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Viability of bacteria in unused air filter media
Pergamon
Armspheric Enuironment Vol. 31, No. 15, pp. 2305-2310, 1997 ‘0 1997 Elsevier Science Ltd All rights reserved. Printed in Great Bntam
PII: s1352-231q97)ooo20-4
VIABILITY
1352-2310/97
%17.Ml f 0.00
OF BACTERIA IN UNUSED AIR FILTER MEDIA
R. MAUS,*
A. GOPPELSRODER
and H. UMHAUER
Institut fiir Mechanische Verfahrenstechnik und Mechanik, Universitat Karlsruhe (TH), 76128 Karlsruhe, Germany (First received 18 July 1996 and infinalform
28 November 1996. Published May 1997)
Abstract-Different experimental techniques were applied to determine the effects of different air filter media on the viability of bacteria. Rinse suspensions of unused filter media were employed in standard inhibition tests to determine the effects of filter ingredients on bacterial growth under ideal nutritional conditions. Furthermore, a new test procedure was proposed and validated to determine the survival of viable microorganisms in fibrous air filters as a function of different parameters. Samples of filter media were challenged with microbial aerosols in an experimental set-up designed for measuring the collection efficiencies of fibrous filters. The loaded filter samples were then challenged with clean air under controlled conditions for a definite time span and numbers of viable microorganisms in the filter media were determined as colony forming units. The filter samples were retrieved from unused filter media usually employed in common air conditioning and ventilation systems. Under ideal nutritional and moisture conditions, growth of investigated microorganisms in nutrient broth and on nutrient agar was not inhibited by the inclusion of filter samples or rinse solutions of different filters in the growth medium with one exception. M. luteus and E. coli collected in air filter media and exposed to low air humidity (RH = 30-60%) showed a decline in their viability as a function of time (within 1 h). The decline rate was dependent on the type of bacteria employed and also the filter material itself. 0 1997 Elsevier Science Ltd. Key word index: Air filters, bioaerosols, viability, filter material.
1. INTRODUCTION
Fibrous filter media are often used in heating, ventilation and air conditioning (HVAC) systems as well as for automobile cabin filters or respirator filters to separate particulate matter from the air flow. These fibrous filter media exhibit a relatively small pressure drop compared to alternative dust collection devices while maintaining a high collection efficiency. Fibrous filter media are inexpensive and can be used without replacement for a long period of time (up to several months or years) at normal particle concentrations in the raw aerosol. The media are nonwovens consisting of synthetic or glass fibers. Fiber materials like cellulose or cotton are rarely used nowadays. If these filters are employed for separating atmospheric dust, not only mineral and soot particles are collected in the filter, but also particles of biological origin. These bioaerosols consist of particles emitted directly or indirectly from living organisms and include plant debris, fragments of insects, skin scales and hairs of mammals, microorganisms (bacteria, fungal spores) and pollen. In recent times the public awareness of airborne biological particles has increased because of their possible substantial health
*Author to whom correspondence should be addressed.
impacts even if number concentrations are significantly below the dust particle concentrations. Various pathogenic or infectious microorganisms are transmitted via airborne route and may cause a wide variety of illnesses when inhaled and deposited in the respiratory tract of humans or animals. Diseases like tuberculosis, diphtheria or legionellosis (Nevalainen et al., 1993; Brousseau er al., 1994; Lacey and Dutkiewicz, 1994) are proliferated in the abovementioned manner. To be infectious or pathogenic microorganisms need to be viable which is not a prerequisite for organisms that cause allergic effects. Many fungal conidia or spores and pollen can cause allergic reactions such as hay fever, rhinitis, asthma or pneumonitis by contact or inhalation (Bernstein et ul., 1983; Lacey and Crook, 1988; Lacey and Dutkiewicz, 1994). Bioaerosols in indoor environments decrease the air quality and affect the human health as described above. Typical symptoms like headaches, nausea and mucous membrane irritation which are associated with a minor indoor air quality are called sick building syndrome (Patterson, 1991; Nevalainen et al., 1993). Increased bioaerosol levels in indoor environments may come from different sources. Building material, e.g. wallpaper, insulation material, gypsum boards, may be a source of airborne microorganisms when microbial growth is supported by sufficient moisture and nutrients (Nevalainen, 1993). It has been
2305
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R. MAUS
shown that fibrous insulation materials or wallpaper contain substances which serve as nutrients for various microorganisms (Ezeonu et al., 1994; Nikulin et al., 1994; Chang et al., 1995). It has been shown in different studies that the air conditioning or ventilation system itself contributes to an increased concentration of microorganisms in ventilated rooms (Ager and Tickner, 1983; Bernstein et al., 1983; Sverdrup and Nyman, 1990; Hugenholtz and Fuerst, 1992; Pasanen et al., 1993). Different parts of the ventilation system were identified as sources of the increased bioaerosol content of air. For instances, humidification units (Macher and Girman, 1990) and air filters (Elixmann ef al., 1987) have been identified as sources of such air contaminants. In these installations, an environment can develop which is favourable for microbial growth. Over a long period of time and with insufficient maintenance of the units, growing bacteria and fungi were able to resuspend in the air stream by droplet formation and the dry dispersion of fungal spores. These phenomena have led to discussions of the role of filter units in air conditioning systems as potential growth sites for microorganisms and as sources of airborne microorganisms. Several investigations have dealt with the question of whether microorganisms may develop in high efficiency (HEPA) filters (Riiden and Thofern, 1976; Ohgke et al., 1993). Most have revealed that microorganisms do not multiply in unused filter media at low relative humidities (RH < 70%). Microbial growth was only induced when sufficient moisture and additional nutrients were supplied (Schmidt-Lorenz et al., 1981; Ohgke et al., 1993; Kemp et ul., 1995). Some investigators performed experiments with used filter material as well. They showed that atmospheric dust deposited in the used filter media impairs microbial growth when it is suspended in liquid growth media (Riiden et al., 1975). Results of other experimental investigations (Kemp et al., 1995; Simmons and Crow, 1995), performed under more realistic conditions, showed that atmospheric dust and/or cellulose fibers in air filter media may support fungal growth when the air humidity is sufficiently high (RH > 70-80%). These findings led to the present study which deals with the behaviour of bacteria in fibrous filter media employed for general ventilation purposes. The goal of this study was to describe the effects of fiber material on the survival of selected microorganisms after deposition in fibrous filter media. Experiments were performed in vitro but also under realistic conditions by challenging unused filter media with airborne bacteria. This paper presents the methods to determine the viability of bacteria in fibrous filters together with the results obtained with different unused filter media and different types of bacteria and yeasts. 2. MATERIALS To
AND METHODS
determine the influence of the fiber material on sur-
vival, i.e. viability
of bacteria
in air filters, different
experi-
