Method to Determine the Number of Bacterial Spores Within Aerosol ...

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We describe methodology to reveal the number of microbial spores within aerosol particles. The procedure involves visualiza- tion under differential- ...
Aerosol Science and Technology, 39:960–965, 2005 c American Association for Aerosol Research Copyright  ISSN: 0278-6826 print / 1521-7388 online DOI: 10.1080/02786820500352098

Method to Determine the Number of Bacterial Spores Within Aerosol Particles Monica Carrera2 , Jana Kesavan1 , Ruben Zandomeni2 , and Jose-Luis Sagripanti1 1

Research & Technology Directorate, Edgewood Chemical Biological Center, U.S. Army, Aberdeen Proving Ground, Maryland, USA 2 GeoCenters Inc., Gunpowder Branch, Aberdeen Proving Ground, Maryland, USA

We describe methodology to reveal the number of microbial spores within aerosol particles. The procedure involves visualization under differential- interference-contrast microscopy enhanced by high-resolution photography and further analysis by computerassisted imaging. The method was used to analyze spore of Bacillus globigii in aerosols generated by a small (pressured metered-dose inhaler type) generator. Particles consisting in 1 or 2 spores accounted for 85% of all generated particles. This percentage rose to 91% when the same aerosol was collected on an Andersen cascade impactor that collected particles larger than 0.65 µm and was even higher (96%) when particles larger than 3.3 µm were also eliminated. These results demonstrate that the imaging analysis of aerosol particles collected on glass slides is sensitive to even relatively small changes in aerosol particle composition. The accuracy of the enhanced microscopic method described herein (differences between visual and computer analysis were approximately 3% of the total particle counts) seems adequate to determine the spore composition of aerosols of interest in biodefense.

INTRODUCTION The extent of the dispersal of anthrax spores from contaminated envelopes (shortly after September 11, 2001) was surprising. Before this attack, most scientists (and likely also the perpetrators) would have not expected that spores could disperse through envelopes and buildings at the extent that anthrax spores did. Although studies abound on the physical principles governing aerosols and on the characteristics of many aerosol collectors (Placencia et al. 1982; Jensen et al. 1992; Nevalainen et al. 1992; Mainelis et al. 1999; Griffiths and Steward 1999; Aizenberg et al. 2000), quantifying the bacterial content of individual aerosols particles needs to be examined further. The goal of this study was to determine the number of microbial spores inside aerosol particles, since this property could

Received 28 October 2004; accepted 19 April 2005. Address correspondence to Jose-Luis Sagripanti, Dr. Sc., US Army RDECOM, Attn: AMSRD-ECB-RT, Bldg. 3150, Aberdeen Proving Ground, MD 21010-5424, USA. E-mail: joseluis.sagripanti@ us.army.mil

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be relevant to dispersion, infectivity, and survival of aerosolized spores. Establishing the number of spores inside each aerosol particle is different than studying the size distribution of the same aerosol. The size distribution of aerosol particles containing Bacillus subtilis aerosolized by a CO2 pressure sprayer was previously determined and a model developed to predict deposition trajectories as a function of particle size (Lighthart et al. 1991). Colony formation has been the method of choice to enumerate airborne bacteria. However serious drawbacks with this approach have been documented. Colony counting of collected particles can quantify how many particles contained culturable microorganisms but not how many organisms were in each particle and can introduce an error due to indistinguishable colony overlap (Chang et al. 1994). Although the error due to colony masking can be reduced by correction factors obtained by computer simulation, the corrective approach applies to specific conditions and does not account completely for the error of elongated or irregular colonies. (Chang et al. 1994). In addition, colony formation counting is subject to error because bacteria may remain viable yet lose their ability to form colonies during aerosolization (Heidelberg et al. 1977). METHODOLOGY AND RESULTS We produced aerosols with a small, pressurized metered dose inhaler and collected the expelled particles as a dry residue on the surface of uncoated glass slides (Figure 1A). To microscopically resolve single spores in unstained, live specimens, it was necessary to increase the optical magnification to a total of 1000 × and to use the Differential Interference Contrast (DIC) mode. The DIC mode gave a three-dimensional image without the distortion of the diffracted light produced by phase contrast and clearly resolved spores within particles (Figure 1B). Although common glass slides gave satisfactory results, Teflon or any other transparent coating over the slides could be used without interfering with the microscopic analysis. We attempted to estimate the number of spores within particles simply by correlating the size of spores with the size and the shape of each particle. We obtained images of aerosol

