EPA/600/R-19/082 | August 2019
www.epa.gov/homeland-security-research
United States
Environmental Protection
Agency
&EPA
Sample Analysis of Native Air Filters
for Characterization and Extent
Mapping of Biological Incidents
Office of Research and Development
Homeland Security Research Program
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SAMPLE ANALYSIS OF NATIVE AIR FILTERS FOR
CHARACTERIZATION AND EXTENT MAPPING OF
BIOLOGICAL INCIDENTS
U.S. Environmental Protection Agency
Cincinnati, OH 45268
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Disclaimer
The United States 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
United States Environmental 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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TABLE OF CONTENTS
Page
EXECUTIVE SUMMARY xi
1.0 INTRODUCTION 1
1.1 Background 1
1.2 Objective 2
1.3 Scope 2
2.0 MATERIALS AND METHODS 4
2.1 Filter Categories and Selection 4
2.1.1 AQ Filters 4
2.1.2 Non-AQ Filters 5
2.2 Test Matrix 6
2.3 Microbiological Methods 9
2.3.1 Spore Bank 10
2.3.2 Spore Loading (Spiking) 11
2.3.3 Spore Recovery 12
2.3.4 Culture Method 13
2.3.5 RV-PCRMethod 14
2.4 Method Implementation 18
2.5 Data Reduction and Analysis 19
2.5.1 Culture - Percent Recovery 19
2.5.2 RV-PCR 20
2.5.3 Presentation of Results 20
3.0 RESULTS AND DISCUSSION 22
3.1 AQ Filter Analyses Results 22
3.1.1 Culture Method 22
3.1.2 RV-PCR Method 34
3.2 Non-AQ Filter Analyses Results 43
3.2.1 Culture Method 43
3.2.2 RV-PCR Method 49
3.3 Summary of Detection Accuracy 55
3.4 Ancillary Results 58
3.4.1 PES vs PVDF Membrane Filter Vials 58
4.0 Quality Assurance/Quality Control 60
4.1 Equipment Calibration 60
4.2 QC Results 60
4.3 Operational Parameters 60
4.4 Audits 61
4.4.1 Performance Evaluation Audit 61
4.4.2 Technical Systems Audit 61
4.4.3 Data Quality Audit 61
4.5 QA/QC Reporting 62
4.6 Data Review 62
5.0 SUMMARY OF METHOD OBSERVATIONS AND EXPERIENCES 63
6.0 CONCLUSIONS AND RECOMMENDATIONS 65
7.0 REFERENCES 67
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LIST OF TABLES
Page
Table 1. Test Matrix for AQ Filters 8
Table 2. Test Matrix for Non-AQ Filters 9
Table 3. Target B. a. Sterne Spore Loading Levels onto Each Filter Substrate 11
Table 4. Recovery Efficiencies for Presumptive B. a. Sterne Spores from PM2.5 Air Quality
Filters Cultured in the SB A Medium 23
Table 5. Recovery Efficiencies for Presumptive B. a. Sterne Spores from PM10 Air Quality
Filters Cultured in the SB A Medium 24
Table 6. RV-PCR Analyses of PM2.5 Air Quality Filters for Detection of B. a. Sterne
Spores Using Chromosomal and pXOl Gene Targets (N > 3 Replicates) 37
Table 7. RV-PCR Analyses of PM10 Air Quality Filters for Detection of B. a. Sterne Spores
Using Chromosomal and pXOl Gene Targets (N > 3 Replicates) 38
Table 8. Recovery Efficiencies for Presumptive B. a. Sterne Spores from Non-Air Quality
Filters Cultured in the SBA Medium (N > 3 Replicates) 44
Table 9. RV-PCR Analyses of Non-Air Quality Filters for Detection of B. a. Sterne Spores
Using Chromosomal and pXOl Gene Targets (N > 3 Replicates) 50
Table 10. Summary of the Accuracy of the Method Response to Detect B. a. Sterne 56
Table 11. Positive and Negative B. a. Sterne Detection Frequency for Culture and Molecular
Analysis Methods 57
Table 12. Test Matrix for Comparing PES and PVDF Membranes 58
Table 13. Performance Evaluation Audits 61
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LIST OF FIGURES
Page
Figure 1. Air Quality Filter Types: PM2.5 (Left: High and Avg Particulate Loads) and PM10
(Right: New Media) 5
Figure 2. Non-AQ Filter Types: Bus Filter (Top Left); Building HVAC Filter (Top Right);
Subway Platform Filter (Bottom Left); and Subway Rolling Stock Filter (Bottom
Right) 6
Figure 3. Photographs of Metro Bus Engine Filter (left) and PM10 Air Quality Filter (right)
After Spiking with the B. a. Sterne Suspension 12
Figure 4. From left to right: B. a. Sterne on SBA, MYP, and BBCA 14
Figure 5. Top: Manifold Containing 16 Filter Vials; Middle: Capping Tray; Bottom: Capped
Filter Vials Containing RHIR 15
Figure 6. Process Flow Chart Depicting Key Method Process Steps in Chronological Order 18
Figure 7. Percent Recovery Efficiencies (Average ± One Standard Deviation of N > 3
Replicates) of Presumptive B. a. Sterne Spores from PM2.5 Filters from Arizona
Using the SBA Medium (New, Avg, and High refer to relative ambient particulate
loads) 26
Figure 8. Percent Recovery Efficiencies (Average ± One Standard Deviation of N > 3
Replicates) of Presumptive B. a. Sterne Spores from PM2.5 Filters from Florida
Using the SBA Medium (New, Avg, and High refer to relative ambient particulate
load) 26
Figure 9. Percent Recovery Efficiencies (Average ± One Standard Deviation of N > 3
Replicates) of Presumptive B. a. Sterne Spores from PM2.5 Filters from
Massachusetts Using the SBA Medium (New, Avg, and High refer to relative
ambient particulate loads) 27
Figure 10. Percent Recovery Efficiencies (Average ± One Standard Deviation of N > 3
Replicates) of Presumptive B. a. Sterne Spores from PM2.5 Filters from Wisconsin
Using the SBA Medium (New, Avg, and High refer to relative ambient particulate
loads) 27
Figure 11. Percent Recovery Efficiencies (Average ± One Standard Deviation of N > 3
Replicates) of Presumptive B. a. Sterne Spores from PM10 Filters from California
Using the SBA Medium (New, Avg, and High refer to relative ambient particulate
loads) 29
Figure 12. Percent Recovery Efficiencies (Average ± One Standard Deviation of N > 3
Replicates) of Presumptive B. a. Sterne Spores from PM10 Filters from New
Hampshire Using the SBA Medium (New, Avg, and High refer to relative ambient
particulate loads) 30
Figure 13 Percent Recovery Efficiencies (Average ± One Standard Deviation of N > 3 Three
Replicates) of Presumptive B. a. Sterne Spores from PM10 Filters from South
Carolina Using the SBA Medium (New, Avg, and High refer to relative ambient
particulate loads) 30
Figure 14 Percent Recovery Efficiencies (Average ± One Standard Deviation of N > 3
Replicates) of Presumptive B. a. Sterne Spores from PM10 Filters from Wisconsin
Using the SBA Medium (New, Avg, and High refer to relative ambient particulate
loads) 31
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LIST OF FIGURES (CONT.)
Page
Figure 15. Colonies Recovered from PM10 California Filters Contained Background with B.
a. Sterne Morphology (the colony on the left was confirmed negative and the
colony on the right was confirmed positive for B. a. Sterne) 34
Figure 16. RV-PCR Analysis of B. a. Sterne Spores Recovered from PM2.5 Filters from
Arizona Using Chromosomal and pXOl Gene Targets (Average ± One Standard
Deviation for N > 3 Replicates); Positive Response Equals ACt > 9 (New, Avg, and
High refer to relative ambient particulate loads) 39
Figure 17. RV-PCR Analysis of B. a. Sterne Spores Recovered from PM2.5 Filters from
Florida Using Chromosomal and pXOl Gene Targets (Average ± One Standard
Deviation for N > 3 Replicates); Positive Response Equals ACt > 9 (New, Avg, and
High refer to relative ambient particulate loads) 39
Figure 18. RV-PCR Analysis of B. a. Sterne Spores Recovered from PM2.5 Filters from
Massachusetts Using Chromosomal and pXOl Gene Targets (Average ± One
Standard Deviation for N > 3 Replicates); Positive Response Equals ACt > 9 (New,
Avg, and High refer to relative ambient particulate loads) 40
Figure 19. RV-PCR Analysis of B. a. Sterne Spores Recovered from PM2.5 Filters from
Wisconsin Using Chromosomal and pXOl Gene Targets (Average ± One Standard
Deviation for N > 3 Replicates); Positive Response Equals ACt > 9 (New, Avg, and
High refer to relative ambient particulate loads) 40
Figure 20. RV-PCR Analysis of B. a. Sterne Spores Recovered from PM10 Filters from
California Using Chromosomal and pXOl Gene Targets (Average ± One Standard
Deviation for N > 3 Replicates); Positive Response Equals ACt > 9 (New, Avg, and
High refer to relative ambient particulate loads) 41
Figure 21. RV-PCR Analysis of B. a. Sterne Spores Recovered from PM10 Filters from
New Hampshire Using Chromosomal and pXOl Gene Targets (Average ± One
Standard Deviation for N > 3 Replicates); Positive Response Equals ACt > 9 (New,
Avg, and High refer to relative ambient particulate loads) 41
Figure 22. RV-PCR Analysis of B. a. Sterne Spores Recovered from PM10 Filters from
South Carolina Using Chromosomal and pXOl Gene Targets (Average ± One
Standard Deviation for N > 3 Replicates); Positive Response Equals ACt > 9 (New,
Avg, and High refer to relative ambient particulate loads) 42
Figure 23. RV-PCR Analysis of B. a. Sterne Spores Recovered from PM10 Filters from
Wisconsin Using Chromosomal and pXOl Gene Targets (Average ± One Standard
Deviation for N > 3 Replicates); Positive Response Equals ACt > 9 (New, Avg, and
High refer to relative ambient particulate loads) 42
Figure 24. Subway Rolling Stock End-of-Service Filter Spiked with 30, 300, or 3,000
Spores (from left to right, respectively) 46
Figure 25. Percent Recovery Efficiencies (Average ± One Standard Deviation of N > 3
Replicates) of Presumptive B. a. Sterne Spores from Bus Engine Filters Using the
SBA Medium (New, Mid, and End refer to service life or duty cycle of the filter) 46
Figure 26. Percent Recovery Efficiencies (Average ± One Standard Deviation of N > 3
Replicates) of Presumptive B. a. Sterne Spores from Building HVAC Filters Using
the SBA Medium (New, Mid, and End refer to service life or duty cycle of the
filter) 47
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LIST OF FIGURES (CONT.)
Page
Figure 27. Percent Recovery Efficiencies (Average ± One Standard Deviation of N > 3
Replicates) of Presumptive B. a. Sterne Spores from Subway Platform Filters Using
the SBA Medium (New, Mid, and End refer to service life or duty cycle of the
filter) 47
Figure 28. Percent Recovery Efficiencies (Average ± One Standard Deviation of N > 3
Replicates) of Presumptive B. a. Sterne Spores from Subway Rolling Stock Filters
Using the SBA Medium (New, Mid, and End refer to service life or duty cycle of
the filter) 48
Figure 29. Bus Engine End-of-Service Life Filter Deconstructed Prior to Spore Recovery
(left) and Suspension After Recovered from Bus Engine Filters New (unused) in
Top Row and End-of-Service Life in Bottom Row of RV-PCR Vacuum Manifold 48
Figure 30. RV-PCR Analysis of B. a. Sterne Spores Recovered from Bus Engine Filters
Using Chromosomal and pXOl Gene Targets (Average ± One Standard Deviation
for N > 3 Replicates); Positive Response Equals ACt > 9 (New, Mid, and End refer
to service life or duty cycle of the filter) 53
Figure 31. RV-PCR Analysis of B. a. Sterne Spores Recovered from Building HVAC Filters
Using Chromosomal and pXOl Gene Targets (Average ± One Standard Deviation
for N > 3 Replicates); Positive Response Equals ACt > 9 (New, Mid, and End refer
to service life or duty cycle of the filter) 53
Figure 32. RV-PCR Analysis of B. a. Sterne Spores Recovered from Subway Platform
Filters Using Chromosomal and pXOl Gene Targets (Average ± One Standard
Deviation for N > 3 Replicates); Positive Response Equals ACt > 9 (New, Mid, and
End refer to service life or duty cycle of the filter) 54
Figure 33. RV-PCR Analysis of B. a. Sterne Spores Recovered from Subway Rolling Stock
Filters Using Chromosomal and pXOl Gene Targets (Average ± One Standard
Deviation for N > 3 Replicates); Positive Response Equals ACt > 9 (New, Mid, and
End refer to service life or duty cycle of the filter) 54
Figure 34. Side-by-Side Analysis of ACt Values Generated During the RV-PCR using PES
and PVDF Filter Vials 59
Figure 35. Side-by-Side Analysis of Final Ct Values Generated During the RV-PCR using
PES and PVDF Filter Vials 59
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LIST OF APPENDICES
Page
APPENDIX A. FORMULATIONS OF RECIPES USED IN BIOLOGICAL TEST
METHODS A-l
APPENDIX B. DUPLEX VERSUS SINGLEPLEX REAL-TIME PCR SPOT REPORT B-l
APPENDIX C. WORK INSTRUCTION FOR DOSING FILTER SWATCHES WITH
BACILLUS ANTHRACIS SPORES C-l
APPENDIX D. WORK INSTRUCTION FOR BACILLUS ANTHRACIS SPORE
RECOVERY D-l
APPENDIX E. WORK INSTRUCTION FOR CULTURE OF BACILLUS
ANTHRACIS SPORES RECOVERED FROM AIR FILTERS E-l
APPENDIX F. WORK INSTRUCTION FOR MANUAL DNA EXTRACTION AND
PURIFICATION FROM BACILLUS ANTHRACIS SPORES F-l
APPENDIX G. WORK INSTRUCTION DRV-PCR IO R BA CI 1.1. IS A \ 11 IRA CIS
SPORES G-l
APPENDIX H. WORK INSTRUCTION FOR SELECTING PRESUMPTIVE
BACILLUS ANTHRACIS STERNE COLONIES FOR QPCR
CONFIRMATION H-l
APPENDIX I. CULTURE RESULTS FOR AIR QUALITY FILTERS USING SHEEP
BLOOD AGAR MEDIUM 1-1
APPENDIX J. CULTURE RESULTS FOR AIR QUALITY FILTERS USING MYP
MEDIUM J-l
APPENDIX K CULTURE RESULTS FOR AIR QUALITY FILTERS USING BBCA
MEDIUM K-l
APPENDIX L. RV-PCR RESULTS FOR AIR QUALITY FILTERS USING
CHROMOSOMAL AND PXOl GENE TARGETS L-l
APPENDIX M. CULTURE RESULTS FOR NON-AIR QUALITY FILTERS USING
SHEEP BLOOD AGAR MEDIUM M-l
APPENDIX N. CULTURE RESULTS FOR NON-AIR QUALITY FILTERS USING
MYP MEDIUM N-l
APPENDIX O. RV-PCR RESULTS FOR NON-AIR QUALITY FILTERS USING
CHROMOSOMAL AND PXOl GENE TARGETS O-l
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Abbreviations and Acronyms
Acronym
Definition
AQ
Air Quality
Avg
Filters selected that had relatively moderate (average) particulate loadings for the site
B. a. Sterne
Bacillus anthracis Sterne
B. anthracis
Bacillus anthracis
BBCA
Brilliance Bacillus cereus Agar
BHIB
Brain Heart Infusion Broth
BSC
Biological Safety Cabinet
°C
Degree(s) Celsius
CFU
Colony Forming Unit(s)
Ct
Threshold Cycle
dH20
Distilled Water
DNA
Deoxyribonucleic Acid
End
Filers removed at the end of their service life as defined by the scheduled
maintenance schedule
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
High
Filters selected that had relatively high particulate loadings for the site
HVAC
Heating, Ventilation, and Air Conditioning
L
Liter(s)
UL
Microliter(s)
Mid
Filters that were in use for approximately half of their scheduled service life
mL
Milliliter(s)
mM
Millimolar
ModG
Modified G
MYP
Mannitol Egg Yolk Polymyxin
NAF
Native Air Filter
New
Filters that were not yet used; no ambient particulate matter collected
NTC
No Template Control
NYCT
New York City Transit
PBS
Phosphate Buffered Saline
PBST
PBS plus 0.05% Tween
PC
Positive Control
PCR
Polymerase Chain Reaction
PE
Performance Evaluation
PES
Polyethersulfone
PMP
Paramagnetic Particles
PVDF
Polyvinyldiene Difluoride
OA
Quality Assurance
QAPP
Quality Assurance Project Plan
QC
Quality Control
QMP
Quality Management Plan
qPCR
quantitative PCR
rcf
Relative Centrifugal Force
rpm
Revolution(s) per Minute
Vlll
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Abbreviations and Acronyms (Cont.)
Acronym
Definition
RV-PCR
Rapid Viability PCR
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 System Audit
TSA
Technical Systems Audit
UTR
Underground Transport Restoration
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 Environmental Protection Agency's (EPA's) Homeland
Security Research Program (HSRP) within EPA's Office of Research and Development.
Dr. Worth Calfee was the project lead for this document. Contributions of the following
individuals and organizations to the development of this document are acknowledged.
United States Environmental Protection Agency
Dr. 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
The United States Environmental Protection Agency (EPA) is the federal lead supporting
remediation of land and public infrastructure following the release of a hazardous substance to
the environment that threatens public health. EPA's remediation responsibility includes
responding to a bioterrorism incident, such as the release of Bacillus anthracis (B. anthracis) in
an urban area. EPA, in coordination with other Government agencies, National Laboratories, and
Stakeholders have conducted studies to support preparation for response and remediation
following such a release. These studies have included releases of surrogates for B. anthracis
spores in outdoor environments and subway stations to better understand the transport of
aerosols and to assess models to predict their behavior. Those studies have also been used to
establish and assess air and surface sampling methods.
In addition to sampling methods, EPA has also pioneered the development of new analytical
methods supporting response and remediation efforts. EPA has developed 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) (EPA, 2012 and Shah, 2017). The
analytical methods used in this current study were based on EPA's 2012 version of "Protocol for
Detection of B. anthracis Spores from Environmental Samples During the Remediation Phase of
an Anthrax Event," but with some updates based on EPA's 2017, Second Edition.
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. A
previous study demonstrated that a sampling strategy involving native air samplers could be
implemented in an urban area with the cooperation and collaboration of the public-private sector
(Ackelsberg, et al., 2011). Particulate filters indigenous to the affected area, and although
intended for other applications (e.g., ambient air quality particulate sampler or building heating,
ventilation, and air conditioning [HVAC] filter), may be operating during and/or immediately
after an incident. Thus, those ubiquitous native air filters (NAFs) offer the potential to better map
an incident by having a potentially higher quantity of organisms collected and/or provide a
higher fidelity of mapping. Inherently, those NAFs will have or will be collecting ambient
particulate matter prior to and/or during the incident. The impact of the ambient particulate load
on the NAFs may interfere with the current analytical methods to recover, identify, and quantify
B. anthracis. In this study, EPA seeks to assess the feasibility of using NAFs for potential use in
biological incident extent mapping. If feasible, it could facilitate subsequent sampling plans,
increase the speed of a response, and potentially save cost.
The objective of this study was to identify and evaluate the compatibility of currently deployed
NAF devices (e.g., PM10 samplers, building HVAC filters) with current 5. anthracis analytical
methods (culture and RV-PCR), for the ultimate goal of characterizing and mapping the extent of
contamination following a biological contamination incident involving B. anthracis spores.
Literature containing pertinent information related to field air sampling equipment was surveyed
to identify sources of filters and their filter types associated with native air samplers. Two classes
of native air samplers were defined and from which filters were sought: 1) air quality (AQ)
samplers such as those used at air quality monitoring sites around the United States and 2) non-
air quality (non-AQ) filters such as those in a building HVAC system or various types of air
filters associated with transportation vehicles that would be ubiquitous and likely operating in an
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urban setting. These filter types were utilized in laboratory-based testing whereby a known
quantity of Bacillus anthracis Sterne (B. a. Sterne) (non-pathogenic strain of B. anthracis, used
as a surrogate for fully virulent strains) spores were spiked onto the filter media. Currently-
recommended B. anthracis analytical methods, culture and RV-PCR, were attempted on the
spiked media.
The key findings, conclusions, and recommendations from this research are:
• The foremost conclusion is that filters recovered from both AQ and non-AQ filters may
be useful and beneficial to analyze for B. anthracis to help map the extent of biological
incidents, recognizing there are limitations to their use. This conclusion is made based on
the data showing that, even in the presence of other particulate matter having been
collected on filters, B. a. Sterne spores that were spiked onto the filters could be
recovered and successfully detected; however, the study results clearly indicate that the
background flora and other particulate matter can adversely impact the method sensitivity
and accuracy. Consequently, the NAF could be used to supplement results from other
sampling plans but should not be relied upon solely as the definitive biological release
incident mapping tool. The overall accuracy of the method properly detecting B. a. Sterne
(combined true positives and true negatives) across all filter types was 82% for culture
and 85% for RV-PCR.
• RV-PCR can be used to positively identify viable B. a. Sterne in the presence of complex,
dirty sample matrices of NAFs. However, background flora and grime collected can
impact the lower limit of detection and/or reduce the response to B. a. Sterne.
• Background flora and non-living material (dirt/grime) interferes with identification and
quantifying B. a. Sterne using the traditional plate culture method, particularly for non-
AQ filters. Presumptive B. a. Sterne colonies may not actually be the target organism
because background flora can have an indistinguishable colony morphology, leading to
false positives and an overestimate of the detection of the B. a. Sterne. Conversely, the
apparent B. a. Sterne quantity recovered can be biased low due to suppression of
B. a. Sterne growth with competing background flora. It is possible for so much
background flora to be present on NAFs such that the presence of B. a. Sterne cannot be
made, potentially leading to false negatives.
• The RV-PCR method requires great care and diligence to implement effectively. Most
notably, glove changes were required between samples for each step, which is onerous
and time consuming; however, it was found to be necessary to minimize cross-
contamination.
• The primary recommendation is to assess the impact that spiking of B. a. Sterne spores
onto the NAF 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
generating an aerosol of B. a. Sterne and then pulling the aerosol-laden air through the
NAF rather than applying spores via a liquid suspension spike. The method would then
be applied to recover and analyze for B. a. Sterne. This approach is expected to primarily
affect spore recovery, which then may impact detection limits and or accuracy to identify.
• Priority should be placed on analyzing filters having the lowest loading of background
particulate matter, to the extent that can be determined by the shortest duty cycle of non-
AQ filters or by gravimetric analysis of AQ filters.
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1.0 INTRODUCTION
1.1 Background
Under Emergency Support Function #10 of the National Response Framework, 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
(https://www.fema.gOv/media-library/assets/documents/l 17791. EPA, in coordination with other
Government agencies, National Laboratories, and Stakeholders have conducted studies to
support preparation for that role. These studies have included releases of surrogates for B.
anthracis in outdoor environments and subway stations to better understand the transport of
aerosol releases and to assess models to predict the spread of the particles. Those studies have
also been used to establish and assess air and surface sampling methods.
In addition to sampling methods, EPA has also pioneered the development of new analytical
methods supporting response and remediation efforts. EPA has developed 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) (EPA, 2012 and Shah, 2017). The
analytical methods used in this study were based on EPA's 2012 version of "Protocol for
Detection of B. anthracis Spores from Environmental Samples During the Remediation Phase on
an Anthrax Event," but with some updates based on EPA's 2017, Second Edition.
In the case of a biological contamination incident, EPA must characterize the extent of the spread
of the biological threat agent using established sampling and analytical methods such as those
noted above. Although a biological contamination incident may occur where BioWatch samplers
are operated, they may not be present in sufficient numbers and optimally spaced or located to
adequately characterize the biological threat agent spread. A previous study demonstrated that a
sampling strategy involving native air samplers could be implemented in an urban area with the
cooperation and collaboration of the public-private sector (Ackelsberg, et al., 2011). Particulate
filters indigenous to the affected area, and although intended for other applications (e.g., ambient
air quality particulate sampler or building heating, ventilation, air conditioning [HVAC] filter)
are likely to be more prevalent, widely dispersed, and operating during and/or immediately after
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an incident. Thus, those ubiquitous native air filters (NAFs) offer the potential to better map an
incident by having a potentially higher quantity of organisms collected and/or provide a higher
fidelity of mapping through their higher abundance/density in comparison to purposefully
deployed counterterrorism air samplers. Inherently, those NAFs will have or will be collecting
ambient particulate matter prior to and/or during the incident. The impact of the ambient
particulate load on the NAFs may interfere with the current analytical methods to recover,
identify, and quantify B. anthracis. EPA seeks to assess the feasibility of using NAFs for
potential use in biological incident extent mapping by assessing their compatibility with current
B. anthracis analytical approaches. If feasible, it could facilitate subsequent sampling plans,
increase the speed of a response, and potentially save cost.
1.2 Objective
The objective of this study was to identify and evaluate the compatibility of B. anthracis
analytical methods with currently deployed NAF devices (e.g., PM10 samplers, building HVAC
filters) for the ultimate purpose of assessing whether NAFs are feasible to use for characterizing
and mapping the extent of contamination following a biological contamination incident
involving B. anthracis spores.
1.3 Scope
The scope of the research reported here was to assess the EPA methods to recover and
subsequently analyze for the positive identification of B. anthracis spores - specifically, Bacillus
anthracis Sterne (B. a. Sterne) spores that were spiked by applying droplets of a stock spore
suspension onto both air quality (AQ) filters (PM2.5 and PM10 filters from ambient air quality
monitoring sites) and non-air quality (non-AQ) filters from bus engine air intake filters, building
HVAC filters, subway platform filters, and subway rolling stock filters. A total of 377 filter
samples were spiked with B. a. Sterne, recovered per EPA protocols and analyzed using both
culture and molecular methods based on those previously developed by EPA (EPA, 2012).
(Initially, EPA's 2012 version of "Protocol for Detection of B. anthracis Spores from
Environmental Samples During the Remediation Phase of an Anthrax Event" was used because
the 2017 version was not yet finalized.) Elements of the EPA 2017, Second Edition version
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(Shah, 2017) were incorporated as noted in this report in order to ensure the results reflected the
state-of-the-art of the methods and gave the best indication of method capability.
The performance of the culture method was assessed by determining percent recovery efficiency
of presumptive B. a. Sterne spores spiked onto the filter, 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.
It is important to note that this study was not solely an assessment of the analytical method to
identify and/or quantify B. anthracis, but rather an assessment of the method end-to-end, to
include physical recovery from the filter media (and other grime or flora associated with the
filter 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 Filter Categories and Selection
Two categories of NAFs were assessed: those used in AQ samplers (for PM2.5 and PM10
collection for ambient air quality monitoring) and those used in non-AQ applications (e.g., bus
filter, building HVAC filter, subway platform filter, and subway rolling stock filter).
2.1.1 AQ Filters
The selection of PM2.5 and PM10 samples for use during this NAF project was based on a query
of EPA's Air Quality System, which revealed that there are 2,585 active PM2.5 monitors and
4,719 active PM10 monitors located across the United States. Therefore, if an incident involving
a biological aerosol release occurred, one or more of those samplers may be able to be used to
help determine the extent of the release (i.e., the area of contamination). The AQ filters used
were 47-mm diameter Teflon for the PM2.5 and 8- x 10-inch (20 x 25 cm) glass fiber filter for
the PM10. Representative images of each AQ type is shown in Figure 1. The 47-mm-diameter
filters were cut into quarters to prepare test coupons for testing. Coupons, 4x4 cm, were cut
from the flat sheet PM10 filters, avoiding the edges of the filter. The filters were recovered from
four targeted geographic regions, nominally from the Northeast, Southeast, North, and West to
the extent possible. The PM2.5 filters recovered for use were from Arizona (AZ), Florida (FL),
Massachusetts (MA), and Wisconsin (WI). The PM10 filters recovered were from California
(CA), New Hampshire (NH), South Carolina (SC), and Wisconsin (WI).
AQ filters with relative ambient particulate loading levels of average (Avg) and high (High), as
determined by gravimetric analysis data accompanying the filters, were obtained from each
geographic region for each filter type. The relative particulate load descriptors of Avg and High
were assigned by comparing the mass loading for filters from that region. Filters with the highest
mass loading were selected and denoted as High and those near the middle of the mass loadings
were denoted as Avg. Filters that had not been used to sample ambient air and thus had no
ambient particulate loading were denoted as New filters or New media. (Because the same filter
media was used, the New media was obtained independent of the geographic region and
represented all.) Sample availability and project scope did not allow for selecting filters for
specific times of the year.
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Figure 1. Air Quality Filter Types: PM2.5 (Left: High and Avg Particulate Loads)
and PM10 (Right: New Media)
2.1.2 Non-AQ Filters
Representative images of each non-AQ filter type - building HVAC filter, bus filter, subway
platform filter, and the subway rolling stock filter - are shown in Figure 2. All four filter types
were pleated, but no additional information regarding the media type, fiber properties, or fiber
density were provided. The swatches of filter media cut from the filters and used as coupons
were generally obtained from the flat surface of the pleat, avoiding the pleat peaks and valleys.
The bus filter, subway platform filter, and the subway rolling stock filter were all obtained from
the New York City Transit (NYCT) system. The NYCT Authority has been involved in prior,
related studies such as the Underground Transport Restoration (UTR) 2017 project, so there are
additional data related to those filters that may complement these results (Serre and Oudejans,
2017), but are beyond the scope of this project. EPA requested that NYCT provide filters from
each of the applications/locations that were unused (New), in the middle portion of their duty
cycle (Mid), and at the end-of-duty cycle (End) as determined by their maintenance schedule.
NYCT provided filters following these guidelines, so filters were obtained that contained varying
quantities of ambient particulate matter. No specific duty cycle information (time, mileage) was
provided from NYCT.
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_P»Uj: i I.
Figure 2. Non-AQ Filter Types: Bus Filter (Top Left); Building HVAC Filter (Top Right);
Subway Platform Filter (Bottom Left); and Subway Rolling Stock Filter (Bottom Right)
2.2 Test Matrix
Each of the filter samples described in Section 2.1 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 methods using culture analysis and RV-PCR to determine the percent recovery for each
of the three ambient particulate loading conditions.
The completed test matrix for the AQ filters and non-AQ filters are provided in Table 1 and
Table 2, respectively. In total, 377 filter samples were analyzed, comprising 108 PM2.5 filters,
111 PM10 filters, and 158 non-AQ filters (approximately 40 for each of the four non-AQ filter
types). The columns of Particle Loads in Table 1 and Duty Stage in Table 2 are as defined in
Section 2.1 and represent new filters (New), average (Avg), or high (High) particle loading for
AQ filters (Table 1) and new filters (New), middle-of-duty cycle (Mid), and end-of-duty cycle
(End) for non-AQ filters (Table 2). The target spore load of 0, 30, 300, or 3,000 was the number
of spores intended to be spiked onto the filters. Following physical extraction, the sample volume
was split nominally in half to result in 0, 15, 150, 1,500 spore load challenges for each of two
detection assays (culture and RV-PCR). These details are discussed further in Section 2.3.2.
Generally, three replicates were completed for each target B. a. Sterne spore loading quantity,
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but there were instances of higher number of replicates, especially early in the study as the
analytical methods were being refined and implemented. The 0-spore load (no purposeful
application of B. a. Sterne onto the test filter) served as a negative control. Use of New filter
media 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.
The analytical methods of culture and RV-PCR were used to quantify or identify recovered
B. a. Sterne spores spiked and subsequently recovered in the sample extracts. Sheep Blood Agar
(SB A) was the primary medium used for all culture analyses; Mannitol Egg Yolk Polymyxin
(MYP) and/or Brilliance Bacillus cereus agar (BBCA) were used for a subset of samples. MYP
and BBCA are both chromogenic media that have been developed to aid in differentiating target
pathogen microbial growth from background flora. They were assessed early in the study
analyses to determine whether there was a benefit to unambiguously quantifying B. a. Sterne. As
will be discussed in Section 3.1.1, it was decided to discontinue the culture assays with the
chromogenic agar and continue using only the SB A media because no benefit was gained using
the chromogenic media (Calfee, 2017). Details regarding the analytical methods are discussed in
Section 2.3.
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Table 1. Test Matrix for AQ Filters
Filter
Type
Geographic
Region
Particle
Loads(a)
(jig/cm2)
Target Spore
Loads onto
Filter(b)
Nominal
Spores
Available per
Analytical
Method(c)
Replicates
Analytical Method(d)
Culture
Molecular
PM2.5
N/A
New (0)
0/30/300/3000
0/15/150/1500
3/3/3/3
SBA
RV-PCR
Wisconsin
(WI)
Avg (14)
0/30/300/3000
0/15/150/1500
3/3/3/3
SBA/BBCA
RV-PCR
High (47)
0/30/300/3000
0/15/150/1500
3/3/3/3
SBA/BBCA
RV-PCR
Florida
(FL)
Avg (13)
0/30/300/3000
0/15/150/1500
3/3/3/3
SBA
RV-PCR
High (39)
0/30/300/3000
0/15/150/1500
3/3/3/3
SBA
RV-PCR
Arizona
(AZ)
Avg (16)
0/30/300/3000
0/15/150/1500
3/3/3/3
SBA
RV-PCR
High (39)
0/30/300/3000
0/15/150/1500
3/3/3/3
SBA
RV-PCR
Massachusetts
(MA)
Avg (12)
0/30/300/3000
0/15/150/1500
3/3/3/3
SBA
RV-PCR
High (29)
0/30/300/3000
0/15/150/1500
3/3/3/3
SBA
RV-PCR
PM10
N/A
New (0)
0/30/300/3000
0/15/150/1500
3/3/3/3
SBA/MYP
RV-PCR
Wisconsin
(WI)
Avg (41)
0/30/300/3000
0/15/150/1500
3/3/3/3
SBA
RV-PCR
High (104)
0/30/300/3000
0/15/150/1500
5/4/3/3
SBA/MYP
RV-PCR
New
Hampshire
(NH)
Avg (69)
0/30/300/3000
0/15/150/1500
3/3/3/3
SBA
RV-PCR
High (199)
0/30/300/3000
0/15/150/1500
3/3/3/3
SBA
RV-PCR
California
(CA)
Avg (118)
0/30/300/3000
0/15/150/1500
3/3/3/3
SBA/BBCA
RV-PCR
High (277)
0/30/300/3000
0/15/150/1500
3/3/3/3
SBA/BBCA
RV-PCR
South
Carolina
(SC)
Avg (65)
0/30/300/3000
0/15/150/1500
3/3/3/3
SBA
RV-PCR
High (132)
0/30/300/3000
0/15/150/1500
3/3/3/3
SBA
RV-PCR
(a) Ambient particle load reported from the air quality station from which the filters were received.
(b) Target number of spores spiked onto filter - See Section 2.3.2 for discussion.
(c) Nominally half of the target quantity of spores spiked onto the filter were available for each of the two
analytical filter - See Section 2.3.3 for discussion.
(d) BBCA (selective); SB A; MYP agar (chromogenic); RV-PCR assay, chromosomal and pXOl gene targets.
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Table 2. Test Matrix for Non-AQ Filters
Filter Type
Duty
Stages
Target Spore
Loads onto Filter*3'
Nominal Spores
Available per
Analytical
Method(b)
Replicates
Analytical Method*0'
Culture
Molecular
Bus
New
0/30/300/3000
0/15/150/1500
5/3/3/3
SBA/MYP
RV-PCR
Mid
0/30/300/3000
0/15/150/1500
3/3/3/3
SBA/MYP
RV-PCR
End
0/30/300/3000
0/15/150/1500
5/5/5/3
SBA/MYP
RV-PCR
Building
HVAC
New
0/30/300/3000
0/15/150/1500
4/4/3/3
SBA/MYP
RV-PCR
Mid
0/30/300/3000
0/15/150/1500
3/3/3/3
SBA/MYP
RV-PCR
End
0/30/300/3000
0/15/150/1500
4/4/4/4
SBA/MYP
RV-PCR
Subway
Rolling
Stock
New
0/30/300/3000
0/15/150/1500
3/3/3/3
SBA/MYP
RV-PCR
Mid
0/30/300/3000
0/15/150/1500
3/3/3/3
SBA/MYP
RV-PCR
End
0/30/300/3000
0/15/150/1500
3/3/3/3
SBA/MYP
RV-PCR
Subway
Platform
New
0/30/300/3000
0/15/150/1500
3/3/3/3
SBA/MYP
RV-PCR
Mid
0/30/300/3000
0/15/150/1500
3/3/3/3
SBA/MYP
RV-PCR
End
0/30/300/3000
0/15/150/1500
3/3/3/3
SBA/MYP
RV-PCR
(a) Target number of spores spiked onto filter - See Section 2.3.2 for discussion.
(b) Nominally half of the target spore loaded onto the filter were available for each of the two analytical methods -
See Section 2.3.3 for discussion.
(c) SBA; MYP agar (chromogenic); RV-PCR assay, chromosomal and pXOl gene targets.
2.3 Microbiological Methods
All sample processing and analytical methods used were from those provided by the EPA
Protocol for Detection of Bacillus cmthracis in Environmental Samples During the Remediation
Phase of an Anthrax Incident (EPA, 2012), with any differences or revisions noted. Both a
culture and molecular analytical method were assessed as will be discussed in Sections 2.3.4 and
2.3.5.
Early application of the RV-PCR method to samples not purposely spiked with B. a. Sterne
resulted in measurable levels of B. a. Sterne. There were instances of solution leakage of the
capping tray and poor welds of the membrane in the filter vials that were potentially the source
or contributor to the contamination. The capping tray was replaced with a new unit and filter vial
leakage did not persist. Also, with subsequent refinement of glove change frequency and
technique of equipment operation, cross-contamination was reduced and ultimately eliminated.
Following are sections that summarize specific procedures and steps applied to conduct the
study.
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2.3.1 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
11 agent following the Biosafety in Microbiological and Biomedical Laboratories 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 (BEINR-
1400) was used to grow an overnight culture on Tryptic Soy Agar. 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) sporulation broth (see Appendix A, 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 A, 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) at 4°C 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 at 4°C 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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2.3.2 Spore Loading (Spiking)
PM2.5 filters were received as circular 47-mm swatches and were quartered prior to spiking;
PM10 and all non-AQ filter types were cut to 4-cm2 swatches. Note that the EPA protocol
(EPA, 2012) was originally developed for processing of 37-mm vacuum filter cassette samples,
thus the area of filter sample analyzing the AQ and non-AQ filters was about 2.5 times less than
that of the 37-mm filters. 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 SBA 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# 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 filter samples affected the sensitivity or lower limits of the analytical
method.
Table 3. Target B. a. Sterne Spore Loading Levels onto Each Filter Substrate
Loading
Level
Stock Concentration
(CFU/mL)
Target Total
CFU per
Filter(a)
Extract
Volume (mL)
Theoretical
Concentration in
Extract (CFU/mL)
High
3.0 x 104
3,000
25
120
Medium
3.0 x 103
300
25
12
Low
3.0 x 102
30
25
1.2
(a) 100 |iL of stock suspension applied (20, 5-|iL drops).
Each swatch to be spiked with B. a. Sterne spores was transferred to a Petri dish and 20 5-|iL
droplets were pipetted onto the surface of each filter swatch (see Figure 3) for a total of 100 [xL
of stock suspension applied. Negative control swatches that were included in each batch were
transferred directly into sealed 50-mL conical tubes prior to spiking swatches with B. a. Sterne.
The spiked swatches then dried overnight inside of a BSC.
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Figure 3. Photographs of Metro Bus Engine Filter (left) and PM10 Air Quality Filter
(right) After Spiking with the B. a. Sterne Suspension
2.3.3 Spore Recovery
Throughout the recovery procedure, gloves were changed between handling samples to limit the
likelihood of cross-contamination between samples.
Following spiking and drying, the filter swatches were placed into a 50-mL tube and a mesh
support was placed over the filter swatch. Fifteen (15) mL of cold (4°C) extraction buffer with
Tween® 20 (0.22 jam polyethersulfone (PES) filter sterilized 700 mL IX phosphate buffered
saline (PBS), 0.05% Tween 20, pH 7.4, and 300 mL 200 proof ethanol) was added to each
sample and the lids were sealed using Parafilm. Samples were vortexed for 20 minutes on a
platform vortex set to speed 7 (VWR, Cat. 945057). After vortexing on a single-tube vortex for 3
to 5 seconds each, the samples sat for 2 minutes to enable large particles to settle prior to
transferring -12.5 mL of the suspension into a corresponding labeled 50-ml conical collection
tube. A second spore extraction was then completed by addition of 10 mL of cold (4°C)
extraction buffer without Tween 20 (0.22 jam PES filter sterilized 700 mL IX PBS, pH 7.4 and
300 ml. 200 proof ethanol) to each sample tube and the lids were sealed using Parafilm. Samples
were vortexed for 10 minutes on a platform vortex set to speed 7. After vortexing on a single-
tube vortex for 3 to 5 seconds, the samples sat for 2 minutes to enable large particles to settle
prior to transferring the remaining -12.5 mL of the suspensi on into the corresponding labeled
50-ml conical collection tube. After vortex mixing, 10.5 mL of the recovered suspension aliquot
was transferred into a labeled 15-ml conical tube to be used for the culture-based microbial
analysis described in Section 2.3.4, and the remaining volume (nominally -12.5 ml) was
transferred into a labeled filter vial for RV-PCR analysis as described in Section 2.3.5.
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2.3.4 Culture Method
Culture-based microbiological analysis was performed on each sample by filtering the recovered
extract through filter funnels and filter media (Pall, Cat. 4804) then placing the filters onto solid
bacterial growth media and incubating. Serial dilution and spread-plating procedures, as
prescribed by the 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 undiluted
extract from a sample 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 PBS with 0.05% Tween (PBST),
then 10 mL of PBST was added to each MicroFunnel filter to suspend 1 mL or 4 mL of the
sample extract followed by vacuum filtration. The walls of each filter funnel were rinsed with
10 mL of PBST and filtered through the MicroFunnel, then the filter membrane was removed
and placed onto MYP, BBCA, and/or SBA media. (As shown in Table 1 and Table 2, MYP and
BBCA were used in a subset of the sample conditions in the test matrix and SBA was the only
culture medium carried throughout all test conditions.)
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. When B. a. Sterne grows on
MYP, the expected colony color is pink, and when grown on BBCA, the expected colony color
is turquoise green. Figure 4 shows representative images of B. a. Sterne colonies on each culture
medium used in this study.
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. The samples were not blinded for analysis. The
microbiologists were aware of which samples should and should not contain B. a. Sterne.
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Nonetheless, as the results demonstrated, 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).
Figure 4. From left to right: B. a. Sterne on SB A, MYP, and BBC A
2.3.5 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 DNA. 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 or
Polyethersulfone [PES] membrane; Whatman Cat. AV125NPUPSU), 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. Cold
(4°C) Brain Heart Infusion Broth (BH1B) (5 mL) was then added to each filter vial, the vials
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were capped, and then vortex-mixed for 10 minutes on a setting of 7. Images of the manifold and
capping tray are depicted in Figure 5. 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.
Figure 5. Top: Manifold Containing 16 Filter Vials; Middle: Capping Tray;
Bottom: Capped Filter Vials Containing BHIB
Following overnight incubation of the filter vials with BHIB, the vials were mixed on the
platform vortex for 10 minutes with speed set to 7. (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.
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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
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 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 pellets in remaining 200 |iL 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 contacted 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
16
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EPA/600/R-19/082
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vortexed another 10 seconds and incubated in the heating block for 1 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
tube was briefly vortexed and then centrifuged at 2,000 rpm 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 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
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.
Consequently, it was agreed to use the duplex assay method for this study. A summary report of
assessment with details of the approach and supporting results is provided in Appendix B.
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 A. Each sample DNA extract was assayed in triplicate
reactions. Controls consisted of four positive control wells containing 50 pg of DNA extracted
from B. a. Sterne 34F2 (NR-1400, BEI Resources) and four no template controls 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:
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EPA/600/R-19/082
August 2019
• Stage 1: 1 cycle at 95°C for 20 seconds
• Stage 2: 45 cycles at 95°C for 3 seconds followed by 60°C for 30 seconds
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).