et al. mental techniques were employed. First the effect of fiber material itself on bacterial reproduction was tested under ideal moisture and nutritional conditions. With another experimental technique, the effect on viability of exposing bacteria collected in fibrous filters to an air flow (RH < 60%) was studied. Filter media: Filter media classified into different filter categories according to EN 779 (EN = European Norm) or ASHRAE 52 (1992),,(ASHRAE = American Societv of Heating, Refrigerating and Air Conditioning Engineers) were studied. The classification is based on the total collection efficiency obtained experimentally with synthetic or atmospheric dust. In this investigation, the following unused coarse and fine dust filter media (filter class EU4 to EU7; efficiency for atmospheric dust E < 80%) were applied: one glass fiber medium (G), two polyester media (PES), one polypropylene medium (PP) and two media consisting of 3 layers of different fiber materials (PES, PP, PC = polycarbonate). The fibers in all filter media used, except the glass fiber medium, were thermally consolidated and include no binder materials. Bacteria: Micrococcus luteus (ATCC# 4698), Escherichia 6051) coli (ATCC # 23716) and Bacillus subtilis (ATCC# were chosen as typical bacteria (ATCC = American Type Culture Collection, USA). Species of this genera are common constituents of bioaerosols in different environments (Fannin et al., 1985; Nevalainen et al., 1993; Laitinen et al.. 1994) and react in different ways to environmental stress (Cox, 1987). For specific tests (see Section 2.1), the yeasts Hansenula holstii (DSM # 70764) and Saccharomyces cerevisiae were used in addition (DSM = Deutsche Sammlung von Mikroorganismen und Zellkulturen, Germany). For cultivation of bacteria Standard I nutrient broth solution (Merck No. 7882) or Standard I nutrient agar (Merck No. 7881) was used. Yeasts were cultivated in a 2% malt extract broth solution (Merck No. 5397) or on 2% malt extract agar. 2.1.
Testsfor inhibitory substances
in jlilter
mediu
2.1.1. Diffusion tests on nutrient agar. Circular filter samples (diameter = 58 mm) were stamped out of filter mats and each rinsed in 50 ml of a 0.9% NaCl solution within plastic bags by the agitation of a lab-blender (Stomacher 400, Seward Medical, U.K.) for 5 min. The resulting solutions were pasteurized at 80°C. A solution of NaCl(O.9%) without a filter sample was identically treated in the lab-blender and was used as control. The solutions were pasteurized rather than sterilized for significantly reducing the number of viable microorganisms while minimizing thermal degradation of chemical compounds originating from the filter material. Tests on agar plates did not show any contamination of the pasteurized solutions. Bacteria (E. co/i, B. subtilis, M. luteus) and yeasts (S. cereuisiae, H. holstii) were precultivated in nutrient broth solution for 48 h at 30°C. One ml of this microbial suspension was blended with 200 ml of the corresponding nutrient agar before solidifying. This agar was poured over a solid layer of cell free agar in Petri dishes and immediately after the second layer hardened, round pieces (diameter = 8 mm) were stamped out. The holes were filled with rinse solutions or the control solution. After incubation at 30°C for 48 h, the growth of the microorganisms was observed during a period of 5 days. Growth inhibition, caused by diffusion of active substances into the agar, resulted in colony free zones around the holes (Creager et al., 1990). 2.1.2. Growth tests in nutrient broth containingjlter samples. Identical samples of filter media (halves of circular samples, 58 mm) were pasteurized at 8O”C, submerged in 25 ml sterile nutrient broth in Erlenmeyer flasks and inoculated with 0.1 ml of a suspension of microorganisms. After incubation for 48 h at 3O”C, 1 ml of the suspension
Viability
of bacteria
in unused
air filter media
2307
AerosolGeneration
Rinsing Filter Sam le (Lab-Blender STOMA 8 HER)
Plating
1
nun Petri Dishes with Nutrient
Agar
4
Colony Foning Units (CFU) per Filter Sample Fig. 1. Schematic
diagram
of the experimental setup and procedure for determination bacteria in fibrous filter samples.
was taken to determine the relative cell concentration expressed as the extinction at 580 nm by a filter photometer. For each set of parameters (filter medium, bacteria species) two replicate experimental trials and one control trial (without filter sample) were performed. 2.2. Viability
in
dry filter
media
aerosol. To determine the viability of bacteria after collection in a fibrous filter medium an experimental setup was used which allowed the defined challenging and loading of filter samples with bacterial aerosols (Maus and Umhauer, 1996). It consisted of three main components: an aerosol generation unit, a flow channel with measurement chambers and a measuring unit (Fig. 1). For generating a bacterial aerosol a suspension nebulizer was used which disperses a bacterial suspension prepared as follows: Erlenmeyer flasks containing 200ml of a sterile Standard I nutrient broth solution were each inoculated with a bacteria colony. After a 48 h incubation at 30°C in a water bath on a rotary shaker the bacteria were separated in a centrifuge at about lSOOg, and then washed with sterile demineralized water. This procedure was repeated twice to remove as much salt as possible (Juozaitis et al., 1994). The numbers of viable cells in the resulting suspension were about 108-1010 cfuml-‘. The final suspension was stored where necessary at about 4”C, but for no longer than two weeks. Preliminary investigations had shown that there were no marked changes in the numbers of viable cells during that storage time. The generated primary aerosol was mixed with a conditioned air flow, to evaporate the water and regulate the relative humidity. The aerosol was then channelled through a neutralizer (s’Kr, 10 mCi), to neutralize the particle charge caused during the aerosol-generation process. Through a mixing area the airborne particles passed into a channel (inner diameter d, = 50 mm) with the filter sample to be examined (Fig. 1). The particles passing through the test filter into the clean gas flow were removed by a high-efficiency filter (HEPA) to prevent the release of particles into the environment. The total air flow through the setup was generated by a pump and monitored by a rotameter (FIC). 2.2.1. Loading
offilter
Pump
samples with microbial
of the viability
of
The main feature of the setup consisted of the filter holder with the filter sample and the two optical particle counters (Fig. 1). The filter holder carried the filter sample and was positioned in the vertical flow channel. Both optical particle counters, which work with a purely optically defined measuring volume (Umhauer, 1983), were identical and allowed the determination of particle flux as well as particle size ranging between 0.5 and 26pm. Thus, the measuring range covered the typical size range of airborne bacteria. The measuring chambers of the optical particle counters were located before and behind the filter sample and were an integrated part of the flow channel. Therewith the challenging of the filter samples could be monitored and controlled, hence allowing the loading of different filter samples with similar numbers of bacterial particles. The filtration velocity, i.e. the filter face velocity, during filter challenge was kept at 1 m s-l for all experiments. After the loading process (Z 2 min) the filter sample was challenged with clean air (RH = 30-60%; temperature = 19-23°C) for a definite time t. 2.2.2. Evaluation of viable bacteria in jilter sample. After above-described treatment the filter sample was retrieved and submerged in 100ml sterile sodium chloride solution (0.9% NaCl + 0.04% TWEEN 80) in a plastic bag. The bag with the filter sample was agitated in the above mentioned lab-blender for 5 min to resuspend the bacteria attached to filter fibers. The resulting bacterial suspension was serially diluted and samples of 0.1 ml were streaked onto Standard I nutrient agar. After incubation for 48 h at 30°C the colonyforming units per filter sample were determined by the plate count method and their viability was calculated. The viability is defined as the fraction of the number of viable bacteria at time t and the number of viable bacteria at t = 0, which is immediately after the loading process. Consequently, for all sets of parameters the viability data at t = 0 equals 100%. The experimentally determined viability is compared with calculations according to a model proposed by Cox (1987). This model accounts for the fact that the viability of airborne bacteria is affected by dehydration and by toxic reactions with oxygen present in air. These effects result in a loss of viability depending on the type of bacteria and exposure time. Cox (1987) obtained a function which depended on
2308
R. MAUS
time and two constants which cannot be determined theoretically but have to be derived from the experiment. In the present study the constants were fitted to the experimental viability data.
