EPA/600/R-10/048 | May 2010 | www.epa.gov/nhsrc
United States
Environmental Protection
Agency
INVESTIGATION REPORT
INVESTIGATION OF SIMULATED
SUNLIGHT IN THE INACTIVATION
OF B. ANTHRACIS AND B. SUBTILIS
ON OUTDOOR MATERIALS
Office of Research and Development
National Homeland Security Research Cen-
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EPA/600/R-10/048 | May 2010 | www.epa.gov/nhsrc
INVESTIGATION REPORT
INVESTIGATION OF SIMULATED
SUNLIGHT IN THE INACTIVATION
OF B. ANTHRACIS AND B. SUBTILIS
ON OUTDOOR MATERIALS
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
Contract No. GS23F0011L-3
Office of Research and Development
National Homeland Security Research Center
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DISCLAIMER
The U.S. Environmental Protection Agency (EPA), through its Office of Research and Development, funded and
directed this investigation through a Blanket Purchase Agreement (BPA) under General Services Administration
contract number GS23F0011L-3 with Battelle. 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 (NG16)
U.S. Environmental Protection Agency
Mail Code E343-06
Research Triangle Park, NC 27711
(919) 541-5029
wood.joe@epa.gov
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ACKNOWLEDGMENTS
The US EPA's National Homeland Security Research Center (NHSRC) would like to acknowledge the following
organizations and individuals for their support in the development of this report, whether in contributing to the
research or providing a peer review.
Contributors (Battelle)
Thomas Kelly
Young Choi
James Rogers
Karen Riggs
Zachary Willenberg
Peer reviewers
Worth Calfee (EPA NHSRC), Leroy Mickelson (EPA National Decontamination Team), and Jonathon
Thornburg (RTI International)
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EXECUTIVE SUMMARY
The U.S. Environmental Protection Agency's (EPA)
National Homeland Security Research Center (NHSRC)
helps protect human health and the environment
by carrying out investigations on the persistence of
biological agents in the environment. This report
describes an investigation of the effect of UV-A/B
light (representing sunlight) for inactivating spores of
Bacillus anthracis (Ames) and Bacillus subtilis on test
coupons of glass, bare pine wood, unpainted concrete,
and topsoil.
Test Procedures. Test coupons were 1.9 cm by 7.5
cm, except for topsoil, which was prepared by filling
a Parafilm®-lined 3.5 cm diameter by 1 cm deep Petri
dish with uncompacted topsoil. For testing, coupons
were "contaminated" by inoculation with approximately
108 colony forming units per coupon of the biological
warfare agent B. anthracis (Ames), or the surrogate
organism B. subtilis (American Type Culture Collection
[ATCC] 19659). Test coupons were exposed to UV-
A/B light, and the number of viable spores on each
coupon was determined after a predefined duration of
exposure. Positive control coupons were spiked in the
same way and subjected to the same test environment
except for being shielded from exposure to UV-A/B
light. Five replicates of both test and positive control
coupons were used for each coupon material in each
test. Four different durations of UV-A/B exposure were
used: 24, 168, 336, and 672 h. Those total exposures
were accumulated by alternating 12-h periods of UV-
A/B light and darkness, resulting in 12 h of UV-A/B
exposure on each test day. For example, 168-h UV-A/B
exposure was accumulated over 14 test days. Each UV-
A/B exposure test with each organism was conducted
separately and involved coupon inoculation, UV-A/B
exposure, spore extraction, and enumeration, i.e.,
UV-A/B exposures were separate, non-simultaneous,
and non-overlapping tests. All test procedures were
conducted according to a peer-reviewed test/quality
assurance (QA) plan.
UV-C at the positive control coupons. Temperature and
RH at the positive control coupons were within about
3 °C and within about 10% RH, respectively, of the
temperature and RH at the test coupons when the UV-
A/B lights were on, and within 0.5 °C and 5% RH when
the lights were off.
The effectiveness of UV-A/B exposure for inactivating
spores was quantified as mean Iog10 reduction relative to
the positive control coupons for each material with each
spore type at each time point.
Results. Table ES-1 shows the mean log reduction
values for B. anthracis (Ames) and B. subtilis on the four
test materials at each of the four UV-A/B exposure time
points. The 95% confidence interval (CI) is also shown
for each mean log reduction value. UV-A/B inactivation
was most effective for both B. anthracis and B. subtilis
on glass, and least effective on topsoil. The maximum
mean log reduction results on glass exceeded 5 logs for
both B. anthracis and B. subtilis. Mean log reductions
achieved on bare pine wood and unpainted concrete
were primarily in the range of about 1 to 2 log reduction,
and those mean log reductions were similar across both
materials and both organisms. The topsoil mean log
reductions varied around zero and never reached 1 log
reduction for either organism. Table ES-1 indicates (by
bold values) those cases in which statistically significant
differences in mean log reduction were found between
the two organisms. Of the 16 such comparisons, seven
show significant differences in mean log reduction
between B. subtilis and B. anthracis. No clear pattern
is evident in the occurrence of these seven cases with
coupon material or UV-A/B exposure duration.
UV-A/B/C intensities, temperature, and relative
humidity (RH) were monitored at the locations of both
the test and positive control coupons throughout all
testing. Those measurements confirmed UV-A/B levels
at the test coupons of approximately 100 uW/cm2UV-A
and 70 uW/cm2 UV-B, with no detectable UV-C (i.e., <1
uW/cm2). There was also no detectable UV-A, UV-B, or
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EXECUTIVE SUMMARY (CONT.)
Table ES-1. Summary of Mean Log Reduction Results with UV-A/B (Simulated Sunlight) Exposure on Four
Materials
Material
Glass
Bare Pine Wood
Unpainted Concrete
Topsoil
Mean Log Reduction (± 95% CI) by UV-A/B Exposure Time3
24 h
B.a.
3.77
(±0.29)
0.69
(±0.15)
0.81
(±0.24)
-1.10b
(±0.09)
B.s.
2.59
(±0.24)
0.92
(±0.23)
0.44
(±0.37)
0.31
(±0.50)
168 h
B.a.
5.25
(±1.61)
1.16
(±0.34)
0.65
(±0.28)
0.09
(±0.08)
B.s.
3.84
(±0.15)
0.11
(±0.23)
2.44
(±0.59)
0.75
(±0.19)
336 h
B.a.
5.81
(±1.40)
1.01
(±0.64)
2.21
(±0.83)
0.17
(±0.10)
B.s.
3.99
(±0.37)
0.94
(±0.64)
0.48
(±0.15)
-0.08
(±0.38)
672 h
B.a.
4.72
(±0.88)
1.51
(±0.60)
1.51
(±0.55)
0.20
(±0.15)
B.s.
5.29
(±1.21)
1.27
(±0.20)
2.15
(±0.10)
0.15
(±0.09)
1 B.a. = B. anthracis (Ames), B.s. = B. subtilis. Values in bold forB. subtilis are significantly different from corresponding values
forB. anthracis.
b Unusual spore recoveries seen; see text.
