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Applied and Environmental Microbiology, November 1998, p. 4410-4415, Vol. 64, No. 11
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Analysis of Airborne Actinomycete Spores with Fluorogenic
Substrates
S. V.
Gazenko,
T.
A.
Reponen,*
S. A.
Grinshpun, and
K.
Willeke
Aerosol Research and Exposure Assessment
Laboratory, Department of Environmental Health, University of
Cincinnati, Cincinnati, Ohio 45267-0056
Received 1 May 1998/Accepted 7 August 1998
 |
ABSTRACT |
The reactions between seven fluorogenic substrates and different
groups of enzymes, esterases, lipases, phosphatases, and dehydrogenases, were studied in a search for a new method for the
detection of actinomycete spores. Fluorescence measurement was chosen
as a fast and sensitive method for microbial analysis. The focus of the
research was on the spores of important air contaminants: Streptomyces albus and Thermoactinomyces
vulgaris. For the measurement of the enzymatic activity, the
chosen fluorogenic substrate was added to a mixture of spores and
nutrient media, and the resulting fluorescence was measured with a
spectrofluorometer. Fluorogenic substrates were found to show
enzymatic activities even for dormant spores. Comparison of the
enzymatic activities of dormant spores with those of vegetative cells
showed similarity of the enzymatic profiles but higher activity for
vegetative cells. The increase of enzymatic activity from dormant
spores to vegetative cells was not linear but fluctuating. The largest
fluctuations were found after 4 to 5 h of incubation. The
enzymatic activities of S. albus were 10 to 50 times lower
than those of T. vulgaris, except for the dehydrogenase
activity, which was seven times higher. These results indicate that
analysis with fluorogenic substrates has the potential for becoming a
fast and sensitive method for the enumeration and identification of
airborne actinomycete spores.
| |
INTRODUCTION |
Actinomycetes are recognized as
important indoor air pollutants. Their appearance in indoor air can be
used as an indicator of mold problems (3, 16). Similar
to fungi, actinomycetes can grow on building materials in wet and warm
places and spread their spores into the indoor air. Exposure to
actinomycete spores can cause various adverse health effects (10,
14, 15). The physical diameter of actinomycete spores is about 1 µm, which makes their counting and identification from field samples
difficult with optical microscopes due to masking by background
particles. Therefore, airborne actinomycete spores have traditionally
been analyzed by impaction onto special nutrient media and subsequent colony enumeration (3, 16). However, the forming of
actinomycete colonies takes at least 1 week. In addition, microbial
impaction may cause significant stress to viable microorganisms
resulting from the mechanical interaction with a solid or semisolid
medium, from desiccation, and from insufficient embedding in the
nutrient medium (20). The viable spores of some species have
dormant status and may need activation before the spore development can begin (5, 12). Thus, fast and reliable detection of these spores from air samples, especially without long preliminary growth, is
highly desirable.
In a search for a reliable and faster method, actinomycete spores were
analyzed in this study by measuring their enzymatic activity in the
presence of artificial substrates. Enzymatic activity is an important
feature of any live cell, and while the characteristics of enzymatic
activities of vegetative cells are well known, less is known about that
of spores (17, 18). Actinomycete spores have various
enzymatic systems: enzymes involved in the central metabolism of
carbon, nitrogen, and phosphorous compounds; enzymes catalyzing
dehydrogenation reactions; cytochrome a, b, and
c, and catalase (5). The enzymatic activity
changes during the transformation of the spores from the dormant stage
to emergence of germination tubes and appearance of the vegetative
cells (11). These features were found by traditional
biochemical methods, which are complicated and time-consuming.
The enzymatic activity of microorganisms can be detected and
measured by using artificial substrates. Fluorogenic substrates engage
directly with special enzymes of live cells through biochemical reactions and turn to fluorescent substances inside or outside the cell. The amount of the resulting fluorescence depends on the
enzymatic activity of the cell. This feature is used in cyto- and
histochemistry, as well as in microbiology for the analysis of
vegetative cells (6, 7, 19). However, not much is known about the interaction between fluorogenic substrates and enzymes of
airborne actinomycete spores. Furthermore, the penetration of large
molecules of fluorogenic substrates through a thick and multilayered
spore sheath and their involvement in spore metabolism need study.
