After the final centrifugation, the spores were resuspended to a total volume of 100 mL in
sterile dFhO. The spore bank was assigned a unique lot number and stored refrigerated at 2 to 8°
C.
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2.4.2 Spore Loading (Spiking)
Sponge-Sticks and VFCs were stored at 2 to 8°C prior to being spiked and processed (up to 16
months). All filter manipulations were performed within a surface-decontaminated, certified
biological safety cabinet (BSC) and handled using sterile forceps and scissors.
B. a. Sterne spiking stocks were vortex-mixed and diluted using sterile dFhO to the three spiking
stock target concentrations shown in Table 3. Each spiking stock was spread plated onto SB A on
the day of testing to calculate the actual concentration of spores spiked in colony forming units
(CFU)/mL. The loading levels in Table 3 represent loadings that yielded enough B. a. Sterne
spores in the sample recovery extracts to make meaningful measurements with both the culture
and RV-PCR methods and covered a range that was expected to span their lower limit of
detection or quantification, which was an important consideration to assess whether grime or
flora associated with the samples affected the sensitivity or lower limits of the analytical method.
Table 3. Target B. a. Sterne Spore Loading Levels Spiked onto Each Sample Substrate
Loading
1 .evel
Slock Concentration
(( IT/ml.)
Target Total
( IT per
Sample'1"
Kxtract
Volume
(ml.)
Theoretical
Concentration in
Kxtract (CR/ml.)
nigh
3.U 101
3,000
25
120
Medium
3.0 x 103
300
25
12
Low
3.0 x 102
30
25
1.2
(a) 100 microliter (|iL) of stock suspension applied (20, 5-|iL drops).
Each Sponge-Stick to be spiked with B. a. Sterne spores was positioned in a specimen cup so
that the dirty side was facing up and 20 5-|iL droplets, for a total of 100 |iL of stock suspension,
were pipetted onto the surface of each Sponge-Stick (the sides of the sponge that could contact
the specimen cup wall were not spiked; see Figure 5). For VFCs, the final spiking stock
concentrations were prepared in 50% ethanol and applied as 20 5-|iL droplets distributed over
the surface of collected particulates and filter by removing the plug from the port used to attach
the nozzle and inserting the pipette tip with inoculum (see Figure 5). The spiked Sponge-Sticks
were stored sealed in specimen cups at 2 to 8°C overnight and VFCs were dried overnight inside
of a BSC (fan on) with red inlet plug removed.
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Figure 5. Sponge-Stick (left) and Vacuum Filter Cassette (right)
After Spiking with the B. a. Sterne Suspension
2.4.3 Spore Recovery
Throughout the recovery procedure, gloves were changed between handling samples to limit the
likelihood of cross-contamination between samples.
Sponge-Sticks
Following spiking, 90 mL cold (2 to 8°C) phosphate buffered saline (PBS) extraction buffer with
0.05% Tween 20 and 30% ethanol (final concentrations) was added into a Stomacher® bag. The
remaining handle was removed, and the Sponge-Stick was unfolded and aseptically added to the
Stomacher bag and homogenized for 1 minute at 260 rpm in a Stomacher (Seward). Each sample
then sat for 10 minutes 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. A subset of the
sponges (final 4 trials, 64 samples) were returned to the specimen cup and retained at 2 to 8°C
for TSB enrichment. The remaining -90 mL suspension was gently mixed by pipetting up and
down three times with a sterile 50-mL pipet, then the suspension was split in half and centrifuged
at 3,500 rcf for 15 minutes 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 microbial analysis described in Section 2.4.4, and RV-
PCR analysis as described in Section 2.4.5.
For Sponge-Stick TSB enrichment, the extracted sponge and remaining spore recovery
suspension was saved at 2 to 8°C for the final four trials of analysis. Twenty-five mL of TSB
was added to the saved sponge along with any remaining spore recovery suspension, then
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incubated at 37 ± 2°C for 24 to 48 hours. Samples with turbid TSB broth were then streaked onto
three SBA plates for isolation. Colonies with B. a. Sterne morphology that were isolated on these
streak plates were screened using real-time PCR assays. An aliquot of the TSB broth suspension
(50 |iL) for all TSB-enriched samples was pelleted by centrifugation at 12,000 rcf for 2 minutes,
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 minutes, then screened using real-time PCR assays.
Vacuum Filter Cassettes
Following spiking, 5 mL of extraction buffer with 0.05% Tween 20 and 30% ethanol (extraction
buffer) was added to the conical tube containing the nozzle and tubing and set aside. Six (6) mL
total of extraction buffer was used to rinse and recover particulates collected within the VFC by
adding 2 mL of extraction buffer in three successive rinse steps. Following the second rinse step,
the filter was transferred to the 2 oz. cup containing rinsate. The nozzle and tubing containing
5 mL extraction buffer was sonicated in a sonicating bath for 1 minute, then vortexed for
2 minutes and combined with filter rinsate in the 2 oz. cup. The 2 oz. cup containing filter and
11 mL of extraction buffer were sonicated in a sonicating bath for 3 minutes. 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 microbial analysis described in
Section 2.4.4, and RV-PCR analysis as described in Section 2.4.5.
For VFC TSB enrichment, all filters were enriched following spore recovery within the 2 oz. cup
by adding 30 mL of TSB, then incubated at 37 ± 2°C for 24 to 48 hours. Turbid TSB broth was
then streaked onto three SBA plates for isolation. Colonies with B. a. Sterne morphology that
were isolated on these streak plates were screened using real-time PCR assays. An aliquot of the
TSB broth suspension (50 |iL) for all TSB-enriched samples was pelleted by centrifugation at
12,000 rcf for 2 minutes, 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 minutes, then screened using
real-time PCR assays.
2.4.4 Culture Method
Culture-based microbiological analysis was performed on each sample by filtering the recovered
extract through MicroFunnel filters (Pall, Cat. 4804) then placing the filters onto solid bacterial
growth media and incubating. Serial dilution and spread-plating procedures, as prescribed by the
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full EPA B. anthracis method (EPA, 2012), were not performed since the spike levels were
at/near the detection limit for the assay (i.e., spread-plating 0.1 mL of the undilute extract from a
Sponge-Stick spiked with 3,000 spores would have resulted in -12 CFU if 100% efficient).
Accordingly, milliliter volumes of the recovered extract were captured onto MicroFunnel filters
in the current study.
Initially, each MicroFunnel filter was pre-wetted with 5 mL of phosphate buffered saline (PBS)
with 0.05% Tween (PBST), then 10 mL of PBST was added to each MicroFunnel filter to
suspend aliquots, 2-mL and 8-mL for Sponge-Sticks and 1-mL and up to 4-mL for VFCs, of the
extract followed by vacuum filtration. The walls of each MicroFunnel filter were rinsed with
10 mL of PBST and filtered through the MicroFunnel filter, then the filter membrane was
removed and placed onto SBA media.
For the culture method, colonies with a typical B. a. Sterne morphology following overnight
incubation at 35 to 37°C were counted to determine percent spore recovery. Typical B. a. Sterne
morphology on SBA are 2 to 5 mm in diameter, flat or slightly convex with edges that are
irregular, have a ground-glass appearance, and are not B-hemolytic.
For the final four Sponge-Stick trials, the extracted sponge was retained and stored at 2 to 8°C.
For samples that did not have B. a. Sterne morphology (culture non-detects), 25 mL of TSB was
added to the extracted sponge and incubated at 37 ± 2°C for 24 to 48 hours. Following
incubation, turbid cultures were streaked for isolation onto SBA plates and examined. If no
B. a. Sterne colonies were observed, 50 |iL of the broth was concentrated and analyzed using
real-time PCR assays.
Two different microbiologists enumerated colonies over the course of the project, all of whom
were trained by the lead microbiologist on the project to most consistently identify presumptive
B. a. Sterne 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. There were instances of the presence of presumptive
B. a. Sterne on samples that should not have any (false positive) and no colonies where there
should have been (false negatives). All the results presented for culture analyses are based on
presumptive identification of B. a. Sterne colonies.
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2.4.5 RV-PCR Method
Positive Control Preparation
Genomic deoxyribonucleic acid (DNA) of B. a. Sterne was extracted for use as a positive control
for RV-PCR based analysis. The B. a. Sterne 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 Quant-iT™ PicoGreen™ dsDNA
Assay Kit (Invitrogen, 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.
Sample Processing (per EPA Method 2017, Second Edition (Shah, 2017))
Following filtration of-12.5 mL of recovered extract through the Whatman™ Autovial™ filter
vials (with polyvinyldiene difluoride [PVDF] membrane; Whatman Cat. AV125NPUAQU), two
buffer washes were performed. The first wash was 12.5 mL of cold (4°C) high salt buffer
(10X PBS) followed by 12.5 mL of cold (4°C) low salt wash buffer (IX PBS). The top portion
of the manifold was then removed and placed into a capping tray with pre-filled luer lock caps to
seal the filter vials. Five (5) mL of cold (4°C) Brain Heart Infusion Broth (BHIB) was then
added to each filter vial, the vials capped, and then vortex-mixed for 10 minutes on a setting of 7.
Images of the manifold and capping tray are depicted in Figure 6. Following the vortex step, the
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 hours) in an incubator shaker set to 37 ± 1°C at 230 rpm.
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EPA/6Q0/R-19/083
June 2019
Figure 6. Manifold Containing 16 Filter Vials (Top); Capping Tray (Middle);
Capped Filter Vials Containing BHIB (Bottom)
Following overnight incubation (-16 hours) of the filter vials with BHIB, the vials were mixed
on the platform vortex for 10 minutes with speed set to 7. (Note, the 16-hour incubation was
longer than the 9-hr incubation specified in the "U.S. EPA Protocol for Detection of Bacillus
aiithracis in Environmental Samples During Remediation Phase of an Anthrax Incident" in the
2012 version. The 2017, 2nd Edition of the protocol specified 9 hours, or longer. The 16-hour
incubation allowed for a standard work schedule to be maintained rather than require an
overnight shift that would have been required by a 9-hour 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 (Tfmai) aliquot.
DNA Extraction and Purification
Prior to extraction of DNA, the lysis buffer with anti-foam reagent and the alcohol wash was
added according to the manufacturer's instructions in the Magnesil Blood Genomic, Max Yield
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EPA/600/R-19/083
June 2019
System Kit (Promega, Cat. MD1360) and a heat block was pre-heated to 80°C. All screw-
capped, 1-mL aliquots were thawed and centrifuged at 14,000 rpm (18,188 rcf) for 10 minutes
(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 second pulses for a total of 60 seconds. Each tube was then vortex-
mixed for 10 seconds at low speed directly before the lysate was transferred to a 2-mL labeled
Eppendorf tube. The lysate tube was then incubated at room temperature for 5 minutes.
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 Tfmaitube for 10 seconds
(high, -1,800 rpm), the samples were incubated at room temperature for 5 minutes.
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 seconds, the tubes were
opened, and the liquid removed without disturbing the PMPs. Lysis buffer (360 |iL) was then
added to each To and Tfmaitube, capped, and vortexed for 10 seconds. 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 seconds,
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 seconds, 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 minutes. The open tubes were then heated at 80°C in a heat block
inside a BSC until the PMPs were dry (-20 minutes). 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 seconds, and incubated in the heat block for 80 seconds. The tubes were then
vortexed another 10 seconds and incubated in the heating block for a minute. The vortexing and
heating for 1 minute was 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 minutes. Each
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EPA/600/R-19/083
June 2019
tube was briefly vortexed and then centrifuged at 2,000 rpm (371 rcf) at 4°C for 1 minute. The
tubes were then vortexed and placed on the magnetic stand for at least 30 seconds. The elute was
collected (-80 to 90 |iL) and transferred to clean, labeled, 1.5-mL tubes on a cold block. The
tubes were centrifuged at 14,000 rpm (18,188 rcf) at 4°C for 5 minutes 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 hours.
R V-PCR Assay
The EPA protocol originally provided (EPA, 2012) uses singleplex, real-time PCR assays for
B. anthracis detection and quantification. Battelle assessed the feasibility to combine two
singleplex assays targeting the chromosome and pXOl assays described in the EPA protocol into
a duplex assay to reduce analysis time and cost associated with filter extract analysis. It was
previously demonstrated that the RV-PCR performance was unchanged when conducted using
the duplex assay in a single analysis or using the singleplex assays in two independent analyses.
The duplex TaqMan® real-time PCR assay utilized FAM and VIC reporter dyes for detection of
two B. a. Sterne DNA sequence targets simultaneously in a single reaction. (FAM and VIC are
Applied Biosystems trademark fluorescent reporter dyes on 5' end of PCR probe that emit at
-517 nm and -551 nm, respectively.) The two assays target sequences on the B. anthracis
chromosome and pXOl plasmid and were previously described as singleplex real-time PCR
assays (Letant et al., 2011). The duplex PCR assay Master Mix was prepared using the
conditions provided in Appendix B. Each sample DNA extract was assayed in triplicate
reactions. Controls consisted of four positive control wells containing 50 picogram (pg) of DNA
extracted from B. a. Sterne 34F2 (NR-1400, BEI Resources) and four no template controls
(NTCs) were also included with each assay. 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 95°C for 20 sec
• Stage 2: 45 cycles at 95°C for 3 sec followed by 60°C for 30 sec
Note, the Stage 1 cycle conditions were slightly modified from the original EPA method and
concurrence to proceed with the revisions was provided by EPA (Shah, 2018).
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EPA/600/R-19/083
June 2019
volume was then split between the traditional culture method and RV-PCR. The culture aliquot
was divided into 2-mL or 8-mL aliquots for Sponge-Sticks and 1-mL or < 3-mL aliquots
(remaining volume without transferring settled particulates) for VFCs and plated onto media and
incubated overnight as outlined in the "Work Instruction for Culture of Bacillus anthracis Spores
Recovered - Sponge-Sticks or VFC" in Appendix G or H, respectively. The To RV-PCR aliquot
was stored frozen while the recovered spores enrich 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 Bacillus anthracis" in Appendix I. The extracted
DNA was then analyzed using a duplex real-time PCR assay targeting the chromosome and
pXOl of B. anthracis per "Work Instruction for RV-PCR for Bacillus anthracis Spores -
Sponge-Sticks or VFC" in Appendix J or K, respectively. PCR was also used to confirm or refute
presumptive B. a. Sterne spores selected from the culture analysis per "Work Instruction for
Selecting Presumptive B. a. Sterne Colonies for qPCR Confirmation" in Appendix L. Selected
samples for which the culture was a non-detect were further analyzed using an enrichment
procedure per "Work Instruction for TSB Enrichment for Culture non-Detects - Sponge-Sticks
or VFC," in Appendix M or N, respectively.
2.6 Data Reduction and Analysis
2.6.1 Culture — Percent Recovery
The percent recovery efficiency (Erecovery) of B. a. Sterne from each spiked surface sample was
calculated by dividing the number of presumptive B. a. Sterne CFUs recovered (Nrecover) from the
filter by the actual number of B. a. Sterne spores spiked (Nspike) onto the filter (determined from
the stock suspension titer for each test), then multiplied by 100. Nrecover is a product of the
presumptive B. a. Sterne 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 v extract
Erecovery(./o) = * 100%
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EPA/600/R-19/083
June 2019
Further, the number of presumptive B. a. Sterne spores present in the volume of extract collected
onto the MicroFunnel filter membrane was divided by the extract volume analyzed; 2 mL and
8 mL for Sponge-Sticks or 1 mL and < 3 mL for VFCs to yield a presumptive B. a. Sterne spore
concentration (Crecover) (CFU/mL). The extract volume (Vextract) (~25 mL for Sponge-Sticks and
11 mL for VFCs) were used to determine B. a. Sterne CFUs recovered from the filter sample.
The percent recovery was calculated for both the low-volume (2 mL or 1 mL for Sponge-Sticks
and VFCs, respectively) and high-volume (8 mL or < 3 mL for Sponge-Sticks and VFCs,
respectively) aliquots. The reported percent recovery was determined using the below rules:
1) Report the percent recovery from the aliquot (low-volume or high-volume) that has
between 20 to 80 CFU.
2) Report the high-volume aliquot percent recovery if the CFU counted from both
aliquots is less than 20.
3) Report the high-volume aliquot percent recovery if the CFU counted from both
aliquots is between 20 to 80.
4) Report the low-volume aliquot percent recovery if the background flora on the high-
volume aliquot produces numerous colonies or a lawn of growth, thus complicating the
identification of B. a. Sterne colonies.
The number of CFUs are reported as presumptive B. a. Sterne colonies. PCR analysis of
presumptive colonies was required to positively confirm the presence of B. a. Sterne. To perform
this task, a portion (-10 CFUs per trial) of the presumptive colonies was collected into 100 |iL of
PCR-grade water in microcentrifuge tubes. The colony suspension was then heated for 5 minutes
on a heat block at 95°C. The lysate was cooled and then centrifuged at 14,000 rpm (18,188 rcf)
for 2 minutes and the supernatant was analyzed using the real-time PCR assays targeting the B.
anthracis chromosome and pXOl gene targets.
2.6.2 RV-PCR
The cycle threshold (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 Tfmai aliquot from the average Ct (triplicate reactions) value generated by the To
aliquot. A positive ACt value indicates that viable B. a. Sterne spores were detected in the sample
if all the below acceptance criteria were met:
• The ACt must be greater than or equal to 9 for both the chromosome and pXOl targets
(ACt = Ct (To) - Ct (Tfinal) > 9)
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EPA/600/R-19/083
June 2019
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 (i.e., three PCR targets
(chromosome, pXOl, and pX02) are utilized for RV-PCR analysis of fully virulent Bacillus
anthracis).
2.6.3 Presentation of Results
The method employed to recover B. a. Sterne spores spiked onto the samples was consistent with
current EPA methods, as described in Section 2.4.4. In the instance of an actual biological
release, the entire extract would be analyzed either using a culture method or a RV-PCR method
(Calfee, 2018). In the study performed and reported here, however, the sample extract was split
as described in Section 2.4.4 and 2.4.5, so that approximately half of the sample extract was used
for culture analysis and the other half for RV-PCR analysis. Consequently, neither the culture
nor the RV-PCR had the potential maximum quantity (assuming 100% recovery efficiency from
the filter) of spores available in the extract for analysis. Rather, each split sample extract 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 axes labels denote the nominal maximum number of spores
available in the sample for its respective analysis, which was half of the target spore load.
For example, results were presented in plots of both spore recovery efficiency for the culture
analyses and of ACt for RV-PCR analyses with an x-axis title of "Nominal Spores Available for
Analysis (CFU)" with an x-axis label of 0, 15, 150, and 1,500. 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 filter types and analytical
methods, recognizing that the filters were originally spiked with target quantities of 0, 30, 300,
and 3,000 5. a. Sterne spores.
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EPA/600/R-19/083
June 2019
As described in Section 2.4.2, the samples were spiked with a target quantity of spores by
applying twenty (20) 5-|iL drops of a B. a. Sterne spore stock suspension with a target titer of
30,000 spores/mL, diluted in log increments. The reported spore load for each filter analyzed
was based on the B. a. Sterne spore suspension titer measured for each test trial in CFU. As
expected, there was variability in the measured spore titer for each trial. Consequently, the
summary tables of results also contain the average (± one standard deviation) of the measured or
determined quantity of spores spiked onto the filter, which provides the reader with information
other than the nominal spore load as defined in the test matrices to aide with interpretation of the
results.
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EPA/600/R-19/083
June 2019
3.0 RESULTS AND DISCUSSION
As described in the previous section, all results presented in plots have an x-axis title and labels
of 0, 15, 150, and 1,500 CFU representing the nominal spores available for analysis. Similarly,
the summary results in the tables contain the same nominal quantity of spores available, and the
determined quantity of spores applied to the surface sampler substrate being assessed. This
convention of presenting the results was considered the most accurate and consistent
representation and allowed for the most unambiguous discussion and interpretation of results
across all the filter types and analytical methods, recognizing that the filters were originally
spiked with target quantities of B. a. Sterne spores of 0, 30, 300, and 3,000 but extract samples
were split in approximately equal volume for the two analyses.
Note, the spores available for analysis represent the maximum number of spores available
(assumes 100% recovery from the filter and no physical losses associated with processing of
samples); it is not an absolute indication of the analytical method's limit of identification. Rather,
it is a measure of the method's end-to-end performance to identify B. a. Sterne.
3.1 Sponge-Stick Analyses Results
3.1.1 Culture Method
A summary of the average and standard deviation of the measured recovery efficiencies of
presumptive B. a. Sterne spores recovered from the Sponge-Stick samples spiked with
B. a. Sterne and using SBA medium are presented in Table 4. The determined number of spores
available and the number of presumptive B. a. Sterne spores recovered are tabulated along with
the nominal quantity of spores available for analysis (15, 150 and 1,500 CFU/filter sample). The
presumptive B. a. Sterne recovery efficiencies on the SBA plates are plotted in Figure 8 through
Figure 25, one plot for each surface sampled with a Sponge-Stick. (There is no plot for the Street
Grate surface since the recovery efficiency was 0% for all nominal spores available conditions.)
Note, a percent recovery is not tabulated nor plotted for the 0-spore spike condition since, by
definition, a meaningful recovery efficiency cannot be calculated, even though there could have
been presumptive B. a. Sterne colonies counted based on colony morphology. However, any
presumptive B. a. Sterne CFUs for the 0-spike condition were reported in Table 4. The quantity
of presumptive B. a. Sterne colonies for each Sponge-Stick sample used in the percent recovery
calculations are reported in Appendix O.
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EPA/600/R-19/083
June 2019
The percent recovery efficiency for presumptive B. a. Sterne spores generally range between 10
to 50% for most surface samples for all nominal spores available condition (15, 150, and 1,500)
with no consistent trend related to the quantity of spores available for analysis. The percent
recoveries were within a range of 15 to 40% for the 150 and 1,500 nominal spores available
condition, which includes the field blank samples that had an average percent recovery of 52, 40,
and 36%) for a nominal B. a. Sterne spore load of 15, 150, and 1,500, respectively. The Street
Grate and Painted Crosswalk samples were the only exception to this result. The Street Grate
samples, regardless of the nominal spore load, had no identifiable presumptive B. a. Sterne
recovered. The Painted Crosswalk had no identifiable presumptive B. a. Sterne recovered at 15
nominal spore load and very few at the 150 and 1,500 spores available condition, resulting in
recovery efficiencies < 10%>. In some instances, the culture plates had organism growth (not of a
B. a. Sterne morphology) resulting in > 100 colonies that was likely masking colonies of a
B. a. Sterne morphology. When no presumptive B. a. Sterne spores were identified from spike
samples, the result was noted as a false negative.
Sponge-Stick sample recovery efficiencies associated with a nominal 15 5. a. Sterne spores
available generally had a higher standard deviation than those samples with the 150 and 1,500
nominal spores available condition, which was attributed to the relatively few (< 10) recovered
presumptive B. a. Sterne colonies. For the Steps (Metal) surface sample the average recovery
efficiency exceeded 100%>, which is clearly an over-estimation of recovered spores. The
infeasibly high spore recovery efficiencies (> 100%>) was attributed to presence of background
flora on those filters with a colony morphology that was indistinguishable from B. a. Sterne, and
thus counted as a presumptive B. a. Sterne spore. On average, with 100%> recovery, the 8-mL
aliquot plated would have 5 spores to enumerate. Samples with the 150 and 1,500 spore
condition generally have a lower standard deviation compared to the 15-spore condition because
more actual B. a. Sterne spores are available to enumerate. As discussed in Section 2.4.4, the
method for determining the number of B. a. Sterne spores recovered was determined based on
colony morphology, and thus susceptible to biasing high due to non-5, a. Sterne organisms
exhibiting an indistinguishable morphology to the microbiologist counting the colonies.
-------
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Floor Tile (Sponge Stick)
\
Nominal Spores Available for Analysis (CFU)
Figure 8. Percent Recovery Efficiencies (Average ± One Standard Deviation of N = 5
Replicates) of Presumptive B. a. Sterne Spores from Floor Tile Sponge-Stick Samples Using
SBA Medium
Concrete Floor (Sponge Stick)
100
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Figure 9. Percent Recovery Efficiencies (Average ± One Standard Deviation of N = 5
Replicates) of Presumptive B. a. Sterne Spores from Concrete Floor Sponge-Stick Samples
Using SBA Medium
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EPA/6Q0/R-19/083
June 2019
Steps {Metal Grid) (Sponge Stick)
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Figure 10. Percent Recovery Efficiencies (Average ± One Standard Deviation of N = 5
Replicates) of Presumptive B. a. Sterne Spores from Steps (Metal) Sponge-Stick Samples
Using SBA Medium
Wall Tile Sponge Stick
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Nominal Spores Available for Analysis (CFU)
Figure 11. Percent Recovery Efficiencies (Average ± One Standard Deviation of N = 5
Replicates) of Presumptive/?, a. Sterne Spores from Wall Tile Sponge-Stick Samples Using
SBA Medium
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EPA/6Q0/R-19/083
June 2019
100
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Figure 12. Percent Recovery Efficiencies (Average ± One Standard Deviation of N = 5
Replicates) of Presumptive B. a. Sterne Spores from Glass Window Sponge-Stick Samples
Using SBA Medium
100
E 90
Electronic Display Panel (Below Ground) (Sponge Stick)
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Figure 13. Percent Recovery Efficiencies (Average ± One Standard Deviation of N = 5
Replicates) of Presumptive B. a. Sterne Spores from Electronic Display Panel (Below
Ground) Sponge-Stick Samples Using SBA Medium
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EPA/6Q0/R-19/083
June 2019
100
£ 90
Glass Panel (Sponge Stick)
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70
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50
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Nominal Spores Available for Analysis (CFU)
Figure 14. Percent Recovery Efficiencies (Average ± One Standard Deviation of N = 5
Replicates) of Presumptive B. a. Sterne Spores from Glass Panel Sponge-Stick Samples
Using SBA Medium
100
S 90
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Fluorescent Light Fixture (Sponge Stick)
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70
60
50
40
30
20
10
Nominal Spores Available for Analysis (CFU)
Figure 15. Percent Recovery Efficiencies (Average ± One Standard Deviation of N = 5
Replicates) of Presumptive B. a. Sterne Spores from Fluorescent Light Fixture Sponge-
Stick Samples Using SBA Medium
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EPA/6Q0/R-19/083
June 2019
100
£ 90
Overhead Sign (Sponge Stick)
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70
60
50
40
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Nominal Spores Available for Analysis (CFU)
Figure 16. Percent Recovery Efficiencies (Average ± One Standard Deviation of N = 5
Replicates) of Presumptive B. a. Sterne Spores from Overhead Sign Sponge-Stick Samples
Using SBA Medium
Metro Card Machine (Sponge Stick)
100
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-------
EPA/6Q0/R-19/083
June 2019
Subway Car Air Filter Grille (Sponge Stick)
100
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Figure 18. Percent Recovery Efficiencies (Average ± One Standard Deviation of N = 5
Replicates) of Presumptive B. a. Sterne Spores from Subway Car Filter Grille Sponge-Stick
Samples Using SBA Medium
Field Blanks (Sponge Stick)
100
~ 90
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80
70
60
50
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Figure 19. Percent Recovery Efficiencies (Average ± One Standard Deviation of N = 5
Replicates) of Presumptive B. a. Sterne Spores from Field Blank Sponge-Stick Samples
Using SBA Medium
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EPA/6Q0/R-19/083
June 2019
Concrete Sidewalk {Sponge Stick)
100
90
o
20
10
Nominal Spores Available for Analysis (CFU)
Figure 20. Percent Recovery Efficiencies (Average ± One Standard Deviation of N = 5
Replicates) of Presumptive B. a. Sterne Spores from Concrete Sidewalk Sponge-Stick
Samples Using SBA Medium
Electonic Display Panel (Above Ground) (Sponge Stick)
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Figure 21. Percent Recovery Efficiencies (Average ± One Standard Deviation of N = 5
Replicates) of Presumptive B. a. Sterne Spores from Electronic Display Panel (Above
Ground) Sponge-Stick Samples Using SBA Medium
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EPA/6Q0/R-19/083
June 2019
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Nominal Spores Available for Analysis (CFU)
Figure 22. Percent Recovery Efficiencies (Average ± One Standard Deviation of N = 5
Replicates) of Presumptive B. a. Sterne Spores from Crosswalk Signal Sponge-Stick
Samples Using SBA Medium
100
90
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EPA/6Q0/R-19/083
June 2019
Painted Crosswalk (Sponge Stick)
100
90
80
70
60
50
E 40
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20
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Nominal Spores Available for Analysis (CFU)
Figure 24. Percent Recovery Efficiencies (Average ± One Standard Deviation of N = 5
Replicates) of Presumptive B. a. Sterne Spores from Painted Crosswalk Sponge-Stick
Samples Using SBA Medium
Granite Bench
100
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Figure 25. Percent Recovery Efficiencies (Average ± One Standard Deviation of N = 5
Replicates) of Presumptive B. a. Sterne Spores from Granite Bench Sponge-Stick Samples
Using SBA Medium
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EPA/6Q0/R-19/083
June 2019
A subset of colonies recovered were screened using real-time PCR assays targeting the
chromosomal and pXOl gene targets. A total of 229 colonies isolated from Sponge-Sticks were
screened; of those colonies screened, 93% (213 correct) were confi rmed as correctly identified.
Overall, background flora interfered with identification of presumptive B. a. Sterne from Street
Grating (STGRAT) to a greater degree than the other surfaces. All STGRAT samples had
background flora counts of greater than 83 colonies. A few representative images of SBA plates
from STGRAT are shown in Figure 26.
Figure 26. Sponge-Stick Samples from Street Grating Contained Background Flora that
Interfered with Identification of B. a. Sterne Morphology to a Greater Degree Compared to
Other Surfaces (both images were inoculated with 2 mL of extract).
The presence of material interfering with the analysis is not surprising considering that the
Sponge-Sticks surface samples were noticeably dirty as shown in Figure 27.
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Figure 27. Sponge-Stick Samples: Subway Car Filter Grille (Top Left); Steps (Top Right);
Crosswalk Signal (Bottom Left); Telephone Booth (Bottom Right)
3.1.2 Sponge-Stick TSB Enrichment
For Sponge-Sticks, there were instances where a colony with B. a. Sterne morphology was
isolated from turbid TSB broth when streaked for isolation on SBA; those isolated colonies were
screened using real-time PCR assays. In all cases the screened colonies were real-time PCR
negative. For TSB broth suspension analysis using real-time PCR assays, 3 of the 21 Sponge-
Stick samples that were screened resulted in average Ct values of < 40 for at least 2 of 3
replicates for both chromosome and pXOl real-time PCR assays (positive) and the other 18
samples were negative (Ct value of > 40 for 2 of 3 replicates for both real-time PCR assays). All
3 positive samples were collected from the Street Grating surface, one each spiked with 30, 300,
and 3,000 spore loads. The average Ct values from these three samples ranged from 36.5 to 40.3.
For each of these samples, the Ct values generated from RV-PCR were lower than those
generated during the TSB enrichment broth real-time PCR, indicating that the spore recovery
method for Sponge-Sticks and RV-PCR enrichment of B. a. Sterne spores was more efficient
than TSB enrichment of the extracted sponge. A summary of the PCR results of TSB-enriched
samples is presented in Appendix S.
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3.1.3 RV-PCR Method
A summary of the average and sample standard deviation of the RV-PCR ACt values for the
detection of B. a. Sterne spores recovered from Sponge-Stick surface samples are presented in
Table 5. The ACt results are plotted in Figure 28 through Figure 46 with each plot associated
with 1 of the 19 specific surface samples. The summary table and associated plots follow the
same column header and x-axis labeling convention as used for the presentation of culture
results. Most notably, the nominal number of spores available for analysis of 15, 150, and
1,500 CFU are used; it represents the maximum number of spores available, assuming a 100%
recovery efficiency and half the sample extract is available for RV-PCR analysis. The average
quantity of spores determined available are presented in Table 5. The 0-spore-available condition
is included in the plots because meaningful RV-PCR results can be obtained, unlike that for a
recovery efficiency. 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 both the
chromosomal and pXOl gene target ACt values having to be > 9 to be a positive result. The RV-
PCR results for each Sponge-Stick sample analyzed are presented in Appendix P.
RV-PCR analyses of all the Sponge-Stick samples with a nominal 150 or 1,500 B. a. Sterne
spores available for analysis resulted in a positive RV-PCR response, ACt > 9. The average ACt
for the 1,500 nominal B. a. Sterne spore available condition exceeded 20 for all surface samples,
other than that for the Street Grate, which had a value of 17 (see Figure 46). As shown in
Figure 28 through Figure 46, RV-PCR ACt values were consistently the lowest for the nominal
15-spores-available condition and highest for the 1,500 nominal spore condition. All surface
samples exhibited some suppression of ACt magnitude associated with the previously collected
particulate material, which is apparent when the ACt magnitude for the nominal 15 5. a. Sterne
spores available condition of the field blank samples (ACt = 25) is compared to that of the other
surfaces' samples that typically have a ACt <15, which suggests that the lower limit of detection
of the RV-PCR method is near the nominal 15-spores-available condition for samples that also
have ambient particulate matter also present.
RV-PCR had a positive response to all sponge wipe surface samples with a nominal 15
B. a. Sterne spores available for analysis condition, except for Street Grate and Painted
Crosswalk surfaces. Street Grate and Painted Crosswalk each had at least one sample for which
the RV-PCR response was negative (B. a. Sterne not detected) for the nominal 15-spores-
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available condition, indicating the limit of detection of the method is being approached. Results
showing that sample ACt standard deviations are relatively large and the greatest with the
nominal 15-spores-available condition relative to those measured at the 150 and 1,500 spores
available condition also suggests that the method detection limit is being approached at the 15-
spore load.
There were instances where the 0-spike condition resulted in a measurable ACt value, but in no
instances did ACt exceed 9, meaning that there were no RV-PCR false positive responses and
that cross-contamination was not an issue.
Consistently, throughout all analyses, very good agreement (ACt differed by < 1 between the two
gene targets) was obtained for the chromosomal and pXOl gene targets for all sponge wipe
surface samples and for all nominal spore loads.
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35
30
25 ¦
< 20
s.
Floor Tile (Sponge Stick)
10 -
I Chromosome
IpXOl
o o
• Threshold
Nominal B. a. Sterne Spores Available for Analysis (CFU)
Figure 28. RV-PCR Analysis of B. a. Sterne Spores Recovered from Floor Tile Sponge-
Stick Samples Using Chromosomal and pXOl Gene Targets (Average ± One Standard
Deviation for N > 3 Replicates); Positive Response Equals ACt > 9
35
Concrete Floor (Sponge Stick)
30 ¦
25 ¦
y 20
3 Replicates); Positive Response Equals ACt > 9
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35
30
25
y 20
3 Replicates); Positive Response Equals ACt > 9
Wall Tile (Sponge Stick)
I Chromosome
IpXOl
»Threshold
Nominal B. a. Sterne Spores Available for Analysis (CFU)
Figure 31. RV-PCR Analysis of B. a. Sterne Spores Recovered from Wall Tile Sponge-Stick
Samples Using Chromosomal and pXOl Gene Targets (Average ± One Standard Deviation
for N > 3 Replicates); Positive Response Equals ACt > 9
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35
30
25
Overhead Sign (Sponge Stick)
< 20
<
10
I Chromosome
IpXOl
5 ¦
Nominal B. a. Sterne Spores Available for Analysis (CFU)
Figure 36. RV-PCR Analysis of B. a. Sterne Spores Recovered from Overhead Sign
Sponge-Stick Samples Using Chromosomal and pXOl Gene Targets (Average ± One
Standard Deviation for N > 3 Replicates); Positive Response Equals ACt > 9
35
30
25
^ 20
Q)
00
ro
% 15
<
Metro Card Machine (Sponge Stick)
10
I Chromosome
IpXOl
• Threshold
Nominal B. a. Sterne Spores Available for Analysis (CFU)
Figure 37. RV-PCR Analysis of B. a. Sterne Spores Recovered from Metro Card Machine
Sponge-Stick Samples Using Chromosomal and pXOl Gene Targets (Average ± One
Standard Deviation for N > 3 Replicates); Positive Response Equals ACt > 9
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35
Concrete Sidewalk (Sponge Stick)
I Chromosome
IpXOl
• Threshold
LA
Nominal B. a. Sterne Spores Available for Analysis (CFU)
Figure 40. RV-PCR Analysis of II a. Sterne Spores Recovered from Concrete Sidewalk
Sponge-Stick Samples Using Chromosomal and pXOl Gene Targets (Average ± One
Standard Deviation for N > 3 Replicates); Positive Response Equals ACt > 9
Electronic Display Panel - Above Ground (Sponge Stick)
I Chromosome
I pXOl
• Threshold
rh
%
Nominal B. a. Sterne Spores Available for Analysis (CFU)
Figure 41. RV-PCR Analysis of B. a. Sterne Spores Recovered from Electronic Display
Panel (Above Ground) Sponge-Stick Samples Using Chromosomal and pXOl Gene
Targets (Average ± One Standard Deviation for N > 3 Replicates);
Positive Response Equals ACt > 9
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35
Crosswalk Signal (Sponge Stick)
dh
I Chromosome
IpXOl
» Threshold
* 3 Replicates); Positive Response Equals ACt > 9
Telephone Booth (Sponge Stick)
I Chromosome
IpXOl
»Threshold
so
Nominal B. a. Sterne Spores Available for Analysis (CFU)
%
Figure 43. RV-PCR Analysis of B. a. Sterne Spores Recovered from Telephone Booth
Sponge-Stick Samples Using Chromosomal and pXOl Gene Targets (Average ± One
Standard Deviation for N > 3 Replicates); Positive Response Equals ACt > 9
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though there could have been presumptive B. a. Sterne colonies counted based on colony
morphology.