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CONTENTS
Disclaimer v
Acknowledgements vi
Executive Summary vii
Abbreviations/Acronyms xi
1.0 Introduction 1
2.0 Summary of Test Procedures 3
2.1 Preparation of Test Coupons 3
2.2 UV-A/B Exposure Procedure 3
2.3 Spore Recovery Procedures 5
2.4 Spore Growth and Confirmation 6
2.5 Calculation of Mean Log Reduction 7
3.0 Quality Assurance/Quality Control 9
3.1 Equipment Calibration 9
3.2 QC Results 9
3.3 Audits 9
3.3.1 Performance Evaluation Audit 9
3.3.2 Technical Systems Audit 10
3.3.3 Data Quality Audit 10
3.4 Test/QA Plan Amendments and Deviations 10
3.5 QA/QC Reporting 10
3.6 Data Review 10
4.0 Results 11
4.1 Spore Growth and Confirmation 11
4.2 QC Results 11
4.3 Uniformity of Test Conditions 12
4.4 Mean Log Reduction Results 16
5.0 Summary 25
6.0 References 27
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TABLES
Table ES-1. Summary of Mean Log Reduction Results with UV-A/B (Simulated Sunlight)
Exposure on Four Materials viii
Table 3-1. Performance Evaluation Audit of Solarmeter UV Radiometers 10
Table 4-1. Summary of Ultraviolet (UV-A/B) Light Intensity at the Test Coupons 13
Table 4-2. Summary of Temperature and Relative Humidity Conditions in UV-A/B Exposure
Testing with Bacillus anthracis (Ames) Spores 14
Table 4-3. Summary of Temperature and Relative Humidity Conditions in UV-A/B Exposure
Testing with Bacillus subtilis Spores 15
Table 4-4a. UV-A/B Inactivation of Bacillus anthracis (Ames) Spores - 24 Hour Exposure 17
Table 4-4b. UV-A/B Inactivation of Bacillus subtilis Spores -24 Hour Exposure 18
Table 4-5a. UV-A/B Inactivation of Bacillus anthracis (Ames) Spores - 168 Hour Exposure 19
Table 4-5b. UV-A/B Inactivation of Bacillus subtilis Spores - 168 Hour Exposure 20
Table 4-6a. UV-A/B Inactivation of Bacillus anthracis (Ames) Spores - 336 Hour Exposure 21
Table 4-6b. UV-A/B Inactivation of Bacillus subtilis Spores - 336 Hour Exposure 22
Table 4-7a. UV-A/B Inactivation of Bacillus anthracis (Ames) Spores - 672 Hour Exposure 23
Table 4-7b. UV-A/B Inactivation of Bacillus subtilis Spores - 672 Hour Exposure 24
Table 5-1. Summary of Mean Log Reduction Results with UV-A/B (Simulated Sunlight)
Exposure on Four Materials 26
FIGURES
Figure 2-1. Schematic Representation of Test Chamber 4
Figure 2-2. Schematic of Five Coupon Positions on the Support Trays 4
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ABBREVIATIONS/ACRONYMS
ATCC American Type Culture Collection
B. a. Bacillus anthracis
B.s. Bacillus subtilis
BBRC Battelle Biomedical Research Center
BSC biosafety cabinet
C Celsius
Ct threshold cycle
CFU(s) colony-forming unit(s)
CI confidence interval
cm centimeter
EPA U.S. Environmental Protection Agency
h hour
J Joule
L liter
LR log reduction
min minute
uL microliter
um micrometer
uW microwatts
mL milliliter
mM millimolar
NHSRC National Homeland Security Research Center
nm nanometer
ORD EPA Office of Research and Development
PBS phosphate-buffered saline
PCR polymerase chain reaction
QA quality assurance
QC quality control
QMP quality management plan
RH relative humidity
rpm revolutions per minute
SD standard deviation
SE standard error
SFW sterile filtered water
TOPO Task Order Project Officer
TSA technical systems audit
UV ultraviolet light
UV-A ultraviolet light (320 to 400 nm wavelength)
UV-A/B combination of UV-A and UV-B light used in testing
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ABBREVIATIONS/ACRONYMS (CONTINUED)
UV-B
UV-C
W
ultraviolet light (290 to 320 run wavelength)
ultraviolet light (180 to 290 run wavelength)
watts
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1.0
INTRODUCTION
The U.S. Environmental Protection Agency's (EPA)
National Homeland Security Research Center (NHSRC)
evaluates the role that natural conditions play in
counteracting chemical and biological homeland security
threats, by working with stakeholders and subject matter
experts to develop test plans, conduct evaluations,
collect and analyze data, and prepare peer-reviewed
reports. All evaluations are conducted in accordance
with rigorous quality assurance (QA) protocols to ensure
the generation of high quality data and defensible results.
NHSRC investigated the effect of simulated sunlight
(combination of UV-A (i.e., 320-400 run) and UV-B
(i.e., 290-320 nm) light, referred to as UV-A/B) in
inactivating Bacillus anihracis (Ames) spores and
Bacillus subtilis (American Type Culture Collection or
ATCC 19659) spores on representative surface materials.
The procedures of this investigation are specified in
a peer-reviewed test/QA plan, that was developed
according to the requirements of a previously established
quality management plan (QMP).1 In this investigation,
the mean log reduction in viable spores due to UV-A/B
exposure was determined at four successive non-zero
time points for each of the two test organisms on each
of four test materials: glass, bare pine wood, unpainted
concrete, and topsoil. An initial 24-h UV-A/B exposure
test was conducted; each successive time point was then
chosen by EPA based on the results of the preceding test.
The same four exposure times were used for both B.
subtilis and B. anthracis.
In all tests, UV-A/B exposure was conducted in
alternating 12-h periods of light and dark. Positive
control coupons of the test materials, inoculated with the
test organisms, were placed in the same test environment
as those exposed to UV-A/B light, but were shielded
from any UV-A/B exposure. The temperature and
relative humidity (RH) of the test environment were
monitored at both the position of the test coupons and
the position of the positive control coupons throughout
testing. UV-A and UV-B intensity was monitored at
five locations in the array of test coupons and five
corresponding locations in the array of positive control
coupons. This monitoring was conducted to confirm both
the UV-A and UV-B intensities reaching the test coupons
and the absence of UV-A/B exposure on the positive
control coupons.
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2.0
SUMMARY OF TEST PROCEDURES
Test procedures were performed in accordance with the
test/QA plan and are briefly summarized here.
2.1 Preparation of Test Coupons
B. anthracis Ames and B. subtilis spores were inoculated
onto test coupons of bare wood, glass, unpainted
concrete, and topsoil in an appropriate biosafety cabinet
(BSC-II or -III) according to established Battelle
Biomedical Research Center (BBRC) procedures.
Inoculated coupons were prepared fresh for each day
of experimental work. Test coupons were placed flat in
the BSC and spiked at approximately 1 x 10s colony-
forming units (CPUs) per coupon. This spiking was
accomplished by dispensing a 100 uL aliquot of a
spore stock suspension (approximately 1 x 109 CPUs/
mL) using a micropipette as 10 droplets (each of 10
uL volume) across the surface of the test coupon. This
approach provided more uniform distribution of spores
across the coupon surface than would be obtained
through a single drop of the suspension. It is possible
that the application procedure produced multiple layers
of spores on the test coupons. If this were the case,
spores in the topmost layers could have diminished the
UV-A/B exposure of spores in the underlying layers.
However, this is a hypothesis that would have to be
verified with additional research. After spiking, the test
coupons remained undisturbed overnight in the BSC to
dry. With the exception of topsoil (see Section 2.3), test
coupons were sterilized before use by gamma irradiation
(bare wood) or autoclaving (glass, unpainted concrete).
2.2 UV-A/B Exposure Procedure
On the day following spore inoculation, the test, blank,
and positive control 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 light to which the coupons were exposed.
Five test coupons and five positive control coupons of
each material were used for each UV-A/B exposure
time point. One blank coupon of each material was also
included with the test coupons and with the positive
control coupons for each time point. Test coupons and
associated blank coupons were placed flat on top of the
raised tray below the UV lamps, and positive control
coupons and associated blank coupons were placed flat
beneath that tray, shielded from direct UV-A/B light
(Figure 2-1). The lower portions of the test chamber
walls were covered with black paper to minimize
reflected UV-A/B light.
The 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 test
(or positive control) coupon of each of the four materials
was placed at each of these five positions, 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 test and
positive control trays.
All UV exposure testing started at normal room
temperature and RH: approximately 22 °C and 50% RH.
However, no attempt was made to control the chamber
temperature or RH, and despite circulation of air through
the chamber, the chamber temperature increased a few
degrees Celsius and RH dropped by a few percent during
the UV-A/B exposure periods. Temperature and RH
were recorded at 5-minute intervals throughout all tests
at the locations of both the test and control 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.
Averages, standard deviations, maxima, and minima of
the recorded temperature and RH data over the duration
of each UV-A/B exposure are presented in Section 4 of
this report to document the test conditions.
All testing consisted of alternating 12-h periods of
UV-A/B exposure (lamps on) and darkness (lamps
off). On the basis of mean log reduction results from
an initial 24-h UV-A/B exposure, the next UV-A/B
exposure period was selected by the EPA Task Order
Project Officer (TOPO). This process was repeated upon
completion of each UV-A/B exposure to select a range
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Figure 2-1. Schematic Representation of Test Chamber (not to scale).
Position 2
Position 3
oui
u_u
Position 1
Position 4
Position 5
Figure 2-2. Schematic of Five Coupon Positions on the Support Trays.
