J. Aerosol Sci., Vol 16. No. 3, p p 193 200.

1985 Pnnted in Great Britain

0021 8502i85 $3.00 + 0 0 0 q 1985 Pergamon Press Ltd

USE OF BACTERIA AS M O D E L NONSPHERICAL AEROSOL PARTICLES

A. J. ADAMS, D. E. WENNERSTROM* and M. K. MAZUMDER University of Arkansas, Graduate Institute of Technology, P. O. Box 3017, Little Rock, AR 72203, U.S.A. and Department of Microbiology and Immunology, University of Arkansas lot Medical Sciences, Little Rock, AR 72205, U.S.A.
(Fir.st receired 27 Auyust 1984: and in rerisedJorm I I December 19841

Abstract--Nonviable rod-shaped bacteria Bacillus .suhtilis and E.wherichia coil and the spherical bacterium Staphylococcus epidermidis were aerosolized using the conventional technique o1" pneumatic atomization. Single-particle aerodynamic relaxation time (SPART) analysis was carried out in order to determine the orientation-averaged aerodynamic size distribution for the aerosol. Results indicate that the compressed-airnebulizer used in this study is effectivein generating bacterial aerosols of both uniform nonspherical and monodisperse spherical particles. The aerodynamic diameters as measured by the SPART technique agree with published data for the cell dimensionsand optical microscopic analysis. Further, the SPART method distinguished the two rod-shaped organisms based on their aerodynamic diameter. This study demonstrates that nonviable bacterial cells can serve as a model system in studying aerosols of nonspherical particles and illustrates the potential of the SPART technique in the rapid detection and characterization of airborne biological particles. INTRODUCTION One aspect of aerosol science currently receiving considerable attention is the study of nonspherical particles. N u m e r o u s studies aimed at characterizing the physical properties and dynamic behavior of nonspherical airborne particulates have been reported (Coletti, 1984; Dahneke, 1982; Gallily et al., 1983; Heyder and Scheuch, 1983; Kasper and Shaw, 1983; Liu et al., 1983; Mazumder et al., 1982). These efforts have been prompted by the finding that many natural and man-made aerosols contain nonspherical particles, some of which are regularly shaped (fibers, sea salt-crystals, and cylindrical microorganisms) while others are irregular in geometry (dusts, combustion and industrial aerosols~. The deviation in the dynamic behavior of a nonspherical particle from an equivalent spherical one is modeled by ascribing to the particle a dynamic shape factor 7,, a dimensionless parameter which relates the true drag force to an equivalent volume diameter (see Hinds, 1982, p. 47). It is possible to derive analytical expressions for Z in terms of the particle dimensions only for some simple regular geometric shapes, such as ellipsoids of revolution (Fuchs, 1964). For most of the particle shapes of interest it is necessary to make empirical measurements of ~ and this has been done for various regular and irregular nonspherical particles (Davies, 1979; Niida et al., 1983). One objective in probing the physical properties of nonspherical particles has been the development of instrumentation capable of detecting and characterizing aspherical particles (Detenbeck, 1980). One experimental difficulty which has impeded the systematic characterization of nonspherical particulate aerosols is the lack of suitable model systems. Hinds (1982) has emphasized the importance of monodisperse aerosols of known size, shape, and density in aerosol science and technology. The recent proliferation of data describing aerosols of spherical particles is in large part due to the development of monodisperse latex polymers (for discussion see Mercer, 19731. Although several recent studies have employed linear chains of spherical particles as a model system (Dahneke, 1982; Kasper and Shaw, 1983; Kasper, 1984), there is at present no readily available commercial source of uniform nonspherical particles for use as test aerosols. In this report we describe the generation and characterization of uniform aerosols containing cylindrical particles. We have found that nonviable rod-shaped bacteria in the size range approx. 1 ~4 ~m can serve as suitable regularly shaped non-spherical particles in model 193
AS 16/~-A

