APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1977, p. 1170-1176
Copyright C 1977American Society for Microbiology
Vol. 33, No. 5
Printed in U.S.A.
Effect of Combined Heat and Radiation on Microbial
D. A. FISHER2 AND I. J. PFLUG*
Department ofFood Science and Nutirition and the School ofPublic Health, University ofMinnesota,
St. Paul, Minnesota 55108
Received for publication 22 October 1976
A series of experiments at several levels of relative humidity and radiation
dose rates was carried out using spores ofBacillus subtilis var. niger to evaluate
the effect of heat alone, radiation alone, and a combination of heat and radia-
tion. Combined heat and radiation treatment of microorganisms yields a de-
struction rate greater than the additive rates of the independent agents. The
synergistic mechanism shows a proportional dependency on radiation dose rate,
an Arrhenius dependency on temperature, and a dependency on relative humid-
ity. Maximum synergism occurs under conditions where heat and radiation
individually destroy microorganisms at approximately equal rates. Larger syn-
ergistic advantage is possible at low relative humidities rather than at high
One of the more intriguing subjects of the
Planetary Quarantine research has been the
discovery of the synergistic effect that results
from the combination of heat and radiation for
bacterial spore destruction. Koesterer (2) ob-
served this effect while carrying out explora-
tory sterilization studies for the National Aeu-
ronautics and Space Administration (NASA).
Scientists at the Sandia Laboratories carried
out extensive laboratory and feasibility studies
on the use of combined heat and radiation for
spacecraft sterilization (5).
This report describes studies carried out at
the University of Minnesota to investigate the
sterilization attributes of the thermoradiation
process. Destruction rate tests were carried out
at a number of radiation levels, temperatures,
and relative humidities. Both wet- and dry-
heat conditions were used. We have attempted
not only to develop destruction rate data but
also to determine the mechanistic basis for the
synergism displayed by these seemingly inde-
pendent lethal agents.
MATERIALS AND METHODS
Biological procedures. Bacillus subtilis var. niger
spores grown at 32°C in synthetic sporulation me-
dium-10 (3) were used in this study. The spores were
cleaned by exposure to ultrasonic energy and by
repeated washing with deionized distilled water and
'Minnesota Agricultural Experiment Station Scientific
Journal Series paper no. 9800.
2Present address: E. I. Dupont de Nemours, Richmond,
For the wet-heat studies, the spores were sus-
pended in 5 ml ofSorensen 0.067 M phosphate buffer
(pH 7.0) in screw-capped glass test tubes. The popu-
lation density of the suspension was approximately
106 spores per ml. After inoculation the tubes were
refrigerated at 40C until treated. After treatment
the tubes were placed in an ice bath until assayed.
In the assay procedure, the sample was mixed and a
1-ml portion was diluted in buffered distilled water
(described in references 4), and duplicate portions
were plated using Trypticase soy agar (BBL).
For the dry-heat studies, a 0.01-ml portion of an
ethanol suspension of the spores was deposited on
stainless steel planchets (12.7 by 12.7 mm, 106
spores per planchet). The planchets were then equil-
ibrated at 22°C, 50% relative humidity, for at least
24 h before treatment. Samples were moved as
needed to the University ofMinnesota Gamma Irra-
diation Facility for testing. After treatment, the
planchets were placed in ice-cooled flasks until as-
sayed using NASA Standard Procedures (4). In the
assay procedure, buffered distilled water was added
to each flask, the flask was suspended in an ultra-
sonic (25 kHz, 0.35 W/cm2) tank filled with an
aqueous solution containing 0.3% Tween 80 (de-
scribed in reference 4) for 2 min, and duplicate por-
tions ofthe eluate were plated using Trypticase soy
All inoculation and recovery procedures were car-
ried out in a class 100 clean room. Colony-forming
units were counted after 48 h of incubation at 32°C.
Radiation system. The University of Minnesota
Gamma Irradiation Facility uses a cylindrical array
of cesium-137 sources of approximately 10,000 Ci.
The radiation field was mapped using Fricke Dosi-
metry as a primary reference and a calibrated Victo-
reen rate meter as a secondary reference.
Before biological testing, areas within the radia-
FISHER AND PFLUG
maximum synergism occurs when heat and ra-
diation are approximately equally effective
The maximum synergistic advantage de-
pends on the spore water level. Dry-heat ther-
moradiation gains more synergistic advantage
than does wet heat. SIs of 2.5 are possible with
dry heat, whereas with wet heat, the maximum
SI is limited to about 1.5. An implicit result is
that a larger synergistic advantage is possible
at low relative humidities rather than at high
We believe that the synergistic effect is a
consequence of the need to degrade a single or
pair of vital macromolecules at a multiple of
locations in order to "kill" the microbial spore.
A mathematical model describing such a proc-
ess has been developed which a priori predicts
the experimental phenomena (1).
Conclusions. From this study we conclude
the following. (i) Radiation and heat display a
synergistic effect in the destruction ofmicrobial
spores. (ii) No synergism is possible unless each
physiological stress is great enough to effec-
tively destroy spores by itself. (iii) The syner-
gistic mechanism has characteristics resem-
bling each of the constituent agents; a propor-
tional dependency on radiation dose rate, an
Arrhenius dependency on temperature, and it
is affected by relative humidity. (iv) Maximum
synergism occurs at those conditions where
heat and radiation are equally effective as ster-
These studies were supported in part by National Aero-
nautics and Space Administration grantNGL 24-005-160.
The assistance of Geraldine M. Smith, Rebecca Gove,
Steve Znameroski, and Yvonne Heisserer are gratefully
1. Fisher, D. A., and I. J. Pflug. 1975. Semiannual Prog-
ress report no. 14, NASA grant NGL 24-005-160. En-
vironmental microbiology as related to planetary
quarantine. University ofMinnesota, Minneapolis.
2. Koesterer, M. G. 1965. Thermal death studies on
microbial spores and some considerations for the
sterilization of spacecraft components. Dev. Ind. Mi-
3. Lazzarini, R. A., and E. Santangelo. 1967. Medium-
dependent alteration of lysine transfer ribonucleic
acid in sporulating Bacillus subtilis cells. J. Bacte-
4. National Aeronautics and Space Administration. 1968.
NASA standard procedures for the microbiological
examination of space hardware, NHB 5340.1A. Na-
tional Aeronautics and Space Administration, Wash-
ington, D. C.
5. Sivinski, H. D. 1972. Final report on planetary quaran-
tine activities, N72-30064#; NASA-CR-127835. SC-
RR-72 0516. Sandia Laboratories, Albuquerque, New
6. Webb, R. B., C. F. Ehret, and E. L. Powers. 1958. A
study of the temperature dependence of radiation
sensitivity of dry spores of Bacillus megaterium be-
tween 5°K and 309'K. Experimentia 14:324-326.
APPL. ENVIRON. MICROBIOL.