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Applied and Environmental Microbiology, October 1998, p. 3807-3812, Vol. 64, No. 10
Aerosol Research and Exposure Assessment
Laboratory, Department of Environmental Health, University of
Cincinnati, Cincinnati, Ohio, 45267-0056,1 and
Health Research Services Division, DynCorp, Durham, North
Carolina 277032
Received 27 April 1998/Accepted 5 August 1998
Airborne actinomycete spores, important contaminants in
occupational and residential environments, were studied with respect to
their (i) release into the air, (ii) aerodynamic and physical size
while airborne, and (iii) survival after collection onto agar with an
impactor. Three actinomycete species were selected for the tests to
exemplify the three main spore types: Streptomyces albus
for arthrospores, Micromonospora halophytica for
aleuriospores, and Thermoactinomyces vulgaris for
endospores. The results show that the incubation conditions
(temperature, time, and nutrients) needed for the development of spores
for their release into air are different from the conditions that are
needed for colony growth only. Additional drying of M. halophytica and T. vulgaris cultures was needed
before spores could be released from the culture. The aerodynamic sizes
of the spores, measured with an aerodynamic particle sizer, ranged from
0.57 (T. vulgaris) to 1.28 µm (M. halophytica). The physical sizes of the spores, when measured with a microscope and an image analysis system, were found to be
smaller than previously reported in the literature. The relative recovery of the spores on agar media ranged from 0.5 (T. vulgaris) to 35% (S. albus). The results indicate
that the culturability of the collected airborne actinomycete spores
varies widely and is affected by several variables, such as the species
and the sampling flow rate. Therefore, alternatives to commonly used
cultivation methods need to be developed for the enumeration of
actinomycete spores.
Actinomycetes are a diverse group of
gram-positive bacteria. They resemble fungi because they are adapted to
life on solid surfaces (8) and they can produce mycelium and
dry spores like most fungi (15). Actinomycete spores are
known to be important air contaminants in occupational environments,
such as agriculture and waste composting facilities (18,
26), and have recently gained special attention as indicators of
mold problems in buildings (31). They do not belong to the
normal microbial flora in indoor air but have been found in buildings
suffering from moisture and mold problems (4, 25). In
addition, airborne spores of several actinomycete species (e.g.,
Saccharopolyspora rectivirgula, Micropolyspora faeni, Thermoactinomyces vulgaris, and
Streptomyces albus) have been related to the incidence of
allergic alveolitis and other severe health effects (14, 19,
21, 28, 33). The cellular mechanism of the health effects
caused by actinomycete spores was recently studied by Hirvonen et al.
(11). Their study shows that Streptomyces spp.
are able to stimulate lung macrophage reactions, which can lead to
inflammation and tissue injury.
Actinomycete spores are formed either by subdivision of existing hyphae
by fragmentation or swelling or by endogenous spore formation. The
hyphae that subdivide into spores can be sheathless or have a sheath,
which partly remains in the spores after fragmentation (35).
This leads to three main spore types: arthrospores (subdivision of
sheathed hypha), aleuriospores (subdivision of sheathless hypha), and
endospores. The significance of the differences in the spore structure
is not known, but these differences are expected to cause differences
in the survival and airborne behavior of these spores.
Although actinomycete spores have been detected in air samples, their
release into the air is not well understood. In nature, actinomycete
spores can become airborne by mechanical disturbance of the substance
they are growing on, e.g., by operation of an agricultural implement or
by exposure to gusty wind (22). Only a few laboratory
studies have been performed using airborne actinomycete spores. Lacey
and Dutkiewicz (20) released actinomycete spores from
contaminated hay by mechanical handling, whereas Madelin and Johnson
(24) released actinomycete spores from culture media by air
currents. Actinomycete spores are more difficult to aerosolize than
fungal spores because they are smaller than fungal spores (30). More information needs to be gained on the aerodynamic diameter (da), agglomeration, and hygroscopicity
of airborne actinomycete spores because these properties affect
actinomycete behavior in air, in the human respiratory tract, in
air-purifying filters, and in aerosol samplers.
