SEPA
EPA/600/R-14/150 | July 2014 | www.epa.gov/ord
United States
Environmental Protection
Agency
Environmental Persistence of
Vegetative Bacillus
and Yersinia pestis
Office of Research and Development
National Homeland Security Research Center
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EPA 600/R-14/150
July 2014
Environmental Persistence of
Vegetative Bacillus anthracis
and Yersinia pest is
United States Environmental Protection Agency
Research Triangle Park, North Carolina 27711
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Disclaimer
The U.S. Environmental Protection Agency (EPA), through its Office of Research and
Development's National Homeland Security Research Center (NHSRC), funded and directed
this investigation through Task Order 4 of EPA Contract EP-C-11-038. This document has been
subjected to the Agency's review and has been approved for publication. Note that approval
does not signify that the contents necessarily reflect the views of the Agency. Mention of trade
names or commercial products in this document or in the methods referenced in this document
does not constitute endorsement or recommendation for use.
Questions concerning this document or its application should be addressed to:
Joseph P. Wood
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Mail Code E343-06
Research Triangle Park, NC 27711
(919)541-5029
wood.i oe@epa. gov
in
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Acknowledgments
Contributions of the following individuals and organization to the development of this document
are gratefully acknowledged.
EPA Team Members
Worth Calfee, EPA National Homeland Security Research Center (NHSRC)
Shawn Ryan, EPA NHSRC
Leroy Mickelson EPA Office of Solid Waste and Emergency Response
Peer Reviewers
Erin Silvestri, EPA NHSRC
Martin Page, U.S. Army
Doris Betancourt, EPA National Risk Management Research Laboratory
Battelle Memorial Institute
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Executive Summary
This report describes an investigation of the persistence of vegetative cells of Bacillus anthracis
(Ames) and Yersiniapestis (C092), both with and without exposure to ultraviolet (UV)-A/B
radiation (representing sunlight) on several surfaces and topsoil. The investigation of vegetative
cells of B. anthracis is motivated by the possibility of using germinants to convert spores to
vegetative cells, facilitating inactivation of the cells through natural attenuation or other
decontamination processes.
ES.l Test Procedures
The coupon materials used for B. anthracis testing were glass, bare pine wood, unpainted
concrete, and topsoil. The coupon materials used for Y. pestis included glass, galvanized steel,
painted wallboard paper, and topsoil. Testing was conducted at normal laboratory temperature,
with relative humidity (RH) levels ranging from approximately 35 % to over 90 %. In all
testing, the number of viable cells on each coupon was enumerated at predefined time points
after inoculation of the coupons. In addition, qualitative growth tests were conducted to confirm
the quantitative test results. In testing with B. anthracis, all coupon extracts (except as noted)
were enumerated both with and without application of a heat shock procedure that distinguished
heat-sensitive (presumed vegetative) B. anthracis cells from heat resistant cells (presumed
spores).
ES.2 Results for Vegetative Bacillus anthracis (Ames)
ES.2.1 Vegetative Bacillus anthracis Persistence without UV Exposure
In a test in which the inoculum contained B. anthracis in both vegetative cell and spore form,
both cells and spores were recovered from the test materials 56 days (the maximum duration
tested) after inoculation.
Without the presence of spores, vegetative B. anthracis had minimal persistence on glass, wood
and unpainted concrete. For these materials, B. anthracis cells persisted for less than 1 hour to a
maximum of 12 hours, depending on the material and RH. Vegetative B. anthracis had the
highest persistence in topsoil, which ranged between 4-5 days.
In every persistence test we confirmed that all B. anthracis cells remained vegetative throughout
the entire test duration, with one exception. A different finding occurred in two tests in which the
topsoil was wetted before inoculation, and the RH was maintained above 90 % to prevent drying
of the soil. Results from those tests showed that the initially 100 % vegetative B. anthracis
population grew by approximately a factor of 10 and showed extensive sporulation within one
week after inoculation of the topsoil coupons. Over 10 % of the total B. anthracis population
was in spore form at 48 hours after inoculation.
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ES.2.2 Vegetative Bacillus anthracis Persistence with UV Exposure
The persistence of vegetative B. anthracis on topsoil with exposure to simulated sunlight was
determined at six time points ranging from 1 hour to 120 hours after inoculation on to topsoil.
The longest elapsed time tested in which B. anthracis cells were recovered from the soil was 96
hours. These results showed that exposure to simulated sunlight may have had only a minor
impact on the persistence of vegetative B. anthracis in topsoil, presumably due to shading by soil
particles.
ES.3 Results for Yersiniapestis
ES.3.1 Yersinia pestis Persistence without UV Exposure
The persistence of Y pestis was tested at normal room temperature and RH levels from 54 to
over 90 %. It was found that Y. pestis persisted at one hour after inoculation on all four test
materials, but was completely inactivated within 24 hours after inoculation onto glass, painted
wallboard paper, and galvanized metal. Y. pestis persisted to at least six days on topsoil, but
when the soil was wetted and kept moist with elevated RH, viable Y. pestis was present at seven
days after inoculation. No viable cells were observed at 14 days after inoculation.
ES.3.2 Yersinia pestis Persistence with UV Exposure
One test of the persistence of Y. pestis was conducted on all four test materials under UV-A/B
exposure. In this test, Y. pestis did not persist on galvanized metal at the one-hour time point, but
did persist at the one-hour time point on the other three materials.
Impact of Study
This work provides information on the persistence of vegetative B. anthracis on surfaces and
soil, provided that the organism is completely germinated and sporulation is prevented. Such
results may be useful in the development of wide area remediation plans that consider the
possibility of germination and natural attenuation. To date, research has not shown such required
effective germination of spores on surfaces.1 For soil materials, natural attenuation may also be a
viable decontamination option provided that longer attenuation times (e.g., approximately a
week) are acceptable, and the soil can be kept relatively dry. Additional research is
recommended to confirm that the persistence of B. anthracis cells produced through the
germination of spores on materials is of similar duration to that of cells harvested in the
laboratory during the exponential growth phase and inoculated onto materials.
Similar to B. anthracis, the results of the study on the persistence of Y pestis show that natural
attenuation may also be a viable option for the decontamination of non-soil materials. For soils,
natural attenuation may also be a viable decontamination option provided that longer attenuation
times (e.g., approximately a week) are acceptable, and that soils can be kept reasonably dry.
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Contents
DISCLAIMER Ill
ACKNOWLEDGMENTS IV
EXECUTIVE SUMMARY V
ABBREVIATIONS/ACRONYMS X
1.0 INTRODUCTION 1
2.0 SUMMARY 01 TEST PROCEDURES 3
2.1 Preparation of Test Coupons 3
2.2 Vegetative B. anthracis Production 4
2.3 UV-A/B Exposure Procedure 5
2.4 Non-UV Persistence Testing 8
2.5 Organism Recovery Procedures 8
2.6 Calculation of Quantitative Persistence 9
2.7 Qualitative Growth Testing 10
3.0 QUALITY ASSURANCE/QUALITY CONTROL 12
3.1 Equipment Calibration 12
3.2 QC Results 12
3.3 Audits 12
3.3.1 Performance Evaluation Audit 12
3.3.2 Technical Systems Audit 13
3.3.3 Data Quality Audit 13
3.4 QAPP Amendments and Deviations 13
3.5 QA/QC Reporting 14
3.6 Data Review 14
4.0 RESULTS FOR BACILL US ANTHRACIS 15
4.1 QC Results 15
4.2 Vegetative Bacillus anthracis Persi stence without UV Exposure 15
4.2.1 Uniformity of Test Conditions 16
4.2.2 Results of Persistence Tests with Vegetative Bacillus anthracis 17
4.3 Persistence of Vegetative Bacillus anthracis under Exposure to Simulated
Sunlight 28
4.3.1 Uniformity of Test Conditions 29
4.3.2 Persistence Results in Simulated Sunlight Tests with Vegetative Bacillus
anthracis 30
5.0 RESULTS FOR YERSINIA PESTIS 33
5.1 Recovery Testing 33
5.2 QC Results 33
5.3 Yersinia pestis Persi stence without UV Exposure 34
5.3.1 Uniformity of Test Conditions 34
5.3.2 Results in Persistence Tests with Yersinia pestis 35
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5.4 Persistence of Yersiniapestis under Exposure to Simulated Sunlight 37
5.4.1 Uniformity of Test Conditions 37
5.4.2 Persistence Results in Simulated Sunlight Test with Yersinia pestis 38
6.0 SUMMARY 40
6.1 Results for Vegetative Bacillus anthracis (Ames) 40
6.1.1 Vegetative Bacillus anthracis Persistence without UV Exposure 40
6.1.2 Vegetative Bacillus anthracis Persistence with UV Exposure 41
6.2 Results for Yersinia pestis 42
6.2.1 Yersinia pestis Persistence without UV Exposure 42
6.2.2 Yersinia pestis Persistence with UV Exposure 42
7.0 REFERENCES 44
Appendices
Appendix A: Description of Issues and Test Results Leading to Development of
Procedure for Preparing Purely Vegetative Bacillus anthracis
Appendix B: Data Tables from Testing of Vegetative Bacillus anthracis Persistence
under UV-A/B Exposure
Appendix C: Data Tables From Selected Persistence Tests with Yersinia pestis
Figures
Figure 2-1. Schematic Representation of Test Chamber 6
Figure 2-2. Schematic of Five Coupon Positions on the Support Trays 6
Figure 4-1. Vegetative Bacillus anthracis (Ames) Persists for at Least 96 Hours in Topsoil at
Ambient Laboratory Temperature and Relative Humidity 22
Figure 4-2. Persistence of Vegetative Bacillus anthracis at One Hour on Glass Increases with
RII 24
Figure 4-3. Enumeration of Spores and Total Cells of Bacillus anthracis Shows Growth and
Sporulation in the 48-hour Test on Wet Soil at 93 % RH 28
Figure 4-4. Vegetative Bacillus anthracis Persistence with Exposure to Simulated Sunlight with
a Low UV-A/UV-B Ratio 31
Figure 4-5. Vegetative Bacillus anthracis Persistence with Exposure to Simulated Sunlight with
a High UV-A/UV-B Ratio 32
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Tables
Table 3-1. Performance Evaluation Audit Results 13
Table 4-1. Temperature and Relative Humidity Conditions during Non-UV Exposure
Persistence Tests with Vegetative Bacillus anthracis 17
Table 4-2. Persistence of Vegetative Bacillus anthracis (Four Materials, One Hour, 36
% RH; Test B A1) 18
Table 4-3. Persistence of Vegetative Bacillus anthracis on Glass (Eight Hours, 34 %
RI I;Test BA2) 19
Table 4-4. Persistence of Vegetative Bacillus anthracis (Four Materials, One Hour, 75
% RH; Test BA3) 20
Table 4-5. Persistence of Vegetative Bacillus anthracis on Topsoil (120 Hours, 46 %
RH; l est B A4) 21
Table 4-6. Persistence of Vegetative Bacillus anthracis on Three Materials (24 Hours,
92 % RH; Test BA5) 23
Table 4-7. Persistence of Vegetative Bacillus anthracis on Wetted Topsoil (28 Days, 96
% RH; Test BA6) 25
Table 4-8. Persistence of Vegetative Bacillus anthracis on Wetted Topsoil (48 Hours, 93
% RH; Test BA7) 27
Table 4-7. Ultraviolet Radiation Intensity at the Topsoil Test Coupons in Testing with
Bacillus anthracis 29
Table 4-8. Summary of Temperature and Relative Humidity Conditions in Simulated
Sunlight Testing with Vegetative Bacillus anthracis 30
Table 5-1. Recovery Testing of Yersiniapestis 33
Table 5-2. Temperature and Relative Humidity Conditions during Non-UV Persistence
Tests with Yersinia pestis 35
Table 5-3. Persistence of Yersinia pestis (Four Materials, 144 Hours, 65 % RH; Test
YP3) 36
Table 5-4. Persistence of Yersinia pestis (Wetted Topsoil, 14 Days, 96 % RH; Test YP4) 37
Table 5-5. Ultraviolet Radiation Intensity at the Exposed Coupons in Test YPUV1 with
Yersinia pestis 38
Table 5-6. Summary of Temperature and Relative Humidity Conditions at the
Exposed Coupons in Test YPUV1 with Yersinia pestis 38
Table 5-7. UV-A/B Inactivation of Yersinia pestis on Four Coupon Materials (24-Hour
Exposure at Low UV-A/UV-B Ratio; Test YPUV1) 39
IX
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Abbreviations/Acronyms
BSC biosafety cabinet
C Celsius
CFU colony-forming unit(s)
CI confidence interval
cm centimeter(s)
DNA deoxyribonucleic acid
EPA U.S. Environmental Protection Agency
EAI Etiologic Agent Inventory
ISO International Standards Organization
J Joule
L liter
LR log reduction
|iL microliter(s)
|im micrometer(s)
[j,W microwatt(s)
mL milliliter(s)
NHSRC National Homeland Security Research Center
NIST National Institute of Standards and Technology
nm nanometer(s)
OD600 optical density at 600 nanometers wavelength
PBS phosphate-buffered saline
PE performance evaluation
QA quality assurance
QAPP Quality Assurance Project Plan
QC quality control
QMP quality management plan
RH relative humidity
rpm revolutions per minute
SD standard deviation
SE standard error
SFW sterile filtered water
T temperature
TOPO Task Order Project Officer
TSA technical systems audit
TSB tryptic soy broth
UV ultraviolet
UV-A ultraviolet light (320 to 400 nm wavelength)
UV-A/B combination of UV-A and UV-B light used in testing
UV-B ultraviolet light (290 to 320 nm wavelength)
UV-C ultraviolet light (180 to 290 nm wavelength)
W watt(s)
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1.0 INTRODUCTION
The U.S. Environmental Protection Agency's (EPA's) Homeland Security Research Program
(HSRP) is helping protect human health and the environment from adverse impacts resulting
from the release of chemical, biological, or radiological agents. As part of fulfilling that mission,
the HSRP evaluates the role that natural conditions play in counteracting chemical and biological
homeland security threats. All evaluations are conducted in accordance with quality assurance
(QA) protocols to ensure the generation of high quality data and defensible results.
This study investigated the persistence of Bacillus anthracis (Ames) vegetative cells and
Yersiniapestis (C092) on representative surface materials. The emphasis of this investigation
on vegetative cells (rather than spores) of B. anthracis is driven by the potential to take
advantage of vegetative cells in remediation activities after B. anthracis contamination. That is,
a germinant might be used to convert B. anthracis spores to vegetative cells, which could then be
inactivated without the need for sporicidal decontaminants. Vegetative cells might be
inactivated largely or entirely by natural attenuation, including exposure to sunlight, thereby
reducing or eliminating the need for chemical decontaminants. Thus to assess whether natural
attenuation might be a viable option for remediation, the purpose of this study was to determine
how long vegetative cells of B. anthracis would persist on different materials under various
environmental conditions.
Separate persistence experiments were also conducted with Y. pestis in this study. This work
builds on previous research2 that investigated the inactivation of Y. pestis with chlorine dioxide
and vapor-phase hydrogen peroxide and the persistence of Y. pestis on aluminum, carpet,
computer keys, and wallboard paper. In particular, this study aimed to investigate the impact of
materials not tested previously (such as topsoil) and the effect of simulated sunlight on Y. pestis
persistence. As with B. anthracis, the purpose of the Y pestis portion of the study was to assess
whether natural attenuation might be a feasible remediation option by determining how long this
agent might survive on different materials under various environmental conditions.
Persistence under normal laboratory conditions or simulated sunlight exposure was determined
for each of the two test organisms (in separate experiments) on coupons of four test materials,
which were chosen in part based on organism recovery tests conducted in this study prior to the
persistence testing. Glass, bare pine wood, unpainted concrete, and topsoil coupons were used
for testing with vegetative B. anthracis, to represent both porous and nonporous outdoor
surfaces. For Y. pestis, the same four materials were initially chosen, but based on low recovery
results with bare pine wood and unpainted concrete in the initial recovery tests, galvanized steel
and painted wallboard paper were used instead of those two materials for testing with Y pestis.
The coupon preparation and recovery test results for Y pestis are described in Chapters 2 and 5
of this report. Simulated sunlight testing involved exposure to a combination of ultraviolet
(UV)-A (i.e., 320-400 nanometers [nm]) and UV-B (i.e., 290-320 nm) radiation (referred to as
UV-A/B), and was conducted using alternating 12-hour periods of light and dark. Coupons of
the test materials, inoculated with the test organisms, were placed in the same test environment
as those exposed to UV-A/B radiation, but were shielded from any UV-A/B exposure.
Persistence testing in the absence of simulated sunlight was conducted by exposing the test
organisms only to normal laboratory lighting conditions.
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The persistence of the two test organisms was determined by both quantitative and qualitative
measures. Quantitative testing involved extracting and enumerating viable organisms from test
coupons at each time point in each test. Qualitative testing involved placing an aliquot of the
coupon extract in nutrient broth and assessing whether any growth occurred over periods of up to
seven days of incubation. The qualitative testing provided a more sensitive measure of the
presence of any remaining viable organisms at later time points in a test, when organism counts
fell below the detection limit of the quantitative procedure.
