Dry heat inactivation of bacterial spores and influence of spore moisture content on z values as related to spacecraft sanitatio

Angelotti, R.

Campbell, J. E.

English

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ECOLOGY AND THL.4MAL INACTIVATION OF MICROBES
IN AND ON INTERPLANETARY SPACE VEHICLE
COMPONENTS
Fourteenth Quarterly Report of Progress
Research Project R-36-015-001
July 1 - September 30, 1968
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1171
ECOLOGY AND THERMAL INACTIVATION OF MICROBES
IN AND ON INTERPLANETARY SPACE VEHICLE
COMPONENTS
Fourteenth Quarterly Report of Progress
Research Project R-36-015-001
July 1 - September 30, 1968
Food Microbiology
Robert Angelotti
Herbert E. Hall
T. F. Butler
R. G. Thompson, Jr.
CONTRIBUTORS
Milk Sanitation
R. B. Read, Jr.
A. L. Reyes
R. G. Crawford
A. J. Wehby
Food Chemistry
J. E. Campbell
Statistics
James T. Peeler
Report Submitted by:	 Report Reviewed and Forwarded by:
Robert Angelotti	 J	 Campbel
Microbiologist	 Pr ncipal Investigator
CONTENTS
Page
PART I - KINETICS OF SPORE INACTIVATION BY DRY HEAT
	 1
Introduction	 1
Experimental	 2
Identification of a system for thermal
inactivation studies
	 2
Preparation of spore crop
	 3
Recovery of spores from glass tubes
	 3
Cross contamination between tubes in the tin can
	 6
Projected Research for the Fifteenth Quarter
	 6
PART II - INFLUENCE OF SPORE MOISTURE CONTENT ON Z 
	 7
Introduction	 7
Experimental	 7
Projected Research for the Fifteenth Quarter
	 8
Table 1	 9
Table 2	 10
Table 3	 11
Table 4	 12
Table 5	 13
ECOLOGY AND THERMAL INACTIVATION OF MICROBES
IN AND ON INTERPLANETARY SPACE VEHICLE
COMPONENTS
PART I - KINETICS OF SPORE INACTIVATION BY DRY HEAT
Introduction
The experimental design of research on the dry heat inactiv-
ation of bacterial spores done by others to date has been based
primarily upon the assumption that spore inactivation follows a
first order reaction. This has enabled the generation of D and
z values for dry heat inactivation of spores and from these data,
processes have been calculated for the sterilization of space
hardware. Several reports on microbial inactivation by wet heat
have shown significant deviations from first order during the
final stages of thermal inactivation, wherein there is a tendency
for the inactivation curve to become asymptotic to baseline. The
purpose 'of this study is to determine whether significant devia-
tions from fi_:st order occur during dry heat inactivation and,
if so, to obtain data that can be used to discriminate between
suggested models for thermal inactivation by dry heat.
Destruction rate curves for spores are usually obtained by
conventional plating methods. Since these are generally ineffec-
tive below spore concentrations of about 100/ml of eluting fluid,
this technique cannot be used to identify the nature of what may
be the most significant part of the destruction rate curve from
-1-
a process calculation standpoint - that area where the number of
survivors is from 10° to 10 -3 per test unit. To work in this
range of survival rates, techniques must be used that enabie
the handling of several hundred test units per destruction rate
curve determination so that the most probable number of survivors
can be obtained with a reasonable amount of precision. Initial
experimentation has 10-en centered around the identification of a
suitable experimental system.
Experimental
Identification of a system for thermal inactivation studies
Initial experiments were done to determine the feasibility
of using lyophilization as a technique for drying spores. Spores
of Bacillus subtilis var. niger were dispersed into S ml self-
stopping lyophilization vials, frozen in dry ice and acetone,
dried, and stoppered. A high frequency vacuum tester was used
to check vacuum in the vials after lyophilization. Variations in
final vacuum were found after lyophilization, which suggested the
vial leaked around the stopper. After several attempts to prevent
this leaking, the system was discarded since any gaseous inter-
change with atmosphere could affect the spore a w and the heat
resistance of these spores.
We next examined the feasibility of using 8 x SO mm borosil-
icate glass tubes as test units when sealed in commercial 206 x
300 tin cans. Glass tubes equipped with thermocouples were sealed
-2-
in these cans and they were submerged in a 125°C silicone oil
bath. In cans that were filled with tubes (approximately 70
tubes per can), the rate of heat penetration was dependent upon
the position of the tube in the can (Fig. 1). To obtain a more
uniform rate of heat penetration, an aluminum matrix was made
(capacity, 37 tubes) and heat penetration studies repeated (Fig. 2).
Heat penetration was uniform and this system was judged worthy of
further study.
Preparation of spore crop
Three spore crops of B. subtilis var. ni er were produced
following the methods described in the first quarterly report
(NASA Research Project R-36-015-001, April 1 - June 30, 1965).
All crops had spore concentrations of 5.0 x 10 9 to 1 x 1010/ml