Actinomycetes have been sampled from the air with impactors and have
been analyzed by culturing (4, 7, 25). However, it is not
clear how many of the total number of viable spores can be enumerated
by these techniques. Collection by impaction can cause stress, which
may result in the decrease of microbial culturability. This has been
demonstrated with vegetative cells of bacteria (32), but no
information appears to have been available prior to this study on the
collection stress of spores. The culturability of impacted
microorganisms may also be decreased when they are insufficiently
embedded in the agar (32). When the impaction velocity is
low, the microorganisms may deposit on the top of the collection agar
instead of penetrating into the agar medium. Thus, they may have
limited ability to obtain nutrients and moisture from the agar. On the
other hand, endospores usually need activation, e.g., by heating,
before they are able to germinate and form colonies (15).
In the present study, we investigated the following properties of
actinomycete spores: (i) their release into the air, (ii) their
aerodynamic and physical sizes in the airborne state, and (iii) their
survival after collection onto agar with an impactor. The release of
actinomycete spores was studied semiquantitatively to find the growth
and aerosolization conditions which ensure a sufficient amount of
released spores for the experiments. Information on the properties of
actinomycete spores can be used for the development of new detection
and control methods for them.
Actinomycete species and their preparation for
aerosolization.
Three actinomycete species available from the
American Type Culture Collection (ATCC, Rockville, Md) were selected to
represent the three main spore types: S. albus (ATCC
3004) represented arthrospores, Micromonospora halophytica
(ATCC 27596) represented aleuriospores, and T. vulgaris
(ATCC 43649) represented endospores. S. albus spores
are formed in chains and are slightly ellipsoidal in shape. They have
been reported to be 0.7 to 1.0 µm in length and 0.7 µm in width
(24). M. halophytica spores are formed as
singlets and are spherical. Their physical size has been reported to be about 1.2 µm (16). T. vulgaris spores are
produced as singlets, and they are spherical or slightly ellipsoidal.
They have been reported to be 0.5 to 1.5 µm in physical size
(18) and to have the same morphology as endospores of
Bacillus and Clostridium spp. (5, 8).
As is typical for endospores, Thermoactinomyces spores are
normally dormant and need activation to enhance their germination. In
this study, cold activation was used for T. vulgaris samples by keeping the samples at 20°C for 24 to 48 h before
incubation, as suggested by Kalakoutski and Agre (15).
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Characteristics of Airborne Actinomycete
Spores
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
TABLE 1.
Incubation conditions for actinomycete spores
3) was related to the sensitivity of the
aerodynamic particle sizer, which measured the total concentration of
spores as described below.
Experimental setup for aerosolization of spores.
The
physical and microbiological characteristics of actinomycete spores
were determined by using the experimental setup schematized in Fig.
1. To prevent the release of spores
outside the experimental system, all components were housed inside a
biosafety cabinet (Model 6TX; Baker Company, Inc., Sanford, Maine).
This setup has previously been used for testing different dispersion
techniques for other microorganisms (30). Actinomycete
spores were aerosolized by using our recently developed swirling-flow
disperser (30). In this disperser, agar was placed on the
inner walls of the dispersion vessel. The agar was inoculated, covered,
and incubated as described above. After incubation, HEPA-filtered air
was blown through two nozzles, which were directed tangentially toward
the actinomycete growth on the inner wall of the vessel. This air flow
(15 liters min1) released actinomycete spores directly
from the microbial growth. The spore aerosol was diluted with
HEPA-filtered air ranging from 15 to 40 liters min1,
depending on the airborne spore concentration desired for the test. The
desired concentration was calculated for the optimal colony surface
density on impactor samples with insignificant masking effect
(3). Unless indicated otherwise, the test aerosol flow was
adjusted to 30% ± 5% RH by mixing appropriate portions of desiccated
and humidified air.
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Measurement of spores.
In the measurement chamber of the
setup depicted in Fig. 1, the total concentration and the size
distribution of aerosolized actinomycete spores were measured with an
aerodynamic particle sizer (Aerosizer, API Mach II; Amherst Process
Instruments, Inc., Hadley, Mass.). The Aerosizer measures the
concentration of particles and the health-related aerodynamic
equivalent diameters, which depend on the shape and density of the
particles. We assume that the detection limit of the Aerosizer is
0.0004 airborne spores cm3, which corresponds to the
detection of one particle during a 1-min time interval at a flow rate
of 2.5 liters min1. However, at least 10 particles per
size channel should be measured to achieve reliable size distributions
(10). Assuming that the particle size distribution consists
of 10 size channels and the aerodynamic particle sizer is operated for
1 min at 2.5 liters min1, the minimum total spore
concentration needed for statistically reliable data is 0.04 spores
cm3. The culturable count of actinomycete spores was
measured with the N-6 Andersen impactor (Graseby Andersen, Smyrna, Ga.)