et al.
Table 1. Results of plate diffusion tests for various
Filter medium
Organism
PES I
E. coli M. luteus B. subtilis H. ho&ii S. cerevisiae
PES II
E. coli M. luteus B. subtilis H. holstii S. cerevisiae
PP
E. coli M. luteus B. subtilis H. ho&ii S. cerevisiae
PPlPCJPP
3. RESULTS AND DISCUSSION 3.1. Diffusion tests on nutrient agar and growth tests in nutrient
filter
rinse solutions Control
Rinse solutions
broth
In diffusion tests the microorganisms were not influenced by the rinse solutions with three exceptions (Table 1): M. luteus and H. holstii showed a colony free zone and B. subtilis “flocculent” growth in the vicinity of holes filled with rinse solution of PES I. Therefore, most of the filter media investigated did not contain water-soluble substances that could inhibit the growth of bacteria on nutrient agar. In Fig. 2 the growth of test organisms in nutrient broth containing filter samples is shown. Because of the results obtained in diffusion tests, the trials with B. subtilis and H. holstii were only performed with PES I medium. For all investigated filter media except one, growth was not affected by the presence of the filter sample when compared with the control trial. The fiber materials did not have any notable inhibitory or supportive effect. Only for one filter medium (PES I) inhibition of growth of M. luteus, B. subtilis and H. holstii could be observed, thus confirming diffusion tests (Table 1). A different filter medium consisting of polyester fiber as well (PES II) did not inhibit growth of M. luteus (Fig. 2). Apparently, PES I released a water soluble substance, which hampered cell division of some species. There is no information available about the possible compounds in the fiber material which might be responsible for this phenomena. Detailed analysis and identification of compounds embedded in synthetic fibers is complex (Haslam et al., 1972; Falkai, 1981) and determination of the potential bactericidal effects of isolated substances involve sophisticated microbiological and molecular biological methods which were beyond the scope of the present study. 3.2. Viability
in
dry filter
G
_ _
_ _
_ _ _
_ _
E. coli M. luteus B. subtilis H. holstii S. cerevisiae
_ _
_
_ -
_ _ _ _
E. coli M. luteus B. subtilis H. holstii S. cerevisiae
_ _ _ _
_ _ _
PES = polyester, PP = polypropylene, PC = polycarbonate, G = glass; - = no growth inhibition, + = growth inhibition, f = flocculent growth.
2 ^x .r 2 s =
in Nutrient Broth
media
In Figs 3 and 4 results for the viability of two bacterial species in various fibrous filters are shown. The viability of E. coli and M. luteus clearly declined on both fiber materials within the measuring time of 1 h. However, in PES I the viability was about 4% for E. coli after 1 h while the viability of M. luteus was notably higher ( x 60%) after the same time span. The identical magnitude of differences could be observed for the glass fiber medium (G). In a filter medium consisting of polypropylene and polycarbonate fibers (PP/PC/PP), the viability after 1 h was x20% for E. coli and x95-100% for M. luteus (Fig. 4). The results indicate that bacteria exposed to air flow suffer from environmental stress, i.e. the toxic effects of oxygen and dehydration (Cox, 1987), which led to a decline in
control
PES I
Fig. 2. Growth of various with submerged samples
polyester, G = glass,
PES II
PP
G
PPIPCIPP
microorganisms in nutrient broth of different filter media: PES = PP = polypropylene, PC = polycarbonate.
their viability. The decline rate was dependent on the organisms employed. The inactivation of the more hardy M. luteus (gram-positive) was more slowly than that of the more sensitive E. coli cells (gram negative). In contrast to the results obtained for the filter medium PES I in nutrient broth (Fig. 2), the viability
Viability of bacteria in unused air filter media I
I 30
I 60
J 90
time in min Fig. 3. Viability of E. coli and M. luteus in two unused air filter media as a function of time. Curve fit according to the model by Cox (1987); PES = polyester, G = glass.
1 I_._ 0
/
!
30
60
E. coli
i
1
2309
,
I
I
30
60
90
time in min Fig. 4. Viability of E. coli and M. luteus in a unused air filter medium as a function of time. Curve fit according to the model by Cox (1987); PP = polypropylene, PC = polycarbonate.
I
-
D
time in min Fig. 5. Viability of E. coli in three unused air filter media as a function of time. Curve fit according to the model by Cox (1987); PES = polyester, PP = polypropylene, PC ==polycarbonate.
on viability after the collection process. As mentioned above, volatile and/or water soluble chemical compounds which were introduced in the fiber fabrication process may still be present in the manufactured fibrous filter and affect the viability of bacteria. Depending on the fiber material different compounds may be present that exhibit different magnitudes of damaging effects on the bacteria collected on the filter fibers (Simmons and Crow, 1995). The model calculations according to Cox (1987) showed a good agreement with the experimental data (Figs 3-5) indicating that viability of microorganisms was mainly impaired by the presence of oxygen and dehydration of cells, as proposed in the model by Cox (1987).
