1'0 ABSTRACT

1
COMPARISON OF IMPACTION AND DEAD- SPACE
NEBULIZERS ON THE BIOAEROSOL CHARACTERISTICS OF
VEGETATIVE BACTERIA 
Brittany Goodenow and Chad J. Roy Division of
Toxinology Aerobiology, Department of
Aerobiology Product Evaluation United States
Medical Research Institute of Infectious Diseases
(USAMRIID), Fort Detrick, Maryland, USA
4.0 RESULTS
1.0 ABSTRACT
3.0 MATERIALS AND METHODS (cont.)
Aerosol generation of biological aerosols
(bioaerosols) is an essential part of ongoing
vaccine efficacy and agent pathogenesis research
efforts at USAMRIID. In this study, two
commerically-available nebulizers (Collison 3-jet
and the BioAerosol Nebulizing Generator BANG)
were compared to assess the impact of
aerosolization on the viability of the bacteria
Pseudomonas aeruginosa. Operating procedures
were kept constant between the nebulizers and
aerosol samples were taken continuously during
generation of the experimental atmospheres.
Serial dilutions (107, 108, and 109 CFU/ml) of
the P.aeruginosa were used as starting
concentrations in each of the nebulizers.
Aerosol samples were analyzed using both
conventional culture and nonculture techniques
such as fluorescent microscopy and flow
cytometry. Particle analysis indicated smaller
and less heterodisperse aerosols were generated
by the BANG (MMAD0.54GSD1.2) than the Collison
(MMAD1.0GSD1.4). Results of the culture
analysis indicated the Collison generated
slightly higher viable aerosol concentrations of
P.aeruginosa than the BANG suggested by lower
mean aerosol dilution factors (Collison1.32E-05
? 3.0E-04, BANG5.71E-06?1.6E-06). Flow
cytometric analysis of total organism output was
virtually identical from the nebulizers. In
contrast to the culture data, Live/dead analysis
by flow cytometry indicated a higher percentage
of viable bacterial cells relative to the
concentration of the starting material in the
BANG output. The results of this preliminary
study suggest that these nebulizers are
comparable in total particle output. The
Collison produced a slightly higher viable
bioaerosol, although differential flow cytometric
analysis of viability indicated the BANG produced
biaoerosols with a higher viability ratio. This
disparity of results may be explained by the
viable but nonculturable VBNC phenomena
observed in vegetative bacteria exposed to
unusual environmental stress during
aerosolization.
Bioaerosol Quantitation. The starting solution
were seven-fold diluted in triplicate and 100 ?l
of each dilution was plated on TSA. The AGI
samples were diluted at 110, 1100, and 11000
in triplicate and 100?l of each dilution was
plated on TSA. The plates were incubated at 37?C
and counted at 48 hours. Diluent PBS was also
plated and total CFU were subtracted from colony
counts from the experimental samples. Flow
cytometry was performed on the starting material
and AGI samples for total microorganism count.
The preparation for flow cytometry has been
previously described in the literature (Lange et
al., 1997). A bacterial counting kit (Molecular
Probes, Inc., Eugene, OR) was used for flow
cytometry analysis. Briefly, one ?l of universal
DNA stain was added to a one ml aliquot of either
the starting solution or AGI sample. The
solution was then vortexed, and allowed to
incubate at 37? C for five minutes. A
microsphere standard supplied in the kit was
sonicated for 10 minutes before addition to the
preparation. Exactly ten ?l of the microsphere
standard was added, and the preparation again was
vortexed. A live/dead bacterial viability kit
(Molecular Probes, Inc., Eugene, OR) was used for
flow cytometry analysis. A mixture of a red and
green DNA stain were added at a 11 dilution with
the starting solution or AGI sample. The
solution was vortexed and incubated at room
temperature for 30 minutes. All flow cytometric
analysis was performed using a Becton Dickinson
FACScan with a single excitation argon ion laser
at a wavelength of 488 nanometers. Results were
analyzed using a Multiple Document Interface
program (Version 2.8, J. Trotter, Scripps
Research Institute, CA).

4.0 RESULTS
Figure 2. Dot plots of serial total aerosol
samples for the BANG (A, B, C) and Collison
3-jet (D, E, F) at starting concentrations of
107, 108, 109, respectively. Aerosol samples
analyzed for total organisms indicated minimal
differences in the total particle output between
nebulizers. This is evident in the dot plots
above the total bacteria (R2 green) increases
as the starting concentration increases. Internal
microsphere standards (R1 red) were 10 ?m PSL
beads.
2.0 OBJECTIVE
Figure 1
Figure 2
Figure 3. Live/dead flow cytometric analysis of
aerosol samples. A trend of increased viability
is observed in the aerosols that were performed
with a higher concentration of P.aeruginosa in
the nebulizer starting concentration. Higher
viability counts relative to the total bacterial
cells counted are observed in the aerosols
generated by the BANG compared to the Collison
3-jet at the highest concentration attempted.
The objective of this study was to compare the
Collison and the BANG nebulizer using bacterial
viability as the primary indicator of differences
between the two devices. This was accomplished
by organism enumeration by both culture and
nonculture methods before and after aerosol
generation.
 