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of exposure times that clearly delineated the inactivation
of spores on each material. The four UV-A/B exposure
periods used were the same for all materials and for
both organisms, and consisted of 24, 168, 336, and 672
h, accumulated through successive alternating 12-h
periods of lights on/lights off. For example, the 168-h
UV exposure required 14 calendar days to conduct. At
the conclusion of each UV-A/B exposure period, test and
positive control coupons of all materials were removed
from the test chamber, and the spores were extracted and
enumerated to determine mean log reduction 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, with emphasis on the more
photobiologically active UV-B.3 Wide variations in
natural UV-A/B levels occur due to time of day, day of
the year, location, cloud cover, air pollution levels, and
altitude.4'11 Peak (i.e., noontime) UV-B levels reported
in a few studies range from about 20 to 150 uW/cm2.8'10
The target UV-B level chosen for testing was 70 uW/
cm2, which corresponds to a daily dose of about 3.0 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.jsf). Since
UV-B is more photobiologically important, no specific
UV-A level was targeted. However, the UV-A level was
consistent throughout all experiments, at approximately
100 uW/cm2. This UV-A level corresponds to a daily
UV-A dose of about 4.3 J/cm2 with 12 hours of exposure
per day. The actual total UV-B doses during the 24, 168,
336, and 672 hour exposure periods were 5.8, 43.1, 85.6,
and 169 J/cm2, respectively, in testing with B. anthracis,
and 5.8, 41.9, 83.8, and 169 J/cm2, respectively, in
testing with B. subtilis. The corresponding actual
total UV-A doses during the 24, 168, 336, and 672
hour exposure periods were 8.8, 63.8, 126, and 243 J/
cm2, respectively, in testing with B. anthracis, and
8.7, 58.7, 124, and 243 J/cm2, respectively, in testing
with B. subtilis. (These doses are calculated based on
the measured mean UV intensities in each test, which
are tabulated in Section 4.) A target of zero for UV-C
radiation (i.e., 180-290 nm) was chosen, because of the
absence of this UV component in sunlight at ground
level. The target UV intensities at the positive control
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 test
coupon and positive control coupon trays, at least near
the start and end of every 12-h 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 average, standard deviation (SD),
maximum, and minimum of those position averages are
shown in Section 4.
It must be stressed that each UV-A/B exposure test
with each organism was a separate and unique test,
involving inoculation of the four types of test coupons,
accumulation of UV-A/B exposure in daily 12-hour
doses up to the specified total duration, extraction
of spores from the coupons, and enumeration of the
recovered spores. The eight total tests (four exposure
periods with each of two organisms) were conducted
sequentially over a time period of approximately one
year, and were not simultaneous, overlapping, or nested
in any way. Each test included replicate coupons of
all materials, but there were no replicate runs of the
entire test procedure. Variability in the test results likely
occurred due to variation in procedures, e.g., slightly
different spore inoculations or coupon characteristics
in each test, small variations in temperature, RH, and
UV-A/B conditions from test to test, and variability in
recovery and enumeration processes.
2.3 Spore Recovery Procedures
Following the UV-A/B exposure period, each test,
positive control, 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). With
the exception of bare concrete, the coupons were then
extracted by agitation on an orbital shaker for 15 minutes
at approximately 200 revolutions per minute (rpm) at
room temperature. For bare concrete, recovery of spores
required an alternate procedure in which 45 minutes of
sonication was used instead of the period of agitation.
For all coupons, following extraction 1 mL of the
coupon extract was removed, and a series of dilutions
through 10~7 was prepared in sterile filtered water (SFW).
An aliquot (0.1 mL) of the undiluted extract and of
each serial dilution was then spread plated onto tryptic
soy agar plates and incubated overnight at 35 to 37 °C.
Plates were enumerated within 18 to 24 h of plating. The
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number of CFUs/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.
The use of topsoil as a test coupon required techniques
to ensure adequate recovery of spiked B. anthracis
or B. subtilis spores, and the absence of interference
from native topsoil microorganisms in counting of
recovered spores. A heat shock procedure was found
to minimize interference by native microorganisms.
Specifically, spiked or blank topsoil was extracted in
PBS/Triton X-100 solution as described above, and then
the recovered supernatant was heat-shocked in a water
bath at 65 °C for one hour before being serially diluted
and plated. Topsoil samples spiked with B. anthracis or
B. subtilis spores each showed the presence of a single
homogeneous species, with all colonies of uniform size
and morphologically characteristic of the respective
Bacillus species. Some blank topsoil samples showed
growth of colonies of other native species, which was
not seen with the spiked topsoil samples. Consequently,
although topsoil blanks showed some growth, that
growth did not occur with extracts of spiked topsoil, so
no interference existed in terms of counting recovered
spores. The mechanism by which growth of native
species is suppressed in the extracts of spiked topsoil
was not investigated, but may involve monopolization of
nutrients by the large numbers of spiked spores. By this
procedure, spore recovery trials conducted before testing
showed the recovery of spores spiked onto topsoil to be
approximately 50% for B. anthracis and approximately
34% for B. subtilis.
Blank coupons controlled for viable spores inadvertently
introduced to test coupons, and were spiked with an
equivalent amount of 0.1 mL of "stock suspension" that
did not contain B. anthracis or B. subtilis spores. The
blank coupons underwent the same spore extraction
process as the inoculated coupons, at the same time
as those coupons (i.e., following completion of a UV-
A/B exposure period). To be considered acceptable for
quantitative determination of log reduction, extracts
of blank coupons had to contain no CPUs showing the
morphology characteristic of the respective Bacillus
species. As noted above, the occurrence of native
organisms on uninoculated topsoil coupons did not
violate this blank acceptance criterion. The mean
percent spore recovery from each coupon type was
calculated using results from positive control coupons
(inoculated, not exposed to UV-A/B light), by means of
the following equation:
Mean % Recovery = [Mean CPU ,CFU . * 100 (1)
J L pc/ spike] v '
where Mean CPU c is the mean number of CPUs
recovered from five replicate positive control coupons of
a single type, and CFUspike is the number of CPUs spiked
onto each of those coupons. The value of CFUspike is
known from enumeration of the stock spore suspension.
Spore recovery was calculated for both B. anthracis and
B. subtilis on each coupon type. Spore recoveries from
positive control coupons were within the target range
specified in the test/QA plan in nearly all cases. Sections
2.4 and 3.2 describe the few exceptions.
2.4 Spore Growth and Confirmation
During the 24-h UV-A/B exposure testing, excessively
high spore recoveries were found with B. anthracis
on topsoil test coupons and with B. subtilis on topsoil
positive control coupons. A spore recovery of 370% was
found for B. anthracis on the topsoil test coupons (see
Table 4-4a) and a spore recovery of 260% was found for
B. subtilis on the topsoil positive control coupons (see
Table 4-4b). Although experimental spore recoveries
exceeding 100% can occur due to test variability, these
two results greatly exceeded the expected variability.
To assess whether these results could have been caused
by growth of spore populations after inoculation onto
topsoil coupons, or by misidentification of extracted
spores, a series of tests was done (established by
amendment to the test/QA plan; see Section 3.4). First,
a growth test was conducted by retaining inoculated
topsoil coupons at normal room conditions for one
week before spore extraction. Then tests were done
using qualitative polymerase chain reaction (PCR)
to determine whether the spores found on the topsoil
coupons from the initial testing were in fact B. anthracis
(Ames) or B. subtilis spores, the organisms that were
spiked onto the coupons. This PCR analysis was
implemented to check on the identifications made by
colony morphology.
For the PCR confirmation, the topsoil extracts from
the 24-h UV-A/B exposure tests with B. anthracis
(Ames) and with B. subtilis were replated onto tryptic
soy agar and incubated for 18 to 24 h at 37 °C and 35
°C, respectively. Then 50 individual colonies from the
suspected B. anthracis (Ames) extract and 50 individual
colonies from the suspected B. subtilis extract were
picked with a 1 uL disposable sterile loop. Each picked
colony was suspended in 200 uL of 10 mM Tris-HCl
(pH 8) in a 1.5 mL microcentrifuge tube containing
a 0.22 um filter unit (Millipore, Bedford, MA). Each
-------
microcentrifuge tube was heated at 95 °C for 20 minutes
and then centrifuged at 6,000 x gravity for 2 minutes.
The near-boiling temperature lysed the vegetative cells,
and resulted in release of their DNA. The filter unit was
used to collect cellular debris, and was discarded after
use. The bacterial lysate was stored at -20 °C until PCR
testing.
A commercial off-the-shelf PCR assay (Invitrogen,
Carlsbad, CA) was used to confirm that the DNA
isolated from the suspected B. anthracis (Ames) colonies
was in fact that of B. anthracis origin. This assay utilized
primers specific for the B. anthracis Cap B Domain. A
custom PCR assay (Applied Biosystems, Forster City,
CA) was created by special order to confirm B. sub tills.
Primers were designed that target a conserved region of
B. subtilis chromosomal DNA, since multiple strains of
this bacterium exist. The results of the PCR analysis are
summarized in Section 4.1.
2.5 Calculation of Mean Log Reduction
The effect of UV-A/B on inactivation of spores was
assessed by determining the number of viable organisms
remaining on each test coupon after UV-A/B exposure.