aerosol systems. Although the regularity and uniformity of bacterial particles as ,~=ii a~ their ability to remain intact and in some cases viable when aerosolized have io~: i~cc~ recognized, cylindrically shaped bacteria apparently ha~e not been x~delv exploited il~ aerosol studies. We note that within the field of applied and environmental biology ~i~cre ~ current interest in biological aerosols, specifically the generation, characterizations, and behavior of airborne bacterial and viral particles tSordelli et al.. 1984: Mohr ,'t al.. 19~4i. Here we demonstrate that bacteria of both spherical and rod-shaped morphologies ~at~ be aerosolized by the conventional technique of pneumatic atomization, as e~ idenced by ~ingleparticle aerodynamic relaxation time (SPART) analysis.* EXPERIMENTAL PROCEDURE Biological aerosol studies were carried out with three bacteria. Bacillus subtilis ilong rodt. Escherichia coli (short rod), and Staphylococcus epidermidis {sphere) were obtained from the stock culture collection of the Department of Microbiology and Immunology, University of Arkansas for Medical Sciences. Each organism was maintained at 4 C on slants of Todd Hewitt broth (Difco) solidified with 1.5 o,, agar. For experiments, cells were transferred by sterile loop to 200 cm 3 of Todd Hewitt broth and incubated at 37cC in an oscillating water bath with shaking at t l0cycles/min. After incubation for 18 hr, cells were harvested by centrifugation at 19,000 0 for 10 rain at 5~C, washed once in water and inactivated by the addition of formaldehyde at a final concentration of 0.7 ?o and incubation for 1 hr at room temperature. The cells were then washed one time by centrifugation as above and resuspended in water that had been filtered by passage through a cellulose acetate membrane with an average pore diameter of 0.45/am (Millipore). Gram-stained smears were examined at 1000 X using an American Optical Microscope equipped with an oil immersion objective. Compressed-air nebulization was used to generate aerosols of bacterial particles. The generator, a U-Mid single-use jet nebulizer (Bard-Parker, Rutherford, New Jersey, U.S.A. or Becton Dickinson, Lincoln Park. New Jersey, U.S.A.) has a liquid usable capacity of 500 ml and produces aerosol particles efficiently in the 0.3~,/am diameter range. This type of generator is equipped with a dial for selecting a required oxygen dilution. This was set to 100/o in our studies. Filtered dry air at a typical pressure of 1.03 x 105 Pa (15 psig) was applied to the nebulizer, resulting in an air flow of approximately 7 l/min. Previous studies using polylatex spheres (PLS) in the nebulizer have shown that in the 1 #m diameter particle size range an initial liquid suspension of 10 9 particles/cm 3 results in an aerosol concentration of 104/cm 3. Comparable concentrations were noted with bacterial samples. The principal method for analyzing the bacterial aerosols was single particle aerodynamic relaxation time (SPART) analysis (Mazumder et al., 1979). This technique employs a dualbeam laser Doppler velocimeter to monitor single particle dynamics in an oscillatory acoustic field o f 24 kHz. The aerodynamic diameter of individual particles is determined by measuring the phase lag of the particle motion relative to the driving acoustic excitation. The time measurement used in the phase determination is made in such a way that 0 - rt/2 phase lag is measured to 1 part in 128. A microcomputer is used to record the counts in each of the 128 channels, the total number of counts N, and the total sampling time in seconds. The size distribution is displayed immediately by an on-board printer. A plotting routine generally presents the data as dN/d(logda) as is, or in a normalized form. The phase lag, a direct measure of the particle aerodynamic relaxation time rp, varies significantly with aerodynamic diameter only in the frequency range centered at cozp = 1, where o9 is the angular frequency of the acoustic excitation wave. For an acoustic frequency of 24 kHz the SPART instrument can effectively size particles with high resolution in the 0.3-6/am aerodynamic diameter range. Recently the operating range of the system was

* The results reported here werepresentedin part at the 1984Annual BiophysicalSocietyMeeting,San Antonio, Texas, U.S.A., 19-23 February 1984.