MICROBIAL SPORES WITHIN AEROSOL PARTICLES

FIG. 1. Image of aerosolized spores. Metered dose inhalers were filled with (5 ml) spores of Bacillus globigii 0.05% w/w using Dymel 134a as propellant (DuPont, Wilmington, DE 19898). Applications of aerosols with the metered dosed inhaler were contained inside a disposable 45-gallon disposable polyethylene bag held with tape to a wire skeleton (0.80 m long by 0.50 m wide by 0.40 m high). The structured bag was placed inside a Biosafety Level-II cabinet. The aerosol generator was operated inside the bag by inserting a hand through the bag opening. Aerosol particles from the generator were collected on glass microscope slides, which were cleaned and rinsed with ethanol prior to use. The glass slides were situated at 25 cm from the aerosol generator, in a vertical orientation and centered at the same height with the nozzle. After aerosol impact, the slides were removed from the bag and very gently covered with 2 or 3 cover slips and their corners sealed with washable glue. The slides were analyzed in a Leica DMR Optical Microscope (Leica Microsystems Inc.) under differential interference contrast (DIC). Several pictures were taken randomly across slides in a raster pattern scan to cover representative areas of slides. A) DIC micrograph employing a 100 × oil immersion objective to reach a maximum magnification of 1,000 ×. B) The area framed in A further digitally magnified three times.

particles by DIC micrograph and analyzed them by computerassisted imaging. Aerosol particles observed by DIC microscopy at 1000 × optical magnification and digital micrographs analyzed by the computer software are compared in Figure 2. From several options in the software package, we measured the Ferret’s diameter, which is the longest distance between any two points along the section boundary of each aerosol particle. The boundary of each particle defined with computer assistance (Figure 2B) corresponded with the particles observed microscopically (Figure 2A). We calibrated the computer images of spore particles (provided in pixels) using the mean length of B. globigii (1.22 ± 0.12 µm, Zandomeni et al. 2003) as internal standard to calculate the particle Ferret’s diameter. (Figure 3A and Table 1). First, we simply assumed that particles with Ferret’s diameters within two spore lengths (between 1.2 to 2. 4 µm) would contain 1 or 2 spores. Under this assumption, 93% of the particles consisted in 1 or 2 spores. However, there are obvious geometries where 3 spores could also satisfy this criterion. Therefore, we trained the software to recognize single and multiple spores

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by defining visually the area of particles with single, double, and more spores. We determined that particles containing one, two, or three to five spores had average areas between 0–1.0 µm2 , 1.0–2.0 µm2 , or 2.0–5.0 µm2 , respectively. The distribution of particles obtained by analyzing particle area is shown in Figure 3B. Considering the area of particles, the software estimated that 86% of the particles consisted of either one or two spores. (Table 1). Although computer-assisted image analysis was relatively fast, we were concerned with the error introduced by the presence of debris and broken cells, which were visible in the images. Since aerosols dispersed during a biological attack are likely to contain relatively unpurified (or dirty) spores, we needed a more precise and robust methodology for potential uses other than analyzing clean laboratory preparations. As a result we relied on visual recognition of spores within particles to rule out the debris interpreted as spore particles by the program used in computer-assisted analysis. We complemented the optical magnification provided by the microscope lenses with digital magnification by observing the 5.1 Megapixel pictures taken with the DC-480 CCD camera (Leica Microsystems Inc., Bannockburn, Illinois, USA). (Figure 1 Panel B) This approach made it possible to clearly resolve and count the spore composition of each aerosol particle. The composition of 200 aerosol particles per slide was microscopically analyzed as described in Figure 1. The number of spores in each particle was counted and particles were classified in one of four classes: 1) particles composed of single spores, 2) containing two spores, 3) between three and five spores, and 4) particles consisting of six or more spores. To rule out collecting spores artificially clustered by proximity to the generating nozzle, we collected aerosol particles at 10 cm, 15 cm, and 25 cm from the generating nozzle. At least three independent applications on three different slides were collected for each distance. We then counted the spores within more than 1800 aerosol particles collected in ten different slides. The relative percentage of the number of spores within particles is shown in Figure 4. The similar distribution of spores obtained at different distances demonstrates that even at the shortest collected distance, the number of spores in each particle remains stable in the aerosols without any measurable clumping or dispersion. That is, the number of spores within a particle remains fixed while the particles diverge between each other with increasing distance from the nozzle. By visually counting spores within particles, the proportion of aerosol particles consisting of one individual spore was 70%. Those particles containing two spores accounted for 15% of all analyzed particles. Aerosol particles consisting of three to five spores represented 11% and large particles containing six or more spores accounted for the remaining 4% (Figure 4). This agreement between visual and computer-assisted analysis (Table 1) validates the use of the selected software and the particle area analysis method for particles containing 5 or fewer spores.

