APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 1979, p. 642-643 Vol. 38, No. 4
0099-2240/79/10-0642/02$02.00/0
Evidence for More Than One Division of Bacteria Within Airborne Particles
R. L. DIMMICK,* H. WOLOCHOW, AND M. A. CHATIGNY
Naval Supply Center, Naval Biosciences Laboratory, University of California, Oakland, California 94625
Received for publication 18 May 1979
When the protocol that we had used to demonstrate a single division of bacterial cells in airborne particles was changed
to one that increased the glycerol content of the atomizer fluid from 1 to 5% (vol/vol), thus producing larger
particles, more than two (and nearly three) divisions of bacteria occurred within 6 h of aerosol time.
------------------------------------------------------------------------------------------------------------------------------------------
In a series of papers, we presented evidence that a bacterium (Serratia marcescens) can undergo
at least one division within small (2-,um diameter, more or less), airborne, aqueous particles
containing nutrients (2, 3, 6).
This paper presents evidence that when the droplet size was increased to 4 to 6 jim in diameter,more than one division
occurred.
MATERIALS AND METHODS
The techniques, instrumentation, and species (S. marcescens 8UK) described in the previous papers
were used in this study with the following exceptions:
(i) the glycerol content of the atomizer fluid was increased from 1% (vol/vol) to 5% (vol/vol) for the
purpose of creating larger particles at equilibrium, with the moisture content of air holding the aerosol
(4); and (ii) Coulter Counter studies were not done, since we had satisfied ourselves that the increase in
viable bacterial numbers above that of the initial numbers was not artifactual (not caused by wall effects
within the aerosol chambers) and that the settling rate of the contained particles was measured adequately
by continuous light-scatter (nephelometric) measurements (1).
RESULTS
Data from two replicate runs wherein maximal growth occurred are shown graphically in Fig. 1A and B. Logistical
problems prevented our taking a 12-h sample at night; hence, hypothetical data points at 12 h of aerosol time
(indicated with asterisks) were derived by interpolation of the observed decay of viable cell numbers. We include them
for clarity and for estimating a potentially maximal number of new cells produced.
On this basis, the increase in numbers of viable airborne cells, corrected for fallout, was sixfold in one test (Fig. 1A) and
slightly more than sevenfold in another (Fig. 1B). On the basis of the 24-h samples, the increases were fivefold and
sixfold, respectively. Since samples were taken in impingers, wherein the bacteria are distributed as individual cells
within sampler fluid before the dilution and plating step, the corrected numbers (triangles in Fig. 1) do not represent an
increase in airborne particles, but rather an increase in the number of cells within particles remaining in the volume of air
sampled.
The increased physical decay of the aerosol during the first 6 h, measured as light scattered from particles, showed that
the median particle size (1) was larger than those of aerosols previously tested (3) and was approximately 4 jim in
diameter, with some particles as large as 6 um and some as small as 1 um. The heterogeneity of the particle sizes and
the probability of the distribution of bacteria within the initially formed particles prevented a valid quantitative
analysis of the data, but taken on face value, they point to an initial doubling time of about 1 h. After that time, the
doubling time increased to about 5 h. Under ideal conditions, when media in flasks held in an incubator that provides
aeration by agitation are employed, the doubling time of this species is about 45 min. Either some newly formed cells
began to die after about 2 h of aerosol time, or the environment within the particles became inhibitive; the latter is a more
reasonable explanation since the decay rate of viable cell numbers from 12 to 24 h of aerosol time is the same as the
physical decay for that time, indicating no loss of viable cell numbers.
The results show unequivocally that it is possible for bacteria held in moist; airborne particles to double at least twice
and very likely three times when the particle size is 4 to 6 um in diameter (i.e., the volume is 50 to 200 times that
of the cell volume).
DISCUSSION
Aside from a desire to obtain new fundamental knowledge of the extent of bacterial capabilities for growth in unusual
environments, we are making these studies to determine whether microbial forms might survive and increase in number
in a gas. We have shown that growth beyond a single doubling is possible if the particles are moist and contain
nutrients, although propagation beyond three generations may be limited because of accumulation of waste products
within the particle or lack of nutrients.