Bacteria as aerosols

195

extended to include particles in the 3-20 pm range by operating at an acoustic frequency of 1 kHz (Adams et al., 1984). Prior to each study the SPART analyzer was calibrated using uniform latex particles. Separate suspensions of 0.6 and 1.09 pm diameter PLS were subjected to aerosolization by pneumatic atomization, yielding monodisperse aerosols with concentrations of 104-105 particles/cm 3. These aerosols containing PLS particles of known sizes were used to verify the SPART calibration. Washed bacteria were suspended in distilled water to yield concentrations of approximately 109/cm 3. The resulting cell suspension (50-100 ml) was then added to the reservoir of the nebulizer and compressed air applied. Two sampling schemes were employed: both resulted in similar recorded size distributions. In one case aerosol particles were generated directly into a 4-1. flask and the sample was drawn directly from the flask without drying and channeled through the SPART relaxation chamber within minutes of generation. In the second case aerosol particles were first channeled through a conventional silica gel drying column (outer diameter 5.0 cm, inner screen diameter 2 cm, length 60 cmt and then passed through a 1-m polyvinylchioride tube (5 cm in diameter) to the SPART analyzer. The sample flow rate through the instrument is typically 50 cm3/min and a typical count rate for the SPART system is 1000-2000 per min. In order to obtain initial data concerning the physical state of the aerosolized bacterial particles, a study of cell viability and particle count rate as a function of time following aerosolization in a flask was carried out in separate runs using B. subtilis and E. coil In this study the treatment with formaldehyde was omitted in the preparation procedure. At certain set time intervals the bacterial aerosol was sampled by drawing 10 ml into a plastic syringe containing approximately 1 ml of warm growth medium. The syringe was capped and shaken to entrap the aerosolized bacteria and the sample was immediately plated by adding the medium to molten agar. At the same time, the aerosol was sampled by the SPART analyzer and the number of particles per unit time was determined. Several additional experiments were performed in order to help facilitate data interpretation. The effect of formaldehyde-treatment on the cell size distribution of E. coil was explored by the measurement of 'live' cells which had only been washed in distilled water. In another experiment a suspension of E. coli was sonicated at 200 W for 5 min and then aerosolized and sampled. To verify that the SPART instrument could distinguish the short rod E. coli from the long rod B. subtilis, 1 cm 3 aliquots ofE. coli, standardized to a turbidity of 400 Klett units (Klett-Summerson photometer with blue filter) were added sequentially to 75 cm 3 of B. subtilis diluted 1:4 from an original suspension of 400 Klett units. The size distribution of the particles was determined following aerosolization after each addition. In a separate experiment, the aerosol of B. subtilis particles was collected on a 0.2 pm Nuclepore polycarbonate filter, the particulates resuspended in water by vigorous shaking, and the sediment after centrifugation placed on a optical microscope slide for observation. An additional control experiment in which bacterial suspensions were filtered through 0.2 #m Nuclepore by vacuum filtration and the filtrate used to generate aerosol was also carried out.

R E S U L T S AND D I S C U S S I O N The morphologies of B. subtilis, E. coli, and S. epidermidis are well known. Bergey's Manual of Determinative Bacteriology (Buchanan and Gibbons, 1974) lists B. subtilis as a rod, 0.7-0.8/~m x 2-3 pro, E. coli as a rod 0.4-0.7 ~tm 1-3/~m, and S. epidermidis as a sphere with diameter 0.5-1.5 pm. Optical microscopic analysis of dried stained smears of our preparations confirmed these shapes and dimensions. To calculate predicted mean aerodynamic diameters the following dimensions are assumed: 0.75 x 3.0 #m for B. subtilis, 0.5 x 1.5 pm for E. coli, and 1.00 gm for S. epidermidis. Note that these figures are consistent with Bergey~s data. The aerodynamic diameter d a o f a nonspherical particle can be related to an equivalent volume diameter de by: cl~ = ppd~, poZ, tl)

where pp is the particle mass density, Po is unit density, and Z ts the dynamic shape Iaclor lz~ order to apply equation (li for calculating predicted aerodynamic diameters, ~alues tot mass density and shape factor must be presumed. Specific density values from 107 to i.25 g c m ? have been reported for bacteria (Lamanna et al.. 1973L No attempt to independentl~ determine the mass density of the aerosolized bacterial cells was made. Because t~t the uncertainty in pp we calculated predicted diameters for specific densit 5 values oi ! and. 1.16 g/cm 3 {mean of range cited above). Assuming cylindrical shape for the rod-shaped bacteria, published data for the shape factors characteristic of distinct L D values can be used. In our sampling procedure the Reynolds number is less than 0.1 and thus no orientation is presumed. An orientation average determined by