The intent of the experiments with B. anthracis was to use inoculum containing purely
vegetative cells. However, initial tests showed cell cultures containing predominantly vegetative
cells, but with a few spores also present. Consequently, procedures were developed to ensure a
purely vegetative B. anthracis cell population for subsequent tests. Additionally, a heat shock
procedure was used in all subsequent tests with B. anthracis to confirm that only vegetative cells
were inoculated onto test materials and to distinguish vegetative cells from spores in coupon
extracts.
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2.0
SUMMARY OF TEST PROCEDURES
Test procedures were performed in accordance with the Quality Assurance Project Plan (QAPP;
which is available upon request) and are briefly summarized here. The activities conducted
during this project consisted of persistence testing with either vegetative B. anthracis or with Y.
pestis on four coupon materials, under different humidity conditions and under different levels of
UV-A/B exposure. The following subsections describe the procedures that were used in carrying
out these activities.
2.1 Preparation of Test Coupons
Test coupons of glass, galvanized metal, bare pine wood, and painted wallboard paper were each
cut from larger pieces to 1.9 centimeters (cm) x 7.5 cm in size. Coupons of unpainted concrete
were 1.0 cm x 3.5 cm in size, and were made by pouring into individual molds, rather than by
cutting from a larger piece. The different size of the concrete coupons relative to the other
coupons was solely due to the molds available and is not likely to affect the test results. Topsoil
coupons consisted of 3.5 cm diameter Petri dishes having a height of 1 cm lined with Parafilm®
and filled level with the top of the dish with uncompacted topsoil. The topsoil was obtained at a
retail garden store and was shown by analysis to have approximately 30 % moisture content, 5 %
organic carbon content, and a pH of approximately 7.3. Glass, concrete, and galvanized metal
coupons were sterilized before use by autoclaving. Topsoil was autoclaved in bulk before being
distributed into Petri dishes. Bare pine wood and painted wallboard paper coupons were
sterilized before use by gamma irradiation. Test materials were sterilized to avoid confounding
results from non-target organisms.
The B. anthracis (Ames) used for this testing was prepared from a qualified stock of spores of
the Ames strain at the Battelle Biomedical Research Center (BBRC, West Jefferson, OH). The
spore lot was subject to stringent characterization and qualification processes required by the
laboratory for spore production. Specifically, the spore lot was characterized prior to use by
observation of colony morphology, direct microscopic observation of spore morphology and
size, and determination of percent refractivity and percent encapsulation. Variations in the
expected colony phenotypes were recorded. Endotoxin concentration of each spore preparation
was determined by the Limulus Amebocyte Lysate assay to assess whether contamination from
gram-negative bacteria occurred during the propagation and purification process of the spores.
Genomic deoxyribonucleic acid (DNA) was extracted from the spores and DNA fingerprinting
by the polymerase chain reaction was done by Dr. Paul Keim at Northern Arizona University to
confirm the genotype. The virulence of the spore lot was measured by challenging guinea pigs
intradermally with a dilution series of spore suspensions, and virulence was expressed as the
intradermal median lethal dose. In addition, testing was conducted for robustness of the spores
via hydrochloric acid resistance. The number of viable spores in the stock suspension was
determined by colony count and expressed as colony forming units per milliliter (CFU/mL).
Theoretically, once plated onto bacterial growth media, each viable spore germinates and yields
1 CFU.
The virulent Y. pestis strain C092 was obtained from Etiological Agent Inventory (EAI) number
YUC429, originally obtained from the University of Chicago and stored at -80 °C. Fresh
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cultures were prepared in advance of each day that coupons were inoculated by transferring one
or two colonies from a streak plate (freshly grown on tryptic soy agar (TSA) or stored less than
two weeks at 2 to 8 °C) into 10 to 20 mL of tryptic soy broth (TSB). This culture was then
incubated overnight at 26 ± 2 °C on an orbital shaker set to 200 revolutions per minute
(rpm). The bacterial culture was then diluted with fresh media to an optical density at 600 nm
384
(OD60o) of approximately 0.1 to 0.2 using a SPECTRAmax Plus spectrophotometer
(Molecular Devices, Sunnyvale, CA). A gram stain was performed on the cultured bacteria, and
the colony morphologies were confirmed to be consistent with previous descriptions of Y. pestis
(i.e., grayish-white colonies with a "fried egg" appearance) after approximately 72 hours at 26 ±
2 °C.
Y. pestis and vegetative B. anthracis were inoculated onto test coupons in an appropriate
biosafety cabinet (BSC-II or -III) according to established procedures.3 Inoculated coupons were
prepared fresh for each day of experimental work. Test coupons were placed flat in the BSC and
inoculated with vegetative B. anthracis at approximately 8 x 105 to 5 x 106 CFU per coupon, or
7 8
with Y. pestis at approximately 3x10 to 2x10 CFU per coupon. The different inoculation
levels of the two organisms were a result of the different growth characteristics of the two
cultures. Inoculation was accomplished by dispensing a 100 microliter (|iL) aliquot of a stock
suspension as 10 droplets (each of 10 |iL volume) across the surface of the test coupon using a
micropipette. This approach provided more uniform distribution of cells across the coupon
surface than would be obtained through a single drop of the suspension. A spreader was not used
after application of the ten droplets to the coupon. In nearly all cases, the test coupons remained
undisturbed in the BSC for one hour to dry after inoculation. In a single persistence test with
vegetative B. anthracis, inoculated coupons were extracted before the usual one-hour drying
period elapsed, to assess the persistence of that organism over time intervals shorter than one
hour.
2.2 Vegetative B. anthracis Production
The intent of this project was to test with purely vegetative cells of B. anthracis (Ames).
However, observations from initial tests (summarized in Appendix A) disclosed that B. anthracis
cell cultures contained predominantly vegetative cells but also a few spores, indicating incipient
endospore formation. In one initial persistence test in which the inoculum contained B. anthracis
in both vegetative cell and spore form (distinguished by heat shock), cells were recovered from
these materials at 56 days; refer to Table A-l.
Consequently, an improved procedure was developed that could be conducted reproducibly to
produce a stock solution with a relatively high titer of purely vegetative B. anthracis. That
procedure consisted of the following steps:
• Prepare a 1:100 dilution in sterile filtered water (SFW) of standard B. anthracis (Ames)
spore stock, which is approximately 1 x 109 CFU/mL.
n
• Add 10 |iL of the resulting 1:100 dilution (which is approximately 1x10 CFU/mL) to
200 mL of TSB.
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2
• Allow the resulting culture (which is initially approximately 5x10 CFU/mL) to
incubate at 37 °C while shaking on an orbital shaker at 200 rpm for 9 to 10 hours.
• During heating and shaking of the culture, remove an aliquot at 1-hour intervals for
determination of ODeoo as an indication of the titer of the culture. An OD600 of
approximately 2 is expected at the end of the 9- to 10-hour incubation period.
• Remove the culture from heating and shaking and verify the purity of the vegetative cell
culture by microscopic examination and heat shock treatment.
This procedure was used to prepare a fresh culture of vegetative B. anthracis for the start of each
new test with that organism, and consistently provided a vegetative B. anthracis culture of
7 7
approximately 1x10 to 5xio CFU/mL, free of any detectable spores. Additional details on
this procedure are presented in Appendix A.
2.3 UV-A/B Exposure Procedure
For the simulated sunlight tests with vegetative B. anthracis, blank coupons and inoculated
coupons were transferred into a test chamber equipped with small UV lamps, shown
schematically in Figure 2-1 (figure not to scale). The three UV lamps used were Reptisun® 10.0
UVB (15 Watts, 48 cm long), made by Zoo Med Laboratories, Inc. (San Luis Obispo, CA),
which provided both the UV-A and UV-B radiation to which the coupons were exposed. These
lamps emitted no UV-C (wavelength < 290 nm) radiation, consistent with the absence of UV-C
in sunlight at ground level. In the UV exposure testing with vegetative B. anthracis, only
coupons of topsoil were used. Five UV-exposed coupons and five non-exposed coupons of
topsoil were used for each UV-A/B exposure time point. One blank topsoil coupon was also
included with the exposed coupons and with the non-exposed coupons for each time point. The
exposed coupons and the associated blank coupon were placed flat on top of the raised tray
below the UV lamps, and non-exposed coupons and the associated blank coupon were placed flat
beneath that tray, shielded from direct UV-A/B radiation (Figure 2-1). The lower portions of the
test chamber walls were covered with black paper to minimize reflected UV-A/B radiation.
The topsoil coupons were arrayed in five separate positions on the support trays, as shown
schematically in Figure 2-2, with Position 1 in the center of the tray and Positions 2 through 5
located toward the corners of the tray. One topsoil coupon was placed at each of these five
positions, so that coupons were equally distributed across the support trays. A blank coupon was
also placed at Position 1 of the appropriate tray.
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1L
1L
W Lamps (3)
/
/
\
~stance
Appraximiely
12 certi meters
Chamber
V'.yis
Darkened
HOBO T.'PH
Datalogger (one
on each tray)
a.
lCL
Test Coupons
aid Blar+;s
(5 positions)
Cortrol
CoLpons ard
Blar+;s
(5 positions)
Figure 2-1. Schematic Representation of Test Chamber (not to scale)
Position 2 Position 3
1L K
o UJ
zi
o<
Position 1
Position 4 Positions
Figure 2-2. Schematic of Five Coupon Positions on the Support Trays
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A single UV exposure test was conducted with Y pestis in the same test chamber used for the
UV exposure tests with B. anthracis. However, the Y. pestis UV exposure test used coupons of
all four coupon materials placed in the UV test chamber at once, and only UV- exposed coupons
were used (i.e., no non-exposed coupons were included in the test). Five inoculated UV-exposed
coupons of each material were used, and one blank coupon of each material was also included
with the exposed coupons. For that Y pestis UV exposure test, the coupons were arrayed in the
five separate positions on the UV-exposed support tray, as shown in Figure 2-2, with one
exposed coupon of each of the four coupon materials placed at each of Positions 1 through 5, so
that all coupon materials were equally distributed across the support trays. This approach
ensured that all materials received equivalent UV-A/B exposures during testing. A blank coupon
of one of the four materials was also placed at each of Positions 2 through 5, so that the four
blanks were similarly distributed on both the exposed and non-exposed trays.
All UV exposure testing was conducted at normal room temperature (T) (i.e., approximately 22
°C) and over a range of RH from approximately 50 to 75 % RH. However, the chamber
temperature and RH were not rigidly controlled, and despite circulation of air through the
chamber, the chamber temperature typically increased slightly, and RH decreased slightly during
the UV-A/B exposure periods. Temperature and RH were recorded at 5-minute intervals
throughout the tests at the locations of both the exposed and non-exposed coupons by a HOBO®
Model U12-011 temperature and RH sensor/data logger (Onset, Cape Cod, MA) placed near the
center (Position 1, Figure 2-2) of each coupon support tray. The average and standard deviation
(SD) of the recorded temperature and RH data at each tray over the duration of each UV-A/B
exposure are presented in Sections 4 and 5 of this report to document the test conditions. The
UV testing consisted of alternating 12-hour periods of UV-A/B exposure (lamps on) and
darkness (lamps off), with the first 12-hour period always having lamps on. This alternating UV
exposure schedule was designed to replicate natural diurnal conditions, allowing for potential
repair of photo-induced damage in the test organisms between the periods of UV exposure. At
the conclusion of each UV-A/B exposure period, exposed and non-exposed coupons of all
materials were removed from the test chamber, and the test organisms were extracted and
enumerated to determine persistence due to the UV-A/B exposure.
The UV lamps used for testing simulate natural sunlight by including both UV-A and UV-B
components but without UV-C.4 Wide variations in natural UV-A/B levels occur due to time of
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day, day of the year, location, cloud cover, air pollution levels, and altitude. " Peak (i.e.,
noontime) UV-B levels reported in a few studies range from approximately 20 to 150 microwatts
2 9 11 2
(|iW)/cm . " The target UV-B level chosen for testing was 44 |iW/cm , which corresponds to a
daily dose of approximately 1.9 Joules (J)/cm2 with 12 hours of exposure per day. To put this in
context, this UV-B dose is similar to the daily UV-B dose received during the summer months in
Raleigh, North Carolina (see UV monitoring data at http://uvb.nrel.colostate.edu/UVB/index.isf).
2 2
The UV-A level was set at either approximately 100 |iW/cm or approximately 1,785 |iW/cm ,
to assess the effect of UV-A relative to the more photobiologically active UV-B.4 These UV-A
2 2
levels correspond to a daily UV-A dose of about 4.3 J/cm and 77 J/cm , respectively, with 12
hours of exposure per day. The former is a low UV-A level, and the latter is similar to the daily
UV-A dose received during the summer months in Raleigh, North Carolina (see UV monitoring
data at http://uvb.nrel.colostate.edu/UVB/index.isf). A target of zero for UV-C radiation was
7
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chosen because of the absence of this UV component in sunlight at ground level. The target UV
intensities at the unexposed coupons were zero for UV-A, UV-B, and UV-C.
The actual UV intensities were measured at each of the five positions shown in Figure 2-2 on
both the exposed and non-exposed coupon trays, at least near the start and end of every 12-hour
UV-A/B exposure period. UV intensities were measured using Solarmeter® Digital Ultraviolet
Radiometers, Model 5.7 (UV-A/B) (Serial No. 15957), Model 6.2 (UV-B) (Serial No. 01802),
and Model 8.0 (UV-C) (Serial No. 00275) (Solartech, Inc., Harrison Twp., MI). The UV-A
intensity was determined by subtracting the UV- B reading from the UV-A/B reading. The UV-
A, UV-B, and UV-C intensities at each coupon position over each UV-A/B exposure test were
determined, and the averages and SDs of those intensities are shown in Sections 4 and 5. The
lamps used in testing are designed not to produce UV-C radiation, nevertheless UV-C
measurements were made to confirm the absence of UV-C.
2.4 Non-UV Persistence Testing
The majority of the persistence tests were conducted without using UV-A/B radiation. The
persistence testing was conducted in a laboratory chamber similar to the chamber used for the
UV exposure testing, but with only minimal normal laboratory lighting and no UV lamps or UV
sensors. The laboratory lighting was turned on and off manually by laboratory personnel on
approximately a 12-hours on/12 hours off schedule. Initial tests involved all test materials and
relatively long time points and were intended to provide a broad assessment of the persistence of
the organisms. The conditions for subsequent tests were then chosen based on reviewing the
results of previous tests. The resulting test matrix included targeted testing with individual
coupon materials; determining persistence at multiple time points; varying the test RH; and
varying the moisture content of topsoil coupons used in testing.
In the non-UV persistence testing, the temperature was not controlled but was always close to the
temperature of the normal laboratory environment (i.e., approximately 22 ± 2 °C). The RH in
different tests ranged from approximately 35 % to over 95 %. The RH of the normal laboratory
environment provided sufficient control for tests at RH from approximately 35 % to 65 %;
saturated salt solutions were used to control RH at approximately 75 % to over 95
% in other tests. Both T and RH were monitored in all testing.
2.5 Organism Recovery Procedures
Following the persistence test period or UV-A/B exposure period, each inoculated and associated
blank coupon was transferred aseptically to a sterile 50-mL conical vial containing 10 mL of
extraction solution. The extraction solution consisted of sterile phosphate-buffered saline (PBS)
solution with Triton X-100 surfactant (i.e., 99.9 % PBS solution, 0.1 % Triton X-100 by
volume). The coupons were then extracted by agitation on an orbital shaker for 15 minutes at
approximately 200 rpm at room temperature. For all coupons, 1 mL of the coupon extract was
removed following extraction, and a series of dilutions through 10"7 was prepared in SFW. For
B. anthracis, an aliquot (0.1 mL) of the undiluted extract and of each serial dilution was spread
plated in triplicate onto TSA plates and incubated overnight at 35 to 37 °C. For B. anthracis,
plates were enumerated within 18 to 24 hours of plating. For Y. pestis, an aliquot (0.1 mL) of the
8
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undiluted extract and of each serial dilution was spread plated in triplicate onto TSA plates and
incubated overnight at 26 °C. For Y. pestis, plates were enumerated within 48 to 72 hours of
plating. For both organisms, the number of CFU/mL was determined by multiplying the average
number of colonies per plate by the reciprocal of the dilution and accounting for the 0.1 mL
plated volume.
A heat shock procedure was applied to all coupon extracts in testing with B. anthracis to
distinguish vegetative B. anthracis cells (which were killed by the heat shock) from B. anthracis
spores (which survived the heat shock). Specifically, the 10 mL coupon extract was split into
two equal portions, with one portion heat shocked and the other not, before plating of both
aliquots for enumeration as described above. The heat shock procedure consisted of placing a
small vial containing an aliquot of the extract into a water bath at 65 °C for one hour before the
aliquot was serially diluted and plated.
Blank coupons controlled for viable organisms inadvertently introduced to test coupons, and
were each inoculated with 100 |iL of inoculum carrier liquid that did not contain B. anthracis or
Y. pestis cells. The blank coupons underwent the same extraction process as the inoculated
coupons, at the same time as those coupons. To be considered acceptable, extracts of blank
coupons had to contain no CFU. The mean percent cell recovery from each coupon type was
calculated by means of the following equation:
Mean % Recovery = [Mean CFUc/CFUpike] x 100 (1)
where Mean CFUc is the mean number of CFU recovered from five replicate coupons of a single
type, and CFUpike is the number of CFU inoculated onto each of those coupons. The value of
CFUpike is known from enumeration of the stock suspension by the same dilution, plating,
incubation, and enumeration procedures as were applied to the coupon extracts, as described
above. Recovery was calculated for both B. anthracis and Y. pestis on each coupon type.