The recovery efficiencies of presumptive B. a. Sterne from VFCs were generally low (< 10%)
for all surfaces sampled, which is attributed primarily to spores being retained on the MCE filter
substrate in the VFC and/or to some extent on the particulate matter previously collected on the
VFC. Instances where the average recovery efficiency exceeded 10% were associated with a
large standard deviation, which was the result of few (<10 colonies) being counted. The field
blank recovery efficiencies were 0, 4, and < 1% for nominal 15-, 150-, and 1,500-spore-available
condition, respectively, suggesting the spiked spores may be retained on the VFC MCE
substrate.
Whether the instances of the recovery efficiency from VFCs being less than 100% were due to
less than complete physical recovery of the spore or other physical loss mechanisms such as
retention on processing containers or interference of growth due to the presence of grime or
competing flora, was not resolved. It is also noted that spore/filter surface interactions may
influence the percent recovery measured, which could be affected by the spore spiking method.
The application of spores using droplets of a stock suspension may both assist or hinder the
ability to physically recover the spores from VFCs. Spores collected as an aerosol as expected
during normal field operation may adhere to the filter substrate more strongly than the majority
of spores present when applied as a droplet of suspension. When spores are applied as a droplet
of spore suspension, the spores may more readily disperse into the extract solution either due to
being a large agglomerate or weakly adhered to a surface. Conversely, spores collected as an
individual entity may adhere to the VFC substrate more strongly and be more difficult to
physically recovery. For those reasons, it would be recommended to consider additional research
to conduct similar analyses as performed here, but "spike" the B. a. Sterne by aerosolizing and
collecting onto the filter via air sampling.
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Most notable is the relatively large sample standard deviations and recovery efficiencies
exceeding 100% associated with the nominal 15-spores-available condition that are attributed to
few B. a. spores recovered and/or impact of background flora that could bias the presumptive
B. a. Sterne spore count high or low.
The Subway Car Filters (SCF1LT) appeared (by visual observation) to be the dirtiest of the VFC
filters and had an abundance of background flora that complicated the identification and
quantification of recovered B. a. Sterne. Figure 47 contains photographs of representative
SCFILT samples at various stages, from sample receipt, to spore recovery and culture plates
qualitatively illustrating the dirtiness of the samples and the impact on the culture plates to
quantifying B. a. Sterne.
Figure 47. Subway Car Filter (SCFILT) Representative Images, Sample Receipt, Addition
of Extraction Buffer, and Culture Growth on SBA Filter Spiked with 30, 300, or 3,000
Spores (from left to right, respectively)
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Concrete Floor (Vacuum Filter Cassette)
100
£
90 1
0)
O
Q.
80 :
CO
Qi
C
70 ¦
0>
tn
CO
60 ;
CO
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>
50 ;
Q.
fc
40 -
3
0>
CL
30 -
o
>
20 -
a>
>
o
u
0>
10 -
cc
0
Nominal Spores Available for Analysis (CFU)
Figure 48. Percent Recovery Efficiencies (Average ± One Standard Deviation of N =5
Replicates) of Presumptive B. a. Sterne Spores from Concrete Floor Vacuum Filter
Cassette Samples Using SBA Medium
100
90
o
Cl
00
0)
Ei
CL
<+-
o
£¦
a)
>
o
u
ce
80
70
60
50
40
30
20
10
Steps (Metal Grid) (Vacuum Filter Cassette)
%
Nominal Spores Available for Analysis (CFU)
Figure 49. Percent Recovery Efficiencies (Average ± One Standard Deviation of N = 5
Replicates) of Presumptive B. a. Sterne Spores from Steps (Metal) Vacuum Filter Cassette
Samples Using SBA Medium
-------
100
90
80
70
60
Q.
E
3
«/)
01
a.
*~—
o
40
30
20
10
EPA/6Q0/R-19/083
June 2019
Carpet (Vacuum Filter Cassette)
Nominal Spores Available for Analysis (CPU)
Figure 50. Percent Recovery Efficiencies (Average ± One Standard Deviation of N = 5
Replicates) of Presumptive B. a. Sterne Spores from Carpet Vacuum Filter Cassette
Samples Using SBA Medium
Subway Car Filter (Vacuum Filter Cassette)
100
E
90;
on
(1)
O
GL
80 ¦
so;
4—'
o.
E
40 ;
D
on
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V-
Q-
30;
*¦»—
O
£"
20 ;
a
>
o
u
01
io;
C£
0 :
Nominal Spores Available for Analysis (CFU)
Figure 51. Percent Recovery Efficiencies (Average ± One Standard Deviation of N = 5
Replicates) of Presumptive B. a. Sterne Spores from Subway Car Filter Vacuum Filter
Cassette Samples Using SBA Medium
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Concrete Sidewalk (Vacuum Filter Cassette)
100
VP*
o\
90
50
Q.
E
40
3
0
k-
Q-
30
M—
o
£¦
20
(U
>
o
a
OJ
10
a.
0
°o
Nominal Spores Available for Analysis (CFU)
Figure 52. Percent Recovery Efficiencies (Average ± One Standard Deviation of N = 5
Replicates) of Presumptive B. a. Sterne Spores from Concrete Sidewalk Vacuum Filter
Cassette Samples Using SBA Medium
Pavement (Vacuum Filter Cassette)
100
90
o
o.
ro
CD
Q.
E
3
O
Er
a>
>
o
u
cu
ce
80
70
60
50
40
30
20
10
Nominal Spores Available for Analysis (CFU)
Figure 53. Percent Recovery Efficiencies (Average ± One Standard Deviation of N = 5
Replicates) of Presumptive li. a. Sterne Spores from Pavement Vacuum Filter Cassette
Samples Using SBA Medium
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Figure 55. Vacuum Filter Cassettes (Top Left to Right: Subway Car Filter, Pavement,
Floor Concrete; Bottom Left to Right: Carpet, Steps, Sidewalk Concrete)
3.2.2 Vacuum Filter Cassette TSB Enrichment
For VFC samples, B. a. Sterne morphology was not isolated from turbid TSB broth on SBA
streak plates from non-field blank samples. The TSB broth suspension, in all cases, was screened
using real-time PCR assays. For the Floor (Concrete), Sidewalk Concrete, Pavement (Asphalt)
samples, and field blanks, TSB broth enrichment was PCR positive at a lower spore loading level
than RV-PCR positive, indicating that spores were not being physically removed from the filter
of the VFC samples. This increased sensitivity of TSB-enriched filters compared to RV-PCR
enrichment may be an artifact of liquid inoculation of the filters and may not reflect the natural
collection of spores from surfaces using VFCs. Liquid inoculation of the field blank samples,
where the spore suspension was applied directly to the filter with no particulates present for
spore attachment, had the largest difference in detection limit between TSB enrichment and RV-
PCR, where TSB enrichment was two orders of magnitude more sensitive. This result further
indicates that the spores tend to adhere to the surface of the inoculated filter during spore
recovery steps; however, if spores attached to particulates within the filter cassette, physical
recovery improved, as indicated by a closer positive detection limit between TSB enrichment and
RV-PCR for samples containing particulates. A summary of the PCR results of TSB-enriched
samples is presented in Appendix T.
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3.2.3 RV-PCR Method
A summary of the average and sample standard deviation of the RV-PCR ACt values for the
detection of B. a. Sterne spores recovered from VFC samples are presented in Table 7. The ACt
results are plotted in Figure 56 through Figure 62, with each plot associated with a specific
surface. The summary table and associated plots follow the same column header and x-axis
labeling convention as used for the presentation of culture results. Most notably, the nominal
number of spores available for analysis of 15, 150, and 1,500 CFU are used; it represents the
maximum number of spores available, assuming a 100% recovery efficiency and half the sample
extract is available for RV-PCR analysis. The average quantity of spores determined available
are presented in the summary tables. The 0-spore available condition is included in the plots
because meaningful RV-PCR results can be obtained, unlike that for a recovery efficiency. The
plots all depict an area shaded in red that is the region of a negative confirmation result and an
area of green that is a positive confirmation result, delineated by both the chromosomal and
pXOl gene target ACt values having to be > 9 to be a positive result. The RV-PCR results for
each VFC sample analyzed are presented in Appendix R.
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35
Subway Car Filter (Vacuum Cassette Filter)
I Chromosome
IpXOl
»Threshold
Nominal B. a. Sterne Spores Available for Analysis (CPU)
Figure 59. RV-PCR Analysis of B. a. Sterne Spores Recovered from Subway Car Filter
Vacuum Filter Cassette Samples Using Chromosomal and pXOl Gene Targets (Average ±
One Standard Deviation for N > 3 Replicates); Positive Response Equals ACt > 9
Concrete Sidewalk (Vacuum Cassette Filter)
I Chromosome
I pXOl
• Threshold
i—i—i
i i i
Nominal B. a. Sterne Spores Available (CFU)
%
Figure 60. RV-PCR Analysis of B. a. Sterne Spores Recovered from Concrete Sidewalk
Filter Vacuum Filter Cassette Samples Using Chromosomal and pXOl Gene Targets
(Average ± One Standard Deviation for N > 3 Replicates);
Positive Response Equals ACt > 9
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3.3 Summary of Detection Accuracy
The results presented in Sections 3.1 and 3.2 can be further reduced to a high-level performance
summary of detection accuracy of the two analytical methods and their associated false positive
and negative frequencies. For the culture method, false positive was defined as the identification
(counting) of one or more presumptive B. a. Sterne colonies when none were spiked onto the
surface sampler substrate (Sponge-Stick or VFC); false negative was when no presumptive
B. a. Sterne colonies were counted, yet the sampler substrate was spiked, and an accurate
detection when either no colonies were identified in the 0-spike condition or identified for a
sampler substrate spiked condition. The positive identification for RV-PCR is as defined in
Section 2.4.5 (ACt > 9 for both gene targets). A true positive was defined as correctly detecting
B. a. Sterne in a spiked sample and a true negative as no detection of B. a. Sterne in a sampler
substrate that was not spiked. A summary of those results is presented in Table 8 and Table 9 for
the Sponge-Stick and VFC samples, respectively for each sampled surface.
The RV-PCR analytical method was successful in accurately identifying (97% correct true
detection and 100% correct true negative response) the presence or absence of B. a. Sterne in the
Sponge-Stick samples, meaning the particulate matter previously collected on the Sponge-Stick
had minimal adverse impact on method performance. Combining true positives and true
negatives, the RV-PCR method had an accuracy of 97.3%. There were no false positives for RV-
PCR analyses of Sponge-Stick samples, indicating no cross-contamination. The false negatives
(3.2%) for RV-PCR of Sponge-Stick samples were believed to be due, in part, to poor physical
recovery of organisms from the sampler substrate as well as likely some loss in sensitivity due to
the ambient particulate matter recovered along with the B. a. Sterne. The RV-PCR method was
able to accurately identify the presence of B. a. Sterne in 47% of the VFC samples that were
spiked with B. a. Sterne spores, but was 100% accurate in identifying true negatives. The
relatively low accuracy of RV-PCR to identify the presence of B. a. Sterne from VFC samples
compared to the Sponge-Sticks is attributed to the low (< 10%) recovery efficiency of
B. a. Sterne from the VFC substrate.
The culture method's ability to accurately identify B. a. Sterne was hampered by the presence of
particulate matter previously collected on the Sponge-Stick samples (77% correct true positives).
The true negatives were correctly identified in 96% of the samples. Combining true positives and
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true negatives, the culture method using SBA medium had an accuracy of 80%. The false
negative detections for culture were associated predominantly with samples having a background
flora of competing organisms did not permit identification of any colonies with B. a. Sterne
morphology. The false positives for culture were attributed, and in some instances confirmed
with PCR analysis of selected colonies, to presumptive B. a. Sterne colonies not being correct, as
discussed earlier. The culture method was able to accurately identify the presence of B. a. Sterne
in 54% of the VFC samples that were spiked with B. a. Sterne spores, but was 90% accurate in
identifying true negatives. The relatively low accuracy of the culture method to identify the
presence of B. a. Sterne from VFC samples compared to the Sponge-Sticks is attributed to the
low (< 10%) recovery efficiency of B. a. Sterne from the VFC substrate.
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4.0 QUALITY ASSURANCE/QUALITY CONTROL
Quality assurance (QA)/quality control (QC) procedures were performed in accordance with the
Testing and Evaluation (T&E II) Program Quality Management Plan (QMP), Version 1 and the
TO 09 Quality Assurance Project Plan (QAPP) (Battelle, 2017). 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 RWI testing included positive and negative controls for both spread
plate samples and qPCR. In addition, they included quantification of both the spore bank and
B. a. Sterne spike suspensions to verify either CFU/mL titer or target spike concentrations.
Positive controls (PC) and NTCs were included for each RV-PCR assay and in all cases the 50-
pg PC consistently resulted in Ct values in the mid-20s, as expected. There were two instances
that NTC wells generated a fluorescence signal (Ct of 44.5 and 43.5) that crossed the threshold,
occurring during Sponge-Stick Trial 12 on September 10, 2018 and VFC Trial 7 on January 28,
2019. The signal was detected in the pXOl assay, not the chromosome assay, and all other NTC
wells, zero spike samples, and To samples did not cross the threshold, indicating that this
erroneous signal did not have an impact on the Ct values generated for samples. 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 B. a. Sterne
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. The field blank samples provided a means to baseline the
method performance without competing flora and grime.
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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 (PE) audits were conducted to assess the quality of the results obtained
during these experiments. Table 11 summarizes the PE audits that were performed; equipment
was within acceptable tolerance range.
Table 11. Performance Evaluation Audits
Measurement
Audit
Allow si hie
Acliiitl
Procedure
Tolenince
Tolenince
Volume of liquid from
micropipettes
Gravimetric evaluation
± 10%
Passed calibration as
found/as returned
Time
Compared to independent
clock
± 2 sec/hour
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 (TSA) were documented and
submitted to the laboratory technical lead for response. TSA was conducted on June 15, 2018 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 work
instructions, and data acquisition and handling procedures were reviewed. None of the findings
of the TSA required corrective action.
4.4.3 Data Quality Audit
At least 10% of data acquired during the evaluation were audited. Data were reviewed in
December 2018 and January 2019. A QA auditor traced the data from the initial acquisition,
through 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. Only minor
issues were noted with the data, mostly data transcription errors that were corrected.
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4.5 QA/QC Reporting
Each assessment and audit was documented in accordance with the QAPP and QMP. For these
tests, findings were noted (none were significant) in the data quality audit, and no follow-up
corrective action was necessary. The findings were mostly minor data transcription errors
requiring some recalculation of efficacy results, but none were gross errors in recording. QA/QC
procedures were performed in accordance with the QAPP.
4.6 Data Review
Records and data generated in the evaluation received a QC/technical review before they were
utilized in calculating or evaluating results and prior to incorporation in this report.
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5.0 SUMMARY OF METHOD OBSERVATIONS AND EXPERIENCES
While implementing the method, key observations and experiences were noted that will be useful
to understand and/or take into consideration for future iterations or versions of the method. Key
observations were:
• The RV-PCR method requires great care and diligence to implement effectively. Most
notable, the method required changing gloves between procedural samples for each step,
which is time-consuming. Glove changing is critical to avoid cross-contamination
samples, which would negatively impact key decisions in the response, 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.
• The RV-PCR sample analyses were performed in batches of 16 per trial using a single
system based on initial trials to implement the methods. 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 ERLN if
actual samples were being processed. A single trial was completed over 4 consecutive
days of operation, starting with sample spiking on Day 1. (Had these been actual filters
collected post-biological release, the spiking activity would, obviously, not be performed
by the ERLN.)
• A 16-hour incubation duration for RV-PCR was used in this study, but the EPA method
typically uses a 9-hour incubation duration. It is reasonable to initially use the 9-hour
incubation because the RV-PCR ACt was commonly over 15 for the air quality and non-
air quality filters analyzed if the filters had 150 or more spores available. In practice,
longer incubation times could be implemented for selected samples to confirm a negative
response with a 9-hour incubation time.
• Estimated staff time to process 16 samples was approximately 64 hours and $1,500 of
consumables. The 64 hours of staff time budget was approximately distributed by:
o 8 hours for activities related specifically to the spiking of the filters being
assessed, which was a requirement of the study, but not an activity that would be
performed had these been actual field samples. This task included time to prepare
the stock suspensions, enumerate stock suspension, spike the filters, and
associated documentation.
o 10 hours for spore recovery.
o 10 hours for culture analysis.
o 24 hours for RV-PCR analysis.
o Additionally, 4 hours was needed for PCR confirmation analysis of eight samples,
when performed.
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EPA/600/R-19/083
June 2019
• Had the EPA 2012 method 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 labor hours and $1,000 in
materials to perform culture analysis (with PCR confirmation of at least three colonies
per sample). To process the same number of samples, an estimated 40 hours and $1,200
would be required using RV-PCR analysis. Each of the analytical methods would take 2
or 3 days:
o The benefit to RV-PCR is that B. a. Sterne can be detected in sample matrices
with high amounts of background flora and grime. For culture analysis, the
growth of viable B. a. Sterne spores may be masked by background flora and
grime in environmental samples, and therefore go undetected.
• The extract suspension from Sponge-Sticks tended to 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:
o At 15-minute, 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.
o At 1-hour, 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.
• For Sponge-Sticks, colony PCR screening results indicate that the microbiologist's
identification of colonies were correct 93% of the time, from a total of 229 colonies
screened. There was one instance where the presumptive ID of a colony was negative and
colony PCR was positive, and 15 instances where presumptive B. a. Sterne colonies were
colony PCR negative.
• For VFCs, colony PCR screening results indicate that the microbiologist's identification
of colonies were correct 68% of the time, from a total of 50 colonies screened. There
were 16 instances where presumptive B. a. Sterne colonies were colony PCR negative.
o Seventeen (17) colonies that were streaked and isolated from the TSB enrichment
broth were screened using colony PCR. Only three were positive and each of the
three colonies was isolated from field blank samples that did not have background
flora competition.
• For Sponge-Sticks, TSB enrichment did not lead to the isolation of B. a. Sterne on SBA
streak plates. Real-time PCR analysis of the turbid TSB resulted in three positive samples
from Street Grating surface, although the RV-PCR enrichment Ct values were lower,
indicating that spore recovery method for Sponge-Sticks and RV-PCR enrichment was
more sensitive than TSB enrichment of the extracted sponge. The enrichment analysis
was included for the last four trials of Sponge-Sticks.
• Data suggest that B. a. Sterne spiked onto VFCs are not efficiently removed from the
filter. As a result, TSB enrichment, which is an enrichment of the VFC filter following
spore recovery, is PCR positive at a lower loading level than RV-PCR, which is an
enrichment of the spore recovery suspension:
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EPA/600/R-19/083
June 2019
o Recommend evaluating recovery method and perhaps incorporating a vortex step
in addition to the bath sonication of the VFC.
o Vacuum (VFC) field blank samples containing 0, 30, and 300 spores had no
observed growth (turbidity) in filter vials following overnight incubation in BHIB
(RV-PCR Tfinal enrichment) and resulted in RV-PCR negative samples. The
extracted filter from these field blank samples that was enriched with TSB did
grow during overnight incubation and resulted in TSB enrichment PCR positive
samples.
• For Sponge-Sticks and VFCs, B. a. Sterne was not isolated from streaking turbid TSB
onto SBA plates from non-field blank samples. However, B. a. Sterne was present in the
TSB enrichment broth for many of the VFC samples, as determined by PCR analysis of
an aliquot of the broth.
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EPA/600/R-19/083
June 2019
6.0 CONCLUSIONS AND RECOMMENDATIONS
The foremost conclusion is that the RV-PCR B. anthracis analysis method was > 97% accurate
in correctly identifying the presence or absence of B. a. Sterne in Sponge-Stick samples that had
previously collected background material from real-world surface sampling. The culture method
was less accurate (77%) in correctly identifying the presence or absence of B. a. Sterne in the
same Sponge-Stick samples, meaning the presence of real-world material collected during
surface sampling can hinder the culture method performance. The 18 field blank samples
analyzed - 15 samples spiked with B. a. Sterne and 3 samples not spiked - were 100%
accurately identified by the culture method.
Neither the culture nor molecular analytical methods performed as well with surface samples
collected using the VFCs compared to the Sponge-Stick sampling method. The decrease in
performance as measured by the accuracy of properly identifying the presence or absence of
B. a. Sterne spiked onto the samples was attributed primarily to poor physical recovery of
B. a. Sterne from the VFC MCE collection substrate and, also, possibly, to the collected ambient
particulate matter. Physical recovery of spores could potentially be improved by modification of
the method; the addition of a vortex step after sonication of the VFC MCE collection substrate is
recommended to be assessed. It is possible that the spiking method (drops of a B. a. Sterne
suspension applied directly onto the VFC collection substrate) affected the physical recovery of
organisms for subsequent analysis. Collection of the B. a. Sterne as an aerosol while
simultaneously collecting the ambient particulate matter (as would be the case in an actual
sampling campaign following a bioterrorism incident) may yield improved recovery efficiencies.
This effect, however, may be most apparent when there is an opportunity to collect much
ambient particulate matter.
One recommendation is therefore to assess the impact that spiking of B. a. Sterne spores onto the
VFC substrates has on the recovery and subsequent analyses. The liquid suspension spiking
method may bias the recovery efficiencies favorably (higher efficiency) or unfavorably.
Specifically, it is recommended to expand the study by depositing an aerosol of B. a. Sterne onto
real-world "dirty" surfaces, and then sampling the comingled background material with
deposited B. a. Sterne using the VFC. It is also recommended to perform the same sampling
effort using the Sponge-Stick sampling method for comparison and completeness. RV-PCR can
-------
EPA/600/R-19/083
June 2019
be used to positively identify viable B. a. Sterne in presence of complex, dirty sample matrices
from Sponge-Stick surface samples. The background flora and grime collected on the Sponge-
Sticks can reduce the lower limit of detection and/or suppress the sensitivity of the B. a. Sterne
signal, but samples with as few as a nominal quantity of 15 B. a. Sterne spores could routinely be
positively identified.
The results from this study will be useful to those analyzing samples collected following a
bioterrorism incident by having an understanding that the culture results may be confounded by
an overwhelming presence of background flora, obscuring the presence of B. anthracis spores.
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EPA/600/R-19/083
June 2019
7.0 REFERENCES
Battelle, "Quality Assurance Project Plan (QAPP) for Analysis of Native Filters for
Characterization and Extent Mapping of Biological Incidents," Version 3.2, February 15,
2017, with Amendment July 13, 2017.
Calfee, M. W., et al. (2013). "Comparative evaluation of vacuum-based surface sampling
methods for collection of Bacillus spores." Journal of Microbiological Methods 95(3): 389-
396.
Calfee, W. Personal Communication; Meeting minutes from in-progress review meeting hosted
at Battelle, June 24 and 25, 2018.
Letant, S.E., et al., Rapid viability PCR method for detection of live, virulent Bacillus anthracis
in environmental samples. Appl Environ Microbiol, 2011. 77(18): p. 6570-8.
Rose, L. J., et al. (2011). "National Validation Study of a Cellulose Sponge Wipe-Processing
Method for Use after Sampling Bacillus anthracis Spores from Surfaces." Applied and
Environmental Microbiology 77(23): 8355-8359.
Shah, S., Protocol for Detection of Bacillus anthracis in Environmental Samples During the
Remediation Phase of an Anthrax Incident, 2nd Edition. U.S. Environmental Protection
Agency, Washington, DC, 2017.
Shah, S. Personal Communication; Email to Scott Nelson of Battelle, June 25, 2018.
Tufts, J. A., et al. (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, Protocol for Detection of Bacillus anthracis in
Environmental Samples During the Remediation Phase of an Anthrax Event. December
2012. EPA/600/R-12/577.
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.
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EPA/600/R-19/083
June 2019
EVALUATION OF ANALYTICAL
METHODS FOR THE DETECTION OF
BACILLUS ANTHRACIS SPORES:
COMPATIBILITY WITH REAL-WORLD
SAMPLES COLLECTED FROM
OUTDOOR AND SUBWAY SURFACES
APPENDICES A-T
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EPA/600/R-19/083
June 2019
APPENDIX A: TARGET SURFACES
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EPA/6Q0/R-19/083
June 2019
Figure A-l. Carpet Surface Located by the Jackie O Entrance to Station,
Off 42nd Street - Sampled with Vacuum Filter Cassette
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EPA/6Q0/R-19/083
June 2019
Rtoadway
& St
oldnavy.com
os»"i
Figure A-2. Concrete Floor Located Just Below Times Square Police Station,
One Level Down - Sampled with Sponge-Sticks and Vacuum Filter Cassette
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EPA/6Q0/R-19/083
June 2019
HEL( €M^T€ Tiati Si;
Figure A-3. Crosswalk Signals Located Above Various Intersections on Broadway, 44th, and 7th Street
- Sampled with Sponge-Sticks
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EPA/6Q0/R-19/083
June 2019
Figure A-4. Electronic Display Panels (Below Ground) Located in Times Square 42nd Street Station,
Near Track 3 - Sampled with Sponge-Sticks
-------
EPA/6Q0/R-19/083
June 2019
OUTFRONT,
Times Square
42 St Station
OOO®' a c
& Elevator across 42 St
tELtSS SERVICE AVAILABLE
Visit mta.info/bustime
IF WUSEE SOMETHING. SAY SOMETHING
Figure A-5. Electronic Display Panels (Above Ground) Located Above Subway Entrance Next to the
Times Square Police Station - Sampled with Sponge-Sticks
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EPA/6Q0/R-19/083
June 2019
Figure A-6. Floor Tiles Located at the Grand Central Shuttle Landing,
Between Tracks 1 and 3, Near the Control Booth - Sampled with Sponge-Sticks
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EPA/6Q0/R-19/083
June 2019
Figure A- 7. Fluorescent Light Fixtures Located Near the PSU by the
Record Mart - Sampled with Sponge-Sticks
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EPA/6Q0/R-19/083
June 2019
New Tk City Transit
SuBWay Map
da£G£B
Figure A-8. Glass Panels on Subway Map Displays Located Near Madison and 42nd Street Subway
Exit - Sampled with Sponge-Sticks
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EPA/6Q0/R-19/083
June 2019
Figure A-9. Glass Windows/Facades Located Near the PSU by
the Record Mart - Sampled with Sponge-Sticks
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EPA/6Q0/R-19/083
June 2019
Figure A-10. Granite Bench Located Near the Times Square
Police Station - Sampled with Sponge-Sticks
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EPA/6Q0/R-19/083
June 2019
4STRACK
Figure A-ll. Metro Card Machines Located Near the 42nd and
Park Avenue Subway Exit - Sampled with Sponge-Sticks
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EPA/6Q0/R-19/083
June 2019
Figure A-12. Overhead Signs Located at Various Locations Near the Grand Central Exit to 42nd Street
and Madison Avenue - Sampled with Sponge-Sticks
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EPA/6Q0/R-19/083
June 2019
tf
Figure A-13. Crosswalk Painted Pavement (Asphalt) Located on the Intersection of Broadway and 46th
Street - Sampled with Sponge-Sticks
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EPA/6Q0/R-19/083
June 2019
Figure A-14. Pavement (Asphalt) Located at the Intersection of
Broadway and 46th Street - Sampled with Vacuum Filter Cassettes
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EPA/6Q0/R-19/083
June 2019
Figure A-15. Sidewalk Concrete Located Next to the Green Wall by the Intersection of Broadway and
45th Street - Sampled with Vacuum Filter Cassettes and Sponge-Sticks
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EPA/6Q0/R-19/083
June 2019
Figure A-16. Steps (Metal Grid) Located Near the PSU by the Record Mart - Sampled with Vacuum
Filter Cassettes and Sponge-Sticks
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EPA/6Q0/R-19/083
June 2019
i/'/r/iniiimiiiiiiii
ttvjimmiiniimiiil
///////ifr/Mfirffifiifi
jwwiiimnnnnwl
/.'///////'/f If f If If 11II11
///////#//!/## Hill I KM
////////7///IIIIIIIIIH
f/juwwinmmm
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\\\\\\\\v\\\\v\\^%
mmmmm
%v\\\\\\\\\\vm^
Figure A-17. Street Grate Located by the Intersection of
Broadway and 46th Street - Sampled with Sponge-Sticks
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EPA/6Q0/R-19/083
June 2019
Figure A-18. Subway Car HVAC Filters Taken from the Front and Back HVAC Returns of Cars -
Sampled with Vacuum Filter Cassettes (Offsite)
-------
EPA/6Q0/R-19/083
June 2019
Figure A-19. Subway Car Filter Grille Located on the Front and Back HVAC
Returns of Car 1951 - Sampled with Sponge-Sticks
-------
EPA/6Q0/R-19/083
June 2019
Figure A-20. Telephone Booth (Interior) - Sampled with Sponge-Sticks
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EPA/6Q0/R-19/083
June 2019
® Manhattan Bus Map
Figure A-21. Wall Tiles (Ceramic) Located Just Below the Times Square Police Station,
One Level Down - Sampled with Sponge-Sticks
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EPA/600/R-19/083
June 2019
APPENDIX B: FORMULATIONS OF RECIPES USED IN BIOLOGICAL
TEST METHODS
-------
EPA/600/R-19/083
June 2019
Spore Production
Table 1. Components of Modified G Sporulation Medium
Ingredient
Amount/L
Yeast Extract
2.0 g
(NFlicSO,
2.0 g
CaCli • 21 hO
0.03 g
CuS04 • 5H20
0.005 g
FeSQ4 • 7HiO
0.0005 g
MgSOi • 7H20
0.2 g
M11SO4 • H2O*
0.06 g
/11SO, • 7H20
0.005 g
K2HPO4
0.5 g
dH20
1000 mL
*MnSC>4 • H2O substituted for MnS04 • 4HzO. If M11SO4 • 4H2O is used, add 0.05 g.
Table 2. Components of Leighton-Doi Sporulation Medium
Component
Amount/L
KC1
1.88 g
CaCl2
0.29 g
FcSO, x 7 H20
0.003 g
MnSOi x H2O
0.0017 g
MgSOi x 7 II;()
0.025 g
Dextrose
0.9 g
Nutrient Broth
16.0 g
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EPA/600/R-19/083
June 2019
Table 3. Duplex Assay Conditions
Component (Duplex Assay)
2x FAST PCR Mix
Volume for one reaction (jiL)
12.5
PCR-grade water
.5
pXOl For Primer (25 |iM) 1
pXOl Rev Primer (25 |iM) 1
pXOl Probe (2 jxM) 1
chromosome For Primer (25 |iM) 1
chromosome Rev Primer (25 (xM) 1
chromosome Probe (2 jiM) 1
Template 5
Total volume 25
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EPA/600/R-19/083
June 2019
APPENDIX C: WORK INSTRUCTION FOR SPIKING WITH BACILLUS
ANTHRACIS STERNE SPORES-SPG STICKS
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EPA/600/R-19/083
June 2019
WORK INSTRUCTION FOR SPIKING WITH BACILLUS ANTHRACIS STERNE SPORES - SPG STICKS
I. PURPOSE/SCOPE
To spike Sponge-Sticks for spore recovery testing.
II. Analyst/Reviewers
Role
Name
Initials
Date
Analyst
Analyst
Reviewer
III. MATERIALS/EQUIPMENT
Materials
Item
Manufacturer
Lot Number
Exp.
Date
Temp.
Initials & Date
Bacillus anthracis
34F2spores
Inhouse
34F2101716
TBD
2-8 °C
Sterile Dl water
Teknova
W335019D1701
4/19/20
R.T.
Blood Agar
BBL
2-8 °C
1.5 or 2 mL tubes
Eppendorf
F170109Q
G175420P
N/A
R.T.
Sterile forceps
N/A
N/A
N/A
R.T.
Equipment
Item
Manufacturer
Serial Number
Thermometer
/Rees #
Calibration
Due
Initials &
Date
Biosafety
Cabinet (BSC)
The Baker Company
57544
N/A
8/2019
Micropipette
TypeilOOO
Rainin
N/A
Micropipette
Type:L200
Rainin
N/A
Micropipette
Type:L10 or
120
Rainin
N/A
Refrigerator
Fisher
C3274822
115
3/2019
N/A= Not Applicable
Other Supplies and Equipment
• Micropipette filter tips
• Biohazard bags
• Bench coat
• Filters
Page 1 of 4
RWIs WI-SPG-SPIKE-1-V7 (October 1, 2018)
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EPA/600/R-19/083
June 2019
WORK INSTRUCTION FOR SPIKING WITH BACILLUS ANTHRACIS STERNE SPORES - SPG STICKS
IV. PROCEDURE
A, Decontaminate the BSC with DNA Erase, bleach and isopropanol prior to use.
1. Decontaminated by Date
B. Name filters
1. Label the Stomacher bag with filter ID for each filter.
i. AAA-BBB-CCC-DDD
1. AAA = Sample#
2. BBB = Sample Type
3. CCC = Location
4. DDD = Spore Spike Level
ii. Electronically populate below table with sample names to be prepared on each
day from the Sample Log.
Sample
#
Sample
Type
Location
Filter
Vial
1
Spore
Spike
level
Filter ID
Date
Spiked
/initials
1
SPG
Field Blank
PVDF
0
1-SPG-FLDBLK-A-S15-0
2
SPG
street Grate
PVDF
0
2-S PG -STG RAT-A-S15-0
3
SPG
Field Blank
PVDF
30
3-SPG-FLDBLK-A-S16-30
4
SPG
Street Grate
PVDF
30
4-S PG-STG RAT-A-S16-30
5
SPG
Field Blank
PVDF
300
5-SPG-FLDBLK-A-S17-300
6
SPG
Street Grate
PVDF
300
6-S PG-STG RAT-A-S17-300
7
SPG
Field Blank
PVDF
3000
7-SPG-FLDBLK-A-S18-3000
8
SPG
street Grate
PVDF
3000
8-S PG-STG RAT-A-S18-3000
9
SPG
Granite Bench
PVDF
0
9-SPG-GRNBEN-A-S15-0
10
SPG
Wall Tile
PVDF
0
10-SPG-WLTILE-B-S19-0
11
SPG
Granite Bench
PVDF
30
11-SPG-GRNBEN-A-S16-30
12
SPG
Lab Blank
PVDF
30
12-SPG-LABBLANK-30
13
SPG
Granite Bench
PVDF
300
13-SPG-GRNBEN-A-S17-300
14
SPG
Lab Blank
PVDF
300
14-SPG-LABBLANK-300
15
SPG
Granite Bench
PVDF
3000
15-SPG-GRNBEN-A-S18-3000
16
SPG
Lab Blank
PVDF
3000
16-SPG-LABBLANK-3000
C. Spike Swatches
1. Prepare dosing stocks
i. Fill in information from stock tube.
