Samples of soil collected from Kennedy Space Center near spacecraft assembly facilities were found to contain microorganisms very resistant to conventional sterilization techniques. The inactivation behavior of the naturally occurring spores in soil was investigated using dry heat and ionizing radiation, first separately, then in combination. Dry heat inactivation rates of spores were determined for 105 and 125 C. Radiation inactivation rates were determined for dose rates of 660 and 76 krad/hr at 25 C. Simultaneous combinations of heat and radiation were then investigated at 105, 110, 115, 120, and 125 C. Combined treatment was found to be highly synergistic requiring greatly reduced radiation doses to accomplish sterilization

David, T. J.

Lindell, K. F.

Reynolds, M. C.

English

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TACTIVS ONF ORUACDAy0' IV
ORAI VSOTL (SaL' uCuRPTiG R~73-2951 04~C ndia Labs) p =? 4
/°SCL 08B nca
SLA-73-0161
May 1973
THERMORADIATION INACTIVATION OF
NATURALLY OCCURRING ORGANISMS IN SOIL
Marcel C. Reynolds
Kermit F. Lindell
Thomas J. David
Biosystems Research Department
SANDIA LABORATORIES
OPERATED FbOp THE UNITFD STATES ATOMI C ENERGY COMMISSION BY SANDIA CORPORATIONJ ALBUQUERQUE NEW MEXItC LIVERMORE (,ALIFOPA,
https://ntrs.nasa.gov/search.jsp?R=19730016677 2020-03-11T19:45:03+00:00Z
SL,A-73-0161
THERMORADIATION INACTIVATION OF
NATURALLY OCCURRING ORGANISMS IN SOIL
Marcel (C. Reynolds
Kermit F. Lindell
Thomas .I. I )avid
Biosystems Research l)epartment
Sandia Laboratories
Albuquerque, New Mexico 87115
Printed May 1973
Abstract
Samples of soil collected from Kennedy Space Center near spacecraft assembly
facilities were found to contain microorganisms very resistant to conventional steri-
lization techniques. The inactivation behavior of the naturally occurring spores in soil
was investigated using dry heat and ionizing radiation, first separately, then in com-
bination. Dry heat inactivation rates of spores were determined for 105 and 125 C.
Radiation inactivation rates were determined for dose rates of 660 and 76 krad/hr at
255C. Simultaneous combinations of heat and radiation were then investigated at 105,
110, 115, 120, and 125°C. Combined treatment was found to he highly synergistic
requiring greatly reduced radiation doses to accomplish sterilization.
This work was conducted under Contract No. M -12,853, Planetary Programs, Office
of Space Science and Applications, NASA Headquarters, W ashington, D. C.
I -)
Contents
Page
Introduction 5
Alaterials and Mlethods 5
Experimental Results 7
Conclusions 10
References 10
Figures
1. Equipment Setup for Thermoradiation
Experiment with Controlled Humidity 6
2. Dry Heat Inactivation at 1250 C 7
3. Comparison of Thermoradiation,
Radiation, and Dry Heat Inactivation 8
4. Thermoradiation Inactivation at
Various Temperatures 9
5. D-\Value Versus Temperature at a
D)ose Rate of 76 krad/hr 9
3-4
THERMORAD IATION INACTIVATION OF
NATURALLY OCCURR ING ORGAN ISMS IN SOIL
Introduction
Ior some time it has been recognized that
microbial contamination of spacecraft during
assemblyi at Kennedy Space ('enter (KSC) might
\xell include naturally occurring organisms. 'T'hese
organisms indigenous to the local soil could be
transported via shoes, clothing, tools, etc., into
assembly areas and eventually deposited on space-
craft surfaces. To determine what effect these
natural organisms might have on spacecraft steri-
lization rationale, the Phoenix Laboratory, P'IIS,
began a study using samples of natural soil from
around(i assembly facilities at KSC(, They investi-
gated dry heat sterilization of these naturally
occurring populations and the resistance of isolates
from the hardy sub-population that survived heat
treatment. Their findings indicated extraordi-
narily high resistance to the 125° C dry heat steri-
lization planned for spacecraft.
