C-14 metabolic studies on an aerosol mixture of Escherichia coli and a coliphage are indicative of phage production

Chatigny, M. A.

Dimmick, R. L.

Wolochow, H.

English

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i1. 1 1 t	 . - 1 -.1-
STUDIES ON PROPACATION (IV MICROBES
IN THE AIRBORNE STATE
Annual Report, 1974-75
NASA Contract W13,450
Task No. 193-58-62-13-10
R.L. Dimmick, H. Wolochow, and M.A. Chatigny
aye'.
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NAVAL BIOMEDICAL RESEARCH LABORATORY
Naval Supply Center
Oakland, California 94625
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(NASA-CR-142757) STUDIES ON PROPAGATION OF
	 N75-24294
MICROBES IN THE AIRBORNE STATE Annual
	 £
Report, 1974-1975 (Naval Biomedical Research
Lab., Oakland) 6 p HC $3.25
	 CSCL 06M
	 Unclas
G3/51 22000
	 ff	 (	 ,
Annual Report, 1974-75
NASA Contract 1473,450
'task No. 193-58-62-13-10
STUDIES ON PROPAGATION OF MICROBES IN THE AIRBORNE SPATE
R.L. Dimmick, 11.'Wolochow, and M.A. Chntigny
INTRODUCTION
The third quarterly progress report (a) reiterated the rationale
for attempting to determine whether microbes are able, under any cir-
cumsLance, to propagate in the airborne state, (b) outlined the
experimental approach and (c) defined the continuity of investigative
areas. The document included a report that airborne microbes (a) can
sustain metabolic function, (b) are able tc. ,produce at least one new
gE:cration, and (c) appeared to incorporate small amounts of labelled
DNA base material into the DNA (acid insoluble material).
It was noted that higher levels of incorporation of the label into
DNA must be demonstrated if new DNA production were to be accepted as
fact., and that different, corroborative proof that DNA could be produced
was also required. The best proof: is, obviously, "o find a situation
where the number of airborne cells increases more than 2-fold. However,
if phage is produced by cells in the airborne state, then new DNA is
being formed. However, the conservative viewpoint might hold that this
does not constitute proof of transcription of the specific DNA needed
by the microbe to continue the process of propagation.
We have, the+refore, conducted experiments intended to increase
the sensitivity of the mcae rement of DNA fonnaton, both by the use
of labelled base and by the use of a phage-sensitive species of ubiqui-
tous microbe.
EXPERIMENTAL DETAILS AND RESULTS
A. Incorporatior. Studies
Details of the growth of the test bacteria, (Serra tia marcescens,
SM) the formation, mixing and containment of aerosols, and the tech-
niques for incorporating and measuring 3H-thymidine have been previously
reported.
Aerosols of the test bacteria (8-hr culture) and of 3H-thymidine,
both of which were suspended in tryptone broth plus glycerin and 2-
dcoxy adenosine, were mixed in air prior to filling the 1500 1 aerosol
chamber. A control experiment run simultaneously and in an identical
chmnhcv' utilized the same har_terial suspension, but cells were killed
with	 formtili.n.
A mu:;sive snnplr: was Lhvii talcan after a selected interval of
nnrcr,:ol Limn. 1'ivo i.mpinl;ers containing 20 ml, of lY, formal.in were
oporated in porallel for 20 minutes, sampling 1250 1 of air. Each
AnuunI koport, 107405
NASA Cenlrnal WI4,4S0
Task No. .193-:iY-G_-•.I'1-JO
I
impinger lost about 3 ml of fluid during the sampling period, so the
total volume of collection fluid was 85 ml. Samples of the fluid
were counted for tritium content, the fluid was centrifuged, poured
off and recounted, Material retained in the centrifuge tubes (calls)
was resuspended in 3 ml of the formali.n solution. Aliquots were
removed for counting and 0.6 m1 of 50% trichloroacetic acid (TC) was
added. After 10 minutes the solution was filtered according to the
procedure of Straat (1974) and the radioactivity on the filter pad
was counted.
Results are shown in Table 1. The important finding is the
contrast between the decay per minute (dpm) in the insoluble fraction
from live or dead cells in aerosols. The chi-square score for the
two sets of .`.igures is 207; (a score of 5 or greater represents a
highly significant difference), so there is no doubt that live cells
are incorporating thymidine into new DNA material.
Although the evidence in run 1-24 was, alone, convincing enough
(more than 3-fold difference) we were concerned with the apparently
high count in the dead-cell insoluble fraction. That is, it was
possible that nonbiological interaction had occurred. Hence, the
second run (2-19) was accomplished, and this time the count in the
dead-cell run was essentially background. As an additional test, a
simulated, in vitro, sample, using the same volume of fluid and num-
