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California Institute of Technology 
Pasadena. California 

^ // ff MS-}-/sr^f 

National Aeronautics and Space Administration 
f/V^AfD- Contract No. NASw-6 1 

V/4-s* <ue, ^ Df 7;^ 

External Publ ication No. _698 ) 





Richard W. Davies J f ^r~~^ * , /? S^tuu 

Marcus G. Comuntzis | y^^v/P^ %£i ^ r ._ 


Copy No 

pp. ii, 1-16 

"I J C!aliionuaInstitute of Te chnology 
Pasacleria 3, California r 

August 31, 1959 

Jet Propulsion Laboratory External Publication No. 698 



Introduction 1 

Definition of Biological Contamination 3 

Pollution 3 

Infection 4 

Space-Flight Environment 5 

International Discussion 7 

Operational Tactics 7 

Terminal Sterilization 8 

Sterile Assembly 10 

Built-in Sterilization 10 

Maintaining Sterilization 11 

Testing Procedure 11 

Biological Contamination Tolerances 12 

Recommendations and Conclusions 13 

References 16 

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Jet Propulsion laboratory External Publication No. 698 



Richard W. Davies 
Marcus G. Comuntzis 

Jet Propulsion Laboratory 
California Institute of Technology 
Pasadena, California 

presented at the 
London, England 
August 31 - September 5, 1959 


Speculation on the existence of extraterrestrial life is 
sufficiently commonplace to suggest that the concept is subtly 
imbedded in our social culture. However, it is difficult to verify 
the origin of the extraterrestrial life concept because of a tendency 
to ascribe original authorship of many ideas to antiquity. 1 

The discovery of life on any of the planets would be one of the 
most exciting events in human history. Satisfying society's general 

This paper presents the results of one phase of research carried 
out at the Jet Propulsion Laboratory, California Institute of 
Technology, under Contract No. NASw-6, sponsored by the National 
Aeronautics and Space Administration. 

^According to P. Duhem, Le Syste~me du Monde (Vol. 1, pp. 17-18, 
1913) the Pythagorean Philolaus (5th century B.C.) postulated the 
existence of an inhabited anti-earth which was always in opposition 
to the earth in relation to the sun. 

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Jet Propulsion Lab oratory External Publication No. 698 

curiosity would, however, be only one facet of the discovery. The 
event would also have tremendous scientific interest because, next 
to the synthesis of living matter in the laboratory, it would be the 
most important step that could be made toward understanding of the 
problem of the origin of life. Systems based on nucleic acid and 
proteins as bearers of life may or may not be unique. This is one 
of the fundamental questions that the discovery of extraterrestrial 
life might answer. 

The biological importance of the planets is not limited to 
detecting and studying life on them. Even if no life is found to 
exist, the opportunity to sample organic compounds on the planets 
might give some valuable clues to the origin of life. Sterile worlds 
may provide the information necessary to the understanding of the 
organic chemical processes that preceded the development of life on 
the earth. 

Present knowledge (as well as the lack of it) of the planets Mars 
and Venus is compatible with the possibility both of an indigenous 
life and of the support and rapid proliferation of terrestrial micro- 
organisms. The introduction of terrestrial organisms and contaminants 
might so distort the biology of either planet as to constitute a 
scientific catastrophe. The processes are irreversible and they 
make the search for life on other planets most sensitive to irremediable 
harm. If the earth were sterile, it would require only months or 
years to universally populate it with the descendants of a single 
cell. A common bacterium, E. Coli , has a mass of 10 -J - 2 grans and a 
fission interval of 30 minutes. Ideally, it would take 66 hours for 

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Jet Propulsion Laboratory External Publication No. 698 

the progeny to attain the mass of the earth. The progeny never reach 
this magnitude principally because the food supply is insufficient. 
Nevertheless, this extrapolation illustrates that the exponential 
growth rate of bacteria is truly explosive and, therefore, the 
timescale of planetary biological distortion need not be long. Indeed, 
it could be considerably less than the time interval of earth-planet 
oppositions. Space probes which have any likelihood of a landing, 
intentional or accidental, should be subject to careful sterilization. 


It is convenient to separate biological contamination into two 
kinds, pollution and infection. Biological pollution is meant to 
be a deposit of a large enough number of micro-organisms to be 
scientifically significant, as such, without further growth. Infec- 
tion is meant to describe the growth of one or more viable organisms. 
Likewise, pollution can be divided into two categories; viable 
pollution, which does not grow by nature of its environment, and 
non-viable pollution. 