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FIG. 2. Computer-assisted image analysis of aerosol particles. Imaging analysis of the pictures was performed using the Image Pro Express software (Media Cybernetics L.P Silver Spring, Maryland, USA) and Image J 1.31 (an image processing and analysis software in Java format developed at the National Institutes of Health, Bethesda, Maryland which is in the public domain). A) DIC micrograph of B.globigii spores within aerosol particles observed at 1000 × optical magnification and further enlarged digitally to identify individual and clustered spores. B) Particle boundaries obtained from picture A by computer-assisted analysis. Direct comparison of panels A and B allows correlation between particle size and the number of spores inside each particle.

To assure an unbiased (by size) collection of particles, and to examine the proportion of spores within small respirable particles that will stay suspended for a long time in the atmosphere, and to study the sensitivity of our method, aerosol particles generated by the same pressurized metered inhaler as before were collected with components of an Andersen cascade impactor

(Andersen, 1958; Figure 5). When the cascade impactor with only stage 6 was used to collect particles larger than 0.65 µm, the proportion of particles containing 1, 2, 3 to 5, and more of 5 spores was 80%, 11%, 5%, and 4%, respectively. The proportions shifted to 84% consisting of 1 spore, 11% of 2, 5% of 3 to 5, and 0% with more than 5 spores, in another series of

FIG. 3. Size distribution of aerosol particles determined by computer-assisted analysis. Pictures were obtained and analyzed with the Image-J software as described in Figure 2. A) The Ferret’s diameter of each particle was assigned and the bar heights represent the frequency of particles grouped accordingly to the diameters indicated in the X-axis. B) The area of each particle was calculated and correlated with the number of spores by training the software as described in the text. The bars represent the frequency of particles with the number of spores indicated in the X-axis.

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FIG. 4. Distribution of spores inside aerosol particles at various distances. The glass slides were situated as described in the legend of Figure 1 at various distances from the pressurized metered inhaler. The height of the bars represents the percentage of particles containing the number of spores indicated in the X-axis and collected at 10 cm (light bars), 15 cm (gray bars) and 25 cm (dark bars) from the aerosol source. The bracket on top of the bars represents the standard deviation obtained after counting a total of 1,800 particles.

FIG. 5. Aerosols collected with an Andersen cascade impactor. Spores of B. globigii were delivered into a 0.26 m by 0.26 m by 1.00 m high plexi-glass box. The air in the box was heated using a hot plate for faster drying of the aerosols. The temperature and humidity inside the chamber were 30 C◦ and 36% respectively. The aerosol in the box was mixed using a fan. Aerosol particles were collected with components of a six-stage cascade impactor (Andersen Sampler Inc., Atlanta, GA) using an airflow of 28.3 liters/min, according to manufacturer’s instructions. In normal operation, agar filled petri dishes are placed in the impactor for collecting and culturing bioaerosols, however, in this test particles were collected onto microscope slides for counting. Paraffin wax was used to fill a petri dish to the appropriate height for the correct operation of the impactor and a microscope slide was placed in the petri dish on top of the wax to collect the particles. A micrographic image of a characteristic sample obtained after removing stages 1, 2, 4, and 5 from the cascade impactor and using only stages 3 as a fractionator to remove large particles and 6 to collect particles with aerodynamic diameters between 0.65 µm and 3.3 µm is shown in the Inset. The heights of the bars represent the percentage of particles containing the number of spores indicated on the X-axis. The bracket on top of the bars represents the standard deviation of the mean percentage obtained over three runs counting 200–250 particles on each slide for a total of 700 particles.