(12) and with an agar slide impactor. The latter device is
not commercially available but has been described in detail by
Juozaitis et al. (13). The relative recovery of collected
spores was determined by relating the culturable count of spores
collected on the impactor substrate to the total count measured
upstream and downstream of the impactor with the Aerosizer, as further
described below.
Microscopic size measurements. In order to determine the size distribution of actinomycete spores, a piece of actinomycete culture was taken from an agar plate with a sterilized knife, placed on a microscopic slide (Superfrost/Plus; Fisher Scientific, Pittsburgh, Pa.), and allowed to air dry. A drop of permount medium (Fisher Scientific) was added and then covered with a coverslip. The sample was then digitally imaged with a color video camera (DXC-760MD 3CCD; Sony Electronics, Inc., Tokyo, Japan) attached to the trinocular head of a phase-contrast microscope (Eclipse E800; Nikon Corp., Tokyo, Japan). The phase-contrast optical system consisted of a phase-contrast condenser (0.9 N.A.) and an oil immersion objective (1.25 N.A., CFI DL 100X; Plan Achromat). The video camera control unit was connected to a Matrox Millennium video board. The actinomycete culture sample was analyzed using the Image-1/MetaMorph imaging software system (Universal Imaging Corporation, West Chester, Pa.) running on a Pentium 166 MHz CPU platform. Interactive morphometric analysis of the samples with Image-1/MetaMorph system consisted of decoding the color image, configuring the decoded image "Lookup Table" as a monochrome, and then applying a threshold-based boundary detection algorithm. A calibration scale was then activated and the "Show Regional Statistics" tool was engaged to display the resulting morphological measurements.
Prior to measuring the size of the actinomycete spores, the microscope and image analysis system were calibrated with standard polystyrene latex particles (PSL) of two sizes: 1.02 and 2.43 µm (Bangs Laboratories, Inc., Carmel, Ind.). When determining the size of PSL particles and of each of the actinomycete species, at least 100 particles were measured per sample, from which the averages and standard deviations were calculated.Assessment of the recovery of aerosolized spores.
The
relative recoveries (see definition below) of different actinomycete
species were compared with each other by use of an Andersen impactor,
which is widely used in occupational and public health applications to
collect actinomycete spores (4, 7, 25). Because the spore
size distribution was measured with the Aerosizer, only the sixth stage
of the Andersen sampler (N-6) was utilized for spore collection. The
flow rate for the Andersen impactor was 28.3 liters min1
(air velocity through each of the 400 impaction holes was 24 m
s1). To test the effect of impact stress on the relative
recovery of S. albus spores, the spores were collected
with the agar slide impactor at flow rates of 3.8, 6, 8, 10, 15, 20, 25, and 28 liters min1. These flow rates correspond to
air velocities through the single slit of this impactor of 24, 38, 50, 63, 94, 125, 156, and 175 m s1, respectively. The
velocity of spore impact on the agar surface is approximately equal to
the velocity of the air jet coming from the slit or from the holes of
the impaction plate above the agar surface.
3.
The percent relative recovery was determined as
CCFU/(Ctotal × Ecoll) × 100, where CCFU
is the concentration of spores obtained with the Andersen impactor or
the agar slide impactor (CFU m3),
Ctotal is the total spore concentration obtained
with the Aerosizer (spores m3), and
Ecoll is the collection efficiency of the
bioaerosol impactor. The last parameter was determined for each
actinomycete species and each impactor flow rate by measuring the spore
concentrations up- and downstream of the bioaerosol impactor with the
Aerosizer.
Assessment of the effect of spore embedding in agar. The association between the relative recovery and different levels of spore embedding in agar during collection with the impactor was studied by conducting two experiments with S. albus spores. In both experiments, agar plates were first exposed to spores in the test chamber for 20 s by letting the spores sediment onto the agar. Gravity settling is not a recommended practice for the quantitative sampling of bioaerosols in field situations because it depends on particle size and is influenced strongly by air turbulence. Our experiments were done in the laboratory in a measurement chamber with laminar air flow and with monodisperse spore aerosols. The gravity settling was used in these experiments to minimize the impaction velocity and thus eliminate the embedding of spores during their retrieval on the agar.