Pollution is a type of contamination that applies to the Moon, 
Mars, and Venus. Pollution would be most likely if a mammal were 
splattered on any one of these three bodies. For example, the Moon's 
area is 4x10 3 square meters, and the intestines of a mammal can 
contain 10 12 micro-organisms per kilogram. If the mammal died in 
flight, the putrified tract could contain 10 13 micro-organisms per 
kilogram (l). Present techniques are capable of detecting one 

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External Publication No. 698 

micro-organism per square centimeter. These techniques could be 
immediately extended to detect one micro-organism per square meter. 
Future improvements in technique may increase the detecting 
sensitivity by a few orders of magnitude; therefore, a single probe 
leaving a residue of from 10" to 10 ° dead bacteria could provide a 
misleading background noise for future investigators. ^ 


Infection appears least likely on the Moon because water is the 
lowest common denominator of all known terrestrial organisms, and 
all present evidence of solvents on the Moon's surface is highly 
controversial (2, 3). The hypothesis that beneath the lunar surface 
material one would find both water traces and relics of primitive 
organisms is not so unreasonable as to warrant the immediate dismissal 
of the matter of infection. 

Mars is arid by terrestrial standards; its polar caps consist of 
thin hoar frost, and dense terrestrial type water clouds have never 
been observed. However, the polar caps retreat and the equatorial 
dark areas advance with the onset of the Martian spring. The pressure 
(85 mb, or less) and temperature (200-300°K) are so low it is 
frequently supposed that the presence of liquid water on the surface 
is very rare. This point has been refuted (4, 5). If salts are 
present on the Martian surface, an anti-freeze mechanism can occur. 

These comments would be unnecessary but for the fact that many 
people (not all laymen) presuppose that no significant discoveries 
will be made until a man is landed on the planets. 

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Jet Propulsion Laboratory External Publication No. 698 

It is reasonable to suggest that the dark areas of the planet contain 
salts, perhaps in the form of deposits left behind by dried-up seas. 

Sinton (6, 7) has found three small absorption dips, at 3.43, 
3.57, and 3.67 microns, associated with the dark areas of Mars. This 
suggests the presence of organic matter on Mars, but the question of 
its origin is an open question. 

-These few facts indicate Mars may be a promising subject, both 
for basic biological research and infection . 

Theories of Venus are so varied, and the facts so few, it is 
imperative to be very cautious, at least in the early stages of 


On cursory examination, probe sterilization may appear to be 
unnecessary because the space-flight environment is so hostile to 
terrestrial organisms. Several self-sterilizing mechanisms which 
immediately suggest themselves are: 

1) Ultraviolet radiation from the sun 

2) Space vacuum 

3) High temperatures on the Moon's surface 

4) Heat of impact, or impact explosion on the Moon 

5) Heat of entry into a planetary atmosphere 
We shall discuss these in order. 

Parts of the probe will never be exposed to sunlight. Ultraviolet 
radiation will destroy organisms which are nakedly exposed, but its 
penetrating power is so low that organisms can survive if surrounded 
by only a small group of dead ones. 

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Jet Propulsion Laboratory External Publication No. 698 

Laboratory vacuum is employed t>. help preserve micro-organisms. 
There is, as yet, no knowledge on sp ,ce vacuum being bactericidal. 
Perhaps this question can be answered in the near future by means of 
a satellite experiment. 

The Moon and the Planets most likely have cracks and fissures 
on their surfaces which would protect organisms from exposure to 
high temperatures and ultraviolet radiation, (5, p. 306). 

A probe hard-landed on the Moon would have an impact velocity 
of approximately 3 kilometers per second. This is not sufficient 
kinetic energy to melt or vaporize the probe on impact, but it is 
sufficient to scatter parts of the payload all over the Moon's surface 
if the initial impact were on a hard surface, such as a mountain. 
The orbital velocity of a satellite in the Moon's gravitational field 
is roughly 2 kilometers a second. It is contended that the probe 
would bury itself in the Moon's surface. We do not believe that any 
of the supporting arguments presented thus far are sufficiently 
convincing to be dogmatic. 