2.6 Calculation of Quantitative Persistence
The effect of coupon drying, different T/RH conditions, or UV-A/B exposure on the persistence
of the test organisms was assessed by determining the number of viable organisms recovered
from each test coupon after some period of time and/or UV-A/B exposure. In tests reported here,
there were five test coupons at each time point for each material. When no viable CFU were
found in a coupon extract, a CFU count of 1 was assigned, resulting in a base 10 logarithm (log)
of zero for the CFU count for that coupon. When this occurred, the cell population on the
coupon was considered to be completely inactivated within the detection limit of 33 CFU per
extract sample. The possibility that viable cells still remained on the coupon despite the
quantitative indication of zero CFU recovered was assessed by means of the qualitative testing
described in Section 2.7.
Persistence of an organism on a coupon material was determined as the mean of the logs of the
number of CFU recovered from each of the five replicate coupons of that coupon material at a
test time point. First, the CFU count value from each coupon extract was determined, and then
the mean of the log values was determined for each set of five replicate coupons, i.e.,
9
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Mean of Logs, = (log10 CFUty)
(2)
where logio CFUty refers to the j individual log values of the CFU extracted from the coupons of
material with / = 5 and the overbar indicating a mean value.
Also, the number of CFU of an organism recovered in extracts of test coupons was compared to
the number inoculated onto the coupons to calculate the mean log reduction (LR). Mean LR for
a test organism on the z'th coupon material was calculated as the difference between the mean log
value from Equation 2 and the log of the number of cells inoculated onto the coupons, which is
known from the enumeration of the stock suspension as described in Section 2.5. Mean LR is
calculated as:
where logio CFUi refers to the log of the number of cells inoculated onto the material coupons
and logio CFUty is as defined above. LR was determined for each coupon material at each time
point in the testing.
Whenever the mean of the logs was calculated as in Equations 2 and 3, the SD of the five logio
CFUty values was also calculated. The SD was then used to calculate the standard error (SE) of
the result and in turn the 95% confidence interval (CI) for each result. The 95 % CI is:
The significance of differences in persistence across different coupon materials, test conditions,
or time points was assessed based on the 95 % CI of each result. Differences were judged to be
significant if the 95 % CIs of two results did not overlap.
2.7 Qualitative Growth Testing
In addition to the extraction and quantitative enumeration described in Section 2.5, qualitative
growth testing was conducted in all tests for vegetative B. anthracis and in some tests for Y.
pestis by placing 1 mL of the coupon extract into an individual vial containing 10 mL of TSB.
After one day of incubation, the solution in each vial was inspected visually for turbidity
(cloudiness, indicating bacterial growth), and this inspection was then repeated after seven days
of incubation. After the seven-day incubation a small amount of any solution showing turbidity
was also streaked onto a tryptic soy agar plate, and the morphology of the resulting CFU was
used to confirm the presence of the target organism. The qualitative results presented in Chapter
4 indicate whether bacterial growth was observed after 1 day and after 7 days. A "Y" indicates
growth was visually observed with at least one of five coupon extracts, and an "N" indicates no
growth was observed (complete inactivation of the organism). If at least one of the 7-day streak
plates confirmed the presence of the target organism, this is indicated by using a bold letter "Y".
LR = (log10 CFUi) - (log10 CFUti;)
(3)
95% CI = ±(1.96 x SE) = ± (1.96 x [SD/V5])
(4)
10
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The qualitative growth test procedures provided a more sensitive indication of complete
inactivation of the test organisms than did the extraction and enumeration alone, due to the
inability of the extraction and enumeration to detect very small numbers of viable cells. In a few
cases, the presence of viable cells was not indicated by the one-day qualitative test but was
indicated by the seven-day test. Due to the greater sensitivity of the seven-day qualitative test,
those results are emphasized in this report. The agreement or disagreement between quantitative
enumeration and qualitative test results is noted where appropriate to document the persistence
of the test organisms.
11
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3.0 QUALITY ASSURANCE/QUALITY CONTROL
QA/QC procedures are summarized below.
3.1 Equipment Calibration
All equipment (e.g., pipettes, incubators, BSCs) and monitoring devices (i.e., for T, RH, and UV-
A/B, UV-B, and UV-C intensity) were verified as being certified, calibrated, or validated.
Battelle's Instrumentation Services Laboratory, which is accredited by the American Association
for Laboratory Accreditation to the International Standards Organization (ISO) 17025 standard,
established NIST-traceable calibrations of the T and RH monitors used in this test. The three
Solarmeter UV radiometers were obtained from the manufacturer certified with NIST-traceable
calibrations, and that certification was in effect throughout all testing.
3.2 QC Results
QC efforts conducted during testing included inoculated non-exposed coupons in UV-A/B
exposure testing, procedural blanks (not inoculated, UV-A/B-exposed), laboratory blanks (not
inoculated, not UV-A/B-exposed), and spike control samples (analysis of the stock cell
suspension). The results for these QC samples in each decontaminant evaluation are included in
the results sections (see Sections 4 and 5).
3.3 Audits
3.3.1 Performance Evaluation Audit
Performance evaluation (PE) audits were conducted between January 2 and January 22, 2013, on
equipment and measurements that factored directly into the test results, as specified in the QAPP.
In the PE audit, the delivery of cell suspensions to coupons was audited by gravimetric checking
of the micropipettes used for inoculation. The measurement of the concentration of cell
suspensions by plating and enumeration was audited by recounting of the enumeration plates by
a different laboratory staff member. The T and RH measurements were audited by comparison
to a combination thermometer/hygrometer obtained from the laboratory and traceable to NIST.
Table 3-1 lists the target criteria and results of the PE audits, and shows that all of the PE audit
results met the target tolerances. Measurement of time was not subjected to a PE audit because
the test durations were sufficiently long that high accuracy was not required.
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Table 3-1. Performance Evaluation Audit Results
Measurement
Audit Procedure
Target
Tolerance
Achieved
Tolerance
Volume of cell
suspensions or water
Gravimetric evaluation of micropipettes(a)
±10%
-0.18 to -0.44 %
0.21 to 1.59%
0.02 to 1.54%
-0.14 to-0.62%
Enumeration of cell
concentration
Recounting of enumeration plates by a
different laboratory staff member
±10%
0.8 % (B.a.)
1.7 %{Y.p.)
Temperature
Compared to independent calibrated
thermometer®
±2 °C
0.1 °C(C)
0.2 °C
Relative humidity
Compare to independent calibrated
hygrometer®
±10%
3.2 %c
0.9 %
(a) Gravimetric check was performed by returning pipettes to the manufacturer (Rainin); results shown are from
calibration certificates received for the four pipettes. Range of tolerances for each pipette results from
calibration checks at delivered volumes ranging from 0.1 to 1.0 mL.
(b) Audit standard was a Vaisala (Boulder, CO, USA) Model HMT338 thermometer/hygrometer, serial number
111996775.
(c) Results shown are for the HOBO T/RH sensors positioned with the UV-exposed and shielded (non-exposed)
coupons, respectively.
3.3.2 Technical Systems Audit
Laboratory QA staff conducted a technical systems audit (TSA) during testing on January 15,
2013, to ensure that the evaluation was being conducted in accordance with the QAPP and the
QMP. As part of the TSA, test procedures were compared to those specified in the QAPP, and
data acquisition and handling procedures were reviewed. Observations and findings from the
TSA were documented and submitted to the laboratory lead for response. No adverse findings
resulted from this TSA, but one QAPP amendment and one QAPP deviation were prepared as a
result of this TSA to document changes made to improve the test procedures. The TSA report
was submitted to EPA on January 31, 2013. TSA records were permanently stored with the
laboratory QA Manager.
3.3.3 Data Quality Audit
All of the data acquired during the evaluation were audited. A QA auditor traced the data from
the initial acquisition, through reduction and statistical analysis, to final reporting to ensure the
integrity of the reported results. All calculations performed on the data undergoing the audit
were checked.
3.4 QAPP Amendments and Deviations
Three amendments to the QAPP were prepared, reviewed, approved, and distributed to all parties
involved in this evaluation. The subjects addressed by those three amendments were,
respectively:
13
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• The selection of different coupon materials (i.e., galvanized metal and painted wallboard
paper) for testing with Y. pestis than were used in testing with B. anthracis, and the
performance of UV-A/B exposure testing with only one of these organisms at a time,
instead of with both organisms at once. The former change was made due to low
recoveries observed with Y pestis from concrete and bare pine wood (see Section 5.1).
The latter change was made because the UV exposure system could not achieve a
uniform UV-A/B intensity across the numerous test coupons when both organisms were
tested at once.
• Dilution of stock cell suspensions to a target optical density was done for Y pestis but not
for B. anthracis. This change was made because the purely vegetative B. anthracis stock
suspension was used without dilution to achieve a high enough inoculation of cells onto
test coupons.
• Accommodation of a variety of exploratory test procedures other than UV-A/B exposure
or persistence testing, including cell recovery testing, evaluation of heat shock
procedures, experimentation to achieve purely vegetative B. anthracis cell cultures, and
extraction of inoculated coupons after very short drying times (i.e., < one hour). This
change was made to allow flexibility in investigating vegetative B. anthracis production
procedures and the survivability of vegetative B. anthracis on coupon materials.
One QAPP deviation was prepared. That deviation stated that a saturated salt solution was not
used as originally planned to control the RH in the UV-A/B exposure testing. The reason for this
deviation was that the UV-A/B exposure tests were brief, and as a result the salt solution was
ineffective in controlling the RH. The impact of this deviation on the testing was minimal, as the
UV-A/B exposure tests were conducted at normal laboratory RH, and acceptable control was
maintained by the laboratory climate control systems without the use of the salt solution.
3.5 QA/QC Reporting
Each audit was documented in accordance with the Quality Management Plan (QMP). The
results of the audits were submitted to the EPA.
3.6 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 reports. All data were
recorded by laboratory staff. The person performing the QC/technical review added his/her
initials and the date to a hard copy of the record being reviewed. This hard copy was returned to
the laboratory staff member who stored the record.
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4.0
RESULTS FOR Bacillus anthracis
This section summarizes QC and persistence results for B. anthracis. Data are presented to
document the uniformity of the UV exposure test conditions. Persistence results with and without
UV-A/B exposure are reported for B. anthracis on the test materials.
4.1 QC Results
All procedural and laboratory blanks met the criterion of no observed CFU of the inoculated
B. anthracis organism. Spike control samples were taken from the cell suspension on each day
of testing, and serially diluted, nutrient plated, and counted to establish the cell suspension
density used to inoculate the coupons. This process required approximately 24 hours, so the cell
suspension density was known after completion of each day's testing. The target criterion was to
maintain as high a cell suspension density as possible while producing a 100 % vegetative
B. anthracis suspension. In actuality, B. anthracis suspensions of approximately 8 x 106 to 5 x
n
10 CFU/mL were produced, leading to actual spike values that ranged from approximately 8 x
105 CFU/coupon to 5 x 106 CFU/coupon. An aliquot of each B. anthracis cell suspension used
to inoculate coupons and portions of all coupon extracts were subjected to the heat shock
treatment noted in Section 2.5. No inoculation suspension subjected to the heat shock treatment
showed any viable organisms, indicating the absence of B. anthracis spores and confirming that
all coupons were inoculated with purely vegetative B. anthracis cells within the limit of
detection.
4.2 Vegetative Bacillus anthracis Persistence without UV Exposure
Using the procedure for preparing a purely vegetative B. anthracis cell suspension (Section 2.2)
seven persistence tests were conducted with vegetative B. anthracis without exposure to UV-A/B
radiation. All those tests were conducted at normal laboratory temperatures. As noted in Section
2.4, the conditions for tests were chosen based on reviewing the results of previous tests. Those
seven tests and the rationale for the test conditions in each test are listed below in chronological
order.
• Test BA1 - Persistence on four coupon materials at a mean RH of 36 % (laboratory
ambient RH) after a one-hour exposure time. Test BA1 was an initial scoping test to
investigate relative persistence on the four test materials.
• Test BA2 - Persistence on glass coupons at a mean RH of 34 % (laboratory ambient RH)
at time points from 15 minutes to 8 hours following inoculation. This test was
conducted to obtain data on the persistence on glass coupons with greater time
resolution than in Test BA1, and emphasized qualitative testing at all time points.
• Test BA3 - Persistence on four coupon materials at a mean RH of 75 % after a one-hour
exposure time. This test was conducted for comparison to Test BA1, to investigate the
effect of higher RH on vegetative B. anthracis persistence.
• Test BA4 - Persistence on topsoil coupons at a mean RH of 46 % at six time points from
1 hour to 120 hours following inoculation. Previous tests showed the highest persistence
15
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of vegetative B. anthracis on topsoil, therefore this test focused on that test material with
its normal moisture content of approximately 30 % by weight.
• Test BA5 - Persistence on wood, concrete, and glass coupons at a mean RH of 92 % at
time points of 1, 8, 12, and 24 hours after inoculation. This test was conducted to assess
the effect of high RH on persistence on these three materials before focusing exclusively
on topsoil as the test material in the remaining tests.
• Test BA6 - Persistence on wetted topsoil coupons at a mean RH of 96 % at time points
of 1 hour, and 7, 14, 21, and 28 days after inoculation. This test was designed to
investigate whether sporulation would occur in wetted topsoil. Each topsoil coupon was
wetted with 1 mL of SFW, resulting in wetted soil without producing a muddy
consistency.
• Test BA7 - Persistence on wetted topsoil coupons at a mean RH of 93 % at time points
of 1, 4, 8, 12, 18, 24, and 48 hours after inoculation. This test was conducted to
investigate the onset of sporulation, which was observed to have reached completion by
the seven-day time point in the previous test. Each topsoil coupon was wetted with 1
mL of SFW, resulting in wetted soil without producing a muddy consistency.
In all tests, the heat shock procedure was applied to an aliquot of the vegetative B. anthracis
suspension used to inoculate the coupons and to all extracts of test coupons. Except in the last
two tests conducted with wetted topsoil, no sporulation of the B. anthracis was observed at any
point in the testing (i.e., only vegetative B. anthracis cells were present). Also, in each of these
tests, the qualitative growth procedure described in Section 2.7 was used following the extraction
of test coupons.
4.2.1 Uniformity of Test Conditions
Table 4-1 summarizes the temperature and RH conditions that were maintained during the
persistence tests with vegetative B. anthracis. Not surprisingly, Table 4-1 shows that test
temperature and RH showed a greater range of variation in the longer test periods.
16
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Table 4-1. Temperature and Relative Humidity Conditions during Non-UV Exposure
Persistence Tests with Vegetative Bacillus anthracis
Mean T
T Range
Mean RH
RH Range
Test No.
Persistence Test
(°C)
(°C)
(%)
(%)
BA1
Four Materials/1 hour/36 % RH
21.5
21.1-21.7
36.0
36.0-37.0
BA2
Glass/eight hours/34 % RH
22.1
20.6-23.9
33.9
27.5 -45.3
BA3
Four Materials/one hour/75 % RH
21.8
21.4-22.2
75.4
73.7-76.4
BA4
Topsoil/120 hours/46 % RH
21.1
20.2-27.3
46.4
34.3 -58.4
BA5
Three Materials/24 hours/92 % RH
21.1
20.9-22.3
92.4
92.0-92.6
BA6
Wet Topsoil/28 days/96 % RH(a)
21.0
20.2-22.1
95.7
89.4 -96.8
BA7
Wet Topsoil/48 hours/93 % RH(a)
20.9
20.6-22.0
92.9
91.6-93.2
(a) Each topsoil coupon wetted with 1 mL of SFW at start of test.
4.2.2 Results of Persistence Tests with Vegetative Bacillus anthracis
Tables 4-2 through 4-8 show the test results from the non-UV persistence testing with vegetative
B. anthracis in Tests BA1 through BA7, respectively. Each of these tables shows the vegetative
B. anthracis inoculum onto each coupon, the mean log (± SD) of the recovered CFU, the mean
(± SD) of the CFU percent recovery, the mean log reduction (± CI) relative to the inoculum, and
the subsequent qualitative growth test results. Note that the format of these tables differs
somewhat, in that Tables 4-2 and 4-4 show results with all four coupon materials at a single time
point of one hour, Tables 4-3, 4-5, 4-7, and 4-8 show results from a single coupon material at
multiple time points, and Table 4-6 shows results from three coupon materials at multiple time
points. Also, Tables 4-7 and 4-8 include results from enumeration of aliquots of coupon extracts
that were subjected to the heat shock treatment described in Section 2.5.
Table 4-2 shows results after one hour of exposure of the vegetative B. anthracis inoculum at a
mean RH of 36 % RH and room temperature in Test BA1. No viable cells were recovered from
glass, wood, or concrete in the quantitative testing, and the absence of viable cells on those
materials was confirmed by the qualitative testing. Approximately 5 % of the vegetative B.
anthracis in the inoculum survived the one-hour exposure time on topsoil in this test, and that
result was confirmed by the qualitative results.