bers of cells but with nn increased thymidine content, was run.
Percent entratument of counts in the insoluble fractions compared
favorably to the percentage found in the second run (2-19), so we
conclude that the high count with dead cells in run 1-24 was contam-
ination in some inexplicable manner, and the true count should have
been not more than 4% of the suspension fluid count, i.e. about 240
dpm.
B. Pha;c Production
A phage sensitive strain of Escherichia soli (EC) labelled E3000,
and a T3 c03.iphage, were obtained from the bacteriology department of
the University of California. By successive transfer of the microbe
in the s.lected, chemically-defined minimal medium used to cultivate
SM, we isolated a variant that would grow well with glycerin as an
energy and carbon source, NH 4+ as a nitrogen source and having the
property to produce phage at concentrations approaching 109/ml.
One mixod-nerosol experiment with phage has been conducted, but
the data have not been fully analyzed, However, the ratio of the
phage Gout y nt Lo th:. bacterial content increased during the last 12
hr Inbli'val of the atrosol-time, which is indicative of phage produc-
t Iun.
L	 f
Annual R,:porl	 1(174-75
NASA ContraCL 191;3,450
Titsk Nu. 193-M-02-13-10
C. Discussion and Future Work
With the exception of direct proof that the test microbe can
iniLinte more than 1 division, the evidence now available (that new
DNA can be created and that new phage is apparently formed) is suf-
ficient to leave little doubt that under ideal circumstances, and
wsth a test organism that might be more adaptable than the one used,
true propagation of a microbial species in the airborne state can
occur. We believe that the additional time and effort that might
be involved in finding an ideal circumstance to prove extended
division in air. in a direct manner will be less responsive to the
central. question regarding Pg in the Jovian atmosphere than obtain-
ing Information about anaerobic microbial aerosols in reducing gases.
Nothtnl; is known of the latter., and since this environment is more
nearly equal to the Jovian situation than is air, we propose to con-
duct future biological studies with anaerobes.
We are restricted to work with particles 2 to 3 Um in diameter
in order to obtain the most long lasting aerosols possible within
microbial dimensLons (these particles would, however, be equally
po•slstent in the Jovian atmosphere). Such particles probably do
not contain enough nutrients to support extended growth, so "feed-
ing" the particles in either vapor or particulate form must be done
if growth is to continue. Since nutritional requirements of anaerohi-
osis, as well as rates and mechanics of feeding, are different than
for nurobic growth, the study of anaerobes approaches the primary
question more directly than continued studies with aerobes.
As noted in the Third Quarterly report, if a species is to
remain resident in the Jovian atmosphere, the doubling time must
exceod the half-life time of removal of particles by turbulence_.
We w1ll, study rates of removal of particles as a result of both
gravitational settling and turbulence in an aerosol chamber large
enough to permit the simulation of thermal turbulence as it might
occur in the unenclosed environment. The data so obtained will he
compared to models of turbulence and both the biological and physi-
cal parameters eancetned in simulating microbial growth in Jovian
environments wiiS, he evaluated..
D. Summary ofWork
1. 14C metabolic studies were completed and terminated.
2. In vitro incroporation studies of 311-thymidine were completed,
:uui Lrchni.gtut: applicable to studies of incorporation in the airborne
slate wore evolved.
3. Studles •of both mixed aerosols ; and of pre-incorporated
aot°osnls (soeleing evidouce of dilution of the labe3) were conducted.
D,lei from tl •t• !ormcr, Included in this report, :,how incorporation
ooculrod.
S
,
Annnnl Repoi i , 1.974-75
NASA Contract W13,450
Task No. 193--58-62-13-10
4. During attempts to show dilution of the label, we found
evidence that at least one doubling of airborne cells is possible,''
as shown both by nn increased number of viable cells and an increased
number of cull-size units measured by the Coulter Counter.
5. presumptive evidence of phage formation in the airborne
stnto has been nbtained.
LITERATURE CITED
Straat, Patricia A. 1974. Interim Report to NBRL, Detection of growth
in aerosols, Biospherics, Inc., Rockville, Md.
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Studies on propagation of microbes in the airborne state

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