4. CONCLUSIONS
of M. luteus on PES I and G was less impaired than the viability of E. coli. Therefore the effects of fiber material under ideal nutritional and moisture conditions were different compared to the conditions at low air humidities. In nutrient broth bacterial growth takes place and the number of viable bacteria increases while at low air humidities the bacteria lose their ability to reproduce and the number of viable cells decreases. In Fig. 5 the survival of E. coli in three different filter media of different composition can be seen. The viability of E. coli declined for all three filter media employed but the decline rates were notably different for each filter medium. For PES I the viability declined to about 4% after 1 h and the viability was about 15-fold higher (60%) for the PES/PC/PES medium. These differences must be due to different fiber components which affected the viability of bacteria when attached to the filter fibers. Differences in the physical properties of the filter media (fiber diameter, fiber surface structure, porosity, etc.) only affect particle collection but should not have an effect
In experimental studies it could be shown that all, except one, investigated fiber materials did not exhibit an inhibitory effect on growth kinetics of M. luteus and E. coli in nutrient broth (ideal moisture and nutrient conditions). In addition, no notable growth enhancement due to the presence of fiber materials in nutrient broth could be observed. Tests conducted with bacteria collected in air filter media showed that the viability of bacteria declined within measuring times of 1 h, when relative air humidity was between 30 and 60%. The decline rate was strongly dependent on fiber material and type of microorganisms employed. More hardy organisms, like gram-positive M. luteus, seem to be more resistant to environmental stress factors, i.e. dehydration, toxic action of oxygen, than sensitive gram-negative bacteria (E. coli). In future studies it is planned to investigate the long term viability (during several days and weeks) of bacteria and fungal spores in fibrous filters as a function of air humidity and fiber material. It will be evaluated
R. MAUS et al.
2310
how these factors affect the viability of microorganisms in air filters and under which conditions microbial decay or growth is likely. Beside unused filter media it is further planned to include used filter media in the investigations to elucidate the effect of atmospheric dust collected in filter media on the viability of microorganisms. Acknowledgements-This work was supported by Projekte Europiiisches Forschungszentrumfir MaJnahmen zur Luftrein!-dung (PEF) under grant PEF 3 95 001 from Forschungszentrum Karlsruhe, Germany. The authors are grateful to Prof. K. Willeke and Prof. S. Grinshpun from the University of Cincinnati, U.S.A. for the valuable discussions and helpful suggestions. Special thanks are due to Dr K. Grimm and S. Wiirner from the University of Karlsruhe, Germany for their continuous support in our experimental investigations.
REFERENCES Ager, B. P. and Tickner, J. A. (1983) The control of microbial hazards associated with air conditioning and ventilation systems. Ann. Occup. Hyg. 27, 341-358. ASHRAE 52 (1992) Method of testing cleaning devices used in general ventilation air for removing particulate matter. American Society of Heating Refrigeration and Ventilation Engineers, Atlanta, U.S.A. Bernstein, R. S., Sorensen, W. G., Garabrant, D., Reaux, C. and Treitman, R. D. (1983) Exposures to respirable, airborne penicillium from a contaminated ventilation system: clinical, environmental and epidemiological aspects. Am. lnd. Hyg. Ass. J. 44, 161-169. Brousseau, L., Chen, S.-K., Vesley, D. and Vincent, J. H. (1994) System design and test method for measuring respirator filter efficiency using Mycobacterium aerosols. J. Aerosol Sci. 25, 1567-1577. Chang, J. C. S., Foarde, K. K. and Vanosdell, D. W. (1995) Growth evaluation of fungi on ceiling tiles. Atmospheric Environment 29, 2331-2337. Cox, C. S. (1987). The Aerobiological Pathway of Microorganisms. Wiley, U.K. Creager, J. G., Black, J. G. and Davison, V. E. (1990 ) Micro-
biology. Principles and Applications. Prentice Hall, Englewood Cliff, NJ. Elixmann, J. H., Jorde, W. and Linskens, H. F. (1987) Filters of an airconditioning installation as disseminators of fungal spores. In Advances in Aerobiology, ed. G. Boehm, pp. 283-286. BirkhIuser Verlag, Switzerland. European Norm EN 779 (1994) Particulate air filters for general ventilation; requirements, testing, marking. European Union. Ezeonu, I. M., Noble, J. A., Simons, R. B., Price, D. L.; Crow, S. A. and Ahearn, D. G (1994) Effect of relative humidity on fungal colonization of fiberglass insulation. Appl. Envir. Microbial. 60, 2149-2151. Falkai, B&lavon (1981) Synthesefasern. Verlag Chemie, Ger-
many. Fannin, K. F., Vana, S. C. and Jakubowski, W. (1985) Effect of an activated sludge wastewater treatment plant on ambient air densities of aerosols containing bacteria and viruses. Appl. Envir. Microbial. 49, 1191-l 196. Haslam, J., Willis, H. A. and Squirrell, C.M. (1972) Zdenti$cation and Analysis of Plastics. Iliffe Books, U.K. Hugenholtz, P. and Fuerst, J. A. (1992) Heterotrophic bacteria in an air-handling_ system. ADDI. _ __ Envir. Microbial. 58, 3914-3920. Juozaitis, A., Willeke, K., Grinshpun, S. A. and Donnelly, J. (1994) Impaction onto a glass slide or agar versus impinge-
ment into a liquid for the collection and recovery of airborne microorganisms. Appl. Envir. Microbial. 60, 861-870.
Kemp, S. J., Kuehn, T. H., Pui, D. Y. H., Vesley, D. and Streifel, A. J. (1995) Filter collection efficiency and growth of microorgansims on filters loaded with outdoor air. ASHRAE Trans. 101,228-238. Lacey, J. and Crook, B. (1988) Review: Fungal and actinomycete spores as pollutants of the workplace and occupational allergens. Ann. Occup. Hyg. 32, 515-533. Lacey, J. and Dutkiewicz, J. (1994) Bioaerosols and occupational lung disease. J. Aerosol Sci. 25, 1371-1404. Laitinen, S., Kangas, J., Kotimaa, M., Liesivuori. J., Martikainen, P. J., Nevalainen, A., Sarantila, R. and Husman, K. (1994) Workers exposure to airborne bacterial and endotoxins at industrial wastewater treatment. Am. lnd. Hyg. Ass. J. 55, 1055-1060. Macher, J. M. and Girman, J. R. (1990) Multiplication of microorganism in an evaporative air cooler and possible indoor contamination. Envir. Znt. 16, 203-211. Maus, R. and Umhauer, H. (1996) Determination of the fractional efficiencies of fibrous filter media by optical in-situ measurements. Aerosol Sci. Technol. 24, 161-173. Nevalainen, A. (1993) Microbial contamination of buildings. Proc.