2.4 Method Implementation
The primary microbial methods used to spike/recover/analyze the NAFs, shown as they occurred
in chronological order, are depicted graphically in the process flow diagram of Figure 6.
Filter Sample
Prep
Spore
Spike
Day 1
(Tues)
Spore
Recovery
Day 2
(Wed)
J
Day 2
(Wed)
1
Plate
SBA
MYP
BBCA
Incubate
Overnight
Day 2 —* Day 3
(Wed — Thurs)
Day 3
(Thurs)
I r
j i i
Enumerate
DNA
Extraction
(t0 and tf)
Day 3
(Thurs)
RV-PCR
Analysis
Day 4
(Fri)
Figure 6. Process Flow Chart Depicting Key Method Process Steps in Chronological Order
The methods implemented, in the form of work instructions followed by the analytical staff, are
provided in Appendices C through H. These work instructions also complement those
microbiological methods described in Section 2.3, and emphasize glove-changing schedules that
were implemented to minimize cross-contamination. The work instructions were reviewed in
detail to refine and ensure proper implementation of the methods (Calfee, 2017).
The above method was used to analyze 16 filter samples per trial, with 1 trial conducted per
week. For each weekly test, filters were cut into swatches and spiked using B. a. Sterne spores
suspended in water. Each test consisted of swatches loaded with 0, 30, 300, or 3,000 spores per
filter swatch per "Work Instruction for Dosing Filter Swatches with Bacillus cmthracis Spores"
in Appendix C. The spiked filters were dried overnight before spores being recovered following
the "Work Instruction for Bacillus anthracis Spore Recovery," as detailed in Appendix D. The
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EPA/600/R-19/082
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recovered suspension volume was then split between the traditional culture method (10.5 mL)
and RV-PCR method (12.5 mL) analyses. The aliquot for culture was divided into 1-mL or 4-mL
volumes and filter-plated onto media and incubated overnight as outlined in the "Work
Instruction for Culture of Bacillus anthracis Spores Recovered from Air Filters" in Appendix E.
The To RV-PCR aliquot was stored frozen while the recovered spores enriched overnight, then
the Tfmai aliquot was removed and the DNA was extracted from both To and Tfmai aliquots per
"Work Instruction for Manual DNA Extraction and Purification from Bacillus anthracis" in
Appendix F. 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" in Appendix G. PCR was also used to confirm or refute presumptive
B. a. Sterne colonies selected from the culture analysis per "Work Instruction for Selecting
Presumptive B. a. Sterne Colonies for quantitative PCR (qPCR) Confirmation" in Appendix H.
2.5 Data Reduction and Analysis
2.5.1 Culture — Percent Recovery
The percent recovery efficiency (Erecovery) of B. a. Sterne from each spiked filter 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) and multiplying 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
^recovery (%) = 77 * 100%
^ spike
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 (either 1 mL or
4 mL) to yield a presumptive B. a. Sterne spore concentration (Crecover) (CFU/mL) that was then
multiplied by the extract volume (Vextract) (25 mL) to determine the total presumptive B. a. Sterne
CFUs recovered from the filter sample. The reported percent recovery was determined using the
below rules:
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EPA/600/R-19/082
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1) Report the percent recovery from the aliquot (1-mL or 4-mL) that has between 20 to
80 CFU.
2) Report the 4-mL aliquot percent recovery if the CFU counted from both the 1-mL and
4-mL aliquots is less than 20.
3) Report the 4-mL aliquot percent recovery if the CFU counted from both the 1-mL and
4-mL aliquots is between 20 and 80.
4) Report the 1-mL aliquot percent recovery if the background flora on the 4-mL 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. A portion
of the presumptive colony was collected into 100 |iL of PCR-grade water in microcentrifuge
tubes. The colony suspension was then heated for 5 minutes on a heat block at 95°C. The lysate
was cooled and then centrifuged at 14,000 rpm for 2 minutes and the supernatant was analyzed
using the real-time PCR assays targeting the B. anthracis chromosome and pXOl gene targets.
2.5.2 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 (from triplicate reactions) value generated by
the To aliquot. A positive ACt (> 9) value indicates that viable B. a. Sterne spores were recovered
from the filter. For a sample to be considered positive, the below acceptance criterion had to be
met:
• The ACt must be greater than or equal to 9 for both the chromosome and pXOl targets
(ACt = Ct (To) - Ct (Tfinal) > 9).
Additional criteria exist for the positive confirmation of a sample if analyzing samples obtained
from an actual incident, but for this study the above criterion was used.
2.5.3 Presentation of Results
The method employed to recover B. a. Sterne spores spiked onto the NAFs was consistent with
current EPA methods, as described in Section 2.3.4. The entire extract would be analyzed either
using a culture method or a RV-PCR method, solely, in actual practice and application by EPA if
analyzing samples collected after a biological release incident (Calfee, 2018). In the study
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EPA/600/R-19/082
August 2019
performed and reported here, however, the sample extract was split as described in Sections 2.3.4
and 2.3.5, so that approximately half of the extract sample was used for culture analysis and the
other half for RV-PCR analysis. In this way, results from both methods could be compared in a
pair-wise manner. 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 extract sample 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 B. a. Sterne spores.
As described in Section 2.3.2, the NAFs were spiked with a target quantity of spores by applying
twenty (20) 5-[xL 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-19/082
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3.0 RESULTS AND DISCUSSION
A detailed discussion of the calculations and approach to presenting the results was provided in
Section 2.5. In summary, all results presented in plots have an x-axis title and labels of 0, 15,
150, and 1,500, representing the nominal spores available for analysis (CFU). Similarly, the
summary results in the tables contain the same nominal quantity of spores available, and also the
determined quantity of spores applied to the filter. 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
analysis by culture and RV-PCR methods.
Note that the spores available for analysis represent the maximum number of spores that could
be 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 AQ Filter 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 AQ filter substrates spiked with B. a. Sterne
and using the SBA medium are presented in Table 4 (PM2.5 filters) and Table 5 (PM10 filters).
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 quantity of presumptive B. a. Sterne colonies reported in the
tables is half of the actual total recovered because in the context of the tables, only half of the
extract samples was made available for analysis. The quantity of presumptive B. a. Sterne
colonies for each filter sample, used in the percent recovery calculations, are reported in
Appendix I for the culture method using the SBA medium. When either the MYP chromogenic
agar or the BBCA growth medium was also used in the culture method, the recovery efficiencies
are reported in Appendices J and K, respectively.
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EPA/600/R-19/082
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Table 4. Recovery Efficiencies for Presumptive B. a. Sterne Spores from PM2.5 Air Quality
Filters Cultured in the SBA Medium
Ambient
Spores Available for Analysis
Spores
Spore
Location
Particle
Sample
(CFU)
Recovered
Recovery
Load(a)
(jig/cm2)
Reps
Nominal(b)
Determined
(X ± o)(c)
(CFU)
(X ± o)(d)
Efficiency (%)
(X ±
3
0
0
0
N/A
—
(0)
3
15
18 ±9
6.3 ±0.0
46 ±32
(New)
3
150
180 ± 90
74 ±46
44 ± 15
3
1,500
1,800 ± 900
430 ± 40
33 ±26
3
0
0
1.0 ± 1.8
N/A
Avg
3
15
18 ±9
17 ±4.8
120 ± 80
(16)
3
150
180 ± 90)
91 ±44
52 ±3.4
AZ
3
1,500
1,800 ± 900
720 ± 230
46 ± 16
3
0
0
2.1 ±3.6
N/A
High
3
15
7 ± 0
3.1 ±3.1
42 ±42
(39)
3
150
70 ±3
52 ± 15
72 ±23
3
1,500
700 ± 30
540±150
74 ± 19
3
0
0
0
N/A
Avg
3
15
15 ±9
12 ±9.5
71 ±34
(13)
3
150
150 ± 90
77 ±21
59 ±26
FL
3
1,500
1,500 ± 900
980 ±160
75 ±25
3
0
0
0
N/A
High
3
15
15 ±9
14 ±7.9
110 ±94
(39)
3
150
150 ± 90
110 ±24
81 ±25
3
1,500
1,500 ± 900
960 ± 82
75 ±29
3
0
0
0
N/A
Avg
3
15
14 ±4
6.3 ±5.4
51 ±47
(12)
3
150
140 ± 40
51 ±34
36 ± 19
MA
3
1,500
1,400 ± 400
790 ±210
57 ±6.8
3
0
0
0
N/A
High
3
15
14 ±4
10 ± 1.8
76 ± 18
(29)
3
150
140 ± 40
60 ±40
39 ±21
3
1,500
1,400 ± 400
540 ± 400
34 ±23
3
0
0
0
N/A
Avg
3
15
11± 1
7.3 ±4.8
67 ±42
(14)
3
150
110 ± 6
56 ±27
54 ±28
WI
3
1,500
1,100 ±60
560 ± 220
53 ±23
3
0
0
0
N/A
High
3
15
11± 1
9.4 ±0.0
88 ±5
(47)
3
150
110 ± 6
77 ± 10
73 ± 11
3
1,500
1,100 ±60
820 ± 36
77 ±6.7
(a) Relative ambient particle load on the filter (with measured mass loading per area).
(b) Nominally one-half of the target spore load onto the filter and assuming 100% recovery of spores.
(c) Based on the spiking suspension titer measured each test trial, 100% recovery efficiency, and one-half of extract
used for culture analysis.
(d) Presumptive B. a. Sterne colonies based on morphology, and one-half of extract used for culture analysis.
(e) Calculated using the spore loading on each filter and presumptive B. a. Sterne spores recovered from each filter
sample.
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EPA/600/R-19/082
August 2019
Table 5. Recovery Efficiencies for Presumptive B. a. Sterne Spores from PM10 Air Quality
Filters Cultured in the SBA Medium
Ambient
Particle
Load(a)
(jig/cm2)
Sample
Reps
Spores Available for Analysis
(CFU)
Spores
Recovered
(CFU)
(X ± o)(d)
Spore
Recovery
Location
Nominal(b)
Determined
(X ± o)(c)
Efficiency
(%)
(X±a)
3
0
0
0
N/A
—
(0)
3
15
6 ± 3
0.0 ±0.0
0.0 ±0.0
(New)
3
150
60 ±30
7.3 ±4.8
15 ± 12
3
1,500
700 ±0
57 ± 15
8.8 ±2.4
3
0
0
21 ±3.6
N/A
Avg
3
15
13 ±5
18 ±4.8
150 ±29
(118)
3
150
130 ±50
14 ±9.5
13 ± 10.2
CA
3
1,500
1,300 ±500
68 ± 13
6.2 ±3.6
3
0
0
9.4 ±9.4
N/A
High
3
15
13 ±5
7.3 ±4.8
55 ±22
(277)
3
150
130 ±50
23 ±6.5
22 ± 16
3
1,500
1,300 ±500
59 ±8.3
5.1 ± 1.7
3
0
0
0
N/A
Avg
3
15
14 ± 10
4.2 ±7.2
17 ±29
(69)
3
150
140 ±100
6.3 ±6.3
6.0 ±8.4
NH
3
1,500
1,400 ± 1000
83 ± 15
8.2 ±4.8
3
0
0
0
N/A
High
3
15
14 ± 10
0.0 ±0.0
0.0 ±0.0
(199)
3
150
140 ±100
7.3 ±4.8
5.6 ±2.0
3
1,500
1,400 ± 1000
210±130
24 ±20
3
0
0
1.0 ± 1.8
N/A
Avg
3
15
14 ± 1
4.2 ±4.8
29 ±33
(65)
3
150
140 ±6
14 ±7.2
9.9 ±5.5
SC
3
1,500
1,400 ± 60
84 ±24
6.2 ±2.0
3
0
0
3.1 ± 3.1
N/A
High
3
15
14 ± 1
13 ±3.1
91 ±26
(132)
3
150
140 ±6
18 ±7.2
13 ± 5.1
3
1,500
1,400 ± 60
79 ±49
5.8 ±3.7
3
0
0
0
N/A
Avg
3
15
9 ± 0
0.0 ±0.0
0.0 ±0.0
(41)
3
150
90 ±0
12 ±6.5
13 ±7.2
WI
3
1,500
900 ±0
90 ±71
10 ±7.9
5
0
0
0
N/A
High
4
15
6 ± 1
0.8 ± 1.6
12 ±24
(104)
3
150
60 ±30
4.2 ±7.2
9.3 ± 16
3
1,500
900 ± 500
40 ± 9.5
5.2 ±2.8
(a) Relative ambient particle load on the filter (with measured mass loading per area).
(b) Nominally one-half of the target spore load onto the filter and assuming 100% recovery of spores.
(c) Based on the spiking suspension titer measured each test trial, 100% recovery efficiency, and one-half of
extract used for culture analysis.
(d) Presumptive B. a. Sterne colonies based on morphology and one-half of extract used for culture analysis
(e) Calculated using the spore loading on each filter and presumptive B. a. Sterne spores recovered from each
filter sample.
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EPA/600/R-19/082
August 2019
The presumptive B. a. Sterne recovery efficiencies on the SBA plates are plotted in Figures 7
through 14, one plot for each filter type. Note, a percent recovery is not tabulated or plotted for
the 0-spore spike condition since, by definition, a meaningful recovery efficiency cannot be
calculated, even though there could have been a finite number of presumptive B. a. Sterne
colonies counted based on colony morphology. Nonetheless, there are instances when one or
more colonies were counted as B. a. Sterne for the 0-spike condition, and the presumptive values
are reported in Table 4 and Table 5.
Review of the percent B. a. Sterne spore recovery plots in Figures 7 through 10 for PM2.5 filters
obtained from Arizona, Florida, Massachusetts, and Wisconsin, respectively, indicate that the
percent recovery was lowest for the New filter material and generally higher for the Avg and
High ambient particulate load filter load condition, which may be due to the applied spores
adhering more strongly to the clean filter substrate than to the particulate matter present on the
Avg or High ambient load filters and/or are physically removed with the particulate matter
during recovery. The average percent recovery efficiencies were 35 to 45% for New media and
the nominal B. a. Sterne spores available condition of 150 to 1,500; recovery efficiencies were
typically 40 to 80% for the Avg and High ambient particles loads with the 150 and 1,500
available B. a. Sterne spores condition. The filters with a target of 15 B. a. Sterne spores
generally had a higher standard deviation than the 150 and 1,500 nominal spores available
condition, which was attributed to the relatively few (< 15) recovered presumptive B. a. Sterne
colonies. In most filters analyzed (at least 90% of the filter samples spiked), colonies with a
B. a. Sterne morphology were recovered from all samples.
25
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EPA/600/R-] 9/082
August 2019
120
\0
0s*
£ 100
o
V 80
4-<
to
n5
CO
PM2.5-AZ
E
3
t/>
OJ
u
CL
o
f
HI
>
o
u
0)
a.
60
40
20
New
Avg
High
\
Nominal Spores Available for Analysis (CFU)
Figure 7. Percent Recovery Efficiencies (Average ± One Standard Deviation of N > 3
Replicates) of Presumptive B. a. Sterne Spores from PM2.5 Filters from Arizona Using the
SBA Medium (New, Avg, and High refer to relative ambient particulate loads)
PM2.5-FL
120
* ^ %
"o O
Nominal Spores Available for Analysis (CFU)
Figure 8. Percent Recovery Efficiencies (Average ± One Standard Deviation of N > 3
Replicates) of Presumptive B. a. Sterne Spores from PM2.5 Filters from Florida Using the
SBA Medium (New, Avg, and High refer to relative ambient particulate load)
26
-------
EPA/600/R-] 9/082
August 2019
PM2.5-MA
120
Nominal Spores Available for Analysis (CFU)
Figure 9. Percent Recovery Efficiencies (Average ± One Standard Deviation of N > 3
Replicates) of Presumptive B. a. Sterne Spores from PM2.5 Filters from Massachusetts
Using the SBA Medium (New, Avg, and High refer to relative ambient particulate loads)
PM2.5-Wl
120
a; 100
o
5 80
g 60
a 40 ¦
o
2"
New
Avg
High
* /so %
s? Sf
'o " "O ° °o
Nominal Spores Available for Analysis (CFU)
Figure 10. Percent Recovery Efficiencies (Average ± One Standard Deviation of N > 3
Replicates) of Presumptive B. a. Sterne Spores from PM2.5 Filters from Wisconsin Using
the SBA Medium (New, Avg, and High refer to relative ambient particulate loads)
27
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EPA/600/R-19/082
August 2019
Figures 11 through 14 include B. a. Sterne spore recovery efficiency plots for PM10 filters
obtained from California, New Hampshire, South Carolina, and Wisconsin. They indicate
generally low (5 to 20%) recovery efficiencies associated with the nominal 150 and 1,500
B. a. Sterne spores available. There were no presumptive B. a. Sterne spores recovered from the
New PM10 filter substrate with a nominal 15 spores available for analysis. Additionally, the
nominal 15-spores available condition for the Wisconsin, Avg filter and New Hampshire, High
filter also yielded no quantifiable presumptive B. a. Sterne colonies. In those instances, the
culture plates had other organism growth that was likely masking colonies of a B. a. Sterne
morphology. Like the PM2.5 filters, the nominal 15 5. a. Sterne spore available filters had the
greatest variability of measured percent recovery, attributed to so few (typically <10
presumptive B. a. Sterne colonies) recovered from the spiked filter. When no presumptive
B. a. Sterne colonies were recovered from spiked filters, it was noted as a false negative.
Filters obtained from California and South Carolina had instances of presumptive B. a. Sterne
colonies counted for the 0-spores available condition, resulting in an overestimate of the number
of true B. a. Sterne spores recovered. Figures 11 and 13 demonstrate that an over-estimation of
recovered spores is most likely with the nominal 15-spore available condition. Specifically, the
percent recovery of presumptive B. a. Sterne colonies from PM10 filters from California for Avg
and High particle loads spiked with 30 spores (Figure 11) was 150 and 55%, respectively. The
infeasibly high spore recovery efficiencies (> 100%) were 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 colony.
Subsequent colony screening of presumptive B. a. Sterne colonies using real-time PCR assays
targeting the chromosomal and pXOl gene targets confirmed that there were instances where the
presumed B. a. Sterne colony was shown not to be B. a. Sterne. Likewise, the South Carolina
filters had background flora collected on the filters that had a colony morphology
indistinguishable from B. a. Sterne but shown to not be B. a. Sterne by PCR analysis screening,
and thus, artificially inflated the percent recovery values. These results demonstrate the
importance of selected confirmation screening of presumptive B. a. Sterne when analyzing
unknown samples from an actual incident. The impact of this background organisms/bacteria on
percent recoveries is more apparent for the nominal 15-spore available condition because a few
additional presumptive B. a. Sterne colonies greatly increases the calculated percent recovery.
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EPA/600/R-19/082
August 2019
For the 150 and 1,500 nominal spore level test conditions, the adverse impact of the background
flora was diluted or suppressed because of greater competition from the B. a. Sterne spores.
120
o\
100
a» 80 ¦
CO
£ 60
Q.
£
40
o
® 20
o
u
3
Replicates) of Presumptive B. a. Sterne Spores from PM10 Filters from California Using
the SBA Medium (New, Avg, and High refer to relative ambient particulate loads)
29
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EPA/600/R-19/082
August 2019
120
£
jS 100
o
Q.
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Q)
C
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PM10-NH
80 ¦
a 60
Q.
£
3
to „ _
o
o
0
cc
20 ¦
New
D
Avg
irb
High
Nominal Spores Available for Analysis (CFU)
Figure 12. Percent Recovery Efficiencies (Average ± One Standard Deviation of N > 3
Replicates) of Presumptive B. a. Sterne Spores from PM10 Filters from New Hampshire
Using the SBA Medium (New, Avg, and High refer to relative ambient particulate loads)
120
PM10-SC
VP
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a 60
a
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3 Three
Replicates) of Presumptive B. a. Sterne Spores from PM10 Filters from South Carolina
Using the SBA Medium (New, Avg, and High refer to relative ambient particulate loads)
30
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EPA/600/R-19/082
August 2019
120
PM10-WI
100
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Nominal Spores Available for Analysis (CFU)
°o
Figure 14 Percent Recovery Efficiencies (Average ± One Standard Deviation of N > 3
Replicates) of Presumptive B. a. Sterne Spores from PM10 Filters from Wisconsin Using
the SBA Medium (New, Avg, and High refer to relative ambient particulate loads)
Average spore recovery efficiencies, for both PM2.5 and PM10 filter types, have the largest
standard deviations associated with the spike condition resulting in nominally 15 spores available
for analysis. This result is, in part, attributed to relatively few (<10 colonies counted per plate)
and/or the confounding effect of the presence of other colonies that have similar morphology that
affect the estimated (presumptive) B. a. Sterne colonies. On average, with 100% recovery, the
4-mL aliquot plated would have five colonies to enumerate. Filters with the 150 and 1,500
nominal spore condition generally have a lower standard deviation as compared to the 15
nominal spore condition because more actual B. a. Sterne spores were recovered. As discussed in
Section 2.3.4, 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-
B. a. Sterne organisms exhibiting an indistinguishable morphology to the microbiologist
counting the colonies.
The fact that the recovery efficiency of the 15-spore available condition for the New media was
the lowest compared to the filters with an ambient particulate load could also indicate that the
applied B. a. Sterne spores were not as efficiently physically recovered from the filter substrate.
31
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EPA/600/R-19/082
August 2019
The existence of the ambient particulate matter of grime or flora on the filter substrate may
reduce the adherence of spiked B. a. Sterne spores to the filter substrate.
The higher recovery efficiency of B. a. Sterne from PM2.5 filters compared to the PM10 filters is
likely attributed to the spiked B. a. Sterne spores being adhered less strongly to the PM2.5 filter
substrate than to that of the PM10 filter substrate. The PM2.5 filters were Teflon (MTL Corp Cat
No. PT47-EP) and PM10 filters quartz fiber (Whatman Cat No. 1851-8531). One possible
explanation is that the applied spores could distribute into the fiber matrix of the PM10 filter
fibers and thus have an opportunity to contact individual fibers as a spore, whereas in the Teflon
filter substrate, such distribution may not occur. The applied spores may remain present as
agglomerates on the surface of the Teflon substrate and more readily removed. Assessing
recovery mechanisms were beyond the scope of this study.
The lower recovery efficiencies of B. a. Sterne from PM10 filters with collected particulate
matter may be attributed to more flora/grime compared to the PM2.5 filters, which may interfere
with accurate quantification of B. a. Sterne. The ambient particulate loading on the Avg PM2.5
filter ranged from 12 to 16 |ig/cm2 compared to 41 to 118 |ig/cm2 for the PM10. Similarly, the
High PM10 filters had an ambient particulate load 3 to 6 times that of the High PM2.5 filters,
with PM2.5 ranging from 29 to 47 |ig/cm2 and PM10 ranging from 104 to 277 |ig/cm2.
The issue of whether the instances of recovery efficiency being less than 100% were due to less
than complete physical recovery of the spores, other physical loss mechanisms such as retention
on processing containers, or interference of growth due to the presence of grime or competing
flora was beyond the scope of the study. 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 by pipetting droplets of a stock suspension may assist or hinder the
ability to physically recover the spores. Spores collected as an aerosol, as would be expected
during normal field operation, may adhere to the filter fiber or collection surface more strongly
than when applied as a droplet of suspension. Conversely, they may be adhered less strongly
because the spores are present with other inert particles that may benefit physical recovery.
When spores are applied as a droplet spike as done in this study, they may more readily disperse
into the extract solution either due to being a large agglomerate or weakly adhered to a surface.
Conversely, the spores applied as a droplet may penetrate the filter substrate by capillary action
32
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EPA/600/R-19/082
August 2019
and be more difficult to physically recover. For those reasons and uncertainty, it is recommended
to consider future research to assess whether the application method matters - whether "spiking"
the filters with B. a. Sterne by aerosolizing and collecting onto the filter via air sampling yields
different results than those obtained in this study.
The presence of other flora collected on the filters during their intended use can bias the average
efficiency high due to counting actual non-5, a. Sterne colonies as B. a. Sterne or bias low
because the background flora competed with the growth of B. a. Sterne and suppressed or
masked B. a. Sterne growth. There were instances when no distinct B. a. Sterne colony
morphology could be discerned, and in those occurrences, the recovery efficiency was reported
as zero.
The uncertainty associated with or introduced by quantifying B. a. Sterne spore recovery
efficiency based solely on colony morphology was revealed by PCR analysis of few
representative colonies in each trial. A total of 76 colonies were screened using colony PCR.
Fifteen (15) of these presumptive B. a. Sterne colonies were confirmed negative for the
B. anthracis chromosome and pXOl targets even though by morphology, these colonies were
indistinguishable from B. a. Sterne, which highlights the importance for genetic confirmation of
culture results. All 15 colonies that were identified incorrectly by morphology came from PM10
filters from two regions, California and South Carolina. Of the 29 colonies that were screened
using colony PCR from these two regions, 52% were negative by real-time PCR analysis. The
presence of a background flora with a morphology that is indistinguishable from B. a. Sterne
artificially increased the percent spore recoveries for PM10 filters from these two regions. Eight
(8) of these colonies were selected from zero spike samples, and by subtracting the number of
colonies that appeared to have a B. a. Sterne morphology from zero spike samples, we could
account for the number of colonies that contributed to the recovery values on spiked samples.
Figure 15 depicts colonies and their morphology used to determine whether the colony was
B. a. Sterne that was representative throughout the analysis of culture plates.
33
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EPA/600/R-19/082
August 2019
H
Figure 15. Colonies Recovered from PM10 California Filters Contained Background with
B. a. Sterne Morphology (the colony on the left was confirmed negative and the colony on
the right was confirmed positive for B. a. Sterne)
Chromogenic growth media (MYP and BBC A) were used to culture sample extracts from a
subset of trials to assess whether it benefited differentiating B. a. Sterne morphology from
background and to reduce the presence of background with the addition of antibiotics
(Polymyxin B and Trimethoprim). The percent spore recoveries on the SBA and MYP growth
media were equivalent when colonies were isolated on both media types. However, the growth of
B. a. Sterne on MYP commonly spread into a lawn, interfering with the ability to accurately
quantify and identify J?, a. Sterne in many instances. Consequently, the MYP medium was no
longer used. The BBCA percent recoveries were 5 to 7 times less than SBA, so use of the BBCA
medium was also discontinued from the analytical methods at the direction of the EPA TOCOR
(Calfee, 2017). A summary of those results and associated discussion are provided in
Appendices J and K.
3.1.2 RV-PCR Method
A summary of the average and standard deviation of the RV-PCR ACt values for the detection of
B. a. Sterne spores recovered from the AQ filter substrate are presented in Table 6 (PM2.5) and
Table 7 (PM10). The ACt results are plotted in Figures 16 through 23 with each plot associated
with one of the eight specific filter types. The summary tables 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
(TU are used; it represents the maximum number of spores available assuming a 100% recovery
34
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EPA/600/R-19/082
August 2019
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 PCR ACt values having to be > 9 to be a positive result.
The RV-PCR results for each air quality filter sample analyzed are presented in Appendix L.
RV-PCR analyses of the PM2.5 filters generated average ACt values between 26.5 and 28.7
(chromosomal and pXOl gene targets) and sample standard deviations typically < 5 for all
locations and particle loads. Figures 16 through 19 indicate there is little difference in the ACt as
a function of ambient particle loading for the filter and spike conditions assessed. (In
Section 3.1.1, the culture results supported the recovery of B. a. Sterne spores from all the PM2.5
filters.) There were instances for the filters obtained from Florida that had a measurable ACt
associated with the 0-spike condition. It is believed that the generated ACt may have been
associated with low-level cross-contamination as those results were obtained early, within the
first 10% of the matrix being completed and resolved within the first 25% of the matrix being
completed, as the method was being implemented. The main issues were associated with leaking
filter vials on the manifold and a warped manifold that were resolved with equipment change
(new manifold implemented), refinement of technique, and rigorous care in method execution.
The originally purchased filter manifold did not consistently seal well. Lawrence Livermore
National Laboratory provided a filter manifold that was more effective at sealing and helped
reduce occurrences of likely cross-contamination (non-zero ACt values for the 0-spike
condition.) Also, there were instances where the PES filter vials exhibited leakage or by-pass
flow that could have led to sample contamination. The impact was only apparent in the 0-spike
condition of the filters analyzed because all other samples purposely (via spiking) contained the
B. a. Sterne target organism.
As shown in Figures 20 through 23, RV-PCR analyses of the PM10 filters typically resulted in
an average ACt > 20 at nominal spores available conditions of 150 or 1,500 and sample standard
deviations < 5. Filters from three of the four geographic regions (South Carolina, California, and
Wisconsin) all exhibited some suppression of ACt magnitude associated with a higher relative
ambient particulate load. Attenuation of the ACt magnitude was more pronounced for the
35
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EPA/600/R-19/082
August 2019
nominal 15 B. a. Sterne spores available condition relative to that of the 150 or 1,500 nominal
spores available, which suggests that the lower limit of detection of the RV-PCR method is near
the nominal 15-spores available condition. Once the nominal B. a. Sterne spore load is at or
above 150, there was little difference in measured ACt for the PM10 filters assessed. 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. Lastly, there were cases with the 15-spore load condition where one or two of the
three replicates had a ACt > 9 and the remainder < 9, further indicating the limit of detection of
the method is being approached.
Nearly half of the 0-spike conditions for all four regions of PM10 filters had at least one sample
with a non-zero ACt. There were instances where TO was a value other than 45, indicating
potential initial contamination, which occurred early in the application of the method. Most all of
these non-zero results were associated with analyses performed early in the study before some
low-level cross-contamination had been minimized (e.g., sample handling technique and
increased glove changes) as the method implementation progressed.
Comparing the ACt values of the PM2.5 filters versus the PM10 shows that the RV-PCR method
was less adversely affected when applied to the PM2.5 filters to that of the PM10 filters. There
was little difference between ACt values measured with the Avg or High ambient particulate
loading condition compared to the New filter for PM2.5 filters, whereas a noticeable attenuation
in ACt was observed for selected PM10 filters, which would suggest that the higher ambient
particulate load on the PM10 filters compared to the PM2.5 filters was interfering with the
method detection limit. Also, there appears to be some consistency in the culture results with that
of the RV-PCR in that the recovery efficiencies for the PM10 were generally lower than that of
the PM2.5. That low recovery efficiency could also partly explain why the RV-PCR detection
limit appears to be approached around the nominal 15 spores available for the PM10 filters but
lower with the PM2.5 filters.
Consistently, throughout all analyses, very good agreement (ACt differed by < 3 between the two
gene targets) was obtained for the chromosomal and pXOl gene targets for both the PM2.5 and
PM10 filters and for all of the nominal spore loads.
36
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EPA/600/R-19/082
August 2019
Table 6. RV-PCR Analyses of PM2.5 Air Quality Filters for Detection of B. a. Sterne
Spores Using Chromosomal and pXOl Gene Targets (N > 3 Replicates)
Ambient
Particle
Load(a)
Spores Available for Analysis
(CFU)
ACt (X ± a)
Location
Nominal(b)
Determined(c)
Chromosomal
pXOl
(jig/cm2)
(X ± o)
Gene Target
Gene Target
0
N/A
0.0 ±0.0
-0.8 ± 1.3
—
(0)
15
18 ±9
27.9 ±0.4
27.1 ± 1.6
(New)
150
180 ± 90
27.5 ±0.8
27.5 ±0.6
1,500
1,800 ± 900
27.1 ±0.2
27.4 ±0.2
0
N/A
-0.1 ±0.2
-0.4 ±0.7
Avg
15
18 ±9
26.5 ± 1.0
25.3 ±3.5
(16)
150
180 ± 90
27.2 ± 1.1
24.8 ±2.4
AZ
1,500
1,800 ± 900
27.0 ± 1.3
26.1 ±2.6
0
N/A
0.0 ±0.0
0.0 ±0.0
High
15
7 ± 0
26.5 ±3.0
26.5 ±2.9
(39)
150
70 ±3
27.7 ±0.9
27.8 ± 1.0
1,500
700 ± 30
27.5 ±0.7
27.9 ±0.8
Avg
(13)
0
N/A
4.8 ±6.5
4.3 ±7.4
15
15 ±9
27.6 ± 1.0
27.9 ± 1.0
150
150 ± 90
27.7 ±0.9
27.9 ±0.9
FL
1,500
1,500 ± 900
26.9 ± 1.4
27.8 ±0.7
0
N/A
3.9 ±6.7
4.1 ±7.1
High
15
15 ±9
27.8 ± 1.4
28.0 ± 1.4
(39)
150
150 ± 90
28.7 ±0.6
29.0 ±0.6
1,500
1,500 ± 900
27.4 ±0.7
27.8 ±0.7
0
N/A
0.0 ±0.0
0.0 ±0.0
Avg
15
14 ±4
27.2 ±0.3
27.4 ±0.4
(12)
150
140 ± 40
27.4 ±0.5
27.5 ±0.5
MA
1,500
1,400 ± 400
27.5 ± 1.0
27.7 ±0.9
0
N/A
0.0 ±0.0
0.0 ±0.0
High
15
14 ±4
27.9 ±0.7
28.0 ±0.7
(29)
150
140 ± 40
27.7 ±0.6
27.9 ±0.4
1,500
1,400 ± 400
27.4 ±0.6
27.7 ±0.4
0
N/A
0.0 ±0.0
0.0 ±0.0
Avg
15
11± 1
26.7 ±3.1
26.8 ±3.2
(14)
150
110 ± 6
28.2 ±0.8
28.4 ±0.7
WI
1,500
1,100 ±60
27.7 ±0.2
28.0 ±0.2
0
N/A
0.0 ±0.0
0.0 ±0.0
High
15
11± 1
27.8 ±0.9
27.9 ± 1.1
(47)
150
110 ± 6
28.2 ±0.4
28.4 ±0.4
1,500
1,100 ±60
27.9± 0.5
28.2 ±0.5
(a) Relative ambient particle load (with measured mass loading per area).
(b) Nominally one-half of the target spore load onto the filter and assuming 100% recovery of spores.
(c) Based on the spiking suspension titer measured each test trial, 100% recovery efficiency, and one-half of
extract used for RV-PCR analysis.
37
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EPA/600/R-19/082
August 2019
Table 7. RV-PCR Analyses of PM10 Air Quality Filters for Detection of B. a. Sterne Spores
Using Chromosomal and pXOl Gene Targets (N > 3 Replicates)
Location
Ambient
Particle
Spores Available for Analysis
(CFU)
ACt (X ± a)
Load(a)
(jig/cm2)
Target(a)
Determined(c)
(X ± o)
Chromosomal
Gene Target
pXOl
Gene Target
0
N/A
2.7 ±4.7
3.0 ±5.3
—
(0)
15
6 ± 3
14.3 ± 11.7
15.5 ± 10.7
(New)
150
60 ±30
26.8 ± 1.1
26.1 ±2.8
1,500
700 ±0
26.6 ±0.5
26.8 ±0.2
0
N/A
0.0 ±0.0
0.0 ±0.0
Avg
15
13 ±5
9.8 ± 17.0
10.0 ± 17.4
(118)
150
130 ±50
23.1 ± 1.8
23.5 ± 1.9
CA
1,500
1,300 ± 500
26.3 ±3.9
26.7 ±4.1
0
N/A
0.0 ±0.0
0.6 ± 1.0
High
15
13 ±5
11.5 ± 15.8
12.4 ± 15.6
(277)
150
130 ±50
26.5 ±3.5
26.8 ±3.2
1,500
1,300 ±500
28.0 ± 1.2
28.4 ±0.9
0
N/A
1.7 ±2.9
5.4 ±4.2
Avg
15
14 ± 10
21.8 ± 11.3
22.1 ± 10.7
(69)
150
140 ±100
26.7 ± 1.3
26.9 ± 1.3
NH
1,500
1,400 ± 1000
27.5 ±0.2
27.8 ±0.4
0
N/A
0.0 ±0.0
3.3 ±5.7
High
15
14 ± 10
14.1 ± 11.7
15.7 ± 10.1
(199)
150
140 ±100
25.5 ±3.0
25.6 ±3.0
1,500
1,400 ± 1000
25.8 ±0.9
26.1 ±0.8
0
N/A
0.0 ±0.0
0.0 ±0.0
Avg
15
14 ± 1
13.8 ± 12.2
14.0 ± 12.4
(65)
150
140 ±6
28.1 ±0.5
28.2 ±0.4
SC
1,500
1,400 ± 60
25.0 ±3.4
25.2 ±3.3
0
N/A
0.0 ±0.0
0.0 ±0.0
High
15
14 ± 1
10.8 ±9.4
10.9 ±9.5
(132)
150
140 ±6
22.5 ±6.0
22.8 ±6.0
1,500
1,400 ± 60
23.8 ±1.5
24.2 ± 1.4
0
N/A
0.0 ±0.0
0.0 ±0.1
Avg
15
9 ± 0
14.5 ± 10.9
17.0 ±7.5
(41)
150
90 ±0
24.0 ± 1.2
24.6 ± 1.4
WI
1,500
900 ±0
27.5 ± 1.4
28.1 ± 1.5
0
N/A
12.1 ± 1.6
10.2 ±2.9
High
15
6 ± 1
16.5 ±9.2
14.1 ±9.1
(104)
150
60 ±30
23.3 ±4.2
23.6 ±3.9
1,500
900 ± 500
26.3 ± 1.2
26.3 ±0.8
(a) Relative ambient particle load (with measured mass loading per area).
(b) Nominally one-half of the target spore load onto the filter and assuming 100% recovery of spores.
(c) Based on the spiking suspension titer measured each test trial, 100% recovery efficiency, and one-half of
extract used for RV-PCR analysis.
38
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EPA/600/R-] 9/082
August 2019
35
PM2.5-AZ
New
Avg
High
30
25
< 20
10 ¦
ill fi
ih
r
AX
rinrh Hhrh
I Chromosome i i pXOl Threshold
so
%
/ft /ft- /* o
Nominal B. a. Sterne Spores Available for Analysis (CFU)
Figure 16. RV-PCR Analysis of B. a. Sterne Spores Recovered from PM2.5 Filters from
Arizona Using Chromosomal and pXOl Gene Targets (Average ± One Standard Deviation
for N > 3 Replicates); Positive Response Equals ACt > 9 (New, Avg, and High refer to
relative ambient particulate loads)
35
30
25
< 20 ^
CD
> 15
PM2.5-FL
10
New
-Jl rnr+i
.Eli
Avg
i¥i
High
I Chromosome
IpXOl
- - Threshold
I.I ii II ii i.i ii I.I ii i.i ii II ii I.I
\
o
/ft
/<
\
Nominal B. a. Sterne Spores Available for Analysis (CFU)
Figure 17. RV-PCR Analysis of B. a. Sterne Spores Recovered from PM2.5 Filters from
Florida Using Chromosomal and pXOl Gene Targets (Average ± One Standard Deviation
for N > 3 Replicates); Positive Response Equals ACt > 9 (New, Avg, and High refer to
relative ambient particulate loads)
39
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EPA/600/R-] 9/082
August 2019
35
30
25
4 20
PM2.5- MA
15
10 •
New
ifl
Avg
pir^i dbF"
High
irfl ninFh irti
5 ¦
I Chromosome
IpXOl
Threshold
<5- °
\
X
Nominal B. a. Sterne Spores Available for Analysis (CFU)
Figure 18. RV-PCR Analysis of B. a. Sterne Spores Recovered from PM2.5 Filters from
Massachusetts Using Chromosomal and pXOl Gene Targets (Average + One Standard
Deviation for N > 3 Replicates); Positive Response Equals ACt > 9 (New, Avg, and High
refer to relative ambient particulate loads)
PM2.5-Wl
New
r*l rL rtirfi
Avg
r-
High
Aril
J
I Chromosome
I pXOl
%o °
Nominal B. a. Sterne Spores Available for Analysis (CFU)
%
%
Figure 19. RV-PCR Analysis of B. a. Sterne Spores Recovered from PM2.5 Filters from
Wisconsin Using Chromosomal and pXOl Gene Targets (Average ± One Standard
Deviation for N > 3 Replicates); Positive Response Equals ACt > 9 (New, Avg, and High
refer to relative ambient particulate loads)
40
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EPA/600/R-19/082
August 2019
35
30
25 ¦
< 20
15 ¦
PM10-CA
10
5 ¦
CD
New
Avg
±10
High
I pXOl — — Threshold
-i.
¦ i
r —i—* i—1 i—1 i 1 i—1 ¦—r
iV o
S S0 "Sq.
Nominal B. a. Sterne Spores Available for Analysis (CFU)
Figure 20. RV-PCR Analysis of B. a. Sterne Spores Recovered from PM10 Filters from
California Using Chromosomal and pXOl Gene Targets (Average ± One Standard
Deviation for N > 3 Replicates); Positive Response Equals ACt > 9 (New, Avg, and High
refer to relative ambient particulate loads)
35
30
25 ¦
y 20
o>
00
p
PM10-NH
10 -
New
in A
Avg
High
ifirh
—.I—i in
no
I Chromosome
I pXOl
~
n
so "%>,
'¦s>
<<¦
'o °o
Nominal B. a. Sterne Spores Available for Analysis (CFU)
<$•
X
Figure 21. RV-PCR Analysis of B. a. Sterne Spores Recovered from PM10 Filters from
New Hampshire Using Chromosomal and pXOl Gene Targets (Average ± One Standard
Deviation for N > 3 Replicates); Positive Response Equals ACt > 9 (New, Avg, and High
refer to relative ambient particulate loads)
41
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EPA/600/R-] 9/082
August 2019
35
PM10-SC
30 -
25 ¦
^ 20
15 -
10 -
New
Avg
High
ir. *
r*ir*i
5 ¦
JH
I Chromosome
TT
I pXOl — — Threshold
1,1111, III
i 11 r
i—1 r
O A A O /a ^ /a o a /a
s •$> '% s so -bQ s so s-o0
Nominal B. a. Sterne Spores Available for Analysis (CPU)
Figure 22. RV-PCR Analysis of B. a. Sterne Spores Recovered from PM10 Filters from
South Carolina Using Chromosomal and pXOl Gene Targets (Average ± One Standard
Deviation for N > 3 Replicates); Positive Response Equals ACt > 9 (New, Avg, and High
refer to relative ambient particulate loads)
35
PM10-Wl
New
Avg
High
30 ¦
25
« 20
0)
5
10 ¦
n
Hfl
r
nh
rfi
5 -
HL
I Chromosome
IpXOl
Threshold
I—1 r
3 Replicates); Positive Response Equals ACt > 9 (New, Avg, and High
refer to relative ambient particulate loads)
42
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EPA/600/R-19/082
August 2019
3.2 Non-AQ Filter Analyses Results
3.2.1 Culture Method
A summary of the average and standard deviation of the measured recovery efficiencies of
presumptive B. a. Sterne spores recovered from the non-AQ filter substrate spiked with
B. a. Sterne spores and using the SB A medium to culture are presented in Table 8. 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 quantity of presumptive B. a. Sterne colonies reported in the
tables is half of the actual total recovered because in the context of the tables, only half of the
extract samples was made available for analysis. The quantity of presumptive B. a. Sterne
colonies for each sample, used in the percent recovery calculations, are reported in Appendix M
for the SBA culture medium. The recovery efficiencies are based on the culture method using the
SBA medium. When the MYP chromogenic agar was also used in the culture method, the
recovery efficiencies are reported in Appendix N.
The recovery efficiencies are plotted in Figures 25 through 28, one plot for each filter type. Note,
a percent recovery is not tabulated or plotted for the 0-spore spike condition since, by definition,
a meaningful recovery efficiency cannot be calculated, even though there could have been a
finite number of presumptive B. a. Sterne colonies counted based on colony morphology.