X., = 2Xh, 3 + X~/3,

(2)

where Xh is the shape factor for cylindrical axis horizontal in a vertical flow field and X, the value for longitudinal orientation, is used to calculate predicted values for d~. Values tbr Zh and X~, were taken following Fuchs 11964, p. 41). Figure 1 presents a typical normalized size distribution for E. coil as recorded by the SPART analyzer. As noted in Table 1, which compiles the results of comparing the measured SPART values with those calculated by (lt and (2), an average aerodynamic diameter of 0.89/Jm was measured. The SPART value compares favorably with the calculated value of 0.83/~m (assuming a density of 1.16). Included in Fig. 1 is the distribution from the sonicated suspension, indicating that (1) in the initial case the peak in the distribution is due to whole cells and (2) under the specific sonication conditions some intact cells remain. We note here that there was no significant difference in the size distribution between qive" cells and those pretreated with formaldehyde, In Fig. 2 a representative normalized distribution for B. subtilis is given. An average value for five preparations of 1.29 ~m was obtained with the SPART analyzer, while calculated

1. O0 O. 900 O. 800.
...d

O. 700
O'1

0.600 O. SO0 O. 400

O. 300
i.-.,

0.:~00,

0.100. O. 0.10

./
0.20 0.50 1.0 2.0 RERODYr4~IlC DIRI1ETER (IIICROMETERS] S.O

10.

Fig. 1. Normalized aerodynamic size distribution [( 1/N )dN/dllog d=) vs d=] for aerosolized E. olt a,~ measured by the SPART analyzer belore (-.-o--} and after [ - - O - - ) sonication of the suspension. The count median aerodynamic diameter, for approximately 1000 particles sampled per minute, was 0.891 ~m before sonication and 0.614#m after sonication. The geometric standard deviations were 1~15 and 1.29, respectively.

Bacteria as aerosols Table 1. Predicted and measured values of orientation-averaged aerodynamic diameter Measured orientation averaged d o in (/lm) 1.29 +- 0.06 0.89+_0.05 0.92 +_ 0.08

197

Sample
B. subtilis E. coli S. epidermidis

N u m b e r of preparations 5 5 2

Geometrical size (/am) 0.75 x 3.0 0.5 x 1.5 1.0

Assumed density (g/am 3) 1.0 1.16 1.0 1.16 1.00 1.16

Predicted d,, (urn) 1.19 1.29 0.76 0.83 1.00 1.08

Difference (':,,~ 7.7 0.0 14.6 6.7 8.(1 15.0

values of 1.19/~m and 1.29 were predicted based on the assumptions previously cited. We note the presence of a secondary peak at approximately 0.85 #m. The microscopic analysis of aerosol particles collected by filtration revealed that particles of this smaller size were present to a minor extent in the aerosol. Rather than cell fragments these particles appeared to be coalesced cytoplasmic material. We did find that when either Todd-Hewitt broth or istonic saline was present in the cell suspension a significant amount of particles in the 0.65-0.85/~m diameter size range was recorded by the SPART instrument. The photomicroscopy of collected aerosol particles also revealed the minor presence of cell coats apparently emptied of intracellular material. These 'shells' may also contribute to the secondary peak at 0.85/~m. A typical SPART distribution for S. epidermidis is given in Fig. 3. The results of generating an aerosol from a mixture ofE. coli and B. subtilis are presented in Fig. 4. The SPART technique is capable of distinguishing the two particles based on aerodynamic behavior. The two curves represent successive additions of E. coli suspension to a suspension ofB. subtilis. We note here that in our control experiment, in which filtrate after passage through a 0.2 #m filter was used to generate aerosol, the particle count rate dropped from 1000 per min to 35 per min, with the bulk of these counts occurring at 0.5 #m.

1.00 O. 900 O. 800

..J

O. 700
Ln

t
, , , , ,

0.600 O. 500 O. 400 O. 300

2
O. 200
Olf

0.100 O. 0. I0

0.20

0.50 1.0 2.0 RRODYHRMIC DIflrlETER (MICROMETERS]

S.O

10.

Fig. 2. Representative normalized aerodynamic size distribution for B. suhtilis as measured by the SPART technique, corresponding to a count median aerodynamic diameter of 1.29~m and a geometric standard deviation of 1.29.

1.00

O. 900
O. 800

O. ?00
O. 600 0.500 0.400.
Z

0.300.
,...,

O. 200
0. I 0 0 O. 0.10

0.'20

0.'S0 1.0 2~ 0 RERODYI~RrlIc DIP,METER CrlIcRorIETERS)

S. 0

10,

Fig. 3. Normalized aerodynamic size distribution for S. epidermidis as measured by the SPART analyzer. The peak in the size distribution occurs at 0.88.um.