In the first experiment, the spores were embedded in agar by manually pressing against each other two identical agar plates with spores deposited on them. The agar plates (5 cm in diameter) were prepared as contact plates so that the surface of the agar was higher than the walls of the agar plates. From 18 agar plates with spores on them, 6 randomly selected agar plates were incubated as controls, and the remaining 12 agar plates were randomly divided into six pairs of agar plates. The two agar plates of each pair were manually pressed against each other, thus embedding the spores deep into the agar. In the second experiment, the depth of embedding was studied. A wedge-shaped agar layer was deposited on top of the spores, as shown in Fig. 2. Thus, the spores were either fully exposed or covered by an agar layer that varied from a depth of 0.1 mm on the thin side of the wedge to a depth of 5 mm on the thick side of the agar wedge. About half of the surface area was left uncovered with agar to confirm the uniformity of the spore deposition.
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Statistical analysis. All statistical tests were conducted in accordance with the general linear model procedures of the Statistical Analysis System (SAS Institute, Inc., Gary, N.C.). The Scheffe's test was used to identify the difference that the general linear model indicated.
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Release of actinomycete spores into air.
The first set of
experiments on the study of actinomycete spore release from surfaces
was conducted by growing the actinomycete cultures under the conditions
recommended by the ATCC (Table 1). It was found that these conditions
are sufficient for colony formation and spore production but are not
optimal for the efficient release of spores from the swirling-flow
disperser. For instance, regular NZA and TSA media turned out to be too
soft: the dispersing air flow produced holes on the agar layer. The
amount of agar in the growth media was therefore increased from 1.5 to
2% (i.e., to the same level as in the ISP2 medium). Microscopic
examination showed that this increase in agar concentration increased
the amount of spores produced by the actinomycete culture. However, the
amount of spores released from any of the three actinomycete cultures
was still below the detection limit of the Aerosizer (0.0004 spores
cm3) when cultures were incubated for 1 to 5 weeks at the
incubation temperatures recommended by the ATCC.
3).
In the third set of experiments, the actinomycete cultures were again
prepared with 2% agar but incubation was tested at three different
temperatures: 26, 37, and 50°C. As expected, mesophilic S. albus and M. halophytica did not grow at 50°C,
and thermophilic T. vulgaris did not grow at 26°C.
Among these temperatures, 37°C turned out to be most suitable for
growing S. albus and M. halophytica for
aerosolization experiments. S. albus released an
average concentration of 1.21 ± 0.43 spores
cm3 (n = 5) after 1 week of incubation at
37°C, which was sufficient for the experiments. When testing
T. vulgaris and M. halophytica, it was
found that drying the culture after incubation was necessary before the
spores could be released from the culture. Incubation of M. halophytica for 3 weeks at 37°C and of T. vulgaris for 3 weeks at 50°C with subsequent drying were found
to be suitable for sufficient spore release. In the subsequent
experiments, the cultures were dried by keeping the culture vessels in
the incubator uncovered for 24 h before aerosolization. The
average concentrations of aerosolized M. halophytica
and T. vulgaris spores were 0.13 ± 0.10 and
0.21 ± 0.10 spores cm3, respectively
(n = 5 for both species).
The conditions found for sufficient release of the three actinomycete
spore species are summarized in Table 1.
Physical characteristics of airborne actinomycete
spores.
Typical size distributions of spores representing
the three actinomycete species are presented in Fig.
3. The Aerosizer measured the number
concentration, N, of spores in equal logarithmic intervals of particle
size, with the size expressed as da (29,
30, 34). For a particle density of 1 g cm3,
the da equals the physical diameter of a
spherical particle (2). As seen, the geometric mean of the
da, dg, ranged from 0.57 µm for T. vulgaris to 1.28 µm for
M. halophytica, and the geometric standard
deviation, 
g, was fairly narrow, ranging from
1.24 for M. halophytica to 1.54 for T. vulgaris. The sizes for the three different actinomycete spores
were all statistically significantly different from each other
(P = 0.0001).