43
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EPA/600/R-19/082
August 2019
Table 8. Recovery Efficiencies for Presumptive B. a. Sterne Spores from Non-Air Quality
Filters Cultured in the SBA Medium (N > 3 Replicates)
Spores Available for Analysis
Spores
Spore
Recovery
Efficiency
(%)
(X±a)
Filter
Duty
Stage(a)
Sample
(CFU)
Recovered
Type
Reps
Nominal(b)
Determined
(X ± a)(c)
(CFU)
(X ± o)(d)
5
0
0
0
N/A
New
3
15
20 ±4
5.2 ±6.5
24 ±30
3
150
200 ± 40
13 ± 11
6.0 ±4.8
3
1,500
2,000 ± 400
100 ±37
5.3 ± 1.7
3
0
0
2.1 ± 1.8
N/A
Bus
Mid
3
15
13 ±7
6.3 ±5.4
50 ±52
Engine
3
150
130 ± 70
21 ±7.9
21 ± 13
3
1,500
1,300 ± 700
170 ±38
14 ±6.0
5
0
0
1.6 ± 1.8
N/A
End
5
15
13 ±8
19 ± 5.1
190 ±110
5
150
130 ± 80
27 ± 10
27 ± 16
3
1,500
2,000 ± 400
340 ±140
17 ±4.4
4
0
0
0
N/A
New
4
15
18 ± 1
8.6 ±3.0
48 ± 16
3
150
160 ± 10
50 ±23
32 ± 13
3
1,500
1,600 ± 100
600 ±81
38 ±7.9
3
0
0
0
N/A
Building
Mid
3
15
13 ±7
2.1 ± 1.8
23 ±20
HVAC
3
150
130 ± 70
47 ±24
38 ± 16
3
1,500
1,300 ± 700
600 ±120
53 ±22
4
0
0
47 ±56
N/A
End
4
15
18 ± 1
31 ± 16
170 ±79
4
150
180 ± 10
69 ±22
39 ± 15
4
1,500
1,800 ± 100
740 ±180
43 ± 13
3
0
0
0
N/A
New
3
15
10 ±3
10 ±3.6
110 ± 59
3
150
100 ± 30
47 ±29
43 ± 16
3
1,500
1,000 ± 300
590 ± 64
61 ± 19
3
0
0
13 ± 13
N/A
Subway
Mid
3
15
17 ±3
9.4 ±3.1
59 ±27
Platform
3
150
170 ± 40
70 ± 12
42 ±9.0
3
1,500
1,700 ± 300
450 ±51
28 ±7.0
3
0
0
21 ±26
N/A
End
3
15
10 ±3
25 ±22
290 ±250
3
150
100 ± 30
58 ±29
57 ±30
3
1,500
1,000 ± 300
500 ± 280
56 ±37
44
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EPA/600/R-19/082
August 2019
Table 8. Recovery Efficiencies for Presumptive B. a. Sterne Spores from Non-Air Quality
Filters Cultured in the SBA Medium (N > 3 Replicates) (Cont.)
Filter
Type
Duty
Stage(a)
Sample
Reps
Spores Available for Analysis
(CFU)
Spores
Recovered
(CFU)
(X ± o)(d)
Spore
Recovery
Nominal(b)
Determined
(X ± a)(c)
Efficiency
(%)
(X ± o)(e)
3
0
0
0
N/A
New
3
15
28 ± 12
14 ± 1.8
57 ±28.6
3
150
280 ± 120
160 ± 46
63 ± 15.1
3
1,500
2,800 ± 1200
1100 ±530
38 ± 3.1
Subway
Rolling
Stock
3
0
0
6.3 ±8.8
N/A
Mid
3
15
17 ±3
13 ±22
60 ± 100
3
150
170 ± 40
50 ±33
31 ±24
3
1,500
1,700 ± 300
250 ±330
16 ±22
3
0
0
8.3 ±7.2
N/A
End
3
15
28 ± 12
29 ±26
130 ±25
3
150
280 ± 120
58 ±71
25 ±20
3
1,500
2,800 ± 1200
UD(fl
UD
(a) Relative ambient particulate loading.
(b) Nominally one-half of the target spore load onto the filter and assuming 100% recovery of spores.
(c) Based on the spiking suspension titer measured each test trial, 100% recovery efficiency, and one-half of extract
used for culture analysis.
(d) Presumptive B. a. Sterne colonies based on morphology and one-half of extract used for culture analysis
(e) Calculated using the actual spore loading on each filter and presumptive B. a. Sterne spores recovered on each
filter sample.
(f) Undetermined (UD) due to excess growth of collected ambient organisms on the filter and/or presumptive
B. a. Sterne.
The basic trends, observations, general results, and discussion provided in Section 3.1.1 for the
culture results (percent recovery efficiencies) for the air quality type filters applies here with the
non-AQ filters. Most notable is the relatively large sample standard deviation and apparent
recovery efficiencies exceeding 100% associated with the nominal 15-spores-available condition
that are attributed to few B. a. Sterne spores recovered and/or impact of background flora that
could bias the presumptive B. a. Sterne spore count high or low.
As examples, the highest overall spore recovery efficiencies approached 60% for the subway
platform filters when loaded with nominal 150 to 1,500 spores available to a low of 0% (no
colonies with B. a. Sterne morphology could be identified). The bus engine filter has the lowest
percent recovery of the non-AQ filters when the filters were New. The subway rolling stock
filters appeared (by visual observation) to be the dirtiest of the non-AQ filters and had an
abundance of background flora that complicated the identification and quantification of
45
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EPA/600/R-] 9/082
August 2019
recovered B. a. Sterne. Figure 24 shows photographs of representative culture plates depicting
the amount of background flora and grime that adversely affected B. a. Sterne quantification.
Figure 24. Subway Rolling Stock End-of-Service Filter Spiked with 30, 300, or 3,000 Spores
(from left to right, respectively)
120
O
a
to
CO
nj
CO
CL
£
3
CO
0)
Sm
CL
100
80
60
40
CL>
cn
Bus Engine
New
Mid
OZL
End
%
Nominal Spores Available for Analysis (CFU)
Figure 25. Percent Recovery Efficiencies (Average ± One Standard Deviation of N > 3
Replicates) of Presumptive B. a. Sterne Spores from Bus Engine Filters Using the SBA
Medium (New, Mid, and End refer to service life or duty cycle of the filter)
46
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EPA/600/R-] 9/082
August 2019
Building HVAC
120
g 100
Q.
IS)
Q)
C
o> 80
*->
to
nj
cd
§ 60
Q.
E
3
ill
cu 40
Q-
H—
o
% 20
o
u
Q)
C£
New
Mid
a
End
^ ^ %
Nominal Spores Available for Analysis (CFU)
Figure 26. Percent Recovery Efficiencies (Average ± One Standard Deviation of N > 3
Replicates) of Presumptive B. a. Sterne Spores from Building HVAC Filters Using the SBA
Medium (New, Mid, and End refer to service life or duty cycle of the filter)
120
Subway Platform
100
c
80
Q.
E
3
Ul
Q) 40
o
g 20
o
New
Mid
^ % %
Nominal Spores Available for Analysis (CFU)
Figure 27. Percent Recovery Efficiencies (Average ± One Standard Deviation of N > 3
Replicates) of Presumptive B. a. Sterne Spores from Subway Platform Filters Using the
SBA Medium (New, Mid, and End refer to service life or duty cycle of the filter)
47
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EPA/600/R-] 9/082
August 2019
120
£ 100
o
a
(/i
0)
=
5 80
Subway Rolling Stock
60
a
E
3
Replicates) of Presumptive B. a. Sterne Spores from Subway Rolling Stock Filters Using
the SBA Medium (New, Mid, and End refer to service life or duty cycle of the filter)
A representative, qualitative illustration of how dirty the filters were, and the associated
suspension extract, is shown in Figure 29 for a bus engine filter. There was a range of observable
discoloration and/or noticeable suspended particulate matter and the bus engine filter represented
one of the dirtier filters. The images demonstrate that the analytical methods were applied to very
challenging matrices and were far from being applied to pristine samples in a laboratory setting
as that associated with method development.
> 1 • * * " v-v* *. •
•
v , Tr / X
Figure 29. Bus Engine End-of-Service Life Filter Deconstructed Prior to Spore Recovery
(left) and Suspension After Recovered from Bus Engine Filters New (unused) in Top Row
and End-of-Service Life in Bottom Row of RV-PCR Vacuum Manifold
48
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EPA/600/R-19/082
August 2019
3.2.2 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 the non-AQ filter substrates are presented in
Table 9. The ACt results are plotted in Figures 30 through 33, with each plot associated with one
of the four specific filter types. The summary tables 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 non-AQ filter sample analyzed are presented in Appendix O.
New non-AQ filters from bus engine, building HVAC, subway platform, and subway rolling
stock generated a ACt value between 20.8 and 28.0 (chromosome and pXOl target PCR assays).
For used filters, both Mid and End, the ACt values increase with spore load, indicating inhibition
from background, either due to growth inhibition during overnight incubation (enrichment step)
or molecular inhibition of the real-time PCR assays, not due to physical recovery because the
new filter material response for all spore loads are consistently above a ACt of 20. (The ACt
values for both real-time PCR assay targets (chromosomal and pXOl) tracked similarly for each
sample extract throughout the study.)
49
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EPA/600/R-19/082
August 2019
Table 9. RV-PCR Analyses of Non-Air Quality Filters for Detection of B. a. Sterne Spores
Using Chromosomal and pXOl Gene Targets (N > 3 Replicates)
Filter
Duty
Spores Available for Analysis
(CFU)
ACt (X ± a)
Type
Stage(a)
Actual(b)
Determined(c)
(X ± o)
Chromosomal
Gene Target
pXOl
Gene Target
0
0
2.8 ±4.2
3.0 ±3.4
New
15
20 ±4
27.7 ± 1.1
27.6 ± 1.0
150
200 ± 40
27.5 ±0.9
27.7 ± 1.0
1,500
2,000 ± 400
28.0 ±0.1
27.6 ±0.8
0
0
0.0 ±0.0
0.4 ±0.7
Bus
Mid
15
13 ±7
14.1 ±4.1
14.1 ±3.3
Engine
150
130 ±70
20.1 ±3.2
20.5 ±2.8
1,500
1,300 ±700
22.0 ±1.7
22.8 ± 1.5
0
0
4.5 ±6.1
4.9 ±6.2
End
15
13 ±8
14.9 ±3.9
15.8 ±3.4
150
130 ±80
19.2 ±4.2
19.9 ± 3.8
1,500
2,000 ± 400
24.1 ±2.0
24.7 ± 1.9
0
0
7.1 ±3.6
9.3 ± 1.8
New
15
18 ± 1
25.7 ± 1.5
26.4 ± 1.3
150
160 ± 10
20.8 ±5.1
21.2 ±5.1
1,500
1,600 ± 100
27.3 ± 1.2
27.6 ± 1.0
0
0
0.0 ±0.0
-0.3 ±0.5
Building
Mid
15
13 ±7
16.4 ± 1.1
16.9 ±1.2
HVAC
150
130 ±70
22.5 ± 1.0
22.9 ±0.8
1,500
1,300 ±700
26.4 ± 1.2
26.9 ±1.1
0
0
6.3 ±6.2
9.2 ±3.6
End
15
18 ± 1
16.9 ±4.0
17.1 ±3.8
150
180 ± 10
19.2 ± 1.7
19.8 ±1.8
1,500
1,800 ± 100
23.8 ± 1.9
24.5 ± 1.9
0
0
3.9 ±3.4
3.7 ±4.3
New
15
10 ±3
22.1 ± 10.9
19.3 ± 13.2
150
100 ±30
24.9 ±5.8
24.9 ±6.3
1,500
1,000 ±300
23.5 ±3.5
22.1 ±4.8
0
0
0.0 ±0.0
0.0 ±0.0
Subway
Mid
15
17 ±3
11.0 ± 9.7
11.2 ± 9.8
Platform
150
170 ±40
19.6 ±0.1
20.0 ±0.2
1,500
1,700 ±300
20.0 ± 1.3
20.5 ± 1.2
0
0
3.0 ±5.2
1.7 ±2.7
End
15
10 ±3
11.6 ± 4.8
11.2 ± 6.4
150
100 ±30
15.8 ± 1.8
13.1 ±3.7
1,500
1,000 ±300
18.6 ±5.2
18.5 ±5.3
50
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EPA/600/R-19/082
August 2019
Table 9. RV-PCR Analyses of Non-Air Quality Filters for Detection of B. a. Sterne Spores
Using Chromosomal and pXOl Gene Targets (N > 3 Replicates) (Cont.)
Filter
Duty
Spores Available for Analysis
(CFU)
ACt (X ± a)
Type
Stage(a)
Actual(b)
Determined(c)
(X ± o)
Chromosomal
Gene Target
pXOl
Gene Target
0
0
5.3 ±5.2
6.7 ±5.9
New
15
28 ± 12
26.5 ±3.5
26.5 ±3.5
150
280±120
27.7 ±0.7
27.9 ±0.7
1,500
2,800± 1200
27.6 ±0.8
27.4 ±0.4
Subway
Rolling
Stock
0
0
0.0 ±0.0
0.0 ±0.0
Mid
15
17 ±3
8.9 ±7.8
9.2 ± 8.1
150
170 ±40
17.8 ±2.7
18.3 ± 2.7
1,500
1,700 ±300
20.3 ± 1.7
20.9 ± 1.9
0
0
9.0 ±7.8
9.1 ±7.9
End
15
28 ± 12
12.0 ±5.3
13.9 ±3.1
150
280±120
17.2 ±3.1
17.9 ± 3.0
1,500
2,800± 1200
18.1 ±4.1
18.8 ± 4.0
(a) Relative ambient particle loading.
(b) Nominally one-half of the target spore load onto the filter and assuming 100% recovery of spores.
(c) Based on the spiking suspension titer measured each test trial, 100% recovery efficiency, and one-half of
extract used for RV-PCR analysis.
In all instances, the ACt value for both chromosomal and pXOl gene targets were > 9 when the
filters had nominally 15 spores available for analysis. Consistently, very good agreement
(average ACt differed by < 3 between the two gene targets) was obtained for the chromosomal
and pXOl gene targets for both all non-AQ filter types and for all of the nominal spore loads.
There were instances (for example, with the subway rolling stock filter and its end-of-service life
condition) where the ACt for the 0-spike condition was > 9, indicating a positive presence of
B. a. Sterne. Those non-zero ACt values were primarily associated with samples tested early in
the study and were likely due to low-level cross-contamination that was subsequently eliminated
through rigorous glove change-out, refinement of method execution technique, and extreme care
in procedure execution. The originally purchased filter manifold did not consistently seal well.
Lawrence Livermore National Laboratory provided a filter manifold that was more effective at
sealing and helped reduce occurrences of likely cross-contamination (non-zero ACt values for
the 0-spike condition.) Also, there were instances where the PES filter vials exhibited leakage or
by-pass flow that could have led to sample contamination. The impact was only apparent in the
0-spike condition of the filters analyzed because all other samples purposely (via spiking)
contained the B. a. Sterne target organism.
51
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EPA/600/R-19/082
August 2019
The RV-PCR method was adversely affected by the collection of ambient particulate matter
collected on the non-AQ filters. The ACt values were highest for the New filters of each filter
type compared to the ACt values for the Mid or End cycle filters for all quantities of B. a. Sterne
spores available for analysis. Although the RV-PR response was attenuated, the RV-PCR method
was able to detect the presence of B. a. Sterne. Only for the subway platform and the subway
rolling stock were there instances where the nominal 15-spores-available condition did not yield
a positive response. (On average, ACt > 9 was achieved for the subway platform filters, but there
was one 15-spore spike condition for the New and one for the Mid service life that had one or
both the chromosomal and pXOl gene targets with ACt < 9, and there were two replicates for the
End service life that had ACt < 9. Similarly, on average, ACt > 9 was achieved for the subway
rolling stock filters, but there was one 15-spore-spike condition for the Mid and one for the End
service life that had one or both the chromosomal and pXOl gene targets with ACt < 9.
(See Appendix O for individual test replicate results.) The ACt values were the highest for the
New filters for all four filter types for all three spore loading levels, which would suggest that the
higher ambient particulate load on the filter had some interfering effect. The RV-PCR method
detection across the four filters was around the 15-spores-available threshold, similar to that for
the PM2.5 AQ filters.
52
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EPA/600/R-] 9/082
August 2019
35
Bus Engine
30 ¦
25 ¦
< 20
Q)
bfl
2
| 15
10 -
New
rinnh nbnh
m
Mid
End
II
In
5 -
~n
E3ET
I Chromosome i i pX03 — — Threshold
rh.l II 1,1 II 1,1 II 1,1 II IJX
A
1 *
I*
"O "O
Nominal B. a. Sterne Spores Available for Analysis (CFU)
%
Figure 30. RV-PCR Analysis of B. a. Sterne Spores Recovered from Bus Engine Filters
Using Chromosomal and pXOl Gene Targets (Average ± One Standard Deviation for
N > 3 Replicates); Positive Response Equals ACt > 9 (New, Mid, and End refer to service
life or duty cycle of the filter)
35
30
25
Building HVAC
< 20
10
New
"I
"in
Mid
rf
r+in
—I
-1
End
n
n
IpXOl
1.1 II II II I.I II II II r
So
0 %
V "o
Nominal B. a. Sterne Spores Available for Analysis (CFU)
Figure 31. RV-PCR Analysis of B. a. Sterne Spores Recovered from Building HVAC Filters
Using Chromosomal and pXOl Gene Targets (Average ± One Standard Deviation for
N > 3 Replicates); Positive Response Equals ACt > 9 (New, Mid, and End refer to service
life or duty cycle of the filter)
53
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EPA/600/R-] 9/082
August 2019
35
30 -
25 -
^ 20
§ 15
<
10 ¦
5 ¦
Subway Platform
New
Mid
End
irfi
IpXOl
~a
_
A "V •?«
A O
s so
Nominal B. a. Sterne Spores Available for Analysis (CFU)
"V
Figure 32. RV-PCR Analysis of B. a. Sterne Spores Recovered from Subway Platform
Filters Using Chromosomal and pXOl Gene Targets (Average ± One Standard Deviation
for N > 3 Replicates); Positive Response Equals ACt > 9 (New, Mid, and End refer to
service life or duty cycle of the filter)
35
Subway Rolling Stock
30 ¦
25 ¦
^ 20
ai
00
n
10 -
New
Mid
End
ir*l ir^i
D
-i
B
5 -
I Chromosome
HI
pXOl — — Threshold
HT
i r
o
t r-1 ¦—t—1 " ¦—r
°
Nominal B. a. Sterne Spores Available for Analysis (CFU)
s s0
Figure 33. RV-PCR Analysis of B. a. Sterne Spores Recovered from Subway Rolling Stock
Filters Using Chromosomal and pXOl Gene Targets (Average ± One Standard Deviation
for N > 3 Replicates); Positive Response Equals ACt > 9 (New, Mid, and End refer to
service life or duty cycle of the filter)
54
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EPA/600/R-19/082
August 2019
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 culture method, false positive was defined as the identification
(counting) of one or more presumptive B. a. Sterne spores when none were spiked onto the filter;
false negative was defined as when no presumptive B. a. Sterne spores were counted, yet the
filter was spiked, and an accurate detection when either no spores were identified in the 0-spike
condition or identified for a filter spike condition. The positive identification for RV-PCR is as
defined in Section 2.3.5 (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
filter that was not spiked. A summary of those results is presented in Table 10 and expressed as
percentage for each filter type assessed.
The false negative detections for culture were associated with either the 0-spike condition or
when 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 false negative for RV-PCR were believed to be due, in part, to poor physical recovery of
spores from the filter as well as likely some loss in sensitivity due to the ambient particulate
matter recovered along with the B. a. Sterne spores. The false positives for RV-PCR are
attributed to likely cross-contamination that was suspected to have occurred early in the study as
the method was being implemented.
Both methods performed poorest for the PM10 filters, which in part was believed due to having
the lowest percent recovery of applied B. a. Sterne spores. The overall accuracy of the method
properly detecting B. a. Sterne (combined true positives and true negatives) were 82% for culture
and 85% for RV-PCR.
Table 11 gives similar summary of method response comparison as Table 10, but as a measure of
the consistency of both methods yielding the same response of whether B. a. Sterne was detected
or not.
55
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EPA/600/R-19/082
August 2019
Table 10. Summary of the Accuracy of the Method Response to Detect B. a. Sterne
Filter
Type
Total Number of Samples
Culture / Molecular Response
Spiked
Not
Spiked
Total
True
Positive(a)
True
Negative(b)
False
Positive(c)
False
Negative(d)
PM2.5
81
27
108
79/81
24/25
3/2
2/0
98%/ 100%
89% / 93%
11%/7.4%
2.5% 10%
PM10
82
29
111
64/68
22/25
7/4
18/14
78% / 83%
76%/86%
24%/ 14%
22%/ 17%
Bus
31
12
43
29/31
8/11
4/2
2/0
94%/ 100%
67%/92%
33%/ 17%
6.5% / 0%
HVAC
31
11
42
30/31
8/8
3/3
1/0
97%/ 100%
73%/73%
27% / 27%
3.2%/0%
Platform
27
9
36
25/23
5/9
4/0
2/4
93%/85%
56%/
100%
44%/0%
7.4%/ 15%
Rolling
Stock
27
9
36
22/25
6/6
3/3
5/2
81%/93%
67%/67%
33%/33%
19%/7.7%
Sum
279
97
376
249 / 259
73/74
24/14
30/20
All Filter
Types
89% / 93%
75%/76%
25%/ 14%
10%/7%
(a) Number of positive responses (for each analytical method) to filters that were spiked with B. a. Sterne;
percentage calculated by dividing number of occurrences by number of spiked samples.
(b) Number of negative responses (for each analytical method) to filters that were not spiked with B. a. Sterne;
percentage calculated by dividing number of occurrences by number of samples not spiked.
(c) Number of positive responses (for each analytical method) to filters that were not spiked with B. a. Sterne;
percentage calculated by dividing number of occurrences by number of samples not spiked.
(d) Number of negative responses (for each analytical method) to filters that were spiked with B. a. Sterne;
percentage calculated by dividing number of occurrences by number of spiked samples.
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Table 11. Positive and Negative B. a. Sterne Detection Frequency for Culture and
Molecular Analysis Methods
Filter
Type
Total Number of Samples
Culture / Molecular Response
Spiked
Not
Spiked
Total
Pos/Pos(a)
Neg/Neg^
Pos/Neg(c)
Neg/Pos(d)
PM2.5
81
27
108
79
22
3
4
97.5%
81.5%
2.8%
3.7%
PM10
82
29
111
59
27
12
13
72.0%
93.1%
10.8%
11.7%
Bus
31
12
43
29
8
2
4
93.5%
66.7%
4.7%
9.3%
HVAC
31
11
42
30
7
2
3
96.8%
63.6%
4.8%
7.1%
Platform
27
9
36
23
7
6
0
85.2%
77.8%
16.7%
0.0%
Rolling
Stock
27
9
36
24
6
1
5
88.9%
66.7%
2.8%
13.9%
Sum
279
97
376
244
77
26
29
Percent
87.5%
79.4%
6.9%
7.7%
(a) Both the culture and molecular responses positively identified the presence of B. a. Sterne correctly in samples
that were spiked; percentage calculated by dividing number of occurrences by number of spiked samples.
(b) Both the culture and molecular responses correctly gave a negative response to the presence of B. a. Sterne in
samples that were not spiked; percentage calculated by dividing number of occurrences by number of samples
not spiked.
(c) Culture yielded a positive response for B. a. Sterne and molecular response was negative for the presence of
B. a. Sterne; percentage calculated by dividing number of occurrences by number of samples analyzed.
(d) Culture yielded a negative response for B. a. Sterne and molecular response was positive for the presence of
B. a. Sterne; percentage calculated by dividing number of occurrences by number of samples analyzed.
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3.4 Ancillary Results
3.4.1 PES I'.v PVDF Membrane Filter Vials
One unexpected outcome of this project was the determination of the importance of consumable
availability via supply chain. In the case of an actual event, time to results will be of utmost
importance, and a method will need to be flexible enough to endure shortages of supplies. The
method used here was tested in this way because of a back-order situation for the filter vials for
the RV-PCR method that had to be resolved. During testing, an order for 10 boxes of filter vials
with polyethersulfone (PES) membranes was placed in mid-October 2017 (GE Healthcare, Cat.
No. AV125NPUPSU) and placed onto backorder and did not arrive until August 8, 2018. When
the order was placed in mid-October, multiple vendors were showing the item number in stock.
However, follow up with the vendors indicated they were not available, and the expected
shipment date continued to be pushed to a later date. While in communication with the
manufacturer (GE Healthcare), a filter vial with polyvinyldiene difluoride (PVDF) membrane
was identified that was also available for similar applications and is manufactured as a stock item
rather than a made-to-order item.
The filter vials with PES and PVDF membrane were compared side-by-side by spiking spores
directly into the first extraction buffer (PBS with Tween 20 and Ethanol) followed by filtration
then RV-PCR analysis. The test matrix in Table 12 shows the test matrix and number of
replicates used to determine if the two product numbers were equivalent. Figures 34 and 35 show
how the ACt and the final Ct values compare between the two different membranes. The binary
result of "positive" or "negative" was unaffected and all ACt values were > 25 for all spike
levels; the lone exception was a sample that leaked during enrichment (ACt was 23). All matched
pair Tfmai Ct values were within ±1.1 with the PVDF Tfmai Ct generally higher compared to its
PES mate, indicating slightly less sensitivity. After this evaluation, testing continued using PES
filter vials.
Table 12. Test Matrix for Comparing PES and PVDF Membranes
Target Spore Loading
PES Membrane (replicates)
PVDF Membrane (replicates)
0
2
2
1
4
4
150
5
5
1,500
5
5
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EPA/600/R-19/082
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PVDF vs PES ACt (pXOl Assay)
Target Spore Load (CFU)
Figure 34. Side-by-Side Analysis of ACt Values Generated During the RV-PCR using
PES and PVDF Filter Vials
Final Ct Value (pXOl Assay) for PVDF and PES Membrane
20.0
19.5
19.0
18.5
4-»
18.0
u
17.5
c
'll.
17.0
16.5
16.0
15.5
15.0
¦ KVUh (puxij
¦ PES (pXOl)
¦
¦
¦
¦
¦
1
¦
¦
1
1
m
¦
¦
i—
i—
H
t—
H
H
rH
rsl
ro
t
LO
r<
LO
LO
o
O
o
O
o
o
rH
rH
ui
un
LO
o
o
o
rH
rH
rH
LH
rH
in
rH
LO
rH
Target Spore Load (CFU)
Figure 35. Side-by-Side Analysis of Final Ct Values Generated During the RV-PCR using
PES and PVDF Filter Vials
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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 NAF testing included positive and negative controls for both spread
plate samples and qPCR. In addition, both the spore bank and B. a. Sterne spike suspension
concentrations (CFU/mL) were measured for each test so that known quantities of spores spiked
onto the filter sample could be made.
Positive controls (PC) and no template controls (NTC) were included for each RV-PCR assay
and in all cases performed as expected, with Ct values consistently in the mid-20s for the 50 pg
PC and no Ct value generated for NTCs. 7500 Fast system performance was assessed according
to internal standard operating procedure (SOPs) and maintained at regular intervals, monthly
(optical and background calibration), every 6 months (dye calibration), and annual (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.
4.3 Operational Parameters
Micropipettes, thermometers, and timers used were calibrated against a traceable standard at
regular intervals (every 6 months or annual) and used only within acceptable calibration interval
established by internal SOPs.
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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 13 summarizes the PE audits that were performed. A 20-|iL
pipette (C20267) used for Master Mix addition and sample addition was found out of tolerance
on 13 August 2018. Volumes pipetted were 5 |iL (sample addition) and 20 |iL (Master Mix
addition) for RV-PCR analysis. The pipette was evaluated at three set volumes, 2 |iL, 10 |iL, and
20 |iL and measurements ranged from 2.207 to 2.247; 10.183 to 10.334; and 20.247 to 20.407
for each of the set volumes, which is outside of specifications for internal SOPs. Controls on
RV-PCR assay performed as expected and the pipette was adjusted before being returned to
service.
Table 13. Performance Evaluation Audits
Measurement
Audit
Allowable
Actual
Procedure
Tolerance
Tolerance
Passed calibration as
Volume of liquid from
micropipettes
Gravimetric evaluation
± 10%
found/as returned with one
exception (C20267), as
described above
Time
Compared to independent
clock
± 2 seconds/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. The 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 the 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
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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.
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 PES membrane type of filter vial syringeless filter vials (GE Healthcare, Cat.
AV125NPUPSU) was not always readily available from distributors and the lead time to
manufacture was quoted to be 45 business days. The PES membrane type is a made-to-
order product. The PVDF membrane type was a stock item and thus more readily
available. Ten (10) boxes of PES filter vials were backordered from October 2017 to
August 2018 (10 months). Both filter types were determined suitable for the RV-PCR
assay.
• The RV-PCR method requires great care and diligence to implement effectively. Most
notably, the RV-PCR method required changing gloves between samples for each step,
which is onerous and time consuming. However, it was found necessary to minimize
cross-contamination.
• Mixing the samples in the platform vortex resulted in loose lids. The method was revised
from the original recommended 30-mL tubes to BD Falcon brand 50-mL conical tubes,
and Parafilm was used to seal the lids during platform vortex steps.
• 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 filter analyses were performed in batches of 16 filter samples per trial using a single
system based on initial trials to implement the methods. The 16 filter 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 four consecutive
days of operation, starting with filter sample spiking during the day and drying overnight.
(Had these been actual filters collected post-biological release, this spiking activity
would, obviously, not be performed by the ERLN.)
• A 16-hour incubation 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 AQ and non-AQ 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
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performed had these been actual field samples. This task included time to prepare
the stock suspensions, enumerate stock suspension, spike the filters, and complete
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.
• 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.
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6.0 CONCLUSIONS AND RECOMMENDATIONS
The foremost conclusion is that filters recovered from both AQ and non-AQ filters may be useful
to analyze for B. anthracis to help map the extent of contamination from biological incidents,
recognizing there are limitations to their use. This conclusion is made based on the data showing
that, even in the presence of other particulate matter having been collected on filters, B. a. Sterne
spores that were spiked onto the filters could be recovered and successfully analyzed; however,
the study results clearly indicate that the background flora and other particulate matter can
adversely impact the method sensitivity and accuracy. Consequently, the NAF could be used to
supplement results from other sampling plans but should not be relied upon solely as the
definitive biological warfare incident mapping tool.
Both the culture and molecular methods can be adversely affected by the presence of ambient
particulate matter on the filter being assessed. The methods' sensitivities were impacted by the
presence of collected ambient particulate matter.
The filter substrate composition and/or structure may be important factor in the end-to-end
performance of the methods because it could affect the physical recovery of organisms from the
NAF being assessed. Furthermore, the results reported in this study are caveated by the fact that
the NAFs were spiked by applying suspension droplets of B. a. Sterne, and that application
method could impact physical recovery of B. a. Sterne spores.
The foremost recommendation is to assess the impact that spiking of B. a. Sterne spores onto the
NAF 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 generating an aerosol of B. a. Sterne and
then pulling the aerosol-laden air through the NAF rather than applying spores via a liquid
suspension spike. The EPA method would then be implemented to recover and analyze for
B. a. Sterne. This approach is expected to primarily affect spore recovery, which then may
impact detection limits and or identification accuracy.
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Other key conclusions and recommendations from the study include:
• RV-PCR can be used to positively identify viable B. a. Sterne in the presence of complex,
dirty sample matrices of NAFs; however, background flora and grime also collected and
present on the filter can impact the lower limit of detection and/or reduce the response to
B. a. Sterne.
• Background flora and non-living material (dirt/grime) interferes with identification and
quantifying B. a. Sterne using the traditional plate culture method, particularly for non-
AQ filters. Presumptive B. a. Sterne colonies may not actually be B. a. Sterne because
background flora can have an indistinguishable colony morphology, leading to false
positives and an over-estimation of the number of actual B. a. Sterne spores. Conversely,
the apparent B. a. Sterne quantity recovered can be biased low due to suppression of
B. a. Sterne growth with competing background flora. It is possible for so much
background flora to be present on NAFs such that the presence of B. a. Sterne cannot be
made, potentially leading to false negatives.
• Priority should be placed on analyzing filters having the lowest loading of background
particulate matter, to the extent that can be determined by the shortest duty cycle of non-
AQ filters or by gravimetric analysis of AQ filters.
• Priority should be placed on use of PM2.5 filters over PM10 filters when both are
available from the same area and would have been operating at an appropriate time
relative to an incident.
• Recommend that the consumable supplies to execute the method be assessed for their
availability in sufficient quantities to process the number of anticipated samples from an
event. An alternative source of the consumable or qualifying an acceptable alternative
material is recommended so that there is no single point failure in the supply chain.
• There was no apparent benefit of using a chromogenic agar for the filters tested. MYP
media yielded results that were comparable to those obtained with SBA. BBCA media
containing the selective supplement (Oxoid Cat. No. SR0230) yielded percent recovery
of B. a. Sterne 5 to 7 times less than the counts from SBA.
The results from this study may be useful for sample plan development and interpretation of
results following a large-scale biological incident where native air sample types provide utility or
are available.
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7.0 REFERENCES
Ackelsberg, J., et al. (2011). "The NYC Native Air Sampling Pilot Project: Using HVAC Filter
Data for Urban Biological Incident Characterization." Biosecur Bioterror 9(3): 213-224.
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, W. Personal Communication; Meeting minutes from in-progress review meeting hosted
at Battelle, September 24 and 25, 2017.
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.
Serre, S. and L. Oudejans. Underground Transport Restoration (UTR) Operational Technology
Demonstration. U.S. Environmental Protection Agency, Washington, D.C., EPA/600/R-
17/272, 2017.
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.
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.
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SAMPLE ANALYSIS OF NATIVE AIR
FILTERS FOR CHARACTERIZATION
AND EXTENT MAPPING OF
BIOLOGICAL INCIDENTS
APPENDICES A-0
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LIST OF APPENDICES
Page
APPENDIX A. FORMULATIONS OF RECIPES USED IN BIOLOGICAL TEST
METHODS A-Error! Bookmark not defined.
APPENDIX B. DUPLEX VERSUS SINGLEPLEX REAL-TIME PCR SPOT REPORT.. B-
Error! Bookmark not defined.
APPENDIX C. WORK INSTRUCTION FOR DOSING FILTER SWATCHES WITH
BACILLUS ANTHRACIS SPORES C-Error! Bookmark not defined.
APPENDIX D. WORK INSTRUCTION FOR BACILLUS ANTHRACIS SPORE
RECOVERY D-Error! Bookmark not defined.
APPENDIX E. WORK INSTRUCTION FOR CULTURE OF BACILLUS
ANTHRACIS SPORES RECOVERED FROM AIR FILTERS E-Error!
Bookmark not defined.
APPENDIX F. WORK INSTRUCTION FOR MANUAL DNA EXTRACTION AND
PURIFICATION FROM BACILLUS ANTHRACIS SPORES F-Error!
Bookmark not defined.
APPENDIX G. WORK INSTRUCTION DRV-PCR FOR BACILLUS ANTHRACIS
SPORES G-Error! Bookmark not defined.
APPENDIX H. WORK INSTRUCTION FOR SELECTING PRESUMPTIVE
BACILLUS ANTHRACIS STERNE COLONIES FOR QPCR
CONFIRMATION H-Error! Bookmark not defined.
APPENDIX I. CULTURE RESULTS FOR AIR QUALITY FILTERS USING SHEEP
BLOOD AGAR MEDIUM I-Error! Bookmark not defined.
APPENDIX J. CULTURE RESULTS FOR AIR QUALITY FILTERS USING MYP
MEDIUM J-Error! Bookmark not defined.
APPENDIX K CULTURE RESULTS FOR AIR QUALITY FILTERS USING BBCA
MEDIUM K-Error! Bookmark not defined.
APPENDIX L. RV-PCR RESULTS FOR AIR QUALITY FILTERS USING
CHROMOSOMAL AND PXOl GENE TARGETS L-Error! Bookmark not
defined.
APPENDIX M. CULTURE RESULTS FOR NON-AIR QUALITY FILTERS USING
SHEEP BLOOD AGAR MEDIUM M-Error! Bookmark not defined
APPENDIX N. CULTURE RESULTS FOR NON-AIR QUALITY FILTERS USING
MYP MEDIUM N-Error! Bookmark not defined.
APPENDIX O. RV-PCR RESULTS FOR NON-AIR QUALITY FILTERS USING
CHROMOSOMAL AND PXOl GENE TARGETS O-Error! Bookmark not
defined.
69
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APPENDIX A. FORMULATIONS OF RECIPES USED
IN BIOLOGICAL TEST METHODS
A-l
-------
Spore Prod uction
Table 1. Components of Modified G Sporulation Medium
Ingredient
Amount/L
Yeast Extract
2,0 g
(Xllit-SO,
2,0 g
CaCh • 2H2O
0,03 g
CuSOi • 5H2O
0.005 g
I cS()., • 7H20
0.0005 g
MgS04 • 7H20
0.2 g
M11SO4 • 11 ;•<) ¦•
0.06 g
Z11SO4 • 7H20
0.005 g
K2HPO4
0.5 g
dH20
1000 111L
* \IiiS( >i • H2O substituted for M11SO1 • 4H2O. If MnS04 • 4H2O is used, add 0.05 g.
Table 2. Components of Leighton-Doi Sporulation Medium
Component
Amount/L
KC1
1.88 g
CaCh
0.29 g
FeSG4 x 7 H20
0.003 g
M11SO1 x H2O
0.0017 g
MgSO, x 7 !!;()
0.025 g
Dextrose
0.9 g
Nutrient Broth
16.0 g
A-2
-------
Table 3. Duplex Assay Conditions
Component (Duplex Assay) Volume for one reaction (fiL)
2x FAST PGR Mix 12.5
PCR-grade water 1.5
pXOl For Primer (25 p.M) 1
pXOl Rev Primer (25 |iM) 1
pXOl Probe (2 jiM) 1
chromosome For Primer (25 (0.M) 1
chromosome Rev Primer (25 u \ 1 i 1
cliromosome Probe (2 |iM) 1
Template 5
Total volume 25
A-3
-------
APPENDIX B. DUPLEX VERSUS SINGLEPLEX
REAL-TIME PCR SPOT REPORT
B-l
-------
SPOT REPORT
on
Task 4 Analysis Method Comparison: Duplex versus Singleplex PCR
Prepared under
Contract Number EP-C-15-002
Task Order Number 0009
EPA Task Order Contracting Officer Representative
Worth Calfee
Prepared by
Battelle
Columbus, Ohio 43201
11/23/16
B-2
-------
Purpose
The purpose of this experiment was to determine whether the Bacillus anthracis chromosome
and pXOI real-time PCR assays from Letant et al. (2011) and the EPA protocol EPA/600/R-
12/577 (2012) can be performed as a duplex assay. This experiment will also determine the
LOQ of the DNA (DNA) extracted from B. anthracis Sterne 34F2 (NR-1400, BEI Resources) and
the limit of detection of the spore suspension that will be used to dose filters for this study. If
successful, the duplex assay will produce the same quality data as the assays run in singleplex,
and thus the duplex assay can be used instead of the singleplex assays, increasing the
efficiency of analysis. The duplex method was the analytical method proposed, but does differ
from the EPA's current singleplex approach. The duplex assay proposed by Battelle also
differed from the EPA's RV-PCR method in that the DNA is not extracted prior to analysis, and
instead the assay is run directly from spores. If the assay is successful in detecting and
quantifying directly from spores, this will also increase the efficiency of the assay by removal of
the DNA extraction step from the analytical process.
Materials and Methods
Materials
The materials used in the conduct of the assay are listed below:
• Bacillus anthracis Sterne 34F2 (NR-1400, BEI Resources) DNA.
• Bacillus anthracis Sterne 34F2 (NR-1400, BEI Resources) spore suspension.
• TaqMan Fast Advanced PCR Master Mix (4444557, Life Technologies).
• Unlabeled Sequence Detection Primers, (4304971, Life Technologies).
• Custom TaqMan Probe-MGBNFQ, (4316034, Life Technologies).
• MicroAmp Fast Optical 96-well Reaction Plate with Barcode (4346903, Life
Technologies).
• MicroAmp Optical 8-Cap strips (4323032, Life Technologies).
Methods
The duplex master mix (Table 1) and singleplex master mixes (Table 2) were distributed into a
single 96-well reaction plate. The master mix was prepared using the conditions described in
the 2012 EPA protocol except that the non-fluorescent quencher of the DNA probes for this
method were minor groove binder (MGB) groups rather than Black Hole Quencher.
B-3
-------
Table 1. Duplex Assay Conditions
Component (Duplex Assay)
Volume for One Reaction (pi)
2x FAST PCR Mix
12.5
PCR-qrade water
1.5
pXOI For Primer (25 pM)
1
pXOI Rev Primer (25 pM)
1
pXOI Probe (2 uM)
1
chromosome For Primer (25 pM)
1
chromosome Rev Primer (25 mM)
1
chromosome Probe (2 pM)
1
DNA Template
5
Total volume
25
Table 2. Singleplex Assay Conditions
Component (Singleplex Assay)
Volume for One Reaction (pL)
2x FAST PCR Mix
12.5
PCR-qrade water
4.5
pXOI or Chromosome For Primer (25 pM)
1
pXOI or Chromosome Rev Primer (25 pM)
1
pXOI or Chromosome Probe (2 pM)
1
DNA Template
5
Total volume
25
A 10-fold dilution series of DNA extracted from Bacillus anthracis Sterne 34F2 (NR-1400, BEI
Resources) ranging from 50 ferntograms to 5 x 10® femtograms per 5 |jL was used as a
standard curve for the singleplex assays and the duplex assay. An additional four reactions of
the DNA were included for each assay at 5 x 104 femtograms (50 pg) because the EPA method
specified running positive controls at this concentration, A 10-fold dilution series of unextracted
Bacillus anthracis Sterne 34F2 spores ranging from 5 spores to 5 x 10s spores per 5 pL was
also included on each assay. No template controls (negative controls) were run in quadruplicate
for each assay.
The reaction plate was run on a 7500 Fast Dx Real-Time PGR Instrument (Applied Biosystems)
using the conditions outlined in Table 3 and data was analyzed using 7500 Fast System
Software v1.4.0. The cycling conditions were set as described in the 2012 EPA method except
that the extension time was increased from 20 seconds to 30 seconds in this method. This
change was made because the thermocycler would not accept an extension time of less than 24
seconds and the Battelle real-time PGR assay for BaS is conducted with 30s extension times.
B-4
-------
Table 3. Thermocycler Conditions on 7500 Fast
Stage
Temperature (°C)
Time (Min:Sec)
Repeat
Ramp Rate
1
50
2:00
1
Auto (Fast 7500 Mode)
95
10:00
Auto (Fast 7500 Mode)
2
95
0:05
45
Auto (Fast 7500 Mode)
60
0:30*
Auto (Fast 7500 Mode)
*7500 Fast does not allow an extension time of less than 24 seconds. The 2012 EPA method specifies an
extension time of 20 seconds.
Limit of detection was defined as the lowest concentration of spores that resulted in a signal that
crossed the threshold for both replicates. The limit of quantification was defined as the lowest
quantification was the lowest concentration of the DNA standard curve that resulted in a signal
that crossed the threshold for both replicates. A Student's paired t-test was performed on the Ct
values from the singleplex compared to the duplex and duplex assay was considered successful
when there was no significant difference found between the duplex and singleplex (P-value
greater than 0.05 = no significant difference between the assays). Ct values of 36 or higher
were excluded from analysis or when one of the two replicates was undetected.
B-5
-------
I- " ults ar, ' r'i .cussion
The results of this experiment demonstrate that the Bacillus anthracis chromosome and pXOI
real-time PCR assays can be performed in a duplex format to reduce the reagent cost and labor
associated with screening B. anthracis Sterne DNA (Table 4; chromosome, paired Student's t-
test P-value = 0.747; Table 5: pXOI, paired Student's t-test P-value = 0.354) and unextracted
spores (Table 6: chromosome, paired Student's t-test P-value = 0.554;
Table 7: pXOI, paired Student's t-test P-value = 0.305).
Table 4. Chromosome Real-Time PCR Assay with DISIA
Concentration
(DNA in fg)
Singleplex
Ct Values
Singleplex
Average
Ct
Singleplex
Standard
Deviation
Duplex Ct
Values
Duplex
Average
Ct
Duplex
Standard
Deviation
%ACt ((singleplex-
duplex)/singleplex)
5 x 106
15.3
15.3
0.0
15.1
15.2
0.1
0.6%
5 x 10e
15.3
15.3
5 x 105
18.7
18.6
0.1
18.5
18.5
0.0
0.4%
5 x 105
18.5
18.5
5x 104
21.8
22.1
0.2
22.1
22.0
0.2
0.2%
5x10*
22.0
22.0
5x 104*
22.1
22,2
5 x 104*
22.1
22.2
5 x 104*
22.2
22,1
5 x 104*
22.3
21,8
5 x 103
25.6
25.6
0.1
25.5
25.5
0.1
0.1%
5 x 103
25,5
25,6
5 x 102
29.0
29.0
0.0
29.4
29.4
0.1
-1.1%
5 x 102
29.0
29,3
5 x 101
33.5
32.8
1.0
32.7
32.5
0.2
0.9%
5 x 101
32.1
32,3
*These replicates were run as unknown samples, so were not included in the standard curve. The EPA method recommended
running three positive controls at 50 pg (5 x 104fg) per 96- well plate, so an additional four wells were included at this
concentration per assay.