This report describes our efforts at Sandia
Laboratories to apply the thermoradiation process
for more effective sterilization of tihe naturally
occurring organisms.
Materials and Methods
Preparation of Samples
Approximately six pounds of soil were col-
9lected2 near Hanger AO at Cape Kennedy and sent
to Phoenix on June 17, 1970. The sample con-
sisted of equal amounts from the following three
locations: (1) a ditch about 50 yards west of the
IHigh Bay doors (a sandy type soil with small sea-
shells); (2) a landscaped area at the main (east)
entrance of the hanger (a sandy loam soil); and
(3) the south end of the hanger near the water
cooling tower (a dry sandy-clay soil), The sample
was spread in a thin layer on sterilepaper, covered,
and allowed to dry for 48 hours at room temper-
ature. Dry soil (2771 g) was then dry processed
through a stainless steel sieve series (W. S. Tyler
Co. , Cleveland) down to 0. 124 mm screen size to
remove rocks, shell particles, and plant particles.
A series of rinses in 95%o ethanol and then a final
sieving with 0. 043 mm screen resulted in 4900 ml
of soil-spore suspensions in 95% ethanol. Serial
dilutions were plated with TSA supplemented with
0. 1% soluble starch and 0. 2°/,, east exi:ract aind
incubated for 1 week at 35°'(C'. The viable con-
centration was 2. 7 x 105 organisms and the ap-
proximate weight of soil in the suspension was
0. 03 g/ml.
'T'he soil suspension described above was ulsed
as the inoculum for all the experiments. The sus-
pension was maintained at 4° C during storage.
Prior to use, the soil-spore suspension was iiin-
sonated for two minutes to break up clumps within
the ethanol suspension and was contiinuously agi-
tated during inoculation to prevent settling. The
samples were prepared by pipeting 0. I ml of the
suspension onto the surface of aluminum foil discs.
The discs were 1. 25-inch diameter cut from tbio-
logical grade aluminum foil 0. 0015-inch thick. 'T'ho
samples were then allowed to air Idry until all of
the ethanol had evaporated. When dry, the inocu-
lated discs were assembled on aluminum strips,
1. 50 inches wide by 0. 020 inch thick. Four sample
discs were placed on each strip, a single clean
foil disc was placed over each sample, and then
another aluminum strip placed on top and held in
place with wire clamps. This assembly of the
discs clamped between two strips permitted con-
siderable handling and suspending of the assembly
in a vertical position without loss or damage to the
sample discs. The assembled sample strips wvere
then placed in a desiccator over lDrierite in a vac-
uum for 15 hours prior to exposure to the steri-
lization environment. All of the inoculation and
assembly operations were performed in a (Class 100
laminar airflow clean room.
The use of covered foil as a substrate for the
inoculum was evaluated and the inactivation rates
using this substrate was compared to rates using
open coupons. This was done in order to relate
this study to previous work by PHIS Phoenix L.ab-
oratory. A comparative experiment was performed
using the covered foils, 1-inch square by 0.020-
inch thick coupons of aluminum, and stainless steel.
The coupons were attached to 1. 5-inch wide alu-
minum strips with small wvire hooks, leaving the
inoculated surface exposed. This type of assembly
required extreme care in handling to prevent sample
loss or damage. W ith all other experimental pro-
cedures being identical, no differences in the re-
sulting inactivation rates was detected between the
covered foil discs or the open coupons of either
aluminum or stainless steel.
)
L ..... RH&TEMP ____
| RECORDER
Figure 1. lquipment Setup for Thermoradiation
Experiment with Controlled Tlumidity
Exposure Mlethods
Thle thermal environment wvas provided hy a
recirculating air temperature chamber having a
volume of 0. 578 ft 3 with a rail arrangement in the
door to hold the aluminum strips. The temperature
was controlled and recorded to an accuracy of
±+0. 2°C. The radiation environment was provided
by the Sandia Gamma Irradiation Facility (GIF).
