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Method for the Determination or Rates of Spore Inactivation at Ultra-High Temperatures1
TECHNICAL
NOTES
METHOD FOR THE DETERMINATION INACTIVATION AT ULTI~A-HI~H I n a procedure for determining the rates of destruction of heat-resistant bacterial spores at temperatures between 119-130 C in 3:1 whole milk concentrate or 0.067 ~ phosphate buffer, thermal inactivation tubes (Figure 1, 6 mm by 100 ram) were constructed from cleaned pyrex glass tubing. ]~aeh tube was sealed and constricted in an oxygen flame about 15 mm from one end, to facilitate opening the tube and removing its contents at the time of subeulturing. Sterilized tubes were filled aseptically by means
OR R A T E S O F S P O R E TEMPERATURES 1
of an automatic syringe adjusted to deliver 0.5 ml of the spore-inoculated medium. After filling, the remaining open end of each tube was heated in an oxygen flame until sealed. The spore inoeulmn, sufficient to provide 106 spores per milliliter, was uniformly dispersed by mixing for 20 rain with a magnetic stirring device. Incorporation of air in the milk concentrate was reduced by mixing the inoeulum in a partial vacuum. To prevent premature spore germination, filled tubes were held in an ice bath until they were heated. Published with the approval of the Director of F o u r sealed tubes, containing the inoculated the Wisconsin Agricultural Experiment Station. suspending medium, were placed in a coiled This work was supported in part by u grant from steel wire holder and immersed in a constant the U. S. Steel Corporation. temperature oil bath at 100 C. After the contents of the tubes had reached bath temperature~ they were transferred quickly to a second constant4emperature oil bath (American Instrument Company, Inc., Silver Spring, Maryland) capable of maintaining a constant temperature up to 150 C, with a maximum variation of not more than 0.06 C according to the manufacturer. At designated intervals, the tubes were removed from the bath and cooled rapidly I!. J in an ice bath. To provide satisfactory replication, a second set of four tubes was heated similarly for each time-temperature studied. To determine the number of surviving spores, the unopened tubes were rinsed in 95% ethanol to remove the oily heating medium film, then rinsed in distilled water. Each tube was scratched with a triangular file about 15 mm from the top and also from the bottom of the tube. The tube was sterilized in 70% ethanol and dried with a sterile paper towel before the top of the tube was snapped off and discarded. The contents of the vial were transferred aseptically to a 20-ml screw-cap tube by snapping off the bottom of the pyrex ampule and dropping it and the vial into 4.5 ml of buffered distilled water (2). The 1:10 diluted sample was shaken thoroughly and 4 ml were introduced in 1-ml portions into four sterile petri dishes; or the original sample was diluted further according to the expected number of survivors. About 25 ml of spore recovery medium were poured into each plate, mixed thoroughly, and II allowed to solidify. The incubation tinms and temperatures for recovery of the viable survlving spores were chosen to obtain maximmn colony counts. According to the preceding method, the number of survivors at each hold.!t r; r 1 ~~ ing time and at three different lethal bath temperatures represented the average count of 32 plates. Survivor curves show that it is theoretically PIG. 1. ~Ietal holder, A; heat-penetration tube, B; Thermistor Probe, C; and pyrex thermal in- impossible to attain complete destruction, because the number of smwivors never reaches activation tube, D. 1392
t-----A
D
II
i
i
I
W
TECHNICAL, ~NOTES
zero. Therefore, the inactivation time for each of the three bath temperatures arbitrarily was assumed to be the time to reduce the spore population by six decimal reduction times or six D-values. Thermal death time curves were established by plotting the inactivation times on the logarithmic axis against the corresponding lethal bath temperature on the arithmetic axis on semilog paper. Thus, thermal death time curves indicated the absolute heating time to reduce the spore concentration from a million spores per milliliter to one viable spore per milliliter, irrespective of the come-up time and cooling lag. The rate of heat transfer between the 3:1 milk concentrate or phosphate buffer at 100 C and the oil bath at 119.2 C was determined by means of the Thermistor Probe (Yellow Springs Instrument Company, Inc., Yellow Springs, Ohio) shown in Figure 1. The probe, 22-gauge and 3 inches in length, was inserted into the heat penetration tube (6 mm O.D. by 85 ram) that was sealed only at one end. The probe was connected to a Yellow Springs Instrument Company single-channel Telethermometer and Model 80 Laborato~T Recorder. This temperature-registering equipment provided a sensitive indicating and recording system in the temperature range of 100-150 C. Heat penetration curves (Figure 2) show the rates of heat exchange between two grades of propylene glycol used in the constant-temperature baths and the two spore-suspending menstrua. One of the glycols, designated lowtemperature glycol, has a flash point of 163 C and a fire point of 190 C; the other glycol, referred to as high-temperature glycol, has a flash point of 260 C and a fire point of 315 C. The latter heating medium was more desirable for thermal inactivation tests, because it was not volatile at temperatures above 100 C. The lag factor (j) and the slope of heating curves (f~), presented in Table 1, were calculated according to the procedure given by Ball and Olson (1). The lag factor indicates the time before the temperature differential between the heating medium and the material being heated assume straight-line heating characteristics; whereas, the symbol f~ refers to the time for
1393
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l-
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i4 t'--
~,3,.,~_
i78,
~E h=
236.E
I0
°
I..226.6F-
P,Jg/
"120
30
140
of ~
156.6
,b ,~s 2~ 21s 3'0 /5 ,;o
t,oo 8O
TIME IN SECONDS
FIG. 2. tCates of heat. transfer in vial of phosphate buffer and 3:1 whole milk concentrate in low-temperature glycol, shown by open and closed circles, respectively, and in high-temperature glycol, shown by open and closed triangles (bath temperature = 319.2 C or 246.6 F). the temperature of the materiM being heated to increase tenfold. W. P. SEGNER W. C. FRAZIER AND It. E. CALBERT Departments of Dairy and Food Industries and Bacteriology University of Wisconsin, Madison REFERENCES (1) BAhia, C. O., A.~TDOLSON, F. C. W. Sterilization in Food Technology. McGraw-Hill Book Co., Inc., New York. 1957. ( 2 ) STA~q~DAR~D M]iiTHODS FO~ TI:IFJ EXA~[INATION OF DATg¥ PRODUCTS. ] l t h ed. American Public HcMth Association, New York. 1960.