17
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Table 4-2. Persistence of Vegetative Bacillus anthracis(A)
(Four Materials, One Hour, 36 % RH; Test BA1)
Quantitative Testing
Test Material
Inoculum
(CFU)
Mean of Logs
of Observed
CFU
Mean %
Recovery
Mean Log
Reduction
±CI
Qualitative
Growth(b)
One Seven
day days
Topsoil
Test Coupons 1.21 * 106
Procedural Blank 'c;' 0
Glass
Test Coupons 1.21 * 106
Procedural Blank 0
Bare Pine Wood
Test Coupons 1.21 * 106
Procedural Blank 0
Unpainted Concrete
Test Coupons 1.21 * 106
Procedural Blank 0
4.79 ±0.10
0
0
0
0
0
0
0
5.2 ± 1.3
0
0
0
0
0
0
0
1.30 ±0.09
6.08
6.08
6.08
Y
N
N
N
N
N
N
(a)
Data are expressed as mean (± SD) of the logs of total number of cells (CFU) observed on individual coupons,
percent recovery (± SD), and mean log reduction (± CI).
Growth in nutrient medium at one day and seven days after coupon extraction. Y indicates growth visually
observed with at least one of five coupon extracts, Y indicates growth confirmed to be B. anthracis with at least
one of five coupon extracts, N indicates no growth (complete inactivation).
Procedural Blank = Not inoculated, placed with test coupons during persistence test.
CI = Confidence interval (± 1.96 x SE).
Not Applicable.
(b)
(c)
Table 4-3 shows the results of Test BA2 with vegetative B. anthracis on glass coupons. In that
test, coupons were removed after exposure for 15, 30, and 45 minutes and for 1, 2, 4, 6, and 8
hours after inoculation. Thus in this test, three sets of five coupons each were removed and
extracted before one hour had elapsed (i.e., at 15, 30, and 45 minutes after inoculation). All
coupon extracts were subjected to qualitative growth testing, but only extracts of coupons removed
at one hour after inoculation were also quantitatively enumerated. Table 4-3 shows that after one
hour of exposure, no viable cells were found in the quantitative testing. However, the seven-day
qualitative growth testing showed the presence of viable B. anthracis at all drying times from 15
minutes through 6 hours after inoculation. Qualitative testing of coupon extracts from the eight-
hour exposure showed no growth. These results indicate persistence of vegetative B. anthracis
for at least six hours, but less than eight hours, on glass coupons at the conditions of Test BA2.
While the quantitative persistence results for glass at 1 hour for Tests BA1 and BA2 are the same
(no spores detected), the qualitative results for glass for the two tests are somewhat different (no
persistence at 1 hour vs. persistence at 6 hours, respectively). This difference in qualitative
results for the two tests may be attributed to slight differences in experimental procedures or
conditions that may occur when performing separate experiments.
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Table 4-3. Persistence of Vegetative Bacillus anthracis^ on Glass
(Eight Hours, 34 % RH;Test BA2)
Time Point
Quantitative Testing
Inoculum
(CFU)
Mean of Logs
of Observed
CFU
Mean %
Recovery
Mean Log
Reduction
±CI
Qualitative
Growth(b)
One Seven
day days
15 Minutes
Test Coupons 9.43 x 105
Procedural Blank(c) 0
30 Minutes
Test Coupons 9.43 x 105
Procedural Blank 0
45 Minutes
Test Coupons 9.43 x 105
Procedural Blank 0
1 Hour
Test Coupons 9.43 x 105
Procedural Blank 0
2 Hours
Test Coupons 9.43 x 105
Procedural Blank 0
4 Hours
Test Coupons 9.43 x 105
Procedural Blank 0
6 Hours
Test Coupons 9.43 x 105
Procedural Blank 0
8 Hours
Test Coupons 9.43 x 105
Procedural Blank 0
0
0
0.00
0
5.97
Y
Y
N
N
N
N
N
N
N
(a)
Data are expressed as mean (± SD) of the logs of total number of cells (CFU) observed on individual coupons,
percent recovery (± SD), and mean log reduction (± CI).
Growth in nutrient medium at one day and seven days after coupon extraction. Y indicates growth visually
observed with at least one of five coupon extracts, Y indicates growth confirmed to be B. anthracis with at least
one of five coupon extracts, N indicates no growth (complete inactivation).
Procedural Blank = Not inoculated, placed with test coupons during persistence test.
CI = Confidence interval (± 1.96 x SE).
Not Applicable.
(b)
(c)
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Table 4-4 shows the results from one hour of exposure of the vegetative B. anthracis inoculum
on all four materials at 75 % RH in Test BA3. The quantitative testing showed that
approximately 10 % of the vegetative B. anthracis in the inoculum survived the one-hour
exposure on topsoil, and approximately 2 % survived on glass. No viable cells were detected in
the quantitative testing on wood and concrete coupons, but the qualitative testing showed the
presence of viable cells on those materials. These results show that vegetative B. anthracis
persisted on all four materials for at least one hour under 75 % RH conditions. Comparison of
the quantitative results from Tests BA1 and BA3 (Tables 4-2 and 4-4) shows a significant
increase in persistence at 75 % RH relative to 36 % RH on topsoil (based on comparison of 95 %
CIs) and a significant increase on glass. The qualitative results suggest greater persistence on
wood, glass, and concrete at the higher RH.
Table 4-4. Persistence of Vegetative Bacillus anthracis^
(Four Materials, One Hour, 75 % RH; Test BA3)
Quantitative Testing
Test Material
Inoculum
(CFU)
Mean of Logs
of Observed
CFU
Mean %
Recovery
Mean Log
Reduction
±CI
Qualitative
Growth®
One
day
Seven
days
Topsoil
Test Coupons 7.87 x 105
Procedural Blank(c) 0
Glass
Test Coupons 7.87 x 105
Procedural Blank 0
Bare Pine Wood
Test Coupons 7.87 x 105
Procedural Blank 0
Unpainted Concrete
Test Coupons 7.87 x 105
Procedural Blank 0
4.88 ± 0.20 10.4 ±4.1 1.01 ±0.17
0 0
4.15 ±0.05 1.8 ±0.24 1.74 ±0.05
0 0
0
0
0
0
5.90
5.90
Y
Y
N
N
(a)
Data are expressed as mean (± SD) of the logs of total number of cells (CFU) observed on individual coupons,
percent recovery (± SD), and mean log reduction (± CI).
Growth in nutrient medium at one day and seven days after coupon extraction. Y indicates growth visually
observed with at least one of five coupon extracts, Y indicates growth confirmed to be B. anthracis with at least
one of five coupon extracts, N indicates no growth (complete inactivation).
Procedural Blank = Not inoculated, placed with test coupons during persistence test.
CI = Confidence interval (± 1.96 x SE).
Not Applicable.
(b)
(c)
Because of the much greater persistence of vegetative B. anthracis on topsoil than on the other
coupon materials, most subsequent testing focused on that material. Table 4-5 shows the results of
Test BA4, a 120-hour test with vegetative B. anthracis on topsoil coupons, in which coupons were
removed and extracted at 1, 24, 48, 72, 96, and 120 hours after inoculation. The quantitative test
20
-------
results in Table 4-5 show that vegetative B. anthracis persisted on topsoil coupons at least 96
hours after inoculation, but no viable cells could be detected in the quantitative testing at 120
hours after inoculation. These quantitative test results were confirmed by the qualitative results.
Also, Table 4-5 shows that approximately 9 % of vegetative B. anthracis inoculated on topsoil
coupons were viable at one hour after inoculation, a result that is consistent with the results at
one hour shown in Tables 4-2 and 4-4.
Table 4-5. Persistence of Vegetative Bacillus anthracis^ on Topsoil
(120 Hours, 46 % RH; Test BA4)
Time Point
Quantitative Testing
Qualitative
Growth®
Inoculum
(CFU)
Mean of Logs
of Observed
CFU
Mean %
Recovery
Mean Log
Reduction
±CI
One
day
Seven
days
1 Hour
Test Coupons 8.23 x 105
Procedural Blank(c) 0
24 Hours
Test Coupons 8.23 x 105
Procedural Blank 0
48 Hours
Test Coupons 8.23 x 105
Procedural Blank 0
72 Hours
Test Coupons 8.23 x 105
Laboratory Blank 0
96 Hours
Test Coupons 8.23 x 105
Procedural Blank 0
120 Hours
Test Coupons 8.23 x 105
Procedural Blank 0
4.84 ± 0.15 8.9 ± 3.4 1.08 ±0.15
0 0
2.01 ±0.42 0.018 ±0.015 3.91 ± 0.37
0 0
0.90 ±1.3 0.011 ± 0.017 5.01 ±1.10
0 0
0.67 ± 0.92 0.0025 ±0.0036 5.25 ±0.81
0 0
0.30 ± 0.6 8 0.00091 ±o.ooi8 5.61 ±0.60
0 0
0
0
0
0
5.92
Y
Y
Y
Y
Y
N
N
(a) Data are expressed as mean (± SD) of the logs of total number of cells (CFU) observed on individual coupons,
percent recovery (± SD), and mean log reduction (± CI).
(b) Growth in nutrient medium at one day and seven days after coupon extraction. Y indicates growth visually
observed with at least one of five coupon extracts, Y indicates growth confirmed to be B. anthracis with at least
one of five coupon extracts, N indicates no growth (complete inactivation).
(c) Procedural Blank = Not inoculated, placed with test coupons during persistence test.
CI = Confidence interval (± 1.96 x SE).
Not Applicable.
Figure 4-1 shows the persistence curve of vegetative B. anthracis on topsoil coupons in the 120-
hour Test BA4 summarized in Table 4-5. This figure shows the log of the number of CFU
recovered at each time point in that test, along with error bars showing the 95 % CI of the
recovery results. Figure 4-1 illustrates that much of the loss of vegetative cells in topsoil occurs
in the first 24 hours after inoculation (i.e., less than 0.02 % of the inoculated cells are viable at
the 24-hour time point). As stated in Section 2.6, a heat shock procedure was applied to aliquots
21
-------
of all coupon extracts to distinguish vegetative B. anthracis cells from spores. In Test BA4, the
heat shock procedure confirmed at every time point that the organisms present were vegetative
cells and not spores. Thus, Figure 4-1 and the heat shock results show that the vegetative B.
anthracis either persisted or died off, but did not sporulate in topsoil under the conditions of this
test.
T3
a>
a>
>
o
u
a>
DC
-------
Table 4-6. Persistence of Vegetative Bacillus anthracis^ on Three Materials
(24 Hours, 92 % RH; Test BA5)
Quantitative Testing
Qualitative
Growth®
Mean of Logs
Mean Log
Inoculum
of Observed
Mean %
Reduction
One
Seven
Time Point
(CFU)
CFU
Recovery
±CI
day
days
1 Hour
Test Coupons
Glass
1.43 x
106
5.95 ±0.09
63.0 ± 12.5
0.21 ±0.08
Y
Y
Wood
1.43 x
106
0
0
6.16
N
N
Concrete
1.43 x
106
0.79 ± 1.09
0.0028 ±0.0041
5.37 ±0.95
Y
Y
Laboratory Blank(c)
0
0
0
--
Procedural Blank(d)
0
0
0
—
8 Hours
Test Coupons
Glass
1.43 x
106
1.28 ±2.11
1.05 ±2.34
4.88 ± 1.85
N
Y
Wood
1.43 x
106
0
0
6.16
N
N
Concrete
1.43 x
106
0
0
6.16
N
N
Laboratory Blank
0
0
0
--
Procedural Blank
0
0
0
--
12 Hours
Test Coupons
Glass
1.43 x
106
2.64 ±0.43
0.045 ± 0.047
3.52 ±0.38
N
Y
Wood
1.43 x
106
0
0
6.16
N
N
Concrete
1.43 x
106
0
0
6.16
N
N
Laboratory Blank
0
0
0
--
Procedural Blank
0
0
0
--
24 Hours
Test Coupons
Glass
1.43 x
106
0
0
6.16
N
N
Wood
1.43 x
106
0
0
6.16
N
N
Concrete
1.43 x
106
0
0
6.16
N
N
Laboratory Blank
0
0
0
--
Procedural Blank
0
0
0
--
(a) Data are expressed as mean (± SD) of the logs of total number of cells (CFU) observed on individual coupons,
percent recovery (± SD), and mean log reduction (± CI).
(b) Growth in nutrient medium at one day and seven days after coupon extraction. Y indicates growth visually
observed with at least one of five coupon extracts, Y indicates growth confirmed to be B. anthracis with at least
one of five coupon extracts, N indicates no growth (complete inactivation).
(c) Laboratory Blank = Not inoculated.
(d) Procedural Blank = Not inoculated, placed with test coupons during persistence test.
CI = Confidence interval (± 1.96 x SE).
Not Applicable.
23
-------
The data in Table 4-6 are consistent with a positive effect of higher RH on the persistence of
vegetative B. cmthrcicis on glass, as suggested by the data in Tables 4-2 to 4-4. The results
supporting this RH effect are shown in Figure 4-2, which shows the log of the CFU of vegetative
B. cmthracis recovered after one hour of exposure on glass at RH from 36 % to 92 %. At 36 %
RH, no viable cells were recovered after one hour, but at the higher RH levels, recovery
increased with higher RH. The 95 % CI values are shown as error bars in Figure 4-2, and clearly
illustrate that the persistence on glass at 92 % was significantly greater than at 75 % RH, and that
persistence at both 75 % and 92 % RH was significantly greater than persistence at 36 % RH.
7
Zero Recovery
36 75 92
Percent Relative Humidity
Figure 4-2. Recovery of Vegetative Bacillus anthracis at One Hour
on Glass Increases with RH
Table 4-7 shows the results of persistence testing of vegetative B. anthracis on topsoil coupons
over a 28-day period in Test BA6. In this test, the topsoil coupons were each wetted with 1 mL
of SFW, and the RH in the test chamber was maintained above 95 % throughout the 28-day test
duration. This test was conducted to assess the effect of elevated RH as well as elevated soil
moisture on persistence of vegetative B. anthracis. Coupons were extracted for enumeration at
one hour, and then at 7, 14, 21, and 28 days after inoculation, and also subjected to qualitative
growth assessment at one and seven days after extraction. Table 4-7 shows that in contrast to all
other tests described above, growth and sporulation of B. anthracis occurred in the wetted topsoil
coupons, with the result that the total organism counts in the topsoil coupons increased by more
than an order of magnitude within the first week of the test. Consequently, the recovery values
at all time points longer than one hour in Table 4-7 greatly exceeded 100 % recovery.
24
-------
Table 4-7. Persistence of Vegetative Bacillus anthracis^ on Wetted Topsoil
(28 Days, 96 % RH; Test BA6)
Time Point
Quantitative Testing
Inoculum
(CFU)
Mean of Logs
of Observed
CFU
Mean %
Recovery
Mean Log
Reduction
±CI
Qualitative
Growth(b)
One Seven
day days
1 Hour
Test Coupons
Test Coupons HS(c)
Procedural Blank(d)
7 Days
Test Coupons
Test Coupons HS
Procedural Blank
14 Days
Test Coupons
Test Coupons HS
Procedural Blank
21 Days
Test Coupons
Test Coupons HS
Procedural Blank
28 Days
Test Coupons
Test Coupons HS
Procedural Blank
2.90 x 106
2.90 x 106
0
2.90 x 106
2.90 x 106
0
2.90 x 106
2.90 x 106
0
2.90 x 106
2.90 x 106
0
2.90 x 106
2.90 x 106
0
6.32 ±0.05
0
0
7.37 ±0.01
7.36 ±0.01
0
7.55 ±0.37
7.61 ±0.45
0
7.48 ±0.49
7.72 ±0.39
0
7.21 ±0.69
7.20 ±0.72
0
72.7 ±8.5
0
0
811 ±21
794 ± 19
0
1586 ± 1103
1957± 1406
0
1597± 1400
2290± 1386
0
1110 ±1015
1126 ±1093
0
0.14 ±0.04
6.46
-0.91 ±0.01
-0.90 ±0.01
-1.09 ±0.32
-1.14 ±0.39
-1.02 ±0.43
-1.25 ±0.34
-0.75 ±61
-0.73 ± 63
Y
Y
Y
Y
Y
(a)
Data are expressed as mean (± SD) of the logs of total number of cells (CFU) observed on individual coupons,
percent recovery (± SD), and mean log reduction (± CI).
Growth in nutrient medium at one day and seven days after coupon extraction. Y indicates growth visually
observed with at least one of five coupon extracts, Y indicates growth confirmed to be B. anthracis with at least
one of five coupon extracts, N indicates no growth (complete inactivation).
HS = heat shocked. Each extract of a test coupon is split, and one half is heat shocked prior to enumeration to
inactivate any vegetative B. anthracis cells and leave only spores.
Procedural Blank = Not inoculated, placed with test coupons during persistence test.
CI = Confidence interval (± 1.96 x SE).
Not Applicable.
(b)
(c)
(d)
Table 4-7 also includes the results of enumerating coupon extracts that were subjected to the heat
shock treatment. The heat shock procedure conducted on the coupon extracts at one hour after
inoculation showed no viable organisms, confirming that the cell population was still 100 %
vegetative B. anthracis at that time point. However, the heat shock procedures conducted at all
later time points showed that the organisms present were predominantly to entirely B. anthracis
spores. These results indicate growth and subsequent sporulation of the B. anthracis cell
population within approximately the first seven days of the test. The results of this and previous
tests suggest the impact of moisture in determining whether vegetative B. anthracis cells persist
25
-------
as cells or die out (i.e., under relatively dry conditions), or grow and sporulate (i.e., under wet
conditions as in this test).