Organism
Lot
Prep date
Concentration
Date of
enumeration
Entered/verified
by:
B. anthracis
Sterne
34F2101716
1.8 X 108cfu/mL
September 26,
2018
Page 2 of 4
RWIs WI-SPG-SPIKE-1-V7 (October 1, 2018)
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EPA/600/R-19/083
June 2019
WORK INSTRUCTION FOR SPIKING WITH BACILLUS ANTHRACIS STERNE SPORES - SPG STICKS
ii. Target stock concentration(s).
Stock #
Organism
Lot
Prep
date
Concentration
Total spores
per 100 pL
Entered/verified
by:
1
8.
anthracis
Sterne
34F2101716
3.0 X 10'1
cfu/mL
3,000
2
B.
anthracis
Sterne
34F2101716
3.0 X103
cfu/mL
300
3
B.
anthracis
Sterne
34F2101716
3,0 X102
cfu/mL
30
iii. Prepare dilutions of stock in sterile Dl water. Vortex stock on high for 30
seconds prior to preparing dilutions.
Show calculations:
(1.8 X 10s cfu/mL)*(X)=(3,0 X 107cfu/mL)(lmL) -» 167pL of sample into 833nL H20
(3.0 X 107 cfu/mL)*(X)=(3.0 X 106cfu/mL)(lmL) lOOpL of sample into 900nL H20
(3.0 X 106 cfu/mL)*(X)=(3.0 X 105cfu/mL)(lmL) -> lOOpL of sample into 900(iL H20
(3.0 X 10s cfu/mL)*(X)=(3,0 X 104cfu/mL)(lmL) -> 150nL of sample into 1350nL H20
(3.0 X 104 cfu/mL)*(X)=(3,0 X 103cfu/mL)(lmL) -> 150pL of sample into 1350(iL H20
(3.0 X 103 cfu/mLj*(X)=(3,0 X 102cfu/mL)(lmL) -> 150^1 of sample into 1350jiL H20
Dilutions Prepared By: Date/Initials:
2, Spike Sponge Sticks
i. Position sponge in specimen cup so that the dirty side is facing up. Change
forceps between samples.
ii. Prior to dosing filters, immediately vortex the stock for 30 seconds.
iii. Per sponge, transfer a 120 pL aliquot of the appropriate Stock tube (Low, Med.,
or High) into a 1.5 ml tube.
iv. Place ten 5 (iL droplets onto each side of the sponge stick (twenty 5 |iL droplets
total), being as careful as possible to not have spiked surfaces contact the
specimen cup wall. Position sponge stick as shown in Figure 1. The same pipet
tip can be used to place all twenty droplets, dispose of the 120 pL aliquot once
each swatch has been dosed.
v. Seal the specimen cup and stock overnight @ 2 -8 °C or process immediately to
Spore recovery.
Start time: Date/Initials:
End time: Date/Initials:
3. Enumerate stock
Page 3 of 4
RWIs WI-SPG-SPIKE-1-V7 (October 1, 2018)
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EPA/6Q0/R-19/083
June 2019
WORK INSTRUCTION FOR SPIKING WITH BACILLUS ANTHRACIS STERNE SPORES - SPG STICKS
i. Serially dilute the suspension in Sterile water (if necessary).
1. Fill 2 mL dilution tubes for each sample with 900nL of Sterile water and
label appropriately.
2. Vortex the stock on high for 30 seconds.
3. Transfer 100 |il_ of the stock into the first dilution tube containing 900pL
of Sterile water. Recap the tube and vortex it on high for 30 seconds.
This is the lO^suspension.
ii. Spread 100 |aL aliquots of dilutions onto Blood Agar in triplicate.
iii. Incubate plates
1. Invert the plates and incubate them at 37°C ± 2°Cfor 18 - 24 hours. B.
anthracis produces flat or slightly convex, 2 - 5 mm colonies, with edges
that are slightly irregular and have a "ground glass" appearance.
Incubation start Date/Time: Initials:
Incubation end Date/Time: Initials:
iv. Plate counts
1. Record counts in the below table.
Stock
Media Type
Volume/
(Dilution on
Plate)
Plate Counts
Average
Counts
CFU/mL
Plate
1
Plate
2
Plate
3
1 (3.0 X 104 cfu/mL)
Blood Agar
100 nL/
(10-1)
2 (3.0 X 103cfu/mL)
Blood Agar
100 nL/
(10-')
3 (3.0 X102 cfu/mL)
Blood Agar
100 \xH
(10-1)
Recorded By: Date/Initials:
Position with folded side up or stick side up. Do not spike the sides of the sponge that could contact
the specimen cup wall.
Place ten 5 pL evenly dispersed droplets on each side for a total of twenty 5 pL droplets.
Figure 1. Spiking diagram for sponge sticks.
Page 4 of 4
RWIs WI-SPG-SPIKE-1-V7 (October 1, 2018)
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EPA/600/R-19/083
June 2019
APPENDIX D: WORK INSTRUCTION FOR SPIKING WITH BACILLUS
ANTHRACIS STERNE SPORES-VCF
-------
EPA/600/R-19/083
June 2019
WORK INSTRUCTION FOR SPIKING WITH BACILLUS ANTHRACIS STERNE SPORES - VCF
I. PURPOSE/SCOPE
To spike 37 mm Vacuum Cassette Filters (VCF) for the spore recovery testing.
II. Analyst/Reviewers
Role
Name
Initials
Date
Analyst
Analyst
Reviewer
III. MATERIALS/EQUIPMENT
Materials
Item
Manufacturer
Lot Number
Exp.
Date
Storage
Temp.
Initials & Date
Bacillus anthracis
34F2 spores
Inhouse
34F2101716
TBD
2-8 °C
Sterile Dl water
Teknova
W335017E1701
5/17/202
0
R.T.
Blood Agar
BBL
8313853
2/28/201
9
2-8 °C
15 mL tubes
Falcon
12118014
N/A
R.T.
1.5 or 2 mL tubes
Eppendorf
H176955G
1/28/202
3
R.T.
100% Ethanol
Fisher
184834
11/9/202
3
R.T.
Equipment
Item
Manufacturer
Serial Number
Thermometer
/Rees #
Calibration
Due
Initials &
Date
Biosafety
Cabinet (BSC)
The Baker Company
57544
N/A
8/2019
Micropipette
TypeilOOO
Rainin
C25845
N/A
4/23/2019
Micropipette
Type:L200
Rainin
C19039
N/A
2/28/2019
Micropipette
TypellO or
L20
Rainin
C25835
N/A
6/12/2019
Refrigerator
Fisher
C3274822
115
3/2019
N/A = Not Applicable
Other Supplies and Equipment
• Micropipette filter tips
• Biohazard bags
• Bench coat
• Filters
Page 1 of 5
RWIs WI-VCF-SPIKE-l-v4 (January 23, 2019)
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EPA/600/R-19/083
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WORK INSTRUCTION FOR SPIKING WITH BACILLUS ANTHRACIS STERNE SPORES - VCF
IV, PROCEDURE
A. Decontaminate the BSC with DNA Erase, bleach and isopropanol prior to use.
1. Decontaminated by Date
B. Name filters
1. Label the Stomacher bag with filter ID for each filter.
i. AAA-BBB-CCC-DDD
1, AAA = Sample#
2, BBB = Sample Type
3. CCC = Location
4. DDD = Spore Spike Level
ii. Electronically populate below table with sample names to be prepared on each
day from the Sample Log.
Sample
#
Sample
Type
Location
Filter
Vial
Type
Spore
Spike
level
Filter ID
Date
Spiked
/initials
1
VCF
Floor Cone.
PVDF
0
1-VCF-FLCON-B-S15-0
2
VCF
steps
PVDF
0
2-VCF-STEPS-B-S15-0
3
VCF
Floor Cone.
PVDF
30
3-VCF-FLCON-B-S16-30
4
VCF
Steps
PVDF
30
4-VCF-STEPS-B-S16-30
5
VCF
Floor Cone.
PVDF
300
5-VCF-FLCON-B-S17-300
6
VCF
Steps
PVDF
300
6-VCF-STEPS-B-S17-300
7
VCF
Floor Cone.
PVDF
3,000
7-VCF-FLCON-B-S18-3,000
8
VCF
Steps
PVDF
3,000
8-VCF-STEPS-B-S18-3,000
9
VCF
Sidewalk Cone.
PVDF
0
9-VCF-SWCON-A-S15-0
10
VCF
Pavement
PVDF
0
10-VCF-PAVEMT-A-S15-0
11
VCF
Sidewalk Cone.
PVDF
30
11-VCF-SWCON-A-S16-30
12
VCF
Pavement
PVDF
30
12-VCF-PAVEMT-A-S16-30
13
VCF
Sidewalk Cone.
PVDF
300
13-VCF-SWCON-A-S17-300
14
VCF
Pavement
PVDF
300
14-VCF-PAVEMT-A-S17-300
15
VCF
Sidewalk Cone.
PVDF
3,000
15-VCF-SWCON-A-S18-3.000
16
VCF
Pavement
PVDF
3,000
16-VCF-PAVEMT-A-S18-3,000
C. Spike Swatches
1. Prepare dosing stocks
i. Fill in information from stock tube.
Organism
Lot
Prep date
Concentration
Date of
enumeration
Entered/verified
by:
B. anthracis
Sterne
34F2101716
3.0 X 108cfu/mL
January 21,
2019
ii. Target stock concentration(s).
Page 2 of 5
RWIs WI-VCF-SPIKE-l-v4 (January 23, 2019)
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EPA/600/R-19/083
June 2019
WORK INSTRUCTION FOR SPIKING WITH BACILLUS ANTHRACIS STERNE SPORES - VCF
Stock
Organism
Lot
Prep
date
Cone, in HjO
Cone, in
50% Ethanol
Total
spores
per
100 nL
Spike
Entered/verified
by:
1
B.
anthracis
Sterne
34F2101716
6.0 X 10'
cfu/m L
3,0 X 10'1
cfu/m L
3,000
2
B.
anthracis
Sterne
34F2101716
6.0 X 103
cfu/m L
3,0 X103
cfu/m L
300
3
B.
anthracis
Sterne
34F2101716
6.0 X 102
cfu/m L
3.0 X103
cfu/m L
30
iii. Prepare dilutions of stock in sterile Dl water. Vortex stock on high for 30
seconds prior to preparing dilutions.
Show calculations:
Dilution 1: (3.0 X 108 cfu/m L)*(X)=(3.0 X 107cfu/mL)(lmL) -» lOOpL of sample into 900nLH20
Dilution 2: (3.0 X 107 cfu/m L)*(X)=(3.0 X 106cfu/mL)(lmL) 100|iL of sample into 900(iL HjO
Dilution 3: (3,0 X 106 cfu/mL)*(X)=(3.0 X 105cfu/mL)(lmL) -> 100|jL of sample into 900(iL HjO
Dilution 4: (3.0 X 105 cfu/m L)*(X)=(6.0 X 104cfu/mL)(1.5mL) -» 300(iL of sample into 1200(iL H20
Dilution 5; (6.0X 10*cfu/mL}*(X)=(6,0X 103cfu/mL)(1.5mL) -» ISOnLof sample into 1350nL H20
Dilution 6: (6,0 X 103 cfu/m L)*(X)=(6,0 X 102cfu/mL)(1.5mL) -» ISOuL of sample into 1350(iL H20
Dilutions Prepared By: Date/Initials:
iv. To prepare Stock #1 (3,000 Target Load Spike), add 500 ^L of Dilution 4 and 500
|iL 100% Ethanol to a 1.5 or 2 mLtube and vortex mix thoroughly.
v. To prepare Stock #2 (300 Target Load Spike), add 500 (lLof Dilution 5 and 500
(iL 100% Ethanol to a 1.5 or 2 mLtube and vortex mix thoroughly.
vi. To prepare Stock #3 (30 Target Load Spike), add 500 pL of Dilution 6 and 500 (iL
100% Ethanol to a 1.5 or 2 mL tube and vortex mix thoroughly.
2. Spike VCF
i. Wipe each cassette with 10% bleach solution or bleach wipes followed by a
clean Kimwipe' and discard wipes into an autoclavable biohazard bag.
ii. Prior to dosing filters, immediately vortex the stock for 30 seconds.
iii. Per cassette, transfer a 120 }iL aliquot of the appropriate Stock tube (1 High, 2
Med,, or 3 Low) into a 1.5 mLtube.
Page 3 of 5
RWIs WI-VCF-SPIKE-l-v4 (January 23, 2019)
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EPA/600/R-19/083
June 2019
WORK INSTRUCTION FOR SPIKING WITH BACILLUS ANTHRACIS STERNE SPORES - VCF
iv. Remove the red plug and apply twenty 5 (iL droplets onto each filter as shown
in the below diagram. The same pipet tip can be used to place all twenty
droplets, dispose of the remaining volume once each filter has been dosed.
v. Air dry in BSC overnight with the red plug removed.
Start time: Date/Initials:
End time: Date/Initials:
vi. Firmly replace the top section of each cassette.
3. Enumerate stock
i. Spread 100 |iL aliquots of Dilution #5 and 6 onto Blood Agar in triplicate.
ii. Incubate plates
1. Invert the plates and incubate them at 37°C ± 2°C for 18 - 24 hours. B.
anthracis produces flat or slightly convex, 2-5 mm colonies, with edges
that are slightly irregular and have a "ground glass" appearance.
Incubation start Date/Time: Initials:
Incubation end Date/Time: Initials:
iii. Plate counts
1. Record counts in the below table.
Dilution Tube
Media Type
Volume
Plate (
Plate
1
ounts
Plate
2
Plate
3
Average
Counts
CFU In 50%
Ethanol Stock
5 (6.0 X 10'
cfu/mL)
Blood
Agar
100 fiL/
6 (6.0 X 102
cfu/mL)
Blood
Agar
100 ul
Recorded By: Date/Initials:
Page 4 of 5
RWIs WI-VCF-SPIKE-l-v4 (January 23, 2019)
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EPA/6Q0/R-19/083
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WORK INSTRUCTION FOR SPIKING WITH BACILLUS ANTHRACIS STERNE SPORES - VCF
Place twenty 5 pL evenly dispersed droplets onto filter.
Figure 1. Spiking diagram for VCF.
Page 5 of 5
RWIs WI-VCF-SPIKE-l-v4 (January 23, 2019)
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EPA/600/R-19/083
June 2019
APPENDIX E: WORK INSTRUCTION IOR IS A CILL IS A Ml IRA CIS
STERNE SPORE RECOVERY-SPG STICKS
-------
EPA/600/R-19/083
June 2019
WORK INSTRUCTION FOR BACILLUS ANTHRACIS STERNE SPORE RECOVERY - SPG STICKS
I. PURPOSE/SCOPE
To recover B, anthracis spores from air filters following the EPA/600/R-17/213 published by the EPA July
2017.
II. MATERIALS/EQUIPMENT
Materials
Itom
Manufacture!
Lot Number
Exp.
Date
Storage
Temp.
Initials & Date
extraction buffer with
Tween® 20
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
BHI broth
Inhouse
2-8 °C
Conical tubes, 15 mi-
N/A
R.T.
Falcon Conical Tube,
50mL
N/A
R.T.
Screw top flask, 250 mL
Corning
N/A
R.T.
0.45 |im filter vials
Whatman
N/A
R.T.
2mL screw cap tubes
VWR
70249-813C7-
8102
N/A
R.T.
Sterile disposable
forceps
N/A = Not Applicable
Equipment
Item
Manufacturer
Serial Number
Thermometer/
Rces tf
Calibration
Due
Initials & Date
Biosafety Cabinet
The Baker Company
D/DOO
N/A
8/2019
(BSC)
57544
8/2019
Micropipette
Type ±1000
Rainin
N/A
Incubator Shaker
New Brunswick
590644988
C22712
1/25/19
Refrigerator
Fisher
C3274822
115
3/2019
Swinging Bucket
Centrifuge
Beckman Coulter
X59221
N/A
N/A
Stomacher
Seward
40142
N/A
N/A
N/A = Not Applicable
Filters - Electronically update this table with samples names from the Sample Log
Page 1 of 7
Native Filters WI-SPG SPORE RECOVERY-2-v6 (November 5, 2018)
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WORK INSTRUCTION FOR BACILLUS ANTHRACIS STERNE SPORE RECOVERY - SPG STICKS
Sample #
Sample
type
Sample Location
Filter
Vi.il
1VP-"
Spore
Spike
level
Sample ID
1
SPG
Field Blank
PVDF
0
1-SPG-FLDBLK-A-S15-0
2
SPG
Street Grate
PVDF
0
2-SPG-STG RAT-A-S15-0
3
SPG
Field Blank
PVDF
30
3-SPG-FLDBLK-A-S16-30
4
SPG
Street Grate
PVDF
30
4-SPG-STGRAT-A-S16-30
5
SPG
Field Blank
PVDF
300
5-SPG-FLDBLK-A-S17-300
6
SPG
Street Grate
PVDF
300
6-SPG-STGRAT-A-S17-300
7
SPG
Field Blank
PVDF
3000
7-SPG-FLDBLK-A-S18-3000
8
SPG
Street Grate
PVDF
3000
8-SPG-STGRAT-A-S18-3000
9
SPG
Granite Bench
PVDF
0
9-SPG -GRNBEN-A-S15-0
10
SPG
Lab Blank
PVDF
0
10-SPG-LABBLANK-0
11
SPG
Granite Bench
PVDF
30
11-SPG-GRNBEN-A-S36-30
12
SPG
Lab Blank
PVDF
30
12-SPG-LABBLANK-30
13
SPG
Granite Bench
PVDF
300
13-SPG-GRNBEN-A-S17-300
14
SPG
Lab Blank
PVDF
300
14-SPG-LABBLANK-300
15
SPG
Granite Bench
PVDF
3000
15-SPG-G RNBEN-A-S18-3000
16
SPG
Lab Blank
PVDF
3000
16-SPG-LABBLANK-3000
Other Supplies and Equipment
• Forceps
• Biohazard bags
• Bleach
• 5 ml, 25 mLand 100 mLSerological Pipets
• Pipette aid
• Zipiock bags
III. PROCEDURE
A, RV-PCR Sample Processing: Spore Recovery for Sponge-Stick Samples
Note: Gloves should be used and changed between samples and as indicated below.
1. Prior to sample processing, prepare the following items:
~ Fill sample tube rack with 50 mL screw cap conical tubes and label as appropriate, two 50 mL
conical tubes are required per sample.
~ In a BSC, attach the vacuum manifold to the vacuum trap, waste container (with 400 ml of
bleach), and vacuum source. Attach the filter vials to the manifold, using outer rows first. Verify
that all filter vials are completely pushed down. Place a red pull tab tapered plug in each filter
vial.
~ Document filter vial and sample tube labels.
Performed by: Date:
Page 2 of 7
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EPA/6Q0/R-19/083
June 2019
WORK INSTRUCTION FOR BACILLUS ANTHRACIS STERNE SPORE RECOVERY - SPG STICKS
~ 1,500 mL of Extraction Buffer with Tween" 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 (lx 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 Tween8 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 TSB (See Wl #7: TSB Enrichment for Culture Non-Detects).
9. Follow steps described above for each sample, changing forceps between samples.
10. Gently mix the suspension in the Stomacher bag up and down three times with a sterile 50 mL pipet.
Remove half of the suspension volume (~45-46 mL) and place it in a 50 mL screw cap centrifuge tube
(Aliquot 1). Place the remaining suspension ("45-46 mL) into a second 50 mL tube (Aliquot 2).
Adjust the suspension volumes so that volume is equal in both tubes.
11. Process the suspension for each sample, as described above.
Performed by: Date:
Page 3 of 7
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WORK INSTRUCTION FOR BACILLUS ANTHRACIS STERNE SPORE RECOVERY - SPG STICKS
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
Filter ID
Total volume recovered from
Sponge-Stick
Recorded by:
1
1-SPG-FLDBLK-A-S15-0
2
2-SPG-STGRAT-A-S15-0
3
3-SPG-FLDBLK-A-S16-30
4
4-SPG-STGRAT-A-S16-30
5
5-SPG-FLDBLK-A-S17-300
6
6-SPG-STGRAT-A-S17-300
7
7-SPG-FLDBLK-A-S18-3000
8
8-SPG-STGRAT-A-S18-3000
9
9-SPG-GRNBEN-A-S15-0
10
10-SPG-LABBLANK-0
11
11-SPG-GRNBEN-A-S16-30
12
12-SPG-LABBLANK-30
13
13-SPG-GRNBEN-A-S17-300
14
14-SPG-LABBLANK-300
15
15-SPG-GRNBEN-A-S18-3000
16
16-SPG-LABBLANK-3000
17. Transfer 11 mLof the pooled extract and store on ice or in refrigerator until processed on same day
using Wl #4: Culture of Recovered Spores.
Performed by: Date:
Page 4 of 7
Native Filters WI-SPG SPORE RECOVERY-2-v6 (November 5, 2018)
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WORK INSTRUCTION FOR BACILLUS ANTHRACIS STERNE SPORE RECOVERY - SPG STICKS
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 11 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 offvacuum 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 (5mL) 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
Filter ID
Sample Addition
Volume of Wash
Recorded
#
Buffers
by:
Start
End
10X
IX
Time1
Time2
1
1-SPG-FLDBLK-A-S15-0
2
2-SPG-STG RAT-A-S15-0
3
3-SPG-FLDBLK-A-S16-30
4
4-SPG-STG RAT-A-S16-30
5
5-SPG-FLDBLK-A-S17-300
6
6-SPG-STG RAT-A-S17-300
7
7-SPG-FLDBLK-A-S18-3000
8
8-SPG-STG RAT-A-S18-3000
9
9-SPG-G RN BEN-A-S15-0
10
10-SPG- LAB BLA N K-0
11
11-SPG-GRNBEN-A-S16-30
12
12-SPG-LABBLANK-30
13
13-SPG-GRNBEN-A-S17-300
14
14-SPG- LAB BLA N K-300
15
15-SPG-GRNBEN-A-S18-3000
16
16-SPG- LAB BLA N K-3000
1Record 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 B) below, with filter vial manifold.
B. 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.
Performed by: Date:
Page 5 of 7
Native Filters WI-SPG SPORE RECOVERY-2-v6 (November 5, 2018)
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WORK INSTRUCTION FOR BACILLUS ANTHRACIS STERNE SPORE RECOVERY - SPG STICKS
2. Transfer 12.5 mL of cold (4°C) High salt wash buffer flOx PBS) to each filter-vial using a lOmL
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.
10. Place into the BSC: 5 mL serological pipets, 1000 pL pipet, 1000 pL tips, cold (2-8°C) BHI broth
aliquoted in 50 mL conical tubes, sharps container and orange caps.
11. Pipet 5 ml of cold BHI broth 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 BHI broth addition, this represents To. Bleach wipe the filter vial
Time of BHI 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 To aliquots onto ice in the BSC.
16. After vortexing, transfer filter vials to the BSC. Remove bag.
Performed by: Date:
Page 6 of 7
Native Filters WI-SPG SPORE RECOVERY-2-v6 (November 5, 2018)
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WORK INSTRUCTION FOR BACILLUS ANTHRACIS STERNE SPORE RECOVERY - SPG STICKS
17, Uncap one filter vial at a time and open the corresponding 2 mL tube. Using a 1 mL pipette or
serological pipet (if filter deteriorated), gently pipet upand down ~1G to mix. Transfer 1 mLfrom
each vial to the corresponding pre-chilled (on ice) 2 mL screw cap tube for To. Cap the tube and
place it back onto ice. Wipe the filter vial with a bleach soaked lab wipe. Change gloves between
each sample
After transferring the To aliquots for all samples, place the filter vial rack in a transfer container,
seal, and bleach the container. Store the To aliquot at -20 °C overnight.
To -20 C storage start time: End time: Initial/Date:
18. Transfer the filter vial rack to the shaker incubator. Secure the rack. Incubate at 372C at 230 rpm,
overnight (i.e., 16 hours from the addition of BHI broth to the filter vials). These samples are
referred to as the T, samples. Following incubation record turbidity observation and volume
remaining in the table below.
Start time: End Time: Speed: Temperature:
Sample
Number
Filter ID
Turbid (Yes/No)
Volume remaining
(mL)
Recorded by:
1
1-SPG-FLDBLK-A-S15-0
2
2-SPG-STGRAT-A-S15-0
3
3-SPG-FLDBLK-A-S16-30
4
4-SPG-STGRAT-A-S16-30
5
5-SPG-FLDBIK-A-S17-300
6
6-SPG-STGRAT-A-S17-300
7
7-SPG-FLDBLK-A-S18-3000
8
8-SPG-STGRAT-A-S18-3000
9
9-SPG-GRNBEN-A-S15-0
10
10-SPG-LABBLANK-0
11
11-SPG-GRNBEN-A-S16-30
12
12-SPG-LABBLANK-30
13
13-SPG-GRNBEN-A-S17-300
14
14-SPG-LABBLANK-300
15
15-SPG-GRNBEN-A-S18-3000
16
16-SPG-LABBLANK-3000
19. Proceed to Wl #3: DNA Purification to process To and Ti samples
Technical Review
Performed by: Date:
Page 7 of 7
Native Filters WI-SPG SPORE RECOVERY-2-v6 (November 5, 2018)
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EPA/600/R-19/083
June 2019
APPENDIX F: WORK INSTRUCTION IO R IS A CILL IS A Ml IRA CIS
STERNE SPORE RECOVERY-VCF
-------
EPA/600/R-19/083
June 2019
WORK INSTRUCTION FOR BACILLUS ANTHRACIS STERNE SPORE RECOVERY - VCF
I. PURPOSE/SCOPE
To recover 8, anthracis spores from air filters following the EPA/600/R-17/213 published by the EPA July
2017,
II. MATERIALS/EQUIPMENT
Materials
Item Manufacturer Lot Number Stoiage Initials & Date
Date Temp.
Extraction Buffer with
Tween® 20 + 30%
Ethanol
In house
2-8 °C
10X PBS
Teknova
2-8 °C
IX PBS (pH 7.4)
Teknova
2-8 °C
BHI broth
In house
2-8 °C
Conical tubes, 15 ml
Falcon
12118014
N/A
R.T.
Conical Tube, 50mL
N/A
N/A
N/A
R.T.
Screw top flask, 250
mL
N/A
R.T.
0.45 |j.m filter vials
Whatman
N/A
R.T.
2mL screw cap tubes
VWR
N/A
R.T.
2 oz. cups with lids
(autoclaved)
Container & Packaging
N/A
N/A
R.T.
Sterile Forceps
Unomedical
R.T.
N/A= Not Applicable
Equipment
Item
Manufacturer
Serial Number
Thermometer/
Roes#
Calibiation
Due
Initials & Date
Biosafety Cabinet
(BSC)
The Baker Company
57553
57544
N/A
8/2019
Micropipette
TypeilOOO
Rainin
C25845
C20268
N/A
4/23/2019
6/12/2019
Incubator Shaker
New Brunswick
590644988
Refrigerator
Fisher
C3274822
115
3/2019
Sonicator Bath
Bransonic
RNC010140514E
N/A
N/A
N/A = Not Applicable
Page 1 of 8
Native Filters WI-VCF SPORE RECOVERY-2-v3 (December 5,2018)
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EPA/600/R-19/083
June 2019
WORK INSTRUCTION FOR BACILLUS ANTHRACIS STERNE SPORE RECOVERY - VCF
Filters - Electronically update this table with samples names from the Sample Log
Filter
Spore
Sample
Vial
Spike
Sample #
Type
Location
Type
level
Filter ID
1
VCF
Floor Cone.
PVDF
0
1-VCF-FLCON-B-S15-0
2
VCF
Steps
PVDF
0
2-VCF-STEPS-B-S15-0
3
VCF
Floor Cone.
PVDF
30
3-VCF-FLCON-B-S16-30
4
VCF
Steps
PVDF
30
4-VCF-STEPS-B-S16-30
5
VCF
Floor Cone.
PVDF
300
5-VCF-FLCOIM-B-S17-300
6
VCF
Steps
PVDF
300
6-VCF-STEPS-B-S17-300
7
VCF
Floor Cone.
PVDF
3,000
7-VCF-FLCON-B-S18-3,000
8
VCF
Steps
PVDF
3,000
8-VCF-STEPS-B-S18-3,000
9
VCF
Sidewalk Cone.
PVDF
0
9-VCF-SWCON-A-S15-0
10
VCF
Pavement
PVDF
0
10-VCF-PAVEMT-A-S15-0
11
VCF
Sidewalk Cone.
PVDF
30
11-VCF-SWCON-A-S16-30
12
VCF
Pavement
PVDF
30
12-VCF-PAVEMT-A-S16-30
13
VCF
Sidewalk Cone.
PVDF
300
13-VCF-SWCON-A-S17-300
14
VCF
Pavement
PVDF
300
14-VCF-PAVEMT-A-S17-300
15
VCF
Sidewalk Cone.
PVDF
3,000
15-VCF-SWCON-A-S18-3,000
16
VCF
Pavement
PVDF
3,000
16-VCF-PAVEMT-A-S18-3,000
Other Supplies and Equipment
• Scissors
• Biohazard bags
• Bleach
• 5 mL, 25 mLand 100 mLSerological Pipets
• Pipette aid
• Ziplock bags
• Bench paper
• Stainless Steel SureSeal Cassette Opener, SKC cat. 225-13-5A
III. PROCEDURE
A, RV-PCR Sample Processing; Spore Recovery for Air Filter Samples
Note: Gloves should be used and changed between samples and as indicated below.
1. Prior to sample processing, prepare the following items:
• Fill sample tube rack with 15 mL screw cap conical tubes and label as appropriate, each
containing 11 mL sterile Extraction Buffer with Tween® 20 + Ethanol.
• One labeled 2 oz. sterile cup with lid per sample, sterilized by autoclave (Gravity cycle, 121 °C
for 15 minutes).
Performed by: Date:
Page 2 of 8
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EPA/6Q0/R-19/083
June 2019
WORK INSTRUCTION FOR BACILLUS ANTHRACIS STERNE SPORE RECOVERY - VCF
• In a BSC, attach the vacuum manifold to the vacuum trap, waste container (with 250 ml of
bleach), and vacuum source. Attach the filter vials to the manifold, using outer rows first. Verify
that all filter vials are completely pushed down. Place a red pull tab tapered plug in each filter
vial.
• Document filter vial and sample tube labels.
2. For each 37-mm filter cassette, prepare one 15 ml. conical tube containing 11 ml. of sterile
Extraction Buffer with Tween" 20+ Ethanol and label one 2 oz. sterile cup.
3. In the BSC remove the conical tube containing the nozzle and the cassette from the containment
bags and wipe the outside of the conical tube with a disinfectant and place it into a rack. Aseptically
add 5 ml. of Extraction Buffer with Tween" 20 + Ethanol (from the 11 ml of a pre-measured aliquot
of PBST + Ethanol) 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. Change gloves. 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 mLof Extraction Buffer with Tween" 20+ Ethanol from
the tube now containing the 6 mL into the cassette and replace plug. Roll the cassette around to
allow the liquid to touch all surfaces of the inside of the cassette. If there is a large quantity of
particulate matter, more Extraction Buffer with Tween" 20 + Ethanol may be required. Particulate
matter should be dampened enough to prevent aerosolization.
6. Using the cassette tool pry open the top section of the cassette, using care not to spill the Extraction
Buffer with Tween" 20+ Ethanol inside the cassette and set aside, plug side down as shown in
Figure 1. Set the bottom portion containing the filter aside carefully (filter side up), and using a
pipette rinse the walls of the cassette with 2 mL of Extraction Buffer with Tween" 20 + Ethanol.
Transfer the rinsate using the same pipette to the appropriately labelled 2 oz. sterile cup.
Figure 1. Vacuum Cassette with Top Section Removed
Performed by: Date:
Page 3 of 8
Native Filters WI-VCF SPORE RECOVERY-2-v3 (December 5, 2018)
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EPA/6Q0/R-19/083
June 2019
WORK INSTRUCTION FOR BACILLUS ANTHRACIS STERNE SPORE RECOVERY - VCF
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 2. 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 2. Vacuum Cassette with Top and Middle Sections Removed
8. Use the remainder of the 6 ml. Extraction Buffer with Tween8 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"1 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 Tween8 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 3).
Figure 3. Nozzle Removal Using 1 mL Pipette
Performed by: Date:
Page 4 of 8
Native Filters WI-VCF SPORE RECOVERY-2-v3 (December 5, 2018)
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EPA/6Q0/R-19/083
June 2019
WORK INSTRUCTION FOR BACILLUS ANTHRACIS STERNE SPORE RECOVERY - VCF
12. Seal all of the 2 oz. cups with ParafilrrT. 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 1. Note: Save 2 oz. cups containing
filter. Store at 2-8 °C until enrichment in TSB on same day (See Wl #7: VCFTSB Enrichment for
Culture).
Table 1. Volume of sample recovered from VCF.
Sample
Number
Filter ID
Total volume
recovered from
VCF
Volume available
per analytical
method (Total
Volume -r 2)
Recorded by:
1
1-VCF-FLCON-B-S15-0
2
2-VCF-STEPS-B-S15-0
3
3-VCF-FLCON-B-S16-30
4
4-VCF-STEPS-B-S16-30
5
5-VCF-FLCON-B-S17-300
6
6-VCF-STEPS-B-S17-300
7
7-VCF-FLCON-B-S18-3,000
8
8-VCF-STEPS-B-S18-3,000
9
9-VCF-SWCON-A-S15-0
10
10-VCF-PAVEMT-A-S15-0
11
11-VCF-SWCON-A-S16-30
12
12-VCF-PAVEMT-A-S16-30
13
13-VCF-SWCON-A-S17-300
14
14-VCF-PAVEMT-A-S17-300
15
15-VCF-SWCON-A-S18-3,000
16
16-VCF-PAVEMT-A-S18-3,000
14. Vortex each sample, then allow 3 - S 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 and gloves 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 and gloves between each sample.
Performed by: Date;
Page 5 of 8
Native Filters WI-VCF SPORE RECOVERY-2-v3 (December 5, 2018)
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EPA/600/R-19/083
June 2019
WORK INSTRUCTION FOR BACILLUS ANTHRACIS STERNE SPORE RECOVERY - VCF
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 (lOXand 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
Filter ID
Sample Addition
Volume of Wash
Recorded
#
Buffers
by:
Start
End
10X
IX
Time'
Time-'
1
1-VCF-FLCON-B-S15-0
2
2-VCF-STEPS-B-S15-0
3
3-VCF-FLCON-B-S16-30
4
4-VCF-STEPS-B-S16-30
5
5-VCF-FLCON-B-S17-300
6
6-VCF-STEPS- B-S17-300
7
7-VCF-FLCON-B-S18-3,000
8
8-VCF-STEPS-B-S18-3,000
9
9-VCF-SWCON-A-S15-0
10
10-VCF-PAVEMT-A-S15-0
11
11-VCF-SWCON-A-S16-30
12
12-VCF-PAVEMT-A-S16-30
13
13-VCF-SWCON A-Sl7-300
14
14-VCF-PAVEMT-A-S17-300
15
15-VCF-SWCON-A-S18-3,000
16
16-VCF-PAVEMT-A-S18-3,000
1Record 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.
B. 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 usinga 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 mLscrew
cap bottle.
Performed by: Date:
Page 6 of 8
Native Filters WI-VCF SPORE RECOVERY-2-v3 (December 5,2018)
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EPA/6Q0/R-19/083
June 2019
WORK INSTRUCTION FOR BACILLUS ANTHRACIS STERNE SPORE RECOVERY - VCF
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.
10. Place into the BSC: 5 mL serological pipets, 1000 pL pipet, 1000 pL tips, cold (2-8°C) BHI broth
aliquoted in 50 mL conical tubes, sharps container and orange caps.