The numbers of colony-forming units (CPUs) of B.
anthracis (or B. subtilis) in extracts of test and positive
control coupons were compared to calculate the mean
log reduction attributable to the UV-A/B exposure.
coupon. When no viable CPUs were found in any of the
five extracts of decontaminated coupons, the final mean
log reduction was reported as greater than or equal to (>)
the LR value from Equation 2.
The variances (i.e., the square of the standard deviation)
of the log,n CFUc.. and log,n CFUt.. values were also
G10 ij G10 ij
calculated for both the control and test coupons (i.e., S2c..
and S2t.), and were used to calculate the pooled standard
error (SE) for the mean log reduction value calculated in
Equation 2, as follows:
SE=
(3)
where the number 5 again represents the number y of
coupons in both the control and test data sets. Thus each
mean log reduction result is reported with an associated
SE value.
The significance of differences in mean log reduction
across different coupon materials and spore types was
assessed based on the 95% confidence interval of each
mean log reduction result. The 95% confidence interval
(CI) is:
95%CI = ±(1.96 x SE)
(4)
First, the base 10 logarithm of the CPU count value from
each coupon extract was determined, and then the mean
of those logarithm values was determined for each set of
test and associated control coupons, respectively. Mean
log reduction (LR) due to UV-A/B exposure for a test
organism on the ith coupon material was calculated as
the difference between those mean log values, i.e.:
Differences in mean log reduction were judged as
significant if the 95% CIs of the two mean log reduction
results did not overlap.
(2)
where Iog10 CFt/c refers to they* individual logarithm
values obtained from the positive control coupons and
Iog10 CFUt refers to they* individual logarithm values
obtained from the corresponding test coupons, and the
overbar designates a mean value.
In tests conducted under this plan, there were five
controls and five corresponding test coupons (i.e.,7 =
5) at each time point for each material. When no viable
CPUs were found in a coupon extract, a CPU count of
1 was assigned, resulting in a Iog10 CPU of zero for that
-------
-------
3.0
QUALITY ASSURANCE/QUALITY CONTROL
Quality assurance/quality control (QC) procedures were
performed in accordance with the program QMP1 and the
test/Q A plan for this evaluation, except as noted below.
QA/QC procedures are summarized below.
3.1 Equipment Calibration
All equipment (e.g., pipettes, incubators, biological
safety cabinets) and monitoring devices (i.e., for
temperature, relative humidity, 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
(A2LA) to the ISO 17025 standard, established National
Institute of Standards and Technology (NIST)-traceable
calibrations of the temperature 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
Quality control efforts conducted during testing included
positive control coupons (inoculated with spores, not
UV-A/B-exposed), 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 spore suspension). The results for these QC
samples in each decontaminant evaluation are included
in the results section (see Section 4).
As noted in Section 2.4, excessively high spore
recoveries were observed in the 24-h UV-A/B exposure
test with B. anthracis on topsoil test coupons, and in
that same test with B. subtilis on topsoil positive control
coupons. Those two occurrences were investigated as
described in Sections 2.4 and 4.1. The cause of those
high recovery values was not identified, and the data
were retained in the test results.
3.3 Audits
3.3.1 Performance Evaluation Audit
The test/QA plan called for a Performance Evaluation
(PE) audit of the UV meters used in testing, by
comparison with responses from independent UV
sensors for the same wavelength ranges (UV-A/B,
UV-B, UV-C). However, that PE audit procedure is
unrealistic, as it has been demonstrated that nominally
similar UV meters from different manufacturers may
give very different readings on the same light source,
due to differences in spectral sensitivity, cosine
response, and type of calibration.12'13 In fact, efforts
were largely unsuccessful to identify UV meters from
other manufacturers applicable to the same wavelength
intervals, with nearly the same spectral response curves,
as the Solarmeters used in testing. Nevertheless, a
comparison was made between the Solarmeter Model 5.7
UV-A/B meter (Serial No. 15957) and a Lutron® Model
UV-340A UV-A/B Light Meter (Serial No. AC89597)
using the Reptisun lamps as the light source. The
Model 5.7 reading (174 uW/cm2) was in only qualitative
agreement with the Model UV-340A reading (304
uW/cm2), as expected. A comparison was also made
between the Solarmeter Model 8.0 UV-C meter (Serial
No. 00275) and a UVP Model UVX Digital Radiometer
(Serial No. E28265) with Model UVX-25 UV-C sensor
(Serial No. 31898). That comparison confirmed that the
Reptisun lamps used in testing produced no detectable
UV-C (i.e., less than the 1 uW/cm2 detection limit on
both meters). No appropriate meter could be found with
which to make a corresponding comparison of UV-B
measurements. However, the average UV-B intensity
of 70 uW/cm2 measured at the test coupons during
testing is in reasonable agreement with expected UV-B
intensities at comparable distances from the Reptisun
lamps
(http://www.uvguide.co.uk/fluorescenttuberesults.htm).
To augment the PE audit, the Solarmeter UV-A/B
and UV-B radiometers purchased for this study were
compared to a similar set purchased from the same
manufacturer for another program. Both sets have NIST-
traceable calibrations established by the manufacturer,
but were obtained separately and have different histories
of use. Table 3-1 shows the results for this comparison,
-------
Table 3-1. Qualitative Performance Evaluation Audit of Solarmeter UV Radiometers
Solarmeter Radiometer
Setla
Model 5.7 (UVA+B)
(S. No. 15957)
Model 6.2 (UV-B)
(S. No. 01802)
Solarmeter Radiometer
Set2b
Model 5.7 (UVA+B)
(S. No. 17493)
Model 6.2 (UV-B)
(S. No. 02988)
Setl
Reading
(uW/cm2)
174
76
Set 2
Reading
(uW/cm)
176
77
%
Difference0
-1.1
-1.3
a Used in this study.
b Obtained from a separate study.
c ((Set 1 Reading/Set 2 Reading)-!) x 100.
which was conducted in the test configuration described
in Section 2.2. Table 3-1 indicates agreement within
about 1% between the two sets of meters.
The test/QA plan also called for a PE audit of the
timepiece used to monitor the UV-A/B exposure
periods. The long exposure times in this testing
made high accuracy in the timing of those exposures
unnecessary. A deviation from the test/QA plan,
documenting the decision not to conduct that PE audit,
was prepared, approved, and placed in the study file.
3.3.2 Technical Systems Audit
Battelle QA staff conducted a technical systems audit
(TSA) at the BBRC during testing on July 15, 2009
to ensure that the evaluation was being conducted in
accordance with the test/QA plan and the QMP.' As part
of the TSA, test procedures were compared to those
specified in the test/QA plan, and data acquisition and
handling procedures were reviewed. Observations and
findings from the TSA were documented and submitted
to the Battelle Task Order Leader for response. No
adverse findings resulted from this TSA. TSA records
were permanently stored with the Battelle QA Manager.
3.3.3 Data Quality Audit
All of the data acquired during the evaluation were
audited. A Battelle 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 Test/QA Plan Amendments and
Deviations
One amendment to the test/QA plan was prepared,
reviewed, approved, and distributed to all parties
involved in this evaluation. That amendment called for
three efforts: performance of a one-week spore growth
test on topsoil coupons; confirmation by PCR analysis
of the identity of spores recovered from topsoil coupons
in the 24-h UV-A/B exposure test; and monitoring
of UV intensity at the positive control coupons to
ensure UV-A, UV-B, and UV-C levels were zero.
Two deviations were prepared, one documenting the
acceptance of the three positive control spore recoveries
noted in Section 3.2, which were below the minimum
target recovery of 1% but still suitable for determining
the mean log reduction due to UV exposure, and one
documenting the absence of a PE audit for timing of the
UV-A/B exposure periods.
3.5 QA/QC Reporting
Each audit was documented in accordance with the
QMP.1 The results of the audits were submitted to the
EPA (i.e., to the NHSRC Quality Assurance Manager
and the TOPO).
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
Battelle 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 Battelle staff member who stored the
record.
-------
4.0
RESULTS
In this chapter spore confirmation efforts, QC results,
and spore inactivation (log reduction) results are
summarized. Data are presented documenting the
uniformity of test conditions, and the mean log
reductions due to UV-A/B inactivation are reported for
each of the organisms on each of the test materials.
4.1 Spore Growth and Confirmation
The extraction and enumeration of spores at the end of
the one-week spore growth period did not indicate any
growth of inoculated spores on the topsoil coupons.
Consequently, the high spore recoveries in the 24-h
test could not be attributed to growth of the inoculated
organisms.