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TABLE 1 Spore distribution by computer or visual analysis Computer analysis Spore number per particle

Particle length

Particle area

Visual analysis number of spores

1 2 3–5 6 or more Particles with 1 or 2 spores

58.0% 35.0% 5.5% 1.0% 93.0%

69.0% 17.0% 13.0% 1.0% 86.0%

70.0% 15.0% 11.0% 4.0% 85.0%

experiments where the cascade impactor contained both stages 3 and 6 so that particles between 0.65 µm and 3.3 µm in size were collected on slides below stage 6 (Figure 5). As expected, the proportion of single spores increased when we used stage 3 as an inertial precut to eliminate the large, short-lived aerosol particles. The proportion of aerosol particles consisting of 1 or 2 spores was relatively high in all three experimental settings (horizontal impaction in the structured bag, one stage Andersen, and two stage Andersen). Particles with 1 or 2 spores accounted for 85% of all particles when all particles were collected within the structured bag (Table 1). This percentage rose to 91% when the same aerosol generator was used over an Andersen cascade impactor containing one stage and, as expected, was even higher when large particles were eliminated by using two stages in the impactor. These results demonstrate that the area method of imaging analysis of aerosol particles collected on glass slides is sensitive to even relatively small changes in aerosol particle composition. The accuracy of computer-assisted analysis of particle area when compared to visual counts (discrepancy ranging between 1% and 4%, see Table 1) seems adequate to determine the spore composition of aerosols of interest in biodefense. Our findings demonstrate that the generator that we used produces aerosols where the majority of generated particles are constituted by one or two spores. Although it is likely that different aerosol generators could disperse particles with different spore composition, the spore distribution produced by the aerosol generator used in this study is in agreement with the result presented in the study performed on secondary aerosolization of viable Bacillus anthracis spores in a contaminated US Senate Office in which more than 80% of the B. anthracis particles were within an alveolar respirable size range of 0.95 and 3.5 µm aerodynamic diameter measured with a 6 stage Andersen impactor (Weis et al. 2002). The goal of this study was not to characterize any single aerosol generator but to develop a method to study spores within aerosol particles. The imaging method we used is independent of the aerosol generation system. Therefore, the

approach presented here should be useful to characterize the microbial composition of aerosols generated by a variety of aerosol generators. Characterizing the number of bacterial spores and other microbes in aerosols is important. Aerosols with a high proportion of particles consisting of individual microbes could have high infectious efficacy. In contrast, aerosols with a large proportion of particles containing a high number of aggregated microbes may have increased survival. In this case, a fraction of the aerosolized spores may survive exposure to UV irradiation, desiccation, atmospheric gases, decontamination, and other damaging effects that can be scavenged by microbes in an outer layer while protecting those in the core of the particle. In addition, information on the microbial component within aerosols should help refine dispersion predictions and better define the extent of contamination after release of biological threat agents. Characterization of microbial aerosols with the method described here could assist in better defining the environmental risk after a biological attack.

ACKNOWLEDGEMENTS The authors thank Dr. Edward Stuebing (from the Aerosol Sciences Team, Edgewood Chemical Biological Center, Aberdeen proving Ground, US Army) for advice, encouragement, and for providing the aerosol generators used in this work.

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MICROBIAL SPORES WITHIN AEROSOL PARTICLES Nevalainen, A., Pastuszka, J., Liebhaber, F., and Willeke, K. (1992), Performance of Bioaerosol Samplers: Collection Characteristics and Sampler Design Considerations, Atmospheric Environment. 26A(4):531–540. Placencia, A. M., Peeler, J. T., Oxborrow, G. S., and Danielson, J. W. (1982). Comparison of Bacterial Recovery by Reuter Centrifugal Air Sampler and Slit-to-agar Sampler, Appl. Environ. Microbiol. 44(2):512– 513. Zandomeni, R. O., Fitzgibbon, J. E., Carrera, M., Stuebing, E., Rogers, J. E., and Sagripanti, J.-L. (2003). Spore Size Comparison Between Several Bacillus Species, Proceedings of the 2003 Joint Service Scientific Conference

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on Chemical & Biological Defense Research, Towson, MD. DOD publication ECBC-SP-018, Approved for unlimited distribution. Aberdeen Proving Ground, Maryland 21010-5424, September. Zimmerman, N. J., Reist, P. C., and Turner, A. G. (1987). Comparison of Two Biological Aerosol Sampling Methods, Appl. Environ. Microbiol. 53(1):99– 104. Weis, C. P., Intrepido, A. J., Miller, A. K., Cowin, P. G., Durno, M. A., Gebhardt, J. S., and Bull, R. (2002). Secondary Aerosolization of Viable Bacillus Anthracis Spores in a Contaminated US Senate Office, JAMA. 288(22):2853– 2858.