In this study, we investigated the reactions between seven fluorogenic
artificial substrates and the following groups of enzymes: esterases, lipases, phosphatases, and dehydrogenases. In these biochemical reactions, colorless, nonfluorescent fluorogenic substrates are cleaved by microbial enzymes into highly fluorescent substances (8). Because fluorescence analysis is one of the most
sensitive methods for analytical purposes (6), it was
believed that the fluorogenic substrate method could be developed into
a sensitive, fast, and reliable method for actinomycete spore analysis.
The first phase of this method development was focused on the following aspects: (i) investigation of the enzymatic activities of dormant actinomycete spores by using fluorogenic substrates and (ii) study of
the changes in enzymatic activity of actinomycete spores during their
development from dormant cells to emergence and to vegetative cells.
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MATERIALS AND METHODS |
Microorganisms and growth conditions.
Two actinomycete
species were used in the experiments: Streptomyces albus
(ATCC 3004) and Thermoactinomyces vulgaris (ATCC 43649). The test spores were grown according to the recommendations of
the American Type Culture Collection (1). S. albus was grown on ISP medium 2 (ISP2; Difco Laboratories,
Detroit, Mich.) at 37°C for 7 days. T. vulgaris was
grown on Trypticase soy agar (TSA; Becton Dickinson Microbiological
Systems, Cockeysville, Md.) at 50°C for 7 days. These conditions were
sufficient for spore development.
Preparation of spores.
The spores were removed from the
nutrient media and collected on filters for storage. First, the spores
were washed off the ISP2- or TSA-filled petri plates with 10 to 15 ml
of a 0.01% solution of Tween 80 (Fisher Scientific, Pittsburgh, Pa.).
The resulting suspension consisted primarily of spores but also
included some vegetative cells. The latter were then killed, as live
cells may increase the background fluorescence during the enzymatic
activity measurements of the spores and negatively affect the accuracy of the measurements. This was achieved by filtering the suspension through white cellulosic 47-mm-diameter filters (MSI, Westboro, Mass.)
with a pore size of 0.45 µm and then treating the microorganisms on
the filter with 5 ml of a 30% ethanol solution (in water) for 1 min.
Our preliminary experiments with both species showed that the ethanol
concentration and the treatment time were sufficient to kill the
vegetative cells without killing the spores (data not shown). The
filters with the live spores were stored in an airtight box containing
dry silica gel at 4°C for at least 3 days. Storage at 4°C is
appropriate for long-term storage of dormant spores (4) and
for transformation of the spores from the activated to the dormant
stage (12).
Prior to each experiment, the dormant spores were washed off the
filters by shaking the filters for 10 min in a liquid mixture containing 20% autoclaved nutrient broth and 80% autoclaved NaCl solution in distilled water (9 g of NaCl in 1 liter). The ISP2 broth
was used for S. albus, and TSA broth was used for
T. vulgaris. As all repeats of the tests on the
enzymatic activity needed to be performed at the same spore
concentration, the concentration was adjusted to the same level for
each repetition by measuring the optical transmission of the solution
with a spectrophotometer at a wavelength of 600 nm (Spectronic 21D;
Milton Roy Co., Rochester, N.Y.). Distilled water was used as the
standard for 100% optical transmission. The mixture of spores and
nutrient solution was diluted with nutrient solution until the optical
transmission reached the desired value, as indicated below. After the
appropriate spore concentration was achieved, the mixture was divided
into two parts. The first part (25 to 30 ml) was used for measuring the
enzymatic activity of dormant spores. The second part (180 to 200 ml)
was activated and used for measuring the changes of the spore enzymatic
activity during the development of the spores from the dormant stage to
emergence and to the vegetative cell stage. The spores were activated
by heating the suspension of S. albus for 10 min at
45°C and the suspension of T. vulgaris for 10 min at
70°C (13). The activated mixture was poured into eight
petri plates, 25 ml in each, and was incubated at 37°C for S. albus and at 50°C for T. vulgaris.