All 50 colonies picked from the replated B. anihracis
(Ames) topsoil extracts were positively confirmed as B.
anihracis by PCR (i.e., threshold cycle (C values of 18
to 20). A liquid culture of B. anihracis (Ames) was used
as a positive control, and a liquid culture of B. subiilis
was used as a negative control. The former culture gave
similar positive PCR results (i.e., Ct< 20), and the latter
gave negative PCR results for B. anihracis (i.e., no
positive response through 45 cycles). In addition, blank
culture solutions (sterile filtered water) gave negative
PCR results for B. anihracis.
All 50 colonies picked from the replated B. subiilis
topsoil extracts were also positively confirmed as B.
subiilis by PCR (again, Rvalues of 18 to 20). A liquid
culture of B. subtilis was used as a positive control,
and a liquid culture of B. anihracis (Ames) was used
as a negative control. The former culture gave positive
PCR results (Ct< 20), and the latter gave negative PCR
results for B. subtilis (i.e., no positive response through
45 cycles). In addition, blank culture solutions (sterile
filtered water) gave negative PCR results for B. subtilis.
In summary, the PCR results confirmed that the
organisms found on the topsoil coupons were the
inoculated species, B. anihracis (Ames) or B. subtilis,
and the week-long growth study suggested that growth
of spores on the topsoil coupons was not the cause of the
high spore recoveries observed in the initial testing. As
a result, those initial unusual results may be attributed
to some undisclosed error in inoculation of the topsoil
coupons. No other unusual results were observed in any
other testing throughout this project.
4.2 QC Results
The positive control spore recovery results were within
the target range of 1 to 150% of the spiked spores, with
a few exceptions. As noted in Sections 2.4 and 3.2,
an excessively high spore recovery was observed with
topsoil positive control coupons in the 24-h UV-A/B
exposure test with B. subtilis and also with B. anihracis.
Those two occurrences were investigated as described in
Sections 2.4 and 4.1. The cause of those high recovery
values was not identified, and the data were retained in
the test results. Also, positive control spore recoveries
below 1% were observed with B. subtilis on unpainted
concrete in the 24 h UV-A/B exposure (0.25% recovery),
with B. anihracis on bare pine wood in the 672 h UV-
A/B exposure (0.93% recovery), and with B. subtilis
on unpainted concrete in the 672 h UV-A/B exposure
(0.40% recovery). While those spore recoveries were
lower than the acceptance criterion in the test/QA plan,
they were more than sufficient for determining the mean
log reduction due to UV-A/B exposure, and thus were
retained in the test results. The low recoveries in the 672
h UV-A/B exposure tests may be due in part to some loss
in viability over that extended time period. A test/QA
plan deviation concerning these spore recovery values
was prepared, approved, and retained in the test files.
All procedural and laboratory blanks met the criterion of
no observed CPUs of the inoculated organism. Growth
of native organisms, with colonies morphologically
distinct from those of B. anihracis orB. subtilis, was
observed from some blank topsoil coupons.
Spike control samples were taken from the spore
suspension on each day of testing, and serially diluted,
nutrient plated, and counted to establish the spore
density used to spike the coupons. This process takes
approximately 24 hours, so the spore density is known
after completion of each day's testing. The target
criterion is to maintain a spore suspension density of 1
x 109/mL (± 25%), leading to a spike of 1 x 108 spores
(± 25%) on each test coupon. The actual spike values
for the four UV-A/B exposure periods with B. anihracis
were 9.43 x lOVcoupon, 8.37 x lOVcoupon, 1.11 x 108/
coupon, and 1.04 x lOVcoupon, respectively. The actual
spike values for the four UV-A/B exposure periods with
B. subtilis were 9.93 x lOVcoupon, 1.01 x lOVcoupon,
1.04 x lOVcoupon, and 7.83 x lOVcoupon, respectively.
Thus all the spike values met the target criterion.
-------
4.3 Uniformity of Test Conditions
Tables 4-1 through 4-3 summarize the test conditions of
UV intensity, temperature, and RH monitored during the
testing.
Table 4-1 shows the average (± standard deviation) and
range of intensities (in uW/cm2) of UV-B, UV-A, and
UVA/B measured at five positions in the test coupon
arrays in each of the eight UV-A/B exposure tests. The
intensity of UV-C at both the test and positive control
coupons was below detection (i.e., < 1 uW/cm2) in all
tests, and the intensities of UV-B and UV-A/B at the
positive control coupons were also below detection in
all tests, so these parameters are not shown in Table 4-1.
The data in Table 4-1 show that close consistency of
the average UV-B, UV-A, and UV-A/B intensities was
maintained across all four exposure time periods with
both B. anthracis and B. subtilis. The UV intensities
found at the five different positions in the test coupon
arrays typically ranged from about 5% less than to
10% greater than the average UV intensity over all five
positions.
Table 4-2 shows the average (± standard deviation)
and range of the temperature and RH monitored near
the center of the test coupon array and the positive
control coupon array in all tests with B. anthracis, both
with the UV-A/B lights on and the lights off. Table
4-3 shows the corresponding information for all tests
with B. subtilis. These tables show close consistency
in the test conditions across all four UV-A/B exposure
time periods. The only substantial departure from test
conditions occurred over a three-day period in the
middle of the 672-h exposure with B. subtilis (Table
4-3, right-hand columns), when RH fell to between 10
and 20%. The cause of this occurrence is not known,
and the RH readings had returned to normal before the
occurrence was discovered. The temporarily low RH
values in the 672-h B. subtilis test are not likely to have
any significant effect on the mean log reduction results.
As expected, when the UV-A/B lights were off, the
test and control coupons experienced closely similar
temperature and RH conditions (i.e., within about 0.5
°C and 5% RH). When the lights were on, the test
coupons experienced slightly higher temperatures (i.e.,
by approximately 3 °C) than did the positive control
coupons, and somewhat lower RH (i.e., by about 10%
RH). These small differences are not expected to have
any impact on the mean log reduction due to UV-A/B
exposure.
-------
Table 4-1. Summary of Ultraviolet (UV-A/B) Light Intensity at the Test Coupons3
UV-A/B Exposure Time Points
24 h
168 h
336 h
672 h
Test Condition3
UV-B Average ± SD
UV-B Range
UV-A Average ± SD
UV-A Range
UV-A/B Average ± SD
UV-A/B Range
"All entries are in |iW/cm2 at the test coupons; all UV intensities were zero at the positive control coupons.
bAverage ± SD refers to average over all five measurement positions; range refers to range of the means from the five measurement positions. SD = standard deviation. UV-A
determined by subtraction of UV-B reading from UV-A/B reading.
GB.a. = results from testing with Bacillus anthracis (Ames).
dB.s. = results from testing with Bacillus subtilis.
Test"
67.0 ±4.7
63.3-75.0
102 ±4.3
98.5-108
169 ±8.4
163-83
Control0
66.9 ±4.5
64.3-75.0
101 ±3. 5
99.3-108
168 ±8.0
164-183
Test
71.4 ±3.3
68.5-76.8
106 ±7.7
99.2-119
177 ±10.9
168-196
Control
69.3 ±3.7
66.8-75.8
97.5 ±4.0
92.9-101
167 ±6.3
160-174
Test
70.9 ±3.8
67.2-76.5
105 ±7.1
97.6-114
176 ±10.8
165-190
Control
69.4 ±3.7
66.0-75.2
103 ±6.6
94.1-108
172 ±10.1
160-183
Test
69.6 ±4.4
65.3-75.6
101 ±8.1
90.7-109
171 ±12.3
156-184
Control
70.0 ±3. 9
67.0-76.5
101 ±8.5
91.3-108
171 ±11.7
158-185
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Table 4-2. Summary of Temperature and Relative Humidity Conditions in UV-A/B Exposure Testing with Bacillus anthracis (Ames) Spores
Test Condition3
UVON
T Average ±SD(°C)
T Range (°C)
RH Average ± SD (%)
RH Range (%)
UVOFF
T Average ±SD(°C)
T Range (°C)
RH Average ± SD (%)
RH Range (%)
24 h
Test"
Control0
Test
UV-A/B Exposure Time Points
168 h 336 h
Control Test Control
672 h
Test Control
28.3 ±1.4
23.4-28.9
31.3±3.6
25.5-44.9
23.8 ±1.0
23.3-28.6
32.5 ±0.6
29.7-33.2
25.0 ±0.7
22.8-25.5
41.1±4.6
33.6-57.9
23.4 ±0.5
23.0-25.3
36.5 ±0.8
35.8-39.1
26.3 ±2.8
20.0-29.0
46.2 ±5.4
29.7-60.0
21.6±1.1
20.4-28.4
50.9 ±3. 5
33.7-60.0
23.6 ±1.6
19.8-25.5
57.7 ±5.1
38.1-68.3
21.4 ±0.9
20.3-25.0
56.3 ±4.2
38.2-68.3
26.6 ±1.0
20.9-28.2
35.4 ±2.7
30.7-49.6
21. 8 ±0.9
20.8-27.6
45. 9 ±2.8
33.1-62.4
22.6 ±0.5
20.8-23.8
47.1 ± 1.8
41.4-59.0
21.7 ±0.5
20.7-23.5
49.9 ±1.6
44.8-65.4
28.4 ±1.1
21.7-31.0
32.0 ±3.0
23.6-52.7
22.8 ±0.9
21.4-27.6
41.4 ±3. 3
28.8-54.5
24.4 ±0.6
21.7-27.0
41.0 ±2.9
30.1-57.4
22.6 ±0.6
21.4-25.0
44.6 ±3.0
33.1-58.2
aUV ON and UV OFF refer to 12-h periods of alternating illumination and darkness in test chamber; averages and ranges shown are over all UV ON or UV OFF periods in the
indicated UV exposure period. T = temperature, RH = relative humidity. SD = standard deviation.