A probe that unintentionally enters an atmosphere has a high 
probability of coming in at a shallow angle, which is the ideal 
approach for a successful landing (8, 9). The probe may shed a few 
parts during the planet fall, but the bulk of it would strike the 
surface. A successful descent on Mars would be comparatively simple 
because the tenuous atmosphere of Mars extends so far out from its 
surface. Furthermore, meteors have been found whose interiors show 
no evidence of having been heated appreciably (10). It is evident 
that the rigors of a space journey are not a reliable means of 
preventing biological contamination. 

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CETEX (Committee on Contamination by Extraterrestrial Exploration,} 
representing the International Scientific Unions, has published two 
reports (11, 12) in an attempt to set a tone for developing a code of 
conduct in space research. These reports imply that, particularly 
as regards biological exploration, a purely national program does not 
have much chance of being fruitful. 

The CETEX reports recommend the sterilization of space probes, 
but they do not suggest a procedure for sterilizing probes nor do 
they suggest what tolerances would be acceptable. In this paper, 
we discuss both an operational approach to sterilization and the 
value judgements that will have to be faced by the operational 
agencies responsible for launching space vehicles. 


Sterilizing space probes is an engineering nuisance, however, 
the same ordeal has confronted surgical crews for quite some time. 
In both instances, anticipation of the task is necessary. 

At this time, it is possible to anticipate and recommend four 
phases of payload sterilization for all deep space missions. They 
are, in sequence: 

1. Sterile assembly of components, particularly heat sensitive 

2. Built-in sterilization of parts, particularly where traces 
of water are admissable, 

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Jet Propulsion Laboratory External Publication No. 698 

3. Terminal sterilization, 

4. Maintaining sterilization. 

A microbiological testing procedure must also be integrated into 
the sterilization operations. 

Terminal Sterilization 

Phase three, terminal sterilization, is the most important 
operation and we shall discuss it first. 

All known micro-organisms perish when subjected to dry steam 
at 160°C for twenty minutes (13). There is a time-temperature effect. 
Micro-organisms can survive much higher temperatures over a shorter 
period of time; such as, the flash temperatures in explosions. 
However, approximately 20% of the components that go into payloads 
with which we are now familiar cannot endure 160°C. A more general 
disinfectant for this purpose is ethylene oxide gas (14). 

Ethylene oxide (C2H 4 0) is the simplest of the ethers. It is a 
very small molecule and therefore dissolves in many substances, such 
as rubber, plastic, and oil. As a result of these properties, under 
slight pressure ethylene oxide is quite penetrating, working its way 
into the small interstices of most components. It is non-corrosive, 
and its human toxicity is low. 

Ethylene oxide is a few thousand times more effective as a 
sporicide than other powerful disinfectants (15). Viruses are more 
sensitive to ethylene oxide than many other organisms, whereas, they 
are much more resistant to radiation. 

Ethylene oxide is inflammable in air in concentrations as low 
as 3%. However, a mixture of 10% ethylene oxide and 90% carbon 

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Jet Propulsion Laboratory External Publication No. 698 

dioxide (sometimes called carboxide) is not inflammable even when 
infinitely diluted with air. This mixture at 2 atmospheres pressure 
and 25°C would sterilize most parts of the probe in four hours. The 
sterilization could take place in a polyethylene tent and left there 
to retain its sterility for quite some time. 

This part of the sterilization technique is well established. 
The U. S. Chemical Corps has sterilized many pieces of delicate 
laboratory apparatus without damage. They have also sterilized Air 
Force bombers and a commercial aircraft, in which a via] of live polio 
virus was accidentally broken. 

Gaseous sterilization will not prove effective on certain 
impenetrable components. For these parts (paper capacitors for 
example) heat sterilization or radiation can usually be employed. 

It is impractical to sterilize an entire payload with radiation. 
It is useful for certain small, sealed heat-sensitive components such 
as mylar capacitors. 

The radiation dose required for some specified degree of 
sterilization is proportional to the natural logarithm of the number 
of bacteria. For 10 5 bacteria per gram of material, a dosage of 10^ 
to 10^ rem is required for good sterilization depending upon the 
organism. Actually 10 bacteria is a very high bacteria loading for . 
most payload materials. 

