Microbiological contamination of Gemini 9 spacecraft before and after fligh

Covert, S.

Hotchin, J.

Lorenz, P.

Markusen, A. S.

English

NASA Technical Reports Server

1197 
NASA CR-972 
THE MICROBIOLOGICAL FLORA OF THE GEMINI IX SPACECRAFT 
BEFORE AND AFTER FLIGHT 
By John Hotchin, Peter Lorenz, Aletha Markusen, 
and Scott Covert 
Distribution of this report is provided in the interest of 
information exchange. Responsibility for the contents 
resides in the author or organization that prepared it. 
Prepared under Grant No. NsG-155 by 
THE DUDLEY OBSERVATORY 
Albany, N.Y. 
for 
NATIONAL AERONAUTICS AND SPACE ADMINISTRATION 
For sale by the Clearinghouse for Federal Scientific and Technical Information 
Springfield, Virginia 22151 - CFSTI price $3. 00 
https://ntrs.nasa.gov/search.jsp?R=19680002500 2020-03-12T11:48:35+00:00Z
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ACKNOWLEDGMENTS 
This work was supported by the National Aeronautics and 
Space Administration with NASA grant NSG 155 - 61. 
The authors want to thank Dr. Curtis Hemenway and Dr. Victor 
Tompkins for their encouragement and support, Mr. R. Coon and Mr. A. Laudate 
for their valuable assistance in determining the areas to be swabbed and in 
handling the swabs immediately before and after the swabbing. 
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The Microbiological Flora of the Gemini IX Spacecraft Before and After Flight 
John Hotchin*, Peter Lorenz* *, Aletha Markusen+; and Scott Covert f t 
(From the Division of Laboratories and Research of the 
New York State Department of Health*, Dudley Observatory**, 
State University of New York at Albanyt, and Albany Medical Collegett, 
Albany, New York) 
INTRODUCTION 
In the course of microbiological exposure and collection 
experiments (1) on the Gemini IX spacecraft, it appeared desirable to 
establish the number and type of microorganism within the spacecraft 
immediately before and after flight. For this purpose three locations were 
chosen which were representative of frequently touched, occasionally 
touched, and protected or dust collecting locations. These were swabbed 
at suitable times, and the swabs were returned to the laboratory for 
examination. 
MATERIALS AND METHODS 
Swabbing Technic: Falcon "swube" disposable swabs were used for the 
swabbing of the selected areas within the capsule. The swabs were dispatched 
to Cape Kennedy prior to the start of the spacecraft, after the technicians 
preparing the spacecraft for launch had been instructed in the technic of 
swabbing. Dry swabs were used for the swabbing of three to four locations 
in the space capsule. Four sets of swabs were obtained, called "Preflight 
set No.1", "GT-9A prescrub" set, and "preflight set No.2", which were 
all taken before the flight, and one "postflight" set. The preflight set No. 1 
swabs were taken on May 16, and the GT-9A prescrub set swabs on May 31, 1966. 
These samples resulted from delays in the actual flight schedule and were 
originally intended to be preflight swabs. Preflight set No.2 swabs were 
taken on June 3 before the start of the spacecraft, and the postflight set 
swabs were taken on June 6 after the flight. The swabbing locations (see 
Figs. 1 and 2) of the first set were [1] - R/H door handle; [2J - above center 
panel L/H side; and (3) - behind R/H seat . After being returned to the 
New York State Division of Laboratories and Research at Albany, the 
swabs were stored at 40 C before they were eluted. 
Bacterial Diagnostic Technics 
Elution Procedure: On inspection, the swabs were found to be heavily 
loaded with dirt (see Fig. 3). They were each immersed and squeezed out 
in 5 ml of thioglycollate broth, then eluted for 60 min at 40 C and subsequently 
agitated on a mixer. Samples of all eluted suspensions were then distributed 
among three groups (group 1, 2, and 3) of investigators who obtained their 
results completely independently. 
Group 1: 0.5 to 1 ml quantities of the eluted suspensions 
were diluted in 9 to 9.5 ml of thioglycollate broth, then incubated at 370 C. 
The suspension cultures were checked for bacterial growth after 24 and 