At least one more study is needed before simulated atmospheres should be tested. For the
continued serial propagation of a species, additional nutrients would have to be obtained by
the cell from the atmosphere. Material in the vapor phase contacts airborne particles more
readily than particulate substances can coalesce. 0 Although we have preliminary evidence that
ammonium acetate vapor is readily adsorbed by airborne particulates and does not injure air-
borne cells that were grown in ammonium acetate medium, we have yet to obtain evidence
that these cells obtained nutritional value from the vapor.
------------------------------------------------------------------------------------------------------------------------
Graphs -- skip to bottom
DIVISION OF AIRBORNE BACTERIA 643
A
I7-
5 p__<<
Number, ,
viable cells o
per liter 2
of air,
log scale I0
o uncorrecte
A numbers O
* hypothetia
Ilincrease-ra
B
107-
Number, 3
viable cells
per liter 2
of air.
0g scale 136
IAt_,vAA'-
vO 000
o uncorrected cell
a numbers correct
* hypothetical poini
=increase-ratio, st
F6 pended in a gas. We have shown that growth
----------- A beyond a single doubling is possible if the par- Au .ticlesare moist and contain nutrients, although
° 0 propagation beyond three generations may be
1 limited because of accumulation of waste prod-
Physical Decay, Relative Scale ucts within the particle or lack of nutrients.
e:d cell numfobrefraslloout rreJ Ast leaset one mdore study is needed before p,oint simulated atmospheres should be
tested. For the
atio, start to indicatW time continued aerial propagation of a species, addi-
6 12 18 24 tional nutrients would have to be obtained by
TIME Hrs the cell from the atmosphere. Material in the
vapor phase contacts airborne particles more
readily than particulate substances can coalesce.
0 Although we have preliminary evidence that
0 0 ammonium acetate vapor is readily adsorbed by
17-1 airborne particulates and does not injure air-
Physkal Decay, Relative Scale borne cells that were grown in ammonium acenumbetrs
ed tor laliout- tate medium, we have yet to obtain evidence
tart3to indicated lime that these cells obtained nutritional value from
1|2 ;8 2'4 948 the vapor.
TIME, Hrs.
FIG. 1. Concentration of cells of S. marcescens, as
a function of time, in a rotating-drum, aerosol container
at 31°C and saturated humidity. "Physical
decay" curve depicts the change, with time, of all
aerosolparticles as a result of gravitational removal.
"Uncorrected cell numbers" means concentration of
cells, as measured. "Corrected cell number" means
concentration of cells which would have been present
had no particles that contained cells undergone
"physical decay." "Increase-ratio" is corrected number
of cells, at time t,/number of cells, at time zero.
--------------------------------------------------------------------------------------------------------------------------------
ACKNOWLEDGMENTS
This work was supported in part by the Office of Naval Research and in part by the National Aeronautics and Space
Administration, Office of Planetary Quarantine.
We thank John Hresko, William French, and Irvin Ford for their patient and competent assistance.
LITERATURE CMD
1. Dimmick, R. L, and A. Akers (ed.). 1969. An introduction
to experimental aerobiology. Wiley-Interscience,
New York.
2. Dimmick, R. L., P. A. Straat, H. Wolochow, G. V.
Levin, M. A. Chatigny, and J. R. Schrot. 1975.
Evidence for metabolic activity of airborne bacteria. J.
Aerosol Sci. 6:387-393.
3. Dimmick, R. L, H. Wolochow, and M. A. Chatigny.
1979. Evidence that bacteria can form new cells in
airborne particles. Appl. Environ. Microbiol. 37:924-
927.
4. Green, H. L., and W. R. Lane. 1964. Particulate clouds:
dusts, smokes and mists, 2nd ed. E. and F. N. Spon,
Ltd., London.
5. Ponnamperuma, C. (ed.). 1976. Chemical evolution of
the giant planets. Academic Press Inc., New York.
6. Straat, P. A., H. Wolochow, R. L. Dimmick, and M. A.
Chatigny. 1977. Evidence for incorporation of thymidine
into deoxyribonucleic acid in airborne bacterial
cells. Appl. Environ. Microbiol. 34:292-296.
VOL. 38, 1979