Table 4-8 shows the results of persistence testing of vegetative B. anthracis on topsoil coupons
over a 48-hour period in Test BA7. In this test the topsoil coupons were each wetted with 1 mL
of SFW, and the RH in the test chamber was maintained at approximately 93 % throughout the
48-hour test duration. The purpose of this test was to further investigate the sporulation observed
in the previous test, by attempting to determine more precisely when the onset of sporulation
occurred in the first days after coupon inoculation. Consequently in this test, coupons were
extracted for enumeration at 1, 4, 8, 12, 18, 24, and 48 hours after inoculation, and also subjected
to qualitative growth assessment at one and seven days after extraction. The heat shock
procedure conducted on the coupon extracts showed no viable organisms through the 18-hour
time point, confirming that the initial inoculation and subsequent B. anthracis populations were
100 %vegetative until at least the 18-hour time point. However, Table 4-8 shows that growth of
B. anthracis occurred in the wetted topsoil coupons within approximately eight hours after
inoculation, and the onset of sporulation was observed within 24 hours after inoculation (as
indicated by the heat shocked results). The total B. anthracis population in the topsoil coupons
increased by about a factor of two relative to the inoculated amount within the 48-hour duration
of the test, and more than 10 % of the B. anthracis organisms were in spore form at the end of
the 48-hour time point.
The results of the 48-hour test with B. anthracis on wetted topsoil are illustrated in Figure 4-3,
which shows the total number of recovered B. anthracis CFU (vegetative cells plus spores) and
the number of only spores on the soil coupons at each time point of this test. Until the 24-hour
time point, no spores were present, consistent with the purely vegetative nature of the
inoculation. However, at the 24- and 48-hour time points, the sporulation of the B. anthracis is
clearly evident. The results of this test are consistent with those of the previous 28-day test with
B. anthracis and may show the impact of moisture in determining whether vegetative B.
anthracis cells die out or grow and sporulate.
26
-------
Table 4-8. Persistence of Vegetative Bacillus anthracis^ on Wetted Topsoil
(48 Hours, 93 % RH; Test BA7)
Time Point
Quantitative Testing
Inoculum
(CFU)
Mean of
Logs of
Observed
CFU
Mean %
Recovery
Mean Log
Reduction
±CI
Qualitative
Growth®
One
day
Seven
days
1 Hour
Test Coupons 2.27 x 106 6.10 ± 0.07 56.5 ± 9.3
Test Coupons HS(c) 2.27 x 106 0 0
Procedural Blank(d) 0 0 0
4 Hours
Test Coupons 2.27 x 106 6.36 ±0.03 101 ±6.5
Test Coupons HS 2.27 x 106 0 0
Procedural Blank 0 0 0
8 Hours
Test Coupons 2.27 x 106 6.59 ±0.10 177 ±41.9
Test Coupons HS 2.27 x 106 0 0
Procedural Blank 0 0 0
12 Hours
Test Coupons 2.27 x 106 6.65 ±0.05 198 ±23.7
Test Coupons HS 2.27 x 106 0 0
Procedural Blank 0 0 0
18 Hours
Test Coupons 2.27 x 106 6.61 ±0.05 182 ± 19.3
Test Coupons HS 2.27 x 106 0 0
Procedural Blank 0 0 0
24 Hours
Test Coupons 2.27 x 106 6.71 ±0.07 230 ±41.9
Test Coupons HS 2.27 x 106 3.07 ±1.81 0.48 ±0.69
Procedural Blank 0 0 0
48 Hours
Test Coupons 2.27 x 106 6.66 ±0.05 202 ±21.9
Test Coupons HS 2.27 x 106 5.72 ±0.22 25.9 ±14.1
Procedural Blank 0 0 0
0.25 ±0.06
6.36
0.00 ±0.03
6.36
-0.24 ± 0.09
6.36
-0.29 ± 0.05
6.36
-0.26 ± 0.04
6.36
-0.36 ±0.06
3.28 ± 1.58
-0.30 ±0.04
0.63 ±0.19
Y
Y
Y
Y
Y
Y
Y
(a)
Data are expressed as mean (± SD) of the logs of total number of cells (CFU) observed on individual coupons,
percent recovery (± SD), and mean log reduction (± CI).
Growth in nutrient medium at one day and seven days after coupon extraction. Y indicates growth visually
observed with at least one of five coupon extracts, Y indicates growth confirmed to be B. anthracis with at least
one of five coupon extracts, N indicates no growth (complete inactivation).
HS = heat shocked. Each extract of a test coupon is split, and one half is heat shocked prior to enumeration to
inactivate any vegetative B. anthracis cells and leave only spores.
Procedural Blank = Not inoculated, placed with test coupons during persistence test.
CI = Confidence interval (± 1.96 x SE).
Not Applicable.
(b)
(c)
(d)
27
-------
1.0E+07
1.0E+06
-~-Total CFU
-O- Spores
>
8 1.0E+05
-------
4.3.1 Uniformity of Test Conditions
Tables 4-9 and 4-10 summarize the test conditions of UV intensity, temperature, and RH
monitored during the UV exposure testing with B. anthracis. Table 4-9 shows the average
(± SD) intensities (in (j,W/cm2) of UV-A, UV-B, and UVA/B measured at five positions in the
UV-exposed coupon arrays in each of the two 120-hour UV-A/B exposure tests with
B. anthracis. The intensity of UV-C at both the UV-exposed and non-exposed coupons was
below detection (i.e., < 1 [j,W/cm ) in all tests, and the intensities of UV-B and UV-A/B at the
non-exposed coupons were also below detection in all tests.
Table 4-10 shows the average (± SD) of the temperature and RH monitored near the center of the
UV-exposed coupon array and the non-exposed coupon array in the two 120-hour UV exposure
tests with vegetative B. anthracis. Table 4-10 shows data both with the UV lights on and the
lights off, and shows consistency in the test conditions. When the UV lights were off, the UV-
exposed and non-exposed coupons experienced closely similar temperature conditions (i.e.,
within about 0.7 °C). When the lights were on, the UV-exposed coupons experienced slightly
higher temperatures than did the non-exposed coupons, by 1.6 °C with a low UV-A/UV-B ratio,
and by 4.4 °C with a high UV-A/UV-B ratio (and overall much higher UV intensity). In both
UV tests, the RH at the non-exposed coupons was higher than that at the UV-exposed coupons,
both with the lights on and lights off. The reason is not certain but is probably related to the
temperature of the coupon arrays. All the mean RH values were in the range of approximately
55 to 72 % RH, and the RH difference between UV-exposed and non-exposed coupons is not
expected to affect persistence significantly.
Table 4-9. Ultraviolet Radiation Intensity at the Topsoil Test Coupons in
Testing with Bacillus anthracis^
UV Condition(b)
UV-A/B Exposure Test
Test BAUV1: 120 Hour
Low UV-A to UV-B Ratio
Test BAUV2: 120 Hour
High UV-A to UV-B Ratio
UV-A Average ± SD
99.3 ±4.3
1,824 ±53
UV-B Average ± SD
46.9 ±3.4
44.9 ± 1.7
UV-A/B Average ± SD
146.2 ±6.6
1,869 ±54
(a) All entries are in |i\V/cm at the test coupons; all UV intensities were zero at the non-exposed coupons.
(b) Average ± SD refers to average over all five measurement positions.
SD = standard deviation.
UV-A determined by subtracting UV-B reading from UV-A/B reading.
29
-------
Table 4-10. Summary of Temperature and Relative Humidity Conditions in Simulated
Sunlight Testing with Vegetative Bacillus anthracis
Test Condition'3'
UV-A/B Ex
)osure Test
Test BAUV1:
120 Hour Low UV-A/UV-B Ratio
Test BAUV2:
120 Hour High UV-A/UV-B Ratio
Test(b)
Non-Exposed(c)
Test
Non-Exposed
UV ON
T Average ± SD (°C)
22.1 ±0.8
20.5 ±0.2
25.2 ±2.0
20.8 ±0.5
RH Average ± SD (%)
55.6 ±3.7
71.5 ±4.0
55.2 ± 10.6
63.1 ±3.2
UV OFF
T Average ± SD (°C)
19.8 ±0.7
20.5 ±0.2
20.5 ± 1.7
20.7 ±0.6
RH Average ± SD (%)
57.1 ±2.8
71.9 ± 3.6
57.8 ±5.7
64.6 ±5.0
(a) UV ON and UV OFF refer to 12-h periods of alternating illumination and darkness in test chamber; averages
shown are over all UV ON or UV OFF periods in the indicated UV exposure period. T = temperature, RH =
relative humidity,
(b) Test coupons (UV-A/B-exposed); readings taken at a central position in the coupon array.
(c) Non-exposed coupons (not exposed to UV-A/B); readings taken at a central location in the coupon array.
SD = standard deviation.
4.3.2 Persistence Results in Simulated Sunlight Tests with Vegetative Bacillus anthracis
In both Tests BAUV1 and BAUV2, topsoil coupons were extracted at time points of 1, 24, 48,
72, 96, and 120 hours, and coupon extracts were subjected to both quantitative enumeration and
qualitative growth testing. The heat shock procedure was applied to all coupon extracts and
showed that only vegetative B. anthracis cells were present at all times during these tests. Tables
showing the detailed results from Tests BAUV1 and BAUV2 are included in Appendix B of this
report.
Refer to Figure 4-4 for a comparison of trends in the persistence of vegetative B. anthracis on the
UV-exposed (Test BAUV1; low UV-A) and non-UV exposed topsoil coupons. Viable cells
were recovered from the non-UV exposed topsoil samples at 120 hours but not from the UV
exposed topsoils. In general, the quantitative persistence results in Figure 4-4 show that
vegetative B. anthracis recovery was lower at each time point for the UV-exposed topsoil
coupons than for the non-exposed coupons. However, the 95 % CIs of the UV-exposed and non-
exposed persistence results overlap at all time points except at the one-hour and 24 hour time
points. Thus, within the first 24 hours of exposure the data indicate a significant difference in
persistence due to the simulated sunlight exposure.
We note that per the qualitative results of Test BAUV1, B. anthracis vegetative cells not exposed
to UV persisted to 120 hours in soil, whereas from Test BA4, B. anthracis cells in soil (no UV
exposure) persisted up to 96 hours. This difference in results exemplifies the inherent variability
working with microorganisms, and may also be due to minor uncontrolled differences in
experimental procedures or conditions that may occur when performing separate experiments.
Thus, from these data, the persistence in soil may be best characterized as ranging from 4-5 days.
30
-------
T3
a>
o
u
-------
Non-Exposed
UV Exposed
20
40 60
Time, Hours
80
100
120
Figure 4-5. Vegetative Bacillus anthracis Persistence with Exposure to
Simulated Sunlight with a High UV-A/UV-B Ratio
Overall, the results of Tests BAUV1 and BAUV2 indicate that exposure to simulated sunlight
appears to have minor impact (more so during the first 24 hours) on the persistence of vegetative
B. anthracis on topsoil, presumably due to the shading effect of soil particles.
Comparison of Figures 4-4 and 4-5 also shows that the two persistence curves for the non-
exposed topsoil coupons in Tests BAUV1 and BAUV2 differed significantly at the one hour time
point (with the Test BAUV2 coupons showing greater persistence) and at the 24 hour and 72
hour time points (with the Test BAUV1 coupons showing greater persistence). (The two
persistence curves are compared directly in Figure B-2 in Appendix B.) The reason for these
differences in persistence on non-UV-exposed topsoil coupons is not clear. The slightly higher
RH at the non-exposed coupons in Test BAUV1 relative to Test BAUV2 (see Table 4-10) is not
likely to have been sufficient to cause the observed difference in persistence.
32
-------
5.0 RESULTS FOR Yersinia pestis
This section summarizes QC and persistence results for Y. pestis. Data are presented to
document the uniformity of the UV exposure test conditions, and persistence results with and
without UV-A/B exposure are reported for Y. pestis on the test materials.
5.1 Recovery Testing
In previous studies, recovery of Y. pestis from some coupon materials (including bare wood and
unpainted concrete) immediately after inoculation was found to be very low.2'13 Consequently,
prior to any persistence testing in this project, the recovery of Y. pestis from the coupon materials
was tested. In these tests, the Y. pestis inoculation was allowed to dry for one hour after
inoculation before extraction of the coupons and enumeration of the organisms as CFU.
Although relatively high recovery of Y. pestis from topsoil and glass coupons was observed in
initial tests, the recovery of Y. pestis from wood and concrete coupons was found to be
approximately 0.005 % and 0.001 %, respectively. These recovery values were judged to be too
low for adequate persistence testing, so initial recovery testing was repeated with galvanized
metal ductwork and painted wallboard paper in place of the wood and concrete. The results of
both the initial and further recovery tests with Y. pestis are shown in Table 5-1.
Table 5-1. Recovery Testing of Yersinia pestis^
Inoculum
Mean of Logs of
Mean %
Test Material
(CFU)
Observed CFU
Recovery
Glass
3.97 >
< 107
7.56 ±0.02
91.0 ±3.8
Topsoil
3.97 >
< 107
7.33 ±0.03
54.5 ±3.2
Bare Pine Wood
3.97 >
< 10'
3.28 0.21
0.0052 ±0.0028
Unpainted Concrete
3.97 >
< 10y
2.29 0.77
0.0013 ±0.0019
Galvanized Metal
4.13 >
< 10y
7.29 ±0.10
47.7 ± 10.9
Painted Wallboard Paper
4.13 >
< 107
4.03 ±0.54
0.042 ± 0.047
(a) Data are expressed as mean of the logs of total number of cells (CFU) inoculated onto and recovered
from individual coupons and mean percent recovery.
Table 5-1 shows that recoveries of Y. pestis from glass, topsoil, and galvanized metal were all
near or above 50 %, whereas the recovery from painted wallboard paper was much lower, at
approximately 0.04 %. Nevertheless, the recovery of Y. pestis from painted wallboard paper was
much higher than had been found with wood or concrete, and as a result, that coupon material
was used along with glass, topsoil, and galvanized metal in persistence testing of Y pestis.
5.2 QC Results
All procedural and laboratory blanks met the criterion of no observed CFU of the inoculated Y
pestis organism. Spike control samples were taken from the cell suspension on each day of
testing and serially diluted, nutrient plated, and counted to establish the cell suspension density
used to spike the coupons. This process required approximately 48 to 72 hours, so the cell
suspension density was known after completion of each day's testing. Y pestis suspensions of
33
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8 9
approximately 3x10 to 2x10 CFU/mL were produced, leading to actual spike values that
7 8
ranged from approximately 3 x 10 CFU/coupon to 2 x 10 CFU/coupon.
5.3 Yersinia pestis Persistence without UV Exposure
Four persistence tests were conducted with Y. pestis without exposure to UV-A/B radiation. All
those tests were conducted at normal laboratory temperatures. As noted in Section 2.4, the
conditions for tests were chosen based on reviewing the results of previous tests. Those four
tests and the rationale for the test conditions in each test are listed below in chronological order.
• Test YP1 - Persistence on four coupon materials at a mean RH of 57 % RH, at time
points of one hour and 14 days. This test was an initial scoping test to investigate
relative persistence on the four test materials.
• Test YP2 - Persistence on four coupon materials at a mean RH of 54 % RH, at time
points of one hour and seven days. The lack of recovery of Y. pestis at 14 days in Test
YP1 was the motivation for this shorter test on all four materials.
• Test YP3 - Persistence on four coupon materials at a mean RH of 65 %, at time points of
1, 24, 48, 96, and 144 hours. The absence of persistence of Y. pestis at seven days in
Test YP2 was the motivation for this shorter test on all four materials with multiple time
points after inoculation.
• Test YP4 - Persistence on wetted topsoil coupons at a mean RH of 96 %, at time points
of one hour, seven days, and 14 days. This test was planned for 28 days but was
truncated after 14 days due to inactivation of the Y. pestis. This test focused on topsoil
as the coupon material because of the much greater persistence observed in Test YP3 on
that material relative to the other materials. The test conditions were chosen to assess
persistence of Y pestis under conditions where drying of the organism after inoculation
was not a concern. Each topsoil coupon was wetted with 1 mL of sterile filtered water,
resulting in wetted soil without producing a muddy consistency.
The qualitative growth procedure described in Section 2.7 was used following the extraction of
test coupons in Tests YP3 and YP4 but not in Tests YP1 or YP2.
5.3.1 Uniformity of Test Conditions
Table 5-2 summarizes the temperature and RH conditions that were maintained during the non-
UV persistence tests with Y. pestis.
34
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Table 5-2. Temperature and Relative Humidity Conditions during Non-UV Persistence
Tests with Yersinia pestis
Mean T
T Range
Mean RH
RH Range
Test No.
Persistence Test
(°C)
(°C)
(%)
(%)
YP1
Four Materials/50% RH/14 Days
20.7
20.0-21.4
57.0
44.7-65.1
YP2
Four Materials/50 % RH/7 Days
20.7
20.3-21.2
53.6
45.4-56.5
YP3
Four Materials/65 % RH/144 hours
20.8
20.3-23.5
64.6
52.5-72.6
YP4
Wet Topsoil/90 % RH/28 Days(a)
21.0
20.5-21.5
96.0
91.6-96.5
(a) Each topsoil coupon wetted with 1 mL of SFW at start of test.