1.00,

O. 900 O. 800
_J

O. 700 0.600
e,,t

o. SO0

O. 400
Z

O. 20o o. 200
0. I00 o. 0.10

2,

0.20

0.50 1.0 2.0 RERODYNRrlIC OIRrlETER (tlICROrETER$)

S.O

10.

Fig. 4. Normalized size distributions for aerosols from a mixture o f E. co/i and B. subtilis. The mixtures were prepared by adding a total o f I cm 3 (o) o r 2 cm 3 gl) o f E. coli suspension to 75 cm 3

of B. subtilis.

Bacteria as aerosols Table 2. Time variation of count rate and cell viability Sample
B. suhtilis

199

Time following aerosol generation (mint

< 1

Relative count rate

1.130

Relative viability
1.00

5 12 20
E. coil < 1

0.47 0.32 0.22

1.00

0.17 0.03 0.004

1.00

5 10 15 25

1.00 0.83 0.58 0.38

0.18 0.17 0.05 0.02

The o u t c o m e o f the study o f the variation o f count rate and cell viability is presented in Table 2. Generally bacterial aerosols were generated for approximately 1 min in a 4 liter flask and the sampling times reported in Table 2 refer to the delays following this procedure. Relative count rate and relative viability refer to the initial value. F o r aerosol generation using the U - M i d nebulizer the S P A R T c o u n t rate for B. subtilis decreases moderately fast (to 47 //o in 5 min) and the relative viability drops to 17 ~o. As can be seen, m u c h o f the d r o p in viable counts is due to the reduced particle count. The c o u n t rate for E. coil does not appear to decrease in the first 5 rain while the viable c o u n t falls to 18 ~o. Thus, the data reflect a true loss in viability. The general result o f the greater loss in particle c o u n t for the larger particles (B. subtilis), a l t h o u g h p r o b a b l y influenced by the m e t h o d o f aerosolization and particle shape, is consistent with our empirical observations with polystyrene latex aerosol preparations (unpublished). CONCLUSIONS We have d e m o n s t r a t e d that E. coli, B. subtilis, and S. epidermidis can be aerosolized by pneumatic atomization, that the airborne rod-shaped bacterial particles can serve as test aerosols o f nonspherical particles, that the S P A R T analyzer can aerodynamically size the cells on a single particle basis to within 10 ~o o f the expected value, and that B. subtilis and E. coli can be clearly distinguished based on a e r o d y n a m i c size. In addition, viability o f E. coli and B, subtilis decrease significantly after 5 min. These results show that the S P A R T system rapidly detects and characterizes airborne microbes. Therefore, it has potential in the study o f aerosolization techniques, cell viability, and the morphological response o f bioaerosol particles to varying environmental conditions.
Acknowledgement--We gladly acknowledge helpful discussions with J. D. Wilson, technical help from R. Evans and P. Archer, and secretarial assistance from D. Belk. The authors also wish to thank the reviewers of the manuscript for constructive suggestions.

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Liu, B. Y. H., Pul D. Y. H., Wang, X. Q. and Lefts. ('~ W. I19831 Aeroso/ Set. Tech. 2, 4m~ Mazumder. M. K.. Ware, R. E.. Wilson. J. D., Renninger, R. G., Hiller, F (2, McLeod. P ( R a i b l c ~ ~,~. a~d Testerman, M, K. t1979)J. 4erosoi So1. 10, 561 Mazumder, M. K., Chang, R. J. and Bond, R. L. (19~2) Aerosol S~i, Tech. 1,427. Mercer, T. T. (1973)Aerosol Technology in Hazard Evaluation, p. 329. Academic Press, Nev, York. Mohr, A. J., Adams, D J., Spendlove, R. S. and Spendlove. J. C. ( 19841 Abstract, 1984 Annual Meeting ot American Society of Microbiology. p. 235. St. Louis. Missouri. US.A. Niida, T., Yang, M.. Kasper, G. and Shaw. D, T. ii9831 Aerosol Sti. ?'e~h. 2, 210. Sordelli, D. O., Cerquetti. M. C and Bellanti, J. A {1984) Abstract. 1984 Annual Meeting ol American Society ol Microbiology. p. 30. St, Louis, Missouri, U S A