6th Int. Conf: Indoor Air Quality and Climate,
Helsinki, Vol. 4, pp. l-13. Nevalainen, A., Willeke, K., Liebhaber, F., Pastuszka, J., Burge, H., Heningson, E. (1993) Bioaerosol sampling. In Aerosol Measurement, ed. K. Willeke and P. Baron. van Nostrand Reinhold, New York. Nikulin, M., Pasanen, A.-L., Berg, S. and Hintikka, E.-L. (1994) Stachybotrys atra growth and toxin production in some building materials and fodder under different relative humidities. Appl. Enuir. Microbial. 60, 3421-3424. Ohgke, H., Senkspiel, K. and Beckert, J. (1993) Experimental evaluation of microbial growth and survival in air filters. Proc.
6th Int. Conf
Indoor Air Qua@
and Climate,
Helsinki, Vol. 6, pp. 521-526. Pasanen, A.-L., Nikulin, M., Berg, S. and Hintikka, E.-L. (1994) Stachybotrys atra corda may produce mycotoxins in respirator filters in humid environments. Am. Znd. Hyg. Ass. J. 55, 62-65.
Pasanen, P., Pasanen, A.-L. and Jantunen, M. (1993) Water condensation promotes fungal growth in ventilation ducts. Indoor Air 3, 106-l 12. Patterson, N. R. (1991) Comfort and indoor air quality. Heating, Piping Air Conditioning 63, 107-l 11. Riiden, H. and Thofern, E. (1976) Abscheidung von Schadstoffen und Mikroorganismen in Luftfiltern. Staub-Reinhaltung der Luft 36, 33-36.
Ridden, H., Thofern, E. and Schmitz, M. (1975) Wasserlaslithe Inhaltsstoffe und Mikroorganismen in Feinststaubfiltern von Klimaanlagen. Daub-Reinhaltung der Luft 35, 215-219.
Schmidt-Lorenz, W., Schmid, U., Uehlinger, M. and Blaser, P. (1981) Untersuchungen iiber das Durchwachsen von Schimmelpilzen durch Hochleistungs-Schwebstoffilter. Swiss Med 3, 93-108.
Simmons, R. B. and Crow, S. A. (1995) Fungal colonization of air filters for use in heating, ventilating, and air conditioning (HVAC) systems. J. Znd. Microbial. 14, 41-45. Sverdrup, C. F. and Nyman, E. (1990) A study of microorganisms in ventilation systems of 12 different buildings in Sweden. Proc. 5th Int. Conf: Indoor Air Qualitiy and Climate. Vol. 4, Toronto, pp. 383-588. Umhauer H. (1983) Particle size distribution analvsis bv scatterd light measurements using an optically hefineh measuring volume. J. Aerosol Sci. 14, 765-770. Willeke, K., Qian, Y., Donnelly, J., Grinshpun, S. and Ulevicius, V. (1996) Penetration of airborne microorganisms through a surgical mask and a dust/mist respirator. Am. Ind. Hyg. Ass. J. 57, 348-355.
Armspheric Enuironment Vol. 31, No. 15, pp. 2305-2310, 1997 ‘0 1997 Elsevier Science Ltd All rights reserved. Printed in Great Bntam
PII: s1352-231q97)ooo20-4
VIABILITY
1352-2310/97
%17.Ml f 0.00
OF BACTERIA IN UNUSED AIR FILTER MEDIA
R. MAUS,*
A. GOPPELSRODER
and H. UMHAUER
Institut fiir Mechanische Verfahrenstechnik und Mechanik, Universitat Karlsruhe (TH), 76128 Karlsruhe, Germany (First received 18 July 1996 and infinalform
28 November 1996. Published May 1997)
Abstract-Different experimental techniques were applied to determine the effects of different air filter media on the viability of bacteria. Rinse suspensions of unused filter media were employed in standard inhibition tests to determine the effects of filter ingredients on bacterial growth under ideal nutritional conditions. Furthermore, a new test procedure was proposed and validated to determine the survival of viable microorganisms in fibrous air filters as a function of different parameters. Samples of filter media were challenged with microbial aerosols in an experimental set-up designed for measuring the collection efficiencies of fibrous filters. The loaded filter samples were then challenged with clean air under controlled conditions for a definite time span and numbers of viable microorganisms in the filter media were determined as colony forming units. The filter samples were retrieved from unused filter media usually employed in common air conditioning and ventilation systems. Under ideal nutritional and moisture conditions, growth of investigated microorganisms in nutrient broth and on nutrient agar was not inhibited by the inclusion of filter samples or rinse solutions of different filters in the growth medium with one exception. M. luteus and E. coli collected in air filter media and exposed to low air humidity (RH = 30-60%) showed a decline in their viability as a function of time (within 1 h). The decline rate was dependent on the type of bacteria employed and also the filter material itself. 0 1997 Elsevier Science Ltd. Key word index: Air filters, bioaerosols, viability, filter material.