5.3.2 Results in Persistence Tests with Yersinia pestis
Tests YP1 and YP2 both showed recovery of Y pestis at one hour after inoculation on all four
test materials, with the greatest recovery on topsoil. However, Y pestis was not recovered at the
14-day time point in Test YP1 or at the seven-day time point in Test YP2. Data tables for those
two tests are shown in Appendix C.
Table 5-3 shows persistence results (Test YP3) for Y. pestis on the four coupon materials at 65 %
RH at several time points up to 144 hours (six days) after inoculation. The quantitative test
results indicated that Y. pestis was recovered on all four materials at the one-hour time point.
Recovery at that time point was greatest on topsoil, with approximately 36 % recovered, while
15 % of the cells were recovered on galvanized metal. However, at the 24-hour time point, no Y
pestis was recovered on glass, galvanized metal, or painted wallboard paper. In contrast, Y.
pestis persisted on topsoil at all time points including the 144 hour time point. The qualitative
results from Test YP3 were consistent with the quantitative results, with one exception: the
qualitative growth testing did not confirm the presence of Y pestis on the painted wallboard
paper at the one-hour time point. One possible explanation is the inhibition of growth by some
compound extracted from the wallboard paper. However, this hypothesis was not explored in this
study.
In Table 5-4, the persistence results (Test YP4) are presented for Y pestis on wetted topsoil at
one hour, seven days and 14 days after inoculation. The quantitative test results in Table 5-4
show the agent persists at seven days after inoculation. However, at 14 days after inoculation,
the Y pestis was completely inactivated. These quantitative results were confirmed by the
corresponding qualitative growth tests. The results of Tests YP1 through YP4 indicate that Y
pestis can persist for at least one hour, but less than 24 hours, on glass, galvanized metal, and
painted wallboard paper under normal temperature and RH conditions but can persist for several
days in topsoil. Y. pestis persists for up to six days in topsoil of normal moisture content but
persists for between one and two weeks on wetted topsoil that remains wet due to high RH.
35
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Table 5-3. Persistence of Yersinia pestis^ (Four Materials, 144 Hours, 65 % RH;
Test YP3)
Quantitative Testing
Qualitative
Growth(b)
Test Material
Inoculum
(CFU)
Mean of
Logs of
Observed
CFU
Mean %
Recovery
Mean Log
Reduction
±CI
One
day
Seve
day
Topsoil
Test Coupons 1 Hour
2.70 x
107
6.98 ±0.07
35.7 ±6.4
0.45 ± 0.07
N
Y
Test Coupons 24 Hour
2.70 x
107
3.00 ±0.75
0.0084 ±0.01
4.43 ± 0.66
N
Y
Test Coupons 48 Hour
2.70 x
107
1.72 ± 1.04
0.00059 ±0.00063
5.71 ±0.91
N
Y
Test Coupons 96 Hour
2.70 x
107
1.40 ±0.87
0.00027 ± 0.00040
6.03 ± 0.77
N
Y
Test Coupons 144 Hour
2.70 x
107
1.07 ± 1.02
0.00020 ±0.00031
6.36 ±0.90
N
Y
Laboratory Blank(c)
0
0
0
-
Procedural Blank(d)
0
0
0
-
Glass
Test Coupons 1 Hour
2.70 x
107
4.55 ±0.09
0.13 ±0.029
2.88 ±0.08
N
Y
Test Coupons 24 Hour
2.70 x
107
0
0
7.43
N
N
Laboratory Blank
0
0
0
-
Procedural Blank
0
0
0
-
Galvanized Metal
Test Coupons 1 Hour 2.70 x 107 6.34 ±0.69 15.0 ±14.1 1.09 ±0.61 N Y
Test Coupons 24 Hour 2.70 x 107 0 0 7.43 ^ ^
Laboratory Blank 0 0 0 -
Procedural Blank 0 0 0 -
Painted Wallboard
Paper
Test Coupons 1 Hour 2.70 x 107 3.46 ±0.61 0.028 ±0.047 3.97 ±0.69 N N
Test Coupons 24 Hour 2.70 * 107 0 0 7.43 N N
Laboratory Blank 0 0 0 -
Procedural Blank 0 0 0 -
(a) Data are expressed as mean (± SD) of the logs of total number of cells (CFU) observed on individual coupons,
percent recovery (± SD), and mean log reduction (± CI).
(b) Growth in nutrient medium at one day and seven days after coupon extraction. Y indicates growth visually
observed with at least one of five coupon extracts, Y indicates growth confirmed to be Y. pestis with at least one
of five coupon extracts, N indicates no growth (complete inactivation).
(c) Laboratory Blank = Not inoculated.
(d) Procedural Blank = Not inoculated, placed with test coupons during persistence test.
CI = Confidence interval (± 1.96 x SE).
Not Applicable
36
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Table 5-4. Persistence of Yersinia pestis^ (Wetted Topsoil, 14 Days, 96 % RH; Test YP4)
Time Point
Quantitative Testing
Inoculum
(CFU)
Mean of Logs
of Observed
CFU
Mean %
Recovery
Mean Log
Reduction
±CI
Qualitative
Growth(b)
One Seven
day days
1 Hour
Test Coupons
Laboratory Blank(c)
Procedural Blank(d)
7 Days
Test Coupons
Laboratory Blank
Procedural Blank
14 Days
Test Coupons
Laboratory Blank
Procedural Blank
5.90 x 10'
0
0
5.90 x 107
0
0
5.90 x 107
0
0
7.78 ±0.25
0
0
4.44 ±0.65
0
0
0
0
0
115 ± 51
0
0
0.12 ±0.20
0
0
0
0
0
-0.01 ±0.22
3.33 ±0.64
7.78
Y
N
N
N
(a)
Data are expressed as mean (± SD) of the logs of total number of cells (CFU) observed on individual coupons,
percent recovery (± SD), and mean log reduction (± CI).
Growth in nutrient medium at one day and seven days after coupon extraction. Y indicates growth visually
observed with at least one of five coupon extracts, Y indicates growth visually confirmed to be Y. pestis with at
least one of five coupon extracts, N indicates no growth (complete inactivation).
Laboratory Blank = Not inoculated.
Procedural Blank = Not inoculated, placed with test coupons during persistence test.
CI = Confidence interval (± 1.96 x SE).
Not Applicable.
(b)
(c)
(d)
5.4 Persistence of Yersinia pestis under Exposure to Simulated Sunlight
A single test was conducted to assess the effect of exposure of Y. pestis to UV-A/B radiation.
That test (TestYPUVl) evaluated the recovery of Y. pestis on four coupon materials over 24
hours with a low UV-A/UV-B ratio. In this test, the persistence of Y. pestis on the four coupon
materials was determined after the one hour drying time and after 24 hours of UV exposure of
the coupons. The UV radiation intensity consisted of nominally 100 |iW/cm2 UV-A and 44
|iW/cm UV-B. This test was performed at ambient laboratory temperature and RH
(approximately 40 % RH). As noted in Section 2.3, no non-exposed coupons were used in this
UV test.
5.4.1 Uniformity of Test Conditions
Tables 5-5 and 5-6 summarize the test conditions of UV intensity, temperature, and RH
monitored during the UV exposure test with Y pestis. Table 5-5 shows the average (± SD) of
intensities (in [j,W/cm ) of UV-B, UV-A, and UVA/B measured at five positions in the test
37
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coupon array in the UV-A/B exposure test with Y pestis. The intensity of UV-C was below
detection (i.e., < 1 [j,W/cm ). The data in Table 5-5 show that the consistency of the average UV-
B, UV-A, and UV-A/B intensities was maintained in this test with Y pestis, though the radiation
intensity was not as uniform as in the UV testing with B. anthracis (see Section 4.3).
Table 5-6 shows the average (± SD) of the temperature and RH monitored near the center of the
exposed coupon array in the UV exposure test with Y. pestis. Table 5-6 shows data both with the
UV lights on and the lights off, and shows consistency in the test conditions, i.e., temperature
and RH values when the UV lamps were on were closely similar to the values when the UV
lamps were off.
Table 5-5. Ultraviolet Radiation Intensity at the Exposed Coupons in
Test YPUV1 with Yersinia pestis^
UV Condition®
UV Intensity
UV-A Average ± SD
88.2 ± 10.7
UV-B Average ± SD
39.7 ±3.5
UV-A/B Average ± SD
127.9 ± 13.2
(a) All entries are in (iW/cm at the test coupons; all UV intensities were zero at
the non-exposed coupons.
(b) Average ± SD refers to average over all five measurement positions.
SD = standard deviation. UV-A determined by subtraction of UV-B reading
from UV-A/B reading
Table 5-6. Summary of Temperature and Relative Humidity Conditions at the
Exposed Coupons in Test YPUV1 with Yersinia pestis
Test Condition(a)
Result
UV ON
T Average ± SD (°C)
22.3 ±0.5
RH Average ± SD (%)
41.4 ± 1.1
UV OFF
T Average ± SD (°C)
20.1 ±0.6
RH Average ± SD (%)
42.4 ± 1.4
UV ON and UV OFF refer to 12-hour periods of alternating
illumination and darkness in test chamber; averages shown are
over all UV ON or UV OFF periods in the indicated UV exposure
period.
T = temperature, RH = relative humidity,
SD = standard deviation.
5.4.2 Persistence Results in Simulated Sunlight Test with Yersinia pestis
Table 5-7 shows results from Test YPUV1 in terms of the cell inoculum; the mean log of the
observed cells on the exposed and blank coupons; the cell recovery; and the resulting mean log
reduction (±CI) on the exposed coupons relative to the initial inoculum due to the UV-A/B
38
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exposure. In this test, coupons were extracted at time points of one hour after the initial drying
period and after 24 hours (12 on, 12 off) of UV exposure. Extracted coupons were not subjected
to qualitative growth testing after extraction.
Table 5-7 shows that Y. pestis did not persist on galvanized metal at the one-hour time point. Y.
pestis did persist to that point on the other three materials, with the greatest recovery on glass and
the lowest quantity recovered on painted wallboard paper. Under simulated sunlight exposure, Y.
pestis did not persist at the 24-hour time point on any of the four materials. This result differs
from the observed persistence of Y pestis on topsoil out to at least six days in the absence of UV
exposure (Table 5-3).
Table 5-7. UV-A/B Inactivation of Yersinia pestis on Four Coupon Materials^
(24-Hour Exposure at Low UV-A/UV-B Ratio; Test YPUV1)
Test Material
Quantitative Testing
Inoculum
(CFU)
Mean of Logs of
Observed CFU
Mean %
Recovery
Mean Log
Reduction
±CI
Topsoil
Exposed Coupons 1 Hour
Exposed Coupons 24 Hour
Laboratory Blank(b)
Procedural Blank1-0-1
Glass
Exposed Coupons 1 Hour
Exposed Coupons 24 Hour
Laboratory Blank
Procedural Blank
Galvanized Metal
Exposed Coupons 1 Hour
Exposed Coupons 24 Hour
Laboratory Blank
Procedural Blank
Painted Wallboard Paper
Exposed Coupons 1 Hour
Exposed Coupons 24 Hour
Laboratory Blank
Procedural Blank
2.05
2.05
105
10s
2.05
2.05
105
10s
2.05
2.05
10s
108
2.05
2.05
105
108
6.93 ±0.26
0
0
0
8.15 ±0.02
0
0
0
0
0
0
0
4.43 ±0.38
0
0
0
4.7 ± 1.9
0
0
0
68.9 ±3.7
0
0
0
0
0
0
0
0.018 ±0.016
0
0
0
1.38 ±0.23
8.31
0.16 ±0.02
8.31
8.31
8.31
3.88 ±0.33
8.31
(a) Data are expressed as mean (± SD) of the logs of total number of cells (CFU) observed on individual coupons,
percent recovery (± SD), and mean log reduction (± CI).
(b) Laboratory Blank = Not inoculated.
(c) Procedural Blank = Not inoculated, placed with test coupons during persistence test.
CI = Confidence interval (± 1.96 x SE).
Not Applicable.
39
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6.0 SUMMARY
This project evaluated the persistence of vegetative B. anthracis (Ames) on several materials
under a range of temperature and RH conditions. Persistence testing was conducted both under
normal laboratory lighting conditions and under exposure to controlled levels of UV-A/B
radiation simulating sunlight. Similar persistence tests were also conducted separately for Y.
pestis.
The intent of this project was to test with purely vegetative cells of B. anthracis due to the
possibility of using germinants to convert spores to vegetative cells, thereby facilitating
inactivation of the cells through natural attenuation or decontamination. However, initial tests
showed that B. anthracis cell cultures contained predominantly vegetative cells but also a few
spores. In one initial test in which the inoculum contained B. anthracis in both vegetative cell
and spore form, both cells and spores were recovered from the test materials at 56 days.
Consequently, an improved procedure was developed to reliably produce a stock cell culture
consisting of purely vegetative B. anthracis at a relatively high titer for inoculation onto coupons
(see Appendix A). To ensure purely vegetative cells, heat shock was applied to aliquots of all
stock cell cultures and all coupon extracts in testing with B. anthracis, to clearly distinguish B.
anthracis vegetative cells from spores (heat shock inactivates all cells, but does not affect
spores). The use of these procedures confirmed that in all subsequent tests, only vegetative B.
anthracis cells were inoculated onto test coupons, and allowed clear observation of the
occurrence of B. anthracis sporulation under specific test conditions.
Coupon extracts were subjected to quantitative enumeration of viable cells and to qualitative
growth testing, which provided a more sensitive indication of complete inactivation than did the
quantitative testing. There were several instances when no CFU were detected via quantitative
tests, but bacterial growth did occur in the qualitative tests, thus demonstrating the utility of the
latter test.
The persistence results for B. anthracis (purely vegetative cells, no spores) and Y. pestis, both
with and without UV exposure, are summarized separately below. The persistence of the
vegetative B. anthracis cells produced and used in this study may differ from the persistence of
cells newly germinated from B. anthracis spores by nutrient or non-nutrient germinants in the
environment. Investigation of such potential differences may be a valuable topic for further
research.
6.1 Results for Vegetative Bacillus anthracis (Ames)
6.1.1 Vegetative Bacillus anthracis Persistence without UV Exposure
The persistence of vegetative B. anthracis was tested in the absence of UV radiation on glass,
bare pine wood, unpainted concrete, and topsoil at normal room temperature (approximately 22
°C) and over a range of RH from 34 to over 90 %. The quantitative test results were confirmed
by the qualitative growth testing in all cases. The results of testing were as follows.
40
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• Vegetative B. anthracis persisted for less than one hour after inoculation on bare pine
wood under all test conditions.
• Vegetative B. anthracis also persisted for less than one hour after inoculation on glass
and unpainted concrete when the RH was approximately 50 % or lower.
• Vegetative B. anthracis persistence on glass and unpainted concrete increased when the
RH exceeded approximately 50 %. At 92 % RH, on unpainted concrete, vegetative B.
anthracis persisted for one hour after inoculation; on glass, vegetative B. anthracis
persisted for at least 12 hours after inoculation.
• Vegetative B. anthracis persisted in topsoil for at least 96 hours at 46 % RH. However,
during the simulated sunlight test with low UV-A exposure, CFU were recovered from
the control soil samples (samples not exposed to the UV light) at the 120 hour time point.
In every persistence test summarized above, the heat shock treatment of coupon extracts
confirmed that all B. anthracis cells were vegetative throughout the entire test duration. That is,
no CFU were found in any coupon extract subjected to the heat shock procedure. However, a
different result was found in two tests in which the topsoil was wetted before inoculation, and the
RH was maintained above 90 % to prevent drying of the soil. Results from those tests are
summarized below.
• In wet topsoil, the initially 100 % vegetative B. anthracis population grew by
approximately a factor of 10 and showed extensive sporulation within one week after
inoculation of the topsoil coupons.
• In the second test, we found that the onset of sporulation occurred within 24 hours after
inoculation of the wetted topsoil, and over 10 % of the total B. anthracis population was
in spore form at 48 hours after inoculation.
6.1.2 Vegetative Bacillus anthracis Persistence with UV Exposure
The persistence of vegetative B. anthracis on topsoil under UV-A/B exposure simulating
sunlight was determined at six time points ranging from one hour to 120 hours after inoculation
onto coupons. Two such tests were conducted. One test used levels of UV-A and UV-B
representative of a mid-summer day in Raleigh, North Carolina. The other test used the same
UV-B level but only a minimal level of UV-A. In these tests, all B. anthracis cells were 100 %
vegetative throughout the test duration. The quantitative test results were confirmed by the
qualitative growth testing in all cases; there were a few test results in which CFU were not
recovered quantitatively from soil coupons, but qualitatively showed growth after seven days of
incubation. The results of those tests were as follows.
• From the qualitative results for the UV exposure tests, the longest elapsed time tested in
which B. anthracis cells were recovered from soil was 96 hours, for both the high and
low UV-A test conditions.
41
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• The recovery of vegetative B. anthracis was lower on topsoil coupons exposed to
simulated sunlight than on coupons shielded from that exposure, but the differences in
recovery were generally not significant.
• The results for the CFU recovery from soil when exposed to the high UV-A level are not
significantly different from the CFU recovery from soil when exposed to the low UV-A
level. This finding provides some confirmation of the limited role that UV-A may have
in the inactivation of B. anthracis cells when exposed to sunlight.