These petri plates were removed from the incubator one by one, at the
time intervals indicated below, and the enzymatic activities of the
spores were then determined.
The appearance of vegetative cells at the end of each experiment was
monitored by observing the shape of the cells with a
phase-contrast
microscope (Labophot-2; Nikon Corp., Tokyo,
Japan).
Enzymatic activity measurements.
The measurements of the
fluorescence were performed with a spectrofluorometer (Model LS-5;
Perkin-Elmer Corp., Oak Brook, Ill.). The enzymatic activities of the
spores were measured with seven different fluorogenic substrates. We
used four fluorogenic substrates based on
4-methylumbelliferone: 4-methylumbelliferyl phosphate disodium salt
(MUPh), 4-methylumbelliferyl acetate (MUA), 4-methylumbelliferyl
butyrate (MUB), and 4-methylumbelliferyl propionate (MUPr). Two
fluorogenic substrates were based on fluorescein (fluorescein
diacetate [FDA] and fluorescein dipropionate [FDPr]), and one was
based on resorufin:resazurin (RSZ) (see Table 1). The concentration of
all fluorogenic substrates in the storage solutions was 0.2 mg/ml. The
solvent for the storage solutions was acetone optima (Fisher
Scientific) for MUA, MUB, MUPr, FDA, and FDPr. The distilled water was
utilized as the storage solution for RSZ and MUPh.
The measurements of the enzymatic activities were performed for dormant
spores (time zero) and for activated spores. In the
first set of
experiments, the activated spores were analyzed at
different stages by
incubating the mixture of spores and nutrient
solution on petri plates
at the specified temperature for 2, 4,
and 6 h before the
measurement. In this set of experiments, the
optical transmission of
the suspension containing the spores and
nutrient solution was 95.5%,
which corresponded to a concentration
of approximately 2 · 10
6 CFU/ml for both actinomycete species, as determined by
cultivating
dilutions on agar medium. For the measurements, each of
seven
glass tubes, one for each fluorogenic substrate, received 2.5
ml
of the incubated mixture of spores and nutrient media. Then,
0.25 ml of
the fluorogenic substrate solution was added to each
tube and the
fluorescence was measured immediately. This first
measurement gave the
background fluorescence. After this, the
solutions were incubated for
30 min at 40°C and the fluorescence
was measured again. This
measurement gave the total fluorescence,
which includes the background
fluorescence and the fluorescence
resulting from the enzymatic
activity. The enzymatic activity
of each substrate was determined as
the difference between total
and background
fluorescence.
In the second set of experiments, the time changes in enzymatic
activity of
T. vulgaris spores were measured every 30 min
with two of the fluorogenic substrates, FDPr and RSZ. In these
experiments, a higher spore concentration was used to reduce the
time
of incubation with substrates from 30 to 10 min, and thus,
the
concentration permitted more frequent fluorescence measurements.
The
optical transmission of the mixture of spores and diluted
nutrient
media was 92%.
All measurements were performed under the same conditions of the
spectrofluorometer: the excitation slit was set to the minimum
not
exceeding 0.1 nm, and the emission slit was set to the minimum
of 3 nm.
The fluorescence wavelengths used in this study have
been presented in
Table
1. The results are expressed as
arbitrary
units because the focus of this study was to investigate
relative
changes and differences in fluorescence, not the absolute
fluorescence
values. Each experiment was repeated four times.
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TABLE 1.
Fluorogenic substrates used to detect enzymatic
activities of actinomycete spores with a spectrofluorometer with
the indicated wavelengths
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Statistical analysis.
All statistical tests were conducted
with the analysis of variance procedures of the Statistical Analysis
System (SAS Institute, Inc., Cary, N.C.). Scheffe's test was used to
locate the difference that the analysis of variance indicated. The
acceptance level for statistical significance was P < 0.05.
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RESULTS |
The data on the enzymatic activity of spores obtained at
t = 0, 2, 4, and 6 h are presented in Fig.