1. INTRODUCTION
Fibrous filter media are often used in heating, ventilation and air conditioning (HVAC) systems as well as for automobile cabin filters or respirator filters to separate particulate matter from the air flow. These fibrous filter media exhibit a relatively small pressure drop compared to alternative dust collection devices while maintaining a high collection efficiency. Fibrous filter media are inexpensive and can be used without replacement for a long period of time (up to several months or years) at normal particle concentrations in the raw aerosol. The media are nonwovens consisting of synthetic or glass fibers. Fiber materials like cellulose or cotton are rarely used nowadays. If these filters are employed for separating atmospheric dust, not only mineral and soot particles are collected in the filter, but also particles of biological origin. These bioaerosols consist of particles emitted directly or indirectly from living organisms and include plant debris, fragments of insects, skin scales and hairs of mammals, microorganisms (bacteria, fungal spores) and pollen. In recent times the public awareness of airborne biological particles has increased because of their possible substantial health
*Author to whom correspondence should be addressed.
impacts even if number concentrations are significantly below the dust particle concentrations. Various pathogenic or infectious microorganisms are transmitted via airborne route and may cause a wide variety of illnesses when inhaled and deposited in the respiratory tract of humans or animals. Diseases like tuberculosis, diphtheria or legionellosis (Nevalainen et al., 1993; Brousseau er al., 1994; Lacey and Dutkiewicz, 1994) are proliferated in the abovementioned manner. To be infectious or pathogenic microorganisms need to be viable which is not a prerequisite for organisms that cause allergic effects. Many fungal conidia or spores and pollen can cause allergic reactions such as hay fever, rhinitis, asthma or pneumonitis by contact or inhalation (Bernstein et ul., 1983; Lacey and Crook, 1988; Lacey and Dutkiewicz, 1994). Bioaerosols in indoor environments decrease the air quality and affect the human health as described above. Typical symptoms like headaches, nausea and mucous membrane irritation which are associated with a minor indoor air quality are called sick building syndrome (Patterson, 1991; Nevalainen et al., 1993). Increased bioaerosol levels in indoor environments may come from different sources. Building material, e.g. wallpaper, insulation material, gypsum boards, may be a source of airborne microorganisms when microbial growth is supported by sufficient moisture and nutrients (Nevalainen, 1993). It has been
2305
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R. MAUS
shown that fibrous insulation materials or wallpaper contain substances which serve as nutrients for various microorganisms (Ezeonu et al., 1994; Nikulin et al., 1994; Chang et al., 1995). It has been shown in different studies that the air conditioning or ventilation system itself contributes to an increased concentration of microorganisms in ventilated rooms (Ager and Tickner, 1983; Bernstein et al., 1983; Sverdrup and Nyman, 1990; Hugenholtz and Fuerst, 1992; Pasanen et al., 1993). Different parts of the ventilation system were identified as sources of the increased bioaerosol content of air. For instances, humidification units (Macher and Girman, 1990) and air filters (Elixmann ef al., 1987) have been identified as sources of such air contaminants. In these installations, an environment can develop which is favourable for microbial growth. Over a long period of time and with insufficient maintenance of the units, growing bacteria and fungi were able to resuspend in the air stream by droplet formation and the dry dispersion of fungal spores. These phenomena have led to discussions of the role of filter units in air conditioning systems as potential growth sites for microorganisms and as sources of airborne microorganisms. Several investigations have dealt with the question of whether microorganisms may develop in high efficiency (HEPA) filters (Riiden and Thofern, 1976; Ohgke et al., 1993). Most have revealed that microorganisms do not multiply in unused filter media at low relative humidities (RH < 70%). Microbial growth was only induced when sufficient moisture and additional nutrients were supplied (Schmidt-Lorenz et al., 1981; Ohgke et al., 1993; Kemp et ul., 1995). Some investigators performed experiments with used filter material as well. They showed that atmospheric dust deposited in the used filter media impairs microbial growth when it is suspended in liquid growth media (Riiden et al., 1975). Results of other experimental investigations (Kemp et al., 1995; Simmons and Crow, 1995), performed under more realistic conditions, showed that atmospheric dust and/or cellulose fibers in air filter media may support fungal growth when the air humidity is sufficiently high (RH > 70-80%). These findings led to the present study which deals with the behaviour of bacteria in fibrous filter media employed for general ventilation purposes. The goal of this study was to describe the effects of fiber material on the survival of selected microorganisms after deposition in fibrous filter media. Experiments were performed in vitro but also under realistic conditions by challenging unused filter media with airborne bacteria. This paper presents the methods to determine the viability of bacteria in fibrous filters together with the results obtained with different unused filter media and different types of bacteria and yeasts. 2. MATERIALS To
AND METHODS
determine the influence of the fiber material on sur-
vival, i.e. viability
of bacteria
in air filters, different
experi-
et al. mental techniques were employed. First the effect of fiber material itself on bacterial reproduction was tested under ideal moisture and nutritional conditions. With another experimental technique, the effect on viability of exposing bacteria collected in fibrous filters to an air flow (RH < 60%) was studied. Filter media: Filter media classified into different filter categories according to EN 779 (EN = European Norm) or ASHRAE 52 (1992),,(ASHRAE = American Societv of Heating, Refrigerating and Air Conditioning Engineers) were studied. The classification is based on the total collection efficiency obtained experimentally with synthetic or atmospheric dust. In this investigation, the following unused coarse and fine dust filter media (filter class EU4 to EU7; efficiency for atmospheric dust E < 80%) were applied: one glass fiber medium (G), two polyester media (PES), one polypropylene medium (PP) and two media consisting of 3 layers of different fiber materials (PES, PP, PC = polycarbonate). The fibers in all filter media used, except the glass fiber medium, were thermally consolidated and include no binder materials. Bacteria: Micrococcus luteus (ATCC# 4698), Escherichia 6051) coli (ATCC # 23716) and Bacillus subtilis (ATCC# were chosen as typical bacteria (ATCC = American Type Culture Collection, USA). Species of this genera are common constituents of bioaerosols in different environments (Fannin et al., 1985; Nevalainen et al., 1993; Laitinen et al.. 1994) and react in different ways to environmental stress (Cox, 1987). For specific tests (see Section 2.1), the yeasts Hansenula holstii (DSM # 70764) and Saccharomyces cerevisiae were used in addition (DSM = Deutsche Sammlung von Mikroorganismen und Zellkulturen, Germany). For cultivation of bacteria Standard I nutrient broth solution (Merck No. 7882) or Standard I nutrient agar (Merck No. 7881) was used. Yeasts were cultivated in a 2% malt extract broth solution (Merck No. 5397) or on 2% malt extract agar. 2.1.