6.2 Results for Yersinia pestis
Recovery of Y pestis cells from bare pine wood and unpainted concrete was found to be
unacceptably low in the initial recovery testing conducted before the start of persistence testing.
Further recovery testing showed acceptable recoveries from galvanized metal and painted
wallboard paper, so those materials were used in testing with Y pestis.
6.2.1 Yersinia pestis Persistence without UVExposure
The persistence of Y pestis was tested in the absence of UV-A/B radiation on glass, painted
wallboard paper, galvanized metal, and topsoil, at normal room temperature (approximately 22
°C) and over a range of RH from 54 to over 90 %. The results of that testing were as follows.
• Y. pestis persisted beyond one hour after inoculation on all four test materials.
• Y. pestis was not recovered within 24 hours after inoculation onto glass, painted
wallboard paper, and galvanized metal.
• Y. pestis persisted to at least six days after inoculation on topsoil.
• Persistence of Y. pestis was higher on wetted topsoil and RH maintained above 90 % to
prevent drying of the soil. In that test, viable Y pestis was present at seven days after
inoculation. Complete inactivation (no viable cells) was observed at 14 days after
inoculation.
6.2.2 Yersinia pestis Persistence with UV Exposure
One test was conducted to assess persistence of Y pestis on all four test materials under UV-A/B
exposure. This test used a level of UV-B representative of a mid-summer day in Raleigh, North
Carolina, and a minimal level of UV-A. The results of this test were as follows.
• Under simulated sunlight exposure Y. pestis did not persist on galvanized metal at the
one-hour time point, but did persist at the one-hour time point on the other three
materials.
• Under simulated sunlight exposure, Y pestis did not persist at the 24-hour time point on
any of the four materials.
42
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Impact of Study
This work provides information on the persistence of vegetative B. anthracis on surfaces and
soil, provided that the organism is completely germinated and sporulation is prevented. Such
results may be useful in the development of wide area remediation plans that consider the
possibility of germination and natural attenuation. To date, research has not shown such required
effective germination of spores on surfaces.1 For soil materials, natural attenuation may also be a
viable decontamination option provided that longer attenuation times (e.g., approximately a
week) are acceptable, and the soil can be kept relatively dry. Additional research is
recommended to confirm that the persistence of B. anthracis cells produced through the
germination of spores on materials is of similar duration to that of cells harvested in the
laboratory during the exponential growth phase and inoculated onto materials.
Similar to B. anthracis, the results of the study on the persistence of Y. pestis show that natural
attenuation may also be a viable option for the decontamination of non-soil materials. For soils,
natural attenuation may be a viable decontamination option provided that longer attenuation
times (e.g., approximately a week) are acceptable, and that soils can be kept relatively dry.
43
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7.0 REFERENCES
1. Kane, S., Wollard, J., Bourguet, F., Murphy, G., Alfaro, T., Scher, H. Final Report:
Germination-Lysis Approach for Outdoor Surface Decontamination of Bacillus anthracis
Spores. Lawrence Livermore National Laboratory Report LLNL-TR-625913. March 29,
2103.
2. Persistence Testing and Evaluation of Fumigation Technologies for Decontamination of
Building Materials Contaminated with Biological Agents, EPA/600/R-10/086, U.S. EPA
Office of Research and Development, National Homeland Security Research Center,
August 2010.
3. Rogers, J.V., Sabourin, C.L.K., Choi, Y.W., Richter, W.R., Rudnicki, D.C., Riggs, K.B.,
Taylor, M.L., and Chang, J., Decontamination assessment of Bacillus anthracis, Bacillus
subtilis, and Geobacillus stearothermophilus spores on indoor surfaces using a hydrogen
peroxide gas generator, J. Applied Microbiol. 99(4), 739-748, 2005.
4. Sinha, R.P. and Hader, D.-P., UV-Induced DNA damage and repair: A review,
Photochem. Photobiol. Sci., 1, 225-236, 2002.
5. Mecherikunnel, A.T., Gatlin, J.A., and Richmond, J.C., Data on total and spectral solar
irradiancq, Applied Optics, 22, 1354-1359, 1983.
6. Acra, A., Jurdi, M., Muallem, H., Karahagopian, Y., and Raffoul, Z. Water Disinfection
by Solar Radiation: Assessment and Application, published by International Development
Research Center Canada, Ottawa, Canada, 1990.
7. Diffey, B.L., Sources and measurement of ultraviolet radiation, Methods, 28, 4-13, 2002.
8. Tavakoli, M.B., and Shahi, Z., Solar ultraviolet radiation on the ground level of Isfahan,
Iran. J. Radiat. Res., 5, 101-104, 2007.
9. Jeanmougin, M. and Civatte, J., Dosimetry of solar ultraviolet radiation: Daily and
monthly changes in Paris (in French), Ann. Dermatol. Venereol., 114, 671-676, 1987.
10. Kolari, P. J., Lauharanta, J., and Hoikkala, M., Midsummer solar UV radiation in Finland
compared with the UV radiation from phototherapeutic devices measured by different
techniques, Photodermatol., 3, 340-345, 1986.
11. Balasaraswathy, P., Kumar, U., Srinivas, C.R., and Nair, S., UVA and UVB in Sunlight,
optimal utilization of UV rays in sunlight for phototherapy, Indian J. Dermatol. Venereol.
Leprol., 68, 198-201, 2002.
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Frederick, J.E., Slusser, JR., and Bigelow, D.S., Annual and interannual behavior of
solar ultraviolet irradiance revealed by broadband measurements, Photochem. and
Photobiol., 72, 488-496, 2000.
Calfee, M.W. and Wendling, M., Inactivation of vegetative bacterial threat agents on
environmental surfaces, Science of the Total Environment, 443, 387-396, 2013.
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APPENDIX A:
Description of Issues and Test Results Leading to Development of Procedure
for Preparing Purely Vegetative Bacillus anthracis
-------
Introduction
In cases where the intent is to test with vegetative cells (as in this project), there is the potential
for vegetative cells to sporulate on any surface, including soil, if nutrients are not available.
Thus germination and sporulation have the potential to confound testing with B. anthracis if not
prevented or at least accounted for. These concerns were obviously of great importance for this
project, in which the intent was to test with 100 % vegetative B. anthracis cells.
Initially in this project, only microscopy was used to check for the presence of spores in the
inoculum. To ensure that the inoculum consisted of purely vegetative cells, revised procedures
were implemented. The primary revision made to the test procedures was to apply a heat shock
procedure to aliquots of the inoculum and to aliquots of the extracts of all test coupons to
distinguish B. anthracis spores (which survive the heat shock) from vegetative cells (which do
not survive). That heat shock procedure was well established before the current project (see, for
example, J.V. Rogers et al., Journal of Applied Microbiology, 99, 739-748, 2005) and consists of
subjecting the sample extract to a temperature of 65 °C in a water bath for 60 minutes. The heat
shock procedure was first applied in this project in a test of B. anthracis persistence on four test
materials, and the results clearly indicated that the intent of testing with purely vegetative B.
anthracis cells was not being achieved. That finding motivated efforts to improve the production
method for vegetative B. anthracis, and those efforts in turn led to successful development of a
method for producing a 100 % vegetative cell culture. That method was then used in all
subsequent testing in this project, as described in the body of this report.
The following sections of this Appendix provide more detail on this subject, by presenting 1) the
data from the persistence test that disclosed the lack of a purely vegetative B. anthracis
population, and 2) the development of the successful production method for 100 % vegetative B.
anthracis that was used in all subsequent testing. All test results for B. anthracis in the main
body of this report were obtained using a 100 % vegetative B. anthracis culture to inoculate test
coupons, verified by heat shock, and that every coupon extract in those tests was subjected to the
heat shock treatment.
Test Results Indicating Lack of Purely Vegetative B. anthracis Cells
In early 2013, a test was conducted to assess the persistence (without any UV exposure) of
vegetative B. anthracis on topsoil, glass, bare pine wood, and unpainted concrete at normal room
conditions (i.e., temperature approximately 20 °C and relative humidity approximately 50 %).
The test was of 56 days duration and was the first in which the heat shock procedure was
applied. Coupons of each type were extracted at one hour after inoculation (i.e., immediately
after the inoculation culture had dried) and at 56 days after inoculation. The extract of each
control and test coupon was split in half, and one half was subjected to the heat shock procedure
before both aliquots were diluted, plated, and enumerated as described in Section 2.5 of this
report.
The results from the 56-day test are summarized in Table A-l, which shows for each coupon
material the total number of B. anthracis CFU recovered at the 1-hour and 56-day time points,
the number of B. anthracis spores (determined by the heat shock treatment), and the percentage
of B. anthracis present in spore form.
A-l
-------
Table A-l. Heat Shock Data from 56-Day B. anthracis Persistence Test
Time Point
Parameter
Topsoil
Glass
Wood
Concrete
1 Hour
Total Number of CFU
5.62 x 106
2.40x 104
3.89x 104
3.16x 104
Total Number of Spores
1.74x 104
1.35x 104
1.41x 104
1.35x 104
Percent Spores
0.3
56.2
36.3
42.7
56 Days
Total Number of CFU
7.59x 104
2.75x 104
1.15x 104
5.89x 103
Total Number of Spores
3.31x 104
9.55x 103
7.41x 103
3.63x 103
Percent Spores
43.7
34.7
64.6
61.7
Table A-l shows that at the one-hour time point, the B. anthracis population in the topsoil
coupons was only 0.3 % in spore form, or almost entirely (99.7 %) vegetative. However, on the
other three materials, the percentage of spores ranged from about 36 to 56 % at that time point,
indicating that overall roughly half the B. anthracis on those materials was in spore form.
Similarly at the 56-day time point, a large portion of the total B. anthracis on all four materials
was in spore form, including on the topsoil coupons (approximately 44 % spores). These data
clearly showed the need for improved procedures to assure that testing was conducted with
vegetative B. anthracis cells, leading to the development efforts described in the next section.
Development of Production Procedure for 100 % Vegetative B. anthracis (Ames)
The development effort to assure production of vegetative B. anthracis (Ames) with no spores or
endospores present focused on achieving a sufficiently high cell titer for use in testing and a
purely vegetative cell population. The approach taken was to monitor the growth and nature of
B. anthracis cells closely throughout the 16-hour cell culturing process used previously, to
identify a stopping point that met these goals. Specifically, a growth curve investigation was
conducted as follows:
• A 1:100 dilution was prepared in SFW of standard B. anthracis (Ames) spore stock,
which is approximately 1 x 109 CFU/mL.
n
• Ten |iL of the resulting 1:100 dilution (which is approximately 1x10 CFU/mL) was
added to 200 mL of TSB.
• The resulting culture (which is initially approximately 5x10 CFU/mL) was allowed to
incubate at 37 °C while shaking on an orbital shaker at 200 rpm for up to 16 hours.
• During heating and shaking of the culture, an aliquot was removed at one-hour intervals
for performance of three separate measurements:
o determination of optical density at 600 nm wavelength (ODeoo) as an indication of the
density of the culture.
o microscopic examination to assess the nature of cells in the culture. The focus of this
investigation was to determine at what point in the culturing process the onset of
endospore formation was first observed.
o determination of the concentration of B. anthracis cells in the culture by dilution,
plating, and enumeration.
A-2
-------
The results of the growth curve investigation are shown in Figure A-l, which displays the cell
enumeration results (left vertical axis) and OD600 results (right axis) over 15 hours of the cell
culturing process. Figure A-l shows that at approximately 10 hours into the culturing process,
the log linear (exponential) growth in the log of B. cmthrcicis was near its end, having reached a
concentration of approximately 2 x 107 cells/mL, and the OD6oo value was approximately 2. The
microscopic inspection of the cell culture also showed that at this 10 hour time point, there was
no indication of spore or endospore formation. This observation is illustrated in Figures A-2
through A-6, which show photomicrographs (at 100x magnification) of cells in the culture at 8,
10, 12, 14, and 15 hours into the culturing process. These figures show that at ten hours, only
long chains of vegetative cells are observed (Figure A-3), at 12 hours those chains are starting to
break up (Figure A-4), at 14 hours endospore formation is beginning (Figure A-5), and at 15
hours (Figure A-6) endospores are becoming refractile (and consequently resistant to heat
shock). Based on these observations, for all testing, the incubation of vegetative B. cmthrcicis
was stopped when the OD600 reading reached a value of 2, which occurred reliably after 9 or 10
hours of incubation. As noted in the body of this report, heat shock testing of the cell cultures
was conducted in all tests and confirmed that the identified procedure produced 100 % vegetative
B. cmthracis cells.
Log Cells/mL
OD 600
8 10 12
Hours of Growth
Figure A-l. Results of Growth Curve Investigation of Culturing of Vegetative B. anthracis
A-3
-------
Figure A-2. Photomicrograph of B. anthracis Cell Culture at Eight Hours of Incubation,
Showing Tangling of Long Vegetative Cell Chains (magnification lOOx)
Figure A-3. Photomicrograph of B. anthracis Cell Culture at Ten Hours of Incubation;
Cells Shortening within Long Vegetative Chains, No Sign of Endospores
(magnification lOOx)
A-4
-------
Figure A-4. Photomicrograph of B. anthracis Cell Culture at 12 Hours of Incubation;
Long Vegetative Chains Starting to Break Apart, No Sign of Endospores
(magnification 100x)
Figure A-5. Photomicrograph of B. anthracis Cell Culture at 14 Hours of Incubation;
Long Vegetative Chains Continuing to Break Apart, Endospores Forming
(magnification 100x)
A-5
-------
Figure A-6. Photomicrograph of B. anthracis Cell Culture at 15 Hours of Incubation;
Endospores Forming and Becoming Refractile (magnification 100x)
A-6
-------
APPENDIX B:
Data Tables from Testing of Vegetative Bacillus anthracis Persistence
under UV-A/B Exposure
-------
Tables B-l and B-2 show the results from testing the persistence of vegetative B. anthracis
(Ames) on topsoil over 120 hours in Test BAUV1 and BAUV2, under UV-A/B exposures
involving low and high UV-A/UV-B ratios, respectively. In both tests, topsoil coupons were
extracted at time points of 1, 24, 48, 72, 96, and 120 hours, and extracted coupons were
subjected to qualitative growth testing and assessed for growth at one and seven days after
extraction. These tables show the cell inoculum; the mean log of the observed cells on the non-
exposed, exposed, and blank coupons; the cell recovery; the resulting mean log reduction (±CI)
on the non-exposed and test coupons relative to the initial inoculum; and the results of the
qualitative growth testing at each time point. The test results shown in Tables B-l and B-2 are
the basis for Figures 4-4 and 4-5, respectively, in the body of this report.
Comparison of Tables B-l and B-2 shows that the persistence under exposure to the high UV-
A/UV-B ratio (Table B-2) was similar to that at the same time point under exposure to the low
UV-A/UV-B ratio (Table B-l). This finding is illustrated in Figure B-l, which compares the
persistence on UV-exposed topsoil coupons in the two 120-hour UV tests. At the one hour time
point, recovery of cells at the high UV-A/UV-B condition was significantly greater than with the
low UV-A/UV-B ratio; at 24 hours and at all later time points, the results from the two tests were
not significantly different.
T3
a>
a>
>
o
u
a>
DC
UV Exposed Low UV-A/B Ratio
UV Exposed High UV-A/B Ratio
20
40 60
Time, Hours
80
100
120
Figure B-l. A High UV-A/UV-B Ratio does not Reduce Persistence of Vegetative B.
anthracis Relative to a Low UV-A/UV-B Ratio with the same UV-B level
B-l
-------
Table B-l. UV-A/B Inactivation of Vegetative Bacillus anthracis on Topsoil^
(120-Hour Exposure at Low UV-A/UV-B Ratio; Test BAUV1)
Quantitative Testing
Qualitative
Growth(b)
Mean of Logs
Mean Log
Inoculum
of Observed
Mean %
Reduction
One
Seven
Time Point
(CFU)
CFU
Recovery
±CI
day
days
1 Hour
Nonexposed Coupons(c)
5.03 x
106
4.44 ± 0.42
0.70 ±0.35
2.26 ±0.37
Y
Y
Test Coupons(d)
5.03 x
106
3.11 ± 0.15
0.027 ± 0.009
3.59 ±0.13
Y
Y
Laboratory Blank(e)
0
0
0
-
Procedural Blank®
0
0
0
-
24 Hours
Nonexposed Coupons
5.03 x
106
3.69 ±0.88
0.36 ±0.48
3.01 ±0.77
Y
Y
Test Coupons
5.03 x
10s
0.75 ± 1.06
0.0008 ±0.0014
5.95 ±0.92
N
Y
Laboratory Blank
0
0
0
-
Procedural Blank
0
0
0
-
48 hours
Nonexposed Coupons
5.03 x
106
1.84 ± 1.31
0.021 ±0.045
4.87 ± 1.15
Y
Y
Test Coupons
5.03 x
106
0.44 ±0.99
0.0007 ±0.0015
6.26 ±0.87
N
Y
Laboratory Blank
0
0
0
-
Procedural Blank
0
0
0
-
72 hours
Nonexposed Coupons
5.03 x
106
1.46 ±0.89
0.0016 ±0.0021
5.24 ±0.78
Y
Y
Test Coupons
5.03 x
10s
0.30 ±0.68
0.0002 ± 0.0003
6.40 ± 0.60
N
Y
Laboratory Blank
0
0
0
-
Procedural Blank
0
0
0
-
96 hours
Nonexposed Coupons
5.03 x
106
0.82 ± 1.13
0.0009 ±0.0013
5.88 ±0.99
Y
Y
Test Coupons
5.03 x
106
0
0
6.70
N
Y
Laboratory Blank
0
0
0
-
Procedural Blank
0
0
0
-
120 Hours
Nonexposed Coupons
5.03 x
106
0.40 ±0.89
0.0004 ± 0.0009
6.30 ±0.78
Y
Y
Test Coupons
5.03 x
10s
0
0
6.70
N
N
Laboratory Blank
0
0
0
-
Procedural Blank
0
0
0
-
(a) Data are expressed as mean (± SD) of the logs of total number of cells (CFU) observed on individual coupons,
percent recovery (± SD), and mean log reduction (± CI).