3.0 MATERIALS AND METHODS
Nebulizers and Exposure Chamber. The BANG, a low
flow, dead space, single pass nebulizer, was
specially-designed to incorporate 3 jets to
approximate Collison output (CH Technologies,
Westwood, NJ). The Collison 3-jet nebulizer was
commerically-obtained (BGI Inc., Waltham, MA).
HEPA-filtered, dried air at 25 psi was supplied
to each nebulizer during the aerosol exposures.
Appropriate operating pressure for the BANG has
been previously performed and showed minimal
differences in aerosol output at a supplied
pressure higher than 20 psi (L. Chen, personal
communication). Clean secondary air was also
supplied in the aerosol mixing tube into the
exposure chamber. The primary air flow used with
the BANG was 7 LPM with the total airflow for the
system being 10 LPM. The primary air flow used
with the 3 jet Collison was 7.5 LPM with the
total airfow for the system being 19.5 LPM. The
exposure chamber used was a 0.01 m3 plexiglass
box used in conjunction with ongoing rodent
studies. The average relative humidity and
temperature in the chamber were 35 and 72F
during aerosol runs.  Aerosols. For the
nebulizer characterization, 1X fresh
phosphate-buffered saline (PBS) was used (Gibco,
Grand Island, NY). For the aerosols involving
bacteria, Pseudomonas aeruginosa (ATCC,
Rockville, MD) was grown in Tryptic soy broth
(TSB Difco, Detroit, MI) at 37 degrees C for 24
hours. Thereafter, the broth was centrifuged,
the pellet washed, and resuspended in 1X PBS.
Seven-fold dilution aliquots were plated in
triplicate on Tryptic soy agar (TSA Difco,
Detriot, MI). The 24 hour counts yielded a stock
concentration of 1.0E10 CFU/ml. Dilutions of
1.0E09, 1.0E8, and 1.0E07 CFU/ml were viable
concentrations. Aerosol Characterization.
Particle characterization was performed using an
aerodynamic particle sizer (APS Model 3320, TSI
Products, St. Paul, MN). APS samples were taken
for one minute twice per aerosol run each
aerosol run lasted five minutes. Aerosol
Sampling. 6 LPM all glass impingers (AGI)
containing 10 ml of PBS (Ace Glass, Vineland, NJ)
were used to sample the chamber atmospheres. The
sampling efficiency of the AGI sampler in this
bioaerosol system has been shown to be more than
97 efficient (L. Pitt, personal communication).

5.0 CONCLUSIONS
Table 1. Aerosol Characteristics of the BANG and
3 Jet Collison 1 The spray factor is a unitless
measure of the ratio of starting concentration
(cS) to aerosol concentration (cA) and can be
expressed as SFcS (CFU/?)/cA (CFU/?)

• Nebulizers produced equivalent particle output
  the BANG nebulizer generated a smaller and less
  disperse aerosol
• The process of aerosolization destroys a
  significant number of organisms relative to
  culturable organisms in the starting liquid
  regardless of nebuilzer used
• The Collison nebulizer produced a higher
  culturable bioaerosol the BANG returned a higher
  viable count at high (109) nebulizer starting
  concentrations
• Vegetative bacteria undergo significant
  environmental stress when experimentally-aerosoliz
  ed this process may induce a viable but
  nonculturable state (Hiedelburg, 1997)

Figure 1. Comparison of the BANG and 3-jet by
culture and total organism count by flow
cytometric analysis. Results show minimal
differences between nebulizers regardless of
analysis method, although the Collison 3-jet did
produce slightly higher culturable counts than
the BANG. A consistent 4-log difference between
culturable and total organism aerosol
concentration was detected independent of
starting concentration in the nebulizers.
6.0 REFERENCES
Hiedelburg, J.F., Shahamat, M., Levin, M.,
Rahman, I., Stelma, G., Grim, C., and Colwell,
R.R. (1997). Effect of Aerosolization on
Culturability and Viability of Gram-Negative
Bacteria. Applied and Environmental Microbiology
63(9)3585-3588. Appl Environ Microbiol 1997
Apr63(4)1557-63 Lange, J.L., Thorne, P.S.,
Lynch, N (1997). Application of flow cytometry
and fluorescent in situ hybridization for
assessment of exposures to airborne bacteria
63(4)1557-63.