B-6
-------
Table 5. pX01 Real-Time PGR Assay with DNA
Concentration
(DNA in fg)
Singleplex
Ct Values
Singleplex
Average
Ct
Singleplex
Duplex Ct
Values
Duplex
Average
Ct
Duplex
%ACt ((singleplex-
duplex)/singleplex)
Standard
Deviation
Standard
Deviation
5 x 106
16.3
16.3
0.0
16.0
16.0
0.1
1.6%
5 x 106
16.2
16.0
5 x 105
19.5
19.5
0.0
19.2
19.2
0.1
1.1%
5 x 105
19.5
19.3
5 x 104
22.9
22.9
0.0
22.8
22.7
0.1
0.9%
5 x 104
22.9
22.8
5 x 104*
22.9
22.8
5 x 104*
22.9
22.6
5 x 104*
23.0
22.7
5 x 104*
22.9
22.5
5 x 103
26.4
26.3
0.2
26.2
26.2
0.0
0.2%
5 x 103
26.1
26.2
5 x 102
29.7
29.8
0.1
29.7
29.5
0.3
1.1%
5 x 102
29.9
29.2
5x "ICS1
31.7
31.7
N/A
33.1
32.9
0.3
N/A
5 x 101
Undetected
32.7
*These replicates were run as unknown samples, so were not included in the standard curve. The EPA method
recommended running three positive controls at 50 pg (5 x 104fg) per 96- well plate, so an additional four wells were
included at this concentration per assay.
B-7
-------
Table 6. Chromosome Real-Time PCR Assay Results with Spore Preparation
Concentration
(spores)
Singleplex
Ct Values
Singleplex
Average
Ct
Singleplex
Standard
Deviation
Duplex
Ct
Values
Duplex
Average
Ct
Duplex
Standard
Deviation
%ACt ((singleplex-
duplex)/singleplex)
5 x 105
21.2
21,0
0.3
21.2
21.4
0.3
-2.0%
5 x 105
20,8
21.6
5 x 104
24.8
24.7
0.0
25.0
24.9
0.2
-0.5%
5 x 104
24.7
24.7
5x 10s
28.2
28,3
0.2
28.4
28.3
0,2
0.2%
5 x 103
28.5
28.2
5 x 102
32.1
32.2
0.2
32.0
32.1
0.1
0.4%
5 x 102
32.3
32.1
5 x 101
36,1
35.9
0.3
39.8
39.8
0,1
N/A
5 x 101
35.7
39.7
5x 10°
Undetected
45.0
N/A
37.2
39.2
2.9
N/A
5x 10°
Undetected
41.2
Table 7. pXOI Real-Time PCR Assay Results with Spore Preparation
Concentration
(spores)
Singleplex
Ct Values
Singleplex
Average
Ct
Singleplex
Standard
Deviation
Duplex Ct
Values
Duplex
Average
Ct
Duplex
%ACt ((singleplex-
duplex)/singleplex)
Standard
Deviation
5x 105
21.3
21.3
0.1
21.2
21.2
0.1
0,4%
5 x 10®
21.2
21.1
5 x 104
24.6
24.6
0.0
24.5
24.5
0.0
0.6%
5x10"
24.7
24.5
5 x 103
28.2
28.2
0.0
28.3
28.2
0.2
-0.1%
5 x 103
28.2
28.1
5 x 102
31.5
31.4
0,1
31.6
31.5
0.1
-0.2%
5 x 102
31.3
31.4
5 x 101
35.7
34.6
1.5
36.3
36.3
0.1
N/A
5 x 101
33.5
36.2
5x 10°
Undetected
45.0
N/A
33.6
35.9
3.2
N/A
5x 10°
Undetected
38.1
B-8
-------
The assay performance (slope, intercept and R2 values, Table 8) from the DNA standard curve
did not show significant change nor did the cycle threshold (Ct) values when the assays were
run individually or in the duplex format on either spores or extracted DNA (Figures 1 and 2), All
negative controls were undetected when run in duplex or singleplex assay format. Furthermore,
the percent delta Ct (defined as (singleplex Ct- duplex CT)/singleplex Ct)) for each input (spore
or DNA) was calculated and since all %ACT were within 2% (Tables 4 through 8), this further
supports that the performance of the duplex matches that of the singleplex assay.
B-9
-------
BaS Chromosome Assay
femtograms of gDNA
BaS Chromosome As:
i
5
1.0E+G0 1.0E+01 1.0E+02 1.0E+03 1.0E+04 1.0E+05 1DE+06
Figure 1. Chromosome Real-Time PCR Assay Results. Top: DNA titration. Bottom: spore
titration
B-10
-------
BaS pXOl Assay (gDNA)
femtograms of gDNA
BaS pXOl Assay (spores)
1.0E+00 l.OE+Ol 1.0E+02 1.0E+03 1.0E+04 1.0E+05 1.0E+06
BaS Spores
Figure 2. pXOl Real-Time PCR Assay Results. Top: DNAand Bottom: spores
B-ll
-------
Table 8. Standard Curve (DNA) Performance per Assay
Assay
Slope
Intercept
R2
Singleplex
Chromosome assay
-3,50
38.58
0.997
Duplex Chromosome
assay
-3.50
38.57
0.999
Singleplex pXOI assay
-3.25
38.13
0.996
Duplex pXOI assay
-3.38
38.64
0.999
For the B, anthracis Sterne chromosome target, the limit of quantification was 50 femtograms of
DNA when run in a singleplex or duplex format; and the limit of detection (two replicates) was
500 spores for the singleplex and the duplex format (Table 9).
Table 9. Summary of Limit of Quantification and Limit of Detection for Both Assays When
Run in Singleplex or Duplex Format
Assay
Limit of
quantification
(DNA)
DNA
(P-value*)
Limit of
detection
(spores)
spores
(P-value*)
Singleplex
Chromosome
50 fg
0.747
50
0.554
Duplex
Chromosome
50 fg
5
Singleplex pXOI
500 fg
0.354
50
0.305
Duplex pXOI
50 fg
5
*P-vaiue from paired Student's t-test comparing Ct values between the singleplex and duplex assays on DNA
or spores for each target.
For the B, anthracis Sterne pXOI target, the limit of quantification was 500 femtograms of Total
DNA when run in a singleplex format (one of the replicates at 50 fg being undetected) and 50
femtograms when run in a duplex format; and the limit of detection (two replicates) was 50
spores for both the singleplex format and the duplex format.
B-12
-------
Conclusio ;ommendations
The duplex assay was able to perform as well as the singleplex assays targeting the
B, anthracis chromosome and pXOI targets, detecting as low as 50 ferntograms of extracted
DNA. Both the duplex and singleplex assays were able to amplify directly from spores, without
the need for DNA extraction; with the ability to accurately detect the presence of 500 spores
from the chromosome target or 50 spores from the pXOI target. This difference in detection
between the chromosome and pXOI targets may be due to a difference in copy number present
in the spores.
We recommended to proceed with testing of spiked filters using the duplex RV-PCR method
proposed. The duplex assay produces the same data as the singleplex assay as demonstrated
by Student's paired t-test and will save both time and reagent costs. Assaying directly from
spores produces signal that can be used to reliably detect down to 500 spores. This method
further reduces the time and cost of the assay by removing the DNA extraction step and
reagents. To further determine the LOD for direct analysis from spores, a titration between 500
and 50 spores will need to be conducted, as at 50 and 5 spores some signal is produced,
however, it was not consistent or linear. Therefore, based on the test conducted direct testing
from spores recovered from the filters is recommended for filter swatches spiked with greater
than 500 spores to reduce cost and ensure accurate detection and quantification. Taken
together, the duplex RV-PCR method to directly assay spores recovered from the filter will
streamline the RV-PCR method and is in line with the method originally proposed for this study.
The results of the duplex RV-PCR method are equivalent to that of singleplex RV-PCR, and
both can be used directly on spores for accurate detection of down to 500 spores.
B-13
-------
APPENDIX C. WORK INSTRUCTION FOR DOSING FILTER
SWATCHES WITH BACILLUS ANTHRACIS SPORES
C-l
-------
WORK INSTRUCTION FOR DOSING FILTER SWATCHES WITH BACILLUS ANTHRACIS SPORES
I. PURPOSE/SCOPE
To dose filter swatches 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 anthrads
34F2 spores
Inhouse
34F2101716
TBD
2-8 "C
Petri dishes
R.T.
Sterile Dl water
Teknova
R.T.
Blood Agar
BBL
15 ml tubes
N/A
R.T.
1.5 or 2 ml tubes
Eppendorf
N/A
R.T.
Sterile forceps
N/A
R.T.
Polyester Mesh
McMasterCarr
8218T13
N/A
R.T.
Equipment
Item
Manufacturer
Serial Number
Thermometer
.T.i-i's
Calibration
Due
Initials &
Date
Biosafety
Cabinet (BSC)
The Baker Company
57553
N/A
9/2018
Micropipette
Type±1000
Rainin
N/A
Micropipette
Type;l200
Rainin
N/A
Micropipette
Type120
Rainin
N/A
vortex
VWR
N/A
N/A
N/A
Refrigerator
Fisher
C3274822
115
4/2018
N/A= Not Applicable
Other Supplies and Equipment
• Micropipette filter tips
• Biohazard bags
• Bench coat
Page 1 of 5
Native Filters Wl- DOSE-l-v5
C-2
-------
WORK INSTRUCTION FOR DOSING FILTER SWATCHES WITH BACILLUS ANTHRACIS SPORES
• Filters
IV, PROCEDURE
Decontaminate the BSC with DNA Erase, bleach and isopropanol prior to use.
1. Decontaminated by Date
Cut mesh support and treat with UV.
1. Remove sterile scissors from packaging a wipe with DNA erase, bleach and Isopropanol.
2. Cut 4 cm2 mesh support and expose to UV crosslinker for 5 minutes.
UV start time End time Initials/date
Cut filter swatches
1. Label petri dishes with the corresponding Filter ID,
2. With sterile scissors, cut out the appropriate number of filter swatches from the filter
with the dimensions 4 cm2 (See diagram).
a. For 47 mm filters, these will be cut into quarters, leading to slightly smaller than
4 cm2 swatches.
3. Zero-spike filter swatches will be added directly to 50 mL conical tubes and mesh
support will be placed over the filter swatch prior to opening B. a. Sterne stock tubes in
the Biosafety cabinet
4. Using sterile forceps, transfer the filter swatches to the labeled petri dishes.
Name filters
1. Use the T09Filter_Sample_Log_l\!omenclature added.xlsx (in Native Filters box folder) to
determine the filter ID for each filter.
i. AAA-BBB-CCC-R#-Date
1. AAA = Filter Type
2. BBB = Geographical Region
3. CCC = Particle Loading
4. R#= Replicate number
5. Date= MM/DD/YY
ii. Electronically populate below table with sample names to be prepared on each
day from the Sample Log.
Page 2 of 5
Native Filters Wl- DOSE-l-v5
C-3
-------
WORK INSTRUCTION FOR DOSING FILTER SWATCHES WITH BACILLUS ANTHRACIS SPORES
Sample
#
Filter
type
Geographic
Region
Particle
Load
Filter
Vial
Type
Spore
Spike
level
Filter ID
Date
Cut
/initials
Date
Dosed
/initials
1
PM10
5C
AVG
PVDF
0
l-PMlO-SC-AVG-O
2
PM10
5C
AVG
PVDF
0
2-PM10-SC-AVG-0
3
PM10
SC
AVG
PVDF
30
3-PM10-SC-AVG-30
4
PM10
5C
AVG
PVDF
30
4- P M10-SC-A VG- 30
5
PM10
5C
AVG
PVDF
300
5-PM10-SC-AVG-300
6
PM10
SC
AVG
PVDF
300
6- P M 10-SC-AVG- 300
7
PM10
SC
AVG
PVDF
3,000
7- P M 10-SC-AVG- 3,000
8
PM10
SC
AVG
PVDF
3,000
8-PM10-SC-AVG-3,000
9
PM10
SC
High
PVDF
0
9-PM10-SC-High-0
10
PM10
SC
High
PVDF
0
10-PM10-SC-High-0
11
PM10
SC
High
PVDF
30
ll-PM10-SC-High-30
12
PM10
SC
High
PVDF
30
12-PM10-SC-High-30
13
PM1Q
SC
High
PVDF
300
13-PM10-SC-High-300
14
PM10
SC
High
PVDF
300
14-PM10-SC-High-3(X)
15
PM10
SC
High
PVDF
3,000
15-PM10-SC-High-3,000
16
PM10
SC
High
PVDF
3,000
16-PM10-SC-High-3,000
E. 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
3.2 X 10H cfu/mL
04/09/2018
ii. Target stock concentration(s).
Stock #
Organism
Lot
Prep
date
Concentration
Total spores
per 100 pL
Entered/verified
by:
1
B.
anthracis
Sterne
34F2101716
3.0 X 10*
cfu/mL
3,000
2
B.
anthracis
Sterne
34F2101716
3.0 X 103
ctu/mL
300
3
B.
anthracis
Sterne
34F2101716
3.0 X 102
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:
Page 3 of 5
Native Filters Wl- DOSE-l-v5
C-4
-------
WORK INSTRUCTION FOR DOSING FILTER SWATCHES WITH BACILLUS ANTHRACIS SPORES
(3.2 X 108 cfu/mL)*(X)=(3.0 X 107cfu/mL)(lmL) -» 94pL of sample into 906nL H20
(3.0 X 107 cfu/mL)*(X)=(3.0 X 106cfu/mL)(lmL) -» 100nL of sample into 900(iL H20
(3.0 X106 cfu/mL)*(X)=(3.0 X 10scfu/mL)(lmL) IOOjxL of sample into 900jiL H20
(3.0 X 10s cfu/mL)*(X)=(3.0 X 104cfu/mL)(lmL) IOOjiL of sample into 900nL H20
(3.0 X 104 cfu/mL)*(X)=(3.0 X 103cfu/mL)(lmL) -> 100nL of sample into 900nL H20
(3.0 X 103 cfu/mL)*(X)=(3,0 X 102cfu/mL)(lmL) -> 100nL of sample into 900nL H20
Dilutions Prepared By: Date/Initials:
2. Dose swatches
Geographi
c Region
Filter
Type
Particle
Load
Spike Levels
Filter ID
South
Carolina
PM10
Avg
0/30/300/3000
Q9563526 (04/27/2011) & Q0548769
(09/30/2011). Consume the Q9563526 filter
first, if extra material is needed use Q0548769.
Note Filter IDs used on Wis.
High
0/30/300/3000
Q9563551 (07/02/2011) & Q9563559
(07/20/2011). Consume the Q95635S1 filter
first, if extra material is needed use Q9563559.
Note Filter IDs used on Wis.
i. Prior to dosing filters, immediately vortex the stock for 30 seconds.
ii. Per swatch, transfer a 120 |iL aliquot of the appropriate Stock tube (Low, Med.,
or High) into a 1.5 ml tube.
iii. Place twenty 5 (iL droplets onto each filter swatch as shown in the below
diagram. The same pipet tip can be used to place all twenty droplets, dispose of
the 120 [iL aliquot once each swatch has been dosed.
iv. Air dry in BSC overnight.
Start time: Date/Initials;
End time: Date/Initials:
v. Cover filter swatches with top to petri dish, use Parafilm to seal the petri dishes,
and store at 4°C until ready for recovery.
3. Enumerate stock
i. Serially dilute the suspension in Sterile water (if necessary).
1. Fill 2mL dilution tubes for each sample with 900pL of Sterile water and
label appropriately.
2. Vortex the stock on high for 30 seconds.
3. Transfer 100 pL of the stock into the first dilution tube containing 900(jL
of Sterile water. Recap the tube and vortex it on high for 30 seconds.
This is the 10 suspension.
Page 4 of 5
Native Filters Wl- DOSE-l-v5
C-5
-------
WORK INSTRUCTION FOR DOSING FILTER SWATCHES WITH BACILLUS ANTHRACIS SPORES
ii. Spread 100 aliquots of dilutions onto Blood Agar in triplicate.
iii. 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:
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 10" cfu/mL)
Blood Agar
100 jiL/
no-1)
2 (3.0 X103 cfu/mL)
Blood Agar
100 juL/
(10 ')
3 (3.0 X102cfu/mL)
Blood Agar
100 JUL/
(lO"1)
Recorded By: Date/Initials:
4 cm2 swatch 1/4 of 48 mm filter swatch
Twenty ~5 |il drops
Figure 1. Dispersal layout for dosing filters.
Page 5 of 5
Native Filters Wl- DOSE-l-v5
C-6
-------
EPA/600/R-19/082
10/8/2019
APPENDIX D. WORK INSTRUCTION IOR IS A CILL IS A Ml IRA CIS
SPORE RECOVERY
D-l
-------
EPA/600/R-19/082
10/8/2019
WORK INSTRUCTION FOR BACILLUS ANTHRACIS SPORE RECOVERY
i. PURPOSE/SCOPE
To recover B. anthracis spores from air filters following the BACILLUS Analytical Methods 004 published
by the EPA December 2012.
II. MATERIALS/EQUIPMENT
Materials
Item
extraction buffer with
Tween® 20
Manufacturer
Inhouse
Lot Number
Exp.
Date
Stoiagc
Temp.
2-8 °C
Initials & Date
extraction buffer
without Tween® 20
Inhouse
2-8 °C
10X PBS
Teknova
2-8
IX PBS (pH 7.4)
Teknova
2-8
BHI broth
Inhouse
2-8
Conical tubes, 15 mL
N/A
R.T.
Conical Tube 50mL
N/A
R.T.
Screw top flask, 250
mL
Corning
N/A
R.T.
0.45 urn filter vials
Whatmon
N/A
R.T.
2mL screw cap tubes
VWR
N/A
R.T.
N/A = Not Applicable
Equipment
Item
Manufacturer
Serial Number
Thermometer/
Rees W
Calibration
Due
Initials & Date
Biosafety
Cabinet (BSC)
The Baker Company
5755B
N/A
9/2018
Micropipette
Type:L1000
Rainin
N/A
Incubator
Shaker
New Brunswick
590644988
C25323
7/18/18
Refrigerator
Fisher
35840
115
4/2018
Platform
Vortexer
VWR
5041
N/A
N/A
N/A = Not Applicable
Page lof 7
Native Filters Wl-SPORE RECOVERY-2-v4 (04/09/2018)
D-2
-------
EPA/600/R-19/082
10/8/2019
WORK INSTRUCTION FOR BACILLUS ANTHRACIS SPORE RECOVERY
Filters - Electronically update this table with samples names from the Sample Log
Sample
ft
Filter
type
Geographic
Region
Particle
Load
Filter
Vial
Type
Spore
Spike
level
Filter ID
1
PM10
SC
AVG
PVDF
0
l-PMlO-SC-AVG-O
2
PM10
sc
AVG
PVDF
0
2 - P M10-SC-A VG -0
3
PM10
SC
AVG
PVDF
30
3- P M10-SC-AVG -30
4
PM10
5C
AVG
PVDF
30
4-PM10-SC-AVG-30
5
PM10
5C
AVG
PVDF
300
5-PM10-SC-AVG-300
6
PM10
SC
AVG
PVDF
300
6-PM10-SC-AVG-300
7
PM10
SC
AVG
PVDF
3,000
7-PM10-SC-AVG-3,000
8
PM10
SC
AVG
PVDF
3,000
8-PM10-SC-AVG-3.000
9
PM10
SC
High
PVDF
0
9-PM10-SC-High-0
10
PM10
SC
High
PVDF
0
lO-PMlO-SC-High-O
11
PM10
SC
High
PVDF
30
ll-PM10-SC-High-30
12
PM10
SC
High
PVDF
30
12-PM10-SC-High-30
13
PMIO
SC
High
PVDF
300
13-PM10-SC-High-300
14
PM10
SC
High
PVDF
300
14-PM10-SC-High-300
15
PM10
SC
High
PVDF
3,000
15-PM10-SC-High-3,000
16
PMIO
SC
High
PVDF
3,000
16-PM10-SC-High-3,000
Other Supplies and Equipment
• Forceps
• Biohazard bags
• Bleach
• 25mLSerological Pipettes
• 5mL Serological Pipettes
• Pipette aid
• Ziplock bags
• Bench paper
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 50 mL screw cap conical tubes and label as appropriate.
Performed By: Date:
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WORK INSTRUCTION FOR BACILLUS ANTHRACIS SPORE RECOVERY
~ 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.
~ 15 mL and 50 mL conical tube per sample
~ For each sample prepare one 2 mL Eppendorf tube for enumeration,
n Document filter vial and sample tube labels.
n Prepare aliquots of the Extraction Buffer with Tween and Extraction Buffer without Tween in a
250mL screw capped bottle for washes.
~20mL/samp!e * number of samples = mL
2. Using gloved hands, place mesh support over filter swatches (dirty side facing mesh) in 50 mL tubes
by holding the swatches to the side of the tube with sterile forceps and placing the coiled mesh
support on top. Place swatch so that the spiked side is facing the inside of the tube. Ensure the
sample and mesh are in the bottom half of the tube (avoiding the conical portion). Do not touch any
other surface with gloved hand that was used to position mesh over spiked swatch in conical tube.
Change gloves in between each sample.
Note: The support keeps the swatch from interfering with pipetting activities and improves
efficiency of spore extraction during vortexing.
3. First Extraction: Bleach wipe each tube. Add 15 mL cold (4°C) extraction buffer with Tween8 20 +
Ethanol to samples. Uncap one tube at a time, add extraction buffer, close tube and Parafilm cap
prior to moving to the next sample tube. Bleach wipe each tube.
4. Place tube rack in plastic bag, seal, double bag and bleach the bag prior to transferring to the
platform vortexer located outside the BSC.
5. Vortex samples for 20 minutes on Platform vortexer with the speed set to 7. Make sure to clamp
tube rack from the top and bottom of the vortexer.
Start time: End Time: Speed:
6. After vortexing, transfer sample tube rack to the BSC. Remove tube rack from plastic bag and
discard the bag.
7. Vortex up to 8 sample tubes on a single-tube vortexer in the BSC, for 3 - 5 seconds each. Let sit for
at least 2 minutes to allow large particles to settle prior to aliquoting (for samples containing debris).
If necessary, allow the tubes to settle for up to 5 minutes.
8. Remove Parafilm, bleach wipe the tube, uncap tubes one at a time. Using a 10 mL serological pipet
carefully transfer 12.5 mL to clean clearly labeled 50 mL conical tube (extract pool). Recap 50 mL
conical tube and move to the next sample. Change serological pipets and gloves between samples.
Performed By: Date:
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WORK INSTRUCTION FOR BACILLUS ANTHRACIS SPORE RECOVERY
9. Second Extraction: Add 10 mL of cold (4°C) extraction buffer + Ethanol withoutTween" 20 to each
sample tube, one at a time with a new 10 mL serological pipet for each sample and recapping each
sample tube after buffer addition.
10. After adding extraction buffer to all the tubes, check that all caps are tight and Parafilm each cap.
Place rack in double plastic bags, seal and bleach the outer bag. Transfer double bagged tube rack to
platform vortexer located outside the BSC.
11. Vortex rack for 10 minutes, with speed set to 7.
Start time: End Time: Speed:
12. Move the rack back to the BSC. Discard bags and vortex tubes for 3 - 5 seconds and allow large
particles to settle for at least 2 minutes.
13. Remove Parafilm, bleach wipe the tube, uncap tubes one at a time. Using a 10 m L serological pipet
carefully transfer ~12.5 mL to corresponding extract pool 50 mL conical tube from Step 8, but
carefully avoid settled particles during aliquoting. Recap 50 mL conical tube and move to the next
sample. Change serological pipets and gloves between each sample.
14. Vortex pooled extracts for 10 seconds, allow mixture to settle for approximately 2 minutes to allow
particulates to settle.
15. Transfer 10.5 mL of the pooled extract to a 15 mL conical tube 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.
16. Transfer up to 12 mL of the pooled extract into corresponding filter vial. Recap the vial and move to
the next sample. Change serological pipets and gloves between each sample. Turn on vacuum pump
at 5 -10 psi. Record measured volume in Table 1.
Performed By: Date:
Page 4 of 7
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WORK INSTRUCTION FOR BACILLUS ANTHRACIS SPORE RECOVERY
Table 1. Volume of sample transferred to filter vial
Sample
Number
Filter ID
Total volume transferred to
filter vial for RV-PCR (mL)
Recorded by:
1
1-PMlO-SC-AVG-O
2
2-PM10-SC-AVG-0
3
3- PM10-SC-AVG -30
4
4-PM10-SC-AVG-30
5
5-PM10-SC-AVG-300
6
6-PM10-SC-AVG-300
7
7-PM10-SC-AVG-3,000
8
8-PM10-SC-AVG-3,000
9
9-PM10-SC-High-0
10
10-PM10-SC-High-0
11
ll-PM10-SC-High-30
12
12-PM10-SC-High-30
13
13-PM10-SC-High-300
14
14-PM10-SC-High-300
15
15-PM10-SC-High-3,000
16
16-PM10-SC-High-3,000
17. If necessary, transfer remaining volume of pooled extract into corresponding filter vial. Recap the
vial and move to the next sample. Change serological pipets and gloves between samples. Turn on
vacuum pump at 5 - 10 psi. Complete filtration of liquid through filter vials. Record volume in Table
1.
18. Proceed to RV-PCR processing section (section B) below, with filter vial manifold.
19. Discard tubes that contain filter swatches.
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 2S0 mL screw cap bottle.
2. Transfer 12.5 mL of cold (4°C) High salt wash buffer (lOx PBS) to each filter-vial using a 10 mL
serological pipet. Change pipet and gloves between each sample.
3. Complete filtration of liquid through the filter vials.
Performed By: Date:
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WORK INSTRUCTION FOR BACILLUS ANTHRACIS SPORE RECOVERY
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.
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
Performed By: Date:
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WORK INSTRUCTION FOR BACILLUS ANTHRACIS SPORE RECOVERY
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 37°C at 230 rpm,
overnight (i.e.. 16 hours from the addition of BIII broth to the filter vials). These samples are referred
to as the Tf,„,i 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-PM10-SC-AVG-0
2
2-PM10-SC-AVG-0
3
3-PM10-SC-AVG-30
4
4- P M10-SC-A VG- 30
5
5-PM10-SC-AVG-300
6
6-PM10-SC-AVG-300
7
7-PM10-SC-AVG-3,000
8
8-PM10-SC-AVG-3,000
9
9-PM10-SC-High-0
10
10-PM10-SC-High-0
11
ll-PM10-SC-High-30
12
12-PM10-SC-High-30
13
13-PM10-SC-High-300
14
14-PM10-SC-High-300
15
15-PM10-SC-High-3,000
16
16-PM10-SC-High-3,000
19. Proceed to Wl #3: DNA Purification to process To and T, samples
Performed By: Date:
IV. Technical Review
Performed by: Date:
Page 7 of 7
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APPENDIX E. WORK INSTRUCTION FOR CULTURE OF BACILLUS
ANTHRACIS SPORES RECOVERED FROM AIR FILTERS
E-l
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WORK INSTRUCTION FOR CULTURE OF BACILLUS ANTHRACIS SPORES
RECOVERED FROM AIR FILTERS
I. PURPOSE/SCOPE
Culture of B. anthracis spores recovered from air filters following the BACILLUS Analytical Methods 004
published by the EPA December 2012.
II. MATERIALS/EQUIPMENT
Materials
Item
(U.ub ,'oj
Manufacturer
Lot Number
Exp.
Dato
Storage
Temp.
Initials & Date
Microfunnel filters
PALL
R.T.
Blood Agar
BBL
2-8 °C
N/A= Not Applicable
Equipment
Item
Manufacturer
Thermometer/
Serial Number
Roes #
Calibration
Due
Initials & Date
Biosafety
Cabinet (BSC)
The Baker Company
N/A
Stationary
Incubator
Precision
9509-003
N/A
N/A
Vacuum
manifold
Gelman Sciences
N/A
N/A
N/A
Platform
Vortexer
VWR
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
• Ziplock bags
Page lof 3
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WORK INSTRUCTION FOR CULTURE OF BACILLUS ANTHRACIS SPORES
RECOVERED FROM AIR FILTERS
Filter
Spore
Sample
Filter
Geographic
Particle
Vial
Spike
ft
type
Region
Load
Type
level
Filter ID
1
PM10
SC
AVG
PVDF
0
1-PMlO-SC-AVG-O
2
PMIO
SC
AVG
PVDF
0
2-PM10-SC-AVG-0
3
PM10
sc
AVG
PVDF
30
3-PM10-SC-AVG-30
4
PM10
SC
AVG
PVDF
30
4-PM10-SC-AVG-30
5
PM10
sc
AVG
PVDF
300
5-PM10-SC-AVG-300
6
PM10
sc
AVG
PVDF
300
6-PM10-SC-AVG-300
7
PM10
sc
AVG
PVDF
3,000
7-PM10-SC-AVG-3,000
8
PM10
sc
AVG
PVDF
3,000
8-PM10-SC-AVG-3,000
9
PM10
sc
High
PVDF
0
9-PM10-SC-High-0
10
PMIO
sc
High
PVDF
0
10-PM10-SC-High-0
11
PM10
sc
High
PVDF
30
ll-PM10-SC-High-30
12
PMIO
sc
High
PVDF
30
12-PM10-SC-High-30
13
PMIO
sc
High
PVDF
300
13-PM10-SC-High-300
14
PMIO
sc
High
PVDF
300
14-PM10-SC-High-300
15
PM10
sc
High
PVDF
3,000
15- P M10-SC- H igh- 3,000
16
PM10
sc
High
PVDF
3,000
16-PM10-SC-High-3,000
Filters - Electronically update this table with samples names from the Sample Log
III. PROCEDURE
Note: The following procedure is to be carried out with the 10 mi pooled extract taken from step 14
(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. For each sample, add 1 mLof pooled extract to one filter funnel and 4 mLof pooled
extract to one filter funnel. Apply vacuum.
Performed by: Date:
Page 2 of 3
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WORK INSTRUCTION FOR CULTURE OF BACILLUS ANTHRACIS SPORES
RECOVERED FROM AIR FILTERS
5. Close the vacuum valve and release the vacuum pressure. Rinse the walls of each filter funnel using
10 mL of PBST. Apply vacuum.
6. 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.
7. Incubate plates inverted overnight at 37°C ± 2°C.
Incubation start Date/Time: Initials:
Incubation end Date/Time: Initials:
8. Enter results into the below table.
Filter ID
B. anthracis colonies
Total colonies (all morphologies)
CFU/lmL
CFU/4 mL
CFU/lmL
:FU/4 mL
PBST Negative #1
PBST Negative #2
1-PM10-SC-AVG-0
2-PM10-SC-AVG-0
3- PM10-SC-AVG- 30
4-PM10-SC-AVG-30
5-PM10-SC-AVG-300
6-PM10-SC-AVG-300
7-PM10-SC-AVG-3,000
8-PM10-SC-AVG-3,000
9-PM10-SC-High-0
10-PM10-SC-High-0
ll-PM10-SC-High-30
12-PM10-SC-High-30
13-PM10-SC-High-300
14-PM10-SC-High-300
15-PM10-SC-High-3,000
16-PM10-SC-High-3,000
Counts performed/recorded by:
Performed by:
IV. Technical Review
Reviewed by:
Native Filters WI-Culture-4-v4 (092617)
Date:
Date:
Date:
Page 3 of 3
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APPENDIX F. WORK INSTRUCTION FOR MANUAL DNA
EXTRACTION AND PURIFICATION
FROM BACILLUS ANTHRACIS SPORES
F-l
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WORK INSTRUCTION FOR MANUAL DNA EXTRACTION AND PURIFICATION
FROM BACILLUS ANTHRACIS SPORES
I. PURPOSE/SCOPE
Manual DNA extraction and purification B. anthracis 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. Storage |njtiaIs & Date
Date Temp.
Lysis Buffer
Pro mega
RT
PMPs
Promega
RT
Salt Wash solution
Promega
RT
Alcohol Wash
Promega
RT
70% Ethanol
Inhouse
RT
Elution Buffer
Promega
RT
N/A= Not Applicable
Equipment
Item
Manufacturer Serial Number
Thermometer Calibration Initials &
/Rees # Due Date
Biosafety
Cabinet (BSC)
The Baker Company
57553
N/A
9/2018
Micropipette
Type:L1000
Rainin
N/A
Micropipette
Type: L2 00
Rainin
N/A
Micropipette
Type:L1000
Rainin
N/A
Micropipette
Type; L2 00
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
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WORK INSTRUCTION FOR MANUAL DNA EXTRACTION AND PURIFICATION
FROM BACILLUS ANTHRACIS SPORES
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 T, aliquots in a tube. Do not use 1.5 mL tubes. Transfer Ti aliquot screw
cap tubes to the BSC.
5. Transfer the filter vial rack to the BSC. Uncap one filter vial at a time and transfer 1 mLto
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 nL of the supernatant from each tube, using a 1000 |jL pipet and dispose to waste. Do not
disturb the pellet. Change gloves in between each sample.
8. Add 800 |iL of lysis buffer using a 1000 (iL 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 T0 and T, lysate tubes at room temperature for 5 minutes.
Performed by: Date:
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WORK INSTRUCTION FOR MANUAL DNA EXTRACTION AND PURIFICATION
FROM BACILLUS ANTHRACIS SPORES
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 To and T, lysate
tube.
11. Uncap one tube at a time and add 600 pL of PMPs to each T0 and Ti tube (containing lmL 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 pL 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 pL of 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 To and Ti tube.
Wash Steps:
19. Uncap each tube one at a time and add 360 pL of Salt Wash Solution. Remove tube rack off of
magnetic stand. Vortex on low setting for 5-10 seconds, and transfer to tube rack. Place tube rack
back on magnetic. Invert as described in step A.13. Remove all the liquid as described in Step A.17.
Use a new tip for each To and Ti tube. This is 1st Salt Wash.
20. Uncap each tube one at a time and add 360 pL of Salt Wash Solution. Remove tube rack off of
magnetic stand. Vortex on low setting for 5-10 seconds, and transfer to tube rack. Place tube rack
back on magnetic. Invert as described in step A.13. Remove all the liquid as described in Step A.17.
Use a new tip for each T0 and Ti tube. This is 2nd Salt Wash.
Performed by: Date:
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WORK INSTRUCTION FOR MANUAL DNA EXTRACTION AND PURIFICATION
FROM BACILLUS ANTHRACIS SPORES
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 To and Ti tube. This is 3rd Alcohol Wash.
24. Uncap each tube one at a time and add 500 pL of 70% Ethanol. Remove tube rack off of magnetic
stand. Vortex on low setting for 5-10 seconds, and transfer to tube rack. Place tube rack back on
magnetic. Invert as described in step A.13. Remove all the liquid as described in Step A.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.
28. DNA elution: While they are in the heating block add 200 pL of elution buffer to each To 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:
Temperature:
Start:
End:
Performed by:
Date:
Page 4 of 5
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WORK INSTRUCTION FOR MANUAL DNA EXTRACTION AND PURIFICATION
FROM BACILLUS ANTHRACIS SPORES
32. Briefly vortex each tube (5 - 10 seconds) on low speed and centrifuge at 2000 rpm, 4SC for 1 minute,,
33. Briefly vortex each tube and place on the magnetic stand for at least 30 seconds.
34. Collect liquid from each To and Ti tube and transfer ~80-90 uL to a clean, labeled, 1.5 mL tube on ice
(check tube labels to ensure the correct order). Use a new tip for each tube. Visually verify absence of
PMP carryover during final transfer. If magnetic bead carryover occurred, place 1.5 mL tube on
magnet, collect liquid, and transfer to a clean, labeled, 1.5 mL tube.
35. Centrifuge tubes at 14,000 rpm at 4°C for 5 minutes to pellet any particles remaining with the eluted
DNA; carefully remove supernatant from all samples and transfer to a new 1.5 mL tube using a new tip
for each tube.
Start: End:
36. Store T0 and T, 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 -20SC.
Labeled:
Date/Time:
Storage Temperature:
Storage Location:
Performed by:
Date:
IV. Technical Review
Performed by:
Date:
Comments:
Page 5 of 5
Native Filters Wl-Manual DNA Extraction and Purification-3-v5 (09/26/2017)
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APPENDIX G. WORK INSTRUCTION DRV-PCR FOR
BACILLUS ANTHRACIS SPORES
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WORK INSTRUCTION DRV-PCR FOR BACILLUS ANTHRAC1S SPORES
I. PURPOSE/SCOPE
Duplicate 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-Svl - FORM A
Equipment
Item
Manufacturer
Serial Number
Thermometer/
Roes #
Calibration
Due
Initials & Date
Biosafety
Cabinet {BSC)
N/A
Micropipette
Type:
N/A
Micropipette
Type:
N/A
Micropipette
Type:
N/A
Micropipette
Type:
N/A
Refrigerator
Freezer
Centrifuge
Eppendorf
X58983
N/A
N/A
7500 Fast Dx
Applied Biosystems
275017115
N/A
6/2018
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 lof 6
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WORK INSTRUCTION DRV-PCR FOR BACILLUS ANTHRACIS SPORES
III. PROCEDURE
A. Prepare samples for qPCR
Note: This step must be performed in the BSC outside the PCR clean room set-up area. Prepare a fresh
aliquot of PCR-grade water per sample batch to use for 1:10 dilutions and NTCs.
1. Toand TiDNA extracts: Label 1.5 mLtubes with the sample identifier and "10-fold dilution". Add 90 |iL
of PCR-grade water to the tubes.
2. Mix Toand Ti DNA extracts by vortexing (3-5 seconds), spin at 14,000 rpm for 2 minutes, and transfer
10 (iL of supernatant to 1.5-mL Eppendorf tubes with 90 |iL 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 are to be run. Prepare a sufficient volume of Master Mix to
allow for one extra reaction for every ten reactions, so that there is enough Master Mix regardless of
pipetting variations. For each batch of samples, PCR Master Mix should be made for 4 PCs, 4 NTCs, 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-5vl - FORM A.
3. In a clean PCR-preparation hood, pipet 20 [iLof Master Mix into the wells of the PCR plate. Label four
wells as NTC and four as PC.
4. Add 5 (iL 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 (jL to each sample well. Tightly seal the sample wells with
optical caps.
Performed by: Date:
Page 2 of 6
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WORK INSTRUCTION DRV-PCR FOR BACILLUS ANTHRACIS SPORES
7, Vortex the PC (8. anthracis DNA [10 pg/[iL or 50 pg/5 |iL]) and add 5 jiL 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 Boxj.
Within the Post-Amplification Lab (20-0-48) Load 96-well plates onto 7500 Fast Dx.
1. Set up 7500 Fast Dx
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 d t t to the plate document.
3. Choose ROX™ as the passu I ice 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 T ask (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:
Native Filters Wl-RV-PCR-5-v4 (09/26/17)
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WORK INSTRUCTION DRV-PCR FOR BACILLUS ANTHRACIS SPORES
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 |iL Sample 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 ran, 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
ran.
viii. When run is complete, bum the file to a CD.
ix. Remove 96-well plate from 7500 Fast Dx and dispose
Analysis
1. Open the assay with the most current version of 7500 Fast Dx 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 oil the Results tab in the Well Inspector pane to view the Amplification Plot and
Standard Curve Plot.
Performed by: Date:
Native Filters WI-RV-PCR-5-v4 (09/26/17)
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WORK INSTRUCTION DRV-PCR FOR BACILLUS ANTHRACIS SPORES
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
stan dard 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 le\ el 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 (I/D) every printout.
• Initial, date, and error or otherwise annotate all errors and comments.
• Indicate w hieh, if am, v ells 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
Performed by; Date:
Page 5 of 6
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WORK INSTRUCTION DRV-PCR FOR BACILLUS ANTHRACIS SPORES
j. Repeat the preceding steps to this point for ever}' detector OR each standard curve
associated with this assay.
k. Attach all printouts to the worksheet. This constitutes one data package.
2. After the PCR 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 .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.
IV. 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) 2 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:
V. Technical Review
Performed by: Date:
Comments:
Page 6 of 6
Native Filters Wl-RV-PCR-5-v4 (09/26/17)
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APPENDIX H. WORK INSTRUCTION FOR SELECTING PRESUMPTIVE
BACILLUS ANTHRACIS STERNE COLONIES FOR QPCR
CONFIRMATION
H-l
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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 2012,
II. MATERIALS/EQUIPMENT
Materials
Itom
Manufacturer
Lot Number
Exp. Storage .... „ «
^ _ Initials & Date
Date Temp.
1 jxL loop, 10 }iL loop
or inoculating needles
R.T.
1.5 or 2 mL tubes
R.T.
N/A= Not Applicable
Equipment
Item
Manufacturer
„ . . Thermometer/
Serial Number
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 25mL Serological Pipettes
Page lof 2
Native Filters Wl-Colony Screen-6-vl (041218)
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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
Filters - Record Filter ID and Morphology for Selected Colonies
III. PROCEDURE
A. Selecting colonies
1. Pipette 100 |iL of PCR-grade water into 1.5 or 2 mL tubes.
2. Select colonies. Take pictures of colonies that are selected.
3. Use 1 |iL loop, 10 |iL loop or inoculating needle to select the colony.
4. Immerse needle into PCR-grade water and rotate to dislodge cellular material.
5. 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 2 of 2
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APPENDIX I. CULTURE RESULTS FOR AIR QUALITY FILTERS USING
SHEEP BLOOD AGAR MEDIUM
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PM2.5 New
Sample ID
Spore
Load1
Extraction
Volume
(mL)
Volume
in Filter
Cup
(mL)
Plate
Counts
(CFU)
Average Sample
Concentration
Percent
Recovery
CFU/mL
Total CFU
1-PM2.5-NEW-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
9-PM2.5-NA-NEW-
PES-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
10-PM2.5-NA-NEW-
PVDF-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
3-PM2.5-NEW-30
1.5E+01
25
1
1
1.0
25.0
166.7
1.5E+01
25
4
2
0.5
12.5
83.3
11-PM2.5 -NA-NE W -
PES-30
4.6E+01
25
1
0
0.0
0.0
0.0
4.6E+01
25
4
2
0.5
12.5
27.4
12 -PM2.5 -NA-NE W -
PVDF-30
4.6E+01
25
1
0
0.0
0.0
0.0
4.6E+01
25
4
2
0.5
12.5
27.4
5-PM2.5-NEW-300
1.5E+02
25
1
2
2.0
50.0
33.3
1.5E+02
25
4
12
3.0
75.0
50.0
13 -PM2.5 -NA-NE W -
PES-300
4.6E+02
25
1
9
9.0
225.0
49.2
4.6E+02
25
4
40
10.0
250.0
54.7
14-PM2.5-NA-NEW-
PVDF-300
4.6E+02
25
1
1
1.0
25.0
5.5
4.6E+02
25
4
19
4.8
118.8
26.0
7-PM2.5-NEW-3000
1.5E+03
25
1
38
38.0
950.0
63.3
1.5E+03
25
4
172
43.0
1075.0
71.7
15 -PM2.5 -NA-NE W -
PES-3,000
4.6E+03
25
1
33
33.0
825.0
18.1
4.6E+03
25
4
156
39.0
975.0
21.3
16-PM2.5-NA-NEW-
PVDF-3,000
4.6E+03
25
1
32
32.0
800.0
17.5
4.6E+03
25
4
118
29.5
737.5
16.1
Use values highlighted in green for reporting.
Count based on half filter multiplied by 2
1 Actual number of B. a. Sterne spores spiked onto filter based on spiking suspension titer and volume of suspension applied.
1-2
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PM2.5 Arizona Filter Average
Sample ID
Spore
Load1
Extraction
Volume
(mL)
Volume
in Filter
Cup
(mL)
Plate
Counts
(CFU)
Average Sample
Concentration
Percent
Recovery
CFII/mL
Total CFU
2-PM2.5-AVG-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
1-PM2,5-AZ-AVG-PES-
0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
1
0.3
6.3
#DIV/0!