48 hours of incubation, and blood agar plates were inoculated with the turbid 
suspensions and incubated under aerobic and anaerobic conditions. The 
organisms isolated were characterized by group 2. In addition, 0.5 ml 
aliquots of the eluted suspensions were diluted in 4 ml each of an 0.6 g % 
agar suspension, and 1.5 ml aliquots of the mixture were poured on a 
--~--- - - ---
· Sa·bouraud agar plate which was incubated for 3 days at room temperature 
and 2 blood agar plates which were incubated for 2 days at 37° C, one 
under aerobic and one under anaerobic conditions. 
Group 2: The eluted suspensions investigated by group 2 were 
studied by the methods of determinative bacteriology (2). Apart from 
morphological studies, the isolates were tested for hemolysis, starch 
hydrolysis (3), production of acetylmethylcarbinol from dextrose (3), 
gelatin hydrolysis (3), growth in thioglycollate (Difco) broth over an 
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observation period o..!.;tlays, growth in litmus milk (Difco) and Pennassay 
broth (Difco), H2S (3) and indole production (3), nitrate reduction (4), 
ureae hydrolysis (5), fermentation of glucose, maltose, xylose, sucrose, 
mannitol, lactose and galactose were also studied. 
Methods used by group 3 included incubation at room temperature 
1 . : 
on trypticase soy (BBL) blood agar for 19 days and on Sabouraud dextrose 
agar (BBL) for 26 days, and suspensions in thioglycollate medium (BBL) 
were incubated for 50 days. The identifications of all growth forms discovered 
were based on standard methods of determinative bacteriology (2). 
RESULTS 
The results are given in Tables 1 and 2. The group 1 blood agar 
plates show that the preflight set No.1, swabs Nos. 1 and 2, the preflight 
set No.2, swabs Nos. 1 and 3, the GT-9A prescrub set, swabs Nos. 2 
and 3, and the postflight set, swab No.4, had picked up numbers of micro-
organisms which varied between approximately 20 to 140 per swab (see Table 1). 
IBaltimore Biological Laboratory, Inc., P.O. Box 6711, 
Baltimore, Maryland 21204. 
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The Sabouraud plates of preflight set No.1, swabs Nos. 1 and 2, showed 
growth of a Penicillium species, and an unidentified fungus was found on 
GT-9A prescrub set, swab. No.3. The thioglycollate broth suspensions 
showed growth of Micrococcus ureae, Bacillus cereus and Bacillus megaterium 
of preflight set No.1, swabs Nos. 1 and 2 (see Table 2). The thioglycollate 
broth results, in contrast to the agar plate results, did not show any 
growth of the preflight set No.2, swab No.1, the GT-9A prescrub set, 
swab No.3, and the postflight set, swab No.4. 
The results of group 2 include long-term observations of the 
cultivated suspensions up to 21 days as well as diagnostic studies of the 
microbial growth forms found. After longer incubation periods, the 
primary thioglycollate suspensions showed growth of microorganisms 
which were identified as a Penicillium species in the preflight set No.1, 
swab No.3, as B. megaterium in the GT -9A prescrub set, swab No.3, 
as B. pumilis in the postflight set, swab No.3, and as an Aspergillus species 
in the postflight set, swab No.4 (see Table 2). During the further studies, 
hemolytic B. cereus and Micrococcus ureae were found on preflight set No.1, 
swab No.1, which agrees with the resul ts of group 1. B. subtilis was 
isolated from preflight set No.1, swab No.2, of which a Penicillium 
species, B. megaterium and B. cereus were found by group 1. 
Corynebacterium acne was found on preflight set No.2, swab No.2. 
Swab No.3 of this set showed B. subtilis and Micrococcus ureae. 
The corresponding group 1 result showed microbial growth under conditions 
of aerobic and anaerobic incubation which was not further identified. No 
growth was found in preflight set No.2, swab No.1, in GT-9A prescrub 
set, swabs Nos. 1 and 2, and in postflight set, swabs Nos. 1 and 2. In 
comparison, preflight set No.2, swab No.1, and GT-9A prescrub set, 
swab No.2, showed growth of microorganisms in the results of group 1. 