The Jet Propulsion Laboratory selected some sealed heat-sensit: -x 
components for radiation treatment by the General Electric Corpora tio 
Two packages of identical parts were exposed to 10& and 107 rem fron; 



Jet Propulsion Laboratory External Publication No. 698 

a Co 60 source of gamma rays. A majority of these components withstood 
10' rem. The most important exceptions were transistors and mercury 
cell batteries. 

We estimate that, between gas, heat, and radiation (terminal 
sterilization), 95% of the payload parts can be readily sterilized 
without fear of degrading their performance characteristics. 

Sterile Assembly 

The removal of dust and foreign particles from the space probe 
eliminates a major source of biological pollution and it is, at the 
same time, an engineering virtue. (Most atmospheric pollution is 
borne by dust particles, except perhaps in closed rooms crowded with 
human beings.) 

The washing and scrubbing of parts of the payload with water and 
detergents (or other more acceptable solvents) can reduce the number 
of microbes on the probe by three orders of magnitude. 

Other aspects of sterile assembly include using compounds that 
are made sterile. Parts such as screws and bolts can be dipped in 
any of a number of sterilizing solutions. If screws and fitting 
holes are made to fit exactly, then care must be taken to sterilize 
before joining. Such fittings will remain sterile. If the fittings 
are not perfectly joined, the ethylene oxide gas will penetrate and 
sterilize these interstices. 

Built-in Sterilization 

Wherever possible, substances which are inimical to the well 
being of micro-organisms should be employed. Certainly, substances of 
biological origin, such as casein glue or shellac, should be avoided. 

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Jet Propulsion Laboratory Externa l Publication No. 698 

Recently, germicides that contain organo-metallic compounds as 
active ingredients have been used to disinfect hospitals. These 
substances might prove valuable during the fabricating of sealed 
components with parts that get slightly contaminated with handling. 
This reduction of the contamination load during the initial stages, 
provides an opportunity to, attempt terminal sterilization by radiation 
at a considerably reduced dosage, something of the order of 10 4 rem. 

Built-in sterilization is not so much a specific technique as it 
is a philosophy of preparation for terminal sterilization. 

Maintaining Sterilization 

Once the space probe is sterilized, it will be necessary to 
mount it on the rocket boosters. The technical problem is then one 
of keeping microbes from coming into contact with the probe. 

The probe is encased in a protective metal shroud during the 
launch phase of the space flight. The shroud can be employed to 
house a disinfectant atmosphere throughout the count down and flight 
through the atmosphere. The disinfectant can be either carboxide, 
employed in the terminal phase, or a faster acting but less penetrating 
gas, such as beta-propiolactone or ethylene imine. 

Testing Procedure 

In the past, several identical payloads were made for each 
mission. If this policy can be continued, it will not be difficult 
to produce convincing statistical arguments as to whether or not the 
payload meets the desired sterilization standard. Difficulties may 
arise, however, when the payloads become larger and more expensive. 

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Jet Propulsion Laboratory External Publication No. 698 

In this respect, it would be most practical to turn terminal 
sterilization and the sterilization certification over to an 
organization outside the space-flight groups. It would still be the 
space agency's responsibility to integrate this independent statistical 
estimate of sterilization with the other probabilities involved. This 
brings us to the problem of determining acceptable contamination 


Now we get to the heart of the matter as it is not practical to 
pursue codes of conduct and to employ testing techniques unless the 
community places a subjective value upon what the biologists want to 
protect. Discussions of the ethics of contamination are made confusing 
by people who persist in believing that sterility is an absolute, to 
which only a yes or no answer applies. 

The answer to the question of probe sterility can be given only 
in terms of probabilities. When large numbers of micro-organisms are 
subjected to lethal treatment, the live count drops off exponentially 
with time, or approximately so. The process is mathematically similar 
to the radioactive decay of an unstable nucleus. The death of a 
micro-organism has no clear-cut definition. 

A group of biologists in the United States, including some of the 
nation's most eminent microbiologists, biochemists, and biophysicists , 
who are also sensitive to the engineering areas in space research, 
have given this problem some intensive thought. 

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Jet Propulsion Laboratory External Publication No. 698 

For Mars and Venus, the consensus is that the probability of 
landing one viable organism should be less than one in a million. 
This means that if the probability of successfully impacting a probe 
were judged a_ priori to be one in a hundred it would be necessary to 
sterilize the payload to a tolerance of one chance in ten thousand 
that it have a live organism. We are investigating what degrees of 
sterilization can be expected as the space program evolves. 