11. Pipet 5 ml of cold BHI broth 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 BHI broth addition, this represents To. Bleach wipe the filter vial
Time of BHI 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 mL tube. Using a 1 mL pipette or
serological pipet (if filter deteriorated), gently pipet up and down ~10 to mix. Transfer 1 mL from
each vial to the corresponding pre-chilled (on ice) 2 mL screw cap tube for To. Cap the tube and
place it back onto ice. Wipe the filter vial with a bleach soaked lab wipe. Change gloves between
each sample
After transferring the To aliquots for all samples, place the filter vial rack in a transfer container,
seal, and bleach the container. Store the T0 aliquot at -20 °C overnight.
T0 -20 C storage start time: End time: Initial/Date:
Performed by: Date:
Page 7 of 8
Native Filters WI-VCF SPORE RECOVERY-2-v3 (December 5, 2018)
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EPA/600/R-19/083
June 2019
WORK INSTRUCTION FOR BACILLUS ANTHRACIS STERNE SPORE RECOVERY - VCF
18. Transfer the filter vial rack to the shaker incubator. Secure the rack. Incubate at 378C at 230 rpm,
overnight (i.e., 16 hours from the addition of BHI broth to the filter vials). These samples are
referred to as the Timai samples. Following incubation record turbidity observation and volume
remaining in the table below.
Start time: End Time: Speed: Temperature:
Sample
Number
Filter ID
Turbid (Yes/No)
Volume remaining
(mL)
Recorded by:
1
1-VCF-FLCON-B-S15-0
2
2-VCF-STEPS- B-S15-0
3
3-VCF-FLCON-B-S16-30
4
4-VCF-STEPS-B-S16-30
5
5-VCF-FLCON-B-S17-300
6
6-VCF-STEPS-B-SI 7-300
7
7-VCF-FLCON-B-S18-3,000
8
8-VCF-STEPS-B-S18-3,000
9
9-VCF-SWCON-A-S15-0
10
10-VC F- PAV E MT-A-S15-0
11
11-VCF-SWCON-A-S16-30
12
12-VCF-PAVEMT-A-S16-30
13
13-VCF-SWCON-A-S17-300
14
14-VCF-PAVEMT-A-S17-300
15
15-VCF-SWCON-A-S18-3,000
16
16-VCF-PAVEMT-A-S18-3,000
19. Proceed to Wl #3: DNA Purification to process To and Ti samples
Performed by: Date:
IV. Technical Review
Performed by: Date:
Page 8 of 8
Native Filters WI-VCF SPORE RECOVERY-2-v3 (December 5,2018)
-------
EPA/600/R-19/083
June 2019
APPENDIX G: WORK INSTRUCTION FOR CULTURE OF BACILLUS
ANTHRACIS SPORES RECOVERED FROM SPG STICKS
-------
EPA/600/R-19/083
June 2019
WORK INSTRUCTION FOR CULTURE OF BACILLUS ANTHRACIS SPORES
RECOVERED FROM AIR FILTERS - SPG STICKS
I. PURPOSE/SCOPE
Culture of B. anthrvcis spores recovered from air filters following the EPA/600/R-17/213 published by
the EPA July 2017.
II. MATERIALS/EQUIPMENT
Materials
item
\U-Uj.-oJ
Manufacturer
Lot Number
Exp.
Date
Storage
Temp.
Initials & Date
Microfunnel filters
PALL
R.T.
Blood Agar
BBL
2-8 °C
N/A= Not Applicable
Equipment
Item
Manufacturer
Serial Number
Thermometer/
Roes #
Calibration
Due
Initials & Date
Biosafety
Cabinet (BSC)
The Baker Company
N/A
Stationary
Incubator
Precision
9509-003
N/A
N/A
Vacuum
manifold
Gel man 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
RWIs WI-Culture-4-v4 (November 5, 2018)
Page 1 of 3
-------
EPA/6Q0/R-19/083
June 2019
WORK INSTRUCTION FOR CULTURE OF BACILLUS ANTHRACIS SPORES
RECOVERED FROM AIR FILTERS - SPG STICKS
Filters - Electronically update this table with samples names from the Sample Log
Sample #
Sample
type
Sample Location
Filter
Vial
Fype
Spore
Spike
level
Sample ID
1
SPG
Field Blank
PVDF
0
1-SPG-FLDBLK-A-S15-0
2
SPG
Street Grate
PVDF
0
2-SPG-STGRAT-A-S15-0
3
SPG
Field Blank
PVDF
30
3-SPG-FLDBLK-A-S16-30
4
SPG
Street Grate
PVDF
30
4-SPG-STGRAT-A-S16-30
5
SPG
Field Blank
PVDF
300
5-SPG-FLDBLK-A-S17-300
6
SPG
Street Grate
PVDF
300
6-SPG-STGRAT-A-S17-300
7
SPG
Field Blank
PVDF
3000
7-SPG-FLDBLK-A-S18-3000
8
SPG
Street Grate
PVDF
3000
8-SPG-STG RAT-A-S18-3000
9
SPG
Granite Bench
PVDF
0
9-SPG-GRNBEN-A-S15-0
10
SPG
Lab Blank
PVDF
0
10-SPG-LABBLANK-0
11
SPG
Granite Bench
PVDF
30
11-SPG-GRNBEN-A-S16-30
12
SPG
Lab Blank
PVDF
30
12-SPG-LABBLANK-30
13
SPG
Granite Bench
PVDF
300
13-SPG-GRNBEN-A-S17-300
14
SPG
Lab Blank
PVDF
300
14-SPG-LABBLANK-300
15
SPG
Granite Bench
PVDF
3000
15-SPG-G RIM BEN-A-S18-3000
16
SPG
Lab Blank
PVDF
3000
16-SPG-LABBLANK-3000
III. PROCEDURE
Note: The following procedure is to be carried out with the 10 mL pooled extract taken from step 17
(refer to Wl #2 for Bacillus anthracis spore recovery). Process 2-3 PBST only negative control filter
funnels alongside samples.
A. Culture Method
1. Label two filter funnels per sample, one with 2 mL and one with 8 mL.
2. Place the filter funnels onto the vacuum manifold in a Class II BSC,
3. Add 5 mLof 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, then allow 3-5 minutes of settle time to avoid loading large particulates into
filter funnel. For each sample, add 2 mL of pooled extract to one filter funnel and 8 mL of pooled
extract to one filter funnel. Apply vacuum. Save remaining ~1 mLof culture aliquot and store at 2 -
8 °C until processing using Wl #7: TSB Enrichment for Culture Non-Detects.
Performed by: Date:
Page 2 of 3
RWIs WI-Culture-4-v4 (November 5, 2018)
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EPA/6Q0/R-19/083
June 2019
WORK INSTRUCTION FOR CULTURE OF BACILLUS ANTHRACIS SPORES
RECOVERED FROM AIR FILTERS-SPG STICKS
6. Close the vacuum valve and release the vacuum pressure. Rinse the walls of each filter funnel using
10 mL of PBST. Apply vacuum.
7. With the vacuum valve closed and the vacuum pressure released, remove the membrane from the
filter funnel and place onto Blood Agar. Dispose of filter bases and then change glove.
8. Incubate plates inverted overnight at 37°C ± 2°C.
Incubation start Date/Time: Initials:
Incubation end Date/Time: Initials:
9. Enter results into the below table.
Filter ID
B. anthracis colonies
Total colonies (all morphologies)
CFU/2mL
CFU/8mL
CFU/2 mL
:FU/8 mL
PBST Negative #1
PBST Negative #2
1-SPG-FLDBLK-A-S15-0
2-SPG-STGRAT-A-S15-0
3-SPG-FLDBLK-A-S16-30
4-SPG-STGRAT-A-S16-30
5-SPG-FLDBLK-A-S17-300
6-SPG-STGRAT-A-S17-300
7-SPG-FLDBLK-A-S18-3000
8-SPG-STGRAT-A-S18-3000
9-SPG-GRNBEN-A-S15-0
10-SPG-LABBLANK-0
11-SPG-GRNBEN-A-S16-30
12-SPG-LABBLANK-30
13-SPG-GRNBEN-A-S17-300
14-SPG- LAB BLA N K-300
15-SPG-GRNBEN-A-S18-3000
16-SPG-LABBLANK-3000
Counts performed/recorded by: Date:
Performed by: Date:
IV. Technical Review
Reviewed by: Date:
RWIs WI-Culture-4-v4 (November 5, 2018)
Page 3 of 3
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EPA/600/R-19/083
June 2019
APPENDIX H: WORK INSTRUCTION FOR CULTURE OF BACILLUS
ANTHRACIS SPORES RECOVERED FROM VCF
-------
EPA/600/R-19/083
June 2019
WORK INSTRUCTION FOR CULTURE OF BACILLUS ANTHRACIS SPORES
RECOVERED FROM AIR FILTERS - VCF
I. PURPOSE/SCOPE
Culture of B. anthraeis spores recovered from air filters following the EPA/600/R-17/213 published by
the EPA July 2017.
II. MATERIALS/EQUIPMENT
Materials
Item
(0.05%)
Manufacturer
Lot Number
Exp.
Date
Storage
Temp.
Initials & Date
Microfunnei filters
PALL
R.T.
Blood Agar
BBL
2-8 °C
N/A = Not Applicable
Equipment
Item
Manufacturer
Serial Number
Thermo motor/
Roes #
Calibration
Duo
Initials & Date
Biosafety
Cabinet (BSC)
The Baker Company
N/A
Stationary
Incubator
Precision
9509-003
N/A
N/A
Vacuum
manifold
Gel man Sciences
N/A
N/A
N/A
N/A= Not Applicable
Other Supplies and Equipment
• Forceps
• Bleach
• 5 mL, 10 mL, arid 25mL Serological Pipettes
• Pipette aid
Page 1 of 3
RWIs WI-Culture-VCF-4-v3 (December 10,2018)
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EPA/6Q0/R-19/083
June 2019
WORK INSTRUCTION FOR CULTURE OF BACILLUS ANTHRACIS SPORES
RECOVERED FROM AIR FILTERS - VCF
Filters - Electronically update this table with samples names from the Sample Log
Filter
Spore
Sample
Sample
Vial
Spike
Sample #
type
Location
Type
level
Sample ID
1
VCF
Floor Cone.
PVDF
0
1-VCF-FLCON- B-S15-0
2
VCF
Steps
PVDF
0
2 - VC F-STE PS- B-S15-0
3
VCF
Floor Cone.
PVDF
30
3-VCF-FLCON-B-S16-30
4
VCF
Steps
PVDF
30
4-VCF-STEPS-B-S16-30
5
VCF
Floor Cone.
PVDF
300
5-VCF-FLCON- B-S17-300
6
VCF
Steps
PVDF
300
6-VCF-STEPS-B-S17-300
7
VCF
Floor Cone.
PVDF
3,000
7-VCF-FLCON-B-S18-3,000
8
VCF
Steps
PVDF
3,000
8-VCF-STEPS-B-S18-3,000
9
VCF
Sidewalk Cone.
PVDF
0
9-VCF-SWCON-A-S15-0
10
VCF
Pavement
PVDF
0
10-VCF-PAVEMT-A-S15-0
11
VCF
Sidewalk Cone.
PVDF
30
11-VCF-SWCON-A-S16-30
12
VCF
Pavement
PVDF
30
12-VCF-PAVEMT-A-S16-30
13
VCF
Sidewalk Cone.
PVDF
300
13-VCF-SWCON-A-S17-300
14
VCF
Pavement
PVDF
300
14-VCF-PAVEMT-A-S17-300
15
VCF
Sidewalk Cone.
PVDF
3,000
15-VCF-SWCON-A-S18-3,000
16
VCF
Pavement
PVDF
3,000
16-VCF-PAVEMT-A-S18-3,000
III. PROCEDURE
Note: The following procedure is to be carried out with the ~S mL extract taken from step 15 (refer to
Wl #2 for Bacillus anthracis spore recovery). Process 2-3 PBST only negative control filter funnels
alongside samples.
A. Culture Method
1. Label two filter funnels per sample, one with 1 mL and one with 4 mL.
2. Place the filter funnels onto the vacuum manifold in a Class II BSC,
3. Add 5 mLof 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, then allow 3-5 minutes of settle time to avoid loading large particulates into
filter funnel. For each sample, add 1 mL of pooled extract to one filter funnel and "4mL (remaining
volume) of pooled extract to one filter funnel. Apply vacuum. Enter the remaining volume added to
the 4 mL aliquot in below table.
Performed by: Date:
Page 2 of 3
RWIs WI-Culture-VCF-4-v3 (December 10, 2018)
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EPA/6Q0/R-19/083
June 2019
WORK INSTRUCTION FOR CULTURE OF BACILLUS ANTHRACIS SPORES
RECOVERED FROM AIR FILTERS - VCF
6. Close the vacuum valve and release the vacuum pressure. Rinse the walls of each filter funnel using
10 mL of PBST. Apply vacuum.
7. With the vacuum valve closed and the vacuum pressure released, remove the membrane from the
filter funnel and place onto Blood Agar. Dispose of filter bases and then change glove.
8. Incubate plates inverted overnight at 37°C + 2°C.
Incubation start Date/Time: Initials:
Incubation end Date/Time: Initials:
9. Enter results into the below table.
Filter ID
B. anthracis
colonies
Total colonies (all
morphologies)
Remaining
Volume (4
mL Aliquot)
CFU/l
mL
CFU/4
mL
CFU/l mL
:FU/4 mL
PBST Negative #1
N/A
PBST Negative #2
N/A
1-VCF-FLCON-B-S15-0
2-VCF-STEPS-B-S1S-0
3-VCF-FLCON-B-S16-30
4-VCF-STE PS- B-S16-30
5-VCF-FLCON-B-S17-300
6-VCF-STEPS-B-S17-300
7-VCF-FLCON-B-S18-3,000
8-VCF-STEPS-B-S18-3,000
9-VCF-SWCON-A-S15-0
10-VCF-PAVEMT-A-S15-0
11-VCF-SWCON-A-S16-30
12-VCF-PAVEMT-A-S16-30
13-VCF-SWCON-A-S17-300
14-VCF-PAVEMT-A-S17-300
15-VCF-SWCON-A-S18-3,000
16-VCF-PAVEMT-A-S18-3,000
Counts performed/recorded by:
Performed by:
IV. Technical Review
Reviewed by:
RWIs WI-Culture-VCF-4-v3 (December 10, 2018)
Date:
Date:
Date:
Page 3 of 3
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EPA/600/R-19/083
June 2019
APPENDIX I: WORK INSTRUCTION FOR
MANUAL DNA EXTRACTION AND PURIFICATION FROM
BACILLUS ANTHRACIS SPORES
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EPA/600/R-19/083
June 2019
WORK INSTRUCTION FOR MANUAL DNA EXTRACTION AND PURIFICATION
FROM BACILLUS ANTHRACIS SPORES - RWI
I. PURPOSE/SCOPE
Manual DNA extraction and purification B. arithracis spores from recovered from air filters following the
BACILLUS Analytical Methods 004 published by the EPA December 2012,
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
Thermometer Calibration Initials &
Serial Number
/Rees # Due Date
Biosafety
Cabinet (BSC)
The Baker Company
57544
N/A
8/2019
Micropipette
Type: L2 00
Rainin
N/A
Micropipette
Type:L200
Rainin
N/A
Micropipette
Type: LI 000
Rainin
N/A
Micropipette
Type: LI 000
Rainin
N/A
Ultra-low
Freezer
Woods
X34664
10
4/1/18
Refrigerator
Thermo Fisher
35840
115
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
Page 1 of 5
RWIs Wl-Manual DNA Extraction and Purification-3-v5 (09/26/2017)
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EPA/6Q0/R-19/083
June 2019
WORK INSTRUCTION FOR MANUAL DNA EXTRACTION AND PURIFICATION
FROM BACILLUS ANTHRACIS SPORES - RWI
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°C prior to Section 10.4.8.
NOTE: Process samples from zero spike level to 3,000 spike level. Change gloves when moving from a
spiked sample to a sample containing a lower spike 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 Ti aliquots in a tube. Do not use 1.5 mL tubes. Transfer Taliquot screw
cap tubes to the BSC.
5. Transfer the filter vial rack to the BSC. Uncap one filter vial at a time and transfer 1 mL to
corresponding 2 mL tube after gently pipetting up and down ~10 to mix. Change gloves in between
each sample.
6. Centrifuge 2 mL screw cap tubes (both To and Ti) at 14,000 rpm for 10 minutes (4°C).
Start: End: Speed:
7. Remove 800 pLof the supernatant from each tube, using a 1000 pL pipet and dispose to waste. Do not
disturb the pellet. Change gloves in between each sample.
8. Add 800 pL of lysis buffer using a 1000 pL 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. Change gloves in
between each sample.
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). Change gloves in
between each sample. Incubate the To and Ti lysate tubes at room temperature for 5 minutes.
Performed by: Date:
Page 2 of 5
RWIs Wl-Manual DNA Extraction and Purification-3-v5 (09/26/2017)
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June 2019
WORK INSTRUCTION FOR MANUAL DNA EXTRACTION AND PURIFICATION
FROM BACILLUS ANTHRACIS SPORES - RWI
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 T lysate
tube.
11. Uncap one tube at a time and add 600 pLof PMPs to each To and Ti tube (containing 1 mL sample).
Change gloves in between each sample.
12. Vortex each To and Ti 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 |^L 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 pLof lysis buffer using a 1000 pL 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.17. Use a new tip for each T0 and Ti tube.
Wash Steps:
19. Uncap each tube one at a time and add 360 pLof 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.17.
Use a new tip for each T0 and T tube. This is 1st Salt Wash.
20. Uncap each tube one at a time and add 360 nLof 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.17.
Use a new tip for each To and Ti tube. This is 2nd Salt Wash.
Performed by: Date:
Page 3 of 5
RWIs Wl-Manual DNA Extraction and Purification-3-v5 (09/26/2017)
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EPA/6Q0/R-19/083
June 2019
WORK INSTRUCTION FOR MANUAL DNA EXTRACTION AND PURIFICATION
FROM BACILLUS ANTHRACIS SPORES - RWI
21. Uncap each tube one at a time and add 500 pL 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.17.
Use a new tip for each To and Ti tube. This is Is' Alcohol Wash.
22. Uncap each tube one at a time and add 500 pL 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.17.
Use a new tip for each To and Ti tube. This is 2nd Alcohol Wash.
23. Uncap each tube one at a time and add 500 pL 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.17.
Use a new tip for each T0 and T, tube. This is 3rd Alcohol Wash.
24. Uncap each tube one at a time and add 500 pLof 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.17. Use a new
tip for each To and Ti 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 To and Ti tubes and air dry for 2 minutes.
27. Close tubes and transfer to heat block. Re open tubes once placed on the heat block at 80°C until the
PMPs are dry (~20 minutes, or until dry). Allow all the alcohol solution to evaporate since alcohol may
interfere with analysis. If residual condensation is present, do not remove, leave it in place.
Start: End: Temperature:
28. DNA elution: While they are in the heating block add 200 pL of elution buffer to each T0 and Ti 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:
Performed by: Date:
Page 4 of 5
RWIs Wl-Manual DNA Extraction and Purification-3-v5 (09/26/2017)
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June 2019
WORK INSTRUCTION FOR MANUAL DNA EXTRACTION AND PURIFICATION
FROM BACILLUS ANTHRACIS SPORES - RWI
32. Briefly vortex each tube (5 - 10 seconds) on low speed and centrifuge at 2000 rpm, 42C 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 T, tube and transfer ~80-90 uL to a clean, labeled, 1.5 mLtube on ice
(check tube labels to ensure the correct order). Use a new tip for each tube. Visually verify absence of
PMP carryover during final transfer. If magnetic bead carryover occurred, place 1.5 ml. tube on
magnet, collect liquid, and transfer to a clean, labeled, 1.5 mL tube.
35. Centrifuge tubes at 14,000 rpm at 4°C for 5 minutes to pellet any particles remaining with the eluted
DNA; carefully remove supernatant from all samples and transfer to a new 1.5 mL tube using a new tip
for each tube.
Start: End:
36. Store T0 and Ti DNA extract tubes at 4°C until PCR analysis. Continue to WI-RV-PCR-Native Filters.
Note: If PCR cannot be performed within 24 hours, freeze DNA extracts at -20?C.
Labeled:
Date/Time:
Storage Temperature:
Storage Location:
Performed by:
Date:
IV. Technical Review
Performed by:
Date:
Comments:
Page 5 of 5
RWIs Wl-Manual DNA Extraction and Purification-3-v5 (09/26/2017)
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EPA/600/R-19/083
June 2019
APPENDIX J: WORK INSTRUCTION RV-PCR FOR BACILLUS
ANTHRACIS SPORES-SPG STICKS
-------
EPA/600/R-19/083
June 2019
WORK INSTRUCTION RV-PCR FOR BACILLUS ANTHRACIS SPORES - SPG
I. PURPOSE/SCOPE
Duplex Rapid Viability (DRV)-PCR for B. anthracis spores from recovered from air filters modified from
the single-plex RV-PCR described in BACILLUS Analytical Methods 004 published by the EPA December
2012,
II. MATERIALS/EQUIPMENT
Materials
Enter materials used into Native Filters WI-RV-PCR-Sv2 - FORM A
Equipment
Itom
Manufacturer
Serial Number
Thermometer/
Reos #
Calibration
Due
Initials & Date
Biosafety
Cabinet (BSC)
Baker Thermo Forma
Micropipette
Type;L10
Rainin
N/A
Micropipette
Type:L2Q
Rainin
N/A
Micropipette
Type:L200
Gilson
N/A
Micropipette
Type:L1000
Rainin
N/A
Refrigerator
Kolpak
X57533
Rees #65
2/2019
Freezer
Kelvinator
Centrifuge
Eppendorf
X58983
N/A
N/A
7500 Fast
Applied Biosystems
275017115
N/A
12/2018
N/A = Not Applicable
Other Supplies and Equipment
• Micropipette tips
• 96-well 0.1 ml FAST plates
• Optical caps
• Biohazard bags
• Bleach
• DNase Away
• 70% Isopropanol
Page 1 of 6
RWIs WI-RV-PCR-5-v6 (07/09/18)
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EPA/6Q0/R-19/083
June 2019
WORK INSTRUCTION RV-PCR FOR BACILLUS ANTHRACIS SPORES - SPG
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 Ti DNA extracts'. Label 1.5 mL tubes with the sample identifier and "10-fold dilution". Add 90
|iL 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 pL of supernatant to 1.5-mL Eppendorf tubes with 90 pL 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 a DNA erase, 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 a re 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, 3
PNCs (Method blank) and 6 DNA extracts per sample (3 for TO and 3 forTi DNA extracts). Record
sample names and reaction numbers on WI-RV-PCR-Sv2 - FORM A.
3. In a clean PCR-preparation hood, pipet 20 pLof Master Mix into the wells of the PCR plate. Label four
wells as NTC and four as PC.
4. Add 5 pL of PCR-grade water into the NTC wells.
5. Tightly 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 pL to each sample well. Tightly seal the sample wells with
optical caps.
Performed by: Date:
Page 2 of 6
RWIs WI-RV-PCR-5-v6 (07/09/18)
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June 2019
WORK INSTRUCTION RV-PCR FOR BACILLUS ANTHRACIS SPORES - SPG
7. Vortex the PC (8. anthracis DNA [10 pg/|iL or 50 pg/5 |j.L]) and add 5 |iL to each of the PC wells. Tightly
seal the PC wells with optical caps,
8, Note: This step must be performed in the BSC outside the PCR clean room set-up area (Dead Air Box).
C. Within the Post-Amplification Lab, Load 96-well plates onto 7500 Fast,
1. Set up 7500 Fast
a. Open the 7500 Fast Software and a new file,
i. Configure the New Document dialog box:
1. Assay - Select Standard Curve (Absolute Quantification)
2. Container - Select 96 Wells Clear
3. Template—Select Blank, or Browse for a previously saved file,
4. Choose the Run mode—7500 FAST.
5. Operator—Enter your name.
6. Comments—Enter any comments pertaining to the run.
7. Plate Name—Enter a plate name.
8. Click Next,
ii. Choose the Detector for this assay
1. Choose 6-FAM-MGB from the list, or create a new one now by clicking the
New Detector button. Multiple detectors can be selected by using the Ctrl
key.
2. Choose Add »to add the detectors to the plate document,
3. Choose ROXIM as the passive reference from the Passive Reference drop down
box.
4. Click Next.
iii. The setup window is split into two panes. Use the layout grid to select indiviual wells,
and the Setup tab of the Well Inspector pane to apply detectors and designate well
assignments. As parameters are chosen in the Setup tab, they are recorded in the
Table pane at the bottom of the window.
1. Highlight the wells desired in the layout grid.
2. Check the Use box next to each detector to be analyzed for each well.
3. Click in the Sample Name box, and type in the name.
4. Choose the Task (Standard or NTCj from the dropdown list. If the task is
Standard, you must enter the concentration before proceeding to the next
well. Proceed until all the wells have been assigned all of the appropriate
detectors, names, task, and concentrations (standards only).
5. Click Finish.
iv. Set the method parameters by clicking on the Instrument tab of the Well Inspector
pane. All parameters must be changed to match Table 1.
1. To delete a stage, click on the bar separating the stages and drag to highlight
the stage to be deleted. Click [Delete Step].
Performed by: Date:
Page 3 of 6
RWIs WI-RV-PCR-5-v6 (07/09/18)
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WORK INSTRUCTION RV-PCR FOR BACILLUS ANTHRACIS SPORES - SPG
2. To add a parameter, click the step to the left of the location where you want
to place the new parameter, and choose [Add Cycle], [Add Hold], or [Add
Step].
3, To change cycle times and temperatures, click in the respective boxes in each
stage and type in the appropriate settings.
Table 1. 7500 FAST Method Parameters.
Temperature (X)
Time
Cvcles
95.0
0:20
1
95.0
0:03
45
60.0
0:30
25 Total Volume
v. Save the file. Only files with the (*.sds) extension can be run.
1. From the top, choose File, Save As,
2. If the document is a new plate, go to drive D, Applied Biosystems, SDS,
Documents, and the appropriate folder. Name the file. From the drop down,
"Files of type:" choose ABI Prism SDS Single Plate (*,sds).
3. If the document is a template, follow the same procedure. This file will
already have a name and the (*.sdt) file extension. Change the name to
identify this particular run, and change the file type to ABI Prism SDS Single
Plate (*.$ds).
4. Close the file.
vi. Centrifuge the plate at 300 xG for about 1-2 minutes at room temperature or in
Labnet's MPS-1000 Mini Plate Spinner.
vii. Open the file created in step C. 1. a. v., load 96-well plate into 7500 and start run.
viii. When run is complete, burn the file to a CD.
ix. Remove 96-well plate from 7500 Fast and dispose
D. Analysis
1. Open the assay with the most current version of 7500 Fast software.
a. Analysis can be performed using automatic settings. If required to manually set threshold and
baseline, from the menu bar choose Analysis, and then choose Analysis Settings. Select
Manual Ct, and Manual Baseline.
b. From the menu bar choose Analysis, then choose Analyze from the drop down menu, or click
on the large green triangle icon button in the toolbar.
c. Highlight the unknowns, standards (only one set if there is more than one), and NTCs either in
the Plate Grid pane or in the Table View pane.
d. Click on the Results tab in the Well Inspector pane to view the Amplification Plot and Standard
Curve Plot.
Performed by: Date:
Page 4 of 6
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WORK INSTRUCTION RV-PCR FOR BACILLUS ANTHRACIS SPORES - SPG
e. Ensure that the desired detector appears in the Detector box in both the Amplification Plot
and Standard Curve Plot. If not, chose it from the drop down menus now.
f. If analyzing based on manual threshold and baseline settings,
i. Look at the Ct values for the standards and unknowns in the Report View pane to
determine the lowest Ct value. Change the value in the End (cycle) box on the right
side of the plot to adjust the baseline setting to 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 right cursor should be placed at 20.)
ii. Highlight the standards and NTCs only. Click on the line that represents the threshold
in the amplification plot. Move the threshold so that it is above all background
"noise" generated by non-amplification, and in the lowest part of the exponential
phase of all the standards. Depending on the range of the standard curve, some
standards at the lowest end of the curve may not amplify well; whether or not to
include them will be decided by the Program Manager or Principal Investigator.
iii. Check the values in the Report View pane. Moving the threshold occasionally causes
the Ct values to change. If necessary, readjust the baseline and threshold by
repeating steps 9a through b until the baseline remains at a level two Ct values below
the lowest Ct value in the Report View pane and the threshold is properly set. If
changes are made that would affect the software s response to the data, such as
deleting a well or changing a detector, etc., the software will reset the analysis and
the data will need to be reanalyzed.
iv. Look in the Report View pane to ensure that the Ct value for all of the NTCs is
Undetermined. This means that no amplification was detected in the number of
cycles this analysis ran, and is considered to be a negative result. If any of the wells
show amplification at this point, contact the Program Manager or Principal
Investigator.
v. From the menu bar, choose Tools and then Report Settings. Check the boxes for the
data required to be printed by your project,
vi. Click Print, Done.
g. Highlight each individual well with a Ct less than 45, and check the Multicomponent Plot to
ensure there is actual amplification. Amplification is indicated by an upward curve in the line
representing the reporter dye, and if a quencher is present, a downward curve in the line
representing the quencher.
h. As specified by a project, print Multicomponent Plots.
i. Annotations to be made by the analyst on the printouts:
• Initial and date (l/D) every printout.
• Initial, date, and error or otherwise annotate all errors and comments.
• Indicate which, if any, wells of the Standard Curve were omitted.
• If required by the project, every Multicomponent Plot should indicate which sample
number it represents, and the actual Ct value associated with the well
j. Repeat the preceding steps to this point for every detector OR each standard curve associated
with this assay.
k. Attach all printouts to the worksheet. This constitutes one data package.
Performed by: Date:
Page 5 of 6
RWIs WI-RV-PCR-5-v6 (07/09/18)
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WORK INSTRUCTION RV-PCR FOR BACILLUS ANTHRACIS SPORES - SPG
2. After the PGR run, discard sealed PCR plate.
3. Export the ,csv file
a. Go to File -> Export -> Results,
b. Select folder location -> Native Filters
c. Save results type as xsv.
d. Select save.
i. A dialog box will open.
ii. Check the box, Apply Report Settings for Data Columns.
e. Burn .sds and .csv files onto a CD.
Data Calculations
Calculate an average CT from the replicate reactions for TO and T9 DNA extracts of each sample.
Subtract the average CT of the T9 DNA extract from the average CT of the TO DNA extract. If there is
no CT for the TO DNA extract (i.e., the TO is non-detect), use 45 (total number of PCR cycles used) as
the CT. The change (decrease) in the average CT value from TO to T9 (ACT) > 9 indicates a positive
result suggesting the presence of viable B. anthracis spores in the sample. If an incubation time
longer than 9 hours was used for the RV-PCR, instead of T9, appropriate Tx (incubation time) should
be used. However, (ACT) £ 9 algorithm should still be used for a positive result. Depending upon the
end user's requirement, sample complexity (dirtiness) and the phase of response during an event, a
lower ACT criterion of 2 6 (a two log difference in DNA concentration) and a corresponding higher
endpoint PCR CT of < 39 could be set, A minimum of two out of three TO PCR replicates must result
in CT values < 44 (in a 45-cycle PCR) to calculate the average CT. A minimum of two out of three T9
PCR replicates (or Tx for other incubation time) must result in CT values < 36 to calculate the
average CT for a sample result to be considered positive. Negative controls (NTCs) should not yield
any measurable CT values above the background level. If CT values are obtained as a result of a
possible contamination or cross-contamination, prepare fresh PCR Master Mix and repeat analysis.
In addition, field blank samples should not yield any measurable CT values. If CT values are observed
as a result of a possible contamination or cross-contamination, a careful interpretation of the CT
values for the sample DNA extracts and field blanks must be done to determine if the data is
considered valid or if the PCR analyses must be repeated.
Print RV-PCR results and attach to this Wl package, include relevant calculations and file name.
Performed by: Date:
Technical Review
Performed by: Date:
Page 6 of 6
RWIs WI-RV-PCR-5-v6 (07/09/18)
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EPA/6Q0/R-19/083
June 2019
Real World Interferents WI-RV-PCR-5v2 - FORM A
Project: RWI
DNA ASSAY—96 Well Plate Setup for Fast 7500
Barcode:
Target: Ba Duplex, Chromosome (FAM-MGBNFQ) and pXOl (VIC-MGBNFO)
1. Calculate the total number of reactions per plate:
Sample wells 48 + 4 NTC wells + 4 positive controls + _
_ extras = 61 total rxns/plate (Y)
2. Prepare the Master Mix by combining the following reagents in an appropriate tube according to the following calculation:
Reagent
Manufacturer
Lot No.
Exp. Date
X
Y
Total Volume
OiL)
2X Fast PCR Mix
12.5 jiL
61
762.5
Platinum Taq DNA Polymerase
0.25 jiL
61
15.25
pXOl For. primer (25 uM)
1 jiL
61
61
pXOl Rev. primer (25 uM)
1 jiL
61
61
pXOl Probe (2 jiM)
1 jiL
61
61
Chro. For. primer (25 uM)
1 jiL
61
61
Chro. Rev. primer (25 uM)
1 jiL
61
61
Chro. Probe (2 |nM)
1 jiL
61
61
PCR grade water
1.25 jiL
61
91.5
Total
20 jiL
Distribute 20 jiL of Master Mix into each reaction well, as indicated in the plate layout, below. Loosely cover all wells containing Master
Mix with caps.
Add 5 jliL of PCR-grade water to each of the NTC Wells. Cap wells tightly.
Add 5 jiL of PNC (Method Blank) to the corresponding wells and secure the caps
Add 5 jiL of Sample to the corresponding wells and secure the caps.
Positive Control
Positive Control lot
prep date
8. Centrifuge the plate using Labnef s MPS-1000 Mini Plate Spinner at room temperature, and then load the plate onto the 7500 Fast Dx
1
1 -SPG-TELEBO-A-S01 -0 T0
PC
50 PS
PC
50 PB
PC
?°PS
1 -SPG-TELEBO-A-S01-0 T,
PC
50 p
NTC
10
I 11 I
12
NTC
NTC
NTC
T echnicians
Signature
Date
Master Mix, NTC
Samples
Standards
Analyst
Reviewed By:
Date:
Page 1 of 1
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EPA/600/R-19/083
June 2019
APPENDIX K: WORK INSTRUCTION RV-PCR FOR BACILLUS
ANTHRACIS SPORES-VCF
-------
EPA/600/R-19/083
June 2019
WORK INSTRUCTION RV-PCR FOR BACILLUS ANTHRACIS SPORES - VCF
I. PURPOSE/SCOPE
Duplex Rapid Viability (DRV)-PCR for B. anthracis spores from recovered from air filters modified from
the single-plex RV-PCR described in BACILLUS Analytical Methods 004 published by the EPA December
2012.
II. MATERIALS/EQUIPMENT
Materials
Enter materials used into Native Filters WI-RV-PCR-Sv2 - FORM A
Equipment
, . ,. Thermometer/ Calibration
Item Manufacturer Serial Number _ _ Initials & Date
Rees It Due
Biosafety
Cabinet (BSC)
Baker Thermo Forma
N/A
Micropipette
Type:L10
Rainin
N/A
Micropipette
Type:L20
Rainin
N/A
Micropipette
Type:L200
Gilson
N/A
Micropipette
Type:L1000
Rainin
N/A
Refrigerator
Kolpak
X57533
Rees #65
2/2019
Freezer
Kelvinator
Centrifuge
Eppendorf
X58983
N/A
N/A
7500 Fast
Applied Biosystems
275017115
N/A
12/2018
N/A = Not Applicable
Other Supplies and Equipment
• Micropipette tips
• 96-well 0.1 mL FAST plates
• Optical caps
• Biohazard bags
• Bleach
• DNase Away
• 70% Isopropanol
Page 1 of 6
RWIs WI-RV-PCR-5-v6 (07/09/18)
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WORK INSTRUCTION RV-PCR FOR BACILLUS ANTHRACIS SPORES - VCF
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 Ti DNA extracts'. Label 1.5 mL tubes with the sample identifier and "10-fold dilution". Add 90
|iL 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 pL of supernatant to 1.5-mL Eppendorf tubes with 90 pL 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 a DNA erase, 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 a re 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, 3
PNCs (Method blank) and 6 DNA extracts per sample (3 for TO and 3 for Ti DNA extracts). Record
sample names and reaction numbers on WI-RV-PCR-5v2 - FORM A.