Testsfor inhibitory substances
in jlilter
mediu
2.1.1. Diffusion tests on nutrient agar. Circular filter samples (diameter = 58 mm) were stamped out of filter mats and each rinsed in 50 ml of a 0.9% NaCl solution within plastic bags by the agitation of a lab-blender (Stomacher 400, Seward Medical, U.K.) for 5 min. The resulting solutions were pasteurized at 80°C. A solution of NaCl(O.9%) without a filter sample was identically treated in the lab-blender and was used as control. The solutions were pasteurized rather than sterilized for significantly reducing the number of viable microorganisms while minimizing thermal degradation of chemical compounds originating from the filter material. Tests on agar plates did not show any contamination of the pasteurized solutions. Bacteria (E. co/i, B. subtilis, M. luteus) and yeasts (S. cereuisiae, H. holstii) were precultivated in nutrient broth solution for 48 h at 30°C. One ml of this microbial suspension was blended with 200 ml of the corresponding nutrient agar before solidifying. This agar was poured over a solid layer of cell free agar in Petri dishes and immediately after the second layer hardened, round pieces (diameter = 8 mm) were stamped out. The holes were filled with rinse solutions or the control solution. After incubation at 30°C for 48 h, the growth of the microorganisms was observed during a period of 5 days. Growth inhibition, caused by diffusion of active substances into the agar, resulted in colony free zones around the holes (Creager et al., 1990). 2.1.2. Growth tests in nutrient broth containingjlter samples. Identical samples of filter media (halves of circular samples, 58 mm) were pasteurized at 8O”C, submerged in 25 ml sterile nutrient broth in Erlenmeyer flasks and inoculated with 0.1 ml of a suspension of microorganisms. After incubation for 48 h at 3O”C, 1 ml of the suspension
Viability
of bacteria
in unused
air filter media
2307
AerosolGeneration
Rinsing Filter Sam le (Lab-Blender STOMA 8 HER)
Plating
1
nun Petri Dishes with Nutrient
Agar
4
Colony Foning Units (CFU) per Filter Sample Fig. 1. Schematic
diagram
of the experimental setup and procedure for determination bacteria in fibrous filter samples.
was taken to determine the relative cell concentration expressed as the extinction at 580 nm by a filter photometer. For each set of parameters (filter medium, bacteria species) two replicate experimental trials and one control trial (without filter sample) were performed. 2.2. Viability
in
dry filter
media
aerosol. To determine the viability of bacteria after collection in a fibrous filter medium an experimental setup was used which allowed the defined challenging and loading of filter samples with bacterial aerosols (Maus and Umhauer, 1996). It consisted of three main components: an aerosol generation unit, a flow channel with measurement chambers and a measuring unit (Fig. 1). For generating a bacterial aerosol a suspension nebulizer was used which disperses a bacterial suspension prepared as follows: Erlenmeyer flasks containing 200ml of a sterile Standard I nutrient broth solution were each inoculated with a bacteria colony. After a 48 h incubation at 30°C in a water bath on a rotary shaker the bacteria were separated in a centrifuge at about lSOOg, and then washed with sterile demineralized water. This procedure was repeated twice to remove as much salt as possible (Juozaitis et al., 1994). The numbers of viable cells in the resulting suspension were about 108-1010 cfuml-‘. The final suspension was stored where necessary at about 4”C, but for no longer than two weeks. Preliminary investigations had shown that there were no marked changes in the numbers of viable cells during that storage time. The generated primary aerosol was mixed with a conditioned air flow, to evaporate the water and regulate the relative humidity. The aerosol was then channelled through a neutralizer (s’Kr, 10 mCi), to neutralize the particle charge caused during the aerosol-generation process. Through a mixing area the airborne particles passed into a channel (inner diameter d, = 50 mm) with the filter sample to be examined (Fig. 1). The particles passing through the test filter into the clean gas flow were removed by a high-efficiency filter (HEPA) to prevent the release of particles into the environment. The total air flow through the setup was generated by a pump and monitored by a rotameter (FIC). 2.2.1. Loading
offilter
Pump
samples with microbial
of the viability
of
The main feature of the setup consisted of the filter holder with the filter sample and the two optical particle counters (Fig. 1). The filter holder carried the filter sample and was positioned in the vertical flow channel. Both optical particle counters, which work with a purely optically defined measuring volume (Umhauer, 1983), were identical and allowed the determination of particle flux as well as particle size ranging between 0.5 and 26pm. Thus, the measuring range covered the typical size range of airborne bacteria. The measuring chambers of the optical particle counters were located before and behind the filter sample and were an integrated part of the flow channel. Therewith the challenging of the filter samples could be monitored and controlled, hence allowing the loading of different filter samples with similar numbers of bacterial particles. The filtration velocity, i.e. the filter face velocity, during filter challenge was kept at 1 m s-l for all experiments. After the loading process (Z 2 min) the filter sample was challenged with clean air (RH = 30-60%; temperature = 19-23°C) for a definite time t. 2.2.2. Evaluation of viable bacteria in jilter sample. After above-described treatment the filter sample was retrieved and submerged in 100ml sterile sodium chloride solution (0.9% NaCl + 0.04% TWEEN 80) in a plastic bag. The bag with the filter sample was agitated in the above mentioned lab-blender for 5 min to resuspend the bacteria attached to filter fibers. The resulting bacterial suspension was serially diluted and samples of 0.1 ml were streaked onto Standard I nutrient agar. After incubation for 48 h at 30°C the colonyforming units per filter sample were determined by the plate count method and their viability was calculated. The viability is defined as the fraction of the number of viable bacteria at time t and the number of viable bacteria at t = 0, which is immediately after the loading process. Consequently, for all sets of parameters the viability data at t = 0 equals 100%. The experimentally determined viability is compared with calculations according to a model proposed by Cox (1987). This model accounts for the fact that the viability of airborne bacteria is affected by dehydration and by toxic reactions with oxygen present in air. These effects result in a loss of viability depending on the type of bacteria and exposure time. Cox (1987) obtained a function which depended on
2308
R. MAUS
time and two constants which cannot be determined theoretically but have to be derived from the experiment. In the present study the constants were fitted to the experimental viability data.
et al.