Test coupons (UV-A/B-exposed); readings taken at a central position in the coupon array.
'Positive control coupons (not exposed to UV-A/B); readings taken at a central location in the coupon array.
-------
Table 4-3. Summary of Temperature and Relative Humidity Conditions in UV-A/B Exposure Testing with Bacillus subtilis Spores
Test Condition3
UVON
T Average ±SD(°C)
T Range (°C)
RH Average ± SD (%)
RH Range (%)
UVOFF
T Average ±SD(°C)
T Range (°C)
RH Average ± SD (%)
RH Range (%)
24 h
Test"
Control0
Test
UV-A/B Exposure Time Points
168 h 336 h
Control Test Control
672 h
Test Control
28.3±1.3
23.6-28.9
33.0±2.9
25.7-44.7
23.8±1.0
23.2-28.4
36.2±1.0
34.4-38.3
25.0±0.7
22.8-25.4
42.1±2.5
33.7-53.6
23.3±0.5
22.9-25.1
40.4±2.0
37.4-44.9
25.0±0.8
21.1-26.6
37.6±2.3
33.4-46.1
21.3±0.5
20.4-24.0
44.2±2.2
38.3-59.7
22.4±0.4
20.8-23.5
45.1±2.5
39.8-51.4
21.3±0.4
20.3-24.0
47.6±2.3
41.6-66.9
26.8±1.0
21.3-28.7
38.4±2.9
32.1-53.0
21.6±1.1
20.1-27.3
46.7±3.1
33.6-61.3
23.2±0.7
20.8-25.1
46.5±2.6
41.4-59.9
21.5±0.8
20.5-24.7
50.9±2.3
42.1-64.2
29.0 ±1.1
21.9-30.8
26.5 ±5.3
8.8-44.2
23.1 ±0.9
21.8-28.5
34.1 ±6.4
9.8-45.0
24.8 ±0.8
22.0-28.9
33.2 ±7.0
8.4-45.9
23.0 ±0.6
21.6-25.5
36.2 ±7.3
9.3-47.6
aUV ON and UV OFF refer to 12-h periods of alternating illumination and darkness in test chamber; averages and ranges shown are over all UV ON or UV OFF
periods in the indicated UV exposure period. T = temperature, RH = relative humidity. SD = standard deviation.
Test coupons (UV-A/B-exposed); readings taken at a central position in the coupon array.
Positive control coupons (not exposed to UV-A/B); readings taken at a central position in the coupon array.
-------
4.4 Mean Log Reduction Results
Tables 4-4 through 4-7 show the inactivation (as mean
log reduction) of B. anthracis (Ames) and B. subtilis
spores by UV-A/B exposures lasting 24, 168, 336, and
672 h, respectively. Each of these tables consists of a
part "a" showing the B. anthracis results, and a part "b"
showing the corresponding B. subtilis results. For each
organism on each of the four test materials, the tables
show the spore inoculum; the mean log of the observed
spores on the test, positive control, and blank coupons;
the spore recovery (to two significant figures); and the
resulting mean log reduction (± 95% CI) due to the UV-
A/B exposure. As appropriate, footnotes to Tables 4-4
through 4-7 denote unusual spore recoveries observed
in the initial test (see Section 2.4) or the presence of
endogenous organisms in uninoculated topsoil blanks
(see Section 2.3). The significance of differences in
mean log reduction results among the four coupon
materials and between the two organisms was assessed
by means of the 95% CI values shown in Tables 4-4
through 4-7. Inspection of Tables 4-4 through 4-7
shows that the mean log reductions found on glass at
any UV-A/B exposure time were always significantly
greater than those found on any other material; mean log
reductions on bare pine wood and unpainted concrete
were almost always significantly greater than those on
topsoil. Tables 4-4 through 4-7 show seven cases in
which the mean log reduction found for B. subtilis was
significantly different from the corresponding mean log
reduction found for B. anthracis. However, there is no
clear dependence of those cases on the type of coupon
material or the duration of UV-A/B exposure.
The main conclusion from the results in Tables 4-4
through 4-7 is that UV-A/B inactivation of B. anthracis
and B. subtilis is partly effective on bare wood and
unpainted concrete, ineffective on topsoil, but relatively
effective on glass surfaces.
Tables 4-4 through 4-7 also show that the mean log
reduction results did not always increase uniformly with
increasing UV-A/B exposure time. In some tests with
a particular microorganism/material combination the
LR remained statistically the same (i.e., the 95% CIs
overlapped) despite increased UV-A/B exposure. For
example, with B. anthracis on glass the LR values were
not significantly different for the 168-, 336-, and
672-h time points. This may be attributed to the
relatively large CIs for these LR values, which are due
to the variability of the coupon replicates. Additionally,
the relatively low effectiveness of UV-A/B exposure on
bare wood, unpainted concrete, and topsoil (i.e., mean
log reductions rarely exceeding 1.5 logs) coupled with
the 95% CIs may contribute to the lack of consistent
increase in LR. An example case is the 336 h test with
B. subtilis on unpainted concrete, for which the LR
is low compared to the 168 h result, and without any
overlap of the 95% CIs. This observation may be due to
variability introduced by the separate and unique nature
of each UV-A/B exposure test (as discussed in Section
2.2).
-------
Table 4-4a. UV-A/B Inactivation of Bacillus anthracis (Ames) Spores3 - 24 Hour Exposure
Test Material
Inoculum
(CFUs)
Mean of Logs of
Observed CFUs
Mean %
Recovery
Mean Log
Reduction ± CI
Glass
Positive Controls*
Test Coupons0
Laboratory Blank4
Procedural Blank6
9.43 x 107
9.43 x 107
0
0
7.87 ±0.13
4.10 ±0.30
0
0
82 ±23
0.016 ±0.011
0
0
3.77 ±0.29
Bare Pine Wood
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
9.43 x 107
9.43 x 107
0
0
6.86 ±0.09
6.17±0.15
0
0
7.9 ±1.5
1.6 ±0.52
0
0
0.69 ±0.15
Unpainted Concrete
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
9.43 x 107
9.43 x 107
0
0
7.60 ± 0.27
6.79 ±0.05
0
0
47 ±21
6.6 ±0.72
0
0
0.81 ±0.24
Topsoil
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
9.43 x 107
9.43 x 107
0
0
7.43 ±0.20
8.53±0.10f
0
0
31±11
370 ± 86f
0
0
-1.10±0.19
"Data are expressed as mean of the logs of total number of spores (CFUs) observed on individual coupons, percent recovery, and
mean log reduction.
Inoculated, not exposed to UV-A/B (spore recovery conducted after conclusion of UV-A/B exposure).
'Inoculated, exposed to UV-A/B. UV-A/B exposure accumulated in alternating 12-h periods of light and darkness.
^Laboratory Blank = Not inoculated, placed with positive control coupons and not exposed to UV-A/B
'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.
'Unusual result; investigation of this result discussed in Section 4.1.
CI = Confidence interval (± 1.96 x SE).
""" Not Applicable.