3. In a clean PCR-preparation hood, pipet 20 pLof Master Mix into the wells of the PCR plate. Label four
wells as NTC and four as PC.
4. Add 5 pL of PCR-grade water into the NTC wells.
5. Tightly 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 pL to each sample well. Tightly seal the sample wells with
optical caps.
Performed by: Date:
Page 2 of 6
RWIs WI-RV-PCR-5-V6 (07/09/18)
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EPA/600/R-19/083
June 2019
WORK INSTRUCTION RV-PCR FOR BACILLUS ANTHRACIS SPORES - VCF
7, Vortex the PC (B. anthracis DNA [10 pg/jiL or 50 pg/5 |iL]) and add 5 nL to each of the PC wells. Tightly
seal the PC wells with optical caps.
8, Note: This step must he performed in the BSC outside the PCR clean room set-up area (Dead Air Box).
C. Within the Post-Amplification Lab, Load 96-well plates onto 7500 Fast.
1. Set up 7500 Fast
a. Open the 7500 Fast Software and a new file.
i. Configure the New Document dialog box;
1. Assay - Select Standard Curve (Absolute Quantification)
2. Container-Select 96 Wells Clear
3. Template—Select Blank, or Browse for a previously saved file.
4. Choose the Run mode—7500 FAST.
5. Operator—Enter your name.
6. Comments—Enter any comments pertaining to the run.
7. Plate Name—Enter a plate name.
8. Click Next.
ii. Choose the Detector for this assay
1. Choose 6-FAM-MGB from the list, or create a new one now by clicking the
New Detector button. Multiple detectors can be selected by using the Ctrl
key.
2. Choose Add »to add the detectors to the plate document.
3. Choose ROX™ as the passive reference from the Passive Reference drop down
box.
4. Click Next.
iii. The setup window is split into two panes. Use the layout grid to select indiviual wells,
and the Setup tab of the Well Inspector pane to apply detectors and designate well
assignments. As parameters are chosen in the Setup tab, they are recorded in the
Table pane at the bottom of the window.
1. Highlight the wells desired in the layout grid.
2. Check the Use box next to each detector to be analyzed for each well.
3. Click in the Sample Name box, and type in the name.
4. Choose the Task (Standard or NTC) from the dropdown list. If the task is
Standard, you must enter the concentration before proceeding to the next
well. Proceed until all the wells have been assigned all of the appropriate
detectors, names, task, and concentrations (standards only).
5. Click Finish.
iv. Set the method parameters by clicking on the Instrument tab of the Well Inspector
pane. All parameters must be changed to match Table 1.
1. To delete a stage, click on the bar separating the stages and drag to highlight
the stage to be deleted. Click [Delete Step],
Performed by: Date:
Page 3 of 6
RWIs WI-RV-PCR-5-v6 (07/09/18)
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EPA/6Q0/R-19/083
June 2019
WORK INSTRUCTION RV-PCR FOR BACILLUS ANTHRACIS SPORES - VCF
2. To add a parameter, click the step to the left of the location where you want
to place the new parameter, and choose [Add Cycle], [Add Hold], or [Add
Step],
3. To change cycle times and temperatures, click in the respective boxes in each
stage and type in the appropriate settings.
Table 1. 7500 FAST Method Parameters.
Temperature (°C)
Time
Cycles
95.0
0:20
1
95.0
0:03
45
60.0
0:30
25 uLTotal Volume
v. Save the file. Only files with the (*.sds) extension can be run.
1. From the top, choose File, Save As.
2. If the document is a new plate, go to drive D, Applied Biosystems, SDS,
Documents, and the appropriate folder. Name the file. From the drop down,
"Files of type:" choose ABI Prism SDS Single Plate (*.sds).
3. If the document is a template, follow the same procedure. This file will
already have a name and the (*.sdt) file extension. Change the name to
identify this particular run, and change the file type to ABI Prism SDS Single
Plate (*.sds).
4. Close the file.
vi. Centrifuge the plate at 300 x G for about 1-2 minutes at room temperature or in
Labnet's MPS-1000 Mini Plate Spinner.
vii. Open the file created in step C. 1. a. v., load 96-well plate into 7500 and start run.
viii. When run is complete, burn the file to a CD.
ix. Remove 96-well plate from 7500 Fast and dispose
D. Analysis
1. Open the assay with the most current version of 7500 Fast software.
a. Analysis can be performed using automatic settings. If required to manually set threshold and
baseline, from the menu bar choose Analysis, and then choose Analysis Settings. Select
Manual Ct, and Manual Baseline.
b. From the menu bar choose Analysis, then choose Analyze from the drop down menu, or click
on the large green triangle icon button in the toolbar.
c. Highlight the unknowns, standards (only one set if there is more than one), and NTCs either in
the Plate Grid pane or in the Table View pane.
d. Click on the Results tab in the Well Inspector pane to view the Amplification Plot and Standard
Curve Plot.
Performed by: Date:
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RWIs WI-RV-PCR-5-V6 (07/09/18)
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EPA/600/R-19/083
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WORK INSTRUCTION RV-PCR FOR BACILLUS ANTHRACIS SPORES - VCF
e. Ensure that the desired detector appears in the Detector box in both the Amplification Plot
and Standard Curve Plot. If not, chose it from the drop down menus now.
f. If analyzing based on manual threshold and baseline settings,
i. Look at the Ct values for the standards and unknowns in the Report View pane to
determine the lowest Ct value. Change the value in the End (cycle) box on the right
side of the plot to adjust the baseline setting to 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 right cursor should be placed at 20.)
ii. Highlight the standards and NTCs only. Click on the line that represents the threshold
in the amplification plot. Move the threshold so that it is above all background
"noise" generated by non-amplification, and in the lowest part of the exponential
phase of all the standards. Depending on the range of the standard curve, some
standards at the lowest end of the curve may not amplify well; whether or not to
include them will be decided by the Program Manager or Principal Investigator.
iii. Check the values in the Report View pane. Moving the threshold occasionally causes
the Ct values to change. If necessary, readjust the baseline and threshold by
repeating steps 9a through b until the baseline remains at a level two Ct values below
the lowest Ct value in the Report View pane and the threshold is properly set. If
changes are made that would affect the software's response to the data, such as
deleting a well or changing a detector, etc., the software will reset the analysis and
the data will need to be reanalyzed.
iv. Look in the Report View pane to ensure that the Ct value for all of the NTCs is
Undetermined. This means that no amplification was detected in the number of
cycles this analysis ran, and is considered to be a negative result. If any of the wells
show amplification at this point, contact the Program Manager or Principal
Investigator,
v. From the menu bar, choose Tools and then Report Settings. Check the boxes for the
data required to be printed by your project.
vi. Click Print, Done.
g. Highlight each individual well with a Ct less than 45, and check the Multicomponent Plot to
ensure there is actual amplification. Amplification is indicated by an upward curve in the line
representing the reporter dye, and if a quencher is present, a downward curve in the line
representing the quencher.
h. As specified by a project, print Multicomponent Plots.
i. Annotations to be made by the analyst on the printouts;
• Initial and date (l/D) every printout.
• Initial, date, and error or otherwise annotate all errors and comments.
• Indicate which, if any, wells of the Standard Curve were omitted.
• If required by the project, every Multicomponent Plot should indicate which sample
number it represents, and the actual Ct value associated with the well
j. Repeat the preceding steps to this point for every detector OR each standard curve associated
with this assay.
k. Attach all printouts to the worksheet. This constitutes one data package.
Performed by: Date:
Page 5 of 6
RWIs WI-RV-PCR-5-v6 (07/09/18)
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EPA/600/R-19/083
June 2019
WORK INSTRUCTION RV-PCR FOR BACILLUS ANTHRACIS SPORES - VCF
2. After the PCR run, discard sealed PCR plate.
3, Export the xsv file
a. Go to File -> Export -> Results.
b. Select folder location -> Native Filters
c. Save results type as .csv.
d. Select save.
i. A dialog box will open.
ii. Check the box, Apply Report Settings for Data Columns.
e. Burn .sds and .csv files onto a CD.
Data Calculations
Calculate an average CT from the replicate reactions for TO and T9 DNA extracts of each sample.
Subtract the average CT of the T9 DNA extract from the average CT of the TO DNA extract. If there is
no CT for the TO DNA extract (i.e., the TO is non-detect), use 45 (total number of PCR cycles used) as
the CT. The change (decrease) in the average CT value from TO to T9 (ACT) a 9 indicates a positive
result suggesting the presence of viable B. anthracis spores in the sample. If an incubation time
longer than 9 hours was used for the RV-PCR, instead of T9, appropriate Tx (incubation time) should
be used. However, (ACT) > 9 algorithm should still be used for a positive result. Depending upon the
end user's requirement, sample complexity (dirtiness) and the phase of response during an event, a
lower ACT criterion of > 6 (a two log difference in DNA concentration) and a corresponding higher
endpoint PCR CT of < 39 could be set. A minimum of two out of three TO PCR replicates must result
in CT values <44 (in a 45-cycle PCR) to calculate the average CT. A minimum of two out of three T9
PCR replicates (or Tx for other incubation time) must result in CT values < 36 to calculate the
average CT for a sample result to be considered positive. Negative controls (NTCs) should not yield
any measurable CT values above the background level. If CT values are obtained as a result of a
possible contamination or cross-contamination, prepare fresh PCR Master Mix and repeat analysis.
In addition, field blank samples should not yield any measurable CT values. If CT values are observed
as a result of a possible contamination or cross-contamination, a careful interpretation of the CT
values for the sample DNA extracts and field blanks must be done to determine if the data is
considered valid or if the PCR analyses must be repeated.
Print RV-PCR results and attach to this Wl package, include relevant calculations and file name.
Performed by: Date:
Technical Review
Performed by: Date:
Page 6 of 6
RWIs WI-RV-PCR-5-V6 (07/09/18)
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EPA/6Q0/R-19/083
June 2019
Real World Interferents WI-RV-PCR-5v2 - FORM A
DNA ASSAY—96 Well Plate Setup for Fast 7500
Project: RWI
Barcode:
Target: Ba Duplex, Chromosome (FAM-MGBNFQ) and pXOl (VIC-MGBNFQ)
1. Calculate the total number of reactions per plate:
Sample wells 48 + 4 NTC wells + 4 positive controls + 5 extras = 61 total rxns/plate (Y)
2. Prepare the Master Mix by combining the following reagents in an appropriate tube according to the following calculation:
Reagent
Manufacturer
Lot No.
Exp. Date
X
Y
Total Volume
(UL)
2X Fast PCR Mix
12.5 jiL
61
762.5
Platinum Taq DNA Polymerase
0.25 jiL
61
15.25
pXOl For. primer (25 uM)
1 |iL
61
61
pXOl Rev. primer (25 uM)
1 jiL
61
61
pXOl Probe (2 jaM)
1 jiL
61
61
Chro. For. primer (25 uM)
1 jiL
61
61
Chro. Rev. primer (25 uM)
1 jiL
61
61
Chro. Probe (2 jiM)
1 j^L
61
61
PCR grade water
1.25 jiL
61
91.5
Total
20 jiL
Distribute 20 jiL of Master Mix into each reaction well, as indicated in the plate layout, below. Loosely cover all wells containing Master
Mix with caps.
Add 5 jiL of PCR-grade water to each of the NTC Wells. Cap wells tightly.
Add 5 jiL of PNC (Method Blank) to the corresponding wells and secure the caps
Add 5 jiL of Sample to the corresponding wells and secure the caps.
Positive Control
Positive Control lot
prep date
8. Centrifuge the plate using Labnef s MPS-1000 Mini Plate Spinner at room temperature, and then load the plate onto the 7500 Fast Dx.
a
10
i
i
12
l-VCF-NEW-0 T0
l-VCF-NEW-0 T,
PC
50 P£
PC PC
50 pg 50 pg
PC
50 p.
NTC
NTC
NTC NTC
T echnicians
Signature
Date
Master Mix, NTC
Samples
Standards
Analyst
Reviewed By:
Date:
Page 1 of 1
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EPA/600/R-19/083
June 2019
APPENDIX L: WORK INSTRUCTION FOR SELECTING PRESUMPTIVE
BACILLUS ANTHRACIS STERNE COLONIES FOR QPCR
CONFIRMATION
-------
EPA/600/R-19/083
June 2019
WORK INSTRUCTION FOR SELECTING PRESUMPTIVE BACILLUS ANTHRACIS STERNE COLONIES
FOR QPCR CONFIRMATION
I. PURPOSE/SCOPE
Select and screen B. anthracis Sterne colonies recovered on culture plates using qPCR following the
BACILLUS Analytical Methods 004 published by the EPA December 2017.
II. MATERIALS/EQUIPMENT
Materials
Item
Manufacturer
Lot Number
Exp.
Date
Storage
Temp.
Initial* & Date
1 (iL loop, 10 (iL loop
or inoculating needles
R.T.
1.5 or 2 mL tubes
R.T.
N/A = Not Applicable
Equipment
Item
Manufacturer
Serial Number
Tliermo meter/
Rees #
Calibration
Due
Initials & Date
Biosafety
Cabinet (BSC)
The Baker Company
N/A
Heat Block
Precision
9509-003
N/A
N/A
Thermometer
N/A
N/A
N/A
Camera
N/A
N/A
N/A
N/A= Not Applicable
Other Supplies and Equipment
• Bleach
• 5 mL, 10 mL, and 25mLSerological Pipettes
Page 1 of 3
Native Filters Wl-Colony Screen-6-v3 (December 3 2018)
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EPA/600/R-19/083
June 2019
WORK INSTRUCTION FOR SELECTING PRESUMPTIVE BACILLUS ANTHRACIS STERNE COLONIES
FOR QPCR CONFIRMATION
Tube #
Filter ID
Volume (mL)
Morphology (B. a. Sterne or
Background)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Filters - Record Filter ID and Morphology for Selected Colonies
III. PROCEDURE
A. Selecting colonies
1. Pipette 100 pLof PCR-grade water into 1.5 or 2 mL tubes.
2. Select colonies. Take pictures of colonies that are selected.
3. Use 1 nL loop, 10 (iL loop or inoculating needle to select the colony.
4. Immerse needle into PCR-grade water and rotate to dislodge cellular material.
Performed by: Date:
Page 2 of 3
Native Filters Wl-Colony Screen-6-v3 (December 3 2018)
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EPA/600/R-19/083
June 2019
WORK INSTRUCTION FOR SELECTING PRESUMPTIVE BACILLUS ANTHRACIS STERNE COLONIES
FOR QPCR CONFIRMATION
5. 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: _
6. Store lysed suspension at - 20 °C for qPCR analysis.
7. Prior to qPCR analysis, thaw tubes, centrifuge @ 14,000 rpm for 2 minutes. Use supernatant for
qPCR.
Performed by: Date:
IV. Technical Review
Reviewed by: Date:
Page 3 of 3
Native Filters Wl-Colony Screen-6-v3 (December 3 2018)
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EPA/600/R-19/083
June 2019
APPENDIX M: WORK INSTRUCTION FOR ENRICHMENT FOR
CULTURE NON-DETECTS-SPG STICKS
-------
EPA/600/R-19/083
June 2019
WORK INSTRUCTION FOR ENRICHMENT FOR CULTURE NON-DETECTS - SPG
I. PURPOSE/SCOPE
Enrich extracted sponge and remaining culture aliquot in TSB for B. anthracis Sterne detection following
EPA/600/R-17/213 published by the EPA December 2017.
II. MATERIALS/EQUIPMENT
Materials
Item
Manufacturer
Lot Number
Fxp.
Date
Storage
Temp.
Initials & Date
10 (iL loop or
inoculating needles
R.T.
1.5 or 2 mL tubes
R.T.
SBA Plates
BD
2-8°C
TSB
In- House
TSB110518
5/5/19
R.T.
N/A = Not Applicable
Equipment
Item
Manufacturer
Seiial Number
The rmo meter/
Rees #
Calibration
Due
Initials & Date
Biosafety
Cabinet (BSC)
The Baker Company
N/A
Incubator
Precision
9509-003
Thermometer
N/A
N/A
N/A
Heat Block
VWR
Refrigerator
Fisher
C3274822
115
3/2019
N/A = Not Applicable
Other Supplies and Equipment
• 25mLSerological Pipettes
III. PROCEDURE
A. Selecting colonies
1. Add 25 mL of TSB to each specimen cup containing the extracted sponge (Wl #2 Step 8) and
remaining ~1 mL from the culture plating (Wl # 4 Step 5) for samples that were culture non-detects.
2. Incubate cups at 37 °C ± 2 °C for 24-48 hours.
Incubation start Date/Time: Initials:
Incubation end Date/Time: Initials:
Page 1 of 3
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WORK INSTRUCTION FOR ENRICHMENT FOR CULTURE NON-DETECTS - SPG
3, Evaluate the TSB Enrichment.
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.
Sample
Filter ID
Growth (G+) or No Growth (NG)
Recorded by:
Number
24 hours
48 hours
1
1-SPG-FLDBLK-A-S1S-0
2
2-SPG-STGRAT-A-S15-0
4
4-SPG-STGRAT-A-S16-30
6
6-SPG-STGRAT-A-S17-300
8
8-SPG-STGRAT-A-S18-3000
9
9-SPG-GRNBEN-A-S15-0
10
10-SPG-WLTILE-B-S19-0
4. Cap tightly and mix TSB with growth for 30 seconds. Remove a loopful of broth with a 10 loop
and streak on a SBA plate for isolation. Repeat two times for a total of three SBA isolation plates.
Store enriched samples at 2 - 8 °C.
5. Incubate the isolation plates and TSB with growth at 37 °C ± 2 °C for a maximum of three days.
Incubation start Date/Time; Initials:
Incubation end Date/Time: Initials;
6. Examine plates for B. anthracis colonies. If any colonies are isolated, proceed to PGR confirmation
(Wl #6 Colony Screen),
Sample
Number
Filter ID
B. a. Sterne Colonies
present (Yes/No)
Number of colonies
screened
Recorded by:
1
1-SPG-FLDBLK-A-S15-0
2
2-SPG-STGRAT-A-S15-0
4
4-SPG-STGRAT-A-S16-30
6
6-SPG-STGRAT-A-S17-300
8
8-SPG-STGRAT-A-S18-3000
9
9-SPG-GRNBEN-A-S15-0
10
10-SPG-WLTILE-B-S19-0
7. If no 8. anthracis colonies are observed, perform PCR on TSB with growth (Section B).
B. PCR Confirmation of TSB Enriched Samples
1. Transfer 50 [iL of broth with growth to a microcentrifuge tube,
2. Centrifuge at 12,000 x g for 2 minutes.
Page 2 of 3
Native Filters WI-TSB Enrich-7-v3 (112618)
-------
EPA/600/R-19/083
June 2019
WORK INSTRUCTION FOR ENRICHMENT FOR CULTURE NON-DETECTS - SPG
3. Remove and discard the supernatant in an autoclavable biohazard container. Add 100 |il_of PCR-
grade water to the tube containing the bacterial pellet,
4. Resuspend the pellet by flicking the tube,
5. Lyse the suspension for 5 minutes on a heat block at 95 ± 2 °C.
Incubation start Date/Time: Initials:
Incubation end Date/Time: Initials:
6. Store lysed suspension at - 20 °C for qPCR analysis or refrigerator if processed same day.
7. Prior to qPCR analysis, thaw tubes, centrifuge @ 14,000 rpm for 2 minutes. Use supernatant for
qPCR.
Performed by: Date:
IV. Technical Review
Reviewed by: Date:
Page 3 of 3
Native Filters WI-TSB Enrich-7-v3 (112618)
-------
EPA/600/R-19/083
June 2019
APPENDIX N: WORK INSTRUCTION
TSB ENRICHMENT FOR CULTURE-VCF
-------
EPA/600/R-19/083
June 2019
WORK INSTRUCTION TSB ENRICHMENT FOR CULTURE - VCF
I. PURPOSE/SCOPE
Enrich extracted sponge and remaining culture aliquot in TSB for B. anthracis Sterne detection following
EPA/600/R-17/213 published by the EPA December 2017.
II. MATERIALS/EQUIPMENT
Materials
Item
Manufacturer
Lot Number
Eyp.
Date
Storage
Temp.
Initials & Date
10 (iL loop or
inoculating needles
R.T.
1.5 or 2 mL tubes
R.T.
SBA Plates
BD
2-8°C
TSB
In- House
2-8°C
N/A = Not Applicable
Equipment
Item
Manufacturer
Seiial Number
The rmo meter/
Rees #
Calibration
Due
Initials & Date
Biosafety
Cabinet (BSC)
The Baker Company
N/A
Incubator
Precision
9509-003
Thermometer
N/A
N/A
N/A
Heat Block
VWR
Refrigerator
Fisher
C3274822
115
3/2019
N/A = Not Applicable
Other Supplies and Equipment
• 25mLSerological Pipettes
III. PROCEDURE
A. Selecting colonies
1. Add 30 mL of TSB to each 2 oz. cup containing the cassette filter (Wl #2 Step 13),
2. Incubate cups at 37 °C ± 2 °C for 24-48 hours.
Incubation start Date/Time; Initials:
Incubation end Date/Time: Initials:
3. Evaluate the TSB Enrichment.
Page 1 of 3
Native Filters WI-VCF-TSB Enrich-7-v2 (December 10 2018)
-------
EPA/600/R-19/083
June 2019
WORK INSTRUCTION TSB ENRICHMENT FOR CULTURE - VCF
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.
Sample
Filter ID
Growth (G+) or No Growth (NG)
Recorded by:
Number
24 hours
48 hours
1
1-VCF-FLCON-B-S15-0
2
2-VCF-STEPS-B-S15-0
3
3-VCF-FLCON-B-S16-30
4
4-VCF-STEPS-B-S16-30
5
5-VCF-FLCON-B-S17-300
6
6-VCF-STEPS-B-S17-300
7
7-VCF-FLCON-B-S18-3,000
8
8-VCF-STEPS-B-S18-3,000
9
9-VCF-SWCON-A-S15-0
10
10-VCF-PAVEMT-A-S15-0
11
11-VCF-SWCON-A-S16-30
12
12-VCF-PAVEMT-A-S16-30
13
13-VCF-SWCON-A-S17-300
14
14-VCF-PAVEMT-A-S17-300
15
15-VCF-SWCON-A-S18-3.000
16
16-VCF-PAVEMT-A-S18-3,000
4. Cap tightly and mix TSB with growth for 30 seconds. Remove a loopful of broth with a 10 |iL loop
and streak on a SBA plate for isolation. Repeat two times for a total of three SBA isolation plates.
Store enriched samples at 2 -8 °C.
5. Incubate the isolation plates and TSB with growth at 37 °C + 2 °C for a maximum of three days.
Incubation start Date/Time: Initials:
Incubation end Date/Time: Initials:
6. Examine plates for B. anthracis colonies. If any colonies are isolated, proceed to PGR confirmation
(Wl #6 Colony Screen).
Sample
Number
Filter ID
B. a. Sterne
Colonies present
(Yes/No)
Number of colonies
screened
Recorded by:
1
1-VCF-FLCON-B-S15-0
2
2-VCF-STEPS-B-S15-0
3
3-VCF-FLCON-B-S16-30
4
4-VCF-STEPS-B-S16-30
5
5-VCF-FLCON-B-S17-300
Page 2 of 3
Native Filters WI-VCF-TSB Enrich-7-v2 (December 10 2018)
-------
EPA/600/R-19/083
June 2019
WORK INSTRUCTION TSB ENRICHMENT FOR CULTURE - VCF
Sample
Number
Filter ID
B. a. Sterne
Colonies present
(Yes/No)
Number of colonies
screened
Recorded by:
6
6-VCF-STEPS-B-S17-300
7
7-VCF-FLCON-B-S18-3,000
8
8-VCF-STEPS-B-S18-3,000
9
9-VCF-SWCON-A-S15-0
10
10-VCF-PAVEMT-A-S15-0
11
11-VCF-SWCON-A-S16-30
12
12-VCF-PAVEMT-A-S16-30
13
13-VCF-SWCON-A-S17-300
14
14-VCF-PAVEMT-A-S17-300
15
15-VCF-SWCON-A-S18-3,000
16
16-VCF-PAVEMT-A-S18-3,000
7. If no 8, anthracis colonies are observed, perform PCR on TSB with growth (Section B).
B. PCR Confirmation of TSB Enriched Samples
1. Transfer 50 |iL of broth with growth to a microcentrifuge tube.
2. Centrifuge at 12,000 x g for 2 minutes,
3. Remove and discard the supernatant in an autoclavable biohazard container. Add 100 of PCR-
grade water to the tube containing the bacterial pellet.
4. Resuspend the pellet by flicking the tube.
5. Lyse the suspension for 5 minutes on a heat block at 95 + 2 °C,
Incubation start Date/Time: Initials:
Incubation end Date/Time: Initials;
6. Store lysed suspension at - 20 °C for qPCR analysis or refrigerator if processed same day.
7. Prior to qPCR analysis, thaw tubes, centrifuge @ 14,000 rpm for 2 minutes. Use supernatant for
qPCR.
Performed by: Date:
IV. Technical Review
Reviewed by: Date:
Page 3 of 3
Native Filters WI-VCF-TSB Enrich-7-v2 (December 10 2018)
-------
EPA/600/R-19/083
June 2019
APPENDIX O: CULTURE RESULTS FOR SPONGE-STICK SAMPLES
USING SHEEP BLOOD AGAR MEDIUM
-------
EPA/600/R-19/083
June 2019
APPENDIX P: RV-PCR RESULTS FOR SPONGE-STICK SAMPLES
USING CHROMOSOMAL AND pXOl GENE TARGETS
-------
EPA/600/R-19/083
June 2019
Siimpk- II)
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1 -SPG-FLDBLK-A-SO1 -0 TO
0
45
0
0
Neg.
45
0
0
Neg.
1 -SPG-FLDBLK-A-SO1 -0 Tf
45
0
45
0
2-SPG-SWCON-A-SO1 -0 TO
0
45
0
0
Neg.
45
0
0
Neg.
2-SPG-SWCON-A-SO1 -0 Tf
45
0
45
0
3-SPG-FLDBLK-A-S02-30 TO
21
45
0
26.9
Pos.
45
0
27.3
Pos.
3-SPG-FLDBLK-A-S02-30 Tf
18.1
0.1
17.7
0
4-SPG-SWCON-A-S02-30 TO
21
45
0
15.5
Pos.
45
0
17.5
Pos.
4-SPG-SWCON-A-S02-30 Tf
29.5
0.2
27.5
0.1
5-SPG-FLDBLK-A-S03-300 TO
210
45
0
25.6
Pos.
45
0
25.8
Pos.
5-SPG-FLDBLK-A-S03-300 Tf
19.4
0.1
19.2
0
6-SPG-SWCON-A-S03-300 TO
210
45
0
20.3
Pos.
45
0
20.9
Pos.
6-SPG-SWCON-A-S03-300 Tf
24.7
0
24.1
0
7-SPG-FLDBLK-A-S04-3000 TO
2,100
45
0
25.7
Pos.
45
0
25.9
Pos.
7-SPG-FLDBLK-A-S04-3000 Tf
19.3
0.1
19.1
0
8-SPG-SWCON-A-S04-3000 TO
2,100
45
0
21.3
Pos.
45
0
22.1
Pos.
8-SPG-SWCON-A-S04-3000 Tf
23.7
0
22.9
0
9-SPG-GLSPAN-B-S01-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
9-SPG-GLSPAN-B-S01-0 Tf
45
0
45
0
10-SPG-WLTILE-B-SO1 -0 TO
0
45
0
0
Neg.
45
0
0
Neg.
10-SPG-WLTILE-B-SO1-0 Tf
45
0
45
0
11-SPG-GLSPAN-B-S02-30 TO
21
45
0
0
Neg.
45
0
0
Neg.
11-SPG-GLSPAN-B-S02-30 Tf
45
0
45
0
12-SPG-WLTILE-B-S02-30 TO
21
45
0
0
Neg.
45
0
0
Neg.
12-SPG-WLTILE-B-S02-30 Tf
45
0
45
0
13-SPG-GLSPAN-B-S03-300 TO
210
45
0
19.4
Pos.
45
0
20.2
Pos.
13-SPG-GLSPAN-B-S03-300 Tf
25.6
0.2
24.8
0.1
14-SPG-WLHLE-B-S03-300 TO
210
45
0
21.8
Pos.
45
0
22.8
Pos.
14-SPG-WLTILE-B-S03-300 Tf
23.2
0.1
22.2
0
15-SPG-GLSPAN-B-S04-3000 TO
2,100
45
0
26.5
Pos.
45
0
27.1
Pos.
15-SPG-GLSPAN-B-S04-3000 Tf
18.5
0.1
17.9
0
16-SPG-WLTILE-B-S04-3000 TO
2,100
45
0
22.5
Pos.
45
0
23.7
Pos.
16-SPG-WLTILE-B-S04-3000 Tf
22.5
0.1
21.3
0
1 -SPG-TELEBO-O TO
0
45
0
0
Neg.
45
0
0
Neg.
1 -SPG-TELEBO-O Tf
45
0
45
0
2-SPG-FLCON-O TO
0
45
0
0
Neg.
45
0
0
Neg.
2-SPG-FLCON-O Tf
45
0
45
0
3-SPG-TELEBO-30 TO
31
45
0
17.4
Pos.
45
0
17.8
Pos.
3-SPG-TELEBO-30 Tf
27.6
0.1
27.2
0
4-SPG-FLCON-30 TO
31
45
0
14.8
Pos.
45
0
15.1
Pos.
4-SPG-FLCON-30 Tf
30.2
0
29.9
0
5-SPG-TELEBO-300 TO
310
45
0
20.9
Pos.
45
0
20.9
Pos.
5-SPG-TELEBO-300 Tf
24.1
0.1
24.1
0
6-SPG-FLCON-300 TO
310
45
0
18.7
Pos.
45
0
18.8
Pos.
6-SPG-FLCON-300 Tf
26.3
0.1
26.2
0.1
7-SPG-TELEBO-3000 TO
3,100
45
0
22.9
Pos.
45
0
23.1
Pos.
7-SPG-TELEBO-3000 Tf
22.1
0.1
21.9
0
8-SPG-FLCON-3000 TO
3,100
45
0
21.4
Pos.
45
0
21.4
Pos.
8-SPG-FLCON-3000 Tf
23.6
0
23.6
0
9-SPG-FLLFIX-O TO
0
45
0
0
Neg.
45
0
2.9
Neg.
9-SPG-FLLFIX-O Tf
45
0
42.1
2.5
10-SPG-FLTILE-O TO
0
45
0
0
Neg.
45
0
0
Neg.
10-SPG-FLTILE-O TF
45
0
45
0
1 l-SPG-FLLFIX-30 TO
31
45
0
16.2
Pos.
45
0
16
Pos.
1 l-SPG-FLLFIX-30 Tf
28.8
0
29
0.1
12-SPG-FLTILE-30 TO
31
45
0
12.6
Pos.
45
0
12.5
Pos.
12-SPG-FLTILE-30 TF
32.4
0.1
32.5
0.1
-------
EPA/600/R-19/083
June 2019
Siimpk- II)
Spun-
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13-SPG-FLLFIX-300 TO
310
45
0
18.8
Pos.
45
0
18.7
Pos.
13-SPG-FLLFIX-300 Tf
26.2
0.1
26.3
0.1
14-SPG-FLTILE-300 TO
310
45
0
20.8
Pos.
45
0
21.2
Pos.
14-SPG-FLTILE-300 TF
24.2
0
23.8
0
15-SPG-FLLFIX-3000 TO
3,100
45
0
23.2
Pos.
45
0
23.2
Pos.
15-SPG-FLLFIX-3000 Tf
21.8
0.1
21.8
0
16-SPG-FLTILE-3000 TO
3,100
45
0
19.4
Pos.
44.5
0.8
19.4
Pos.
16-SPG-FLTILE-3000 TF
25.6
0
25.1
0
1 -SPG-CWSIGN-0 TO
0
45
0
1.9
Neg.
45
0
1.2
Neg.
1 -SPG-CWSIGN-0 Tf
43.1
3.2
43.8
2.2
2-SPG-MCMACH-O TO
0
45
0
1.9
Neg.
45
0
1.2
Neg.
2-SPG-MCMACH-O Tf
43.1
3.3
43.8
2.1
3-SPG-CWSIGN-30 TO
16
45
0
18
Pos.
45
0
18
Pos.
3-SPG-CWSIGN-30 Tf
27
0
27
0
4-SPG-MCMACH-30 TO
16
45
0
17.6
Pos.
45
0
17.7
Pos.
4-SPG-MCMACH-30 Tf
27.4
0
27.3
0.1
5-SPG-CWSIGN-300 TO
160
45
0
22.9
Pos.
45
0
23
Pos.
5-SPG-CWSIGN-300 Tf
22.1
0
22
0.1
6-SPG-MCMACH-300 TO
160
45
0
20.3
Pos.
44.9
0.1
20.7
Pos.
6-SPG-MCMACH-300 Tf
24.7
0
24.2
0
7-SPG-CWSIGN-3000 TO
1,600
45
0
21.3
Pos.
45
0
21.4
Pos.
7-SPG-CWSIGN-3000 Tf
23.7
0.1
23.6
0.1
8-SPG-MCMACH-3000 TO
1,600
45
0
21
Pos.
45
0
21.2
Pos.
8-SPG-MCMACH-3000 Tf
24
0
23.8
0
9-SPG-OHSIGN-O TO
0
45
0
0
Neg.
45
0
1.5
Neg.
9-SPG-OHSIGN-O Tf
45
0
43.5
2.6
10-SPG-GRNBEN-O TO
0
45
0
1.5
Neg.
45
0
1.8
Neg.
10-SPG-GRNBEN-O Tf
43.5
2.6
43.2
3.1
11-SPG-OHSIGN-30T0
16
45
0
16.9
Pos.
45
0
16.7
Pos.
ll-SPG-OHSIGN-30Tf
28.1
0.1
28.3
0
12-SPG-GRNBEN-30 TO
16
45
0
17.3
Pos.
45
0
17.4
Pos.
12-SPG-GRNBEN-30 Tf
27.7
0.1
27.6
0
13-SPG-OHSIGN-300 TO
160
45
0
21.4
Pos.
44.7
0.6
21.1
Pos.
13-SPG-OHSIGN-300 Tf
23.6
0.1
23.6
0
14-SPG-GRBEN-300 TO
160
45
0
20.8
Pos.
45
0
21.1
Pos.
14-SPG-GRBEN-300 Tf
24.2
0
23.9
0
15-SPG-OHSIGN-3000 TO
1,600
45
0
26.9
Pos.
43.7
1.4
25.7
Pos.
15-SPG-OHSIGN-3000 Tf
18.1
0
18
0.1
16-SPG-GRBEN-3000 TO
1,600
45
0
24.1
Pos.
43.5
2.6
23.3
Pos.
16-SPG-GRBEN-3000 Tf
20.9
0
20.2
0
1-SPG-STEPS-B-S01-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
1-SPG-STEPS-B-S01-0 Tf
45
0
45
0
2-SPG-EDPAN(B)-B-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
2-SPG-EDPAN(B)-B-0 Tf
45
0
45
0
3-SPG-STEPS-30 TO
19
45
0
16.9
Pos.
44.3
1.2
16.4
Pos.
3-SPG-STEPS-30 Tf
28.1
0.1
27.9
0
4-SPG-EDPAN(B)-30 TO
19
45
0
17.5
Pos.
45
0
17.5
Pos.
4-SPG-EDPAN(B)-30 Tf
27.5
0.1
27.5
0
5-SPG-STEPS-300 TO
190
45
0
16.8
Pos.