2-PM2.5-AZ-AVG-
PVDF-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
4-PM2.5-AVG-30
1.5E+01
25
1
1
1.0
25.0
166.7
1.5E+01
25
4
5
1.3
31.3
208.3
3-PM2.5-AZ-AVG-PES-
30
4.6E+01
25
1
1
1.0
25.0
54.7
4.6E+01
25
4
4
1.0
25.0
54.7
4-PM2. 5-AZ-AVG-
PVDF-30
4.6E+01
25
1
1
1.0
25.0
54.7
4.6E+01
25
4
7
1.8
43.8
95.7
6-PM2.5-AVG-300
1.5E+02
25
1
5
5.0
125.0
83.3
1.5E+02
25
4
13
3.3
81.3
54.2
5-PM2.5-AZ-AVG-PES-
300
4.6E+02
25
1
7
7.0
175.0
38.3
4.6E+02
25
4
35
OO
OO
218.8
47.9
6-PM2. 5-AZ-AVG-
PVDF-300
4.6E+02
25
1
15
15.0
375.0
82.1
4.6E+02
25
4
39
9.8
243.8
53.3
8-PM2.5-AVG-3,000
1.5E+03
25
1
38
38.0
950.0
63.3
1.5E+03
25
4
99
24.8
618.8
41.3
7-PM2.5 -AZ-A V G-
PVDF-3000
4.6E+03
25
1
61
61.0
1525.0
33.4
4.6E+03
25
4
188
47.0
1175.0
25.7
8-PM2. 5-AZ-AVG-
PVDF-3,000
4.6E+03
25
1
74
74.0
1850.0
40.5
4.6E+03
25
4
222
55.5
1387.5
30.4
Use values highlighted in green for reporting.
Count based on half filter multiplied by 2
1 Actual number of B. a. Sterne spores spiked onto filter based on spiking suspension titer and volume of suspension applied.
1-3
-------
EPA/600/R-19/082
10/8/2019
PM2.5 Arizona Filter High
Sample ID
Spore Load1
Extraction
Volume
(mL)
Volume
in Filter
Cup
(mL)
Plate
Counts
(CFU)
Average Sample
Concentration
Percent
Recovery
CFII/mL
Total CFU
9-PM2.5-HIGH-0
0.0E+00
25
1
1
1.0
25.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
10-PM2.5-HIGH-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
2
0.5
12.5
#DIV/0!
9-PM2.5-AZ-HIGH-O
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
11-PM2.5-HIGH-30
1.5E+01
25
1
0
0.0
0.0
0.0
1.5E+01
25
4
1
0.3
6.3
41.7
12-PM2.5-HIGH-30
1.5E+01
25
1
0
0.0
0.0
0.0
1.5E+01
25
4
2
0.5
12.5
83.3
10-PM2.5-AZ-HIGH-
30
1.4E+01
25
1
0
0.0
0.0
0.0
1.4E+01
25
4
0
0.0
0.0
0.0
13-PM2.5-HIGH-300
1.5E+02
25
1
3
3.0
75.0
50.0
1.5E+02
25
4
15
3.8
93.8
62.5
14-PM2.5-HIGH-300
1.5E+02
25
1
2
2.0
50.0
33.3
1.5E+02
25
4
13
3.3
81.3
54.2
12-PM2.5-AZ-HIGH-
300
1.4E+02
25
1
5
5.0
125.0
89.3
1.4E+02
25
4
22
5.5
137.5
98.2
15-PM2.5-AZ-
HIGHF-3,000
1.4E+03
25
1
41
41.0
1025.0
73.2
1.4E+03
25
4
124
31.0
775.0
55.4
15-PM2.5-HIGH-
3,000
1.5E+03
25
1
56
56.0
1400.0
93.3
1.5E+03
25
4
120
30.0
750.0
50.0
16-PM2.5-HIGH-
3,000
1.5E+03
25
1
33
33.0
825.0
55.0
1.5E+03
25
4
125
31.3
781.3
52.1
Use values highlighted in green for reporting.
1 Actual number of B. a. Sterne spores spiked onto filter based on spiking suspension titer and volume of suspension applied.
1-4
-------
EPA/600/R-19/082
10/8/2019
PM2.5 Florida Average
Sample ID
Spore
Extraction
Volume
(mL)
Volume
in Filter
Plate
Counts
(CFU)
Average Sample
Concentration
Percent
Load1
Cup
(mL)
CFII/mL
Total CFU
Recovery
1-PM2.5-AVG-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
2-PM2.5-AVG-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
1-PM2.5-FL-AVG-
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
PVDF-0
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
3-PM2.5-AVG-30
2.0E+01
25
1
1
1.0
25.0
124.4
2.0E+01
25
4
3
0.8
18.8
93.3
4-PM2.5-AVG-30
2.0E+01
25
1
2
2.0
50.0
248.8
2.0E+01
25
4
1
0.3
6.3
31.1
3-PM2.5-FL-AVG-
5.0E+01
25
1
2
2.0
50.0
99.4
PVDF-30
5.0E+01
25
4
7
1.8
43.8
87.0
5-PM2.5-AVG-300
2.0E+02
25
1
11
11.0
275.0
137.5
2.0E+02
25
4
28
7.0
175.0
87.5
6-PM2.5-AVG-300
2.0E+02
25
1
8
8.0
200.0
100.0
2.0E+02
25
4
17
4.3
106.3
53.1
5-PM2.5-FL-AVG-
5.0E+02
25
1
8
8.0
200.0
39.8
PVDF-300
5.0E+02
25
4
29
7.3
181.3
36.0
7-PM2.5-AVG-3000
2.0E+03
25
1
68
68.0
1700.0
85.0
2.0E+03
25
4
200
50.0
1250.0
62.5
8-PM2.5-AVG-3,000
2.0E+03
25
1
75
75.0
1875.0
93.8
2.0E+03
25
4
288
72.0
1800.0
90.0
7-PM2.5-FL-AVG-
5.0E+03
25
1
93
93.0
2325.0
46.2
PVDF-3000
5.0E+03
25
4
216
54.0
1350.0
26.8
Use values highlighted in green for reporting.
Count based on half filter multiplied by 2
1 Actual number of B. a. Sterne spores spiked onto filter based on spiking suspension titer and volume of suspension applied.
1-5
-------
EPA/600/R-19/082
10/8/2019
PM2.5 Florida High
Sample ID
Spore
Load1
Extraction
Volume
(mL)
Volume
in Filter
Cup
(mL)
Plate
Counts
(CFU)
Average Sample
Concentration
Percent
Recovery
CFII/mL
Total CFU
9-PM2.5-HIGH-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
10-PM2.5-HIGH-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
2-PM2.5-FL-HIGH-
PVDF-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
11-PM2.5-HIGH-30
2.0E+01
25
1
2
2.0
50.0
250.0
2.0E+01
25
4
7
1.8
43.8
218.8
12-PM2.5-HIGH-30
2.0E+01
25
1
0
0.0
0.0
0.0
2.0E+01
25
4
2
0.5
12.5
62.5
4-PM2.5-FL-HIGH-
PVDF-30
5.0E+01
25
1
2
2.0
50.0
99.4
5.0E+01
25
4
4
1.0
25.0
49.7
13-PM2.5-HIGH-300
2.0E+02
25
1
7
7.0
175.0
87.5
2.0E+02
25
4
33
8.3
206.3
103.1
14-PM2.5-HIGH-300
2.0E+02
25
1
3
3.0
75.0
37.5
2.0E+02
25
4
28
7.0
175.0
87.5
6-PM2.5-FL-HIGH-
PVDF-300
5.0E+02
25
1
21
21.0
525.0
104.4
5.0E+02
25
4
43
10.8
268.8
53.4
15-PM2.5 -HIGH-3,000
2.0E+03
25
1
76
76.0
1900.0
95.0
2.0E+03
25
4
242
60.5
1512.5
75.6
16-PM2.5 -HIGH-3,000
2.0E+03
25
1
71
71.0
1775.0
88.8
2.0E+03
25
4
260
65.0
1625.0
81.3
8-PM2.5-FL-HIGH-
PVDF-3,000
5.0E+03
25
1
84
84.0
2100.0
41.7
5.0E+03
25
4
232
58.0
1450.0
28.8
Use values highlighted in green for reporting.
Count based on half filter multiplied by 2
1 Actual number of B. a. Sterne spores spiked onto filter based on spiking suspension titer and volume of suspension applied.
1-6
-------
EPA/600/R-19/082
10/8/2019
PM2.5 Massachusetts Average
Sample ID
Spore
Load1
Extraction
Volume
(mL)
Volume
in Filter
Cup
(mL)
Plate
Counts
(CFU)
Average Sample
Concentration
Percent
Recovery
CFU/mL
Total CFU
1-PM2.5-MA-AVG-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
2-PM2.5-MA-AVG-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
1-PM2.5-MA-AVG-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
3-PM2.5-MA-AVG-30
3.2E+01
25
1
0
0.0
0.0
0.0
3.2E+01
25
4
0
0.0
0.0
0.0
4-PM2.5-MA-AVG-30
3.2E+01
25
1
0
0.0
0.0
0.0
3.2E+01
25
4
3
0.8
18.8
58.0
3-PM2.5-MA-AVG-30
2.0E+01
25
1
0
0.0
0.0
0.0
2.0E+01
25
4
3
0.8
18.8
93.8
5-PM2.5-MA-AVG-300
3.2E+02
25
1
7
7.0
175.0
54.2
3.2E+02
25
4
29
7.3
181.3
56.1
6-PM2.5-MA-AVG-300
3.2E+02
25
1
2
2.0
50.0
15.5
3.2E+02
25
4
10
2.5
62.5
19.3
5-PM2.5-MA-AVG-300
2.0E+02
25
1
1
1.0
25.0
12.5
2.0E+02
25
4
10
2.5
62.5
31.3
7-PM2.5 -MA-A V G-
3000
3.2E+03
25
1
63
63.0
1575.0
48.8
3.2E+03
25
4
156
39.0
975.0
30.2
8-PM2.5-MA-AVG-
PVDF-3,000
3.2E+03
25
1
80
80.0
2000.0
61.9
3.2E+03
25
4
160
40.0
1000.0
31.0
7-PM2.5-MA-AVG-
3000
2.0E+03
25
1
47
47.0
1175.0
58.8
2.0E+03
25
4
103
25.8
643.8
32.2
Use values highlighted in green for reporting.
1 Actual number of B. a. Sterne spores spiked onto filter based on spiking suspension titer and volume of suspension applied.
1-7
-------
EPA/600/R-19/082
10/8/2019
PM2.5 Massachusetts High
Sample ID
Spore Load1
Extraction
Volume
(mL)
Volume
in Filter
Cup
(mL)
Plate
Counts
(CFU)
Average Sample
Concentration
Percent
Recovery
CFU/mL
Total CFU
9-PM2.5-MA-HIGH-
0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
10-PM2.5-MA-
fflGH-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
9-PM2.5-MA-HIGH-
0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
11-PM2.5-MA-
fflGH-30
3.2E+01
25
1
0
0.0
0.0
0.0
3.2E+01
25
4
3
0.8
18.8
58.0
12-PM2.5-MA-
fflGH-30
3.2E+01
25
1
0
0.0
0.0
0.0
3.2E+01
25
4
4
1.0
25.0
77.4
11-PM2.5-MA-
fflGH-30
2.0E+01
25
1
0
0.0
0.0
0.0
2.0E+01
25
4
3
0.8
18.8
93.8
13-PM2.5-MA-
fflGH-300
3.2E+02
25
1
5
5.0
125.0
38.7
3.2E+02
25
4
29
7.3
181.3
56.1
14-PM2.5-MA-
fflGH-300
3.2E+02
25
1
5
5.0
125.0
38.7
3.2E+02
25
4
23
5.8
143.8
44.5
13-PM2.5-MA-
fflGH-300
2.0E+02
25
1
3
3.0
75.0
37.5
2.0E+02
25
4
5
1.3
31.3
15.6
15-PM2.5-MA-
HIGHF-3,000
3.2E+03
25
1
62
62.0
1550.0
48.0
3.2E+03
25
4
124
31.0
775.0
24.0
16-PM2.5-MA-
HIGH-3,000
3.2E+03
25
1
61
61.0
1525.0
47.2
3.2E+03
25
4
140
35.0
875.0
27.1
15-PM2.5-MA-
HIGHF-3,000
2.0E+03
25
1
4
4.0
100.0
5.0
2.0E+03
25
4
25
6.3
156.3
7.8
Use values highlighted in green for reporting.
1 Actual number of B. a. Sterne spores spiked onto filter based on spiking suspension titer and volume of suspension applied.
1-8
-------
EPA/600/R-19/082
10/8/2019
PM2.5 Wisconsin Average
Sample ID
Spore
Load1
Extraction
Volume
(mL)
Volume
in Filter
Cup
(mL)
Plate
Counts
(CFU)
Average Sample
Concentration
Percent
Recovery
CFU/mL
Total CFU
1-PM2.5-WI-AVG-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
2-PM2.5-WI-AVG-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
2-PM2.5-WI-AVG-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
3-PM2.5-WI-AVG-30
2.2E+01
25
1
1
1.0
25.0
113.6
2.2E+01
25
4
2
0.5
12.5
56.8
4-PM2.5-WI-AVG-30
2.2E+01
25
1
0
0.0
0.0
0.0
2.2E+01
25
4
4
1.0
25.0
113.6
4-PM2.5-WI-AVG-30
2.0E+01
25
1
5
5.0
125.0
625.0
2.0E+01
25
4
1
0.3
6.3
31.3
5-PM2.5-WI-AVG-300
2.2E+02
25
1
4
4.0
100.0
45.5
2.2E+02
25
4
17
4.3
106.3
48.3
6-PM2.5-WI-AVG-300
2.2E+02
25
1
1
1.0
25.0
11.4
2.2E+02
25
4
10
2.5
62.5
28.4
6-PM2.5-WI-AVG-300
2.0E+02
25
1
6
6.0
150.0
75.0
2.0E+02
25
4
27
6.8
168.8
84.4
7-PM2.5-WI-AVG-
3,000
2.2E+03
25
1
49
49.0
1225.0
55.7
2.2E+03
25
4
146
36.5
912.5
41.5
8-PM2.5-WI-AVG-
3,000
2.2E+03
25
1
25
25.0
625.0
28.4
2.2E+03
25
4
92
23.0
575.0
26.1
8-PM2.5-WI-AVG-
PVDF-3,000
2.0E+03
25
1
60
60.0
1500.0
75.0
2.0E+03
25
4
108
27.0
675.0
33.8
Use values highlighted in green for reporting.
Count based on half filter multiplied by 2
1 Actual number of B. a. Sterne spores spiked onto filter based on spiking suspension titer and volume of suspension applied.
1-9
-------
EPA/600/R-19/082
10/8/2019
PM2.5 Wisconsin High
Sample ID
Spore Load1
Extraction
Volume
(mL)
Volume
in Filter
Cup
(mL)
Plate
Counts
(CFU)
Average Sample
Concentration
Percent
Recovery
CFU/mL
Total CFU
9-PM2.5-WI-High-O
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
10-PM2.5 -WI-High-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
10-PM2.5 -WI-HIGH-
0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
11-PM2.5 -WI-High-
30
2.2E+01
25
1
3
3.0
75.0
340.9
2.2E+01
25
4
3
0.8
18.8
85.2
12-PM2.5 -WI-High-
30
2.2E+01
25
1
1
1.0
25.0
113.6
2.2E+01
25
4
3
0.8
18.8
85.2
12-PM2.5-WI-HIGH-
30
2.0E+01
25
1
0
0.0
0.0
0.0
2.0E+01
25
4
3
0.8
18.8
93.8
13-PM2.5 -WI-High-
300
2.2E+02
25
1
5
5.0
125.0
56.8
2.2E+02
25
4
27
6.8
168.8
76.7
14-PM2.5 -WI-High-
300
2.2E+02
25
1
12
12.0
300.0
136.4
2.2E+02
25
4
21
5.3
131.3
59.7
14-PM2.5 -WI-HIGH-
300
2.0E+02
25
1
13
13.0
325.0
162.5
2.0E+02
25
4
26
6.5
162.5
81.3
15-PM2.5 -WI-High-
3,000
2.2E+03
25
1
62
62.0
1550.0
70.5
2.2E+03
25
4
142
35.5
887.5
40.3
16-PM2.5 -WI-High-
3,000
2.2E+03
25
1
67
67.0
1675.0
76.1
2.2E+03
25
4
144
36.0
900.0
40.9
16-PM2.5 -WI-HIGH-
3,000
2.0E+03
25
1
67
67.0
1675.0
83.8
2.0E+03
25
4
144
36.0
900.0
45.0
Use values highlighted in green for reporting.
Count based on half filter multiplied by 2
1 Actual number of B. a. Sterne spores spiked onto filter based on spiking suspension titer and volume of suspension applied.
1-10
-------
EPA/600/R-19/082
10/8/2019
PM10 New
Sample ID
Spore
Load1
Extraction
Volume
(mL)
Volume in
Filter Cup
(mL)
Plate
Counts
(CFU)
Average Sample
Concentration
Percent
Recovery
CFU/mL
Total CFU
PM10HV-
New-Blank
0.0E+00
25
1
0
0.0
0.0
N/A
25
4
0
0.0
0.0
N/A
PM10HV-
New-Blank
0.0E+00
25
1
0
0.0
0.0
N/A
25
4
0
0.0
0.0
N/A
1-PM10-
NEW-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
PM10HV-
New-30
9.0E+00
25
1
0
0.0
0.0
0.0
25
4
0
0.0
0.0
0.0
PM10HV-
New-30
9.0E+00
25
1
0
0.0
0.0
0.0
25
4
0
0.0
0.0
0.0
3-PM10-
NEW-30
1.8E+01
25
1
0
0.0
0.0
0.0
1.8E+01
25
4
0
0.0
0.0
0.0
PM10HV-
New-300
9.0E+01
25
1
1
1.0
25.0
27.8
25
4
0
0.0
0.0
0.0
PM10HV-
New-300
9.0E+01
25
1
1
1.0
25.0
27.8
25
4
2
0.5
12.5
13.9
5-PM10-
NEW-300
1.8E+02
25
1
0
0.0
0.0
0.0
1.8E+02
25
4
1
0.3
6.3
3.5
PM10HV-
New-3,000
1.3E+03
25
1
4
4.0
100.0
7.7
25
4
15
3.8
93.8
7.2
PM10HV-
New-3,000
1.3E+03
25
1
5
5.0
125.0
9.6
25
4
24
6.0
150.0
11.5
PM10HV-
New-3,000
1.3E+03
25
1
3
3.0
75.0
5.8
25
4
16
4.0
100.0
7.7
Use values highlighted in green for reporting.
1 Actual number of B. a. Sterne spores spiked onto filter based on spiking suspension titer and volume of suspension applied.
1-11
-------
EPA/600/R-19/082
10/8/2019
PM10 California Average
Sample ID
Spore Load1
Extraction
Volume
(mL)
Volume
in Filter
Cup
(mL)
Plate
Counts
(CFU)
Average Sample
Concentration
Percent
Recovery
CFU/mL
Total CFU
1-PM10-C A-AV G-0
0.0E+00
25
1
1
1.0
25.0
#DIV/0!
0.0E+00
25
4
6
1.5
37.5
#DIV/0!
2-PM10-CA-AVG-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
8
2.0
50.0
#DIV/0!
1-PM10-C A-AV G-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
6
1.5
37.5
#DIV/0!
3-PM10-CA-AVG-30
3.1E+01
25
1
1
1.0
25.0
80.6
3.1E+01
25
4
6
1.5
37.5
121.0
4-PM10-CA-AVG-30
3.1E+01
25
1
3
3.0
75.0
241.9
3.1E+01
25
4
7
1.8
43.8
141.1
3-PM10-CA-AVG-30
1.4E+01
25
1
3
3.0
75.0
535.7
1.4E+01
25
4
4
1.0
25.0
178.6
5-PM10-CA-AVG-
300
3.1E+02
25
1
5
5.0
125.0
40.3
3.1E+02
25
4
1
0.3
6.3
2.0
6-PM10-CA-AVG-
300
3.1E+02
25
1
2
2.0
50.0
16.1
3.1E+02
25
4
7
1.8
43.8
14.1
5-PM10-CA-AVG-
300
1.4E+02
25
1
1
1.0
25.0
17.9
1.4E+02
25
4
5
1.3
31.3
22.3
7-PM10-CA-AVG-
3,000
3.1E+03
25
1
9
9.0
225.0
7.3
3.1E+03
25
4
17
4.3
106.3
3.4
8-PM10-CA-AVG-
3,000
3.1E+03
25
1
16
16.0
400.0
12.9
3.1E+03
25
4
25
6.3
156.3
5.0
7-PM10-CA-AVG-
3000
1.4E+03
25
1
6
6.0
150.0
10.7
1.4E+03
25
4
23
5.8
143.8
10.3
Use values highlighted in green for reporting.
B. a. Sterne morphology present on zero spike sample
1 Actual number of B. a. Sterne spores spiked onto filter based on spiking suspension titer and volume of suspension applied.
1-12
-------
EPA/600/R-19/082
10/8/2019
PM10 California High
Sample ID
Spore
Load1
Extraction
Volume
(mL)
Volume
in Filter
Cup
(mL)
Plate
Counts
(CFU)
Average Sample
Concentration
Percent
Recovery
CFU/mL
Total CFU
9-PM10-C A-High-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
3
0.8
18.8
#DIV/0!
10-PM10-C A-High-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
2-PM10-C A-HIGH-0
0.0E+00
25
1
3
3.0
75.0
#DIV/0!
0.0E+00
25
4
6
1.5
37.5
#DIV/0!
1 l-PM10-CA-High-30
3.1E+01
25
1
2
2.0
50.0
161.3
3.1E+01
25
4
4
1.0
25.0
80.6
12-PM10-CA-High-30
3.1E+01
25
1
0
0.0
0.0
0.0
3.1E+01
25
4
2
0.5
12.5
40.3
4-PM10-CA-HIGH-30
1.4E+01
25
1
0
0.0
0.0
0.0
1.4E+01
25
4
1
0.3
6.3
44.6
13-PM10-CA-High-
300
3.1E+02
25
1
2
2.0
50.0
16.1
3.1E+02
25
4
5
1.3
31.3
10.1
14-PM10-C A-High-
300
3.1E+02
25
1
2
2.0
50.0
16.1
3.1E+02
25
4
0
0.0
0.0
0.0
6-PM10-C A-HIGH-
300
1.4E+02
25
1
0
0.0
0.0
0.0
1.4E+02
25
4
9
2.3
56.3
40.2
15-PM10-CA-High-
3,000
3.1E+03
25
1
9
9.0
225.0
7.3
3.1E+03
25
4
21
5.3
131.3
4.2
16-PM10-C A-High-
3,000
3.1E+03
25
1
5
5.0
125.0
4.0
3.1E+03
25
4
20
5.0
125.0
4.0
8-PM10-C A-HIGH-
PVDF-3,000
1.4E+03
25
1
3
3.0
75.0
5.4
1.4E+03
25
4
16
4.0
100.0
7.1
Use values highlighted in green for reporting.
B. a. Sterne morphology present on zero spike sample
1 Actual number of B. a. Sterne spores spiked onto filter based on spiking suspension titer and volume of suspension applied.
1-13
-------
EPA/600/R-19/082
10/8/2019
PM10 New Hampshire Average
Sample ID
Spore
Load1
Extraction
Volume
(mL)
Volume in
Filter Cup
(mL)
Plate
Counts
(CFU)
Average Sample
Concentration
Percent
Recovery
CFU/mL
Total CFU
1-PM10-NH-AV G-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
2-PM10-NH-AVG-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
9-PM10-NH-AVG-
PVDF-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
3-PM10-NH-AVG-30
1.6E+01
25
1
0
0.0
0.0
0.0
1.6E+01
25
4
0
0.0
0.0
0.0
4-PM10-NH-AVG-30
1.6E+01
25
1
0
0.0
0.0
0.0
1.6E+01
25
4
0
0.0
0.0
0.0
11-PM10-NH-AV G-
PVDF-30
5.0E+01
25
1
1
1.0
25.0
49.7
5.0E+01
25
4
0
0.0
0.0
0.0
5-PM10-NH-AVG-
300
1.6E+02
25
1
0
0.0
0.0
0.0
1.6E+02
25
4
0
0.0
0.0
0.0
6-PM10-NH-AVG-
300
1.6E+02
25
1
1
1.0
25.0
15.6
1.6E+02
25
4
0
0.0
0.0
0.0
13-PM10-NH-AVG-
PVDF-300
5.0E+02
25
1
2
2.0
50.0
9.9
5.0E+02
25
4
2
0.5
12.5
2.5
7-PM10-NH-AVG-
3000
1.6E+03
25
1
9
9.0
225.0
14.1
1.6E+03
25
4
23
5.8
143.8
9.0
8-PM10-NH-AVG-
3,000
1.6E+03
25
1
6
6.0
150.0
9.4
1.6E+03
25
4
32
8.0
200.0
12.5
15-PM10-NH-AVG-
PVDF-3,000
5.0E+03
25
1
4
4.0
100.0
2.0
5.0E+03
25
4
25
6.3
156.3
3.1
Use values highlighted in green for reporting.
2 distinct large growths covering the majority of the filter
1 Actual number of B. a. Sterne spores spiked onto filter based on spiking suspension titer and volume of suspension applied.
1-14
-------
EPA/600/R-19/082
10/8/2019
PM10 New Hampshire High
Sample ID
Spore
Load1
Extraction
Volume
(mL)
Volume
in Filter
Cup
(mL)
Plate
Counts
(cfu)
Average Sample
Concentration
Percent
Recovery
CFU/mL
Total CFU
9-PM10-NH-HIGH-O
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
10-PM10-NH-fflGH-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
10-PM10-NH-HIGH-
PVDF-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
11-PM10-NH-HIGH-30
1.6E+01
25
1
0
0.0
0.0
0.0
1.6E+01
25
4
0
0.0
0.0
0.0
12-PM10-NH-HIGH-30
1.6E+01
25
1
0
0.0
0.0
0.0
1.6E+01
25
4
0
0.0
0.0
0.0
12-PM10-NH-fflGH-
PVDF-30
5.0E+01
25
1
0
0.0
0.0
0.0
5.0E+01
25
4
0
0.0
0.0
0.0
13-PM10-NH-HIGH-
300
1.6E+02
25
1
0
0.0
0.0
0.0
1.6E+02
25
4
1
0.3
6.3
3.9
14-PM10-NH-HIGH-
300
1.6E+02
25
1
0
0.0
0.0
0.0
1.6E+02
25
4
2
0.5
12.5
7.8
14-PM10-NH-HIGH-
PVDF-300
5.0E+02
25
1
1
1.0
25.0
5.0
5.0E+02
25
4
4
1.0
25.0
5.0
15-PM10-NH-HIGH-
3,000
1.6E+03
25
1
28
28.0
700.0
43.8
1.6E+03
25
4
88
22.0
550.0
34.4
16-PM10-NH-HIGH-
3,000
1.6E+03
25
1
13
13.0
325.0
20.3
1.6E+03
25
4
60
15.0
375.0
23.4
16-PM10-NH-HIGH-
PVDF-3,000
5.0E+03
25
1
4
4.0
100.0
2.0
5.0E+03
25
4
30
7.5
187.5
3.7
Use values highlighted in green for reporting.
1 Actual number of B. a. Sterne spores spiked onto filter based on spiking suspension titer and volume of suspension applied.
1-15
-------
EPA/600/R-19/082
10/8/2019
PM10 South Carolina Average
Sample ID
Spore Load1
Extraction
Volume
(mL)
Volume
in Filter
Plate
Counts
(CFU)
Average Sample
Concentration
Percent
Cup
(mL)
CFU/mL
Total CFU
Recovery
1-PMlO-SC-AVG-O
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
2-PM10-SC-AVG-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
1-PMlO-SC-AVG-O
0.0E+00
25
1
7
7.0
175.0
#DIV/0!
0.0E+00
25
4
1
0.3
6.3
#DIV/0!
3-PM10-SC-AVG-30
2.7E+01
25
1
0
0.0
0.0
0.0
2.7E+01
25
4
0
0.0
0.0
0.0
4-PM10-SC-AVG-30
2.7E+01
25
1
0
0.0
0.0
0.0
2.7E+01
25
4
1
0.3
6.3
23.1
3-PM10-SC-AVG-30
2.9E+01
25
1
1
1.0
25.0
86.2
2.9E+01
25
4
3
0.8
18.8
64.7
5-PM10-SC-AVG-
2.7E+02
25
1
1
1.0
25.0
9.3
300
2.7E+02
25
4
7
1.8
43.8
16.2
6-PM10-SC-AVG-
2.7E+02
25
1
0
0.0
0.0
0.0
300
2.7E+02
25
4
3
0.8
18.8
6.9
5-PM10-SC-AVG-
2.9E+02
25
1
1
1.0
25.0
8.6
300
2.9E+02
25
4
3
0.8
18.8
6.5
7-PM10-SC-AVG-
2.7E+03
25
1
13
13.0
325.0
12.0
3,000
2.7E+03
25
4
32
8.0
200.0
7.4
8-PM10-SC-AVG-
2.7E+03
25
1
11
11.0
275.0
10.2
3,000
2.7E+03
25
4
31
7.8
193.8
7.2
7-PM10-SC-AVG-
2.9E+03
25
1
5
5.0
125.0
4.3
3,000
2.9E+03
25
4
18
4.5
112.5
3.9
Use values highlighted in green for reporting.
1 Actual number of B. a. Sterne spores spiked onto filter based on spiking suspension titer and volume of suspension applied.
1-16
-------
EPA/600/R-19/082
10/8/2019
PM10 South Carolina High
Sample ID
Spore
Load1
Extraction
Volume
(mL)
Volume
in Filter
Cup
(mL)
Plate
Counts
(CFU)
Average Sample
Concentration
Percent
Recovery
CFU/mL
Total CFU
9-PM10-SC-fflGH-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
10-PM10-SC-fflGH-0
0.0E+00
25
1
2
2.0
50.0
#DIV/0!
0.0E+00
25
4
1
0.3
6.3
#DIV/0!
2-PM10-SC-fflGH-0
0.0E+00
25
1
2
2.0
50.0
#DIV/0!
0.0E+00
25
4
2
0.5
12.5
#DIV/0!
11-PM10-SC-HIGH-
30
2.7E+01
25
1
0
0.0
0.0
0.0
2.7E+01
25
4
4
1.0
25.0
92.6
12-PM10-SC-fflGH-
30
2.7E+01
25
1
1
1.0
25.0
92.6
2.7E+01
25
4
5
1.3
31.3
115.7
4-PM10-SC-HIGH-30
2.9E+01
25
1
0
0.0
0.0
0.0
2.9E+01
25
4
3
0.8
18.8
64.7
13-PM10-SC-fflGH-
300
2.7E+02
25
1
2
2.0
50.0
18.5
2.7E+02
25
4
7
1.8
43.8
16.2
14-PM10-SC-fflGH-
300
2.7E+02
25
1
0
0.0
0.0
0.0
2.7E+02
25
4
3
0.8
18.8
6.9
6-PM10-SC-fflGH-
300
2.9E+02
25
1
1
1.0
25.0
8.6
2.9E+02
25
4
7
1.8
43.8
15.1
15-PM10-SC-fflGHF-
3,000
2.7E+03
25
1
1
1.0
25.0
0.9
2.7E+03
25
4
23
5.8
143.8
5.3
16-PM10-SC-fflGH-
3,000
2.7E+03
25
1
11
11.0
275.0
10.2
2.7E+03
25
4
42
10.5
262.5
9.7
8-PM10-SC-fflGH-
3,000
2.9E+03
25
1
0
0.0
0.0
0.0
2.9E+03
25
4
11
2.8
68.8
2.4
Use values highlighted in green for reporting.
1 Actual number of B. a. Sterne spores spiked onto filter based on spiking suspension titer and volume of suspension applied.
1-17
-------
EPA/600/R-19/082
10/8/2019
PM10 Wisconsin Average
Sample ID
Spore Load1
Extraction
Volume
(mL)
Volume in
Filter Cup
(mL)
Plate
Counts
(CFU)
Average Sample
Concentration
Percent
Recovery
CFU/mL
Total CFU
2-PM10-
AVG-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
9-PM10-
AVG-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
10-PM10-
AVG-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
4-PM10-
AVG-30
1.8E+01
25
1
0
0.0
0.0
0.0
1.8E+01
25
4
0
0.0
0.0
0.0
11-PM10-
AVG-30
1.8E+01
25
1
0
0.0
0.0
0.0
1.8E+01
25
4
0
0.0
0.0
0.0
12-PM10-
AVG-30
1.8E+01
25
1
0
0.0
0.0
0.0
1.8E+01
25
4
0
0.0
0.0
0.0
6-PM10-
AVG-300
1.8E+02
25
1
2
2.0
50.0
27.8
1.8E+02
25
4
2
0.5
12.5
6.9
13-PM10-
AVG-300
1.8E+02
25
1
2
2.0
50.0
27.8
1.8E+02
25
4
3
0.8
18.8
10.4
14-PM10-
AVG-300
1.8E+02
25
1
0
0.0
0.0
0.0
1.8E+02
25
4
6
1.5
37.5
20.8
8-PM10-
AVG-3,000
1.8E+03
25
1
5
5.0
125.0
6.9
1.8E+03
25
4
14
3.5
87.5
4.9
15-PM10-
AVG-3,000
1.8E+03
25
1
2
2.0
50.0
2.8
1.8E+03
25
4
17
4.3
106.3
5.9
16-PM10-
AVG-3,000
1.8E+03
25
1
15
15.0
375.0
20.8
1.8E+03
25
4
55
13.8
343.8
19.1
Use values highlighted in green for reporting.
80% of plate Lawn
1 Actual number of B. a. Sterne spores spiked onto filter based on spiking suspension titer and volume of suspension applied.
1-18
-------
EPA/600/R-19/082
10/8/2019
PM10 Wisconsin High
Sample ID
Spore
Load1
Extraction
Volume
(mL)
Volume in
Filter Cup
(mL)
Plate
Counts
(CFU)
Average Sample
Concentration
Percent
Recovery
CFU/mL
Total CFU
PM10HV-
High-N/A
0.0E+00
25
1
0
0.0
0.0
N/A
25
4
0
0.0
0.0
N/A
PM10HV-
High-N/A
0.0E+00
25
1
0
0.0
0.0
N/A
25
4
0
0.0
0.0
N/A
PM10HV-
High-N/A
0.0E+00
25
1
0
0.0
0.0
N/A
25
4
0
0.0
0.0
N/A
PM10HV-
High-Blank
0.0E+00
25
1
0
0.0
0.0
N/A
25
4
0
0.0
0.0
N/A
PM10HV-
High-Blank
0.0E+00
25
1
0
0.0
0.0
N/A
25
4
0
0.0
0.0
N/A
PM10HV-
High-30
1.3E+01
25
1
0
0.0
0.0
0.0
25
4
0
0.0
0.0
0.0
PM10HV-
High-30
1.3E+01
25
1
0
0.0
0.0
0.0
25
4
0
0.0
0.0
0.0
PM10HV-
High-30
1.3E+01
25
1
0
0.0
0.0
0.0
25
4
1
0.3
6.3
48.1
PM10HV-
High-30
9.0E+00
25
1
0
0.0
0.0
0.0
25
4
0
0.0
0.0
0.0
PM10HV-
High-300
9.0E+01
25
1
0
0.0
0.0
0.0
25
4
0
0.0
0.0
0.0
PM10HV-
High-300
9.0E+01
25
1
1
1.0
25.0
27.8
25
4
4
1.0
25.0
27.8
7-PM10-
High-300
1.8E+02
25
1
0
0.0
0.0
0.0
1.8E+02
25
4
0
0.0
0.0
0.0
PM10HV-
High-3,000
1.3E+03
25
1
5
5.0
125.0
9.6
25
4
12
3.0
75.0
5.8
PM10HV-
High-3,000
1.3E+03
25
1
5
5.0
125.0
9.6
25
4
16
4.0
100.0
7.7
14-PM10-
WI-fflGH-
3000
2.9E+03
25
1
4
4.0
100.0
3.4
2.9E+03
25
4
10
2.5
62.5
2.2
Use values highlighted in green for reporting.
1 Actual number of B. a. Sterne spores spiked onto filter based on spiking suspension titer and volume of suspension applied.
1-19
-------
EPA/600/R-19/082
10/8/2019
APPENDIX J. CULTURE RESULTS FOR AIR QUALITY FILTERS USING
MYP MEDIUM
J-l
-------
EPA/600/R-19/082
10/8/2019
July 10, 2017 Trial - PM10 Wisconsin Filters
Sample ID
Spore Load1
Extraction
Volume
(mL)
Volume in
Filter Cup
(mL)
Plate
Counts
(CFU)
Average Sample
Concentration
Percent
Recovery
CFII/mL
Total CFU
1-PM10HV-High-
N/A
0.0E+00
25
1
0
0.0
0.0
N/A
25
3
0
0.0
0.0
N/A
2-PM10HV-High-
N/A
0.0E+00
25
1
0
0.0
0.0
N/A
25
4
0
0.0
0.0
N/A
3-PM10HV-High-
N/A
0.0E+00
25
1
0
0.0
0.0
N/A
25
4
0
0.0
0.0
N/A
4-PM1OHV-High-
30
1.3E+01
25
1
1
1.0
25.0
192.3
25
4
0
0.0
0.0
0.0
5-PM10HV-High-
30
1.3E+01
25
1
0
0.0
0.0
0.0
25
4
0
0.0
0.0
0.0
6-PM1 OHV-High-
30
1.3E+01
25
1
0
0.0
0.0
0.0
25
4
0
0.0
0.0
0.0
7-PM10HV-High-
3,000
1.3E+03
25
1
5
5.0
125.0
9.6
25
4
10
2.5
62.5
4.8
8-PM1 OHV-High-
3,000
1.3E+03
25
1
2
2.0
50.0
3.8
25
4
5
1.3
31.3
2.4
9-PM1 OHV -New-
3,000
1.3E+03
25
1
1
1.0
25.0
1.9
25
4
20
5.4
135.1
10.4
10-PM1 OHV -New-
3,000
1.3E+03
25
1
0
0.0
0.0
0.0
25
4
20
5.3
131.6
10.1
11-PM1 OHV -New-
3,000
1.3E+03
25
1
3
3.0
75.0
5.8
25
4
20
5.0
125.0
9.6
Green highlighted cells are the values reported for percent recovery
1 Actual number of B. a. Sterne spores spiked onto filter based on spiking suspension titer and volume of suspension applied.
J-2
-------
EPA/600/R-19/082
10/8/2019
July 17, 2017 Trial - PM10 Wisconsin Filters
Sample ID
Spore
Load1
Extraction
Volume
(mL)
Volume in
Filter Cup
(mL)
Plate
Counts
(CFU)
Average Sample
Concentration
Percent
Recovery
CFII/mL
Total CFU
1-PM10HV-
New-Blank
0.0E+00
25
1
0
0.0
0.0
N/A
25
4
0
0.0
0.0
N/A
2-PM10HV-
New-Blank
0.0E+00
25
1
0
0.0
0.0
N/A
25
4
0
0.0
0.0
N/A
3-PM10HV-
New-30
9.0E+00
25
1
0
0.0
0.0
0.0
25
4
0
0.0
0.0
0.0
4-PM10HV-
New-30
9.0E+00
25
1
0
0.0
0.0
0.0
25
4
2
0.5
12.5
138.9
5-PM10HV-
New-300
9.0E+01
25
1
2
2.0
50.0
55.6
25
4
4
1.0
25.0
27.8
6-PM10HV-
New-300
9.0E+01
25
1
0
0.0
0.0
0.0
25
4
1
0.3
6.3
6.9
7-PM10HV-
High-Blank
0.0E+00
25
1
0
0.0
0.0
N/A
25
4
0
0.0
0.0
N/A
8-PM10HV-
High-Blank
0.0E+00
25
1
0
0.0
0.0
N/A
25
4
0
0.0
0.0
N/A
9-PM10HV-
High-30
9.0E+00
25
1
0
0.0
0.0
0.0
25
4
0
0.0
0.0
0.0
10-PM10HV-
High-30
9.0E+00
25
1
0
0.0
0.0
0.0
25
4
1
0.3
6.6
73.1
11-PM10HV-
High-300
9.0E+01
25
1
0
0.0
0.0
0.0
25
4
0
0.0
0.0
0.0
12-PM10HV-
High-300
9.0E+01
25
1
2
2.0
50.0
55.6
25
4
6
1.5
37.5
41.7
Green highlighted cells are the values reported for percent recovery
Spilled during spore recovery
1 Actual number of B. a. Sterne spores spiked onto filter based on spiking suspension titer and volume of suspension applied.
J-3
-------
EPA/600/R-19/082
10/8/2019
APPENDIX K. CULTURE RESULTS FOR AIR QUALITY FILTERS
USING BBCA MEDIUM
K-l
-------
EPA/600/R-19/082
10/8/2019
April 9, 2018 Trial - PM10 California Filters
Sample ID
Spore
Load1
Extraction
Volume
(mL)
Volume in
Filter Cup
(mL)
Plate
Counts
(CFU)
Average Sample
Concentration
Percent
Recovery
CFII/mL
Total CFU
PBSTNeg.
0
25
4
0
0.0
0.0
#DIV/0!
25
4
0
0.0
0.0
#DIV/0!
1-PM10-C A-AV G-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
2-PM10-CA-AVG-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
3-PM10-CA-AVG-
30
3.1E+01
25
1
0
0.0
0.0
0.0
3.1E+01
25
4
0
0.0
0.0
0.0
4-PM10-CA-AVG-
30
3.1E+01
25
1
0
0.0
0.0
0.0
3.1E+01
25
4
0
0.0
0.0
0.0
5-PM10-CA-AVG-
300
3.1E+02
25
1
1
1.0
25.0
8.1
3.1E+02
25
4
Lawn
#VALUE!
#VALUE!
#VALUE!
6-PM10-CA-AVG-
300
3.1E+02
25
1
0
0.0
0.0
0.0
3.1E+02
25
4
0
0.0
0.0
0.0
7-PM10-CA-AVG-
3,000
3.1E+03
25
1
0
0.0
0.0
0.0
3.1E+03
25
4
1
0.3
6.3
0.2
8-PM10-CA-AVG-
3,000
3.1E+03
25
1
1
1.0
25.0
0.8
3.1E+03
25
4
3
0.8
18.8
0.6
9-PM10-C A-High-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
10-PM10-C A-High-
0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
11-PM10-CA-High-
30
3.1E+01
25
1
0
0.0
0.0
0.0
3.1E+01
25
4
0
0.0
0.0
0.0
12-PM10-CA-High-
30
3.1E+01
25
1
0
0.0
0.0
0.0
3.1E+01
25
4
0
0.0
0.0
0.0
13-PM10-CA-High-
300
3.1E+02
25
1
0
0.0
0.0
0.0
3.1E+02
25
4
0
0.0
0.0
0.0
14-PM10-C A-High-
300
3.1E+02
25
1
1
1.0
25.0
8.1
3.1E+02
25
4
2
0.5
12.5
4.0
15-PM10-CA-High-
3,000
3.1E+03
25
1
0
0.0
0.0
0.0
3.1E+03
25
4
4
1.0
25.0
0.8
16-PM10-C A-High-
3,000
3.1E+03
25
1
0
0.0
0.0
0.0
3.1E+03
25
4
3
0.8
18.8
0.6
Use values highlighted in green for reporting
1 Actual number of B. a. Sterne spores spiked onto filter based on spiking suspension titer and volume of suspension applied.
K-2
-------
EPA/600/R-19/082
10/8/2019
April 16, 2018 Trial - PM2.5 Wisconsin Filters
Sample ID
Spore
Extraction
Volume
(mL)
Volume in
Filter Cup
(mL)
Plate
Counts
(CFU)
Average Sample
Concentration
Percent
Load1
CFII/mL
Total CFU
Recovery
PBSTNeg.
0
25
4
0
0.0
0.0
#DIV/0!
25
4
0
0.0
0.0
#DIV/0!