-------
Table 4-4b. UV-A/B Inactivation of Bacillus subtilis Spores3 - 24 Hour Exposure
Test Material
Glass
Positive Controls*
Test Coupons0
Laboratory Blank4
Procedural Blank6
Bare Pine Wood
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Unpainted Concrete
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Topsoil
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Inoculum
(CFUs)
9.93 x 107
9.93 x 107
0
0
9.93 x 107
9.93 x 107
0
0
9.93 x 107
9.93 x 107
0
0
9.93 x 107
9.93 x 107
0
0
Mean of Logs of
Observed CFUs
7.49 ±0.02
4.90 ±0.27
0
0
6.50 ±0.06
5.58 ±0.25
0
0
5.31 ±0.31
4.87 ±0.29
0
0
8.18±0.56f
7.87 ±0.08
0
0
Mean %
Recovery
31 ±1.7
0.092 ± 0.046
0
0
3.2 ±0.45
0.43 ±0.23
0
0
0.25 ±0.18
0.087 ±0.050
0
0
260 ± 220f
77 ±15
0
0
Mean Log
Reduction ± CI
-
2.59 ± 0.24
-
-
-
0.92 ±0.23
-
-
-
0.44 ±0.37
-
-
-
0.31 ± 0.50
-
~
"Data are expressed as mean of the logs of total number of spores (CFUs) observed on individual coupons, percent recovery, and
mean log reduction. Mean log reductions shown in bold are significantly different from mean log reductions for B. anthracis with
the same UV-A/B exposure and coupon material.
Inoculated, not exposed to UV-A/B (spore recovery conducted after conclusion of UV-A/B exposure).
Inoculated, exposed to UV-A/B. UV-A/B exposure accumulated in alternating 12-h periods of light and darkness.
dLaboratory Blank = Not inoculated, placed with positive control coupons and not exposed to UV-A/B.
'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.
Vnusual result; investigation of this result discussed in Section 4.1.
CI = Confidence interval (± 1.96 x SE).
"•" Not Applicable.
-------
Table 4-5a. UV-A/B Inactivation of Bacillus anthracis (Ames) Spores3 - 168 Hour Exposure
Test Material
Glass
Positive Controls*
Test Coupons0
Laboratory Blank4
Procedural Blank6
Bare Pine Wood
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Inoculum
(CFUs)
8.37 xlO7
8.37 xlO7
0
0
8.37 xlO7
8.37 xlO7
0
0
Mean of Logs of
Observed CFUs
7.20 ±0.17
1.95 ±1.83
0
0
6.41 ±0.26
5.26 ±0.29
0
0
Mean %
Recovery
20 ± 8.6
0.0019 ±
0.0025
0
0
3.6 ±2.0
0.27 ± 0.24
0
0
Mean Log
Reduction ± CI
_
5.25 ±1.61
-
_
-
1.16 ±0.34
-
-
Unpainted Concrete
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
8.37 xlO7
8.37 xlO7
0
0
5.93 ±0.14
5.28 ±0.29
0
0
1.1 ±0.30
0.27 ±0.17
0
0
0.65 ±0.28
Topsoil
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
8.37 xlO7
8.37 xlO7
Of
Of
7.11 ±0.07
7.02 ±0.05
0
0
16 ±2.6
13 ± 1.6
0
0
-
0.09 ±0.08
-
-
"Data are expressed as mean of the logs of total number of spores (CFUs) observed on individual coupons, percent recovery, and
mean log reduction.
Inoculated, not exposed to UV-A/B (spore recovery conducted after conclusion of UV-A/B exposure).
'Inoculated, exposed to UV-A/B. UV-A/B exposure accumulated in alternating 12-h periods of light and darkness.
dLaboratory Blank = Not inoculated, placed with positive control coupons and not exposed to UV-A/B.
'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.
fEndogenous organisms were found in uninoculated topsoil blanks; no organisms other than B. anthracis Ames were found on
inoculated coupons.
CI = Confidence interval (± 1.96 x SE).
"-" NotApplicable.
-------
Table 4-5b. UV-A/B Inactivation of Bacillus subtilis Spores3 - 168 Hour Exposure
Test Material
Inoculum (CFUs)
Mean of Logs of
Observed CFUs
Mean %
Recovery
Mean Log
Reduction ± CI
Glass
Positive Controls'1
Test Coupons0
Laboratory Blank4
Procedural Blank6
1.01 x 10s
1.01 x 10s
0
0
7.85 ±0.14
4.01 ±0.09
0
0
73 ±23
0.010 ±
0.0023
0
0
3.84 ±0.15
Bare Pine Wood
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
1.01 x 10s
1.01 xlO8
0
0
6.00 ±0.09
5.89 ±0.28
0
0
1.0 ±0.21
0.87 ±0.43
0
0
0.11 ± 0.23
Unpainted Concrete
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
1.01 x 10s
1.01 x 10s
0
0
7.03 ± 0.02
4.59 ±0.67
0
0
11 ±0.59
0.094 ±0.12
0
0
2.44 ± 0.59
Top soil
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
1.01 x 10s
1.01 x 10s
Of
Of
7.76 ±0.08
7.02 ± 0.20
0
0
58 ±10
11±5.1
0
0
-
0.75 ± 0.19
-
-
"Data are expressed as mean of the logs of total number of spores (CFUs) observed on individual coupons, percent recovery, and
mean log reduction. Mean log reductions shown in bold are significantly different from mean log reductions for B. anthracis
with the same UV-A/B exposure and coupon material.
Inoculated, not exposed to UV-A/B (spore recovery conducted after conclusion of UV-A/B exposure).
'Inoculated, exposed to UV-A/B. UV-A/B exposure accumulated in alternating 12-h periods of light and darkness.
^Laboratory Blank = Not inoculated, placed with positive control coupons and not exposed to UV-A/B.
'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.
'Endogenous organisms were found in uninoculated topsoil blanks; no organisms other than B. subtilis were found on inoculated
coupons.
CI = Confidence interval (± 1.96 x SE).
"-" Not Applicable.
-------
Table 4-6a. UV-A/B Inactivation of Bacillus anthracis (Ames) Spores3- 336 Hour Exposure
Test Material
Glass
Positive Controls*
Test Coupons0
Laboratory Blank4
Procedural Blank6
Bare Pine Wood
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Inoculum
(CFUs)
l.llxlO8
l.llxlO8
0
0
l.llxlO8
l.llxlO8
0
0
Mean of Logs of
Observed CFUs
6.94 ±0.33
1.14 ±1.56
0
0
6.18 ±0.33
5. 17 ±0.66
0
0
Mean %
Recovery
9.8 ± 6.5
0.00025 ±
0.00035
0
0
1.7 ±1.4
0.35 ±0.56
0
0
Mean Log
Reduction ± CI
_
5. 81 ±1.40
-
_
-
1.01 ±0.64
-
-
Unpainted Concrete
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
l.llxlO8
l.llxlO8
0
0
6.37 ±0.28
4.16 ±0.90
0
0
2.5 ±1.7
0.082 ±0.17
0
0
2.21 ±0.83
Topsoil
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
l.llxlO8
l.llxlO8
0
0
7.35 ±0.11
7.18 ±0.02
0
0
21 ±5.0
14 ±0.54
0
0
0.17 ±0.10
"Data are expressed as mean of the logs of total number of spores (CFUs) observed on individual coupons, percent recovery, and
mean log reduction.
Inoculated, not exposed to UV-A/B (spore recovery conducted after conclusion of UV-A/B exposure).
'Inoculated, exposed to UV-A/B. UV-A/B exposure accumulated in alternating 12-h periods of light and darkness.
dLaboratory Blank = Not inoculated, placed with positive control coupons and not exposed to UV-A/B.
'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.
-------
Table 4-6b. UV-A/B Inactivation of Bacillus subtilis Spores3- 336 Hour Exposure
Test Material
Glass
Positive Controls'1
Test Coupons0
Laboratory Blank4
Procedural Blank6
Bare Pine Wood
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Inoculum
(CFUs)
1.04 x 10s
1.04 x 10s
0
0
1.04 x 10s
1.04 x 108
0
0
Mean of Logs of
Observed CFUs
7.49 ±0.17
3. 50 ±0.39
0
0
6.16±0.31
5.22 ±0.66
0
0
Mean %
Recovery
31 ± 9.1
0.0039 ±
0.0025
0
0
1.7 ±1.3
0.35 ±0.43
0
0
Mean Log
Reduction ± CI
_
3.99 ± 0.37
.
.
-
0.94 ±0.64
-
-
Unpainted Concrete
Positive Controls 1.04 x 10s 6.99 ±0.14 9.8 ±2.7
Test Coupons 1.04 x 10s 6.51 ±0.09 3.2 ±0.80 0.48 ±0.15
Laboratory Blank 0 00-
Procedural Blank 0 00-
Topsoil
Positive Controls 1.04 x 10s 6.95 ±0.40 11 ±6.4
Test Coupons 1.04 x 10s 7.02 ±0.17 11 ±3.8 -0.08 ±0.38
Laboratory Blank Of 00-
Procedural Blank Of 00-
"Data are expressed as mean of the logs of total number of spores (CFUs) observed on individual coupons, percent recovery, and
mean log reduction. Mean log reductions shown in bold are significantly different from mean log reductions for B. anthracis with
the same UV-A/B exposure and coupon material.