45
0
17.1
Pos.
5-SPG-STEPS-300 Tf
28.2
0.1
27.9
0
6-SPG-EDPAN(B)-300 TO
190
45
0
18.7
Pos.
45
0
18.8
Pos.
6-SPG-EDPAN(B)-300 Tf
26.3
0
26.2
0
7-SPG-STEPS-3000 TO
1,900
45
0
21.7
Pos.
45
0
21.5
Pos.
7-SPG-STEPS-3000 Tf
23.3
0
23.5
0
8-SPG-EDPAN(B)-3000 TO
1,900
45
0
26.1
Pos.
45
0
26.2
Pos.
8-SPG-EDPAN(B)-3000 Tf
18.9
0
18.8
0
-------
EPA/600/R-19/083
June 2019
Siimpk- II)
Spun-
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Clin int.
Awr;i»i-
(1
(h ni in.
Sul l)i\
(hum.
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KlSllll
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Awr;i»i-
(1
p\()l
sul
l)l\
|)\OI
C liiiin.
\( 1
|)\OI
(Iiih m.
Kisiill
9-SPG-STGRAT-O TO
0
45
0
0
Neg.
44.7
0.5
-0.3
Neg.
9-SPG-STGRAT-O Tf
45
0
45
0
10-SPG-CWPNTD-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
10-SPG-CWPNTD-0 Tf
45
0
45
0
ll-SPG-STGRAT-30 TO
19
45
0
6.3
Neg.
43.2
3.2
6.5
Neg.
1 l-SPG-STGRAT-30 Tf
38.7
5.5
36.7
3
12-SPG-CWPNTD-30 TO
19
45
0
6.6
Neg.
45
0
6.8
Neg.
12-SPG-CWPNTD-30 Tf
38.4
5.7
38.2
5.9
13-SPG-STGRAT-300 TO
190
45
0
12
Pos.
45
0
12.4
Pos.
13-SPG-STGRAT-300 Tf
33
0.1
32.6
0
14-SPG-CWPNTD-300 TO
190
45
0
13.5
Pos.
45
0
13.8
Pos.
14-SPG-CWPNTD-300 Tf
31.5
0.1
31.2
0.1
15-SPG-STGRAT-3000 TO
1,900
45
0
19
Pos.
45
0
19.7
Pos.
15-SPG-STGRAT-3000 Tf
26
0
25.3
0
16-SPG-CWPNTD-3000 TO
1,900
45
0
16.6
Pos.
45
0
16.8
Pos.
16-SPG-CWPNTD-3000 Tf
28.4
0.1
28.2
0
1-SPG-GLSWIN-B-S01-0 TO
0
45
0
0
Neg.
45
0
0.2
Neg.
1-SPG-GLSWIN-B-S01-0 Tf
45
0
44.8
0.3
2-SPG-EDPAN(A)-S01-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
2-SPG-EDPAN(A)-S01-0 Tf
45
0
45
0
3-SPG-GLSWIN-S02-30 TO
18
45
0
20.4
Pos.
45
0
20.1
Pos.
3-SPG-GLSWIN-S02-30 Tf
24.6
0.1
24.9
0
4-SPG-EDPAN(A)-30 TO
18
45
0
16.6
Pos.
45
0
16.8
Pos.
4-SPG-EDPAN(A)-30 Tf
28.4
0
28.2
0
5-SPG-GLSWIN-300 TO
180
45
0
26.8
Pos.
45
0
27.1
Pos.
5-SPG-GLSWIN-300 Tf
18.2
0
17.9
0
6-SPG-EDPAN(A)-300 TO
180
45
0
23.1
Pos.
45
0
23.1
Pos.
6-SPG-EDPAN(A)-300 Tf
21.9
0
21.9
0
7-SPG-GLSWIN-3000 TO
1,800
45
0
26
Pos.
45
0
26
Pos.
7-SPG-GLSWIN-3000 Tf
19
0
19
0
8-SPG-EDPAN(A)-3000 TO
1,800
45
0
26.7
Pos.
44.7
0.5
26.7
Pos.
8-SPG-EDPAN(A)-3000 Tf
18.3
0
17.9
0
9-SPG-SCGRIL-O TO
0
45
0
0
Neg.
45
0
0
Neg.
9-SPG-SCGRIL-O Tf
45
0
45
0
10-SPG-CWPNTD-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
10-SPG-CWPNTD-0 Tf
45
0
45
0
1 l-SPG-SCGRIL-30 TO
18
45
0
14.3
Pos.
44.5
0.8
14
Pos.
1 l-SPG-SCGRIL-30 Tf
30.7
0.2
30.5
0
12-SPG-CWPNTD-30 TO
18
45
0
2.3
Neg.
45
0
6.8
Neg.
12-SPG-CWPNTD-30 Tf
42.7
3.9
38.2
1
13-SPG-SCGRIL-300 TO
180
45
0
8.6
Neg.
45
0
9.4
Neg.
13-SPG-SCGRIL-300 Tf
36.4
1.1
35.6
0.2
14-SPG-CWPNTD-300 TO
180
45
0
14.8
Pos.
45
0
15.1
Pos.
14-SPG-CWPNTD-300 Tf
30.2
0.1
29.9
0
15-SPG-SCGRIL-3000 TO
1,800
45
0
20.6
Pos.
45
0
21.1
Pos.
15-SPG-SCGRIL-3000 Tf
24.4
0
23.9
0
16-SPG-CWPNTD-3000 TO
1,800
45
0
16.3
Pos.
45
0
16.7
Pos.
16-SPG-CWPNTD-3000 Tf
28.7
0
28.3
0
1-SPG-FLTILE-B-S05-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
1-SPG-FLTILE-B-S05-0 Tf
45
0
45
0
2-SPG-FLCON-B-S05-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
2-SPG-FLCON-B-S05-0 Tf
45
0
45
0
3-SPG-FLTILE-B-S06-30 TO
23
45
0
10.2
Pos.
45
0
9.9
Pos.
3-SPG-FLTILE-B-S06-30 Tf
34.8
0.1
35.1
0.2
4-SPG-FLCON-B-S06-30 TO
23
45
0
14
Pos.
45
0
14.1
Pos.
4-SPG-FLCON-B-S06-30 Tf
31
0
30.9
0
-------
EPA/600/R-19/083
June 2019
Siimpk- II)
Spun-
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Clin int.
Awr;i»i-
(1
(h ni in.
Sul l)i\
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U 1
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KlSllll
|)\OI
Awr;i»i-
(I
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sul
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|)\OI
C liiiin.
\( 1
|)\OI
(Iiih m.
Kisiill
5-SPG-FLTILE-B-S07-300 TO
230
45
0
19.7
Pos.
45
0
19.7
Pos.
5-SPG-FLTILE-B-S07-300 Tf
25.3
0
25.3
0
6-SPG-FLCON-S07-300 TO
230
45
0
15.4
Pos.
43.3
3
14.1
Pos.
6-SPG-FLCON-S07-300 Tf
29.6
0.1
29.2
0
7-SPG-FLTILE-S08-3000 TO
2,300
45
0
22.4
Pos.
45
0
22.4
Pos.
7-SPG-FLTILE-S08-3000 Tf
22.6
0
22.6
0.1
8-SPG-FLCON-S08-3000 TO
2,300
45
0
22
Pos.
45
0
22.2
Pos.
8-SPG-FLCON-S08-3000 Tf
23
0
22.8
0
9-SPG-STEPS-B-S05-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
9-SPG-STEPS-B-S05-0 Tf
45
0
45
0
10-SPG-WLTILE-S05-0 TO
0
45
0
0
Neg.
45
0
0.5
Neg.
10-SPG-WLTILE-S05-0 Tf
45
0
44.5
0.9
11-SPG-STEPS-S06-30 TO
23
45
0
17.2
Pos.
45
0
17.3
Pos.
11-SPG-STEPS-S06-30 Tf
27.8
0.1
27.7
0
12-SPG-WLTILE-S06-30 TO
23
45
0
21.4
Pos.
45
0
21.5
Pos.
12-SPG-WLTILE-S06-30 Tf
23.6
0
23.5
0
13-SPG-STEPS-S07-300 TO
230
45
0
23.2
Pos.
45
0
23.6
Pos.
13-SPG-STEPS-S07-300 Tf
21.8
0.1
21.4
0.1
14-SPG-WLTILE-07-300 TO
230
45
0
25.8
Pos.
45
0
26.2
Pos.
14-SPG-WLTILE-07-300 Tf
19.2
0
18.8
0
15-SPG-STEPS-08-3000 TO
2,300
45
0
23.6
Pos.
45
0
23.4
Pos.
15-SPG-STEPS-08-3000 Tf
21.4
0
21.6
0
16-SPG-WLTILE-3000 TO
2,300
45
0
22.8
Pos.
45
0
23.1
Pos.
16-SPG-WLTILE-3000 Tf
22.2
0
21.9
0
1-SPG-GLSWIN-B-S05-0 TO
0
45
0
3.1
Neg.
45
0
1.5
Neg.
1-SPG-GLSWIN-B-S05-0 Tf
41.9
3.4
43.5
1.3
2-SPG-GLSWIN-B-S06-30 TO
34
45
0
23.6
Pos.
45
0
23.2
Pos.
2-SPG-GLSWIN-B-S06-30 Tf
21.4
0.1
21.8
0.1
3-SPG-GLSWIN-B-S07-30 TO
34
45
0
18.2
Pos.
45
0
18.2
Pos.
3-SPG-GLSWIN-B-S07-30 Tf
26.8
0.1
26.8
0
4-SPG-GLSWIN-B-S08-30 TO
34
45
0
18
Pos.
45
0
18.1
Pos.
4-SPG-GLSWIN-B-S08-30 Tf
27
0
26.9
0
5-SPG-GLSWIN-B-S09-300 TO
340
45
0
24.2
Pos.
45
0
24.3
Pos.
5-SPG-GLSWIN-B-S09-300 Tf
20.8
0.1
20.7
0.1
6-SPG-GLSWIN-B-S10-300 TO
340
45
0
17
Pos.
45
0
17
Pos.
6-SPG-GLSWIN-B-S10-300 Tf
28
0.1
28
0.1
7-SPG-GLSWIN-B-S11-3000 TO
3,400
45
0
25.3
Pos.
45
0
25.4
Pos.
7-SPG-GLSWIN-B-S11-3000 Tf
19.7
0.1
19.6
0.1
8-SPG-GLSWIN-B-S12-3000 TO
3,400
45
0
27.6
Pos.
45
0
27.9
Pos.
8-SPG-GLSWIN-B-S12-3000 Tf
17.4
0
17.1
0
9-SPG-EDPAN(B)-S05-0 TO
0
45
0
0
Neg.
45
0
1.3
Neg.
9-SPG-EDPAN(B)-S05-0 Tf
45
0
43.7
2.1
10-SPG-EDPAN(B)-S06-30 TO
34
45
0
19.8
Pos.
45
0
19.6
Pos.
10-SPG-EDPAN(B)-S06-30 Tf
25.2
0
25.4
0
ll-SPG-EDPAN(B)-S07-30 TO
34
45
0
23.7
Pos.
45
0
23.9
Pos.
ll-SPG-EDPAN(B)-S07-30 Tf
21.3
0
21.1
0
12-SPG-EDPAN(B)-S08-30 TO
34
45
0
21
Pos.
45
0
21.1
Pos.
12-SPG-EDPAN(B)-S08-30 Tf
24
0
23.9
0
13-SPG-EDPAN(B)-S09-300 TO
340
45
0
20
Pos.
44.5
0.8
19.6
Pos.
13-SPG-EDPAN(B)-S09-300 Tf
25
0
25
0
14-SPG-EDPAN(B)-S10-300 TO
340
45
0
22.3
Pos.
45
0
22.4
Pos.
14-SPG-EDPAN(B)-S10-300 Tf
22.7
0
22.6
0
15-SPG-EDPAN(B)-S11-3000 TO
3,400
45
0
19.9
Pos.
45
0
20
Pos.
15-SPG-EDPAN(B)-S11-3000 Tf
25.1
0
25
0
16-SPG-EDPAN(B)-S12-3000 TO
3,400
45
0
24.7
Pos.
45
0
25.1
Pos.
16-SPG-EDPAN(B)-S12-3000 Tf
20.3
0.1
19.9
0
-------
EPA/600/R-19/083
June 2019
Siimpk- II)
Spun-
I.ii;kI
Clin int.
Awr;i»i-
(1
(h ni in.
Sul l)i\
(hum.
U 1
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KlSllll
|)\OI
Awr;i»i-
(1
p\()l
sul
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|)\OI
('hum.
\( I
|)\OI
(Iiih m.
Kisiill
1-SPG-GLSPAN-B-S05-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
1-SPG-GLSPAN-B-S05-0 Tf
45
0
45
0
2-SPG-GLSPAN-B-S06-30 TO
48
45
0
20
Pos.
45
0
19.9
Pos.
2-SPG-GLSPAN-B-S06-30 Tf
25
0.1
25.1
0
3-SPG-GLSPAN-B-S07-30 TO
48
45
0
17.6
Pos.
45
0
17.5
Pos.
3-SPG-GLSPAN-B-S07-30 Tf
27.4
0
27.5
0
4-SPG-GLSPAN-B-S08-30 TO
48
45
0
23.4
Pos.
45
0
23.3
Pos.
4-SPG-GLSPAN-B-S08-30 Tf
21.6
0.1
21.7
0
5-SPG-GLSPAN-B-S09-300 TO
480
45
0
25.2
Pos.
45
0
25.1
Pos.
5-SPG-GLSPAN-B-S09-300 Tf
19.8
0.1
19.9
0
6-SPG-GLSPAN-B-S10-300 TO
480
45
0
22.2
Pos.
45
0
22.1
Pos.
6-SPG-GLSPAN-B-S10-300 Tf
22.8
0
22.9
0
7-SPG-GLSPAN-B-S11-3000 TO
4,800
40.5
3.9
20.2
Pos.
44.5
0.9
24
Pos.
7-SPG-GLSPAN-B-S11-3000 Tf
20.3
0
20.5
0
8-SPG-GLSPAN-B-S12-3000 TO
4,800
45
0
24.9
Pos.
45
0
24.8
Pos.
8-SPG-GLSPAN-B-S12-3000 Tf
20.1
0
20.2
0
9-SPG-FLLFIX-B-S05-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
9-SPG-FLLFIX-B-S05-0 Tf
45
0
45
0
10-SPG-FLLFIX-B-S06-30 TO
48
45
0
15.1
Pos.
45
0
15.2
Pos.
10-SPG-FLLFIX-B-S06-30 Tf
29.9
0.1
29.8
0
11-SPG-FLLFIX-B-S07-30 TO
48
45
0
25.8
Pos.
45
0
25.6
Pos.
11-SPG-FLLFIX-B-S07-30 Tf
19.2
0
19.4
0
12-SPG-FLLFIX-B-S08-30 TO
48
45
0
16.1
Pos.
45
0
16.2
Pos.
12-SPG-FLLFIX-B-S08-30 Tf
28.9
0.1
28.8
0
13-SPG-FLLFIX-B-S09-300 TO
480
42.7
4
18.9
Pos.
42.6
3.2
19
Pos.
13-SPG-FLLFIX-B-S09-300 Tf
23.7
0
23.7
0
14-SPG-FLLFIX-B-S10-300 TO
480
42.9
3.6
16.4
Pos.
40.5
2.6
14.1
Pos.
14-SPG-FLLFIX-B-S10-300 Tf
26.5
0.1
26.4
0
15-SPG-FLLFIX-B-S11-3000 TO
4,800
45
0
23.9
Pos.
44.6
0.7
23.5
Pos.
15-SPG-FLLFIX-B-S11-3000 Tf
21.1
0.1
21.1
0
16-SPG-FLLFIX-B-S12-3000 TO
4,800
45
0
24
Pos.
45
0
24.5
Pos.
16-SPG-FLLFIX-B-S12-3000 Tf
21
0
20.5
0
1-SPG-OHSIGN-B-S05-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
1-SPG-OHSIGN-B-S05-0 Tf
45
0
45
0
2-SPG-OHSIGN-B-S06-30 TO
17
43
3.4
18.5
Pos.
45
0
20.4
Pos.
2-SPG-OHSIGN-B-S06-30 Tf
24.5
0
24.6
0
3-SPG-OHSIGN-B-S07-30 TO
17
45
0
12.8
Pos.
45
0
12.9
Pos.
3-SPG-OHSIGN-B-S07-30 Tf
32.2
0.2
32.1
0.1
4-SPG-FLDBLK-A-S05-30 TO
17
45
0
26.1
Pos.
42.1
2.6
23.1
Pos.
4-SPG-FLDBLK-A-S05-30 Tf
18.9
0
19
0
5-SPG-OHSIGN-B-S08-300 TO
170
45
0
21.5
Pos.
45
0
21.7
Pos.
5-SPG-OHSIGN-B-S08-300 Tf
23.5
0.1
23.3
0
6-SPG-OHSIGN-B-S09-300 TO
170
45
0
25.5
Pos.
45
0
25.5
Pos.
6-SPG-OHSIGN-B-S09-300 Tf
19.5
0.2
19.5
0.2
7-SPG-OHSIGN-B-S10-3000 TO
1,700
45
0
26.2
Pos.
45
0
26.1
Pos.
7-SPG-OHSIGN-B-S10-3000 Tf
18.8
0
18.9
0
8-SPG-OHSIGN-B-S11-3000 TO
1,700
45
0
22.7
Pos.
45
0
22.7
Pos.
8-SPG-OHSIGN-B-S11-3000 Tf
22.3
0.7
22.3
0.7
9-SPG-MCMACH-B-S05-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
9-SPG-MCMACH-B-S05-0 Tf
45
0
45
0
10-SPG-MCMACH-B-S06-30 TO
17
45
0
12.1
Pos.
45
0
12
Pos.
10-SPG-MCMACH-B-S06-30 Tf
32.9
0.2
33
0.2
11-SPG-MCMACH-B-S07-30 TO
17
45
0
9.9
Pos.
45
0
10
Pos.
11-SPG-MCMACH-B-S07-30 Tf
35.1
0.3
35
0.1
12-SPG-MCMACH-B-S08-30 TO
17
45
0
15.6
Pos.
45
0
15.7
Pos.
12-SPG-MCMACH-B-S08-30 Tf
29.4
0
29.3
0.1
-------
EPA/600/R-19/083
June 2019
Siimpk- II)
Spun-
I.ii;kI
Clin int.
Awr;i»i-
(1
(h ni in.
Sul l)i\
(hum.
U 1
(h ni ill.
KlSllll
|)\OI
Awr;i»i-
(I
p\()l
sul
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|)\OI
C liiiin.
\( 1
|)\OI
(Iiih m.
Kisiill
13-SPG-MCMACH-B-S09-300 TO
170
45
0
18.2
Pos.
45
0
18.2
Pos.
13-SPG-MCMACH-B-S09-300 Tf
26.8
0
26.8
0
14-SPG-MCMACH-B-S10-300 TO
170
45
0
18
Pos.
45
0
18.5
Pos.
14-SPG-MCMACH-B-S10-300 Tf
27
0
26.5
0
15-SPG-MCMACH-B-S11-3000 TO
1,700
45
0
19.2
Pos.
45
0
19.5
Pos.
15-SPG-MCMACH-B-S11-3000 Tf
25.8
0
25.5
0.1
16-SPG-MCMACH-B-S12-3000 TO
1,700
45
0
20.2
Pos.
45
0
20.5
Pos.
16-SPG-MCMACH-B-S12-3000 Tf
24.8
0
24.5
0
1-SPG-SCGRIL-B-S05-0 TO
0
45
0
0
Neg.
45
0
1.5
Neg.
1-SPG-SCGRIL-B-S05-0 Tf
45
0
43.5
2.6
2-SPG-SCGRIL-B-S06-30 TO
33
45
0
17.8
Pos.
45
0
17.9
Pos.
2-SPG-SCGRIL-B-S06-30 Tf
27.2
0.1
27.1
0.1
3-SPG-SCGRIL-B-S08-30 TO
33
45
0
14.9
Pos.
45
0
14.9
Pos.
3-SPG-SCGRIL-B-S08-30 Tf
30.1
0.1
30.1
0.1
4-SPG-SCGRIL-B-S07-300 TO
330
45
0
21.7
Pos.
45
0
21.9
Pos.
4-SPG-SCGRIL-B-S07-300 Tf
23.3
0
23.1
0
5-SPG-SCGRIL-B-S09-300 TO
330
45
0
15.6
Pos.
45
0
15.8
Pos.
5-SPG-SCGRIL-B-S09-300 Tf
29.4
0
29.2
0
6-SPG-FLDBLK-S06-300 TO
330
45
0
26
Pos.
45
0
26.2
Pos.
6-SPG-FLDBLK-S06-300 Tf
19
0
18.8
0
7-SPG-SCGRIL-B-S10-3000 TO
3,300
45
0
21
Pos.
45
0
20.4
Pos.
7-SPG-SCGRIL-B-S10-3000 Tf
24
0
24.6
0
8-SPG-SCGRIL-B-S11-3000 TO
3,300
45
0
19.7
Pos.
45
0
19.8
Pos.
8-SPG-SCGRIL-B-S11-3000 Tf
25.3
0.1
25.2
0
9-SPG-SWCON-A-S05-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
9-SPG-SWCON-A-S05-0 Tf
45
0
45
0
10-SPG-SWCON-A-S06-30 TO
33
45
0
14.8
Pos.
45
0
14.8
Pos.
10-SPG-SWCON-A-S06-30 Tf
30.2
0.1
30.2
0.1
11-SPG-SWCON-A-S07-30 TO
33
45
0
13.3
Pos.
45
0
13.1
Pos.
11-SPG-SWCON-A-S07-30 Tf
31.7
0
31.9
0.1
12-SPG-SWCON-A-S08-30 TO
33
45
0
17.4
Pos.
45
0
17.6
Pos.
12-SPG-SWCON-A-S08-30 Tf
27.6
0
27.4
0
13-SPG-SWCON-A-S09-300 TO
330
45
0
13.6
Pos.
45
0
14.1
Pos.
13-SPG-SWCON-A-S09-300 Tf
31.4
0.1
30.9
0
14-SPG-SWCON-A-S10-300 TO
330
45
0
18.3
Pos.
45
0
19.1
Pos.
14-SPG-SWCON-A-S10-300 Tf
26.7
0
25.9
0
15-SPG-SWCON-A-S11-3000 TO
3,300
45
0
21.6
Pos.
45
0
21.9
Pos.
15-SPG-SWCON-A-S11-3000 Tf
23.4
0
23.1
0
16-SPG-SWCON-A-S12-3000 TO
3,300
45
0
23.5
Pos.
45
0
24.1
Pos.
16-SPG-SWCON-A-S12-3000 Tf
21.5
0
20.9
0
l-SPG-EDPAN(A)-A-S05-0 TO
0
45
0
0
Neg.
45
0
3
Neg.
l-SPG-EDPAN(A)-A-S05-0 Tf
45
0
42
2.6
2-SPG-EDPAN(A)-A-S06-30 TO
31
45
0
17.5
Pos.
45
0
17.2
Pos.
2-SPG-EDPAN(A)-A-S06-30 Tf
27.5
0.1
27.8
0.1
3-SPG-EDPAN(A)-A-S07-30 TO
31
45
0
24.1
Pos.
45
0
24.1
Pos.
3-SPG-EDPAN(A)-A-S07-30 Tf
20.9
0.1
20.9
0
4-SPG-EDPAN(A)-A-S08-300 TO
310
45
0
21.9
Pos.
45
0
21.7
Pos.
4-SPG-EDPAN(A)-A-S08-300 Tf
23.1
0
23.3
0.1
5-SPG-EDPAN(A)-A-S09-300 TO
310
45
0
20.1
Pos.
45
0
19.7
Pos.
5-SPG-EDPAN(A)-A-S09-300 Tf
24.9
0
25.3
0.1
6-SPG-EDPAN(A)-A-S 10-3000 TO
3,100
45
0
23.4
Pos.
45
0
23.3
Pos.
6-SPG-EDPAN(A)-A-S 10-3000 Tf
21.6
0
21.7
0
7-SPG-EDPAN(A)-A-Sl 1-3000 TO
3,100
45
0
24.2
Pos.
45
0
24.1
Pos.
7-SPG-EDPAN(A)-A-Sl 1-3000 Tf
20.8
0.1
20.9
0
8-SPG-FLDBLK-A-S07-3000 TO
3,100
45
0
25.4
Pos.
45
0
25.4
Pos.
8-SPG-FLDBLK-A-S07-3000 Tf
19.6
0
19.6
0
-------
EPA/600/R-19/083
June 2019
Siimpk- II)
Spun-
I.ii;kI
Clin int.
Awr;i»i-
(1
(h ni in.
Sul l)i\
(hum.
U 1
(h ni ill.
KlSllll
|)\OI
Awr;i»i-
(1
p\()l
sul
l)l\
|)\OI
('hum.
\( I
|)\OI
(Iiih m.
Kisiill
9-SPG-CWSIGN-A-05-0 TO
0
45
0
0
Neg.
45
0
0.8
Neg.
9-SPG-CWSIGN-A-05-0 Tf
45
0
44.2
1.3
10-SPG-CWSIGN-A-06-30 TO
31
45
0
18.8
Pos.
45
0
19
Pos.
10-SPG-CWSIGN-A-06-30 Tf
26.2
0.1
26
0.1
1 l-SPG-CWSIGN-A-07-30 TO
31
45
0
12.8
Pos.
45
0
13
Pos.
1 l-SPG-CWSIGN-A-07-30 Tf
32.2
0.1
32
0
12-SPG-CWSIGN-A-08-30 TO
31
45
0
16.7
Pos.
45
0
17
Pos.
12-SPG-CWSIGN-A-08-30 Tf
28.3
0
28
0
13-SPG-CWSIGN-A-09-300 TO
310
45
0
24.9
Pos.
45
0
24.6
Pos.
13-SPG-CWSIGN-A-09-300 Tf
20.1
0.1
20.4
0
14-SPG-CWSIGN-A-10-300 TO
310
45
0
16.3
Pos.
45
0
16.3
Pos.
14-SPG-CWSIGN-A-10-300 Tf
28.7
0
28.7
0.1
15-SPG-CWSIGN-A-l 1-3000 TO
3,100
45
0
24.5
Pos.
45
0
24.6
Pos.
15-SPG-CWSIGN-A-l 1-3000 Tf
20.5
0
20.4
0
16-SPG-CWSIGN-A-12-3000 TO
3,100
45
0
26.1
Pos.
45
0
26.2
Pos.
16-SPG-CWSIGN-A-12-3000 Tf
18.9
0
18.8
0
1-SPG-TELEBO-A-S05-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
1-SPG-TELEBO-A-S05-0 Tf
45
0
45
0
2-SPG-FLDBLK-A-S08-30 TO
30
45
0
27.4
Pos.
45
0
27.1
Pos.
2-SPG-FLDBLK-A-S08-30 Tf
17.6
0
17.9
0
3-SPG-TELEBO-A-S06-30 TO
30
45
0
18.2
Pos.
45
0
18.1
Pos.
3-SPG-TELEBO-A-S06-30 Tf
26.8
0.1
26.9
0
4-SPG-TELEBO-A-S07-30 TO
30
45
0
19.8
Pos.
45
0
19.9
Pos.
4-SPG-TELEBO-A-S07-30 Tf
25.2
0
25.1
0
5-SPG-TELEBO-A-S08-300 TO
300
45
0
15.3
Pos.
43.6
2.5
14.2
Pos.
5-SPG-TELEBO-A-S08-300 Tf
29.7
0.1
29.4
0
6-SPG-TELEBO-A-S09-300 TO
300
45
0
17.9
Pos.
45
0
17.8
Pos.
6-SPG-TELEBO-A-S09-300 Tf
27.1
0
27.2
0
7-SPG-TELEBO-A-S10-3000 TO
3,000
45
0
23
Pos.
45
0
22.9
Pos.
7-SPG-TELEBO-A-S10-3000 Tf
22
0
22.1
0
8-SPG-TELEBO-A-S11-3000 TO
3,000
42.1
5
21.9
Pos.
42.2
4.9
22.2
Pos.
8-SPG-TELEBO-A-S11-3000 Tf
20.2
0
19.9
0
9-SPG-STGRAT-A-S05-0 TO
0
45
0
7.5
Neg.
45
0
7
Neg.
9-SPG-STGRAT-A-S05-0 Tf
37.5
0.9
38
0.7
10-SPG-STGRAT-A-S06-30 TO
30
45
0
10.1
Pos.
45
0
10.3
Pos.
10-SPG-STGRAT-A-S06-30 Tf
34.9
0.3
34.7
0.1
11-SPG-STGRAT-A-S07-30 TO
30
41.3
6.5
7.8
Neg.
45
0
12.1
Neg.*
11-SPG-STGRAT-A-S07-30 Tf
33.5
0.3
32.9
0.1
12-SPG-STGRAT-A-S08-30 TO
30
42.7
4.1
7.9
Neg.
42.6
4.1
8.7
Neg.
12-SPG-STGRAT-A-S08-30 Tf
34.7
0.1
33.9
0.1
13-SPG-STGRAT-A-S09-300 TO
300
45
0
14.7
Pos.
45
0
15.1
Pos.
13-SPG-STGRAT-A-S09-300 Tf
30.3
0.2
29.9
0.1
14-SPG-STGRAT-A-S10-300 TO
300
45
0
12.5
Pos.
45
0
13
Pos.
14-SPG-STGRAT-A-S10-300 Tf
32.5
0
32
0
15-SPG-STGRAT-A-S11-3000 TO
3,000
45
0
18.3
Pos.
45
0
18.7
Pos.
15-SPG-STGRAT-A-S11-3000 Tf
26.7
0
26.3
0.1
16-SPG-STGRAT-A-S12-3000 TO
3,000
45
0
16.8
Pos.
45
0
17.6
Pos.
16-SPG-STGRAT-A-S12-3000 Tf
28.2
0.1
27.4
0
1 -SPG-CWPNTD-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
1 -SPG-CWPNTD-0 Tf
45
0
45
0
2-SPG-CWPNTD-30 TO
22
45
0
20.3
Pos.
42.9
2.1
18
Pos.
2-SPG-CWPNTD-30 Tf
24.7
0.1
24.9
0
3-SPG-CWPNTD-30 TO
22
45
0
12.6
Pos.
45
0
12.5
Pos.
3-SPG-CWPNTD-30 Tf
32.4
0.2
32.5
0
4-SPG-CWPNTD-300 TO
220
45
0
23.4
Pos.
45
0
23.6
Pos.
4-SPG-CWPNTD-300 Tf
21.6
0
21.4
0
-------
EPA/600/R-19/083
June 2019
Siimpk- II)
Spun-
I.ii;kI
Clin int.
Awr;i»i-
(1
(h ni in.
Sul l)i\
(hum.
U 1
(h ni ill.
KlSllll
|)\OI
Awr;i»i-
(I
p\()l
sul
l)l\
|)\OI
C liiiin.
\( 1
|)\OI
(Iiih m.
Kisiill
5-SPG-CWPNTD-300 TO
220
45
0
21.3
Pos.
45
0
21.4
Pos.
5-SPG-CWPNTD-300 Tf
23.7
0.1
23.6
0
6-SPG-FLDBLK-300 TO
220
45
0
25.3
Pos.
45
0
25.2
Pos.
6-SPG-FLDBLK-300 Tf
19.7
0
19.8
0
7-SPG-CWPNTD-3000 TO
2,200
45
0
25.5
Pos.
45
0
25.3
Pos.
7-SPG-CWPNTD-3000 Tf
19.5
0
19.7
0
8-SPG-CWPNTD-3000 TO
2,200
43.2
3.2
21.1
Pos.
44.4
1
22.3
Pos.
8-SPG-CWPNTD-3000 Tf
22
0
22.1
0
9-SPG-GRNBEN-O TO
0
45
0
0
Neg.
45
0
0
Neg.
9-SPG-GRNBEN-O Tf
45
0
45
0
10-SPG-GRNBEN-30 TO
22
45
0
11.7
Pos.
45
0
11.3
Pos.
10-SPG-GRNBEN-30 Tf
33.3
0.2
33.7
0.2
ll-SPG-GRNBEN-30 TO
22
45
0
9.8
Neg.*
43.5
2.6
7.9
Neg.
1 l-SPG-GRNBEN-30 Tf
35.2
0.3
35.6
0.1
12-SPG-GRNBEN-30 TO
22
45
0
15.4
Pos.
45
0
15.3
Pos.
12-SPG-GRNBEN-30 Tf
29.6
0
29.7
0
13-SPG-GRNBEN-300 TO
220
45
0
18.1
Pos.
45
0
17.9
Pos.
13-SPG-GRNBEN-300 Tf
26.9
0.1
27.1
0
14-SPG-GRNBEN-300 TO
220
45
0
17.8
Pos.
45
0
18.1
Pos.
14-SPG-GRNBEN-300 Tf
27.2
0.1
26.9
0
15-SPG-GRNBEN-3000 TO
2,200
45
0
19.2
Pos.
45
0
19.2
Pos.
15-SPG-GRNBEN-3000 Tf
25.8
0.1
25.8
0
16-SPG-GRNBEN-3000 TO
2,200
45
0
19.7
Pos.
45
0
19.9
Pos.
16-SPG-GRNBEN-3000 Tf
25.3
0
25.1
0
1 -SPG-FLTILE-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
1 -SPG-FLTILE-0 Tf
45
0
45
0
2-SPG-FLTILE-30 TO
24
45
0
11.7
Pos.
45
0
11.3
Pos.
2-SPG-FLTILE-30 Tf
33.3
0.3
33.7
0.2
3-SPG-FLTILE-30 TO
24
45
0
13.5
Pos.
45
0
13.3
Pos.
3-SPG-FLTILE-30 Tf
31.5
0
31.7
0
4-SPG-FLTILE-300 TO
240
45
0
20.3
Pos.
45
0
20.3
Pos.
4-SPG-FLTILE-300 Tf
24.7
0
24.7
0
5-SPG-FLTILE-300 TO
240
45
0
14
Pos.
45
0
13.3
Pos.
5-SPG-FLTILE-300 Tf
31
0
31.7
0.1
6-SPG-FLTILE-3000 TO
2,400
45
0
19.6
Pos.
45
0
19
Pos.
6-SPG-FLTILE-3000 Tf
25.4
0
26
0
7-SPG-FLTILE-3000 TO
2,400
45
0
20.8
Pos.
45
0
20.8
Pos.
7-SPG-FLTILE-3000 Tf
24.2
0
24.2
0
8-SPG-FLDBLK-3000 TO
2,400
45
0
25.2
Pos.
42.1
2.6
22.1
Pos.
8-SPG-FLDBLK-3000 Tf
19.8
0
19.9
0
9-SPG-FLCON-30 TO
24
45
0
17.1
Pos.
45
0
17.1
Pos.
9-SPG-FLCON-30 Tf
27.9
0
27.9
0.1
10-SPG-FLCON-30 TO
24
45
0
16.6
Pos.
45
0
16.6
Pos.
10-SPG-FLCON-30 Tf
28.4
0.1
28.4
0.1
ll-SPG-STEPS-30 TO
24
45
0
14
Pos.
45
0
14
Pos.
ll-SPG-STEPS-30 Tf
31
0.1
31
0
12-SPG-STEPS-300 TO
240
45
0
20
Pos.
45
0
20.1
Pos.
12-SPG-STEPS-300 Tf
25
0
24.9
0
13-SPG-FLCON-300 TO
240
45
0
20.8
Pos.
45
0
20.8
Pos.
13-SPG-FLCON-300 Tf
24.2
0
24.2
0
14-SPG-FLCON-300 TO
240
45
0
20.7
Pos.
45
0
21
Pos.
14-SPG-FLCON-300 Tf
24.3
0.1
24
0
15-SPG-FLCON-3000 TO
2,400
45
0
21
Pos.
45
0
21.2
Pos.
15-SPG-FLCON-3000 Tf
24
0
23.8
0
16-SPG-FLCON-3000 TO
2,400
45
0
22.8
Pos.