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100% (n = 3), which was a statistically significant
size increase (P = 0.0001).
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Spore survival during sampling. The relative recovery values for spores of the three different actinomycete species were as follows: 35.3% ± 11.6% for S. albus (n = 4), 7.4% ± 5.2% for M. halophytica (n = 3), 0.5% ± 0.1% for T. vulgaris without activation (n = 3), and 4.6% ± 1.0% for T. vulgaris (n = 5) with cold activation. The relative recovery of S. albus was statistically significantly higher than the recoveries of the two other actinomycetes (P = 0.0001).
The effect of the impaction process on the relative recovery of actinomycete spores was studied more closely with the agar slide impactor. The relative recovery of S. albus spores varied from 30 to 86%, depending on the flow rate (Table 3). The lowest air flow rate, 3.8 liters min1 resulted in the lowest relative recovery,
30%. The recovery rate reached its maximum at an air flow rate of 20 liters min1 and decreased with further increases in
the flow rate.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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The experiments on the release of actinomycete spores showed that the incubation conditions needed for the development of spores for their release into air are different from the conditions needed for colony growth only. An increase in agar concentration from 1.5 to 2% was found to increase the production of spores such as M. halophytica and T. vulgaris. An increase in spore production with increasing agar concentration has also been observed by Kalakoutski and Agre (15) in their tests with Streptomyces spp. They showed that lower agar concentrations favor vegetative growth, whereas higher agar concentrations enhance the sporulation of Streptomyces spp. However, in the present experiments, spore release was not enhanced with increasing amounts of spores in the actinomycete culture. S. albus needed elevating the incubation temperature, T. vulgaris needed drying of the culture, and M. halophytica needed both of these treatments for sufficient spore release. One explanation for the need for these specific conditions to enhance spore release may be found in the spore maturation process. S. albus and M. halophytica are formed by the fragmentation of hyphae. If the fragmentation process is not totally completed, e.g., due to insufficient temperature, the spores may not be readily released even when they have grown into spore chains that are observable under the microscope. T. vulgaris is formed inside the hyphae and may also need specific conditions before the spores can be effectively released from the hyphae.
Only limited information on the concentrations of individual
actinomycete species in air is available in the literature. Madelin and
Johnson (24) aerosolized three different actinomycete
species that were growing on a mixture of agar and hay and
measured the airborne concentrations of their spores with
an aerodynamic particle sizer. The concentrations of
S. albus spores in their experiments were below
4.5 spores cm3, while the concentrations of two
thermophilic actinomycetes, Faenia rectivirgula and
Saccharomonospora viridis, were as high as 170 spores
cm3. Nevalainen et al. (25) measured
mesophilic actinomycetes in moldy buildings and found concentrations
close to 106 CFU cm3. In some occupational
environments, thermophilic actinomycete concentrations have been
reported to be as high as 108 CFU cm3
(7, 17). In the present laboratory study, S. albus spores were aerosolized by the air currents in the
swirling-flow disperser at concentrations of up to 1.8 spores
cm3; the concentrations of airborne M. halophytica and T. vulgaris were about 0.3 spores
cm3. We conclude that the latter two spore species were
not tested at their optimal maturation conditions. One reason for the
low concentrations of laboratory-generated spores in this study,
relative to those found in the nature, is that the natural
aerosolization of actinomycete spores is rather complex and involves
other mechanisms in addition to mechanical release by air currents. In
outdoor environments, actinomycete spores can be released from the soil by gusty winds or rain drops (22). In indoor environments,
the release may be enhanced by mechanical disturbance of the
actinomycete growth by people or animals. In this study, however, the
laboratory-generated spore concentrations were considered sufficient
for adequate measurements of the particle size distributions and the
determination of the microbial recovery of actinomycete spores. More
research is needed to better understand the release mechanisms and the
optimum release conditions for actinomycete spores from biocontaminated
building materials in indoor environments.
The aerodynamic spore size distributions of S. albus and M. halophytica were found to be
close to monodisperse: the gs were 1.33 and
1.24, respectively (Fig. 3). The dg of the
T. vulgaris spores was the smallest of the three tested
species; these spores also showed the largest variation in spore size.