1-PM2.5-WI-
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
AVG-0
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
2-PM2.5-WI-
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
AVG-0
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
3-PM2.5-WI-
2.2E+01
25
1
0
0.0
0.0
0.0
AVG-30
2.2E+01
25
4
0
0.0
0.0
0.0
4-PM2.5-WI-
2.2E+01
25
1
0
0.0
0.0
0.0
AVG-30
2.2E+01
25
4
0
0.0
0.0
0.0
5-PM2.5-WI-
2.2E+02
25
1
0
0.0
0.0
0.0
AVG-300
2.2E+02
25
4
3
0.8
18.8
8.5
6-PM2.5-WI-
2.2E+02
25
1
0
0.0
0.0
0.0
AVG-300
2.2E+02
25
4
2
0.5
12.5
5.7
7-PM2.5-WI-
2.2E+03
25
1
8
8.0
200.0
9.1
AVG-3,000
2.2E+03
25
4
26
6.5
162.5
7.4
8-PM2.5-WI-
2.2E+03
25
1
1
1.0
25.0
1.1
AVG-3,000
2.2E+03
25
4
20
5.0
125.0
5.7
9-PM2.5-WI-
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
High-0
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
10-PM2.5-WI-
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
High-0
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
11-PM2.5-WI-
2.2E+01
25
1
0
0.0
0.0
0.0
High-30
2.2E+01
25
4
0
0.0
0.0
0.0
12-PM2.5-WI-
2.2E+01
25
1
0
0.0
0.0
0.0
High-30
2.2E+01
25
4
0
0.0
0.0
0.0
13-PM2.5-WI-
2.2E+02
25
1
1
1.0
25.0
11.4
High-300
2.2E+02
25
4
3
0.8
18.8
8.5
14-PM2.5-WI-
2.2E+02
25
1
2
2.0
50.0
22.7
High-300
2.2E+02
25
4
5
1.3
31.3
14.2
15-PM2.5-WI-
2.2E+03
25
1
12
12.0
300.0
13.6
High-3,000
2.2E+03
25
4
37
9.3
231.3
10.5
16-PM2.5-WI-
2.2E+03
25
1
1
1.0
25.0
1.1
High-3,000
2.2E+03
25
4
7
1.8
43.8
2.0
Use values highlighted in green for reporting
1 Actual number of B. a. Sterne spores spiked onto filter based on spiking suspension titer and volume of suspension applied.
K-3
-------
EPA/600/R-19/082
10/8/2019
APPENDIX L. RV-PCR RESULTS FOR AIR QUALITY FILTERS USING
CHROMOSOMAL AND PXOl GENE TARGETS
L-l
-------
EPA/600/R-19/082
10/8/2019
1 rinl l);ilc
S;ini|ilr II)
SpiilV
1 Mllll1
< IlK Illl.
\\rr:i!ir
( 1
( hi'niii.
Mild
IV\
( hum.
\< l
( Iii'iiiii.
kl'Mlll
|i\()l
\\rr;i!ir
( I
|i\()l
SI ml
l)r\
|i\()l
( hum.
\< 1
|i.\()l ( hi'niii.
kl'Mlll
07/10/17
PM10
1-PM10-WI-
High-0 TO
0
45
0
10.7
Neg.*
39.3
1.4
5.8
Neg.
Wisconsin
1-PM10-WI-
High-0 Tf
34.3
0.4
33.6
0.2
2-PM10-WI-
High-0 TO
0
45
0
13.1
Pos.
43.3
2.9
11.9
Pos.
2-PM10-WI-
High-0 Tf
31.9
0.1
31.4
0.1
3-PM10-WI-
High-0 TO
0
45
0
14.4
Pos.
43.5
2.7
13.3
Pos.
3-PM10-WI-
High-0 Tf
30.6
0.2
30.2
0
1-PM10-WI-
High-30 TO
13
45
0
22.7
Pos.
43.4
2.8
21.5
Pos.
1-PM10-WI-
High-30 Tf
22.3
0.1
21.9
0
2-PM10-WI-
High-30 TO
13
40.7
3.7
7
Neg.
39.3
2.6
6.4
Neg.
2-PM10-WI-
High-30 Tf
33.8
0.4
32.9
0
3-PM10-WI-
High-30 TO
13
44.7
0.5
10.5
Neg.*
39.3
0.5
6
Neg.
3-PM10-WI-
High-30 Tf
34.2
0.3
33.2
0.1
1-PM10-WI-
High-3000 TO
1,300
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
1-PM10-WI-
High-3000 Tf
N/A
N/A
N/A
N/A
N/A
N/A
2-PM10-WI-
High-3000 TO
1,300
45
0
27.4
Pos.
44.1
1.5
26.8
Pos.
2-PM10-WI-
High-3000 Tf
17.6
0
17.3
0
3-PM10-WI-
High-3000 TO
1,300
45
0
25.1
Pos.
45
0
25.3
Pos.
3-PM10-WI-
High-3000 Tf
19.9
0
19.7
0
1-PM10-WI-
NEW-3000
TO
1,300
45
0
26.7
Pos.
45
0
26.9
Pos.
1-PM10-WI-
NEW-3000
Tf
18.3
0
18.1
0
2-PM10-WI-
NEW-3000
TO
1,300
45
0
26
Pos.
45
0
26.5
Pos.
2-PM10-WI-
NEW-3000
Tf
19
0.1
18.5
0.1
3-PM10-WI-
NEW-3000
TO
1,300
45
0
27
Pos.
44.7
0.5
26.9
Pos.
3-PM10-WI-
NEW-3000
Tf
18
0
17.8
0.1
07/17/17
PM10
1-PM10-WI-
NEW-0 (TO)
0
45
0
8.1
Neg.
45
0
9.1
Neg.*
Wisconsin
1-PM10-WI-
NEW-0 (Tf)
36.9
0.7
35.9
0.2
2-PM10-WI-
NEW-0 (TO)
0
45
0
N/A
N/A
44.3
1.1
N/A
N/A
2-PM10-WI-
NEW-0 (Tf)
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
L-2
-------
EPA/600/R-19/082
10/8/2019
1 rinl l);ilc
S;ini|ilr II)
SpiilV
1 Mllll1
< IlK Illl.
\\rr:i!ir
( 1
( hi'niii.
Mild
IV\
( hum.
\< l
( Iii'iiiii.
kl'Mlll
Il\()l
\\rr;i!ir
( I
|i\()l
SI ml
l)r\
|i\()l
( hum.
\< 1
|i.\()l ( hi'niii.
kl'Mlll
PM10-WI-
NEW-30 1
(TO)
9
42.8
3.8
6.7
Neg.
45
0
9.3
Neg.*
PM10-WI-
NEW-30 1
(Tf)
36.1
0.2
35.7
0.2
PM10-WI-
NEW-30 2
(TO)
9
45
0
8.5
Neg.
45
0
9.3
Neg.*
PM10-WI-
NEW-30 2
(Tf)
36.5
0.3
35.7
0.7
PM10-WI-
NEW-300 1
(TO)
90
42.9
3.7
25.5
Pos.
40.1
1.2
22.9
Pos.
PM10-WI-
NEW-300 1
(Tf)
17.4
0
17.2
0
PM10-WI-
NEW-300 2
(TO)
90
45
0
27.6
Pos.
45
0
28
Pos.
PM10-WI-
NEW-300 2
(Tf)
17.4
0
17
0
PM10-WI-
HIGH-0 1
(TO)
0
42.4
4.4
10.8
Pos.
40.2
4.2
9.2
Pos.
PM10-WI-
HIGH-0 1
(Tf)
31.7
0.1
31
0.1
PM10-WI-
HIGH-0 2
(TO)
0
45
0
11.7
Pos.
43.3
2.9
10.7
Pos.
PM10-WI-
HIGH-0 2
(Tf)
33.3
0.1
32.6
0.2
PM10-WI-
HIGH-30 1
(TO)
9
45
0
25.8
Pos.
41.1
3.4
22.3
Pos.
PM10-WI-
HIGH-30 1
(Tf)
19.2
0
18.8
0
PM10-WI-
HIGH-300 1
(TO)
90
45
0
21.1
Pos.
44.8
0.3
21.7
Pos.
PM10-WI-
HIGH-300 1
(Tf)
23.9
0
23.1
0
PM10-WI-
HIGH-300 2
(TO)
90
45
0
28.1
Pos.
44.7
0.6
28.1
Pos.
PM10-WI-
HIGH-300 2
(Tf)
16.9
0
16.6
0
10/09/17
PM10
1-PM10-
NEW-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
Wisconsin
1-PM10-
NEW-0 Tf
45
0
45
0
2-PM10-
AVG-0 TO
0
45
0
0
Neg.
44.9
0.2
-0.1
Neg.
2-PM10-
AVG-0 Tf
45
0
45
0
3-PM10-
NEW-30 TO
18
45
0
27.8
Pos.
45
0
27.9
Pos.
L-3
-------
EPA/600/R-19/082
10/8/2019
1 rinl l);ilc
S;ini|ilr II)
SpiilV
1 Mllll1
< IlK Illl.
\\rr:i!ir
( 1
( hi'niii.
Mild
IV\
( hum.
\< l
( Iii'iiiii.
kl'Mlll
|i\()l
\\rr;i!ir
( I
|i\()l
SI ml
l)r\
ll\()l
( hum.
\< 1
|i.\()l ( hi'niii.
kl'Mlll
3-PM10-
NEW-30 Tf
17.2
0.1
17.1
0
4-PM10-
AVG-30 TO
18
45
0
22.4
Pos.
45
0
22.9
Pos.
4-PM10-
AVG-30 Tf
22.6
0
22.1
0
5-PM10-
NEW-300 TO
180
45
0
27.2
Pos.
45
0
27.5
Pos.
5-PM10-
NEW-300 Tf
17.8
0.1
17.5
0
6-PM10-
AVG-300 TO
180
45
0
24.5
Pos.
45
0
25.1
Pos.
6-PM10-
AVG-300 Tf
20.5
0
19.9
0
7-PM10-
High-300 TO
180
45
0
20.6
Pos.
45
0
21.1
Pos.
7-PM10-
High-300 Tf
24.4
0.1
23.9
0
8-PM10-
AVG-3,000
TO
1,800
45
0
26.7
Pos.
45
0
27.1
Pos.
8-PM10-
AVG-3,000
Tf
18.3
0
17.9
0
9-PM10-
AVG-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
9-PM10-
AVG-0 Tf
45
0
45
0
10-PM10-
AVG-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
10-PM10-
AVG-0 Tf
45
0
45
0
11-PM10-
AVG-30 TO
18
45
0
2.1
Neg.
45
0
8.6
Neg.
11-PM10-
AVG-30 Tf
42.9
3.6
36.4
0.4
12-PM10-
AVG-30 TO
18
45
0
19.1
Pos.
45
0
19.6
Pos.
12-PM10-
AVG-30 Tf
25.9
0
25.4
0
13-PM10-
AVG-300 TO
180
45
0
22.6
Pos.
45
0
23
Pos.
13-PM10-
AVG-300 Tf
22.4
0
22
0.1
14-PM10-
AVG-300 TO
180
45
0
24.8
Pos.
45
0
25.6
Pos.
14-PM10-
AVG-300 Tf
20.2
0
19.4
0
15-PM10-
AVG-3,000
TO
1,800
45
0
26.7
Pos.
45
0
27.3
Pos.
15-PM10-
AVG-3,000
Tf
18.3
0
17.7
0
16-PM10-
AVG-3,000
TO
1,800
45
0
29.2
Pos.
45
0
29.8
Pos.
16-PM10-
AVG-3,000
Tf
15.8
0
15.2
0
10/16/17
PM2.5
1-PM2.5-FL-
AVG -0 TO
0
45
0
0
Neg.
45
0
0
Neg.
Florida
1-PM2.5-FL-
AVG -0 Tf
45
0
45
0
L-4
-------
EPA/600/R-19/082
10/8/2019
1 rinl l);ilc
S;ini|ilr II)
SpiilV
1 Mllll'
< IlK Illl.
\\rr:i!ir
( 1
( hi'niii.
Mild
IV\
( hum.
\< l
( Iii'iiiii.
kl'Mlll
Il\()l
\\rr;i!ir
( I
|i\()l
SI ml
l)r\
ll\()l
( hum.
\< 1
|i.\()l ( hi'niii.
kl'Mlll
2-PM2.5-FL-
AVG -0 TO
0
45
0
2.2
Neg.
45
0
0.1
Neg.
2-PM2.5-FL-
AVG -0 Tf
42.8
3.9
44.9
0.2
3-PM2.5-FL-
AVG -30 TO
20
45
0
27.8
Pos.
45
0
28
Pos.
3-PM2.5-FL-
AVG -30 Tf
17.2
0
17
0
4-PM2.5-FL-
AVG -30 TO
20
45
0
28.5
Pos.
45
0
28.8
Pos.
4-PM2.5-FL-
AVG -30 Tf
16.5
0.1
16.2
0.1
5-PM2.5-FL-
AVG -300 TO
200
45
0
28.2
Pos.
45
0
28.4
Pos.
5-PM2.5-FL-
AVG -300 Tf
16.8
0.1
16.6
0
6-PM2.5- FL-
AVG -300 TO
200
45
0
28.2
Pos.
45
0
28.5
Pos.
6-PM2.5- FL-
AVG -300 Tf
16.8
0
16.5
0
7-PM2.5-FL-
AVG -3,000
TO
2,000
45
0
27.5
Pos.
45
0
27.8
Pos.
7-PM2.5-FL-
AVG -3,000
Tf
17.5
0
17.2
0
8-PM2.5-FL-
AVG -3,000
TO
2,000
45
0
27.8
Pos.
45
0
28.2
Pos.
8-PM2.5-FL-
AVG -3,000
Tf
17.2
0
16.8
0
9-PM2.5-FL-
HIGH-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
9-PM2.5-FL-
HIGH-0 Tf
45
0
45
0
10-PM2.5-
FL-HIGH -0
TO
0
45
0
0
Neg.
45
0
0
Neg.
10-PM2.5-
FL-HIGH -0
Tf
45
0
45
0
11-PM2.5-
FL-HIGH -30
TO
20
45
0
28.8
Pos.
45
0
28.8
Pos.
11-PM2.5-
FL-HIGH -30
Tf
16.2
0.1
16.2
0
12-PM2.5-
FL-HIGH -30
TO
20
45
0
0
Neg.
45
0
0
Neg.
12-PM2.5-
FL-HIGH -30
Tf
45
0
45
0
13-PM2.5-
FL-HIGH -
300 TO
200
45
0
29.4
Pos.
45
0
29.6
Pos.
13-PM2.5-
FL-HIGH -
300 Tf
15.6
0
15.4
0
14-PM2.5-
FL-HIGH -
300 TO
200
45
0
28.2
Pos.
45
0
28.5
Pos.
L-5
-------
EPA/600/R-19/082
10/8/2019
1 rinl l);ilc
S;ini|ilr II)
SpiilV
1 Mllll'
< IlK Illl.
\\rr:i!ir
( 1
( hi'niii.
Mild
IV\
( hum.
\< l
( Iii'iiiii.
kl'Mlll
Il\()l
\\rr;i!ir
( I
|i\()l
SI ml
l)r\
ll\()l
( hum.
\< 1
|i.\()l ( hi'niii.
kl'Mlll
14-PM2.5-
FL-HIGH -
300 Tf
16.8
0.1
16.5
0
15-PM2.5-
FL-HIGH -
3,000 TO
2,000
45
0
27.8
Pos.
45
0
28.1
Pos.
15-PM2.5-
FL-HIGH -
3,000 Tf
17.2
0
16.9
0
16-PM2.5-
FL-HIGH -
3,000 TO
2,000
45
0
27.9
Pos.
45
0
28.2
Pos.
16-PM2.5-
FL-HIGH -
3,000 Tf
17.1
0
16.8
0
10/23/17
PM10 New
1-PM10-NH-
AVG-0 TO
0
45
0
0
Neg.
45
0
0.7
Neg.
Hampshire
1-PM10-NH-
AVG-0 Tf
45
0
44.3
1.1
2-PM10-NH-
AVG-0 TO
0
45
0
5
Neg.
45
0
8.8
Neg.
2-PM10-NH-
AVG-0 Tf
40
4.4
36.2
0.6
3-PM10-NH-
AVG-30 TO
16
45
0
27.9
Pos.
45
0
28
Pos.
3-PM10-NH-
AVG-30 Tf
17.1
0
17
0
4-PM10-NH-
AVG-30 TO
16
45
0
8.8
Neg.
45
0
9.7
Neg.*
4-PM10-NH-
AVG-30 Tf
36.2
0.4
35.3
0.2
5-PM10-NH-
AVG-300 TO
160
45
0
27.2
Pos.
45
0
27.5
Pos.
5-PM10-NH-
AVG-300 Tf
17.8
0.1
17.5
0
6-PM10-NH-
AVG-300 TO
160
45
0
27.5
Pos.
45
0
27.7
Pos.
6-PM10-NH-
AVG-300 Tf
17.5
0
17.3
0
7-PM10-NH-
AVG-3,000
TO
1,600
44.5
0.8
27.7
Pos.
45
0
28.3
Pos.
7-PM10-NH-
AVG-3,000
Tf
16.8
0.1
16.7
0
8-PM10-NH-
AVG-3,000
TO
1,600
45
0
27.5
Pos.
45
0
27.6
Pos.
8-PM10-NH-
AVG-3,000
Tf
17.5
0.1
17.4
0
9-PM10-NH-
HIGH-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
9-PM10-NH-
HIGH-0 Tf
45
0
45
0
10-PM10-
NH-HIGH-0
TO
0
45
0
0
Neg.
45
0
0
Neg.
10-PM10-
NH-HIGH-0
Tf
45
0
45
0
11-PM10-
NH-HIGH-30
TO
16
45
0
10.9
Pos.
45
0
11.7
Pos.
L-6
-------
EPA/600/R-19/082
10/8/2019
1 rinl l);ilc
S;ini|ilr II)
SpiilV
1 Mllll1
< IlK Illl.
\\rr:i!ir
( 1
( hi'niii.
Mild
IV\
( hum.
\< l
( Iii'iiiii.
kl'Mlll
Il\()l
\\rr;i!ir
( I
|i\()l
SI ml
l)r\
ll\()l
( hum.
\< 1
|i.\()l ( hi'niii.
kl'Mlll
11-PM10-
NH-HIGH-30
Tf
34.1
0.5
33.3
0
12-PM10-
NH-HIGH-30
TO
16
45
0
4.3
Neg.
45
0
8.1
Neg.
12-PM10-
NH-HIGH-30
Tf
40.7
3.7
36.9
0.5
13-PM10-
NH-HIGH-
300 TO
160
45
0
27.5
Pos.
45
0
27.5
Pos.
13-PM10-
NH-HIGH-
300 Tf
17.5
0.1
17.5
0
14-PM10-
NH-HIGH-
300 TO
160
45
0
27
Pos.
45
0
27.1
Pos.
14-PM10-
NH-HIGH-
300 Tf
18
0
17.9
0
15-PM10-
NH-HIGH-
3,000 TO
1,600
45
0
25.1
Pos.
45
0
25.4
Pos.
15-PM10-
NH-HIGH-
3,000 Tf
19.9
0
19.6
0
16-PM10-
NH-HIGH-
3,000 TO
1,600
45
0
26.8
Pos.
45
0
27
Pos.
16-PM10-
NH-HIGH-
3,000 Tf
18.2
0.3
18
0
10/30/17
PM2.5
1-PM2.5-NA-
New-0 TO
0
45
0
0
Neg.
42.7
3.9
-2.3
Neg.
Arizona
1-PM2.5-NA-
New-0 Tf
45
0
45
0
2-PM2.5-AZ-
AVG-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
2-PM2.5-AZ-
AVG-0 Tf
45
0
45
0
3-PM2.5-NA-
New-30 TO
15
45
0
28
Pos.
45
0
27.9
Pos.
3-PM2.5-NA-
New-30 Tf
17
0.1
17.1
0
4-PM2.5-AZ-
AVG-30 TO
15
45
0
27.6
Pos.
45
0
28
Pos.
4-PM2.5-AZ-
AVG-30 Tf
17.4
0
17
0
5-PM2.5-NA-
New-300 TO
150
45
0
27.9
Pos.
45
0
27.9
Pos.
5-PM2.5-NA-
New-300 Tf
17.1
0
17.1
0
6-PM2.5-AZ-
AVG-300 TO
150
45
0
27.1
Pos.
45
0
27.5
Pos.
6-PM2.5-AZ-
AVG-300 Tf
17.9
0
17.5
0
7-PM2.5-NA-
New-3,000
TO
1,500
45
0
27.1
Pos.
45
0
27.2
Pos.
7-PM2.5-NA-
New-3,000 Tf
17.9
0
17.8
0.1
L-7
-------
EPA/600/R-19/082
10/8/2019
1 rinl l);ilc
S;ini|ilr II)
SpiilV
1 Mllll'
< IlK Illl.
\\rr:i!ir
( 1
( hi'niii.
Mild
IV\
( hum.
\< l
( Iii'iiiii.
kl'Mlll
|i\()l
\\rr;i!ir
( I
|i\()l
SI ml
l)r\
ll\()l
( hum.
\< 1
|i.\()l ( hi'niii.
kl'Mlll
8-PM2.5-AZ-
AVG-3,000
TO
1,500
45
0
28.1
Pos.
45
0
28.6
Pos.
8-PM2.5-AZ-
AVG-3,000
Tf
16.9
0
16.4
0
9-PM2.5-AZ-
High-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
9-PM2.5-AZ-
High-0 Tf
45
0
45
0
10-PM2.5-
AZ-High-0
TO
0
45
0
0
Neg.
45
0
0
Neg.
10-PM2.5-
AZ-High-0 Tf
45
0
45
0
11-PM2.5-
AZ-High-30
TO
15
45
0
28.5
Pos.
45
0
28.3
Pos.
11-PM2.5-
AZ-High-30
Tf
16.5
0.1
16.7
0
12-PM2.5-
AZ-High-30
TO
15
45
0
27.9
Pos.
45
0
28
Pos.
12-PM2.5-
AZ-High-30
Tf
17.1
0
17
0
13-PM2.5-
AZ-High-300
TO
150
45
0
27.3
Pos.
45
0
27.3
Pos.
13-PM2.5-
AZ-High-300
Tf
17.7
0.1
17.7
0
14-PM2.5-
AZ-High-300
TO
150
45
0
27.1
Pos.
45
0
27.2
Pos.
14-PM2.5-
AZ-High-300
Tf
17.9
0
17.8
0
15-PM2.5-
AZ-High-
3,000 TO
1,500
45
0
27.1
Pos.
45
0
27.3
Pos.
15-PM2.5-
AZ-High-
3,000 Tf
17.9
0.1
17.7
0
16-PM2.5-
AZ-High-
3,000 TO
1,500
45
0
27.1
Pos.
45
0
27.6
Pos.
16-PM2.5-
AZ-High-
3,000 Tf
17.9
0
17.4
0
03/26/18
PM2.5
Arizona
1-PM2.5-AZ-
AVG-PES-0
TO
0
44.6
0.7
-0.4
Neg.
43.8
2
-1.2
Neg.
1-PM2.5-AZ-
AVG-PES-0
Tf
45
0
45
0
2-PM2.5-AZ-
AVG-PVDF-
0 TO
0
45
0
0
Neg.
45
0
0
Neg.
2-PM2.5-AZ-
AVG-PVDF-
0 Tf
45
0
45
0
L-8
-------
EPA/600/R-19/082
10/8/2019
1 rinl l);ilc
S;ini|ilr II)
SpiilV
1 Mllll1
< IlK Illl.
\\rr:i!ir
( 1
( hi'niii.
Mild
IV\
( hum.
\< l
( Iii'iiiii.
kl'Mlll
Il\()l
\\rr;i!ir
( I
|i\()l
SI ml
l)r\
ll\()l
( hum.
\< 1
|i.\()l ( hi'niii.
kl'Mlll
3-PM2.5-AZ-
AVG-PES-30
TO
23
43.7
2.2
25.8
Pos.
44.2
1.4
26.5
Pos.
3-PM2.5-AZ-
AVG-PES-30
Tf
17.9
0.1
17.7
0
4-PM2.5-AZ-
AVG-PVDF-
30 TO
23
43.3
2.9
26
Pos.
38.2
0.2
21.3
Pos.
4-PM2.5-AZ-
AVG-PVDF-
30 Tf
17.3
0
16.9
0
5-PM2.5-AZ-
AVG-PES-
300 TO
230
43.4
2.7
26.2
Pos.
41.1
3.4
23.8
Pos.
5-PM2.5-AZ-
AVG-PES-
300 Tf
17.2
0
17.3
0
6-PM2.5-AZ-
AVG-PVDF-
300 TO
230
45
0
28.4
Pos.
39.3
1.2
23
Pos.
6-PM2.5-AZ-
AVG-PVDF-
300 Tf
16.6
0
16.3
0
7-PM2.5-AZ-
AVG-PES-
3,000 TO
2,300
45
0
27.2
Pos.
43.8
1.6
26.3
Pos.
7-PM2.5-AZ-
AVG-PES-
3,000 Tf
17.8
0
17.5
0
8-PM2.5-AZ-
AVG-PVDF-
3,000 TO
2,300
44
1.8
25.6
Pos.
41.3
3.2
23.4
Pos.
8-PM2.5-AZ-
AVG-PVDF-
3,000 Tf
18.3
0
17.9
0
9-PM2.5-NA-
NEW-PES-0
TO
0
45
0
0
Neg.
45
0
0
Neg.
9-PM2.5-NA-
NEW-PES-0
Tf
45
0
45
0
10-PM2.5-
NA-NEW-
PVDF-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
10-PM2.5-
NA-NEW-
PVDF-0 Tf
45
0
45
0
11-PM2.5-
NA-NEW-
PES-30 TO
23
45
0
28.2
Pos.
45
0
28.1
Pos.
11-PM2.5-
NA-NEW-
PES-30 Tf
16.8
0
16.9
0
12-PM2.5-
NA-NEW-
PVDF-30 TO
23
45
0
27.5
Pos.
42.5
3.2
25.3
Pos.
12-PM2.5-
NA-NEW-
PVDF-30 Tf
17.5
0
17.2
0
13-PM2.5-
NA-NEW-
PES-300 TO
230
45
0
27.9
Pos.
45
0
27.8
Pos.
L-9
-------
EPA/600/R-19/082
10/8/2019
1 rinl l);ilc
S;ini|ilr II)
SpiilV
1 Mllll1
< IlK Illl.
\\rr:i!ir
( 1
( hi'niii.
Mild
IV\
( hum.
\< l
( Iii'iiiii.
kl'Mlll
|i\()l
\\rr;i!ir
( I
|i\()l
SI ml
l)r\
|i\()l
( hum.
\< 1
|i.\()l ( hi'niii.
kl'Mlll
13-PM2.5-
NA-NEW-
PES-300 Tf
17.1
0
17.2
0
14-PM2.5-
NA-NEW-
PVDF-300
TO
230
45
0
26.6
Pos.
45
0
26.8
Pos.
14-PM2.5-
NA-NEW-
PVDF-300 Tf
18.4
0.1
18.2
0
15-PM2.5-
NA-NEW-
PES-3,000 TO
2,300
45
0
27.3
Pos.
45
0
27.5
Pos.
15-PM2.5-
NA-NEW-
PES-3,000 Tf
17.7
0.1
17.5
0
16-PM2.5-
NA-NEW-
PVDF-3,000
TO
2,300
45
0
27
Pos.
45
0
27.5
Pos.
16-PM2.5-
NA-NEW-
PVDF-3,000
Tf
18
0
17.5
0
04/02/18
PM2.5 FL &
1-PM2.5-FL-
AVG-0 TO
0
45
0
12.2
Pos.
45
0
12.8
Pos.
PM10 New
Hampshire
1-PM2.5-FL-
AVG-0 Tf
32.8
0.3
32.2
0.2
2-PM2.5-FL-
High-0 TO
0
45
0
11.6
Pos.
45
0
12.3
Pos.
2-PM2.5-FL-
High-0 Tf
33.4
0.1
32.7
0.1
3-PM2.5-FL-
AVG-30 TO
25
45
0
26.6
Pos.
45
0
26.8
Pos.
3-PM2.5-FL-
AVG-30 Tf
18.4
0.1
18.2
0.1
4-PM2.5-FL-
High-30 TO
25
45
0
26.1
Pos.
45
0
26.4
Pos.
4-PM2.5-FL-
High-30 Tf
18.9
0.1
18.6
0.1
5-PM2.5-FL-
AVG-300 TO
250
45
0
26.6
Pos.
45
0
26.9
Pos.
5-PM2.5-FL-
AVG-300 Tf
18.4
0
18.1
0
6-PM2.5-FL-
HIGH-300 TO
250
45
0
No Data
No Data
45
0
No Data
No Data
6-PM2.5-FL-
HIGH-300 Tf
No Data
No Data
No Data
No Data
7-PM2.5-FL-
AVG-3,000
TO
2,500
45
0
25.3
Pos.
45
0
25.4
Pos.
7-PM2.5-FL-
AVG-3,000
Tf
19.7
0
19.6
0.1
8-PM2.5-FL-
HIGH-3,000
TO
2,500
45
0
26.6
Pos.
45
0
27
Pos.
8-PM2.5-FL-
HIGH-3,000
Tf
18.4
0
18
0
9-PM10-NH-
AVG-0 TO
0
45
0
0
Neg.
45
0
6.8
Neg.
9-PM10-NH-
AVG-0 Tf
45
0
38.2
0.2
L-10
-------
EPA/600/R-19/082
10/8/2019
1 rinl l);ilr
S;ini|ilr II)
SpiilV
1 Mllll1
< IlK Illl.
\\rr:i!ir
( 1
( hi'niii.
Mild
IV\
( hum.
\< l
( Iii'iiiii.
kl'Mlll
Il\()l
\\rr;i!ir
( I
|i\()l
SI ml
l)r\
|i\()l
( hum.
\< 1
|i.\()l ( hi'niii.
kl'Mlll
10-PM10-
NH-HIGH-0
TO
0
45
0
0
Neg.
45
0
9.8
Neg.
(chromosome
assay ACt is
10-PM10-
NH-HIGH-0
Tf
45
0
35.2
0.5
negative)
11-PM10-
NH-AVG-30
TO
25
No Data
No Data
No Data
No Data
No Data
No Data
No Data
No Data
11-PM10-
NH-AVG-30
Tf
41.6
3.2
33.5
0.4
12-PM10-
NH-HIGH-30
TO
25
45
0
27
Pos.
45
0
27.2
Pos.
12-PM10-
NH-HIGH-30
Tf
18
0.1
17.8
0.1
13-PM10-
NH-AVG-
300 TO
250
45
0
25.3
Pos.
45
0
25.4
Pos.
13-PM10-
NH-AVG-
300 Tf
19.7
0.1
19.6
0.1
14-PM10-
NH-HIGH-
300 TO
250
45
0
22
Pos.
45
0
22.2
Pos.
14-PM10-
NH-HIGH-
300 Tf
23
0
22.8
0
15-PM10-
NH-AVG-
3,000 TO
2,500
45
0
27.4
Pos.
45
0
27.6
Pos.
15-PM10-
NH-AVG-
3,000 Tf
17.6
0
17.4
0
16-PM10-
NH-HIGH-
3,000 TO
2,500
45
0
25.5
Pos.
45
0
25.9
Pos.
16-PM10-
NH-HIGH-
3,000 Tf
19.5
0
19.1
0
04/09/18
PM10
1-PM10-CA-
AVG-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
California
1-PM10-CA-
AVG-0 Tf
45
0
45
0
2-PM10-CA-
AVG-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
2-PM10-CA-
AVG-0 Tf
45
0
45
0
3-PM10-CA-
AVG-30 TO
16
44.9
0.2
29.5
Pos.
45
0
30.1
Pos.
3-PM10-CA-
AVG-30 Tf
15.4
0.1
14.9
0
4-PM10-CA-
AVG-30 TO
16
45
0
0
Neg.
45
0
0
Neg.
4-PM10-CA-
AVG-30 Tf
45
0
45
0
5-PM10-CA-
AVG-300 TO
160
45
0
23.3
Pos.
45
0
23.5
Pos.
5-PM10-CA-
AVG-300 Tf
21.7
0.1
21.5
0
6-PM10-CA-
AVG-300 TO
160
45
0
24.7
Pos.
45
0
25.4
Pos.
L-ll
-------
EPA/600/R-19/082
10/8/2019
1 rinl l);ilc
S;ini|ilr II)
SpiilV
1 Mllll1
< IlK Illl.
\\rr:i!ir
( 1
( hi'niii.
Mild
IV\
( hum.
\< l
( Iii'iiiii.
kl'Mlll
|i\()l
\\rr;i!ir
( I
|i\()l
SI ml
l)r\
|i\()l
( hum.
\< 1
|i.\()l ( hi'niii.
kl'Mlll
6-PM10-CA-
AVG-300 Tf
20.3
0.1
19.6
0
7-PM10-CA-
AVG-3,000
TO
1,600
45
0
22
Pos.
45
0
22.2
Pos.
7-PM10-CA-
AVG-3,000
Tf
23
0
22.8
0.1
8-PM10-CA-
AVG-3,000
TO
1,600
45
0
29.5
Pos.
45
0
30.1
Pos.
8-PM10-CA-
AVG-3,000
Tf
15.5
0
14.9
0
9-PM10-CA-
High-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
9-PM10-CA-
High-0 Tf
45
0
45
0
10-PM10-
CA-High-0
TO
0
45
0
0
Neg.
45
0
1.7
Neg.
10-PM10-
CA-High-0
Tf
45
0
43.3
2.8
11-PM10-
CA-High-30
TO
16
45
0
5.1
Neg.
45
0
7.4
Neg.
11-PM10-
CA-High-30
Tf
39.9
4.5
37.6
1.4
12-PM10-
CA-High-30
TO
16
45
0
29.5
Pos.
45
0
29.9
Pos.
12-PM10-
CA-High-30
Tf
15.5
0
15.1
0
13-PM10-
CA-High-300
TO
160
45
0
28.9
Pos.
45
0
29.1
Pos.
13-PM10-
CA-High-300
Tf
16.1
0
15.9
0
14-PM10-
CA-High-300
TO
160
45
0
28
Pos.
45
0
28.2
Pos.
14-PM10-
CA-High-300
Tf
17
0.1
16.8
0
15-PM10-
CA-High-
3,000 TO
1,600
45
0
28.7
Pos.
45
0.1
28.8
Pos.
15-PM10-
CA-High-
3,000 Tf
16.3
0.1
16.2
0
16-PM10-
CA-High-
3,000 TO
1,600
45
0
26.6
Pos.
45
0
27.4
Pos.
16-PM10-
CA-High-
3,000 Tf
18.4
0.1
17.6
0
04/16/18
PM2.5 WI
1-PM2.5-WI-
AVG-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
1-PM2.5-WI-
AVG-0 Tf
45
0
45
0
L-12
-------
EPA/600/R-19/082
10/8/2019
1 rinl l);ilc
S;ini|ilr II)
SpiilV
1 Mllll1
< IlK Illl.
\\rr:i!ir
( 1
( hi'niii.
Mild
IV\
( hum.
\< l
( Iii'iiiii.
UrMlll
|i\()l
\\rr;i!ir
( I
|i\()l
SI ml
l)r\
|i\()l
( hum.
\< 1
|i.\()l ( hi'niii.
UrMlll
2-PM2.5-WI-
AVG-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
2-PM2.5-WI-
AVG-0 Tf
45
0
45
0
3-PM2.5-WI-
AVG-30 TO
22
45
0
#DIV/0!
No Data
45
0
#DIV/0!
No Data
3-PM2.5-WI-
AVG-30 Tf
#DIV/0!
#DIV/0!
#DIV/0!
#DIV/0!
4-PM2.5-WI-
AVG-30 TO
22
45
0
27.96
Pos.
45
0
28.27
Pos.
4-PM2.5-WI-
AVG-30 Tf
17.04
0.05
16.73
0.01
5-PM2.5-WI-
AVG-300 TO
220
45
0
27.84
Pos.
45
0
28.02
Pos.
5-PM2.5-WI-
AVG-300 Tf
17.16
0.06
16.98
0.02
6-PM2.5-WI-
AVG-300 TO
220
45
0
27.81
Pos.
45
0
28.04
Pos.
6-PM2.5-WI-
AVG-300 Tf
17.19
0.09
16.96
0.05
7-PM2.5-WI-
AVG-3,000
TO
2,200
45
0
27.6
Pos.
45
0
27.84
Pos.
7-PM2.5-WI-
AVG-3,000
Tf
17.4
0.06
17.16
0.02
8-PM2.5-WI-
AVG-3,000
TO
2,200
45
0
27.69
Pos.
45
0
28.02
Pos.
8-PM2.5-WI-
AVG-3,000
Tf
17.31
0.04
16.98
0.03
9-PM2.5-WI-
High-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
9-PM2.5-WI-
High-0 Tf
45
0
45
0
10-PM2.5-
WI-High-0
TO
0
45
0
0
Neg.
45
0
0
Neg.
10-PM2.5-
WI-High-0 Tf
45
0
45
0
11-PM2.5-
WI-High-30
TO
22
45
0
28
Pos.
45
0
28.3
Pos.
11-PM2.5-
WI-High-30
Tf
17
0.03
16.7
0.05
12-PM2.5-
WI-High-30
TO
22
45
0
28.55
Pos.
45
0
28.85
Pos.
12-PM2.5-
WI-High-30
Tf
16.45
0.01
16.15
0.03
13-PM2.5-
WI-High-300
TO
220
45
0
#DIV/0!
No Data
45
0
#DIV/0!
No Data
13-PM2.5-
WI-High-300
Tf
#DIV/0!
#DIV/0!
#DIV/0!
#DIV/0!
14-PM2.5-
WI-High-300
TO
220
45
0
28.07
Pos.
45
0
28.47
Pos.
L-13
-------
EPA/600/R-19/082
10/8/2019
1 rinl l);ilc
S;ini|ilr II)
SpiilV
1 Mllll1
< IlK Illl.
\\rr:i!ir
( 1
( hi'niii.
Mild
IV\
( hum.
\< l
( Iii'iiiii.
kl'Mlll
Il\()l
\\rr;i!ir
( I
|i\()l
SI ml
l)r\
ll\()l
( hum.
\< 1
|i.\()l ( hi'niii.
kl'Mlll
14-PM2.5-
WI-High-300
Tf
16.93
0.06
16.53
0.06
15-PM2.5-
WI-High-
3,000 TO
2,200
45
0
27.61
Pos.
45
0
28
Pos.
15-PM2.5-
WI-High-
3,000 Tf
17.39
0.04
17
0.03
16-PM2.5-
WI-High-
3,000 TO
2,200
45
0
#DIV/0!
No Data
45
0
#DIV/0!
No Data
16-PM2.5-
WI-High-
3,000 Tf
#DIV/0!
#DIV/0!
#DIV/0!
#DIV/0!
04/30/18
PM2.5
Massachusetts
1-PM2.5-
MA-AVG-0
TO
0
45
0
0
Neg.
45
0
0
Neg.
1-PM2.5-
MA-AVG-0
Tf
45
0
45
0
2-PM2.5-
MA-AVG-0
TO
0
45
0
0
Neg.
45
0
0
Neg.
2-PM2.5-
MA-AVG-0
Tf
45
0
45
0
3-PM2.5-
MA-AVG-30
TO
32
45
0
27.3
Pos.
45
0
27.6
Pos.
3-PM2.5-
MA-AVG-30
Tf
17.7
0.1
17.4
0
4-PM2.5-
MA-AVG-30
TO
32
45
0
27.5
Pos.
45
0
27.7
Pos.
4-PM2.5-
MA-AVG-30
Tf
17.5
0
17.3
0
5-PM2.5-
MA-AVG-
300 TO
320
45
0
27.1
Pos.
45
0
27.2
Pos.
5-PM2.5-
MA-AVG-
300 Tf
17.9
0.1
17.8
0
6-PM2.5-
MA-AVG-
300 TO
320
45
0
27.1
Pos.
45
0
27.2
Pos.
6-PM2.5-
MA-AVG-
300 Tf
17.9
0
17.8
0
7-PM2.5-
MA-AVG-
3,000 TO
3,200
45
0
27.2
Pos.
45
0
27.4
Pos.
7-PM2.5-
MA-AVG-
3,000 Tf
17.8
0
17.6
0
8-PM2.5-
MA-AVG-
3,000 TO
3,200
45
0
26.6
Pos.
45
0
26.9
Pos.
8-PM2.5-
MA-AVG-
3,000 Tf
18.4
0
18.1
0
L-14
-------
EPA/600/R-19/082
10/8/2019
1 rinl l);ilc
S;ini|ilr II)
SpiilV
1 Mllll'
< IlK Illl.
\\rr:i!ir
( 1
( hi'niii.
Mild
IV\
( hum.
\< l
( Iii'iiiii.
kl'Mlll
Il\()l
\\rr;i!ir
( I
|i\()l
SI ml
l)r\
ll\()l
( hum.
\< 1
|i.\()l ( hi'niii.
kl'Mlll
9-PM2.5-
MA-High-0
TO
0
45
0
0
Neg.
45
0
0
Neg.
9-PM2.5-
MA-High-0
Tf
45
0
45
0
10-PM2.5-
MA-High-0
TO
0
45
0
0
Neg.
45
0
0
Neg.
10-PM2.5-
MA-High-0
Tf
45
0
45
0
11-PM2.5-
MA-High-30
TO
32
45
0
27.1
Pos.
45
0
27.2
Pos.
11-PM2.5-
MA-High-30
Tf
17.9
0.1
17.8
0
12-PM2.5-
MA-High-30
TO
32
45
0
28.2
Pos.
45
0
28.4
Pos.
12-PM2.5-
MA-High-30
Tf
16.8
0
16.6
0
13-PM2.5-
MA-High-
300 TO
320
45
0
27.1
Pos.
45
0
27.5
Pos.
13-PM2.5-
MA-High-
300 Tf
17.9
0.1
17.5
0
14-PM2.5-
MA-High-
300 TO
320
45
0
27.8
Pos.
45
0
27.9
Pos.
14-PM2.5-
MA-High-
300 Tf
17.2
0
17.1
0
15-PM2.5-
MA-High-
3,000 TO
3,200
45
0
27.1
Pos.
45
0
27.6
Pos.
15-PM2.5-
MA-High-
3,000 Tf
17.9
0
17.4
0
16-PM2.5-
MA-High-
3,000 TO
3,200
45
0
27
Pos.
45
0
27.3
Pos.
16-PM2.5-
MA-High-
3,000 Tf
18
0.1
17.7
0
05/07/18
PM2.5
Massachusets
1-PM2.5-
MA-AVG-0
TO
0
45
0
0
Neg.
45
0
0
Neg.
and
Wisconsin
1-PM2.5-
MA-AVG-0
Tf
45
0
45
0
2-PM2.5-WI-
AVG-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
2-PM2.5-WI-
AVG-0 Tf
45
0
45
0
3-PM2.5-
MA-AVG-30
TO
20
45
0
26.9
Pos.
45
0
26.9
Pos.
3-PM2.5-
MA-AVG-30
Tf
18.1
0.1
18.1
0
L-15
-------
EPA/600/R-19/082
10/8/2019
1 rinl l);ilc
S;ini|ilr II)
SpiilV
1 Mllll1
< IlK Illl.
\\rr:i!ir
( 1
( hi'niii.
Mild
IV\
( hum.
\< l
( Iii'iiiii.
kl'Mlll
Il\()l
\\rr;i!ir
( I
|i\()l
SI ml
l)r\
ll\()l
( hum.
\< 1
|i.\()l ( hi'niii.
kl'Mlll
4-PM2.5-WI-
AVG-30 TO
20
45
0
28.9
Pos.
45
0
28.9
Pos.
4-PM2.5-WI-
AVG-30 Tf
16.1
0.1
16.1
0
5-PM2.5-
MA-AVG-
300 TO
200
45
0
27.9
Pos.
45
0
28
Pos.
5-PM2.5-
MA-AVG-
300 Tf
17.1
0.1
17
0
6-PM2.5-WI-
AVG-300 TO
200
45
0
29.1
Pos.
45
0
29.2
Pos.
6-PM2.5-WI-
AVG-300 Tf
15.9
0
15.8
0
7-PM2.5-
MA-AVG-
3,000 TO
2,000
45
0
28.6
Pos.
45
0
28.7
Pos.