45
0
23.2
Pos.
16-SPG-FLCON-3000 Tf
22.2
0
21.8
0
-------
EPA/600/R-19/083
June 2019
Siimpk- II)
Spun-
I.ii;kI
Clin int.
Awr;i»i-
(1
(h ni in.
Sul l)i\
(hum.
U 1
(h ni ill.
KlSllll
|)\OI
Awr;i»i-
(1
p\()l
sul
l)l\
|)\OI
C liiiin.
\( 1
|)\OI
(Iiih m.
Kisiill
1-SPG-GLSWIN-B-S13-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
1-SPG-GLSWIN-B-S13-0 Tf
45
0
45
0
2-SPG-GLSWIN-B-S14-30 TO
38
45
0
14.2
Pos.
45
0
14.1
Pos.
2-SPG-GLSWIN-B-S14-30 Tf
30.8
0.1
30.9
0.1
3-SPG-STEPS-B-S11-30 TO
38
45
0
12.9
Pos.
45
0
13.1
Pos.
3-SPG-STEPS-B-S11-30 Tf
32.1
0.1
31.9
0.1
4-SPG-STEPS-B-S12-300 TO
380
45
0
17.7
Pos.
45
0
17.2
Pos.
4-SPG-STEPS-B-S12-300 Tf
27.3
0.1
27.8
0
5-SPG-GLSWIN-B-S15-300 TO
380
45
0
25.3
Pos.
45
0
25
Pos.
5-SPG-GLSWIN-B-S15-300 Tf
19.7
0.2
20
0.1
6-SPG-GLSWIN-B-S16-300 TO
380
45
0
21.8
Pos.
45
0
21.8
Pos.
6-SPG-GLSWIN-B-S16-300 Tf
23.2
0.2
23.2
0.1
7-SPG-GLSWIN-B-S17-3000 TO
3,800
45
0
25.5
Pos.
43.3
2.9
23.4
Pos.
7-SPG-GLSWIN-B-S17-3000 Tf
19.5
0.1
19.9
0
8-SPG-GLSWIN-B-S18-3000 TO
3,800
45
0
25.2
Pos.
45
0
25.1
Pos.
8-SPG-GLSWIN-B-S18-3000 Tf
19.8
0
19.9
0
9-SPG-WLTILE-B-S09-30 TO
38
45
0
17.9
Pos.
45
0
17.2
Pos.
9-SPG-WLTILE-B-S09-30 Tf
27.1
0.1
27.8
0
10-SPG-WLTILE-B-S10-30 TO
38
45
0
17.3
Pos.
45
0
17
Pos.
10-SPG-WLTILE-B-S10-30 Tf
27.7
0.1
28
0.1
11-SPG-WLTILE-B-S11-300 TO
380
45
0
22.6
Pos.
45
0
22.5
Pos.
11-SPG-WLTILE-B-S11-300 Tf
22.4
0.1
22.5
0
12-SPG-WLTILE-B-S12-300 TO
380
45
0
23.9
Pos.
45
0
23.8
Pos.
12-SPG-WLTILE-B-S12-300 Tf
21.1
0
21.2
0
13-SPG-WLTILE-B-S13-3000 TO
3,800
45
0
23
Pos.
45
0
22.9
Pos.
13-SPG-WLTILE-B-S13-3000 Tf
22
0.1
22.1
0
14-SPG-WLTILE-B-S14-3000 TO
3,800
45
0
24.1
Pos.
45
0
24.2
Pos.
14-SPG-WLTILE-B-S14-3000 Tf
20.9
0.1
20.8
0
15-SPG-STEPS-B-S13-3000 TO
3,800
45
0
21.9
Pos.
45
0
21.6
Pos.
15-SPG-STEPS-B-S13-3000 Tf
23.1
0.1
23.4
0
16-SPG-STEPS-B-S14-3000 TO
3,800
45
0
20.1
Pos.
45
0
20
Pos.
16-SPG-STEPS-B-S14-3000 Tf
24.9
0.1
25
0
1 -SPG-FLDBLK-A-S11 -0 TO
0
45
0
7.5
Neg.
45
0
7.5
Neg.
1 -SPG-FLDBLK-A-S11 -0 Tf
37.5
0.8
37.5
0.4
2-SPG-GLSPAN-B-S13-0 TO
0
45
0
0
Neg.
45
0
3.4
Neg.
2-SPG-GLSPAN-B-S13-0 Tf
45
0
41.6
3.1
3-SPG-GLSPAN-B-S14-30 TO
36
45
0
22.1
Pos.
45
0
21.9
Pos.
3-SPG-GLSPAN-B-S14-30 Tf
22.9
0
23.1
0
4-SPG-FLTILE-B-S16-30 TO
36
45
0
16.2
Pos.
45
0
16.2
Pos.
4-SPG-FLTILE-B-S16-30 Tf
28.8
0.1
28.8
0
5-SPG-GLSPAN-B-S15-300 TO
360
45
0
26.7
Pos.
45
0
26.7
Pos.
5-SPG-GLSPAN-B-S15-300 Tf
18.3
0.1
18.3
0.1
6-SPG-GLSPAN-B-S16-300 TO
360
45
0
26
Pos.
45
0
26
Pos.
6-SPG-GLSPAN-B-S16-300 Tf
19
0.1
19
0
7-SPG-GLSPAN-B-S17-3000 TO
3,600
45
0
24.6
Pos.
45
0
24.4
Pos.
7-SPG-GLSPAN-B-S17-3000 Tf
20.4
0.1
20.6
0.1
8-SPG-GLSPAN-B-S18-3000 TO
3,600
45
0
24.4
Pos.
45
0
24.5
Pos.
8-SPG-GLSPAN-B-S18-3000 Tf
20.6
0
20.5
0
9-SPG-EDPAN(B)-B-S13-0 TO
0
44.9
0.2
2
Neg.
43.6
2.4
4.1
Neg.
9-SPG-EDPAN(B)-B-S13-0 Tf
42.9
3.6
39.6
0.5
10-SPG-EDPAN(B)-B-S14-30 TO
36
45
0
23.1
Pos.
45
0
23
Pos.
10-SPG-EDPAN(B)-B-S14-30 Tf
21.9
0.1
22
0
11-SPG-EDPAN(B)-B-S 15-300 TO
360
45
0
23.9
Pos.
45
0
23.7
Pos.
11-SPG-EDPAN(B)-B-S 15-300 Tf
21.1
0
21.3
0.1
12-SPG-EDPAN(B)-B-S16-300 TO
360
45
0
26
Pos.
45
0
26.1
Pos.
12-SPG-EDPAN(B)-B-S16-300 Tf
19
0
18.9
0
-------
EPA/600/R-19/083
June 2019
S.impli- II)
Spun-
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Clin int.
Awr;i»i-
(1
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Sul l)i\
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Kisiill
13-SPG-FLTILE-B-S17-300 TO
360
43.3
2.9
20
Pos.
44.7
0.6
21.2
Pos.
13-SPG-FLTILE-B-S17-300 Tf
23.3
0.1
23.4
0
14-SPG-EDPAN(B)-B-S17-3000 TO
3,600
45
0
25
Pos.
45
0
25
Pos.
14-SPG-EDPAN(B)-B-S17-3000 Tf
20
0.1
20
0
15-SPG-EDPAN(B)-B-S 18-3000 TO
3,600
45
0
28
Pos.
45
0
28.1
Pos.
15-SPG-EDPAN(B)-B-S 18-3000 Tf
17
0
16.9
0
16-SPG-FLTILE-B-S18-3000 TO
3,600
45
0
24
Pos.
45
0
24.2
Pos.
16-SPG-FLTILE-B-S18-3000 Tf
21
0.1
20.8
0
1 -SPG-FLLFIX-B-S13-0 TO
0
45
0
0
Neg.
45
0
1.2
Neg.
1-SPG-FLLFIX-B-S13-0 Tf
45
0
43.8
2.2
2-SPG-FLLFIX-B-S14-30 TO
26
45
0
16.5
Pos.
45
0
16.3
Pos.
2-SPG-FLLFIX-B-S14-30 Tf
28.5
0.1
28.7
0
3-SPG-FLLFIX-B-S15-300 TO
260
45
0
21.7
Pos.
45
0
21.3
Pos.
3-SPG-FLLFIX-B-S15-300 Tf
23.3
0.1
23.7
0.1
4-SPG-FLLFIX-B-S16-300 TO
260
45
0
20
Pos.
45
0
19.8
Pos.
4-SPG-FLLFIX-B-S16-300 Tf
25
0.1
25.2
0
5-SPG-MCMACH-B-S13-300 TO
260
45
0
17.4
Pos.
45
0
17.2
Pos.
5-SPG-MCMACH-B-S13-300 Tf
27.6
0.1
27.8
0
6-SPG-FLLFIX-B-S17-3000 TO
2,600
45
0
24.7
Pos.
45
0
24.9
Pos.
6-SPG-FLLFIX-B-S17-3000 Tf
20.3
0
20.1
0
7-SPG-MCMACH-B-S14-3000 TO
2,600
45
0
23.1
Pos.
45
0
23.1
Pos.
7-SPG-MCMACH-B-S14-3000 Tf
21.9
0.1
21.9
0
8-SPG-FLLFIX-B-S18-3000 TO
2,600
45
0
20.1
Pos.
45
0
20.1
Pos.
8-SPG-FLLFIX-B-S18-3000 Tf
24.9
0
24.9
0
9-SPG-OHSIGN-B-S12-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
9-SPG-OHSIGN-B-S12-0 Tf
45
0
45
0
10-SPG-OHSIGN-B-S13-30 TO
26
45
0
18.6
Pos.
45
0
18.4
Pos.
10-SPG-OHSIGN-B-S13-30 Tf
26.4
0.1
26.6
0
11-SPG-OHSIGN-B-S14-30 TO
26
45
0
18.4
Pos.
45
0
18.3
Pos.
11-SPG-OHSIGN-B-S14-30 Tf
26.6
0.1
26.7
0
12-SPG-OHSIGN-B-S15-300 TO
260
45
0
21
Pos.
45
0
21
Pos.
12-SPG-OHSIGN-B-S15-300 Tf
24
0
24
0
13-SPG-OHSIGN-B-S16-300 TO
260
45
0
20.9
Pos.
45
0
21.1
Pos.
13-SPG-OHSIGN-B-S16-300 Tf
24.1
0.1
23.9
0
14-SPG-SCGRIL-B-S12-300 TO
260
45
0
21.3
Pos.
45
0
21.7
Pos.
14-SPG-SCGRIL-B-S12-300 Tf
23.7
0
23.3
0.1
15-SPG-OHSIGN-B-S17-3000 TO
2,600
45
0
22.3
Pos.
45
0
22.3
Pos.
15-SPG-OHSIGN-B-S17-3000 Tf
22.7
0.1
22.7
0
16-SPG-OHSIGN-B-S18-3000 TO
2,600
45
0
22.5
Pos.
45
0
22.7
Pos.
16-SPG-OHSIGN-B-S18-3000 Tf
22.5
0
22.3
0
1 -SPG-SWCON-A-S13-0 TO
0
45
0
4.6
Neg.
45
0
4.2
Neg.
1 -SPG-SWCON-A-S13-0 Tf
40.4
4
40.8
3.4
2-SPG-SWCON-A-S14-30 TO
33
45
0
13
Pos.
45
0
13.4
Pos.
2-SPG-SWCON-A-S14-30 Tf
32
0.1
31.6
0.1
3-SPG-SWCON-A-S15-300 TO
330
45
0
21.8
Pos.
45
0
22
Pos.
3-SPG-SWCON-A-S15-300 Tf
23.2
0
23
0
4-SPG-SWCON-A-S16-300 TO
330
45
0
19.5
Pos.
45
0
19.9
Pos.
4-SPG-SWCON-A-S16-300 Tf
25.5
0
25.1
0
5-SPG-STGRAT-A-S13-300 TO
330
45
0
17
Pos.
43.4
2.8
15.8
Pos.
5-SPG-STGRAT-A-S13-300 Tf
28
0.1
27.6
0
6-SPG-STGRAT-A-S14-3000 TO
3,300
45
0
12.8
Pos.
45
0
13.1
Pos.
6-SPG-STGRAT-A-S14-3000 Tf
32.2
0.3
31.9
0
7-SPG-SWCON-A-S17-3000 TO
3,300
45
0
21.8
Pos.
45
0
22.1
Pos.
7-SPG-SWCON-A-S17-3000 Tf
23.2
0
22.9
0
8-SPG-SWCON-A-S18-3000 TO
3,300
45
0
24.1
Pos.
45
0
24.4
Pos.
8-SPG-SWCON-A-S18-3000 Tf
20.9
0
20.6
0
-------
EPA/600/R-19/083
June 2019
Siimpk- II)
Spun-
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Clin int.
Awr;i»i-
(1
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Sul l)i\
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Awr;i»i-
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C liiiin.
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(Iiih m.
Kisiill
9-SPG-EDPAN(A)-A-S 12-0 TO
0
45
0
1
Neg.
45
0
1.4
Neg.
9-SPG-EDPAN(A)-A-S12-0 Tf
44
1.8
43.6
2.4
10-SPG-EDPAN(A)-A-S 13-30 TO
33
45
0
20.8
Pos.
45
0
20.8
Pos.
10-SPG-EDPAN(A)-A-S 13-30 Tf
24.2
0.1
24.2
0
1 l-SPG-EDPAN(A)-A-S14-30 TO
3
45
0
9.9
Pos.
45
0
10.2
Pos.
1 l-SPG-EDPAN(A)-A-S14-30 Tf
35.1
0.2
34.8
0.1
12-SPG-EDPAN(A)-A-S15-300 TO
330
45
0
24.6
Pos.
45
0
24.7
Pos.
12-SPG-EDPAN(A)-A-S15-300 Tf
20.4
0.1
20.3
0
13-SPG-EDPAN(A)-A-S 16-300 TO
330
45
0
21.9
Pos.
45
0
22.1
Pos.
13-SPG-EDPAN(A)-A-S 16-300 Tf
23.1
0.1
22.9
0
14-SPG-LABBLANK-300 TO
330
45
0
27
Pos.
45
0
27.1
Pos.
14-SPG-LABBLANK-300 Tf
18
0.1
17.9
0
15-SPG-EDPAN(A)-A-S 17-3000 TO
3,300
45
0
24
Pos.
45
0
24.1
Pos.
15-SPG-EDPAN(A)-A-S 17-3000 Tf
21
0
20.9
0
16-SPG-EDPAN(A)-A-S 18-3000 TO
3,300
45
0
26.9
Pos.
45
0
27.1
Pos.
16-SPG-EDPAN(A)-A-S 18-3000 Tf
18.1
0
17.9
0
1 -SPG-CWSIGN-A-S13-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
1 -SPG-CWSIGN-A-S13-0 Tf
45
0
45
0
2-SPG-CWSIGN-A-S14-30 TO
37
45
0
20.3
Pos.
45
0
20.3
Pos.
2-SPG-CWSIGN-A-S14-30 Tf
24.7
0.1
24.7
0
3-SPG-CWSIGN-A-S15-300 TO
370
45
0
18.9
Pos.
45
0
18.8
Pos.
3-SPG-CWSIGN-A-S15-300 Tf
26.1
0.1
26.2
0
4-SPG-CWSIGN-A-S16-300 TO
370
45
0
18
Pos.
45
0
18.1
Pos.
4-SPG-CWSIGN-A-S16-300 Tf
27
0
26.9
0
5-SPG-GRNBEN-A-S13-300 TO
370
45
0
21.5
Pos.
45
0
21.4
Pos.
5-SPG-GRNBEN-A-S13-300 Tf
23.5
0.1
23.6
0
6-SPG-GRNBEN-A-S14-3000 TO
3,700
45
0
21.1
Pos.
45
0
21.3
Pos.
6-SPG-GRNBEN-A-S14-3000 Tf
23.9
0
23.7
0
7-SPG-CWSIGN-A-S17-3000 TO
3,700
45
0
25.8
Pos.
45
0
25.8
Pos.
7-SPG-CWSIGN-A-S17-3000 Tf
19.2
0
19.2
0
8-SPG-CWSIGN-A-S18-3000 TO
3,700
45
0
23.1
Pos.
45
0
22.9
Pos.
8-SPG-CWSIGN-A-S18-3000 Tf
21.9
0
22.1
0
9-SPG-TELEBO-A-S12-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
9-SPG-TELEBO-A-S12-0 Tf
45
0
45
0
10-SPG-TELEBO-A-S13-30 TO
37
45
0
12.9
Pos.
45
0
13.2
Pos.
10-SPG-TELEBO-A-S13-30 Tf
32.1
0.2
31.8
0
11-SPG-TELEBO-A-S14-30 TO
37
45
0
23.3
Pos.
45
0
23.2
Pos.
11-SPG-TELEBO-A-S14-30 Tf
21.7
0
21.8
0
12-SPG-LABBLANK-30 TO
37
45
0
25.7
Pos.
45
0
25.5
Pos.
12-SPG-LABBLANK-30 Tf
19.3
0.1
19.5
0.1
13-SPG-TELEBO-A-S15-300 TO
370
45
0
21.8
Pos.
45
0
22
Pos.
13-SPG-TELEBO-A-S15-300 Tf
23.2
0.1
23
0
14-SPG-TELEBO-A-S16-300 TO
370
45
0
23.2
Pos.
45
0
23.5
Pos.
14-SPG-TELEBO-A-S16-300 Tf
21.8
0.1
21.5
0
15-SPG-TELEBO-A-S17-3000 TO
3,700
45
0
23.4
Pos.
45
0
23.5
Pos.
15-SPG-TELEBO-A-S17-3000 Tf
21.6
0
21.5
0
16-SPG-TELEBO-A-S18-3000 TO
3,700
45
0
21.3
Pos.
45
0
21.4
Pos.
16-SPG-TELEBO-A-S18-3000 Tf
23.7
0.1
23.6
0
1-SPG-FLCON-B-S15-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
1-SPG-FLCON-B-S15-0 Tf
45
0
45
0
2-SPG-STEPS-B-S15-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
2-SPG-STEPS-B-S15-0 Tf
45
0
45
0
3-SPG-FLCON-B-S16-30 TO
34
45
0
15.6
Pos.
45
0
15.6
Pos.
3-SPG-FLCON-B-S16-30 Tf
29.4
0
29.4
0.1
4-SPG-STEPS-B-S16-30 TO
34
45
0
14
Pos.
45
0
13.8
Pos.
4-SPG-STEPS-B-S16-30 Tf
31
0.1
31.2
0
-------
EPA/600/R-19/083
June 2019
Siimpk- II)
Spun-
I.ii;kI
Clin int.
Awr;i»i-
(1
(h ni in.
Sul l)i\
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Kisiill
5-SPG-FLCON-B-S17-300 TO
340
45
0
16.3
Pos.
45
0
16.5
Pos.
5-SPG-FLCON-B-S17-300 Tf
28.7
0.1
28.5
0.1
6-SPG-STEPS-B-S17-300 TO
340
45
0
17
Pos.
45
0
16.7
Pos.
6-SPG-STEPS-B-S17-300 Tf
28
0
28.3
0.1
7-SPG-FLCON-B-S18-3000 TO
3,400
45
0
20.1
Pos.
45
0
20
Pos.
7-SPG-FLCON-B-S18-3000 Tf
24.9
0.1
25
0
8-SPG-STEPS-B-S18-3000 TO
3,400
45
0
19.4
Pos.
45
0
19.6
Pos.
8-SPG-STEPS-B-S18-3000 Tf
25.6
0
25.4
0
9-SPG-SCGRIL-B-S13-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
9-SPG-SCGRIL-B-S13-0 Tf
45
0
45
0
10-SPG-LABBLANK-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
10-SPG-LABBLANK-O Tf
45
0
45
0
1 l-SPG-LABBLANK-30 TO
34
45
0
25.3
Pos.
45
0
25.2
Pos.
1 l-SPG-LABBLANK-30 Tf
19.7
0.1
19.8
0
12-SPG-SCGRIL-B-S14-30 TO
34
45
0
13.6
Pos.
45
0
13.5
Pos.
12-SPG-SCGRIL-B-S14-30 Tf
31.4
0.1
31.5
0.1
13-SPG-SCGRIL-B-S15-30 TO
34
45
0
10.9
Pos.
45
0
11.1
Pos.
13-SPG-SCGRIL-B-S15-30 Tf
34.1
0.2
33.9
0.1
14-SPG-SCGRIL-B-S16-300 TO
340
45
0
19.1
Pos.
45
0
19.2
Pos.
14-SPG-SCGRIL-B-S16-300 Tf
25.9
0
25.8
0
15-SPG-SCGRIL-B-S17-3000 TO
3,400
45
0
18.4
Pos.
45
0
18.1
Pos.
15-SPG-SCGRIL-B-S17-3000 Tf
26.6
0.1
26.9
0
16-SPG-SCGRIL-B-S18-3000 TO
3,400
45
0
25.2
Pos.
45
0
25.6
Pos.
16-SPG-SCGRIL-B-S18-3000 Tf
19.8
0
19.4
0
1 -SPG-WLTILE-B-S15-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
1 -SPG-WLTILE-B-S15-0 Tf
45
0
45
0
2-SPG-MCMACH-B-S15-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
2-SPG-MCMACH-B-S15-0 Tf
45
0
45
0
3-SPG-WLTILE-B-S16-30 TO
24
45
0
14.7
Pos.
45
0
14.2
Pos.
3-SPG-WLTILE-B-S16-30 Tf
30.3
0.2
30.8
0.1
4-SPG-MCMACH-B-S16-30 TO
24
45
0
19.2
Pos.
45
0
19.3
Pos.
4-SPG-MCMACH-B-S16-30 Tf
25.8
0.1
25.7
0.1
5-SPG-WLTILE-B-S17-300 TO
240
45
0
18.6
Pos.
45
0
18.3
Pos.
5-SPG-WLTILE-B-S17-300 Tf
26.4
0.1
26.7
0
6-SPG-MCMACH-B-S17-300 TO
240
45
0
19.6
Pos.
45
0
19.4
Pos.
6-SPG-MCMACH-B-S17-300 Tf
25.4
0.1
25.6
0
7-SPG-WLTILE-B-S18-3000 TO
2,400
45
0
19.6
Pos.
45
0
19
Pos.
7-SPG-WLTILE-B-S18-3000 Tf
25.4
0.1
26
0
8-SPG-MCMACH-B-S18-3000 TO
2,400
45
0
23
Pos.
45
0
23
Pos.
8-SPG-MCMACH-B-S18-3000 Tf
22
0
22
0
9-SPG-FLDBLK-A-S12-30 TO
24
45
0
25.2
Pos.
45
0
24.8
Pos.
9-SPG-FLDBLK-A-S12-30 Tf
19.8
0.1
20.2
0.1
10-SPG-CWPNTD-A-S16-30 TO
24
45
0
15
Pos.
45
0
14.8
Pos.
10-SPG-CWPNTD-A-S16-30 Tf
30
0
30.2
0
11-SPG-FLDBLK-A-S13-300 TO
240
45
0
26.1
Pos.
45
0
25.7
Pos.
11-SPG-FLDBLK-A-S13-300 Tf
18.9
0
19.3
0
12-SPG-CWPNTD-A-S17-300 TO
240
45
0
19
Pos.
45
0
18.7
Pos.
12-SPG-CWPNTD-A-S17-300 Tf
26
0.1
26.3
0
13-SPG-LABBLANK-300 TO
240
45
0
25.4
Pos.
45
0
25.1
Pos.
13-SPG-LABBLANK-300 Tf
19.6
0.1
19.9
0
14-SPG-FLDBLK-A-S14-3000 TO
2,400
45
0
25
Pos.
45
0
24.9
Pos.
14-SPG-FLDBLK-A-S14-3000 Tf
20
0
20.1
0
15-SPG-CWPNTD-A-S18-3000 TO
2,400
45
0
19.2
Pos.
45
0
18.6
Pos.
15-SPG-CWPNTD-A-S18-3000 Tf
25.8
0
26.4
0.1
16-SPG-LABBLANK-3000 TO
2,400
45
0
25.2
Pos.
45
0
25.1
Pos.
16-SPG-LABBLANK-3000 Tf
19.8
0
19.9
0
-------
EPA/600/R-19/083
June 2019
Siimpk- II)
Spun-
I.ii;kI
Clin int.
Awr;i»i-
(1
(h ni in.
Sul l)i\
(hum.
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C liiiin.
\( 1
|)\OI
(Iiih m.
Kisiill
1 -SPG-FLDBLK-A-S15-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
1 -SPG-FLDBLK-A-S15-0 Tf
45
0
45
0
2-SPG-STGRAT-A-S15-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
2-SPG-STGRAT-A-S15-0 Tf
45
0
45
0
3-SPG-FLDBLK-A-S16-30 TO
31
45
0
26.8
Pos.
45
0
26.4
Pos.
3-SPG-FLDBLK-A-S16-30 Tf
18.2
0
18.6
0
4-SPG-STGRAT-A-S16-30 TO
31
45
0
10.5
Pos.
45
0
11
Pos.
4-SPG-STGRAT-A-S16-30 Tf
34.5
0.2
34
0
5-SPG-FLDBLK-A-S17-300 TO
310
45
0
25.5
Pos.
45
0
25.2
Pos.
5-SPG-FLDBLK-A-S17-300 Tf
19.5
0.1
19.8
0
6-SPG-STGRAT-A-S17-300 TO
310
45
0
14.7
Pos.
45
0
15.3
Pos.
6-SPG-STGRAT-A-S17-300 Tf
30.3
0.1
29.7
0.1
7-SPG-FLDBLK-A-S18-3000 TO
3,100
45
0
26.5
Pos.
45
0
26.2
Pos.
7-SPG-FLDBLK-A-S18-3000 Tf
18.5
0
18.8
0
8-SPG-STGRAT-A-S18-3000 TO
3,100
45
0
16.2
Pos.
45
0
18.3
Pos.
8-SPG-STGRAT-A-S18-3000 Tf
28.8
3
26.7
0.2
9-SPG-GRNBEN-A-S15-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
9-SPG-GRNBEN-A-S15-0 Tf
45
0
45
0
10-SPG-WLTILE-B-S19-0 TO
0
45
0
0
Neg.
45
0
0.1
Neg.
10-SPG-WLTILE-B-S19-0 Tf
45
0
44.9
0.2
11-SPG-GRNBEN-A-S16-30 TO
31
45
0
20.2
Pos.
43.6
2.4
18.7
Pos.
11-SPG-GRNBEN-A-S16-30 Tf
24.8
0.1
24.9
0
12-SPG-LABBLK-30 TO
31
45
0
26
Pos.
45
0
25.6
Pos.
12-SPG-LABBLK-30 Tf
19
0
19.4
0
13-SPG-GRNBEN-A-S17-300 TO
310
45
0
18.1
Pos.
45
0
18.1
Pos.
13-SPG-GRNBEN-A-S17-300 Tf
26.9
0.1
26.9
0
14-SPG-LABBLK-300 TO
310
45
0
26
Pos.
45
0
25.7
Pos.
14-SPG-LABBLK-300 Tf
19
0
19.3
0
15-SPG-GRNBEN-A-S18-3000 TO
3,100
45
0
24.1
Pos.
45
0
24.1
Pos.
15-SPG-GRNBEN-A-S18-3000 Tf
20.9
0
20.9
0
16-SPG-LABBLK-3000 TO
3,100
45
0
26.2
Pos.
43.4
2.7
24.4
Pos.
16-SPG-LABBLK-3000 Tf
18.8
0
19
0
-------
EPA/600/R-19/083
June 2019
APPENDIX Q: CULTURE RESULTS FOR VCF SAMPLES USING SHEEP
BLOOD AGAR MEDIUM
-------
EPA/600/R-19/083
June 2019
APPENDIX R: RV-PCR RESULTS FOR VCF SAMPLES USING
CHROMOSOMAL AND pXOl GENE TARGETS
-------
EPA/600/R-19/083
June 2019
S;mi|)k' II)
Spun-
l.o;i(l
Clin Mil.
Awr;i»i-
CI
Clin Mil.
Sliul
l)i\
Chum.
U 1
Clin mi.
Ri'sull
|>XOI
Awr;i»i-
CI
|)\OI
Si ml
l)i-\
|)\OI
(111 Mil.
\< 1
|>\()l
Clin mii.
kl-Mlll
1-VCF-STEPS-B-
S01-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
1-VCF-STEPS-B-
S01-0 Tf
45
0
45
0
2-VCF-SWCON-A-
S01-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
2-VCF-SWCON-A-
S01-0 Tf
45
0
45
0
3-VCF-STEPS-B-
S02-30 TO
21
45
0
0
Neg.
45
0
0
Neg.
3-VCF-STEPS-B-
S02-30 Tf
45
0
45
0
4-VCF-SWCON-A-
S02-30 TO
21
45
0
0
Neg.
45
0
0
Neg.
4-VCF-SWCON-A-
S02-30 Tf
45
0
45
0
5-VCF-STEPS-B-
S03-300 TO
210
45
0
0
Neg.
45
0
1.1
Neg.
5-VCF-STEPS-B-
S03-300 Tf
45
0
43.9
1.9
6-VCF-SWCON-A-
S03-300 TO
210
45
0
11.9
Pos.
44.2
1.3
11.2
Pos.
6-VCF-SWCON-A-
S03-300 Tf
33.1
0.1
33
0.1
7-VCF-STEPS-B-
S04-3,000 TO
2,100
45
0
12.1
Pos.
45
0
12.2
Pos.
7-VCF-STEPS-B-
S04-3,000 Tf
32.9
0.1
32.8
0.1
8-VCF-SWCON-A-
S04-3,000 TO
2,100
45
0
17.2
Pos.
44.6
0.6
17.3
Pos.
8-VCF-SWCON-A-
S04-3,000 Tf
27.8
0.1
27.3
0
1-VCF-FLCON-B-
S01-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
1-VCF-FLCON-B-
S01-0 Tf
45
0
45
0
2-VCF-FLCON-B-
S02-30 TO
27
45
0
0
Neg.
45
0
1
Neg.
2-VCF-FLCON-B-
S02-30 Tf
45
0
44
1.8
3-VCF-CARPET-
A-S01-30 TO
27
45
0
0
Neg.
45
0
0
Neg.
3-VCF-CARPET-
A-S01-30 Tf
45
0
45
0
4-VCF-SCFILT-B-
SO1-30 TO
27
45
0
0
Neg.
45
0
0
Neg.
4-VCF-SCFILT-B-
S01-30 Tf
45
0
45
0
5-VCF-FLCON-B-
S03-300 TO
270
45
0
18.6
Pos.
45
0
19
Pos.
5-VCF-FLCON-B-
S03-300 Tf
26.4
0.1
26
0
6-VCF-CARPET-
A-S02-300 TO
270
45
0
14.4
Pos.
43.5
1.7
13.1
Pos.
6-VCF-CARPET-
A-S02-300 Tf
30.6
0.1
30.4
0
-------
EPA/600/R-19/083
June 2019
S;mi|)k' II)
Spun-
l.oiid
Clin Mil.
Awr;i»i-
CI
Clin Mil.
Sliul
l)i-\
Chum.
U I
Clin mi.
Ri'sull
|>XOI
Awr;i»i-
CI
|)\OI
Si ml
l)i-\
|)\OI
(111 Mil.
\< 1
|>\()l
Clin mii.
kl-Mlll
7-VCF-FLCON-B-
S04-3,000 TO
2,700
45
0
14.3
Pos.
45
0
14.6
Pos.
7-VCF-FLCON-B-
S04-3,000 Tf
30.7
0.1
30.4
0.1
8-VCF-CARPET-
A-S03-3,000 TO
2,700
45
0
20.2
Pos.
45
0
20.5
Pos.
8-VCF-CARPET-
A-S03-3,000 Tf
24.8
0.1
24.5
0.1
9-VCF-PAVEMT-
A-S01-30 TO
27
45
0
0
Neg.
45
0
0
Neg.
9-VCF-PAVEMT-
A-S01-30 Tf
45
0
45
0
10-VCF -FLDBLK-
A-S01-30 TO
27
45
0
0
Neg.
45
0
0
Neg.
10-VCF -FLDBLK-
A-S01-30 Tf
45
0
45
0
11-VCF-SCFILT-
B-S02-300 TO
270
45
0
0
Neg.
45
0
0
Neg.
11-VCF-SCFILT-
B-S02-300 Tf
45
0
45
0
12-VCF-PAVEMT-
A-S02-300 TO
270
45
0
0
Neg.
45
0
0
Neg.
12-VCF-PAVEMT-
A-S02-300 Tf
45
0
45
0
13-VCF-FLDBLK-
A-S02-300 TO
270
45
0
0
Neg.
43.8
2.1
-1.2
Neg.
13-VCF-FLDBLK-
A-S02-300 Tf
45
0
45
0
14-VCF-SCFILT-
B-S03-3000 TO
2,700
45
0
13.2
Pos.
44.4
1
13
Pos.
14-VCF-SCFILT-
B-S03-3000 Tf
31.8
0
31.4
0.1
15-VCF-PAVEMT-
A-S03-3000 TO
2,700
45
0
0.9
Neg.
45
0
5.3
Neg.*
15-VCF-PAVEMT-
A-S03-3000 Tf
44.1
1.5
39.7
2.3
16-VCF -FLDBLK-
A-S03-3000 TO
2,700
45
0
25.9
Pos.
44
1.8
24.8
Pos.
16-VCF -FLDBLK-
A-S03-3000 Tf
19.1
0
19.1
0
1-VCF-FLCON-B-
S05-30 TO
29
45
0
0
Neg.
45
0
0.7
Neg.
1-VCF-FLCON-B-
S05-30 Tf
45
0
44.3
1.2
2-VCF-FLCON-B-
S06-30 TO
29
45
0
0
Neg.
44.3
1.2
-0.7
Neg.
2-VCF-FLCON-B-
S06-30 Tf
45
0
45
0
3-VCF-STEPS-B-
S05-30 TO
29
45
0
0
Neg.
45
0
0
Neg.
3-VCF-STEPS-B-
S05-30 Tf
45
0
45
0
4-VCF-STEPS-B-
S06-30 TO
29
45
0
12.7
Pos.
45
0
12.8
Pos.
4-VCF-STEPS-B-
S06-30 Tf
32.3
0.1
32.2
0
-------
EPA/600/R-19/083
June 2019
S;mi|)k' II)
Spun-
l.oiid
(li io m.
CI
Clin ini.
Sliul
l)i-\
Chum.
\Cl
Clin mi.
Ri'sull
|>XOI
CI
|)\OI
Si ml
l)i-\
|)\OI
(111 Mil.
\< 1
|>\()l
Clin mii.
kl-Mlll
5-VCF-FLCON-B-
S07-300 TO
290
45
0
0
Neg.
45
0
0
Neg.
5-VCF-FLCON-B-
S07-300 Tf
45
0
45
0
6-VCF-FLCON-B-
S08-300 TO
290
45
0
17.9
Pos.
45
0
18
Pos.
6-VCF-FLCON-B-
S08-300 Tf
27.1
0
27
0
7-VCF-FLCON-B-
S09-3,000 TO
2,900
45
0
N/A*
N/A*
45
0
N/A*
N/A*
7-VCF-FLCON-B-
S09-3,000 Tf
N/A*
N/A*
N/A*
N/A*
8-VCF-FLCON-B-
S10-3,000 TO
2,900
45
0
16.9
Pos.
45
0
17
Pos.
8-VCF-FLCON-B-
S10-3,000 Tf
28.1
0
28
0
9-VCF-CARPET-
A-S04-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
9-VCF-CARPET-
A-S04-0 Tf
45
0
45
0
10-VCF-CARPET-
A-S05-30 TO
29
45
0
9.3
Pos.
45
0
9.2
Pos.
10-VCF-CARPET-
A-S05-30 Tf
35.7
0.2
35.8
0.3
11-VCF-CARPET-
A-S06-300 TO
290
45
0
14.2
Pos.
45
0
14.1
Pos.
11-VCF-CARPET-
A-S06-300 Tf
30.8
0.2
30.9
0.1
12-VCF-STEPS-B-
S07-300 TO
290
45
0
18.4
Pos.