after treatment at 80°C for 10 minutes and were stable during
storage at 5°C. The heat resistance of these crops was compared
to that of the original spore suspension prepared in 1965 using
stainless steel strips as a vehicle. All had a resistance either
equal to or greater than the original crop. Further determinations
of heat resistance when on glass are planned using the 8 x 50 mm
tube-tin can system.
Recovery of spores from glass tubes
Since enumeration of viable spores by plate count or by most
probable number procedures requires dilution in the beginning
portion of the destruction rate curve, methods had to be developed
-3-
WON
for eluting dry spores from the 8 x 50 mm glass tubes. Of the
methods available, insonation has been shown to have the greatest
promise and we evaluated the effect of tube position, insonation
time, and use of two different makes of insonators on spore
elution efficacy.
Glass tubes were inoculated with spores of B. subtilis var.
ni er and dried overnight at 50°C. Sterile buffered dilution
water was added to each tube after drying and the tubes were
insonated, the elution fluid plated, and recovery efficiency
calculated (Table 1). Since only 85% recovery of spores was
obtained under the best conditions tested, this system of elu-
tion was discarded because of low efficiency.
The small diameter of the tubes (8 mm) was thought to
inhibit th.- scrubbing action of the insonation. In an attempt
to improve elution efficiency, we tested the effect of crushing
the tubes before insonation. The 8 x 50 tubes were inoculated
with spores of B. subtilis var. niger, dried, and each tube was
placed in a bottle containing sterile peptone water. The tubes
were crushed with a sterile, stainless steel blade and insonated.
The elution efficiency with crushed tubes was compared with that
for intact tubes insonated under identical conditions (Table 2).
Excellent elution occurred with the Sonagen insonator when
crushed tubes were treated for 24 minutes.
-4-
Since we were primarily concerned with the elution of spores
that had been heated to inactivation temperatures when in contact
with glass, experiments were extended to include spores that had
been heated before elution. A technique was developed for direct
plating of the crushed glass tube which enabled the counting of
spores that had resisted elution by insonation. In this proce-
dure, the number of eluted spores is determined by conventional
plating of a portion of the peptone water after insonation.
Residual spores on the glass tube were counted by first rinsing
the crushed fragments of the glass tube with one 500-m1 and one
50-m1 volume of sterile buffered dilution water. The 50-n1 rinse
water was collected separately in a sterile flank and a portion
of this rinse plated. This would serve as a control of rinsing
efficiency. The rinsed glass fragment° were then plated in tryp-
tone glucose extract agar by placing the fragments in a petri
dish and cove ing these fragments with agar. Residual spores
are counted as colonies after incubation. Elution experiments
with spores heated in the 8 x 50 mm tubes showed that the heating
did not affect elution efficiency when the Sonagen isonator was
used for a treatment period of 24 minutes (Table 3). Our elution
procedure has been standardized at 24 minutes of isonation with
a 300-watt power output at 25 KC/second by a Sonagen apparatus
^.LT80-6, Branson Instrument Inc., Stanford, Conn.).
F'
-5-
Cross contamination between tubes in the tin can
The system proposed for exposure of many individual popula-
tions of spores to heat consists of 37 unstoppered tubes in one
tin can. After heating, these tubes are filled with a recovery
broth and incubated for 7 days and scored as to whether or not
gro,Yth occurred in the Individual tubes. Cross contamination
during the filling operation or during incubation would affect
the apparent recovery rate and cause error in computing the most
probable number of survivors.
Cross contamination has been evaluated and found not to
occur when the tubes in the open can were covered by a piece of
sterile filter paper and the can itself covered with one half of
a petri dish during incubation. All filling was done in a hori-
zontal laminar flow cabinet.
Projected Research for Next Quarter
During the next quarter, we plan to (a) obtain destruction
rate curves for B. subtilis var. niger spores on glass for use
in correcting D values obtained in the matrix-can system for
heating and cooling times, (b) to establish the effect of equili-
bration time at a 45% RH on D value at 125'C, and (c) to obtain
preliminary destruction rate curve data for B. subtilis var.
ni er spores on glass by use of most probable number techniques.
-u-
PART II - INFLUENCE OF SPORE MOISTURE CONTENT ON ZD
Introduction
The effect of spore moisture content on D has been demon-
strated in previous reports. Some data were presented last
quarter which indicated that little, if any, effect on z could be
attributed to spore moisture contents obtained by equilibrating