As previously indicated, the status of the Moon as a biologically 
interesting target is considerably more doubtful than that of the 
planets; therefore, it is more difficult to get an intuitive grasp 
of what tolerances are acceptable. We tenatively suggest that one 
chance in ten (perhaps one hundred) of a viable organism remaining 
on the probe be an acceptable infection tolerance. We also suggest 
that pollution be kept less than 10^ dead organisms per probe for 
Moon and planetary shots. 

These tolerance levels are submitted here for general evaluation, 
with the understanding that, as more information on the celestial 
bodies becomes available, the levels should be revised. 


Planetary biology is one of the most exciting areas of space 
exploration. The unnecessary destruction of potential information 
in this research field by contamination would be an uncultural event. 
It is feasible to sterilize probes in such a manner that the loss of 
information to future investigators is minimized. This can be 

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Jet Propulsion Laboratory External Publication No. 698 

accomplished utilizing ethylene oxide, heat and radiation, accompanied 
by the sterile assembly of special components, as sterilizing agents. 

Pollution tolerances should be kept to 10 8 dead bacteria per 
missile. Infection tolerances should be kept to less than 10 per 
missile for the planets and 10 -1 for the Moon. 

A molecular inventory, preferably in the form of payload 
duplicates, should be kept for each space flight. More information 
on the chemical composition of space-probe materials should be 

An agency specially qualified to handle sterilization should 
perform the terminal disinfection and ascertain the degree of 

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Jet Propulsion Laboratory External Publication No. 698 


American scientists have been very patient while rocket 
technicians have picked their brains for information of value to 
space research. It has been precisely by this technique that we 
accumulated the facts contained in this paper. We hope that, by 
recognizing the gravity of the problem, we have partially compensated 
for our lack of originality. 

Numerous people working with the National Academy of Sciences 
have assisted us in formulating our ideas and we thank them all. 
In particular we want to mention Joshua Lederberg for his 
characteristic insight, and Charles Phillips for making his work 
known to us. 

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Lederberg, J., and Cowie, D. B., "Moondust, " Science , 127: 
1473, 1958. 

Alter, D. , "The Alphonsus Story" , Proceedings of Lunar and 
Planetary Exploration Colloquim , North American Aviation, 
Incorporated, Los Angeles, California, January 12, 1959. 

Kozyrev, N. A., "Volcanic Activity on the Moon?", Sky and 
Telescope , 18(3): 123, 131, January 1959; also, "Observations of 
a Volcanic Process on the Moon," 18(4) :184-186, February 1959. 

Sederholm, Weaver, Sagan, Private Communication, 1959. 

De Vaucouleurs, G. , Physics of the Planet Mars , Faber 
and Faber, Limited, London, 1954 I p. 269). 

Sinton, W. M. , "Martian Vegetation, American Astronomers Report", 
Sky and Telescope , 18(5) :252, March 1959; also, "Spectroscopic 
Evidence for Vegetation on Mars", Astrophysics Journal : 231, 
September 1957. 

Tikhov, G. A t , "Is Life Possible on Other Planets?" Journal of 
the British Astronomical Association , 65(3) :193, 1955. 

Gazley, C, The Penetration of Planetary Atmospheres , Rand Report, 
P-1322. The Rand Corporation, Santa Monica, California, 
February 24, 1958; also, Private Communication. 

Chapman, D. R. , An Approximate Analytical Method for Studying 
Entry into Planetary Atmospheres, TN 4276. National Advisory 
Committee on Aeronautics, Washington, D.C. , May 1958. 

Whipple, F., Private Conversation. 

"Contamination by Extraterrestrial Exploration," Science , 
128:887, 1958. 

"Contamination by Extra-Terrestrial Exploration," Nature , 
pp. 925-928, April 4, 1959. 

Halvorsen, H. 0., Spores , American Institute of Biological 
Sciences, Washington, D.C, 1957 (164 pp.). 

Phillips, C. R. , and Kaye, S. , "The Sterilizing Action of Gaseous 
Ethylene Oxide," American Journal of Hygiene , 50:270, 1949. 

Phillips, C. R. , and Warshowsky, B. , "Chemical Disinfectants," 
The Annual Review of Microbiology 12:525-550, 1958. 

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