(b) Growth in nutrient medium at one day and seven days after coupon extraction. Y indicates growth visually
observed with at least one of five coupon extracts, Y indicates growth confirmed to be B. anthracis with at least
one of five coupon extracts, N indicates no growth (complete inactivation).
(c) Inoculated, not exposed to UV-A/B (recovery conducted after conclusion of UV-A/B exposure).
(d) Inoculated, exposed to UV-A/B. UV-A/B exposure accumulated in alternating 12-h periods of light and
darkness.
(e) Laboratory Blank = Not inoculated, placed with positive control coupons and not exposed to UV-A/B.
(f) Procedural Blank = Not inoculated, placed with test coupons and exposed to UV-A/B. UV-A/B exposure
accumulated in alternating 12-h periods of light and darkness.
CI = Confidence interval (± 1.96 x SE).
Not Applicable.
B-2
-------
Table B-2. UV-A/B Inactivation of Vegetative Bacillus anthracis on Topsoil^
(120-Hour Exposure at High UV-A/UV-B Ratio; Test BAUV2)
Quantitative Testing
Qualitative
Growth®
Mean of Logs
Mean Log
Inoculum
of Observed
Mean %
Reduction
One
Seven
Time Point
(CFU)
CFU
Recovery
±CI
day
days
1 Hour
Nonexposed Coupons(c)
1.09 x
106
5.58 ±0.14
36.3 ± 12.3
0.46 ±0.12
Y
Y
Test Coupons(d)
1.09 x
106
5.33 ±0.006
19.8 ±0.3
0.70 ±0.01
Y
Y
Laboratory Blank(e)
0
0
0
-
Procedural Blank®
0
0
0
-
24 Hours
Nonexposed Coupons
1.09 x
106
1.13 ± 1.03
0.0043 ±0.0041
4.91 ±0.91
N
Y
Test Coupons
1.09 x
10s
0.70 ±0.98
0.0025 ±0.0039
5.33 ±0.86
N
Y
Laboratory Blank
0
0
0
-
Procedural Blank
0
0
0
-
48 hours
Nonexposed Coupons
1.09 x
106
1.13 ± 1.03
0.0043 ±0.0041
4.91 ±0.91
Y
Y
Test Coupons
1.09 x
106
0.97 ±0.90
0.0025 ± 0.0025
5.06 ±0.79
N
Y
Laboratory Blank
0
0
0
-
Procedural Blank
0
0
0
-
72 hours
Nonexposed Coupons
1.09 x
106
0
0
6.04
N
Y
Test Coupons
1.09 x
10s
0
0
6.04
N
Y
Laboratory Blank
0
0
0
-
Procedural Blank
0
0
0
-
96 hours
Nonexposed Coupons
1.09 x
106
0.80 ± 1.10
0.0037 ±0.0050
5.24 ±0.96
N
Y
Test Coupons
1.09 x
106
0
0
6.04
N
Y
Laboratory Blank
0
0
0
-
Procedural Blank
0
0
0
-
120 Hours
Nonexposed Coupons
1.09 x
106
0
0
6.04
N
N
Test Coupons
1.09 x
10s
0
0
6.04
N
N
Laboratory Blank
0
0
0
-
Procedural Blank
0
0
0
-
(a) Data are expressed as mean (± SD) of the logs of total number of cells (CFU) observed on individual coupons,
percent recovery (± SD), and mean log reduction (± CI).
(b) Growth in nutrient medium at one day and seven days after coupon extraction. Y indicates growth visually
observed with at least one of five coupon extracts, Y indicates growth confirmed to be B. anthracis with at least
one of five coupon extracts, N indicates no growth (complete inactivation).
(c) Inoculated, not exposed to UV-A/B (recovery conducted after conclusion of UV-A/B exposure).
(d) Inoculated, exposed to UV-A/B. UV-A/B exposure accumulated in alternating 12-h periods of light and
darkness.
(e) Laboratory Blank = Not inoculated, placed with positive control coupons and not exposed to UV-A/B.
(f) Procedural Blank = Not inoculated, placed with test coupons and exposed to UV-A/B. UV-A/B exposure
accumulated in alternating 12-h periods of light and darkness.
CI = Confidence interval (± 1.96 x SE).
Not Applicable.
B-3
-------
The primary conclusion to be drawn from the UV test results shown above is that UV-A/B has a
small effect, at most, on the persistence of vegetative B. cmthrcicis on topsoil. Consideration of
these data raised the question of how reproducible persistence results are with vegetative B.
cmthrcicis. To address this question, a comparison was made of the persistence of B. cmthrcicis
on topsoil (no UV exposure) in three nominally identical sets of topsoil coupons.
• Test 1 - the test coupons used in the 120-hour persistence test with no UV exposure
(Table 4-5 and Figure 4-1);
• Test 2 - the non-exposed coupons (controls) used in the 120-hour UV exposure test with
the low UV-A/UV-B ratio (Table B-l and Figure 4-4); and
• Test 3 - the non-exposed coupons (controls) used in the 120-hour UV exposure test with
the high UV-A/UV-B ratio (Table B-2 and Figure 4-5).
Figure B-2 shows the persistence curves of vegetative B. cmthrcicis on these three sets of topsoil
coupons. Overall, the three persistence curves are quite similar, all reaching or nearing complete
inactivation of the vegetative B. cmthrcicis within 120 hours. The 95 % CI intervals are plotted at
each time point for each of the three tests. The CI intervals of at least two data points overlap at
every time point, and in most cases all three overlap. Thus the three persistence curves are not
consistently significantly different from one another.
T3
a>
a>
>
o
u
a>
DC
5
4
3
2
1
¦Test 1
Test 2
¦Test 3
0 20 40 60 80 100 120
Time, Hours
Figure B-2. Recovery of Vegetative B. anthracis in Soil, No UV exposure
Figure B-3 shows a more quantitative comparison of the persistence results from these three
nominally identical sets of topsoil coupons. In this figure, the mean log CFU recovered results
B-4
-------
from Tests 2 and 3 are plotted against the corresponding results from Test 1 at the same time
points. Figure B-3 includes a one-to-one line indicating perfect agreement, and parallel lines
indicating differences of+1 and -1 log. Figure B-3 shows that the persistence results in the three
replicate tests agree within approximately 1 log at all time points. A similar degree of agreement
has been seen in previous decontamination tests conducted by Battelle with B. cmthrcicis (Ames)
spores. The implication of Figure B-3 is that persistence can be replicated within about ±1 log in
testing with vegetative B. cmthracis.
on
o
CM
+->
(/)
.0)
T3
a>
a>
>
o
u
a>
DC
u
bO
O
_i
c
ro
a>
5
4
3
2
~ Test 2
~
~
~
~
~
¦ Test 3
+1L°g.'''
~
~ * * *
~
* * /
''
/ ' /
X' "ILog
''
\
\
\
\
\
\
\
\
¦
\
\
~
~
~
~
*
A \ * *
\
N
\
\
\
\
\
II
\
\
Ml
1
i i i
0 1 2 3 4 5 6
Mean Log CFU Recovered, Test 1
Figure B-3. Persistence Results in Three Comparable 120-hour Tests are
within ± 1 Log
B-5
-------
APPENDIX C:
Data Tables from Selected Persistence Tests with Yersinia pestis
-------
Tables C-l through C-3 show detailed results from testing of Y. pestis persistence without
exposure to simulated sunlight. These tables augment the data shown in Section 5 of this report.
Each of these tables shows the Y. pestis inoculum onto each coupon, the mean log (± SD) of the
recovered CFU, the mean (± SD) of the CFU percent recovery, the mean log reduction (± CI),
and the qualitative results of subsequent growth testing, when performed. All three tables show
results for all four coupon materials used with Y. pestis, for each time point used in the respective
test.
Table C-l shows the results from Test YP1, in which Y pestis persistence was assessed at one
hour and at 14 days after inoculation onto the four coupon materials, at ambient laboratory
temperature and 57 % RH. Y pestis was recovered on all four materials at 1 hour after
inoculation, but no viable cells were present at 14 days after inoculation. Qualitative testing was
not conducted in Test YP1, but the results of which would not be expected to differ from the
quantitative results.
Table C-2 shows the results from Test YP2, in which Y pestis persistence was assessed at one
hour and at seven days after inoculation onto the four coupon materials, at ambient laboratory
temperature and 54 % RH. Y pestis persisted on all four materials at one hour after inoculation,
but no viable cells were present at seven days after inoculation. Qualitative testing was not
conducted in Test YP2, but the results of which would not be expected to differ from the
quantitative results.
Table C-3 shows the complete set of results from Test YP3, in which Y. pestis persistence was
assessed at 1, 24, 48, 96, and 144 hours after inoculation onto the four test materials, at ambient
laboratory temperature and 65 % RH. Some of the data in Table C-3 are shown in Table 5-3 in
the body of this report. Table C-3 shows data for glass, galvanized metal, and painted wallboard
paper at 48, 96, and 144 hours after inoculation; those data are not shown in Table 5-3 because Y
pestis did not persist on those coupons at those time points. Despite the inactivation observed at
24 hours and the later time points, testing was continued through those time points for
consistency with the results on topsoil. Table C-3 shows that the qualitative test results
confirmed the quantitative test results in all cases in Test YP3.
C-l
-------
Table C-l. Persistence of Yersiniapestis^ (Four Materials, 14 Days, 57 % RH; Test YP1)
Quantitative Testing
Mean Log
Inoculum
Mean of Logs of
Mean %
Reduction
Test Material
(CFU)
Observed CFU
Recovery
±CI
Topsoil
Test Coupons 1 Hour
3.67 x
107
7.37 ± 0.11
65.2 ± 17.9
0.20 ±0.10
Test Coupons 14 Days
3.67 x
107
0
0
7.56
Laboratory Blank(b)
0
0
0
-
Procedural Blank1-0-1
0
0
0
-
Glass
Test Coupons 1 Hour
3.67 x
107
7.23 ± 0.03
46.1 ±3.1
0.34 ±0.02
Test Coupons 14 Days
3.67 x
107
0
0
7.56
Laboratory Blank
0
0
0
-
Procedural Blank
0
0
0
-
Galvanized Metal
Test Coupons 1 Hour
3.67 x
107
7.43 ±0.16
77.1 ±27.4
0.14 ± 0.14
Test Coupons 14 Days
3.67 x
107
0
0
7.56
Laboratory Blank
0
0
0
-
Procedural Blank
0
0
0
-
Painted Wallboard Paper
Test Coupons 1 Hour
Test Coupons 14 Days
Laboratory Blank
3.67 x
3.67 x
0
0
r- r-
O O
6.21 ±0.30
0
0
0
5.4 ±3.7
0
0
0
1.35 ±0.26
7.56
Procedural Blank
-
(a) Data are expressed as mean (± SD) of the logs of total number of cells (CFU) observed on individual coupons,
percent recovery (± SD), and mean log reduction (± CI).
(b) Laboratory Blank = Not inoculated.
(c) Procedural Blank = Not inoculated, placed with test coupons during persistence test.
CI = Confidence interval (± 1.96 x SE).
Not Applicable.
C-2
-------
Table C-2. Persistence of Yersinia pestis^a\Four Materials, Seven Days, 54 % RH; Test
YP2)
Test Material
Quantitative Testing
Inoculum
(CFU)
Mean of Logs of
Observed CFU
Mean %
Recovery
Mean Log
Reduction
±CI
Topsoil
Test Coupons 1 Hour
Test Coupons 7 Days
Laboratory Blank(b)
Procedural Blank'01
Glass
Test Coupons 1 Hour
Test Coupons 7 Days
Laboratory Blank
Procedural Blank
3.13
3.13
3.13
3.13
10'
107
107
107
7.08 ±0.03
0
0
0
5.06 ± 0.11
0
0
0
38.7 ±2.4
0
0
0
0.38 ±0.11
0
0
0
0.41 ±0.03
7.50
2.44 ±0.10
7.50
Galvanized Metal
Test Coupons 1 Hour 3.13 x 107 2.58 ±0.23 0 0006 4.92 ±0.21
Painted Wallboard Paper
Test Coupons 1 Hour
Test Coupons 7 Days
Laboratory Blank
Procedural Blank
Test Coupons 7 Days 3.13 x 107 0 ^ 7.50
Laboratory Blank 0 0
Procedural Blank 0 0
0
0
3.13 x 107 4.27 ± 0.45 0.085 ± 0.067 3.23 ±0.39
3.13 x 107 0 0 7.50
0 0 0 -
0 0 0 -
(a) Data are expressed as mean (± SD) of the logs of total number of cells (CFU) observed on individual coupons,
percent recovery (± SD), and mean log reduction (± CI).
(b) Laboratory Blank = Not inoculated.
(c) Procedural Blank = Not inoculated, placed with test coupons during persistence test.
CI = Confidence interval (± 1.96 x SE).
Not Applicable.
C-3
-------
Table C-3. Persistence of Yersinia /;esft's^(Four Materials, 144 Hours, 65 % RH; Test
YP3)
Quantitative Testing
Qualitative
Growth(b)
Test Material
Inoculum
(CFU)
Mean of
Logs of
Observed
CFU
Mean %
Recovery
Mean Log
Reduction
±CI
One
day
Seven
days
Topsoil
2.70 x
i rs.1
Test Coupons 1 Hour
10'
6.98 ±0.07
35.7 ±6.4
0.45 ± 0.07
N
Y
Test Coupons 24 Hour
2.70 x
107
3.00 ±0.75
0.0084 ±0.01
4.43 ± 0.66
N
Y
Test Coupons 48 Hour
2.70 x
107
1.72 ± 1.04
0.00059 ± 0.00063
5.71 ±0.91
N
Y
Test Coupons 96 Hour
2.70 x
107
1.40 ±0.87
0.00027 ± 0.00040
6.03 ± 0.77
N
Y
Test Coupons 144 Hour
2.70 x
107
1.07 ± 1.02
0.00020 ±0.00031
6.36 ±0.90
N
Y
Laboratory Blank(c)
0
0
0
-
Procedural Blank(d)
0
0
0
-
Glass
Test Coupons 1 Hour
2.70 x
107
4.55 ±0.09
0.13 ±0.029
2.88 ±0.08
N
Y
Test Coupons 24 Hour
2.70 x
107
0
0
7.43
N
N
Test Coupons 48 Hour
2.70 x
107
0
0
7.43
N
N
Test Coupons 96 Hour
2.70 x
107
0
0
7.73
N
N
Test Coupons 144 Hour
2.70 x
107
0
0
7.43
N
N
Laboratory Blank
0
0
0
-
Procedural Blank
0
0
0
-
Galvanized Metal
N
Test Coupons 1 Hour
2.70 x
107
6.34 ±0.69
15.0 ± 14.1
1.09 ±0.61
Y
Test Coupons 24 Hour
2.70 x
107
0
0
7.43
N
N
Test Coupons 48 Hour
2.70 x
107
0
0
7.43
N
N
Test Coupons 96 Hour
2.70 x
107
0
0
7.73
N
N
Test Coupons 144 Hour
2.70 x
107
0
0
7.43
N
N
Laboratory Blank
0
0
0
-
Procedural Blank
0
0
0
-
Painted Wallboard
Paper
N
N
Test Coupons 1 Hour
2.70 x
107
3.46 ±0.61
0.028 ± 0.047
3.97 ±0.69
Test Coupons 24 Hour
2.70 x
107
0
0
7.43
N
N
Test Coupons 48 Hour
2.70 x
107
0
0
7.43
N
N
Test Coupons 96 Hour
2.70 x
107
0
0
7.43
N
N
Test Coupons 144 Hour
2.70 x
107
0
0
7.43
N
N
Laboratory Blank
0
0
0
-
Procedural Blank
0
0
0
-
(a) Data are expressed as mean (± SD) of the logs of total number of cells (CFU) observed on individual coupons,
percent recovery (± SD), and mean log reduction (± CI).
(b) Growth in nutrient medium at one day and seven days after coupon extraction. Y indicates growth visually
observed with at least one of five coupon extracts, Y indicates growth confirmed to be Y. pestis with at least one
of five coupon extracts, N indicates no growth (complete inactivation).
(c) Laboratory Blank = Not inoculated.
(d) Procedural Blank = Not inoculated, placed with test coupons during persistence test.
CI = Confidence interval (± 1.96 x SE).
Not Applicable.
C-4
-------
&EPA
United States
Environmental Protection
Agency
PRESORTED STANDARD
POSTAGE & FEES PAID
EPA
PERMIT NO. G-35
Office of Research and Development (8101R)
Washington, DC 20460
Official Business
Penalty for Private Use
$300
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