spores at relative humidities in the range of 10% to 90%. Z
values were obtained this quarter for spore¢ equilibrated at
relative humidities of less than 10% and for spores embedded in
epoxy that contained addtd water as well as for spores suspended
in water.
Experimental
In the Thirteenth Quarterly Report of Progress, D values for
spores heated in this system were reported for temperatures of
125', 135', and 140'C.
The data presented for 140'C last quarter were not corrected
for thermal lag. The corrected data are presented this quarter
in Table 4 along with the data of last quarter for 125 and 135'C.
The new data added to this table that have been collected this
quarter include the D values for lyophilized spores and for spores
equilibrated at 3% RH. The D values presented in Table 4 were
used to construct thermal destruction curves from which z  values
were calculated. Figure 3 and Table 5 present the relationship
between z  and spore moisture content for that range of spore
e
-7-
moisture cont — ts from lyophilized spores through spores sus-
pended in water.
With the exception of the sharp drop in z value for spores
suspended in water (z = 9.7 and 
D 1050 32.2 ' D 1100 - 5.8'
D115'C	
2.9) little, if any, difference in z values are noted
for spores regardless of the initial spore moisture content.
It was pointed out last quarter that large z values should be
expected for dry spores and several interpretations were pre-
sented to explain the inability to obtain large z values for
dry spores. In view of the data presented for spores lyophilized
on glass strips and embedded in epoxy (z = 26), it appears that
the most plausible explanation is that moisture is contributed
to the spores by the epoxy. That is, there is sufficient
moisture associated with the epoxy so that irrespective of the
initial spore moisture content, the final spore moisture content
is determined primarily by the moisture content of the plastic.
Projected Research for the Fifteenth Quarter
The effect of moisture on the dry heat resistance of Bacillus
subtilis var. ni er spores will be continued by emulsifying var-
ious amounts of water in epoxy resin. In addition, a new system
of drying spores utilizing air at various flow rates will be
studied.
-8-
TABLE 1
Recovery of dried spores of B. subtilis var. ni er
from 8 x 50 mm borosilicate glass tubes
under different conditions of insonation
Placement of tube Make of insonator Insonationtime (min)
Recovery
(x)
In rack Blackstone 12 55
In rack Blackstone 24 61
On bottom of bath Blackstone 12 74
On bottom of bath Blackstone 24 82
On bottom of bath Sonagen 12 83
On bottom of bath Sonagen 24 85
-9-
}, :a'"^^
TABLE 2
Recovery of spores of B. subtilis var. niger
from intact and crushed glass tubes by insonation
Make of insonator	
Insonation	 Recover (%
time (min) I Intact tubes I Crushed tubes
Blackstone 12 23 76
Blackstone 24 -- 96
Sonagen 12 23 90
Sonagen 24 -- 100
-10-
TABLE 3
Elution efficiency for B. subtilis var. ni er spores
heated in 8 x 50 mm tubes for varying times
at 125°C and the tubes crushed prior to insonation
Treatment
Number of
spores eluted
from tube
Number of
spores
adhering
Elution
efficiency
M
Unheated 9.8 x 105 4.5 > 99.99
Heated-10 min 5.9 x 105 8 > 99.99
Heated-20 min 3.8 x 10' 5 > 99.99
Heated -30
 min 1.9 x 105 2 > 99.99
-11-
---	
Now
TABLE 4
Influence of initial spore moisture content on
thermal resistance of Bacillus subtilis var. ni er
located on glass strips embedded in epoxy plastic
Spores	
Dry-heat exposure temperature
equilibrated
at RH of:b	
125°C	 135°C	 140°C
•
D	 1 95% C.I. c I	 D	 1 95% C.I.	 I D	 1 95% C.I.
Dry 251.4 227.4-280.2 96.6 87.6-108.0 67.8 61.2-75.6
3 250.2 223.2-285.0 78.6 66.6- 95.4 63.4 58.0-69.8
5 ` 275.4 225.3-303.0 108.6 94.2-127.8 68.4 62.9-74.8
7 292.8 269.4-321.0 102.0 96.6-108.0 63.5 56.3-'1.6
10 292.8 264.6-327.6 95.4 84.0-109.8 59.8 53.0-68.6
20 312.6 287.4-342.6 114.0 103.8-127.2 80.7 71.9-92.1
40 318.6 275.4-378.0 184.8 168.6-204.0 86.7 78.1-97.5
60 364.8 322.2-420.0 147.6 123.0-184.8 65.3 53.3-84.3
80 297.0 267.0-334.2 131.4 121.8-142.8 61.3 57.4-65.9
90 290.4 270.6-313.8 128.4 118.8-139.2 51.3 46.4-57.3
a Inoculum = 1 x 10 6
 spores per strip.
b Spores dried on glass strips were exposed to the selected relative
humidities for 48 hours at 25°C before embeddment in plastic.
C C.I. = Confidence Interval.
d Lyophilized 5 hrs. at 104 Hg at -40°C.
If
-12-
TABLE 5
Influence of initial spore moisture content on
z  of Bacillus subtilis var. ni er located on
glass strips embedded in epoxy plastic
Equilibrium
relative humidity
%
zD
°C
95% C.I.
°C
Correlation
index
R2
O a 26.0 23.7 - 28.8 0.99
3 24.2 19.2 -	 32.9 0.97
5 24.8 24.7 - 24.9 1.00
7 22.5 21.7	 -	 23.3 0.99
10 21.6 20.3 - 22.9 0.99
20 25.1 22.4 - 28.5 0.99
40 28.0 19.9 - 47.2 0.92
60 20.7 16.9 -	 26.7 0.98
80 22.6 18.2 - 29.7 0.97
90 20.8 15.8 - 30.4 0.95
100b 9.7 7.2 -	 14.8 0.94
a = Spores were lyophilized 5 hrs. at 10µ Hg at -40°C.
b = Spores were heated in 0.5 ml H2O and corrected for
thermal lag.
-13-
e
0
I°
123
122
121
120
119
118
LEGEND:
	