Inoculated, not exposed to UV-A/B (spore recovery conducted after conclusion of UV-A/B exposure).
'Inoculated, exposed to UV-A/B. UV-A/B exposure accumulated in alternating 12-h periods of light and darkness.
'Laboratory Blank = Not inoculated, placed with positive control coupons and not exposed to UV-A/B.
'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.
fEndogenous organisms were found in uninoculated topsoil blanks; no organisms other than B. subtilis were found on inoculated
coupons.
CI = Confidence interval (± 1.96 x SE).
"-" Not Applicable.
-------
Table 4-7a. UV-A/B Inactivation of Bacillus anthracis (Ames) Spores3- 672 Hour Exposure
Test Material
Glass
Positive Controls'1
Test Coupons0
Laboratory Blank4
Procedural Blank6
Bare Pine Wood
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Inoculum
(CFUs)
1.04 x 10s
1.04 x 10s
0
0
1.04 xlO8
1.04 x 10s
0
0
Mean of Logs of
Observed CFUs
6.15 ±0.29
1.43 ±0.96
0
0
5.97 ±0.12
4.46 ± 0.67
0
0
Mean %
Recovery
1.6 ±0.71
0.00028 ±
0.00061
0
0
0.93 ± 0.24
0.050 ±0.041
0
0
Mean Log
Reduction ± CI
.
4.72 ±0.88
.
~
-
1.51 ±0.60
-
-
Unpainted Concrete
Positive Controls 1.04 x 10s
Test Coupons 1.04 x 10s
Laboratory Blank 0
Procedural Blank 0
6.59 ±0.10
5.08 ±0.61
0
0
3.9 ±0.95
0.30 ±0.49
0
0
1.51 ±0.55
Topsoil
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
1.04 x 10s
1.04 x 10s
Of
Of
7.93 ±0.14
7.73 ±0.09
0
0
85 ±32
52 ±9.4
0
0
0.20 ±0.15
"Data are expressed as mean of the logs of total number of spores (CFUs) observed on individual coupons, percent recovery, and
mean log reduction.
Inoculated, not exposed to UV-A/B (spore recovery conducted after conclusion of UV-A/B exposure).
'Inoculated, exposed to UV-A/B. UV-A/B exposure accumulated in alternating 12-h periods of light and darkness.
^Laboratory Blank = Not inoculated, placed with positive control coupons and not exposed to UV-A/B.
'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.
'Endogenous organisms were found in uninoculated topsoil blanks; no organisms other than B. anthracis Ames were found on
inoculated coupons.
CI = Confidence interval (± 1.96 x SE).
"-" Not Applicable.
-------
Table 4-7b. UV-A/B Inactivation of Bacillus subtilis Spores3- 672 Hour Exposure
Test Material
Glass
Positive Controls'1
Test Coupons0
Laboratory Blank4
Procedural Blank6
Bare Pine Wood
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Unpainted Concrete
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Top soil
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Inoculum
(CFUs)
7.83 x 107
7.83 x 107
0
0
7.83 x 107
7.83 x 107
0
0
7.83 x 107
7.83 x 107
0
0
7.83 x 107
7.83 x 107
Of
Of
Mean of Logs of
Observed CFUs
6.76 ±0.17
1.47 ±1.37
0
0
6.02 ±0.09
4.76 ±0.21
0
0
5.49 ±0.06
3.34 ±0.09
0
0
7.74 ±0.05
7.59 ±0.09
0
0
Mean %
Recovery
7.8 ± 2.3
0.00027 ±
0.00034
0
0
1.4 ±0.27
0.081 ±0.046
0
0
0.40 ± 0.066
0.0029 ±
0.00059
0
0
70 ± 7.6
50 ±10
0
0
Mean Log
Reduction ± CI
_
5.29 ±1.21
-
.
-
1.27 ±0.20
-
-
2.15 ±0.10
-
~
-
0.15 ±0.09
-
-
"Data are expressed as mean of the logs of total number of spores (CFUs) observed on individual coupons, percent recovery, and
mean log reduction.
Inoculated, not exposed to UV-A/B (spore recovery conducted after conclusion of UV-A/B exposure).
'Inoculated, exposed to UV-A/B. UV-A/B exposure accumulated in alternating 12-h periods of light and darkness.
^Laboratory Blank = Not inoculated, placed with positive control coupons and not exposed to UV-A/B.
'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.
'Endogenous organisms were found in uninoculated topsoil blanks; no organisms other than B. subtilis were found on inoculated
coupons.
CI = Confidence interval (± 1.96 x SE).
"-" Not Applicable.
-------
5.0
SUMMARY
Spores ofB. anthracis (Ames) and B. subtilis were
exposed to UV-A/B radiation simulating normal sunlight
on test surfaces of glass, bare pine wood, unpainted
concrete, and topsoil, for periods of 24, 168, 336, and
672 h. Those UV-A/B exposures were accumulated in
alternating 12-h periods of light and darkness, e.g., the
168-h UV-A/B exposure was accumulated over 14 days.
The numbers of viable spores remaining on each test
coupon after UV-A/B exposure were compared to the
numbers remaining on positive control coupons kept in
the same test environment for the same time period but
not exposed to UV-A/B light. The inactivation of spores
by each UV-A/B exposure was calculated as the mean
log reduction in the number of spores on coupons of
each material.
Table 5-1 shows the mean log reduction results for both
B. anthracis and B. subtilis on each of the four materials
at each of the four successive UV-A/B exposure time
points. The 95% confidence interval (CI) is also shown
for each mean log reduction value. Table 5-1 shows that
UV-A/B inactivation was far more effective for both
B. anthracis and B. subtilis on glass than on any of the
other coupon materials. Maximum mean log reduction
results for both B. anthracis and B. subtilis on glass
exceeded 5 logs. Mean log reductions on bare pine
wood and unpainted concrete were similar across both
materials and both organisms, primarily falling in the
range of about 1 to 2 logs at all UV-A/B exposure time
points. Topsoil exhibited the lowest UV-A/B inactivation
results, with mean log reductions that varied around zero
and never reached 1 log reduction for either organism.
Mean log reduction results did not always increase
significantly with increasing UV-A/B exposure
time, probably because of test coupon replicate
variability (resulting in a large CI relative to the mean
LR observed), and test-to-test variability in spore
inoculation, temperature and RH conditions, and other
test procedures. Furthermore, it is possible that the spore
application procedure did not produce a single layer
of spores on the test coupons, leading to protection of
underlying spores from the UV-A/B exposure. This may
also explain some of the observed variability in results.
Additional research would be needed to investigate this
hypothesis.
Table 5-1 indicates those cases in which statistically
significant differences in mean log reduction were
found between the two organisms. Of the 16 such
comparisons, seven show significant differences in
mean log reduction between B. subtilis and B. anthracis.
No clear pattern is evident in the occurrence of these
seven cases with coupon material or UV-A/B exposure
duration.
-------
Table 5-1. Summary of Mean Log Reduction Results with UV-AB (Simulated Sunlight Exposure on Four
Materials
Material
Glass
Bare Pine Wood
Unpainted Concrete
Topsoil
Mean Log Reduction (± 95% CI) by UV-A/B Exposure Time3
24 h
B.a.
3.77
(±0.29)
0.69
(±0.15)
0.81
(±0.24)
-1.10b
(±0.09)
B.s.
2.59
(±0.24)
0.92
(±0.23)
0.44
(±0.37)
0.31
(±0.50)
168 h
B.a.
5.25
(±1.61)
1.16
(±0.34)
0.65
(±0.28)
0.09
(±0.08)
B.s.
3.84
(±0.15)
0.11
(±0.23)
2.44
(±0.59)
0.75
(±0.19)
336 h
B.a.
5.81
(±1.40)
1.01
(±0.64)
2.21
(±0.83)
0.17
(±0.10)
B.s.
3.99
(±0.37)
0.94
(±0.64)
0.48
(±0.15)
-0.08
(±0.38)
672 h
B.a.
4.72
(±0.88)
1.51
(±0.60)
1.51
(±0.55)
0.20
(±0.15)
B.s.
5.29
(±1.21)
1.27
(±0.20)
2.15
(±0.10)
0.15
(±0.09)
*B.a. = B. anthracis (Ames), B.s. = B. subtilis. Values
in bold for B. subtilis are significantly different from
corresponding values for B. anthracis.
bUnusual spore recoveries seen; see text.
-------
6.0
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