Table 1. Results of plate diffusion tests for various
Filter medium
Organism
PES I
E. coli M. luteus B. subtilis H. ho&ii S. cerevisiae
PES II
E. coli M. luteus B. subtilis H. holstii S. cerevisiae
PP
E. coli M. luteus B. subtilis H. ho&ii S. cerevisiae
PPlPCJPP
3. RESULTS AND DISCUSSION 3.1. Diffusion tests on nutrient agar and growth tests in nutrient
filter
rinse solutions Control
Rinse solutions
broth
In diffusion tests the microorganisms were not influenced by the rinse solutions with three exceptions (Table 1): M. luteus and H. holstii showed a colony free zone and B. subtilis “flocculent” growth in the vicinity of holes filled with rinse solution of PES I. Therefore, most of the filter media investigated did not contain water-soluble substances that could inhibit the growth of bacteria on nutrient agar. In Fig. 2 the growth of test organisms in nutrient broth containing filter samples is shown. Because of the results obtained in diffusion tests, the trials with B. subtilis and H. holstii were only performed with PES I medium. For all investigated filter media except one, growth was not affected by the presence of the filter sample when compared with the control trial. The fiber materials did not have any notable inhibitory or supportive effect. Only for one filter medium (PES I) inhibition of growth of M. luteus, B. subtilis and H. holstii could be observed, thus confirming diffusion tests (Table 1). A different filter medium consisting of polyester fiber as well (PES II) did not inhibit growth of M. luteus (Fig. 2). Apparently, PES I released a water soluble substance, which hampered cell division of some species. There is no information available about the possible compounds in the fiber material which might be responsible for this phenomena. Detailed analysis and identification of compounds embedded in synthetic fibers is complex (Haslam et al., 1972; Falkai, 1981) and determination of the potential bactericidal effects of isolated substances involve sophisticated microbiological and molecular biological methods which were beyond the scope of the present study. 3.2. Viability
in
dry filter
G
_ _
_ _
_ _ _
_ _
E. coli M. luteus B. subtilis H. holstii S. cerevisiae
_ _
_
_ -
_ _ _ _
E. coli M. luteus B. subtilis H. holstii S. cerevisiae
_ _ _ _
_ _ _
PES = polyester, PP = polypropylene, PC = polycarbonate, G = glass; - = no growth inhibition, + = growth inhibition, f = flocculent growth.
2 ^x .r 2 s =
in Nutrient Broth
media
In Figs 3 and 4 results for the viability of two bacterial species in various fibrous filters are shown. The viability of E. coli and M. luteus clearly declined on both fiber materials within the measuring time of 1 h. However, in PES I the viability was about 4% for E. coli after 1 h while the viability of M. luteus was notably higher ( x 60%) after the same time span. The identical magnitude of differences could be observed for the glass fiber medium (G). In a filter medium consisting of polypropylene and polycarbonate fibers (PP/PC/PP), the viability after 1 h was x20% for E. coli and x95-100% for M. luteus (Fig. 4). The results indicate that bacteria exposed to air flow suffer from environmental stress, i.e. the toxic effects of oxygen and dehydration (Cox, 1987), which led to a decline in
control
PES I
Fig. 2. Growth of various with submerged samples
polyester, G = glass,
PES II
PP
G
PPIPCIPP
microorganisms in nutrient broth of different filter media: PES = PP = polypropylene, PC = polycarbonate.
their viability. The decline rate was dependent on the organisms employed. The inactivation of the more hardy M. luteus (gram-positive) was more slowly than that of the more sensitive E. coli cells (gram negative). In contrast to the results obtained for the filter medium PES I in nutrient broth (Fig. 2), the viability
Viability of bacteria in unused air filter media I
I 30
I 60
J 90
time in min Fig. 3. Viability of E. coli and M. luteus in two unused air filter media as a function of time. Curve fit according to the model by Cox (1987); PES = polyester, G = glass.
1 I_._ 0
/
!
30
60
E. coli
i
1
2309
,
I
I
30
60
90
time in min Fig. 4. Viability of E. coli and M. luteus in a unused air filter medium as a function of time. Curve fit according to the model by Cox (1987); PP = polypropylene, PC = polycarbonate.
I
-
D
time in min Fig. 5. Viability of E. coli in three unused air filter media as a function of time. Curve fit according to the model by Cox (1987); PES = polyester, PP = polypropylene, PC ==polycarbonate.
on viability after the collection process. As mentioned above, volatile and/or water soluble chemical compounds which were introduced in the fiber fabrication process may still be present in the manufactured fibrous filter and affect the viability of bacteria. Depending on the fiber material different compounds may be present that exhibit different magnitudes of damaging effects on the bacteria collected on the filter fibers (Simmons and Crow, 1995). The model calculations according to Cox (1987) showed a good agreement with the experimental data (Figs 3-5) indicating that viability of microorganisms was mainly impaired by the presence of oxygen and dehydration of cells, as proposed in the model by Cox (1987).
4. CONCLUSIONS
of M. luteus on PES I and G was less impaired than the viability of E. coli. Therefore the effects of fiber material under ideal nutritional and moisture conditions were different compared to the conditions at low air humidities. In nutrient broth bacterial growth takes place and the number of viable bacteria increases while at low air humidities the bacteria lose their ability to reproduce and the number of viable cells decreases. In Fig. 5 the survival of E. coli in three different filter media of different composition can be seen. The viability of E. coli declined for all three filter media employed but the decline rates were notably different for each filter medium. For PES I the viability declined to about 4% after 1 h and the viability was about 15-fold higher (60%) for the PES/PC/PES medium. These differences must be due to different fiber components which affected the viability of bacteria when attached to the filter fibers. Differences in the physical properties of the filter media (fiber diameter, fiber surface structure, porosity, etc.) only affect particle collection but should not have an effect
In experimental studies it could be shown that all, except one, investigated fiber materials did not exhibit an inhibitory effect on growth kinetics of M. luteus and E. coli in nutrient broth (ideal moisture and nutrient conditions). In addition, no notable growth enhancement due to the presence of fiber materials in nutrient broth could be observed. Tests conducted with bacteria collected in air filter media showed that the viability of bacteria declined within measuring times of 1 h, when relative air humidity was between 30 and 60%. The decline rate was strongly dependent on fiber material and type of microorganisms employed. More hardy organisms, like gram-positive M. luteus, seem to be more resistant to environmental stress factors, i.e. dehydration, toxic action of oxygen, than sensitive gram-negative bacteria (E. coli). In future studies it is planned to investigate the long term viability (during several days and weeks) of bacteria and fungal spores in fibrous filters as a function of air humidity and fiber material. It will be evaluated
R. MAUS et al.
2310
how these factors affect the viability of microorganisms in air filters and under which conditions microbial decay or growth is likely. Beside unused filter media it is further planned to include used filter media in the investigations to elucidate the effect of atmospheric dust collected in filter media on the viability of microorganisms. Acknowledgements-This work was supported by Projekte Europiiisches Forschungszentrumfir MaJnahmen zur Luftrein!-dung (PEF) under grant PEF 3 95 001 from Forschungszentrum Karlsruhe, Germany. The authors are grateful to Prof. K. Willeke and Prof. S. Grinshpun from the University of Cincinnati, U.S.A. for the valuable discussions and helpful suggestions. Special thanks are due to Dr K. Grimm and S. Wiirner from the University of Karlsruhe, Germany for their continuous support in our experimental investigations.
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