Group 3. Analogous to the results of groups 1 and 2, preflight set 
No. I, swabs Nos. 1 and 2 were found contaminated (see Tables 1 and 2) 
and Staphylococcus albus and B. cereus were isolated from swab No.1, 
and B. subtilis from swab No. 2 (see Table 2). Flavobacterium breve and 
and a Streptococcus species were isolated from preflight set No.2, swab No.3. 
This swab also showed contamination of the corresponding swabbed area 
in the results of groups 1 and 2. The GT-9A prescrub set, swab No.2, 
showed contamination of the swabbed area with B. subtilis. The results of 
group 1 also showed growth of this swab whereas no microorganisms were 
isolated by group 2. B. coagulans was isolated of postflight set, swab No.3, 
of which B. pumilis was isolated by group 2, whereas no growth was found by 
group 1. No growth was found of preflight set No.1, swab No.3, preflight 
set No.2, swabs Nos. 1 and 2, of GT-9A prescrub set, swabs Nos. 1 and 3 
and of swabs Nos. 1, 2 and 4 of the postflight set. 
In comparison, a Penicillium species was isolated by group 2 
of preflight set No.1, swab No.3, and no growth of this sample was found 
by group 1. Bacterial growth was found by group 1 of preflight set No.2, 
swab No.1, whereas the results of groups 2 and 3 show no growth. 
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Preflight set No.2, swab No.2, showed contamination of the swabbed area 
with Corynebacterium acne according to group 2, whereas the group 1 and 
3 results showed no growth. No microbial growth at all was found by 
groups 1, 2, and 3 of the GT-9A prescrub set, swab No.1, and of the 
postflight set, swabs Nos. 1 and 2. The GT-9A prescrub set, swab No.3, 
showed microbiological growth in the results of group I, and B. megaterium 
was identified by group 2 of this swab in contrast to the group 3 result. 
Whereas the group 1 result did not show any growth of the postflight set, 
swab No.3, B. pumllis was isolated from this swab by group 2 and B. coagulans 
by group 3. In contrast to the group 3 result, bacterial growth was found on 
postflight set, swab No.4, by group 1, and an Aspergillus species was 
identified by group 2. 
CONCLUSIONS 
While the detailed results described here, because of the obvious 
variation, demand careful interpretation, this study clearly shows that the 
Gemini IX spacecraft, as would be expected, was contaminated with an 
assortment of microorganisms before and after the space journey in the 
locations tested by swabbing. As shown in Fig . 3 most of the swabs were 
heavily loaded with dust. The differing spectra of microorganisms found 
by the three examining groups is of interest in view of the different approaches 
used. Group 1, a biological research laboratory group, merely attempted 
a quantitative assay with little identification. Group 2 also was a biological 
research laboratory group, and group 3 a hospital diagnostic laboratory 
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· , 
group. It is evident that groups 2 and 3 found different types of organisms, 
the differences presumably having been determined by the choice of methods. 
This shows that one is likely to find only those organisms for which one is 
specifically equipped to look. These results also bear on the need for 
spacecraft sterilization if existing strictures (6, 7) regarding contamination 
by terrestrial microorganisms are to be implemented. 
SUMMARY 
Three sites within the Gemini IX space capsule were investigated for 
microbiological contamination by swabbing before and after the flight. 
Bacterial or mold growth was observed in three sets of swabs taken before 
the flight and one set of swabs taken after the flight, and most of the 
swabs were then found heaVily covered with dust. The results were obtained 
by the completely independent study of the eluted suspensions of the swabs 
by three research groups and, despite the considerable variation of the 
detailed results, they show that the inside of the Gemini IX space capsule 
was contaminated with microbiological materials both before and after the 
flight. 
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, . 
