BIOTECHNOLOGY AND BIOENGINEERING

T’OL. TIII, ISSITE 1 (1966)

NOTE

Heat Sterilization of Activated Carbon

During the past 6 years there have been several conflicting reports concerning
heat sterilization of activated carbon. Osowitz, in an unpublished report which
was widely circulated, claimed that “small amounts of loose activated carbon dust
will remain viable through any autoclave cycle or 1000°F. furnace.” Because of
the inconsistency of this report and its anachronistic style, most microbiologists
did not consider i t seriously. Osowitz’s results were refuted, in part, by
Koestererl (1964) who showed that viable microorganisms could not be detected in
5CLmg. portions of activated carbon subjected to dry heat treatments of 120°C.
for 6 hr. or 135°C. for 3 hr. However, when similar samples were autoclaved a t
121°C. and 15 lb. pressure for 15, 30, or 45 min., a significant number of tubes
showed the presence of viable organisms. Since he used screw-cap tubes
(Koesterer, personal communication, 1965), there is a strong possibility that proper
exhaustion of air did not occur so that true steam sterilization was not achieved.
I n a review of the biological and physical factors involved in dry-heat steriliza-
tion, Bruch2 (1964) noted that he had come upon information which indicated
that microorganisms in activated carbon can resist dry heat a t temperatures over
260°C. (500°F.)for 1 hr.
Our interest in this problem stems from the many questions about these data
posed by engineering personnel involved in the field of spacecraft sterilization.
The National Aeronautics and Space Administration has established stringent
regulations to prevent the introduction of terrestrial microorganisms on other
planets. One of the requirements for spacecraft impacting Mars is that the proba-
bility of depositing viable organisms be less than 1 in 10,000 (Jaffe,3 1963). I n
implementing this policy, spacecraft required to be sterile will be subjected to a
terminal dry-heat treatment of 135°C. for 24 hr. Consequently, we performed
a series of tests to determine whether the results described above were due to
microorganisms associated with activated carbon being unusually resistant to
heat or were the result of a technique-induced artifact.
Fifty-mg. amounts of activated carbon (Darco G-60, Atlas Chemical Indus-
tries*) were placed into either sterile screw-cap test tubes or sterile culture tubes
with cotton plugs. Samples were autoclaved a t 121°C. and 15 lb. pressure for
15 min., or heated in a dry-heat oven a t 165OC. for 3 hr. or 135°C. for 24 hr.
The results showed that when activated carbon was autoclaved in screw-cap
* The use of trade names is for identification purposes only and does not con-
stitute endorsement by the Public Health Service or by the U.S. Department of
Health, Education, and Welfare.
63 1
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632 NOTE

tubes with the caps loose, 90% (143/160) of the tubes exhibited microbial growth
after the addition of trypticase soy broth and subsequent incr1bat)ion.
When the caps of the tubes were completely tightened, microorganisms were
detected in all cases (60/60). However, no viable microorganisms were detect.ed
in cotton-plugged tubes autoclaved in the same manner (O/ZOO). In addition,
when spore strips containing about lo6 spores of Bacillus subtilis var. niger were
placed in the same type of tubes, viable microorganisms were detected only in the
screw-cap tubes.
It is obvious that screw caps, t,iglitened or loose, prevented the proper ex-
haustion of air so that optimum conditions for steam sterilization were not
achieved.
I n the dry-heat tests no viable microorganisms were detected after the samples
had been heated a t 135 or 165°C. for 24 or 3 hr., respectively.
Consequently, failure to sterilize activated carbon by aut,oclaving, as reported
by other investigators, cannot be attributed to any special property of the carbon
or its microbial flora, but rather is due to inadequate exposure to saturat.ed steam.
What most people familiar with sterilization processes have predicted has been
clearly demonstrated experimentally.
With respect to Osowit,z’s original claim that activated carbon could not be
sterilized at temperatures of IOOO’F., we must assume that, poor microbiological
techniqiies were employed.

References
1. &I. G. Koesterer, Studies for sterilization of space probe components, Report
No. 2 on Contract NASA 879, National Aeronautics and Space Administration,
Washington, D.C., 1964.
2. C. W. Bruch, “Some biological and physical factors in dry-heat sterilization:
a general review,’’ L i f e Sciences and Space Research 11, M. Florkin and A. Dollfus,
Eds., North Holland, Amsterdam, 1964, pp. 357-371.
3. L. D. Jaffe, “Sterilizing unmanned spacecraft,” Astronautical and Aeyospace
Engineering, August, 1963, pp. 22-29.

This research was supported by the National Aeronautics and Space Adminis
tration, under contract No. R-137.

R. PULEO
JOHN
MARTINS. FAVERO

Phoenix Field Station

Technology Branch
Communicable Disease Center
Public Health Service
Department of Health, Education, and Welfare
Phoenix, Arizona 85014

Received August 17, 1966

RIOTECHNOLOGY AND BIOENGINEERING, VOL. VIII, ISSUE 4