45
0
18.4
Pos.
12-VCF-STEPS-B-
S07-300 Tf
26.6
0.1
26.6
0
13-VCF-STEPS-B-
S08-300 TO
290
45
0
11.8
Pos.
45
0
11.9
Pos.
13-VCF-STEPS-B-
S08-300 Tf
33.2
0.1
33.1
0.1
14-VCF-STEPS-B-
S09-3,000 TO
2,900
45
0
13.9
Pos.
45
0
14.1
Pos.
14-VCF-STEPS-B-
S09-3,000 Tf
31.1
0.1
30.9
0
15-VCF-STEPS-B-
S10-3,000 TO
2,900
45
0
17.8
Pos.
45
0
17.8
Pos.
15-VCF-STEPS-B-
S10-3,000 Tf
27.2
0.1
27.2
0
16-VCF-CARPET-
A-S07-3,000 TO
2,900
45
0
20.1
Pos.
45
0
20
Pos.
16-VCF-CARPET-
A-S07-3,000 Tf
24.9
0
25
0
1-VCF-SCFILT-B-
S04-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
1-VCF-SCFILT-B-
S04-0 Tf
45
0
45
0
2-VCF-SCFILT-B-
S05-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
2-VCF-SCFILT-B-
S05-0 Tf
45
0
45
0
-------
EPA/600/R-19/083
June 2019
S;mi|)k' II)
Spun-
l.o;i(l
Clin Mil.
Awr;i»i-
CI
Clin Mil.
Sliul
l)i\
Chum.
U I
Clin mi.
Ri'sull
|>XOI
Awr;i»i-
CI
|)\OI
Si ml
l)i-\
|)\OI
(111 Mil.
\< 1
|>\()l
Clin mii.
kl-Mlll
3-VCF-SCFILT-B-
S06-30 TO
32
45
0
0
Neg.
45
0
0
Neg.
3-VCF-SCFILT-B-
S06-30 Tf
45
0
45
0
4-VCF-SCFILT-B-
S07-30 TO
32
45
0
0
Neg.
45
0
0
Neg.
4-VCF-SCFILT-B-
S07-30 Tf
45
0
45
0
5-VCF-SCFILT-B-
S08-300 TO
320
45
0
0
Neg.
45
0
9.8
Neg.
5-VCF-SCFILT-B-
S08-300 Tf
45
0
35.2
0.3
6-VCF-SCFILT-B-
S09-300 TO
320
45
0
0
Neg.
45
0
0
Neg.
6-VCF-SCFILT-B-
S09-300 Tf
45
0
45
0
7-VCF-SCFILT-B-
S10-3,000 TO
3,200
45
0
2.7
Neg.
45
0
7.9
Neg.
7-VCF-SCFILT-B-
S10-3,000 Tf
42.3
4.8
37.1
0.5
8-VCF-SCFILT-B-
S11-3,000 TO
3,200
45
0
1.1
Neg.
45
0
0
Neg.
8-VCF-SCFILT-B-
S11-3,000 Tf
43.9
1.9
45
0
9-VCF-SWCON-A-
S05-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
9-VCF-SWCON-A-
S05-0 Tf
45
0
45
0
10-VCF-SWCON-
A-S06-30 TO
32
45
0
0
Neg.
45
0
0
Neg.
10-VCF-SWCON-
A-S06-30 Tf
45
0
45
0
11-VCF-SWCON-
A-S07-30 TO
32
45
0
0
Neg.
45
0
0
Neg.
11-VCF-SWCON-
A-S07-30 Tf
45
0
45
0
12-VCF-SWCON-
A-S08-300 TO
320
45
0
20.2
Pos.
45
0
20.1
Pos.
12-VCF-SWCON-
A-S08-300 Tf
24.8
0.1
24.9
0
13-VCF-SWCON-
A-S09-300 TO
320
45
0
0
Neg.
45
0
0
Neg.*
13-VCF-SWCON-
A-S09-300 Tf
45
0
45
0
14-VCF-SWCON-
A-S10-3,000 TO
3,200
45
0
20.3
Pos.
45
0
20.2
Pos.
14-VCF-SWCON-
A-S10-3,000 Tf
24.7
0.1
24.8
0
15-VCF-SWCON-
A-Sl 1-3,000 TO
3,200
45
0
17
Pos.
45
0
17.3
Pos.
15-VCF-SWCON-
A-Sl 1-3,000 Tf
28
0
27.7
0.1
16-VCF -LABBLK-
3,000 TO
3,200
45
0
0
Neg.
45
0
0
Neg.*
16-VCF -LABBLK-
3,000 Tf
45
0
45
0
-------
EPA/600/R-19/083
June 2019
S;mi|)k' II)
Spun-
l.oiid
Clin Mil.
Awr;i»i-
CI
Clin Mil.
Sliul
l)i\
Chum.
U I
Clin mi.
Ri'sull
|>XOI
Awr;i»i-
CI
|)\OI
Si ml
l)i-\
|)\OI
(111 Mil.
\< 1
|>\()l
Clin mii.
kl-Mlll
1-VCF-PAVEMT-
A-S04-0 TO
0
45
0
7.2
Neg.
45
0
5.7
Neg.
1-VCF-PAVEMT-
A-S04-0 Tf
37.8
0.1
39.3
2
2-VCF-PAVEMT-
A-S05-0 TO
0
45
0
5.1
Neg.
45
0
8
Neg.
2-VCF-PAVEMT-
A-S05-0 Tf
39.9
4.4
37
0.1
3-VCF-PAVEMT-
A-S06-30 TO
26
45
0
7.9
Neg.
45
0
8.7
Neg.
3-VCF-PAVEMT-
A-S06-30 Tf
37.1
0.9
36.3
0.5
4-VCF-PAVEMT-
A-S07-30 TO
26
45
0
13.8
Pos.
45
0
13.9
Pos.
4-VCF-PAVEMT-
A-S07-30 Tf
31.2
0.1
31.1
0
5-VCF-PAVEMT-
A-S08-300 TO
260
45
0
8
Neg.
45
0
7.7
Neg.
5-VCF-PAVEMT-
A-S08-300 Tf
37
0.8
37.3
0.5
6-VCF-PAVEMT-
A-S09-300 TO
260
45
0
2
Neg.
45
0
2.3
Neg.
6-VCF-PAVEMT-
A-S09-300 Tf
43
3.5
42.7
3.9
7-VCF-PAVEMT-
A-S10-3,000 TO
2,600
45
0
20.5
Pos.
45
0
20.6
Pos.
7-VCF-PAVEMT-
A-S10-3,000 Tf
24.5
0.1
24.4
0
8-VCF-PAVEMT-
A-Sl 1-3,000 TO
2,600
45
0
20.7
Pos.
45
0
20.8
Pos.
8-VCF-PAVEMT-
A-Sl 1-3,000 Tf
24.3
0.1
24.2
0
9-VCF-CARPET-
A-S08-0 TO
0
45
0
6.7
Neg.
45
0
6.6
Neg.
9-VCF-CARPET-
A-S08-0 Tf
38.3
0.6
38.4
1.3
10-VCF -FLDBLK-
A-S04-0 TO
0
45
0
7.7
Neg.
45
0
8.3
Neg.
10-VCF -FLDBLK-
A-S04-0 Tf
37.3
1.2
36.7
0.5
11-VCF-CARPET-
A-S09-30 TO
26
45
0
10.9
Pos.
45
0
10.9
Pos.
11-VCF-CARPET-
A-S09-30 Tf
34.1
0.2
34.1
0.2
12-VCF -FLDBLK-
A-S05-30 TO
26
45
0
5
Neg.
45
0
6.3
Neg.
12-VCF -FLDBLK-
A-S05-30 Tf
40
4.3
38.7
1
13-VCF-CARPET-
A-S10-300 TO
260
45
0
18.4
Pos.
44
1.8
17.5
Pos.
13-VCF-CARPET-
A-S10-300 Tf
26.6
0.1
26.5
0
14-VCF -FLDBLK-
A-S06-300 TO
260
45
0
0.6
Neg.
45
0
5.4
Neg.
14-VCF -FLDBLK-
A-S06-300 Tf
44.4
1
39.6
0.6
-------
EPA/600/R-19/083
June 2019
S;mi|)k' II)
Spun-
l.o;i(l
Clin Mil.
Awr;i»i-
CI
Clin Mil.
Sliul
l)i\
Chum.
U 1
Clin mi.
Ri'sull
|>XOI
Awr;i»i-
CI
|)\OI
Si ml
l)i-\
|)\OI
(111 Mil.
\< 1
|>\()l
Clin mii.
kl-Mlll
15-VCF-CARPET-
A-Sl 1-3,000 TO
2,600
45
0
12
Pos.
45
0
12.1
Pos.
15-VCF-CARPET-
A-Sl 1-3,000 Tf
33
0.1
32.9
0.1
16-VCF -FLDBLK-
A-S07-3,000 TO
2,600
45
0
26.5
Pos.
44.6
0.8
26.1
Pos.
16-VCF -FLDBLK-
A-S07-3,000 Tf
18.5
0
18.4
0
1-VCF-FLCON-B-
Sll-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
1-VCF-FLCON-B-
Sll-0 Tf
45
0
45
0
2-VCF-STEPS-B-
Sll-0 TO
0
45
0
0
Neg.
44.5
0.8
-0.5
Neg.
2-VCF-STEPS-B-
Sll-0 Tf
45
0
45
0
3-VCF-FLCON-B-
S12-30 TO
51
45
0
0
Neg.
45
0
0
Neg.
3-VCF-FLCON-B-
S12-30 Tf
45
0
45
0
4-VCF-STEPS-B-
S12-30 TO
51
45
0
0
Neg.
45
0
1.7
Neg.
4-VCF-STEPS-B-
S12-30 Tf
45
0
43.3
3
5-VCF-FLCON-B-
S13-300 TO
510
45
0
22.5
Pos.
45
0
23
Pos.
5-VCF-FLCON-B-
S13-300 Tf
22.5
0
22
0
6-VCF-STEPS-B-
S13-300 TO
510
45
0
18.7
Pos.
45
0
18.4
Pos.
6-VCF-STEPS-B-
S13-300 Tf
26.3
0
26.6
0
7-VCF-FLCON-B-
S14-3,000 TO
5,100
45
0
21.1
Pos.
45
0
21.4
Pos.
7-VCF-FLCON-B-
S14-3,000 Tf
23.9
0
23.6
0
8-VCF-STEPS-B-
S14-3,000 TO
5,100
45
0
21.6
Pos.
45
0
21.7
Pos.
8-VCF-STEPS-B-
S14-3,000 Tf
23.4
0.1
23.3
0.1
9-VCF-CARPET-
A-S12-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
9-VCF-CARPET-
A-S12-0 Tf
45
0
45
0
10-VCF-SCFILT-
B-S12-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
10-VCF-SCFILT-
B-S12-0 Tf
45
0
45
0
11-VCF-CARPET-
A-S13-30 TO
51
45
0
0
Neg.
45
0
0
Neg.
11-VCF-CARPET-
A-S13-30 Tf
45
0
45
0
12-VCF-SCFILT-
B-S13-30 TO
51
45
0
0
Neg.
45
0
0.7
Neg.
12-VCF-SCFILT-
B-S13-30 Tf
45
0
44.3
1.2
-------
EPA/600/R-19/083
June 2019
S;mi|)k' II)
Spun-
l.oiid
Clin Mil.
Awr;i»i-
CI
Clin Mil.
Sliul
l)i-\
Chum.
U I
Clin mi.
Ri'sull
|>XOI
Awr;i»i-
CI
|)\OI
Si ml
l)i-\
|)\OI
(111 Mil.
\< 1
|>\()l
Clin mii.
kl-Mlll
13-VCF-CARPET-
A-S14-300 TO
510
45
0
17.9
Pos.
45
0
18.2
Pos.
13-VCF-CARPET-
A-S14-300 Tf
27.1
0
26.8
0
14-VCF-SCFILT-
B-S14-300 TO
510
45
0
0
Neg.
45
0
0
Neg.
14-VCF-SCFILT-
B-S14-300 Tf
45
0
45
0
15-VCF-CARPET-
A-S15-3,000 TO
5,100
45
0
21
Pos.
45
0
21.1
Pos.
15-VCF-CARPET-
A-S15-3,000 Tf
24
0
23.9
0
16-VCF-SCFILT-
B-S15-3,000 TO
5,100
45
0
0
Neg.
44.4
1.1
-0.2
Neg.
16-VCF-SCFILT-
B-S15-3,000 Tf
45
0
44.5
0.8
1-VCF-FLDBLK-
A-S08-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
1-VCF-FLDBLK-
A-S08-0 Tf
45
0
45
0
2-VCF-FLDBLK-
A-S09-30 TO
30
45
0
0
Neg.
45
0
0
Neg.
2-VCF-FLDBLK-
A-S09-30 Tf
45
0
45
0
3 - VCF -PA VEMT -
A-S12-30 TO
30
45
0
0
Neg.
45
0
0
Neg.
3 - VCF -PA VEMT -
A-S12-30 Tf
45
0
45
0
4-VCF-CARPET-
A-S16-30 TO
30
45
0
0
Neg.
45
0
0
Neg.
4-VCF-CARPET-
A-S16-30 Tf
45
0
45
0
5-VCF-FLDBLK-
A-S10-300 TO
300
45
0
0
Neg.
45
0
0
Neg.
5-VCF-FLDBLK-
A-S10-300 Tf
45
0
45
0
6-VCF-PAVEMT-
A-S13-300 TO
300
45
0
7.4
Neg.
45
0
7.4
Neg.
6-VCF-PAVEMT-
A-S13-300 Tf
37.6
0.3
37.6
0.5
7-VCF-FLDBLK-
A-Sl 1-3,000 TO
3,000
45
0
0
Neg.
45
0
0
Neg.
7-VCF-FLDBLK-
A-Sl 1-3,000 Tf
45
0
45
0
8-VCF-PAVEMT-
A-S14-3,000 TO
3,000
45
0
14.1
Pos.
45
0
14.4
Pos.
8-VCF-PAVEMT-
A-S14-3,000 Tf
30.9
0
30.6
0
9-VCF-SCFILT-B-
S16-30 TO
30
45
0
0
Neg.
45
0
0
Neg.
9-VCF-SCFILT-B-
S16-30 Tf
45
0
45
0
10-VCF-SWCON-
A-S12-30 TO
30
45
0
2.4
Neg.
45
0
2.6
Neg.
10-VCF-SWCON-
A-S12-30 Tf
42.6
4.2
42.4
2.5
-------
EPA/600/R-19/083
June 2019
S;mi|)k' II)
Spun-
l.oiid
C'li ni ni.
Awr;i»i-
CI
Clin Mil.
Sliul
l)i-\
Chum.
U I
Clin mi.
Ri'sull
|>XOI
Awr;i»i-
CI
|)\OI
Si ml
l)i-\
|)\OI
(111 Mil.
\< 1
|>\()l
Clin mii.
kl-Mlll
11-VCF-CARPET-
A-S17-300 TO
300
45
0
17.1
Pos.
45
0
16.9
Pos.
11-VCF-CARPET-
A-S17-300 Tf
27.9
0.1
28.1
0
12-VCF-SCFILT-
B-S17-300 TO
300
45
0
0
Neg.
45
0
0
Neg.
12-VCF-SCFILT-
B-S17-300 Tf
45
0
45
0
13-VCF-SWCON-
A-S13-300 TO
300
45
0
18.8
Pos.
45
0
18.8
Pos.
13-VCF-SWCON-
A-S13-300 Tf
26.2
0.1
26.2
0
14-VCF-CARPET-
A-S18-3,000 TO
3,000
45
0
21.1
Pos.
45
0
21.1
Pos.
14-VCF-CARPET-
A-S18-3,000 Tf
23.9
0
23.9
0
15-VCF-SCFILT-
B-S18-3,000 TO
3,000
45
0
0
Neg.
45
0
0
Neg.
15-VCF-SCFILT-
B-S18-3,000 Tf
45
0
45
0
16-VCF-SWCON-
A-S14-3,000 TO
3,000
45
0
27.3
Pos.
45
0
27.6
Pos.
16-VCF-SWCON-
A-S14-3,000 Tf
17.7
0
17.4
0
1-VCF-FLCON-B-
S15-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
1-VCF-FLCON-B-
S15-0 Tf
45
0
45
0
2-VCF-STEPS-B-
S15-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
2-VCF-STEPS-B-
S15-0 Tf
45
0
45
0
3-VCF-FLCON-B-
S16-30 TO
29
45
0
2
Neg.
45
0
2
Neg.
3-VCF-FLCON-B-
S16-30 Tf
43
3.5
43
3.5
4-VCF-STEPS-B-
S16-30 TO
29
45
0
1.8
Neg.
45
0
0
Neg.
4-VCF-STEPS-B-
S16-30 Tf
43.2
3.1
45
0
5-VCF-FLCON-B-
S17-300 TO
290
45
0
18.8
Pos.
45
0
18.6
Pos.
5-VCF-FLCON-B-
S17-300 Tf
26.2
0
26.4
0
6-VCF-STEPS-B-
S17-300 TO
290
45
0
14.3
Pos.
45
0
14.8
Pos.
6-VCF-STEPS-B-
S17-300 Tf
30.7
0.1
30.2
0.2
7-VCF-FLCON-B-
S18-3,000 TO
2,900
45
0
21.3
Pos.
45
0
21.2
Pos.
7-VCF-FLCON-B-
S18-3,000 Tf
23.7
0.1
23.8
0
8-VCF-STEPS-B-
S18-3,000 TO
2,900
45
0
14
Pos.
45
0
14.1
Pos.
8-VCF-STEPS-B-
S18-3,000 Tf
31
0.1
30.9
0
-------
EPA/600/R-19/083
June 2019
S;mi|)k' II)
Spun-
l.oiid
Clin Mil.
Awr;i»i-
CI
Clin Mil.
Sliul
l)i\
Chum.
U I
Clin mi.
Ri'sull
|>XOI
Awr;i»i-
CI
|)\OI
Si ml
l)i-\
|)\OI
(111 Mil.
\< 1
|>\()l
Clin mii.
kl-Mlll
9-VCF-SWCON-A-
S15-0 TO
0
45
0
0
Neg.
45
0
2.8
Neg.
9-VCF-SWCON-A-
S15-0 Tf
45
0
42.2
2.5
10-VCF-PAVEMT-
A-S15-0 TO
0
45
0
2.3
Neg.
45
0
1.3
Neg.
10-VCF-PAVEMT-
A-S15-0 Tf
42.7
4.1
43.7
2.2
11-VCF-SWCON-
A-S16-30 TO
29
45
0
0
Neg.
45
0
2.2
Neg.
11-VCF-SWCON-
A-S16-30 Tf
45
0
42.8
2.1
12-VCF-PAVEMT-
A-S16-30 TO
29
45
0
0
Neg.
45
0
3.7
Neg.
12-VCF-PAVEMT-
A-S16-30 Tf
45
0
41.3
3.3
13-VCF-SWCON-
A-S17-300 TO
290
45
0
22.9
Pos.
45
0
23.1
Pos.
13-VCF-SWCON-
A-S17-300 Tf
22.1
0.1
21.9
0
14-VCF-PAVEMT-
A-S17-300 TO
290
45
0
4
Neg.
45
0
7.8
Neg.
14-VCF-PAVEMT-
A-S17-300 Tf
41
3.5
37.2
0.3
15-VCF-SWCON-
A-S18-3,000 TO
2,900
45
0
29.1
Pos.
45
0
29.3
Pos.
15-VCF-SWCON-
A-S18-3,000 Tf
15.9
0
15.7
0
16-VCF-PAVEMT-
A-S18-3,000 TO
2,900
45
0
10.6
Pos.
45
0
11.1
Pos.
16-VCF-PAVEMT-
A-S18-3,000 Tf
34.4
0.3
33.9
0.2
1-VCF-FLDBLK-
A-S12-0 TO
0
45
0
2.4
Neg.
45
0
4.4
Neg.
1-VCF-FLDBLK-
A-S12-0 Tf
42.6
4.2
40.6
3.8
2-VCF-FLDBLK-
A-S13-30 TO
19
45
0
0
Neg.
45
0
0
Neg.
2-VCF-FLDBLK-
A-S13-30 Tf
45
0
45
0
3-VCF-FLDBLK-
A-S14-30 TO
19
45
0
0
Neg.
41.8
2.8
-1.5
Neg.
3-VCF-FLDBLK-
A-S14-30 Tf
45
0
43.3
2.9
4-VCF-FLDBLK-
A-S15-300 TO
190
45
0
0
Neg.
45
0
0
Neg.
4-VCF-FLDBLK-
A-S15-300 Tf
45
0
45
0
5-VCF-FLDBLK-
A-S16-300 TO
190
45
0
0
Neg.
45
0
0
Neg.
5-VCF-FLDBLK-
A-S16-300 Tf
45
0
45
0
6-VCF-FLDBLK-
A-S17-3000 TO
1,900
45
0
24.2
Pos.
45
0
24.1
Pos.
6-VCF-FLDBLK-
A-S17-3000 Tf
20.8
0
20.9
0
-------
EPA/600/R-19/083
June 2019
S;mi|)k' II)
Spun-
l.oiid
(li io m.
C 1
Clin ini.
Sliul
l)i\
Clmm.
U I
Clin Mil.
Ri'sull
p\OI
(1
|)\OI
St ml
l)i-\
|)\OI
(111 Mil.
\< 1
p\()l
Clin mii.
kl-Mlll
7-VCF-FLDBLK-
A-S18-3000 TO
1,900
45
0
24.1
Pos.
43.3
3
22.3
Pos.
7-VCF-FLDBLK-
A-S18-3000 Tf
20.9
0
21
0
-------
EPA/600/R-19/083
June 2019
APPENDIX S: TSB ENRICHMENT PCR RESULTS
FOR SPONGE-STICK SAMPLES
-------
EPA/600/R-19/083
June 2019
Siiinplo II)
Chromosome Ass;i\
p.XOI Asssij
Trial
A\0r;il»0
(I
Sid l)o\
Kosu II
A\emtio
<1
SKI l)o\
Kosu II
2-SPG-STGRAT-A-S15-0*
45.0
0.0
Neg
41.4
3.1
Pos
22
4-SPG-STGRAT-A-S16-30*
38.6
5.6
Pos
38.4
5.8
Pos
22
6-SPG-STGRAT-A-S17-300*
40.3
4.1
Pos
38.4
1.1
Pos
22
8-SPG-STGRAT-A-S18-3000*
36.8
0.8
Pos
36.5
0.7
Pos
22
10 - SPG-C WPNTD - A-S16-30
45.0
0.0
Neg
45.0
0.0
Neg
21
1 -SPG-FLDBLK-A-S15-0*
44.7
0.5
Neg
38.5
0.4
Pos
22
2-SPG-MCMACH-B -S15 -0
45.0
0.0
Neg
45.0
0.0
Neg
21
4-SPG-MCMACH-B-S16-30
45.0
0.0
Neg
45.0
0.0
Neg
21
1-SPG-WLTILE-B-S15-0
45.0
0.0
Neg
45.0
0.0
Neg
21
10-SPG-WLTILE-B-S19-0
45.5
0.0
Neg
44.6
0.7
Neg
22
9-SPG-SCGRIL-B-S13-0
45.0
0.0
Neg
45.0
0.0
Neg
20
13-SPG-SCGRIL-B-S15-30
45.0
0.0
Neg
45.0
0.0
Neg
20
2-SPG-STEPS-B-S15-0
45.0
0.0
Neg
45.0
0.0
Neg
20
6-SPG-STEPS-B-S17-300
45.0
0.0
Neg
45.0
0.0
Neg
20
1 - SPG-FL CON -B - S15 -0
45.0
0.0
Neg
45.0
0.0
Neg
20
3-SPG-FLCON-B-S16-30
45.0
0.0
Neg
45.0
0.0
Neg
20
10-SPG-LABBLANK-0
45.0
0.0
Neg
45.0
0.0
Neg
20
9-SPG-GRNBEN-A-S15-0
45.0
0.0
Neg
40.8
3.7
Pos
22
9-SPG-TELEBO-A-S12-0
45.0
0.0
Neg
45.0
0.0
Neg
19
10-SPG-TELEBO-A-S13-30
45.0
0.0
Neg
45.0
0.0
Neg
19
1 -SPG-CW SIGN-A-S13-0
45.0
0.0
Neg
45.0
0.0
Neg
19
*Note, these enrichment samples PCR analyzed the week of December 3, 2018. The Tfinal 0 spike
samples also had Ct in of ~ 37, therefore contamination may have caused these positive results.
-------
EPA/600/R-19/083
June 2019
APPENDIX T: TSB ENRICHMENT PCR RESULTS
FOR VACUUM FILTER CASSETTES
-------
EPA/600/R-19/083
June 2019
Carpet VCF Samples
Siiinplo II)
Chromosome Ass;i\
p\()l Asssij
Trial
A\crii*io
(1
Sid l)e\
Kosu II
A\cr;i»c
<1
SKI l)c\
Kcsull
'Mci-c \ki,i:r-\-S()4-u
45 u
00
Vg.
45 u
on
Vg.
3
9-VCF-CARPET-A-S08-0
45.0
0.0
Neg.
45.0
0.0
Neg.
5
9 -V CF-CARPET - A- S12 -0
45.0
0.0
Neg.
45.0
0.0
Neg.
6
3 - VCF-C ARPET-A-SO1-30
36.6
0.7
Pos.
35.6
0.5
Pos.
2
10 -V CF-CARPET -A-S05-30
36.2
0.6
Pos.
36.2
0.8
Pos.
3
11-VCF-CARPET-A-S09-30
45.0
0.0
Neg.
40.4
4.0
Pos.
5
11 -VCF-C ARPET-A-S13-30
41.0
3.7
Pos.
37.7
0.2
Pos.
6
4 -V CF-CARPET -A-S16-30
38
0.8
Pos.
38.4
0.8
Pos.
7
6-VCF-CARPET-A-S02-300
42.9
3.7
Neg.
40.0
4.3
Pos.
2
11-VCF-CARPET-A-S06-300
34.7
0.2
Pos.
34.1
0.4
Pos.
3
13-VCF-CARPET-A-S10-300
32.8
0.0
Pos.
32.6
0.1
Pos.
5
13-VCF-CARPET-A-S14-300
37.0
1.2
Pos.
35.6
0.6
Pos.
6
11-VCF-CARPET-A-S17-300
36.4
0.6
Pos.
35.8
0.1
Pos.
7
8-VCF-CARPET-A-S03 -3,000
34.9
0.1
Pos.
34.1
0.3
Pos.
2
16-VCF-CARPET-A-S07-3,000
31.4
0.3
Pos.
30.8
0.1
Pos.
3
15 - V CF-CARPET - A- S11 -3,000
32.1
0.1
Pos.
31.7
0.1
Pos.
5
15 - VCF-CARPET-A-S 15-3,000
32.9
0.1
Pos.
32.7
0.1
Pos.
6
14-VCF-CARPET-A-S18-3,000
32.1
0.1
Pos.
31.9
0.1
Pos.
7
Subway Car Filter VCF Samples
Siiiliplc II)
ChromosoiiK* Ass;i\
n\()l Ass;i\
Iriiil
A\cr;iiie
(1
Sid l)e\
Result
A\crsi}*c
(1
SKI l)c\
Kosu II
1-VCF-SCFILT-B-S04-0
41.5
3.0
Pos.
35.4
0.3
Pos.
4
2-VCF-SCFILT-B-S05-0
45.0
0.0
Neg.
45.0
0.0
Neg.
4
10-VCF-SCFILT-B-S12-0
45.0
0.0
Neg.
45.0
0.0
Neg.
6
4 -V CF - S CFILT -B-S01-30
45.0
0.0
Neg.
45.0
0.0
Neg.
2
3 -V CF - S CFILT -B-S06-30
42.7
3.9
Neg.
33.7
0.2
Pos.
4
4 -V CF - S CFILT -B-S07-30
42.3
2.5
Neg.
35.7
0.4
Pos.
4
12-VCF-SCFILT-B-S13-30
44.1
1.5
Neg.
41.1
3.4
Pos.
6
9-VCF-SCFILT-B-S16-30
45
0
Neg.
45
0
Neg.
7
11-VCF-SCFILT-B-S02-300
36.9
0.8
Pos.
36.3
0.4
Pos.
2
5-VCF-SCFILT-B-S08-300
45.0
0.0
Neg.
45.0
0.0
Neg.
4
6-VCF-SCFILT-B-S09-300
45.0
0.0
Neg.
44.0
1.8
Neg.
4
14-VCF-SCFILT-B-S14-300
45.0
0.0
Neg.
45.0
0.0
Neg.
6
12-VCF-SCFILT-B-S17-300
41
3.5
Pos.
40.7
1.6
Neg.
7
14-VCF-SCFILT-B-S03-3000
35.2
0.3
Pos.
34.7
0.1
Pos.
2
7-VCF-SCFILT-B-S10-3,000
45.0
0.0
Neg.
45.0
0.0
Neg.
4
8-VCF-SCFILT-B-S11-3,000
43.9
1.8
Neg.
39.5
4.8
Pos.
4
16-VCF-SCFILT-B-S15-3,000
41.2
3.4
Pos.
40.8
3.7
Pos.
6
15-VCF-SCFILT-B-S18-3,000
42.4
4.4
Neg.
36.8
0.3
Pos.
7
-------
EPA/600/R-19/083
June 2019
Sidewalk Concrete VCFSamples
Siimpk' II)
Chromosome Ass;i\
|)\OI Ass;i\
Trial
A\er;ijie
(I
Sid l)e\
Kosu II
A\er;iiie
(1
SKI
l)e\
Kosull
lM CI '-S\VC( )\- \-Su5-u
45 u
0 0
Vg.
45 u
00
Vg.
4
9-VCF-SWCON-A-S15-0
45.0
0.0
Neg.
45.0
0.0
Neg.
8
10-VCF-SWCON-A-S06-30
36.4
1.1
Pos.
35.9
0.7
Pos.
4
11-VCF-SWCON-A-S07-30
36.5
1.6
Pos.
36.2
0.5
Pos.
4
10-VCF-SWCON-A-S12-30
35.0
2.3
Pos.
35.6
2.7
Pos.
7
11-VCF-SWCON-A-S16-30
36.1
0.7
Pos.
36.1
0.2
Pos.
8
12-VCF-SWCON-A-S08-300
32.7
0.4
Pos.
32.5
0.4
Pos.
4
13-VCF-SWCON-A-S09-300
29.2
0.9
Pos.
28.9
0.9
Pos.
4
13-VCF-SWCON-A-S13-300
31.2
0.1
Pos.
30.8
0.1
Pos.
7
13-VCF-SWCON-A-S17-300
33.0
1.2
Pos.
33.1
1.4
Pos.
8
14-VCF-SWCON-A-S 10-3,000
26.2
0.1
Pos.
25.9
0.1
Pos.
4
15 -VCF-S WCON-A-S 11-3,000
30.0
0.2
Pos.
30.0
0.2
Pos.
4
16-VCF-SWCON-A-S14-3,000
29.4
0.1
Pos.
29.7
0.1
Pos.
7
15-VCF-S WCON-A-S18-3,000
28.6
0.3
Pos.
28.7
0.3
Pos.
8
(a) Sample 14 was analyzed held at 2 - 8 C for 1 week before being analyzed
Pavement VCF Samples
Siimpk* II)
Chromosome Ass;i\
pXOI Asssij
Trial
A\cr;i*io
(1
Siml
l)c\
Result
A\cr;i»c
(1
Sinri
l)e\
Ki'sull
1-VCF-PAVEMT-A-S04-0
42.5
4.4
Neg.
45.0
0.0
Neg.
5
2-VCF-PAVEMT-A-S05-0
45.0
0.0
Neg.
43.0
3.5
Neg.
5
10-VCF-PAVEMT-A-S15-0
45
0
Neg.
45
0
Neg.
8
9-VCF-PAVEMT-A-S01-30
36.8
0.3
Pos.
36.0
0.2
Pos.
2
3-VCF-PAVEMT-A-S06-30
43.0
3.5
Neg.
42.7
2.9
Neg.
5
4-VCF-PAVEMT-A-S07-30
35.6
0.3
Pos.
35.0
0.2
Pos.
5
3-VCF-PAVEMT-A-S12-30
39
0.5
Pos.
39.2
0.6
Pos.
7
12-VCF-PAVEMT-A-S16-30
33
0.2
Pos.
32.9
0
Pos.
8
12-VCF-PAVEMT-A-S02-300
32.1
0.7
Pos.
32.0
0.7
Pos.
2
5-VCF-PAVEMT-A-S08-300
33.4
0.4
Pos.
32.7
0.4
Pos.
5
6-VCF-PAVEMT-A-S09-300
36.9
1.5
Pos.
36.6
0.3
Pos.
5
6-VCF-P AVEMT-A-S13 -300
33.7
0.6
Pos.
33.8
0.4
Pos.
7
14-VCF-PAVEMT-A-S17-300
30.6
0.4
Pos.
30.8
0.4
Pos.
8
15-VCF-PAVEMT-A-S03-3000
30.9
0.4
Pos.
31.0
0.4
Pos.
2
7-VCF-PAVEMT-A-S10-3,000
30.7
0.1
Pos.
30.5
0.0
Pos.
5
8-VCF-PAVEMT-A-S11-3,000
29.5
0.2
Pos.
29.1
0.2
Pos.
5
8-VCF-PAVEMT-A-S14-3,000
29.3
0.1
Pos.
29
0
Pos.
7
16-VCF-PAVEMT-A-S18-3,000
28.2
0.1
Pos.
28.1
0.2
Pos.
8
-------
EPA/600/R-19/083
June 2019
Field Blank VCF Samples
Siiinplo II)
C hromosome Ass;i\
p\()l Asssij
Trial
A\or«i}»o
(I
Sid l)o\
Kosu II
A\Ol'illiO
(1
SKl l)o\
Kosu II
|u-\CI'-l'l.l)i:i.K-\-S()4-u
45 u
00
Vg.
44 X
<14
Vg.
5
1-VCF-FLDBLK-A-S08-0
45.0
0.0
Neg.
45.0
0.0
Neg.
7
1-VCF-FLDBLK-A-S12-0
45.0
0.0
Neg.
45.0
0.0
Neg.
9
12-VCF-FLDBLK-A-S05-30
22.4
0.8
Pos.
22.4
1.0
Pos.
5
10-VCF-FLDBLK-A-S01 -30
24.2
0.1
Pos.
24.2
0.1
Pos.
2
2-VCF-FLDBLK-A-S09-30
25.7
0.4
Pos.
25.6
0.4
Pos.
7
2 -V CF -FLDBLK-A- S13-30
27.9
0.0
Pos.
28.1
0.0
Pos.
9
3-VCF-FLDBLK-A-S14-30
26.5
0.1
Pos.
26.8
0.1
Pos.
9
5-VCF-FLDBLK-A-S10-300
21.3
0.4
Pos.
21.3
0.5
Pos.
7
13-VCF-FLDBLK-A-S02-300
20.1
0.1
Pos.
20.1
0.1
Pos.
2
14-VCF-FLDBLK-A-S06-300
25.2
0.4
Pos.
25.4
0.5
Pos.
5
4-VCF-FLDBLK-A-S15-300
26.1
0.3
Pos.
26.2
0.3
Pos.
9
5-VCF-FLDBLK-A-S 16-300
26.1
0.2
Pos.
26.3
0.4
Pos.
9
16-VCF-FLDBLK-A-S03-3000
21.1
1.3
Pos.
21.1
1.4
Pos.
2
16-VCF-FLDBLK-A-S07-3,000
21.3
0.1
Pos.
21.4
0.1
Pos.
5
7-VCF-FLDBLK-A-S11-3,000
21.9
0.2
Pos.
21.9
0.3
Pos.
7
6-VCF-FLDBLK-A-S 17-3000
26.1
0.1
Pos.
26.3
0.1
Pos.
9
7-VCF-FLDBLK-A-S18-3000
25.6
0.1
Pos.
25.8
0.2
Pos.
9
------- |