REFERENCES 
1. Lorenz, P., Hotchin, J., Markusen, A., Orlob, G., HememoTay, C., and 
Hallgren, D. 
Survival of microorganisms in space. Results of Gemini IXA, 
Gemini XII, and Agena VIII satellite borne exposure and collection 
experiments (In preparation). 
2. Breed, R.S., Murray, E.G.D., and Smith, N. Bergey's Manual of 
Determinative Bacteriology; 7th ed. Baltimore, Williams and 
'.1ilkins, 1957. 
3. Seeley, H.W., and Vandenmark, P.J. Microbes in Action: A Laboratory 
Manual of Microbiology. San Francisco, W.R. Freeman and Co., 1962. 
4. Pelczar, M.J. Laboratory Exercises in Microbiology; 2d ed. New York, 
McGraw Hill, 1965. 
5. Difco Manual of Dehydrated Culture Media and Reagents for Microbiology 
and Clinical Laboratory Procedure; 9th ed. Detroit, Difco 
Laboratories, Inc., 1953. 
6. Hall, L.B., and Bruch, C.l-1. 
Procedures necessary for the prevention of planetary contamination 
Life Sciences and Space Research, 1965, 1, 48-62. 
7. Sagan, C., and Coleman, S. 
Spacecraft sterilization standards and contamination of Mars. 
Astronautics and Aeronautics, 1965, 1, 22-27. 
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· . 
Table 1 
Number of Microorganism Eluted from Swab Used for Sampling 
Different Areas of the Gemini IX Spacecraft 
No. of Colonies* Results of** 
Swab Set Swab No. (Group 1) Group 2 Group 3 
1 69 + + 
Preflight Set 
No.1 2 138 + + 
3 0 + 0 
1 0 0 0 
GT-9A 
Prescrub Set 2 23 0 + 
3 46 + 0 
1 17 0 0 
Preflight Set 
No. 2 2 0 + 0 
3 68 + + 
1 0 0 0 
Postflight 2 0 0 0 
Set 
3 0 + + 
4 68 + 0 
*Group 1 results after plating suspensions eluted from the swabs on blood 
and Sabouraud agar. The blood agar plates were incubated aerobically or 
anaerobically at 36° C.; the Sabouraud agar plates were aerobically incubated 
at room temperature of approximately 22° C. The figures were obtained by 
multiplying the colonies counted per swab by the dilution factors. 
**The group 2 and 3 results, which did not include a quantitative assay, 
are mentioned for comparison; + = positive growth, 0 = no growth. 
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· . 
Table 2 
Microorganisms on the Gemini IX Space Capsule Before and After the Flight 
Species of Microorganism 
Swab Set Swab No. Group 1 Group 2 Group 3 
1 M. ureae, B. cereus B. cere~s, M. ureae S. albus, B. cereus 
Penicillium 
--
Preflight Set 2 B. rnegaterium B. subtilis B. subtilis 
No.1 B. cereus, Penicillium 
3 0 Penicillium 0 
1 0 0 0 
GT-9A 2 +* 0 B. subtilis 
Prescrub Set 
3 Fungus * B. megaterium 0 
1 + 0 0 
Preflight Set 2 0 Corynebacterium acne 0 
No.2 
3 + B. subtllis, M. ureae Flavobacterium brev 
Streptococcus specie 
1 0 0 0 
Postflight 2 0 0 0 
Set 
3 0 B. Eumllis B. coagulans 
4 + Aspergillus 0 
* Unidentified 
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e 
s 
NO. J SWAB POINT S-12 EXPERIMENT 
RIGHT AFT STOWAGE CONTAINER 
SIDEWAll STOWAGE CONTAINER EXTENSION 
SIDEWALL STOWAGE CONTAINER 
. ' \ 
IOMM CAMERA MOUNTING BRACKET 
VOICE TAPE RECORDER AND MOUNTING .... f'KFT_J 
LEfT SIDE DRV STOW"GE lAG 
VIEW LOOKING INTO COMMAND PILOTS SIDI 
Spacecraft Interior Stowage Areas (Sheet 1 of 2) 
Figure 1.- Swabbing location of swabs Nos . 1 and 3 in a diagram of the 
interior of Gemini IX spacecraft. 
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rJO . 
< • 
AFT STOWAG E CONTAINER 
RIGHI SIDEWAll CONTAINER 
RI HT HAN l> POUC H STOWAG E "C'VISIO" ..... 
RIG HT SIDEWAll rDN""N"-~ 
. ,. 
VIEW LOOKING INTO PlLgTS SIDE 
Spacecraft Interior Stowage Areas (Sheet 2 of 2) 
Figure 2 .- Swabbi ng l ocation of swab No . 2 i n a di agram of the i nteri or 
of Gemi ni IX spacecraft; thi s location is seen from the s i de oppos i te 
to that of Fi gure 1 . 
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. , 
. 
Figure 3.- Swabs representing the GT-9A prescrub set (left swab), pre -
flight set No . 1 (center swab), and postflight set (right swab) . 
NASA- Langle y. 1967 - 4 CR- 972 13 
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The Microbiological Flora of the Gemini 9 Spacecraft Before and After Flight

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