This facility contained remote handling equipment
to introduce and remove the source and included
xisual, physical and electrical access with neces-
sary safety controls. 'The cobalt-GO60 source was
introduced in a corner of the cell, which is 7 by 8
by 8.5 feet. The dose rates ranged from I x 106
rad/hr to 4 x 103 rad/hr depending on the location
of the sample within the cell.
Moisture content of the air in the temperature
chamber was controlled I)y a system4 of flowing
pressurized air through a saturator in a warm water
hath and then through a condenser coil and wailer
trap in a cold water hath. The air was allowed to
expand into a coil in the warm wvater hbath and then
introduced into the temperature chamber. Adjust-
ments of the air pressure and the temperature of
the cold water bath provided the relative humidity
(Ill) control desired. 'T'he rel ativ c humidity for
these experiments was controlled at 30" 1-111 at 25( ' .
Ieclative humidity measurements of the air were
made at the input to the temperature chamber with
a dewpoint indicator. In addition, continuous meas-
urements were provided by lithium chloride sensors
and a strip c(hart recorder. The RP1 value was con-
trolled within ±I°', 1II during the experiments. 'The
rate of air F'low into the temperature chamniber was
controlled to 0.5 cfm and the air pressure in the
chamber was regulated at about 0. 8 in. v. g. bh
adjustment of a bleeder valve on the chamber.
'or each experiment, thetemperaturechamber
was placed in the Gil' cell at the appropriate dis-
tance from the cobalt-60 source for the desired
dose rate. The chamber was positioned so the sam-
ple strips assembled with the foil discs were ver-
tical and the face of the strips was perpendicular to
the direction of the gamma rays. The temperature
chamber controller and temperature recorder were
located outside the cell with necessary cable con-
nections passed through the cell wall. The humidity
control system was also located outside the cell
with the input air to the temperature chamber pen-
etrating the cell wvall. A block diagram of the
equipment setup is shown in F"igure 1. Silver phos-
phate oc cobalt glass dosimeters, depending on the
dlose r:irge, were placed on selected sample strips
to X erifY the compuled dose rates.
li ccover> Methods
E:ach sample strip when removed from the
temperature chamniber was wrapped in sterile ali-
mninim foil and returined to the clean room facilities
for recoxvery operations. lFrom 20 to 30 minutes
were required to transport the samples from the
remote reactor area where the GIl is located to
the ('lass 100 clean room facilities.
6
1
I:ach sample strip containing four replicate
samples represents a single data point. Iarch of
the samples from the strips was placed in a sep-
arate 50 mi beaker containing 10 ml of sterile, 0. 1
percent Tween 80 water. The samples were then
insonated for 2 minutes to remove the organisms
from the foil discs. Care was exercised in placing
the foil discs in the beakers to assure separation of
the inoculated disc and cover disc, and complete
wetting and submersion of both discs in the water.
During the ultrasonic treatment, occasional agi-
tation of the beakers kept the discs separated and
prevented cold welding together of the two discs.
This protocol assured good recovery of the
organisms from the foil discs. The insonation is
accomplished with the beakers immersed in the
ultrasonic water bath to a level just above the
water level in the beaker.
A\dditional ten-fold serial dilutions were made
as required. Dilutions were plated out in duplicate
on Trypticase Soy Agar supplemented with 0. 1%
soluble starch and 0. 2% yeast extract underlay,
ovellayed with the same type of media and then
placed in an incubator maintained at 35 ° C. Plates
were counted after 1 w eek in the incubator.
Experimental Results
lExperimentation with the naturally occurring
spores in soil was directed primarily toward the
response of natural organisms to dry heat treat-
ment, radiation treatment, then the combination
of dry heat and gamma radiation.