1. These measurements cover the time
needed for the spore development from the dormant stage to the
emergence of the vegetative cell. Enzymatic activity is shown in
dormant spores with fluorogenic substrates. Both S. albus and T. vulgaris showed esterase, lipase, and
dehydrogenase activity with MUA, MUB, MUPr, FDA, FDPr, and RSZ (Fig. 1a
to f). Phosphatase activity (MUPh) was found to be very weak for both S. albus and T. vulgaris in the dormant
stage (Fig. 1g). With most of the substrates, the fluorescence, i.e.,
the enzymatic activities of spores, increased with increasing
incubation time. However, the magnitude of enzymatic activity changes
was different for different fluorogenic substrates. Esterase and lipase
activities (MUA, MUB, MUPr, FDA, and FDPr) showed modest increases,
about 1.1 to 3.0 times, from the dormant stage until the emergence
stage (Fig. 1a to e), whereas dehydrogenase (RSZ) activity increased 3.5 times in S. albus spores and 17.3 times in
T. vulgaris spores (Fig. 1f). Some measurable amounts
of phosphatase activity (MUPh) were found only after 4 and 6 h of
incubation of T. vulgaris. All enzymatic activities,
except dehydrogenase activity (RSZ), were higher in T. vulgaris spores than in S. albus spores.
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FIG. 1.
Enzymatic activities of S. albus and
T. vulgaris spores with seven different fluorogenic
substrates during spore development. Measurements at time zero were
with dormant spores; other measurements were with activated spores
after 2, 4, and 6 h of incubation. Error bars indicate the
standard deviations from the means of four repeats. Statistical
analysis with analysis of variance and Scheffe's test indicated that
a < b < c (P < 0.05).
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The enzymatic activity changes measured every 0.5 h with
T. vulgaris by using FDPr (lipase activity) and RSZ
(dehydrogenase activity) are presented in Fig.
2. Activation of the dormant
T. vulgaris spores done at t = 0.5 h
(Fig. 2) slightly increased the dehydrogenase activity. After that, the
activities remained fairly constant between 1 and 3.5 h. Large
fluctuations appeared between t = 3.5 and 5 h,
especially with RSZ (dehydrogenase activity).
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FIG. 2.
Lipase (FDPr) and dehydrogenase (RSZ) enzymatic
activities of T. vulgaris measured every 30 min. The
enzymatic activity of dormant spores is seen at t = 0;
that of activated spores is seen at t = 0.5 h. The
measurements between 1 and 5 h show the enzymatic activity during
the spore development from activated spore status toward the vegetative
cell status. Error bars indicate the standard deviations from the means
of four repeats. Statistical analysis with analysis of variance and
Scheffe's test indicated that a < b (P < 0.05).
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After 7 to 7.5 h of incubation, all spores were transformed into
vegetative cells, which was confirmed by examining the shape of the
cells under a phase-contrast microscope. Differences in enzymatic
activities between dormant spores and vegetative cells are shown in
Fig. 3 for S. albus and
in Fig. 4 for T. vulgaris. The enzymatic activities of S. albus
vegetative cells were two to seven times higher than those of dormant
spores. The same ratio for T. vulgaris ranged from 1 to
25, except for the phosphatase activity, which was almost absent in the
dormant stage. S. albus had the largest increases from
dormant spore to vegetative cell in esterase (FDA), lipase (FDPr),
dehydrogenase (RSZ), and phosphatase (MUPh) activities. T. vulgaris had the largest increases in lipase (FDPr),
dehydrogenase (RSZ), and phosphatase (MUPh) activities. Similar
to the spores, all enzymatic activities of the S. albus vegetative cells, except for the dehydrogenase
activity (RSZ), were lower than those of T. vulgaris.
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FIG. 3.
Enzymatic activities of dormant spores and vegetative
cells of S. albus. Error bars indicate the standard
deviations from the means of four repeats.
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FIG. 4.
Enzymatic activities of dormant spores and vegetative
cells of T. vulgaris. Error bars indicate the standard
deviations from the means of four repeats.
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Information about the difference between background fluorescence and
the fluorescence caused by enzymatic activity is very important for
creating a method of actinomycete spore enumeration based on
fluorescence measurements. This difference is shown for dormant spores
of S. albus in Fig. 5 and
dormant spores of T. vulgaris in Fig.