All the physical sizes of the actinomycete spores measured in
this study with the microscope and image analysis system were
smaller than those reported by Madelin and Johnson for
S. albus (24) by Kawamoto for M. halophytica (16), and by Lacey for T. vulgaris (18). By measuring monodisperse standard PSL
particles with this system, we found that the measurement system used
was suitable for determining the physical size of spores, as the error
rate was 
3%. The differences between the physical sizes
of the spores determined in this study and those reported in the
literature appear to be related to the preparation technique for
microscopic analysis. Typically, slides are prepared by
staining the spores with water-based stains. Water may be
absorbed by the spores, causing them to extend to their maximum
diameter. However, hygroscopicity tests with S. albus spores showed that large size increases of the spores are not likely to
occur during the normal human breathing cycle. For a growth time of
2 s, typical for the residence time of spores in the humid air
environment of the human respiratory tract, their size increased by no
more than 26%. When spores are exposed to dry conditions, e.g.,
in normal indoor air environments, the water inside the spores
evaporates and the spores shrink to their minimum size.
Therefore, the size measurements of dry spores, as shown in this study,
appear to be more appropriate for human health-related studies.
Comparison of the relative spore recoveries for the three actinomycete species obtained after collection from the air showed that S. albus spores have the highest survival level. The difference in survival level between S. albus and M. halophytica appears to be due to the different structure of the spore wall. S. albus spores have an outer sheath, which protects them against physical damage and drying, whereas M. halophytica spores do not have such a sheath. The low relative recovery of T. vulgaris spores is probably related to the dormant nature of these spores, i.e., they need activation before they can germinate. Although the conventionally used mechanism for bacterial spore activation is heating (15), we used cold activation in this study instead of heat activation because the heat would have melted the agar. If the spores had been collected on a filter, activation by heating could also have been used. However, heat activation was not tested in this study, and therefore, the change in relative recovery by heat activation remains to be determined. When T. vulgaris was activated at 20°C for 24 h before incubation, the relative recovery increased about 10-fold but was still lower than those of the other two tested actinomycete species.
The effect of impact velocity was studied with S. albus spores. The lowest relative recovery was measured at
the lowest sampling air flow rate (3.8 liters min1).
Similar results have been found in an earlier study with vegetative bacterial cells (32) and can be explained as being due to
insufficient embedding of the spores at low sampling flow rates.
After reaching a maximum at about 20 liters
min1, the relative recovery decreased with higher air
flow rates. We attribute this to mechanical injury of the inner
structures of spores by their impaction onto the agar medium. At a flow
rate of 28 liters min1 the impaction velocity is very
high, about 175 m s1, resulting in injury or excess
embedding or a combination of both. Our results showed that the growth
of S. albus spores can be decreased or totally
prevented if they are too deeply embedded in agar. This result appears
to be related to the high oxygen need of actinomycetes (6).
If the spores are embedded too deeply in the agar, they may not get the
amount of oxygen needed for their germination and growth. At flow rates
of 10 to 15 liters min1, the relative recovery rate seems
to decrease. If this observation is not due to measurement error,
an explanation may be sought in the two competing processes of
insufficient embedding and excessive embedding or injury of spores. A
third competing process might be that of spore activation due to
impaction. These competing processes deserve further study.
Our results show that the recovery of actinomycete spores varies considerably depending on the species, the sampling flow rate, and other sampling parameters. Surprisingly low recovery rates were measured for T. vulgaris. To increase the accuracy and precision of spore enumeration, new noncultivation methods need to be developed for the analysis of actinomycete spores. Among the conventional methods, direct microscopic counting with bright-field or epifluorescence microscopy is used for fungal spores in situations when the total (not culturable) count is of interest. This method is not appropriate for actinomycete spores due to their small size, which makes them difficult to distinguish if the sample also contains a high concentration of fungal spores or other particles.
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ACKNOWLEDGMENTS |
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This work was initiated by T.A.R. during postdoctoral research supported by the U.S. Center for Indoor Air Research (CIAR) and was continued in collaboration with S.V.G., who was also supported by a postdoctoral fellowship through the CIAR.
The microscope and the image analysis facility for the measurement of the physical spore sizes were made available by Marshall Anderson, Jonathan Wiest, and Kenneth Conwell; helpful advice on actinomycete cultivation and spore production was given by Pamela Dulaney. We are thankful for their assistance.
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FOOTNOTES |
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* 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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REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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