7-PM2.5-
MA-AVG-
3,000 Tf
16.4
0.1
16.3
0
8-PM2.5-WI-
AVG-3,000
TO
2,000
45
0
27.9
Pos.
45
0
28.1
Pos.
8-PM2.5-WI-
AVG-3,000
Tf
17.1
0
16.9
0
9-PM2.5-
MA-High-0
TO
0
45
0
0
Neg.
45
0
0
Neg.
9-PM2.5-
MA-High-0
Tf
45
0
45
0
10-PM2.5-
WI-High-0
TO
0
45
0
0
Neg.
45
0
0
Neg.
10-PM2.5-
WI-High-0 Tf
45
0
45
0
11-PM2.5-
MA-High-30
TO
20
45
0
28.3
Pos.
45
0
28.3
Pos.
11-PM2.5-
MA-High-30
Tf
16.7
0.1
16.7
0
12-PM2.5-
WI-High-30
TO
20
45
0
26.8
Pos.
45
0
26.7
Pos.
12-PM2.5-
WI-High-30
Tf
18.2
0
18.3
0
13-PM2.5-
MA-High-
300 TO
200
45
0
28.2
Pos.
45
0
28.2
Pos.
13-PM2.5-
MA-High-
300 Tf
16.8
0.1
16.8
0
14-PM2.5-
WI-High-300
TO
200
45
0
27.8
Pos.
45
0
27.9
Pos.
14-PM2.5-
WI-High-300
Tf
17.2
0
17.1
0
15-PM2.5-
MA-High-
3,000 TO
2,000
45
0
28.1
Pos.
45
0
28.1
Pos.
L-16
-------
EPA/600/R-19/082
10/8/2019
1 rinl l);ilc
S;ini|ilr II)
SpiilV
1 Mllll1
< IlK Illl.
\\rr:i!ir
( 1
( hi'niii.
Mild
IV\
( hum.
\< l
( Iii'iiiii.
kl'Mlll
Il\()l
\\rr;i!ir
( I
|i\()l
SI ml
l)r\
ll\()l
( hum.
\< 1
|i.\()l ( hi'niii.
kl'Mlll
15-PM2.5-
MA-High-
3,000 Tf
16.9
0
16.9
0
16-PM2.5-
WI-High-
3,000 TO
2,000
45
0
27.7
Pos.
45
0
27.9
Pos.
16-PM2.5-
WI-High-
3,000 Tf
17.3
0
17.1
0
05/14/18
PM2.5
1-PM10-CA-
AVG-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
Arizona,
Florida,
1-PM10-CA-
AVG-0 Tf
45
0
45
0
Wisconsin &
PM10
2-PM10-CA-
HIGH-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
California
2-PM10-CA-
HIGH-0 Tf
45
0
45
0
3-PM10-CA-
AVG-30 TO
14
45
0
0
Neg.
45
0
0
Neg.
3-PM10-CA-
AVG-30 Tf
45
0
45
0
4-PM10-CA-
HIGH-30 TO
14
45
0
0
Neg.
45
0
0
Neg.
4-PM10-CA-
HIGH-30 Tf
45
0
45
0
5-PM10-CA-
AVG-300 TO
140
45
0
21.2
Pos.
45
0
21.6
Pos.
5-PM10-CA-
AVG-300 Tf
23.8
0
23.4
0.1
6-PM10-CA-
HIGH-300 TO
140
45
0
22.5
Pos.
45
0
23.1
Pos.
6-PM10-CA-
HIGH-300 Tf
22.5
0
21.9
0
7-PM10-CA-
AVG-3,000
TO
1,400
45
0
27.5
Pos.
45
0
27.8
Pos.
7-PM10-CA-
AVG-3,000
Tf
17.5
0.1
17.2
0.1
8-PM10-CA-
HIGH-3,000
TO
1,400
45
0
28.8
Pos.
45
0
29.1
Pos.
8-PM10-CA-
HIGH-3,000
Tf
16.2
0.1
15.9
0.1
9-PM2.5-AZ-
High-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
9-PM2.5-AZ-
High-0 Tf
45
0
45
0
10-PM2.5-
AZ-High-30
TO
14
45
0
23.1
Pos.
45
0
23.1
Pos.
10-PM2.5-
AZ-High-30
Tf
21.9
0
21.9
0
11-PM2.5-
WI-AVG-30
TO
14
45
0
23.2
Pos.
45
0
23.1
Pos.
11-PM2.5-
WI-AVG-30
Tf
21.8
0.1
21.9
0
12-PM2.5-
AZ-HIGH-
300 TO
140
45
0
28.7
Pos.
45
0
28.9
Pos.
L-17
-------
EPA/600/R-19/082
10/8/2019
1 rinl l);ilc
S;ini|ilr II)
SpiilV
1 Mllll1
< IlK Illl.
\\rr:i!ir
( 1
( hi'niii.
Mild
IV\
( hum.
\< l
( Iii'iiiii.
kl'Mlll
Il\()l
\\rr;i!ir
( I
|i\()l
SI ml
l)r\
ll\()l
( hum.
\< 1
|i.\()l ( hi'niii.
kl'Mlll
12-PM2.5-
AZ-HIGH-
300 Tf
16.3
0
16.1
0
13-PM2.5-
FL-High-300
TO
140
45
0
28.6
Pos.
45
0
28.8
Pos.
13-PM2.5-
FL-High-300
Tf
16.4
0
16.2
0
14-PM2.5-
WI-High-300
TO
140
45
0
28.6
Pos.
45
0
28.7
Pos.
14-PM2.5-
WI-High-300
Tf
16.4
0
16.3
0
15-PM2.5-
AZ-High-
3,000 TO
1,400
45
0
28.3
Pos.
45
0
28.8
Pos.
15-PM2.5-
AZ-High-
3,000 Tf
16.7
0.1
16.2
0.1
16-PM2.5-
WI-High-
3,000 TO
1,400
45
0
28.5
Pos.
45
0
28.8
Pos.
16-PM2.5-
WI-High-
3,000 Tf
16.5
0
16.2
0
05/21/18
PM10 South
1-PM10-SC-
AVG-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
Carolina
1-PM10-SC-
AVG-0 Tf
45
0
45
0
2-PM10-SC-
AVG-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
2-PM10-SC-
AVG-0 Tf
45
0
45
0
3-PM10-SC-
AVG-30 TO
27
45
0
18.1
Pos.
45
0
18.2
Pos.
3-PM10-SC-
AVG-30 Tf
26.9
0.1
26.8
0
4-PM10-SC-
AVG-30 TO
27
45
0
23.3
Pos.
45
0
23.8
Pos.
4-PM10-SC-
AVG-30 Tf
21.7
0
21.2
0
5-PM10-SC-
AVG-300 TO
270
45
0
27.9
Pos.
45
0
28.1
Pos.
5-PM10-SC-
AVG-300 Tf
17.1
0.1
16.9
0
6-PM10-SC-
AVG-300 TO
270
45
0
28.6
Pos.
45
0
28.7
Pos.
6-PM10-SC-
AVG-300 Tf
16.4
0
16.3
0
7-PM10-SC-
AVG-3,000
TO
2,700
45
0
28.7
Pos.
45
0
28.8
Pos.
7-PM10-SC-
AVG-3,000
Tf
16.3
0.1
16.2
0.1
8-PM10-SC-
AVG-3,000
TO
2,700
45
0
24.3
Pos.
45
0
24.7
Pos.
8-PM10-SC-
AVG-3,000
Tf
20.7
0
20.3
0
L-18
-------
EPA/600/R-19/082
10/8/2019
1 rinl l);ilr
S;ini|ilr II)
SpiilV
1 Mllll'
< IlK Illl.
\\rr:i!ir
( 1
( hi'niii.
Mild
IV\
( hum.
\< l
( Iii'iiiii.
kl'Mlll
Il\()l
\\rr;i!ir
( I
|i\()l
SI ml
l)r\
ll\()l
( hum.
\< 1
|i.\()l ( hi'niii.
kl'Mlll
9-PM10-SC-
High-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
9-PM10-SC-
High-0 Tf
45
0
45
0
10-PM10-SC-
High-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
10-PM10-SC-
High-0 Tf
45
0
45
0
11-PM10-SC-
High-30 TO
27
45
0
16.9
Pos.
45
0
17.1
Pos.
11-PM10-SC-
High-30 Tf
28.1
0.1
27.9
0
12-PM10-SC-
High-30 TO
27
45
0
15.4
Pos.
45
0
15.6
Pos.
12-PM10-SC-
High-30 Tf
29.6
0.1
29.4
0.1
13-PM10-SC-
High-300 TO
270
45
0
25.4
Pos.
45
0
25.6
Pos.
13-PM10-SC-
High-300 Tf
19.6
0.1
19.4
0
14-PM10-SC-
High-300 TO
270
45
0
15.6
Pos.
45
0
15.9
Pos.
14-PM10-SC-
High-300 Tf
29.4
0.1
29.1
0
15-PM10-SC-
High-3,000
TO
2,700
45
0
22.4
Pos.
45
0
22.9
Pos.
15-PM10-SC-
High-3,000
Tf
22.6
0.3
22.1
0.1
16-PM10-SC-
High-3,000
TO
2,700
45
0
23.6
Pos.
45
0
24
Pos.
16-PM10-SC-
High-3,000
Tf
21.4
0
21
0
05/28/18
PM10 South
1-PM10-SC-
AVG-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
Carolina and
Various Filter
1-PM10-SC-
AVG-0 Tf
45
0
45
0
types
2-PM10-SC-
HIGH-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
2-PM10-SC-
HIGH-0 Tf
45
0
45
0
3-PM10-SC-
AVG-30 TO
29
45
0
0
Neg.
45
0
0
Neg.
3-PM10-SC-
AVG-30 Tf
45
0
45
0
4-PM10-SC-
HIGH-30 TO
29
45
0
0
Neg.
45
0
0
Neg.
4-PM10-SC-
HIGH-30 Tf
45
0
45
0
5-PM10-SC-
AVG-300 TO
290
45
0
27.7
Pos.
45
0
27.9
Pos.
5-PM10-SC-
AVG-300 Tf
17.3
0.1
17.1
0
6-PM10-SC-
HIGH-300 TO
290
45
0
26.6
Pos.
45
0
26.9
Pos.
6-PM10-SC-
HIGH-300 Tf
18.4
0.1
18.1
0
7-PM10-SC-
AVG-3,000
TO
2,900
45
0
22.1
Pos.
45
0
22.2
Pos.
L-19
-------
EPA/600/R-19/082
10/8/2019
1 rinl l);ilc
S;ini|ilr II)
SpiilV
1 Mllll1
< IlK Illl.
\\rr:i!ir
( 1
( hi'niii.
Mild
IV\
( hum.
\< l
( Iii'iiiii.
UrMlll
Il\()l
( I
|i\()l
SI ml
l)r\
ll\()l
( hum.
\< 1
|i.\()l ( hi'niii.
kl'Mlll
7-PM10-SC-
AVG-3,000
Tf
22.9
0.1
22.8
0
8-PM10-SC-
HIGH-3,000
TO
2,900
45
0
25.4
Pos.
45
0
25.6
Pos.
8-PM10-SC-
HIGH-3,000
Tf
19.6
0
19.4
0
9-PM10-N/A-
New-0 TO
0
45
0
0
Neg.
45
0
0
Neg.
9-PM10-N/A-
New-0 Tf
45
0
45
0
10-PM10-
NH-AVG-30
TO
29
45
0
28.7
Pos.
45
0
28.5
Pos.
10-PM10-
NH-AVG-30
Tf
16.3
0.1
16.5
0.1
14-PM10-
WI-High-
3,000 TO
2,900
45
0
26.3
Pos.
45
0
26.7
Pos.
14-PM10-
WI-High-
3,000 Tf
18.7
0.1
18.7
0.1
1 Actual number of B. a. Sterne spores spiked onto filter based on spiking suspension titer and volume of suspension applied.
L-20
-------
EPA/600/R-19/082
10/8/2019
APPENDIX M. CULTURE RESULTS FOR NON-AIR QUALITY FILTERS
USING SHEEP BLOOD AGAR MEDIUM
M-l
-------
EPA/600/R-19/082
10/8/2019
Bus Filter New
Sample ID
Spore Load1
Extraction
Volume
(mL)
Volume in
Filter Cup
(mL)
Plate
Counts
(CFU)
Average Sample
Concentration
Percent
Recovery
CFII/mL
Total CFU
Bus-New-
Blank
0.0E+00
25
1
0
0.0
0.0
N/A
25
4
0
0.0
0.0
N/A
Bus-New-
Blank
0.0E+00
25
1
0
0.0
0.0
N/A
25
4
0
0.0
0.0
N/A
Bus-New-
Blank
0.0E+00
25
1
0
0.0
0.0
N/A
25
4
0
0.0
0.0
N/A
1-BUS-
NEW-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
2-BUS-
NEW-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
3-BUS-
NEW-30
4.4E+01
25
1
1
1.0
25.0
56.8
4.4E+01
25
4
0
0.0
0.0
0.0
4-BUS-
NEW-30
4.4E+01
25
1
0
0.0
0.0
0.0
4.4E+01
25
4
1
0.3
6.3
14.2
11-BUS-
NEW-30
2.9E+01
25
1
0
0.0
0.0
0.0
2.9E+01
25
4
0
0.0
0.0
0.0
12-BUS-
NEW-300
2.9E+02
25
1
0
0.0
0.0
0.0
2.9E+02
25
4
1
0.3
6.3
2.2
5-BUS-
NEW-300
4.4E+02
25
1
0
0.0
0.0
0.0
4.4E+02
25
4
3
0.8
18.8
4.3
6-BUS-
NEW-300
4.4E+02
25
1
1
1.0
25.0
5.7
4.4E+02
25
4
8
2.0
50.0
11.4
15-BUS-
NEW-3,000
2.9E+03
25
1
14
14.0
350.0
12.1
2.9E+03
25
4
28
7.0
175.0
6.0
7-BUS-
NEW-3,000
4.4E+03
25
1
13
13.0
325.0
7.4
4.4E+03
25
4
24
6.0
150.0
3.4
8-BUS-
NEW-3,000
4.4E+03
25
1
15
15.0
375.0
8.5
4.4E+03
25
4
46
11.5
287.5
6.5
Use values highlighted in green for reporting.
1 Actual number of B. a. Sterne spores spiked onto filter based on spiking suspension titer and volume of suspension applied.
M-2
-------
EPA/600/R-19/082
10/8/2019
Bus Filters Mid
Sample ID
Spore Load1
Extraction
Volume
(mL)
Volume in
Filter Cup
(mL)
Plate
Counts
(CFU)
Average Sample
Concentration
Percent
Recovery
CFII/mL
Total CFU
1-BUS-
MID-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
2-BUS-
MID-0
0.0E+00
25
1
1
1.0
25.0
#DIV/0!
0.0E+00
25
4
1
0.3
6.3
#DIV/0!
1-BUS-Mid-
0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
1
0.3
6.3
#DIV/0!
3-BUS-
MID-30
1.8E+01
25
1
1
1.0
25.0
138.9
1.8E+01
25
4
3
0.8
18.8
104.2
4-BUS-
MID-30
1.8E+01
25
1
0
0.0
0.0
0.0
1.8E+01
25
4
0
0.0
0.0
0.0
3-BUS-Mid-
30
4.2E+01
25
1
0
0.0
0.0
0.0
4.2E+01
25
4
3
0.8
18.8
44.6
5-BUS-
MID-300
1.8E+02
25
1
2
2.0
50.0
27.8
1.8E+02
25
4
7
1.8
43.8
24.3
6-BUS-
MID-300
1.8E+02
25
1
0
0.0
0.0
0.0
1.8E+02
25
4
9
2.3
56.3
31.3
5-BUS-Mid-
300
4.2E+02
25
1
3
3.0
75.0
17.9
4.2E+02
25
4
4
1.0
25.0
6.0
7-BUS-
MID-3,000
1.8E+03
25
1
15
15.0
375.0
20.8
1.8E+03
25
4
113
28.3
706.3
39.2
8-BUS-
MID-3,000
1.8E+03
25
1
21
21.0
525.0
29.2
1.8E+03
25
4
39
9.8
243.8
13.5
7-BUS-Mid-
3,000
4.2E+03
25
1
13
13.0
325.0
7.7
4.2E+03
25
4
60
15.0
375.0
8.9
Use values highlighted in green for reporting.
1 Actual number of B. a. Sterne spores spiked onto filter based on spiking suspension titer and volume of suspension applied.
M-3
-------
EPA/600/R-19/082
10/8/2019
Bus Filters End
Sample ID
Spore
Load1
Extraction
Volume
(mL)
Volume in
Filter Cup
(mL)
Plate Counts
(CFU)
Average Sample
Concentration
Percent
Recovery
CFII/mL
Total CFU
BUS-END-
Blank
0.0E+00
25
1
0
0.0
0.0
N/A
25
4
1
0.3
6.3
N/A
BUS-END-
Blank
25
1
0
0.0
0.0
N/A
25
4
1
0.3
6.3
N/A
BUS-END-
Blank
25
1
0
0.0
0.0
N/A
25
4
0
0.0
0.0
N/A
9-BUS-
END-0
0.0E+00
25
1
Not plated
#VALUE!
#VALUE!
#VALUE!
0.0E+00
25
4
Not plated
#VALUE!
#VALUE!
#VALUE!
10-BUS-
END-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
BUS-END-
30
1.5E+01
25
1
2
2.0
50.0
333.3
25
4
5
1.3
31.3
208.3
BUS-END-
30
1.5E+01
25
1
2
2.0
50.0
333.3
25
4
7
1.8
43.8
291.7
BUS-END-
30
1.5E+01
25
1
3
3.0
75.0
500.0
25
4
7
1.8
43.8
291.7
11-BUS-
END-30
4.4E+01
25
1
1
1.0
25.0
56.8
4.4E+01
25
4
0
0.0
0.0
0.0
12-BUS-
END-30
4.4E+01
25
1
2
2.0
50.0
113.6
4.4E+01
25
4
0
0.0
0.0
0.0
BUS-END-
300
1.5E+02
25
1
3
3.0
75.0
50.0
25
4
1
0.3
6.3
4.2
BUS-END-
300
1.5E+02
25
1
2
2.0
50.0
33.3
25
4
8
2.0
50.0
33.3
BUS-END-
300
1.5E+02
25
1
2
2.0
50.0
33.3
25
4
6
1.5
37.5
25.0
13-BUS-
END-300
4.4E+02
25
1
6
6.0
150.0
34.1
4.4E+02
25
4
12
3.0
75.0
17.0
14-BUS-
END-300
4.4E+02
25
1
6
6.0
150.0
34.1
4.4E+02
25
4
7
1.8
43.8
9.9
15-BUS-
END-3,000
4.4E+03
25
1
28
28.0
700.0
15.9
4.4E+03
25
4
69
17.3
431.3
9.8
16-BUS-
END-3,000
4.4E+03
25
1
38
38.0
950.0
21.6
4.4E+03
25
4
90
22.5
562.5
12.8
16-BUS-
END-3,000
2.9E+03
25
1
24
24.0
600.0
20.7
2.9E+03
25
4
60
15.0
375.0
12.9
Use values highlighted in green for reporting.
No discemable colonies due to grime/dirt on filter.
1 Actual number of B. a. Sterne spores spiked onto filter based on spiking suspension titer and volume of suspension applied.
M-4
-------
EPA/600/R-19/082
10/8/2019
HVAC Filters New
Sample ID
Spore Load1
Extraction
Volume
(mL)
Volume in
Filter Cup
(mL)
Plate
Counts
(CFU)
Average Sample
Concentration
Percent
Recovery
CFII/mL
Total CFU
9-HVAC-
NEW-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
10-HVAC-
NEW-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
1-HVAC-
NEW-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
2-HVAC-
NEW-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
11-HVAC-
NEW-30
3.8E+01
25
1
1
1.0
25.0
65.8
3.8E+01
25
4
2
0.5
12.5
32.9
12-HVAC -
NEW-30
3.8E+01
25
1
0
0.0
0.0
0.0
3.8E+01
25
4
4
1.0
25.0
65.8
3-HVAC-
NEW-30
3.3E+01
25
1
0
0.0
0.0
0.0
3.3E+01
25
4
3
0.8
18.8
56.8
4-HVAC-
NEW-30
3.3E+01
25
1
1
1.0
25.0
75.8
3.3E+01
25
4
2
0.5
12.5
37.9
5-HVAC-
NEW-300
3.3E+02
25
1
7
7.0
175.0
53.0
3.3E+02
25
4
24
6.0
150.0
45.5
6-HVAC-
NEW-300
3.3E+02
25
1
4
4.0
100.0
30.3
3.3E+02
25
4
10
2.5
62.5
18.9
13-HVAC -
NEW-300
2.9E+02
25
1
2
2.0
50.0
17.2
2.9E+02
25
4
14
3.5
87.5
30.2
17-hvac-new-
3000
2.9E+03
25
1
54
54.0
1350.0
46.6
2.9E+03
25
4
152
41.6
1039.1
35.8
7-HVAC-
NEW-3,000
3.3E+03
25
1
48
48.0
1200.0
36.4
3.3E+03
25
4
138
34.5
862.5
26.1
8-HVAC-
NEW-3,000
3.3E+03
25
1
41
41.0
1025.0
31.1
3.3E+03
25
4
124
31.0
775.0
23.5
Pieces of HVAC filter present on plated filter.
Use values highlighted in green for reporting.
1 Actual number of B. a. Sterne spores spiked onto filter based on spiking suspension titer and volume of suspension applied.
M-5
-------
EPA/600/R-19/082
10/8/2019
HVAC Filters Mid
Sample ID
Spore Load1
Extraction
Volume
(mL)
Volume in
Filter Cup
(mL)
Plate
Counts
(CFU)
Average Sample
Concentration
Percent
Recovery
CFII/mL
Total CFU
9-HVAC-
MID-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
GRIME
GRIME
GRIME
GRIME
10-HVAC-
MID-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
LAWN
LAWN
LAWN
LAWN
2-HVAC-
MID-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
12-HVAC-
MID-30
1.8E+01
25
1
0
0.0
0.0
0.0
1.8E+01
25
4
1
0.3
6.3
34.7
4-HVAC-
MID-30
1.8E+01
25
1
0
0.0
0.0
0.0
1.8E+01
25
4
1
0.3
6.3
34.7
4-HVAC-
MID-30
4.2E+01
25
1
0
0.0
0.0
0.0
4.2E+01
25
4
0
0.0
0.0
0.0
13-HVAC-
MID-300
1.8E+02
25
1
4
4.0
100.0
55.6
1.8E+02
25
4
LAWN
LAWN
LAWN
LAWN
14-HVAC-
MID-300
1.8E+02
25
1
4
4.0
100.0
55.6
1.8E+02
25
4
7
1.8
43.8
24.3
6-HVAC-
MID-3 00
4.2E+02
25
1
8
8.0
200.0
47.6
4.2E+02
25
4
22
5.5
137.5
32.7
15-HVAC-
MID-3000
1.8E+03
25
1
37
37.0
925.0
51.4
1.8E+03
25
4
117
29.3
731.3
40.6
16-HVAC-
MID-3000
1.8E+03
25
1
54
54.0
1350.0
75.0
1.8E+03
25
4
114
28.5
712.5
39.6
8-HVAC-
MID-3,000
4.2E+03
25
1
53
53.0
1325.0
31.5
4.2E+03
25
4
102
25.5
637.5
15.2
Use values highlighted in green for reporting.
1 Actual number of B. a. Sterne spores spiked onto filter based on spiking suspension titer and volume of suspension applied.
M-6
-------
EPA/600/R-19/082
10/8/2019
HVAC Filters End
Sample ID
Spore Load1
Extraction
Volume
(mL)
Volume in
Filter Cup
(mL)
Plate
Counts
(CFU)
Average Sample
Concentration
Percent
Recovery
CFII/mL
Total CFU
1-HVAC-END-
0
0.0E+00
25
1
4
4.0
100.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
2-HVAC-END-
0
0.0E+00
25
1
10
10.0
250.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
9-HVAC-END-
0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
10-HVAC-
END-0
0.0E+00
25
1
1
1.0
25.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
3-HVAC-END-
30
3.8E+01
25
1
3
3.0
75.0
197.4
3.8E+01
25
4
0
0.0
0.0
0.0
4-HVAC-END-
30
3.8E+01
25
1
4
4.0
100.0
263.2
3.8E+01
25
4
0
0.0
0.0
0.0
11-HVAC-End-
30
3.3E+01
25
1
1
1.0
25.0
75.8
3.3E+01
25
4
0
0.0
0.0
0.0
12-HVAC-
END-30
3.3E+01
25
1
2
2.0
50.0
151.5
3.3E+01
25
4
0
0.0
0.0
0.0
5-HVAC-END-
300
3.8E+02
25
1
5
5.0
125.0
32.9
3.8E+02
25
4
0
0.0
0.0
0.0
6-HVAC-END-
300
3.8E+02
25
1
4
4.0
100.0
26.3
3.8E+02
25
4
0
0.0
0.0
0.0
13-HVAC-
END-300
3.3E+02
25
1
5
5.0
125.0
37.9
3.3E+02
25
4
0
0.0
0.0
0.0
14-HVAC-
END-300
3.3E+02
25
1
8
8.0
200.0
60.6
3.3E+02
25
4
0
0.0
0.0
0.0
7-HVAC-END-
3,000
3.8E+03
25
1
45
45.0
1125.0
29.6
3.8E+03
25
4
0
0.0
0.0
0.0
8-HVAC-END-
3,000
3.8E+03
25
1
56
56.0
1400.0
36.8
3.8E+03
25
4
0
0.0
0.0
0.0
15-HVAC-
END-3,000
3.3E+03
25
1
80
80.0
2000.0
60.6
3.3E+03
25
4
0
0.0
0.0
0.0
16-HVAC-
3.3E+03
25
1
57
57.0
1425.0
43.2
END-3,000
3.3E+03
25
0.0
0.0
0.0
Grime prevents identification/counting of morphologies.
Use values highlighted in green for reporting.
1 Actual number of B. a. Sterne spores spiked onto filter based on spiking suspension titer and volume of suspension applied.
M-7
-------
EPA/600/R-19/082
10/8/2019
Platform Filters New
Sample ID
Spore Load1
Extraction
Volume
(mL)
Volume in
Filter Cup
(mL)
Plate
Counts
(CFU)
Average Sample
Concentration
Percent
CFII/mL
Total CFU
Recovery
9-PLAT-
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
NEW-0
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
10-PLAT-
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
NEW-0
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
1-PLAT-
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
NEW-0
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
11-PLAT-
1.7E+01
25
1
1
1.0
25.0
147.1
NEW-30
1.7E+01
25
4
0
0.0
0.0
0.0
12-PLAT-
1.7E+01
25
1
0
0.0
0.0
0.0
NEW-30
1.7E+01
25
4
4
1.0
25.0
147.1
3-PLAT-
2.8E+01
25
1
0
0.0
0.0
0.0
NEW-30
2.8E+01
25
4
2
0.5
12.5
44.6
13-PLAT-
1.7E+02
25
1
6
6.0
150.0
88.2
NEW-300
1.7E+02
25
4
7
1.8
43.8
25.7
14-PLAT-
1.7E+02
25
1
2
2.0
50.0
29.4
NEW-300
1.7E+02
25
4
13
3.3
81.3
47.8
5-PLAT-
2.8E+02
25
1
7
7.0
175.0
62.5
NEW-300
2.8E+02
25
4
25
6.3
156.3
55.8
15-PLAT-
1.7E+03
25
1
43
43.0
1075.0
63.2
NEW-3,000
1.7E+03
25
4
208
52.0
1300.0
76.5
16-PLAT-
1.7E+03
25
1
53
53.0
1325.0
77.9
NEW-3,000
1.7E+03
25
4
220
55.0
1375.0
80.9
7-PLAT-
2.8E+03
25
1
46
46.0
1150.0
41.1
NEW-3,000
2.8E+03
25
4
146
36.5
912.5
32.6
Count is from half the plate filter multiplied by 2
Use values highlighted in green for reporting.
1 Actual number of B. a. Sterne spores spiked onto filter based on spiking suspension titer and volume of suspension applied.
M-8
-------
EPA/600/R-19/082
10/8/2019
Platform Filters Mid
Sample ID
Spore Load1
Extraction
Volume
(mL)
Volume in
Filter Cup
(mL)
Plate
Counts
(CFU)
Average Sample
Concentration
Percent
CFII/mL
Total CFU
Recovery
1-PLAT-
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
MID-0
0.0E+00
25
4
4
1.0
25.0
#DIV/0!
2-PLAT-
0.0E+00
25
1
2
2.0
50.0
#DIV/0!
MID-0
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
9-PLAT-
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
Mid-0
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
3-PLAT-
3.0E+01
25
1
1
1.0
25.0
83.3
MID-30
3.0E+01
25
4
0
0.0
0.0
0.0
4-PLAT-
3.0E+01
25
1
0
0.0
0.0
0.0
MID-30
3.0E+01
25
4
3
0.8
18.8
62.5
11-PLAT-
4.2E+01
25
1
0
0.0
0.0
0.0
Mid-30
4.2E+01
25
4
2
0.5
12.5
29.8
5-PLAT-
3.0E+02
25
1
4
4.0
100.0
33.3
MID-3 00
3.0E+02
25
4
25
6.3
156.3
52.1
6-PLAT-
3.0E+02
25
1
5
5.0
125.0
41.7
MID-300
3.0E+02
25
4
18
4.5
112.5
37.5
13-PLAT-
4.2E+02
25
1
6
6.0
150.0
35.7
Mid-300
4.2E+02
25
4
LAWN
LAWN
LAWN
#VALUE!
7-PLAT-
3.0E+03
25
1
41
41.0
1025.0
34.2
MID-3,000
3.0E+03
25
4
0
0.0
0.0
0.0
8-PLAT-
3.0E+03
25
1
34
34.0
850.0
28.3
MID-3,000
3.0E+03
25
4
0
0.0
0.0
0.0
15-PLAT-
4.2E+03
25
1
34
34.0
850.0
20.2
Mid-3,000
4.2E+03
25
4
TNTC
#VALUE!
#VALUE!
#VALUE!
Grime makes counting impossible
50% lawn
TNTC/Grime
Use values highlighted in green for reporting.
1 Actual number of B. a. Sterne spores spiked onto filter based on spiking suspension titer and volume of suspension applied.
M-9
-------
EPA/600/R-19/082
10/8/2019
Platform Filters End
Sample ID
Spore
Load1
Extraction
Volume
(mL)
Volume in
Filter Cup
(mL)
Plate
Counts
(CFU)
Average Sample
Concentration
Percent
Recovery
CFII/mL
Total CFU
1-PLAT-END-
0
0.0E+00
25
1
4
4.0
100.0
#DIV/0!
0.0E+00
25
4
lawn
#VALUE!
#VALUE!
#VALUE!
2-PLAT-END-
0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
9-PLAT-END-
0
0.0E+00
25
1
1
1.0
25.0
#DIV/0!
0.0E+00
25
4
GRIME
#VALUE!
#VALUE!
#VALUE!
3-PLAT-END-
30
1.7E+01
25
1
3
3.0
75.0
441.2
1.7E+01
25
4
Lawn
#VALUE!
#VALUE!
#VALUE!
4-PLAT-END-
30
1.7E+01
25
1
3
3.0
75.0
441.2
1.7E+01
25
4
Lawn
#VALUE!
#VALUE!
#VALUE!
11-PLAT-
END-30
2.8E+01
25
1
0
0.0
0.0
0.0
2.8E+01
25
4
0
0.0
0.0
0.0
5-PLAT-END-
300
1.7E+02
25
1
6
6.0
150.0
88.2
1.7E+02
25
4
Lawn
#VALUE!
#VALUE!
#VALUE!
6-PLAT-END-
300
1.7E+02
25
1
2
2.0
50.0
29.4
1.7E+02
25
4
Lawn
#VALUE!
#VALUE!
#VALUE!
13-PLAT-
END-300
2.8E+02
25
1
6
6.0
150.0
53.6
2.8E+02
25
4
GRIME
#VALUE!
#VALUE!
#VALUE!
7-PLAT-END-
3,000
1.7E+03
25
1
57
57.0
1425.0
83.8
1.7E+03
25
4
Lawn
#VALUE!
#VALUE!
#VALUE!
8-PLAT-END-
3,000
1.7E+03
25
1
48
48.0
1200.0
70.6
1.7E+03
25
4
Lawn
#VALUE!
#VALUE!
#VALUE!
15-PLAT-
END-3,000
2.8E+03
25
1
15
15.0
375.0
13.4
2.8E+03
25
4
GRIME
#VALUE!
#VALUE!
#VALUE!
50% lawn
Use values highlighted in green for reporting.
1 Actual number of B. a. Sterne spores spiked onto filter based on spiking suspension titer and volume of suspension applied.
M-10
-------
EPA/600/R-19/082
10/8/2019
Rolling Stock New
Sample ID
Spore Load1
Extraction
Volume
(mL)
Volume in
Filter Cup
(mL)
Plate
Counts
(CFU)
Average Sample
Concentration
Percent
Recovery
CFU/mL
Total CFU
1-ROLL-
NEW-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
2-ROLL-
NEW-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
2-ROLL-
NEW-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
3-ROLL-
NEW-30
7.0E+01
25
1
3
3.0
75.0
107.6
7.0E+01
25
4
4
1.0
25.0
35.9
4-ROLL-
NEW-30
7.0E+01
25
1
0
0.0
0.0
0.0
7.0E+01
25
4
5
1.3
31.3
44.8
4-ROLL-
NEW-30
2.8E+01
25
1
1
1.0
25.0
89.3
2.8E+01
25
4
4
1.0
25.0
89.3
5-ROLL-
NEW-300
7.0E+02
25
1
15
15.0
375.0
53.6
7.0E+02
25
4
63
15.8
393.8
56.3
6-ROLL-
NEW-300
7.0E+02
25
1
11
11.0
275.0
39.3
7.0E+02
25
4
59
14.8
368.8
52.7
6-ROLL-
NEW-300
2.8E+02
25
1
5
5.0
125.0
44.6
2.8E+02
25
4
36
9.0
225.0
80.4
7-ROLL-
NEW-3,000
7.0E+03
25
1
114
114.0
2850.0
40.7
7.0E+03
25
4
TNTC
#VALUE!
#VALUE!
#VALUE!
8-ROLL-
NEW-3,000
7.0E+03
25
1
111
111.0
2775.0
39.6
7.0E+03
25
4
TNTC
#VALUE!
#VALUE!
#VALUE!
8-ROLL-
NEW-3,000
2.8E+03
25
1
39
39.0
975.0
34.8
2.8E+03
25
4
196
49.0
1225.0
43.8
Count is from half the plate filter multiplied by 2
Use values highlighted in green for reporting.
1 Actual number of B. a. Sterne spores spiked onto filter based on spiking suspension titer and volume of suspension applied.
M-ll
-------
EPA/600/R-19/082
10/8/2019
Rolling Stock Mid
Sample ID
Spore
Load1
Extraction
Volume
(mL)
Volume in
Filter Cup
(mL)
Plate
Counts
(CFU)
Average Sample
Concentration
Percent
Recovery
CFU/mL
Total CFU
9-ROLL-
MID-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
10-ROLL-
MID-0
0.0E+00
25
1
1
1.0
25.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
10-ROLL-
Mid-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
GRIME
GRIME
GRIME
#VALUE!
11-ROLL-
MID-30
3.0E+01
25
1
0
0.0
0.0
0.0
3.0E+01
25
4
0
0.0
0.0
0.0
12-ROLL-
MID-30
3.0E+01
25
1
0
0.0
0.0
0.0
3.0E+01
25
4
0
0.0
0.0
0.0
12-ROLL-
Mid-30
4.2E+01
25
1
3
3.0
75.0
178.6
4.2E+01
25
4
GRIME
GRIME
GRIME
#VALUE!
13-ROLL-
MID-300
3.0E+02
25
1
7
7.0
175.0
58.3
3.0E+02
25
4
0
0.0
0.0
0.0
14-ROLL-
MID-300
3.0E+02
25
1
2
2.0
50.0
16.7
3.0E+02
25
4
0
0.0
0.0
0.0
14-ROLL-
Mid-300
4.2E+02
25
1
3
3.0
75.0
17.9
4.2E+02
25
4
GRIME
GRIME
GRIME
#VALUE!
15-ROLL-
MID-3,000
3.0E+03
25
1
50
50.0
1250.0
41.7
3.0E+03
25
4
0
0.0
0.0
0.0
16-ROLL-
MID-3,000
3.0E+03
25
1
0
0.0
0.0
0.0
3.0E+03
25
4
0
0.0
0.0
0.0
16-ROLL-
Mid-3,000
4.2E+03
25
1
11
11.0
275.0
6.5
4.2E+03
25
4
GRIME
GRIME
GRIME
#VALUE!
50% lawn
Grime makes counting impossible
75% lawn
TNTC/Grime
Use values highlighted in green for reporting
1 Actual number of B. a. Sterne spores spiked onto filter based on spiking suspension titer and volume of suspension applied.
M-12
-------
EPA/600/R-19/082
10/8/2019
Rolling Stock End
Sample ID
Spore
Load1
Extraction
Volume
(mL)
Volume in
Filter Cup
(mL)
Plate
Counts
(CFU)
Average Sample
Concentration
Percent
Recovery
CFU/mL
Total CFU
9-ROLL-
END-0
0.0E+00
25
1
1
1.0
25.0
#DIV/0!
0.0E+00
25
4
LAWN
#VALUE!
#VALUE!
#VALUE!
10-ROLL-
END-0
0.0E+00
25
1
1
1.0
25.0
#DIV/0!
0.0E+00
25
4
LAWN
#VALUE!
#VALUE!
#VALUE!
10-ROLL-
END-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
GRIME
#VALUE!
#VALUE!
#VALUE!
11-ROLL-
END-30
7.0E+01
25
1
4
4.0
100.0
143.5
7.0E+01
25
4
LAWN
#VALUE!
#VALUE!
#VALUE!
12-ROLL-
END-30
7.0E+01
25
1
3
3.0
75.0
107.6
7.0E+01
25
4
LAWN
#VALUE!
#VALUE!
#VALUE!
12-ROLL-
END-30
2.8E+01
25
1
GRIME
#VALUE!
0.0
0.0
2.8E+01
25
4
GRIME
#VALUE!
#VALUE!
#VALUE!
13-ROLL-
END-300
7.0E+02
25
1
11
11.0
275.0
39.3
7.0E+02
25
4
LAWN
#VALUE!
#VALUE!
#VALUE!
14-ROLL-
END-300
7.0E+02
25
1
3
3.0
75.0
10.7
7.0E+02
25
4
LAWN
#VALUE!
#VALUE!
#VALUE!
14-ROLL-
END-300
2.8E+02
25
1
GRIME
#VALUE!
0.0
0.0
2.8E+02
25
4
GRIME
#VALUE!
#VALUE!
#VALUE!
15-ROLL-
END-3,000
7.0E+03
25
1
TNTC
#VALUE!
#VALUE!
#VALUE!
7.0E+03
25
4
TNTC
#VALUE!
#VALUE!
#VALUE!
16-ROLL-
END-3,000
7.0E+03
25
1
TNTC
#VALUE!
#VALUE!
#VALUE!
7.0E+03
25
4
TNTC
#VALUE!
#VALUE!
#VALUE!
16-ROLL-
END-3,000
2.8E+03
25
1
GRIME
#VALUE!
#VALUE!
#VALUE!
2.8E+03
25
4
GRIME
#VALUE!
#VALUE!
#VALUE!
Use values highlighted in green for reporting
1 Actual number of B. a. Sterne spores spiked onto filter based on spiking suspension titer and volume of suspension applied.
M-13
-------
EPA/600/R-19/082
10/8/2019
APPENDIX N. CULTURE RESULTS FOR NON-AIR QUALITY FILTERS
USING MYP MEDIUM
N-l
-------
EPA/600/R-19/082
10/8/2019
July 24, 2017 Trial - Bus Filters (New and EOL)
Sample ID
Spore
Load1
Extraction
Volume
(mL)
Volume in
Filter Cup
(mL)
Plate
Counts
(CFU)
Average Sample
Concentration
Percent
Recovery
CFII/mL
Total CFU
1-Bus-
High-Blank
0.0E+00
25
1
3
3.0
75.0
N/A
25
3
5
1.6
40.3
N/A
2-Bus-
High-Blank
25
1
0
0.0
0.0
N/A
25
4
4
1.0
25.0
N/A
3-Bus-
High-Blank
25
1
1
1.0
25.0
N/A
25
4
1
0.3
6.3
N/A
4-Bus-
High-30
1.5E+01
25
1
6
6.0
150.0
1000.0
25
4
0
0.0
0.0
0.0
5-Bus-
High-30
1.5E+01
25
1
2
2.0
50.0
333.3
25
4
0
0.0
0.0
0.0
6-Bus-
High-30
1.5E+01
25
1
1
1.0
25.0
166.7
25
4
0
0.0
0.0
0.0
7-Bus-
High-300
1.5E+02
25
1
0
0.0
0.0
0.0
25
4
0
0.0
0.0
0.0
8-Bus-
High-300
1.5E+02
25
1
4
4.0
100.0
66.7
25
4
0
0.0
0.0
0.0
9-Bus-
High-300
1.5E+02
25
1
3
3.0
75.0
50.0
25
4
0
0.0
0.0
0.0
10-Bus-
New-Blank
0.0E+00
25
1
0
0.0
0.0
N/A
25
4
0
0.0
0.0
N/A
11-Bus-
New-Blank
0.0E+00
25
1
0
0.0
0.0
N/A
25
4
0
0.0
0.0
N/A
12-Bus-
New-Blank
0.0E+00
25
1
0
0.0
0.0
N/A
25
4
0
0.0
0.0
N/A
Formation of large lawn
Lawn may be B.A.
Used values highlighted green for percent recovery
1 Actual number of B. a. Sterne spores spiked onto filter based on spiking suspension titer and volume of suspension applied.
N-2
-------
EPA/600/R-19/082
10/8/2019
August 25, 2017 Trial - Bus Filters (New and EOL)
Sample
ID
Spore
Load1
Extraction
Volume
(mL)
Volume in
Filter Cup
(mL)
Plate Counts
(CFU)
Average Sample
Concentration
Percent
Recovery
CFII/mL
Total CFU
1-BUS-
NEW-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
2-BUS-
NEW-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
3-BUS-
NEW-30
4.4E+01
25
1
0
0.0
0.0
0.0
4.4E+01
25
4
1
0.3
6.3
14.2
4-BUS-
NEW-30
4.4E+01
25
1
0
0.0
0.0
0.0
4.4E+01
25
4
0
0.0
0.0
0.0
5-BUS-
NEW-300
4.4E+02
25
1
3
3.0
75.0
17.0
4.4E+02
25
4
7
1.8
43.8
9.9
6-BUS-
NEW-300
4.4E+02
25
1
2
2.0
50.0
11.4
4.4E+02
25
4
9
2.3
56.3
12.8
7-BUS-
NEW-
3,000
4.4E+03
25
1
11
11.0
275.0
6.3
4.4E+03
25
4
54
13.5
337.5
7.7
8-BUS-
NEW-
3,000
4.4E+03
25
1
12
12.0
300.0
6.8
4.4E+03
25
4
41
10.3
256.3
5.8
9-BUS-
END-0
0.0E+00
25
1
Not plated
#VALUE!
#VALUE!
#VALUE!
0.0E+00
25
4
Not plated
#VALUE!
#VALUE!
#VALUE!
10-BUS-
END-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
11-BUS-
NEW-30
4.4E+01
25
1
0
0.0
0.0
0.0
4.4E+01
25
4
0
0.0
0.0
0.0
12-BUS-
END-30
4.4E+01
25
1
3
3.0
75.0
170.5
4.4E+01
25
4
0
0.0
0.0
0.0
13-BUS-
END-300
4.4E+02
25
1
8
8.0
200.0
45.5
4.4E+02
25
4
24
6.0
150.0
34.1
14-BUS-
END-300
4.4E+02
25
1
3
3.0
75.0
17.0
4.4E+02
25
4
0
0.0
0.0
0.0
15-BUS-
END-
3,000
4.4E+03
25
1
27
27.0
675.0
15.3
4.4E+03
25
4
0
0.0
0.0
0.0
16-BUS-
END-
3,000
4.4E+03
25
1
28
28.0
700.0
15.9
4.4E+03
25
4
0
0.0
0.0
0.0
Colonies counted through grime
No discemable colonies due to grime/dirt on filter
Half of filter appears to be unidentified lawn
Used values highlighted green for percent recovery
1 Actual number of B. a. Sterne spores spiked onto filter based on spiking suspension titer and volume of suspension applied.