6. These figures suggest that the
background fluorescence is caused mainly by the nutrient solution and
that the difference between the total fluorescence and the background fluorescence is caused by the enzymatic activity of the dormant spores.
Except for MUPh, the total fluorescence was 1.1 to 3 times higher than
the background for S. albus and 1.4 to 70 times higher for T. vulgaris. The largest increases of the total
fluorescence compared to the background fluorescence were observed with
MUA, MUB, MUPr, and FDA for both species, as well as with RSZ for
S. albus and with FDPr for T. vulgaris
(Fig. 5 and 6).
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FIG. 5.
Background fluorescence and total fluorescence
(background plus fluorescence resulting from enzymatic activity) of
dormant spores of S. albus. Error bars indicate the
standard deviations from the means of four repeats.
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FIG. 6.
Background fluorescence and total fluorescence
(background plus fluorescence resulting from enzymatic activity) of
dormant spores of T. vulgaris. Error bars indicate the
standard deviations from the means of four repeats.
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DISCUSSION |
The data presented in Fig. 1a to g show that all fluorogenic
substrates reacted with spore enzymes. For these experiments, only
those fluorogenic substrates that react with large groups of enzymes
were used: esterases, lipases, phosphatases, and dehydrogenases. Each
group contains tens of specific enzymes that react with the substrate
and create the average esterase, lipase, phosphatase, or dehydrogenase activity.
Unexpectedly, one of the largest and most diverse enzyme groups,
phosphatases, showed very weak activity in the dormant stage of both
species. There are several possible explanations for this phenomenon.
The nutrient solutions used for spore incubation could have contained
many more available soluble phosphates than phosphate of MUPh. The pH
conditions may not have been favorable for MUPh acceptance by
phosphatases, or soluble MUPh joined another reaction(s) before
contacting phosphatases. It is also possible that the spores of
S. albus and T. vulgaris do not contain
phosphatases in the dormant stage and start to exhibit phosphatase
activity only after 3 to 4 h of incubation with nutrient solution.
The last postulate suggests that the phosphatase activity could be used
as a marker between dormant spores and vegetative cells. Almost all
groups of enzymes were more active in T. vulgaris
spores than in S. albus spores. We can connect this
phenomenon with the high level of T. vulgaris
thermoresistance. It is known that organisms developed under a high
temperature have enhanced levels of metabolism (2), i.e.,
enhanced enzymatic activity. This feature suggests that it may be used
to differentiate thermoresistant from mesophilic microorganisms.
However, further experiments with other thermoresistant microorganisms
are needed to prove this hypothesis.
Only dehydrogenase activity (RSZ) was higher in spores of S. albus than in spores of T. vulgaris, but only in
the beginning of incubation. This may be due to the smaller amount
of cytochrome a in thermophilic than in mesophilic
spores in the dormant stage (21), which leaves less
opportunity for respiration and activity of the dehydrogenase system.
The dehydrogenase activity increased faster in T. vulgaris than in S. albus spores during incubation and after 6 h of incubation reached the same level for both
species. We attribute the faster increase of the dehydrogenase activity in thermophilic T. vulgaris spores than in
S. albus spores to the fast increase in the amount of
cytochrome a and c during the transformation of
thermophilic spores into vegetative cells (21).
The fluctuations in enzymatic activity during spore development,
measured every 0.5 h (Fig. 2), can be attributed to the
substantial biochemical changes in the spores during
their transformation from dormant spores to vegetative
cells. The sequence of events in germination includes endogenous
metabolism, excretion of calcium-dipicolinic acid, loss of the spore
cortex, lipid turnover, active RNA synthesis, DNA synthesis, and other
events caused by lipases, proteases, and dehydrogenases (8, 11,
18). Conversion from one phase to another significantly changes
the enzyme composition. In our tests, each fluorogenic substrate
represented a large group of enzymes; therefore, changes inside each
group cannot be distinguished. Nevertheless, increases in the
lipase activity are probably caused by the transformation of spores
into more metabolically active vegetative cells, and increases in
the dehydrogenase activity after 3.5 to 5 h (Fig. 2) can be
explained through the transformation of endogenous into exogenous respiration.