The results of the first baseline experiment
to determine dry heat resistance of the organisms
in soil is shown in Figure 2. The viable sample
population, beginning at 2 x 104 organisms (zero
heat treatment), undergoes a rapid reduction to
about 102 organisms during the first treatment
period. This initial drop, which was found to he
characteristic of organisms in soil, is followed by
a second logarithmic phase of destruction. The
second phase represents a very heat resistant sub-
population of approximately 1. 07% of the original
sample. Excepting radiation inactivation, this hi-
phasic order of death was noted in all dry heat and
thermoradiation experimentation. The second
phase, on the logarithmic part of the survivor curve,
Figure 2. Dry Ieat Inactivation
at 1250 °C
v(
O:
tY
5 20 24 2E 32 36
TIME, HOURS @ 125°C
was used as a basis of comparison for various
treatments. Uising the method of least squares, the
data was fl'it with a straight line in order that slopes
or l)-values might be compared. in Figure 2, then,
RADIATION DOSE, KRADS
54 108 162 216 270 324 378 432 486
100
10-I
-A
10
I I I I I I I I I I
E
DRY HEAT 0 - 29.45 HRS
00
AI"
SYNERGISTIC RADIATION D = 205 KRADS
INACTIVATION N o
a \-
COMPARISON OF THERMORADIATION,
RADIATION AND DRY HEAT INACTIVATION-
OF NATURALLY OCCURRING SPORES IN SOIL=
(PHS PHOENIX STOCK)
I I I I I I I I I i I
-\
- \
I
- I.
- THERMORADIATION
-D * 1.04 HRS
I 2 3 4 5 6 7 8 9 10
TIME, HOURS @ 125 C
the D-value determined for the resisitant sub-
population is 29. 45 hours. This value is roughly
50 to 100 times the D-value for heat resistant
Bacillus subtilis var. niger spores.
The radiation resistance of the naturally oc-
curring populations were next inv estigated. Sam-
ples were exposed at room temperature (25- C)
to gamma radiation from a cobalt-60 source.
Temperature and moisture-conditioned air wvas
supplied to the sample chambers. The rate of
flow provided one air change per minute. The re-
sults of irradiation are shown in Figure 3. In this
figure, the data from Figure 2 dry heat inactixa-
tion is repeated in order to simplify comparisons
of the inactivation rates with various treatments.
The second curve is the results of radiation at
room temperature with 54 krad/lhr, The I)-x alue
was found to be 205 krad (3. 8 hours) for the entire
population which is a very high resistance indeed.
'l)-valute is the time at temperature or the
radiation dose required to reduce tile viable popil-
lation by 90;,.
The third curve on Figure 3 is the results of si-
multaneously combining dry heal and radiation
(thermoradiation). The thermoradiation I)-value
was found to be approximately one hour as corn-
l Figure 3. Comparison of Thermo-
- radiation, Radiation, and
- IDry lieat inactivation
pared to 3. 8 hours for radiation alone or 29. 4 hours
for heat alone. For an example of the synergism
obtained, the singular effects of heat and radiation
when added wvould reduce the population by 1 log
after 3 hours or' treatment. Thermoradiation, for
the same time at temperature and total dose, re-
duced the population by 3 ]logs. If we examine
the slope or I)-x allies of these examples, the sing-
iul ar effects of heat and radiation, if considered in
an adcdili. e sense, wi ould result in a D-v alue of
3. 37 hontrs. 'iThe thermoradcliation I)-value, 1. 04
hours, is less than 1/3 that of the additive effects.
']'his lev\ el of svnergisnl means that sterilization
can be accomplished in 1/3 of the time at temper-
atlire and with 1/3 the normal close.
Additional experiments were performed to
determine the characteristics of thermoradcliation
treatment at temperatures below 1253 C. For this
series a constant dose rate, sampling time, and
moisture environment were maintained throughout
with only temperatures as a variable. The D-values
based on the resistant sub-populations were found
to be 57 minutes at 125" C. 60 minutes at 120- ('C,
20
z
0
I.-r
0
Q,
RADIATION DOSE, KRADS
19 38 76 95 114 133 152 171 190
20 40 60 80 100 120 140 160 180 200
Figure 4. Thermoradiation Inactivation
at Various Temperatures
TIME, MINUTES AT TEMPERATURE
77 minutes at 115°C, 89 minutes at 110°C, and
95 minutes at 105° C (Figure 4). The interesting
aspect of this series is the manner in which the
shape of the inactivation curve changes. As the
temperatures are lowered, the sharp initial drop
usually experienced at 125° C softens, requiring a
longer exposure time to reach the second phase.