N-3
-------
EPA/600/R-19/082
10/8/2019
September 15, 2017 Trial - Bus & HVAC Filters
Sample ID
Spore
Load1
Extraction
Volume
(mL)
Volume in
Filter Cup
(mL)
Plate
Counts
(CFU)
Average Sample
Concentration
Percent
Recovery
CFII/mL
Total CFU
1-BUS-
MID-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
2-BUS-
MID-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
LAWN
LAWN
LAWN
LAWN
3-BUS-
MID-30
1.8E+01
25
1
1
1.0
25.0
138.9
1.8E+01
25
4
LAWN
LAWN
LAWN
LAWN
4-BUS-
MID-30
1.8E+01
25
1
0
0.0
0.0
0.0
1.8E+01
25
4
LAWN
LAWN
LAWN
LAWN
5-BUS-
MID-300
1.8E+02
25
1
0
0.0
0.0
0.0
1.8E+02
25
4
LAWN
LAWN
LAWN
LAWN
6-BUS-
MID-300
1.8E+02
25
1
0
0.0
0.0
0.0
1.8E+02
25
4
LAWN
LAWN
LAWN
LAWN
7-BUS-
MID-3,000
1.8E+03
25
1
15
15.0
375.0
20.8
1.8E+03
25
4
0
0.0
0.0
0.0
8-BUS-
MID-3,000
1.8E+03
25
1
6
6.0
150.0
8.3
1.8E+03
25
4
LAWN
LAWN
LAWN
LAWN
9-HVAC-
MID-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
LAWN
LAWN
LAWN
LAWN
10-HVAC-
MID-0
0.0E+00
25
1
LAWN
LAWN
LAWN
LAWN
0.0E+00
25
4
LAWN
LAWN
LAWN
LAWN
11-HVAC-
MID-30
1.8E+01
25
1
LAWN
LAWN
LAWN
LAWN
1.8E+01
25
4
LAWN
LAWN
LAWN
LAWN
12-HVAC-
MID-30
1.8E+01
25
1
0
0.0
0.0
0.0
1.8E+01
25
4
LAWN
LAWN
LAWN
LAWN
13-HVAC-
MID-300
1.8E+02
25
1
2
2.0
50.0
27.8
1.8E+02
25
4
17
4.3
106.3
59.0
14-HVAC-
MID-300
1.8E+02
25
1
1
1.0
25.0
13.9
1.8E+02
25
4
13
3.3
81.3
45.1
15-HVAC-
MID-3,000
1.8E+03
25
1
36
36.0
900.0
50.0
1.8E+03
25
4
LAWN
LAWN
LAWN
LAWN
16-HVAC-
MID-3,000
1.8E+03
25
1
22
22.0
550.0
30.6
1.8E+03
25
4
LAWN
LAWN
LAWN
LAWN
Used values highlighted green for percent recovery
1 Actual number of B. a. Sterne spores spiked onto filter based on spiking suspension titer and volume of suspension applied.
N-4
-------
EPA/600/R-19/082
10/8/2019
August 1, 2017 Trial - HVAC Filters
Sample ID
Spore
Load1
Extraction
Volume
(mL)
Volume in
Filter Cup
(mL)
Plate
Counts
(CFU)
Average Sample
Concentration
Percent
Recovery
CFII/mL
Total CFU
1-HVAC-
EOL-O
0.0E+00
25
1
1
1.0
25.0
#DIV/0!
0.0E+00
25
3
0
0.0
0.0
#DIV/0!
2-HVAC-
EOL-O
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
3-HVAC-
EOL-30
3.8E+01
25
1
0
0.0
Lawn
#VALUE!
3.8E+01
25
4
0
0.0
0.0
0.0
4-HVAC-
EOL-30
3.8E+01
25
1
0
0.0
0.0
0.0
3.8E+01
25
4
0
0.0
Lawn
#VALUE!
5-HVAC-
EOL-300
3.8E+02
25
1
7
7.0
175.0
46.1
3.8E+02
25
4
0
0.0
0.0
0.0
6-HVAC-
EOL-300
3.8E+02
25
1
0
0.0
0.0
0.0
3.8E+02
25
4
0
0.0
Lawn
#VALUE!
7-HVAC-
EOL-3,000
3.8E+03
25
1
0
0.0
Lawn
#VALUE!
3.8E+03
25
4
0
0.0
0.0
0.0
8-HVAC-
EOL-3,000
3.8E+03
25
1
0
0.0
Lawn
#VALUE!
3.8E+03
25
4
0
0.0
0.0
0.0
9-HVAC-
NEW-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
10-HVAC-
NEW-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
11-HVAC-
NEW-30
3.8E+01
25
1
2
2.0
50.0
131.6
3.8E+01
25
4
3
0.8
18.8
49.3
12-HVAC-
NEW-30
3.8E+01
25
1
0
0.0
0.0
0.0
3.8E+01
25
4
2
0.5
12.5
32.9
Used values highlighted green for percent recovery
1 Actual number of B. a. Sterne spores spiked onto filter based on spiking suspension titer and volume of suspension applied.
N-5
-------
EPA/600/R-19/082
10/8/2019
September 8, 2017 Trial - HVAC Filters
Sample ID
Spore
Load1
Extraction
Volume
(mL)
Volume in
Filter Cup
(mL)
Plate
Counts
(CFU)
Average Sample
Concentration
Percent
Recovery
CFII/mL
Total CFU
1-HVAC-
NEW-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
2-HVAC-
NEW-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
3-HVAC-
NEW-30
3.3E+01
25
1
0
0.0
0.0
0.0
3.3E+01
25
4
2
0.5
12.5
37.9
4-HVAC-
NEW-30
3.3E+01
25
1
2
2.0
50.0
151.5
3.3E+01
25
4
3
0.8
18.8
56.8
5-HVAC-
NEW-300
3.3E+02
25
1
10
10.0
250.0
75.8
3.3E+02
25
4
14
3.5
87.5
26.5
6-HVAC-
NEW-300
3.3E+02
25
1
4
4.0
100.0
30.3
3.3E+02
25
4
22
5.5
137.5
41.7
7-HVAC-
NEW-
3,000
3.3E+03
25
1
38
38.0
950.0
28.8
3.3E+03
25
4
TNTC
#VALUE!
#VALUE!
#VALUE!
8-HVAC-
NEW-
3,000
3.3E+03
25
1
38
38.0
950.0
28.8
3.3E+03
25
4
121
30.3
756.3
22.9
9-HVAC-
END-0
0.0E+00
25
1
LAWN
#VALUE!
#VALUE!
#VALUE!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
10-HVAC-
END-0
0.0E+00
25
1
LAWN
#VALUE!
#VALUE!
#VALUE!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
11-HVAC-
End-30
3.3E+01
25
1
LAWN
#VALUE!
#VALUE!
#VALUE!
3.3E+01
25
4
0
0.0
0.0
0.0
12-HVAC-
END-30
3.3E+01
25
1
LAWN
#VALUE!
#VALUE!
#VALUE!
3.3E+01
25
4
0
0.0
0.0
0.0
13-HVAC-
END-300
3.3E+02
25
1
4
4.0
100.0
30.3
3.3E+02
25
4
0
0.0
0.0
0.0
14-HVAC-
END-300
3.3E+02
25
1
5
5.0
125.0
37.9
3.3E+02
25
4
0
0.0
0.0
0.0
15-HVAC-
END-3,000
3.3E+03
25
1
LAWN
LAWN
#VALUE!
#VALUE!
3.3E+03
25
4
0
0.0
0.0
0.0
16-HVAC-
END-3,000
3.3E+03
25
1
LAWN
LAWN
#VALUE!
#VALUE!
3.3E+03
25
4
0
0.0
0.0
0.0
Used this value in calculation
Pieces of HVAC filter present on plated filter
Grime prevents identification/counting of morphologies
1 Actual number of B. a. Sterne spores spiked onto filter based on spiking suspension titer and volume of suspension applied.
N-6
-------
EPA/600/R-19/082
10/8/2019
September 15, 2017 Trial - HVAC & Bus Filters
Sample ID
Spore
Load1
Extraction
Volume
(mL)
Volume in
Filter Cup
(mL)
Plate
Counts
(CFU)
Average Sample
Concentration
Percent
Recovery
CFII/mL
Total CFU
1-BUS-
MID-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
2-BUS-
MID-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
LAWN
LAWN
LAWN
LAWN
3-BUS-
MID-30
1.8E+01
25
1
1
1.0
25.0
138.9
1.8E+01
25
4
LAWN
LAWN
LAWN
LAWN
4-BUS-
MID-30
1.8E+01
25
1
0
0.0
0.0
0.0
1.8E+01
25
4
LAWN
LAWN
LAWN
LAWN
5-BUS-
MID-300
1.8E+02
25
1
0
0.0
0.0
0.0
1.8E+02
25
4
LAWN
LAWN
LAWN
LAWN
6-BUS-
MID-300
1.8E+02
25
1
0
0.0
0.0
0.0
1.8E+02
25
4
LAWN
LAWN
LAWN
LAWN
7-BUS-
MID-3,000
1.8E+03
25
1
15
15.0
375.0
20.8
1.8E+03
25
4
0
0.0
0.0
0.0
8-BUS-
MID-3,000
1.8E+03
25
1
6
6.0
150.0
8.3
1.8E+03
25
4
LAWN
LAWN
LAWN
LAWN
9-HVAC-
MID-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
LAWN
LAWN
LAWN
LAWN
10-HVAC-
MID-0
0.0E+00
25
1
LAWN
LAWN
LAWN
LAWN
0.0E+00
25
4
LAWN
LAWN
LAWN
LAWN
11-HVAC-
MID-30
1.8E+01
25
1
LAWN
LAWN
LAWN
LAWN
1.8E+01
25
4
LAWN
LAWN
LAWN
LAWN
12-HVAC-
MID-30
1.8E+01
25
1
0
0.0
0.0
0.0
1.8E+01
25
4
LAWN
LAWN
LAWN
LAWN
13-HVAC-
MID-300
1.8E+02
25
1
2
2.0
50.0
27.8
1.8E+02
25
4
17
4.3
106.3
59.0
14-HVAC-
MID-300
1.8E+02
25
1
1
1.0
25.0
13.9
1.8E+02
25
4
13
3.3
81.3
45.1
15-HVAC-
MID-3,000
1.8E+03
25
1
36
36.0
900.0
50.0
1.8E+03
25
4
LAWN
LAWN
LAWN
LAWN
16-HVAC-
MID-3,000
1.8E+03
25
1
22
22.0
550.0
30.6
1.8E+03
25
4
LAWN
LAWN
LAWN
LAWN
Used this value in calculation
1 Actual number of B. a. Sterne spores spiked onto filter based on spiking suspension titer and volume of suspension applied.
N-7
-------
EPA/600/R-19/082
10/8/2019
August 11, 2017 Trial - PLAT Filters
Sample ID
Spore
Load1
Extraction
Volume
(mL)
Volume in
Filter Cup
(mL)
Plate
Counts
(CFU)
Average Sample
Concentration
Percent
Recovery
CFII/mL
Total CFU
1-PLAT-
END-0
0.0E+00
25
1
Lawn
#VALUE!
#VALUE!
#VALUE!
0.0E+00
25
4
Lawn
#VALUE!
#VALUE!
#VALUE!
2-PLAT-
END-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
3-PLAT-
END-30
1.7E+01
25
1
6
6.0
150.0
882.4
1.7E+01
25
4
Lawn
#VALUE!
#VALUE!
#VALUE!
4-PLAT-
END-30
1.7E+01
25
1
2
2.0
50.0
294.1
1.7E+01
25
4
Lawn
#VALUE!
#VALUE!
#VALUE!
5-PLAT-
END-300
1.7E+02
25
1
10
10.0
250.0
147.1
1.7E+02
25
4
Lawn
#VALUE!
#VALUE!
#VALUE!
6-PLAT-
END-300
1.7E+02
25
1
3
3.0
75.0
44.1
1.7E+02
25
4
Lawn
#VALUE!
#VALUE!
#VALUE!
7-PLAT-
END-3,000
1.7E+03
25
1
49
49.0
1225.0
72.1
1.7E+03
25
4
Lawn
#VALUE!
#VALUE!
#VALUE!
8-PLAT-
END-3,000
1.7E+03
25
1
37
37.0
925.0
54.4
1.7E+03
25
4
Lawn
#VALUE!
#VALUE!
#VALUE!
9-PLAT-
NEW-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
10-PLAT-
NEW-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
11-PLAT-
NEW-30
1.7E+01
25
1
0
0.0
0.0
0.0
1.7E+01
25
4
2
0.5
12.5
73.5
12-PLAT-
NEW-30
1.7E+01
25
1
1
1.0
25.0
147.1
1.7E+01
25
4
2
0.5
12.5
73.5
13-PLAT-
NEW-300
1.7E+02
25
1
2
2.0
50.0
29.4
1.7E+02
25
4
11
2.8
68.8
40.4
14-PLAT-
NEW-300
1.7E+02
25
1
2
2.0
50.0
29.4
1.7E+02
25
4
12
3.0
75.0
44.1
15-PLAT-
NEW-
3,000
1.7E+03
25
1
31
31.0
775.0
45.6
1.7E+03
25
4
115
28.8
718.8
42.3
16-PLAT-
NEW-
3,000
1.7E+03
25
1
30
30.0
750.0
44.1
1.7E+03
25
4
130
32.5
812.5
47.8
Used this value in calculation
1 Actual number of B. a. Sterne spores spiked onto filter based on spiking suspension titer and volume of suspension applied.
N-8
-------
EPA/600/R-19/082
10/8/2019
September 18, 2017 Trial - PLAT & ROLL Filters
Sample ID
Spore
Load1
Extraction
Volume
(mL)
Volume in
Filter Cup
(mL)
Plate
Counts
(CFU)
Average Sample
Concentration
Percent
Recovery
CFII/mL
Total CFU
1-PLAT-
MID-0
0.0E+00
25
1
1
1.0
25.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
2-PLAT-
MID-0
0.0E+00
25
1
2
2.0
50.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
3-PLAT-
MID-30
3.0E+01
25
1
6
6.0
150.0
500.0
3.0E+01
25
4
0
0.0
0.0
0.0
4-PLAT-
MID-30
3.0E+01
25
1
3
3.0
75.0
250.0
3.0E+01
25
4
0
0.0
0.0
0.0
5-PLAT-
MID-3 00
3.0E+02
25
1
3
3.0
75.0
25.0
3.0E+02
25
4
0
0.0
0.0
0.0
6-PLAT-
MID-300
3.0E+02
25
1
5
5.0
125.0
41.7
3.0E+02
25
4
0
0.0
0.0
0.0
7-PLAT-
MID-3,000
3.0E+03
25
1
54
54.0
1350.0
45.0
3.0E+03
25
4
0
0.0
0.0
0.0
8-PLAT-
MID-3,000
3.0E+03
25
1
35
35.0
875.0
29.2
3.0E+03
25
4
0
0.0
0.0
0.0
9-ROLL-
MID-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
10-ROLL-
MID-0
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
11-ROLL-
MID-30
3.0E+01
25
1
0
0.0
0.0
0.0
3.0E+01
25
4
0
0.0
0.0
0.0
12-ROLL-
MID-30
3.0E+01
25
1
0
0.0
0.0
0.0
3.0E+01
25
4
0
0.0
0.0
0.0
13-ROLL-
MID-3 00
3.0E+02
25
1
0
0.0
0.0
0.0
3.0E+02
25
4
0
0.0
0.0
0.0
14-ROLL-
MID-300
3.0E+02
25
1
0
0.0
0.0
0.0
3.0E+02
25
4
0
0.0
0.0
0.0
15-ROLL-
MID-3,000
3.0E+03
25
1
0
0.0
0.0
0.0
3.0E+03
25
4
0
0.0
0.0
0.0
16-ROLL-
MID-3,000
3.0E+03
25
1
0
0.0
0.0
0.0
3.0E+03
25
4
0
0.0
0.0
0.0
Used for calculations
100% lawn
50% lawn
75% lawn
Grime makes counting impossible
1 Actual number of B. a. Sterne spores spiked onto filter based on spiking suspension titer and volume of suspension applied.
N-9
-------
EPA/600/R-19/082
10/8/2019
August 14, 2017 Trial - ROLL Filters
Sample ID
Spore
Extraction
Volume
(ml)
Volume in
Filter Cup
(mL)
Plate
Counts
(CFU)
Average Sample
Concentration
Percent
Load1
CFU/mL
Total CFU
Recovery
1-ROLL-
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
NEW-0
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
2-ROLL-
0.0E+00
25
1
0
0.0
0.0
#DIV/0!
NEW-0
0.0E+00
25
4
0
0.0
0.0
#DIV/0!
3-ROLL-
7.0E+01
25
1
4
4.0
100.0
143.5
NEW-30
7.0E+01
25
4
12
3.0
75.0
107.6
4-ROLL-
7.0E+01
25
1
1
1.0
25.0
35.9
NEW-30
7.0E+01
25
4
4
1.0
25.0
35.9
5-ROLL-
7.0E+02
25
1
22
22.0
550.0
78.6
NEW-300
7.0E+02
25
4
58
14.5
362.5
51.8
6-ROLL-
7.0E+02
25
1
16
16.0
400.0
57.1
NEW-300
7.0E+02
25
4
63
15.8
393.8
56.3
7-ROLL-
NEW-
3,000
7.0E+03
25
1
120
120.0
3000.0
42.9
7.0E+03
25
4
TNTC
#VALUE!
#VALUE!
#VALUE!
8-ROLL-
NEW-
3,000
7.0E+03
25
1
98
98.0
2450.0
35.0
7.0E+03
25
4
TNTC
#VALUE!
#VALUE!
#VALUE!
9-ROLL-
0.0E+00
25
1
LAWN
#VALUE!
#VALUE!
#VALUE!
END-0
0.0E+00
25
4
LAWN
#VALUE!
#VALUE!
#VALUE!
10-ROLL-
0.0E+00
25
1
LAWN
#VALUE!
#VALUE!
#VALUE!
END-0
0.0E+00
25
4
LAWN
#VALUE!
#VALUE!
#VALUE!
11-ROLL-
7.0E+01
25
1
LAWN
#VALUE!
#VALUE!
#VALUE!
END-30
7.0E+01
25
4
LAWN
#VALUE!
#VALUE!
#VALUE!
12-ROLL-
7.0E+01
25
1
LAWN
#VALUE!
#VALUE!
#VALUE!
END-30
7.0E+01
25
4
LAWN
#VALUE!
#VALUE!
#VALUE!
13-ROLL-
7.0E+02
25
1
LAWN
#VALUE!
#VALUE!
#VALUE!
END-300
7.0E+02
25
4
LAWN
#VALUE!
#VALUE!
#VALUE!
14-ROLL-
7.0E+02
25
1
LAWN
#VALUE!
#VALUE!
#VALUE!
END-300
7.0E+02
25
4
LAWN
#VALUE!
#VALUE!
#VALUE!
15-ROLL-
7.0E+03
25
1
TNTC
#VALUE!
#VALUE!
#VALUE!
END-3,000
7.0E+03
25
4
TNTC
#VALUE!
#VALUE!
#VALUE!
16-ROLL-
7.0E+03
25
1
TNTC
#VALUE!
#VALUE!
#VALUE!
END-3,000
7.0E+03
25
4
TNTC
#VALUE!
#VALUE!
#VALUE!
Used for calculations
1 Actual number of B. a. Sterne spores spiked onto filter based on spiking suspension titer and volume of suspension applied.
N-10
-------
EPA/600/R-19/082
10/8/2019
APPENDIX O. RV-PCR RESULTS FOR NON-AIR QUALITY FILTERS
USING CHROMOSOMAL AND PXOl GENE TARGETS
0-1
-------
EPA/600/R-19/082
10/8/2019
Triiil l);ik-
S;iiii|)k' II)
S|>( in-
laid
li. :i.
ihriiiiiiisiiiiu-
( I (10)
li. :i.
ihriiiiiiisiiiiu-
( I (ll)
li. :i.
ihriiiiiiisiiiiu-
U 1
Kisull
li. ;i.
|>\()l
(1 (10)
li. :i.
|)\OI (1
(II)
li. ;i.
|>\()l
U 1
Kisull
07/24/2017
BUS
BUS-High-0
0
45
45
0
Neg.
45
45
0
Neg.
BUS-High-0
0
45
45
0
Neg.
45
45
0
Neg.
BUS-High-0
0
45
45
0
Neg.
45
43.37*
1.21
Neg.
BUS-High-30
15
45
34.06
10.94
Pos.
45
31.93
13.07
Pos.
BUS-High-30
15
45
31.4
13.6
Pos.
45
30.27
14.73
Pos.
BUS-High-30
15
45
31.54
13.46
Pos.
45
31.07
13.93
Pos.
BUS-High-
300
150
45
32.83
12.17
Pos.
45
31.45
13.55
Pos.
BUS-High-
300
150
45
25.93
19.07
Pos.
45
25.17
19.83
Pos.
BUS-High-
300
150
45
24.19
20.81
Pos.
45
23.57
21.43
Pos.
BUS-New-0
0
45
45
0
Neg.
45
45
0
Neg.
BUS-New-0
0
45
45
0
Neg.
45
45
0
Neg.
BUS-New-0
0
45
45
0
Neg.
45
43.13*
1.87
Neg.
07/31/17
HVAC
HVAC-New-
0
0
45
36.9 ± 1.9
8.1
Neg.
45
34.0 ±
0.3
11
Neg.1
HVAC-New-
0
0
45
42.9 ± 1.9
2.1
Neg.
45
37.9 ±
2.7
7.1
Neg.
HVAC-New-
30
38
45
19.3 ± 0.1
25.7
Pos.
45
18.8 ±
0.0
26.2
Pos.
HVAC-New-
30
38
45
17.2 ±0.1
27.8
Pos.
45
16.7 ±
0.0
28.3
Pos.
HVAC-END-
0
0
45
44.8 ±0.3
0.2
Neg.
45
40.7 ±
3.7
4.3
Neg.
HVAC-END-
0
0
45
43.2 ±3.2
1.8
Neg.
45
36.1 ±
0.2
8.9
Neg.
HVAC-END-
30
38
45
28.2 ±0.1
16.8
Pos.
45
27.6 ±
0.1
17.4
Pos.
HVAC-END-
30
38
45
25.2 ±0.1
19.8
Pos.
45
24.3 ±
0.0
20.7
Pos.
HVAC-END-
300
380
45
25.8 ±0.0
19.2
Pos.
45
25.2 ±
0.0
19.8
Pos.
HVAC-END-
300
380
45
23.4 ±0.1
21.2
Pos.
45
22.7 ±
0.1
22.3
Pos.
HVAC-END-
3,000
3,800
45
20.2 ±0.1
24.8
Pos.
45
19.5 ±
0.0
25.5
Pos.
HVAC-END-
3,000
3,800
45
19.6 ± 0.1
25.4
Pos.
45
18.9 ±
0.0
26.1
Pos.
9/5/2017
HVAC
HVAC-New-
0
0
45
34.5 ±0.6
10.5
Pos.
45
34.5 ±
0.4
10.5
Pos.
HVAC-New-
0
0
45
37.3 ± 1.8
7.7
Neg.
45
36.2 ±
0.0
8.8
Neg.
HVAC-New-
30
33
45
20.2 ±0.1
24.8
Pos.
45
19.9 ±
0.0
25.1
Pos.
HVAC-New-
30
33
42.6 ±4.2
18.1 ±0.0
24.5
Pos.
43.9 ±
2.0
17.8 ±
0.0
26.1
Pos.
HVAC-New-
300
330
45
26.8 ±0.2
18.2
Pos.
45
26.4 ±
0.0
18.6
Pos.
HVAC-New-
300
330
45
27.4 ±0.1
17.6
Pos.
45
27.0 ±
0.0
18
Pos.
HVAC-New-
3,000
3,300
45
19.1 ±0.1
25.9
Pos.
45
18.5 ±
0.0
26.5
Pos.
0-2
-------
EPA/600/R-19/082
10/8/2019
Triiil l);ik-
S;iiii|)k' II)
S|>( in-
laid
li. :i.
ihriiiiiiisiiiiu-
( I (10)
li. :i.
ihriiiiiiisiiiiu-
( I (ll)
li. :i.
ihriiiiiiisiiiiu-
U 1
Kisull
li. ;i.
|>\()l
(1 (10)
li. :i.
|)\OI (1
(II)
li. ;i.
|>\()l
U 1
Kisull
HVAC-New-
3,000
3,300
45
16.9 ±0.0
28.1
Pos.
45
16.6 ±
0.0
28.4
Pos.
HVAC-END-
0
0
45
32.6 ±0.2
12.4
Pos.
45
32.5 ±
0.2
12.5
Pos.
HVAC-END-
0
0
45
34.3 ±0.5
10.7
Pos.
45
33.8 ±
0.0
11.2
Pos.
HVAC-END-
30
33
45
25.3 ±0.1
19.7
Pos.
43.2 ±
3.2
24.7 ±
0.0
18.5
Pos.
HVAC-END-
30
33
45
33.8 ±0.3
11.2
Pos.
45
33.3 ±
0.0
11.7
Pos.
HVAC-END-
300
330
45
27.0 ±0.0
18
Pos.
45
26.6 ±
0.0
18.4
Pos.
HVAC-END-
300
330
45
27.1 ±0.1
17.9
Pos.
45
26.3 ±
0.0
18.7
Pos.
HVAC-END-
3,000
3,300
45
21.2 ±0.0
23.8
Pos.
45
20.5 ±
0.0
24.5
Pos.
HVAC-END-
3,000
3,300
45
23.9 ±0.1
21.1
Pos.
45
23.2 ±
0.0
21.8
Pos.
08/7/2017
PLAT
PLAT-New-0
0
40.6 ±3.0
34.5 ±0.5
6.1
Neg.
42.3 ±
3.1
33.9 ±
0.3
8.5
Neg.
PLAT-New-0
0
41.3 ±3.5
35.6 ±0.3
5.7
Neg.
36.9 ±
1.1
34.3 ±
0.1
2.6
Neg.
PLAT-New-
30
17
45
35.5 ±0.6
9.5
Neg.1
38.8 ±
2.1
34.5 ±
0.3
4.2
Neg.
PLAT-New-
30
17
45
16.9 ±0.0
28.1
Pos.
41.8 ±
2.8
16.5 ±
0.0
25.2
Pos.
PLAT-New-
300
170
36.2 ± 1.1
18.0 ± 0.1
18.2
Pos.
35.3 ±
1.1
17.7 ±
0.0
17.6
Pos.
PLAT-New-
300
170
45
16.5 ±0.0
28.5
Pos.
45
16.1 ±
0.0
28.9
Pos.
PLAT-New-
3,000
1,700
38.8 ±1.1
16.8 ± 0.1
21.9
Pos.
36.1 ±
1.2
16.3 ±
0.0
19.8
Pos.
PLAT-New-
3,000
1,700
38.1 ± 1.4
17.0 ±0.0
21.1
Pos.
35.3 ±
0.1
16.5 ±
0.0
18.8
Pos.
PLAT-END-
0
0
34.3 ± 1.23
34.3 ±0.8
0
Neg.
33.7 ±
0.4
33.4 ±
0.4
0.3
Neg.
PLAT-END-
0
0
42.3 ±4.5
33.4 ±0.2
9
Neg.1
37.9 ±
1.6
33.1 ±
0.1
4.8
Neg.
PLAT-END-
30
17
34.9 ±0.2
28.3 ±0.0
6.7
Neg.
34.3 ±
0.1
27.9 ±
0.0
6.4
Neg.
PLAT-END-
30
17
43.3 ±3.0
27.0 ±0.0
16.3
Pos.
45
26.5 ±
0.1
18.5
Pos.
PLAT-END-
300
170
38.2 ±1.9
24.5 ±0.0
13.7
Pos.
35.7 ±
0.3
23.9 ±
0.1
11.9
Pos.
PLAT-END-
300
170
45
28.1 ±0.2
16.9
Pos.
37.7 ±
0.2
27.6 ±
0.2
10.2
Pos.
PLAT-END-
3,000
1,700
43.3 ±1.9
21.7 ± 0.1
21.6
Pos.
42.1 ±
2.6
21.1 ±
0.0
21
Pos.
PLAT-END-
3,000
1,700
34.8 ±0.7
22.3 ±0.5
12.5
Pos.
33.9 ±
0.3
21.5 ±
0.4
12.4
Pos.
08/14/2017
ROLL
ROLL-New-O
0
45
34.2 ±0.4
10.8
Pos.
45
34.2 ±
0.1
10.8
Pos.
ROLL-New-O
0
45
39.4 ± 1.5
5.6
Neg.
45
35.7 ±
0.1
9.3
Neg.1
ROLL-New-
30
70
45
16.4 ±0.1
28.6
Pos.
45
16.4 ±
0.0
28.6
Pos.
0-3
-------
EPA/600/R-19/082
10/8/2019
Triiil l);ik-
S;iiii|)k' II)
S|>( in-
laid
li. :i.
ihriiiiiiisiiiiu-
( I (10)
li. :i.
ihriiiiiiisiiiiu-
( I (ll)
li. :i.
ihriiiiiiisiiiiu-
U 1
Kisull
li. ;i.
I>\()l
(1 (III)
li. :i.
|)\OI (1
(II)
li. ;i.
|>\()l
U 1
Kisull
ROLL-New-
30
70
45
16.6 ±0.0
28.4
Pos.
45
16.4 ±
0.0
28.6
Pos.
ROLL-New-
300
700
45
16.8 ±0.0
28.2
Pos.
45
16.6 ±
0.0
28.4
Pos.
ROLL-New-
300
700
45
17.1 ±0.0
27.9
Pos.
45
16.9 ±
0.0
28.1
Pos.
ROLL-New-
3,000
7,000
45
16.6 ±0.1
28.4
Pos.
43.6 ±
2.3
16.3 ±
0.0
27.3
Pos.
ROLL-New-
3,000
7,000
44.1 ± 1.6
16.7 ± 0.1
27.4
Pos.
44.2 ±
1.3
16.3 ±
0.0
27.9
Pos.
ROLL-END-
0
0
45
31.7 ± 0.1
13.3
Pos.
45
31.6 ±
0.1
13.4
Pos.
ROLL-END-
0
0
45
31.4 ± 0.1
13.6
Pos.
45
31.0 ±
0.1
14
Pos.
ROLL-END-
30
70
45
31.7 ± 0.1
13.3
Pos.
45
31.4 ±
0.2
13.6
Pos.
ROLL-END-
30
70
45
28.4 ±0.1
16.6
Pos.
45
27.9 ±
0.0
17.1
Pos.
ROLL-END-
300
700
45
27.3 ±0.1
17.7
Pos.
45
26.8 ±
0.0
18.2
Pos.
ROLL-END-
300
700
45
24.9 ±0.0
20.1
Pos.
45
24.3 ±
0.0
20.7
Pos.
ROLL-END-
3,000
7,000
45
27.4 ±0.1
17.6
Pos.
45
26.7 ±
0.0
18.3
Pos.
ROLL-END-
3,000
7,000
45
22.6 ±0.1
22.4
Pos.
45
21.9 ±
0.0
23.1
Pos.
08/21/17
BUS
BUS-New-0
0
45
35.7 ±0.6
9.3
Neg.1
41.1 ±
1.0
35.3 ±
0.6
5.8
Neg.
BUS-New-0
0
45
40.1 ±4.6
4.9
Neg.
43.4 ±
1.5
36.0 ±
0.3
7.4
Neg.
BUS-New-30
44
45
16.9 ± 0.1
28.1
Pos.
45
16.7 ±
0.0
28.3
Pos.
BUS-New-30
44
45
16.5 ± 0.1
28.5
Pos.
44.2 ±
1.4
16.2 ±
0.0
28
Pos.
BUS-New-
300
440
45
17.0 ±0.0
28
Pos.
45
16.7 ±
0.0
28.3
Pos.
BUS-New-
300
440
45
17.0 ±0.0
28
Pos.
45
16.7 ±
0.0
28.3
Pos.
BUS-New-
3,000
4,400
45
17.1 ±0.1
27.9
Pos.
45
16.8 ±
0.0
28.2
Pos.
BUS-New-
3,000
4,400
45
16.9 ± 0.1
28.1
Pos.
45
16.6 ±
0.0
26.7
Pos.
BUS-END-0
0
45
33.8 ±0.6
11.2
Pos.
45
33.4 ±
0.2
11.6
Pos.
BUS-END-0
0
45
33.9 ±0.0
11.1
Pos.
45
33.1 ±
0.1
11.9
Pos.
BUS-END-30
44
45
29.5 ±0.1
15.5
Pos.
45
29.2 ±
0.1
15.8
Pos.
BUS-END-30
44
45
23.8 ±0.0
21.2
Pos.
45
23.4 ±
0.0
21.6
Pos.
BUS-END-
300
440
45
21.9 ± 0.1
23.1
Pos.
45
21.7 ±
0.0
23.3
Pos.
BUS-END-
300
440
45
24.0 ±0.1
21
Pos.
45
23.6 ±
0.0
21.4
Pos.
BUS-END-
3,000
4,400
45
22.2 ±0.1
22.8
Pos.
45
21.5 ±
0.0
23.5
Pos.
0-4
-------
EPA/600/R-19/082
10/8/2019
Triiil l);ik-
S;iiii|)k' II)
S|>( in-
laid
li. :i.
ihriiiiiiisiiiiu-
( I (10)
li. :i.
ihriiiiiiisiiiiu-
( I (ll)
li. :i.
ihriiiiiiisiiiiu-
U 1
Kisull
li. ;i.
|>\()l
(1 (10)
li. :i.
|)\OI (1
(II)
li. ;i.
|>\()l
U 1
Kisull
BUS-END-
3,000
4,400
45
21.8 ± 0.1
23.2
Pos.
45
21.3 ±
0.0
23.7
Pos.
09/11/17
BUS and
HVAC
BUS-Mid-0
0
45
45
0
Neg.
45
45
0
Neg.
BUS-Mid-0
0
45
45
0
Neg.
45
45
0
Neg.
BUS-Mid-30
18
45
26.9 ±0.5
18.1
Pos.
43.2 ±
3.1
26.3 ±
0.0
16.9
Pos.
BUS-Mid-30
18
45
30.7 ±0.2
14.3
Pos.
45
30.1 ±
0.1
14.9
Pos.
BUS-Mid-
300
180
45
24.9 ±0.1
20.1
Pos.
45
24.6 ±
0.1
20.4
Pos.
BUS-Mid-
300
180
45
28.1 ±0.1
16.9
Pos.
45
27.2 ±
0.0
17.8
Pos.
BUS-Mid-
3,000
1,800
45
24.8 ±0.0
20.2
Pos.
45
23.8 ±
0.0
21.2
Pos.
BUS-Mid-
3,000
1,800
45
22.6 ±0.1
22.4
Pos.
45
21.7 ±
0.0
23
Pos.
HVAC-Mid-0
0
45
45
0
Neg.
45
45
0
Neg.
HVAC-Mid-0
0
45
45
0
Neg.
44.2 ±
1.4
45
-0.8
Neg.
HVAC-Mid-
30
18
45
28.9 ±0.1
16.1
Pos.
45
28.1 ±
0.1
16.9
Pos.
HVAC-Mid-
30
18
45
27.3 ±0.0
17.7
Pos.
45
26.9 ±
0.0
18.1
Pos.
HVAC-Mid-
300
180
45
21.9 ± 0.0
23.1
Pos.
45
21.5 ±
0.1
23.5
Pos.
HVAC-Mid-
300
180
45
23.6 ±0.1
21.4
Pos.
45
23.0 ±
0.0
22
Pos.
HVAC-Mid-
3,000
1,800
45
19.8 ± 0.1
25.2
Pos.
45
19.4 ±
0.0
25.6
Pos.
HVAC-Mid-
3,000
1,800
45
18.4 ±0.1
26.6
Pos.
45
17.8 ±
0.0
27.2
Pos.
09/18/17
PLAT and
ROLL
PLAT-Mid-0
0
45
45
0
Neg.
45
45
0
Neg.
PLAT-Mid-0
0
45
45
0
Neg.
45
45
0
Neg.
PLAT-Mid-
30
30
45
26.9 ±0.1
18.1
Pos.
45
26.7 ±
0.0
18.3
Pos.
PLAT-Mid-
30
30
45
30.1 ±0.0
14.9
Pos.
45
29.6 ±
0.0
15.4
Pos.
PLAT-Mid-
300
300
45
25.5 ±0.1
19.5
Pos.
45
25.1 ±
0.0
19.9
Pos.
PLAT-Mid-
300
300
45
25.3 ±0.1
19.7
Pos.
45
24.8 ±
0.0
20.2
Pos.
PLAT-Mid-
3,000
3,000
45
23.9 ±0.0
21.1
Pos.
45
23.4 ±
0.1
21.6
Pos.
PLAT-Mid-
3,000
3,000
45
24.8 ±0.1
20.2
Pos.
45
24.4 ±
0.0
20.6
Pos.
ROLL-Mid-O
0
45
45
0
Neg.
45
45
0
Neg.
ROLL-Mid-O
0
45
45
0
Neg.
45
45
0
Neg.
ROLL-Mid-
30
30
45
30.1 ±0.1
14.9
Pos.
45
29.8 ±
0.0
15.2
Pos.
ROLL-Mid-
30
30
45
33.3 ± 0.1
11.7
Pos.
45
32.7 ±
0.2
12.3
Pos.
ROLL-Mid-
300
300
45
30.0 ±0.1
15
Pos.
45
29.6 ±
0.1
15.4
Pos.
0-5
-------
EPA/600/R-19/082
10/8/2019
Triiil l);ik-
S;iiii|)k' II)
Spun-
l.llild
15. ;i.
ihriiiiiiisiiiiu-
( I (10)
li. :i.
ihriiiiiiisiiiiu-
( I (ll)
li. :i.
ihriiiiiiisiiiiu-
U 1
Kisull
li. ;i.
|>\()l
(1 (10)
li. :i.
|)\OI (1
(II)
li. ;i.
|>\()l
U 1
Kisull
ROLL-Mid-
3001
300
45
26.8 ±0.1
18.2
Pos.
45
26.2 ±
0.0
18.8
Pos.
ROLL-Mid-
3,000
3,000
45
23.7 ±0.0
21.3
Pos.
45
23.0 ±
0.0
22
Pos.
ROLL-Mid-
3,000
3,000
45
23.7 ± 0.1
21.3
Pos.
45
23.0 ±
0.0
22
Pos.
09/25/17
non-AQ
BUS-Mid-0
0
45
45
0
Neg.
45
43.8 ±
2.0
1.2
Neg.
mid-duty
HVAC-Mid-0
0
45
45
0
Neg.
45
45
0
Neg.
BUS-Mid-30
42
45
35.1 ±0.6
9.9
Pos.
45
34.5 ±
0.2
10.5
Pos.
HVAC-Mid-
30
42
45
29.5 ±0.1
15.5
Pos.
45
29.2 ±
0.1
15.8
Pos.
BUS-Mid-
300
420
45
21.8 ± 0.1
23.2
Pos.
45
21.7 ±
0.0
23.3
Pos.
HVAC-Mid-
300
420
45
22.0 ±0.1
23
Pos.
45
21.8 ±
0.0
23.2
Pos.
BUS-Mid-
3,000
4,200
45
21.5 ± 0.0
23.5
Pos.
45
20.9 ±
0.0
24.1
Pos.
HVAC-Mid-
3,000
4,200
45
17.5 ±0.0
27.5
Pos.
45
17.2 ±
0.0
27.8
Pos.
PLAT-Mid-0
0
45
45
0
Neg.
45
45
0
Neg.
ROLL-Mid-O
0
45
45
0
Neg.
45
45
0
Neg.
PLAT-Mid-
301
42
45
45
0
Neg.
45
45
0
Neg.
ROLL-Mid-
301
42
45
45
0
Neg.
45
45
0
Neg.
PLAT-Mid-
300
420
45
25.4 ±0.1
19.6
Pos.
45
25.2 ±
0.0
19.8
Pos.
ROLL-Mid-
300
420
45
24.7 ±0.0
20.3
Pos.
45
24.3 ±
0.0
20.7
Pos.
PLAT-Mid-
3,000
4,200
45
26.4 ±0.0
18.6
Pos.
45
25.7 ±
0.0
19.3
Pos.
ROLL-Mid-
3,000
4,200
45
26.7 ±0.1
18.3
Pos.
45
26.3 ±
0.0
18.7
Pos.
10/02/17
PLAT &
ROLL
PLAT-New-0
0
45
45
0
Neg.
45
45
0
Neg.
ROLL-New-O
0
45
45
0
Neg.
45
45
0
Neg.
PLAT-New-
30
28
45
16.4 ±0.0
28.6
Pos.
45
16.5 ±
0.0
28.5
Pos.
ROLL-New-
30
28
45
22.6 ±0.0
22.4
Pos.
45
22.5 ±
0.0
22.5
Pos.
PLAT-New-
300
280
45
17.0 ±0.0
28
Pos.
45
16.9 ±
0.0
28.1
Pos.
ROLL-New-
300
280
45
18.1 ±0.0
26.9
Pos.
45
17.9 ±
0.0
27.1
Pos.
PLAT-New-
3,000
2,800
45
17.5 ±0.1
27.5
Pos.
45
17.4 ±
0.1
27.6
Pos.
ROLL-New-
3,000
2,800
45
18.1 ±0.0
26.9
Pos.
45
17.9 ±
0.0
27.1
Pos.
PLAT-END-
0
0
45
45
0
Neg.
45
45
0
Neg.
ROLL-END-
0
0
45
45
0
Neg.
45
45
0
Neg.
0-6
-------
EPA/600/R-19/082
10/8/2019
Triiil l);ik-
S;iiii|)k' II)
Spun-
l.llild
li. :i.
ihriiiiiiisiiiiu-
( I (10)
li. :i.
ihriiiiiiisiiiiu-
( I (ll)
li. :i.
ihriiiiiiisiiiiu-
U 1
Kisull
li. ;i.
p\()l
( I (HI)
li. :i.
|)\OI (1
(II)
li. ;i.
|>\()l
U 1
Ki-miIi
PLAT-END-
30
28
41.0 ± 3.5
29.1 ±0.1
11.9
Neg.1
38.0 ±
1.1
29.1 ±
0.1
8.9
Neg.
ROLL-END-
30
28
45
38.8 ±5.6
6.2
Neg.
45
34.0 ±
1.3
11
Neg.1
PLAT-END-
300
280
45
28.2 ±0.1
16.8
Pos.
45
27.7 ±
0.1
17.3
Pos.
ROLL-END-
300
280
45
31.1 ±0.1
13.9
Pos.
45
30.3 ±
0.1
14.7
Pos.
PLAT-END-
3,000
2,800
45
23.5 ±0.0
21.5
Pos.
45
22.9 ±
0.0
22.1
Pos.
ROLL-END-
3,000
2,800
45
30.8 ±0.1
14.2
Pos.
45
29.9 ±
0.0
15.1
Pos.
05/28/18
PM10
South
Carolina
and Various
Filter types
11-BUS-
NEW-30
29
45
18.6 ± 0.1
26.4
Pos.
45
18.6 ±
0.1
26.4
Pos.
12-BUS-
NEW-300
290
45
18.5 ± 0.1
26.5
Pos.
45
18.5 ±
0.1
26.6
Pos.
13-HVAC-
NEW-300
290
45
18.3 ± 0.1
26.7
Pos.
45
18.3 ±
0.1
27.1
Pos.
15-BUS-
NEW-3,000
2,900
45
17 ± 0.1
28
Pos.
45
17 ± 0.1
28
Pos.
16-BUS-
END-3,000
2,900
45
18.6 ± 0.1
26.4
Pos.
45
18.6 ±
0.1
26.9
Pos.
17-HVAC-
NEW-3,000
2,900
45
17 ±0.0
28
Pos.
45
17 ±0.0
28
Pos.
0-7
-------
vvEPA
United States
Environmental Protection
Agency
PRESORTED STANDARD
POSTAGE & FEES PAID
EPA
PERMIT NO. G-35
Office of Research and Development (8101R)
Washington, DC 20460
Official Business
Penalty for Private Use
$300
------- |