Comparison of the enzymatic activities in spores and vegetative cells
shows higher activity of the vegetative cells (Fig. 3 and 4). This can
be explained by the more active metabolism of vegetative cells, by the
exchange of nutrients with the surrounding environment, and by
reproduction. It appears that the detected large difference
between dormant spores and vegetative cells in dehydrogenase (RSZ) and
phosphatase (MUPh) activities can be used in the future to distinguish
the spores from vegetative cells. Except for the phosphatase and
dehydrogenase activities, the enzymatic activities of dormant spores
followed the pattern of activities of vegetative cells, but at lower
levels. This finding may also be useful in the development of future
identification methods for spore species.
The accuracy and reliability of the fluorescent method
strongly depend on the amount and fluctuations of the
background fluorescence. Figure 5 and 6 show comparisons of the
background fluorescence with the total fluorescence after 0.5 h of
incubation with the substrates. The total fluorescence has to be
sufficiently higher than the background fluorescence for enzymatic
activity measurements. Spores of T. vulgaris show a
greater difference between the background and total fluorescence levels
than do spores of S. albus, with the exception of RSZ
and MUPh. Background and total fluorescence levels for phosphatase
activity (MUPh) were almost the same for both S. albus
and T. vulgaris, indicating that the dormant spores have very low phosphatase activity.
It appears that the background fluorescence may be decreased,
thus increasing the sensitivity of the measurements. In our experiments, the background fluorescence was mainly caused by the
nutrient solution (20% nutrient broth in NaCl solution). It included
different fluorescent substances such as proteins and vitamins and had
maximum fluorescence at 420 nm when excited at 320 nm and at 530 nm
when excited at 480 nm, i.e., close to the wavelengths used in this
study (fluorescence at 450, 515, and 590 nm and excitation at 320, 480, 560 nm, respectively). If the fluorescence measurements are performed
for only dormant spores, there is no need for incubation with a
nutrient solution. Therefore, a buffer that does not give any
background fluorescence could substitute for the nutrient solution.
According to our study, MUA, MUB, MUPr, and FDA appear to
be the most suitable fluorogenic substrates for the analysis of the
live spores of both tested species. These substrates can be used with
macroanalytical instruments, such as fluorometers and microplate
readers, or with microanalytical instruments, such as luminescence
microscopes or flow cytometer systems. The development of this
methodology needs further research. For example, the possible interferences due to environmental debris in field samples have to be
studied. However, the results of this study show that measurements of
the enzymatic activities with fluorogenic substrates can be used for
spore analysis without long preliminary growth. It is hoped that our
data will be useful for the development of fast and sensitive
methods for enumeration and identification of indoor air spores.
Conclusions.
The results of this study indicate that
enzymatic activities of actinomycete spores can be measured by
using artificial fluorogenic substrates. With this method, even dormant
spores show enzymatic activities. The enzymatic activities of spores
were found to increase when the spores develop from the dormant stage
into vegetative cells. This increase with time is not linear and
reflects biochemical changes that occur in spores during their
development. The increase in enzymatic activity is different for
different substrates. The enzymatic profiles of dormant spores
significantly differ from those of vegetative cells. The enzymatic
activities of S. albus and T. vulgaris
were found to have some special characteristics that can be used to
differentiate them. Our results indicate that measurements with
fluorogenic substrates have the potential for development into a fast
and sensitive method for the enumeration and identification of airborne
actinomycete spores.
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ACKNOWLEDGMENT |
This work was supported by the U.S. Center for Indoor Air
Research (CIAR) through a postdoctoral fellowship to Sergey V. Gazenko. We are thankful for this support.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Aerosol Research
and Exposure Assessment Laboratory, Department of
Environmental Health, University of Cincinnati, P.O. Box 670056, Cincinnati, OH 45267-0056. Phone: (513) 558-0571. Fax: (513)
558-2263. E-mail: Tiina.Reponen{at}uc.edu.
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Applied and Environmental Microbiology, November 1998, p. 4410-4415, Vol. 64, No. 11
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