90
Figure 5. D-Value Versus
Temperature at
a Dose Rate of
76 krad/hr
In
z
:E
-J
:f
50-
40
30
As the temperature is further reduced, the thermo-
radiation curve approaches a straight line which is,
in fact, the case for radiation of naturally occurring
spores at room temperatures (Figure 3). The D-
values presented in Figure 4 are plotted as a func-
tion of temperature in Figure 5 to illustrate the
130
120
110 0
100
70
90
80 0
IN INACTIVATION OF 70 '0
RING SPORES IN SOIL 60 o
60q
TEMPERATURE
TANT DOSE RATE 50 _
ADSIHR)
-40 0
30
105 110 115 120 125
TEMPERATURE, °C
(9
0
Ln
80-
70-
601-
THERMORADIATIO
NATURALLY OCCUR
D-VALUE VS
AT A CONS'
(76 KRA
I * * . I
---------
......I .. . .............. II' ' 'LU
, . . I
lemperature/dose relationship. For example, at
105 ° C with a 95 minute I)-value, the radiation dose
per log population reduction would be 121 krad/log.
As the temperature is elevated (constant dose rate),
the 1)-value drops to 57 minutes with a total dose of
72 kinrad per log population reduction or roughly 60%/,
of the radiation required at the lower temperature.
Conclusions
Naturally occurring organisms present avery
difficult but realistic contaminant for spacecraft
sterilization. Although organisms are frequently
either heat resistant or radiation resistant, the
naturally occurring spores are both. As such,
these organisms have been a desirable adjunct to
our previous studies that were based primarily on
the use of B13. subtilis var. niger as a working or-
ganism. lHowever, the synergistic behavior to
certain combined treatments was in good agreement
with obserx ations of the inactiN ation of ofther bac-
terial spores, bacteriophage, proteins, viruses,
and yeast. 5-8 Although the soil organisms were
found to be highly resistand to both heal and radi-
ation, the degree of synergism appears higher than
for 1. subtilis. We experienced thermoradiation
inactivation rates of 3. 25 times the additive effects
of heat and radiation, whereas for B. subtilis
thermoradiation rates of about 2.6 times the
singular additive rates were found to be the upper
limit. 9 We are presently completing the mathe-
matical characterization of the response of natu-
rally occurring spores to treatment for all tem-
peratures and dose rates of interest. This has
been done for B. subtilis in order that dose rate -
temperature combinations could be selected to
yield the highest level of synergism. The utility
of this approach to determine treatment parameters
is that thermal and radiation exposure can be
minimized.
References
1. Report No. 32, Phoenix Laboratories, lPub)lic
Health Service, January 1971.
2. Report No. 31, Phoenix Laboratories, Public
Ilealth Service, July - September 1970.
3. Report No. 25, Phoenix Laboratories, Public
Health Service, January - March 1969.
4. D. M. Garst and K. F. Lindell, A Precisely
Controlled, Low Range Ilumidity System, SC-
RR-70-775, Sandia Laboratories, November
1970.
5. M. C. Reynolds, The Feasibility of Thermo-
radiation Sterilization of Spacecraft - A Pre-
liminary Report, SC-RR-69-857, Sandia
Laboratories, December 1969.
6. M. C. Reynolds, K. F. Lindell, and N. Laible,
A Study of the Effectiveness of Thermoradiation
Sterilization, SC-RR-70-423, Sandia Labo-
ratories, June 1970.
7. M. ('. Reynolds and D. M. Garst, "Optimizing
Thermal and Radiation Effects for Bacterial
Inactivation, " Space Life Sciences, 2, (3),
December 1970.
8. \V. L. Dugan and R. Trujillo, Biological In-
activation by Thermoradiation, SC-RR-71
0616, Sandia Laboratories, October 1971.
9. Ml. C. Reynolds and J. P. Brannen, "Thermal
Enhancement of Radiosterilization, " Proce-
edings, International Atomic Energy Agency,
International Symposium on Radiation Pres-
ervation of Food, Bombay, India, November
1972.
10


Thermoradiation inactivation of naturally occurring organisms in soil

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