117
116
p TOP TUBE
	 115
O CENTER TUBE
• BOTTOM TUBE
05
95
85
75
1
40	 50	 60
55
55
1.5
35
25
e
0.	 124.9 c
1
0.
0.,
0.L
0.'.
CIA
0.,
O.E
0.^
I . C
ro
C)
a
8
a^
H
41
ro
PQ
30
s	 4
U	
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N	 6
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"	 8
9
10
20
30
40
50
60
70
80
90
100
444•
Id
t o
	
0
p	 0
p•
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0 O
•
p	 O O
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p	 O
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•
0
•
p	 O
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o
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0
I	 I
In	 20
124.8
124.7
124.6
124.5
124.4
124.3
124.2
124.1
124
MINUTES
FIG. 1 TEMPERATURE RECORDED IN GLASS TUBES SEALED IN TIN CANS
0.1
0.2
0.3
0.4
05
0.6
0.7
0.8
0.9
1.0
2
3
4
5
6
8
9
10
20
30
40
50
60
70
80
901!
10	 20	 30	 40	 50	 60
MINUTES
FIG. 2 TEMPERATURE RECORDED IN GLASS TUBES HELD IN ALUMINUM MATRIX
124.9 c
1
124.8
23
75
65
55
-- 45
— 35
—' 25
e
C)
4J
ro
u
c
e
-4H
4J
ro
3
0
a^
m
U
V)
v
vQ
124.7
124.6
124.5
124.4
124.3
124.2
124.1
124
22
fl
20
LEGEND:
• OUTSIDE R(d
O NEXT TO OUTSIDE ROW
q CENTER
--1119
—118
—^II7
— 116
— 115
—105
— 95
— 85
0NR
R 1
^ 9
0N 
8
7
0
6
5
4
3
100
90
80
70
60
50
40
30
20
I
= 95% Confidence Intervals
2
Lyophilized 0.03 0.05 0.07 0.1 	 0.2 0.4
	
0.6	 0.8	 0.9 1.0
WATER ACTIVITY (aw)
FIGURE 3. INFLUENCE OF SPORE MOISTURE CONTENT ON THE Z D VALUE OF
BACILLUS SUBTILIS VAR. NIGER SPORES SUSPENDED IN WATER
OR LOCATED ON GLASS STRIPS EMBEDDED IN EPDXY PLASTIC.
100
90
80
70
60
50
40
30
20
0
NR
R 10}
DN 8
7
6
5
	
= 95% Confidence Intervals
4
3
.	 2
11	 1	 1	 t	 1 	 1	 t	 l	 1	 1	 1	 1
Lyophilized 0.03 0.05 0.07 0.1	 0.2 0.4	 0.6	 0.8	 0.9 1.0
WATER ACTIVITY (aw)
FIGURE 3. INFLUENCE OF SPORE MOISTURE CONTENT ON THE Z V VALUE OF
BACILLUS SUBTILIS VAR. NIGER SPORES SUSPENDED IN WATER
OR LOCATED ON GLASS STRIPS EMBEDDED IN EPDXY PLASTIC.


Ecology and thermal inactivation of microbes in and on interplanetary space vehicle components Quarterly progress report, Jul. 1 - Sep. 30, 1968

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