TABLE i Heat penetration factors in 3:1 milk concentrate and 1:15 ~ phosphate buffer using high temperature and low temperature polyalkylene glycol (bath temperature----119.2 C) Time in seconds Lag factor (j)
Heating curve slope (fh)
Heating medium
Concentrate
Buffer
Concentrate
Buffer
Low-temp. glycol High-temp. glycol
1.59 1.97
1.29 1.73
22.0 22.9
21.2 22.3
NOTES
METHOD FOR THE DETERMINATION INACTIVATION AT ULTI~A-HI~H I n a procedure for determining the rates of destruction of heat-resistant bacterial spores at temperatures between 119-130 C in 3:1 whole milk concentrate or 0.067 ~ phosphate buffer, thermal inactivation tubes (Figure 1, 6 mm by 100 ram) were constructed from cleaned pyrex glass tubing. ]~aeh tube was sealed and constricted in an oxygen flame about 15 mm from one end, to facilitate opening the tube and removing its contents at the time of subeulturing. Sterilized tubes were filled aseptically by means
OR R A T E S O F S P O R E TEMPERATURES 1
of an automatic syringe adjusted to deliver 0.5 ml of the spore-inoculated medium. After filling, the remaining open end of each tube was heated in an oxygen flame until sealed. The spore inoeulmn, sufficient to provide 106 spores per milliliter, was uniformly dispersed by mixing for 20 rain with a magnetic stirring device. Incorporation of air in the milk concentrate was reduced by mixing the inoeulum in a partial vacuum. To prevent premature spore germination, filled tubes were held in an ice bath until they were heated. Published with the approval of the Director of F o u r sealed tubes, containing the inoculated the Wisconsin Agricultural Experiment Station. suspending medium, were placed in a coiled This work was supported in part by u grant from steel wire holder and immersed in a constant the U. S. Steel Corporation. temperature oil bath at 100 C. After the contents of the tubes had reached bath temperature~ they were transferred quickly to a second constant4emperature oil bath (American Instrument Company, Inc., Silver Spring, Maryland) capable of maintaining a constant temperature up to 150 C, with a maximum variation of not more than 0.06 C according to the manufacturer. At designated intervals, the tubes were removed from the bath and cooled rapidly I!. J in an ice bath. To provide satisfactory replication, a second set of four tubes was heated similarly for each time-temperature studied. To determine the number of surviving spores, the unopened tubes were rinsed in 95% ethanol to remove the oily heating medium film, then rinsed in distilled water. Each tube was scratched with a triangular file about 15 mm from the top and also from the bottom of the tube. The tube was sterilized in 70% ethanol and dried with a sterile paper towel before the top of the tube was snapped off and discarded. The contents of the vial were transferred aseptically to a 20-ml screw-cap tube by snapping off the bottom of the pyrex ampule and dropping it and the vial into 4.5 ml of buffered distilled water (2). The 1:10 diluted sample was shaken thoroughly and 4 ml were introduced in 1-ml portions into four sterile petri dishes; or the original sample was diluted further according to the expected number of survivors. About 25 ml of spore recovery medium were poured into each plate, mixed thoroughly, and II allowed to solidify. The incubation tinms and temperatures for recovery of the viable survlving spores were chosen to obtain maximmn colony counts. According to the preceding method, the number of survivors at each hold.!t r; r 1 ~~ ing time and at three different lethal bath temperatures represented the average count of 32 plates. Survivor curves show that it is theoretically PIG. 1. ~Ietal holder, A; heat-penetration tube, B; Thermistor Probe, C; and pyrex thermal in- impossible to attain complete destruction, because the number of smwivors never reaches activation tube, D. 1392
t-----A
D
II
i
i
I
W
TECHNICAL, ~NOTES
zero. Therefore, the inactivation time for each of the three bath temperatures arbitrarily was assumed to be the time to reduce the spore population by six decimal reduction times or six D-values. Thermal death time curves were established by plotting the inactivation times on the logarithmic axis against the corresponding lethal bath temperature on the arithmetic axis on semilog paper. Thus, thermal death time curves indicated the absolute heating time to reduce the spore concentration from a million spores per milliliter to one viable spore per milliliter, irrespective of the come-up time and cooling lag. The rate of heat transfer between the 3:1 milk concentrate or phosphate buffer at 100 C and the oil bath at 119.2 C was determined by means of the Thermistor Probe (Yellow Springs Instrument Company, Inc., Yellow Springs, Ohio) shown in Figure 1. The probe, 22-gauge and 3 inches in length, was inserted into the heat penetration tube (6 mm O.D. by 85 ram) that was sealed only at one end. The probe was connected to a Yellow Springs Instrument Company single-channel Telethermometer and Model 80 Laborato~T Recorder. This temperature-registering equipment provided a sensitive indicating and recording system in the temperature range of 100-150 C. Heat penetration curves (Figure 2) show the rates of heat exchange between two grades of propylene glycol used in the constant-temperature baths and the two spore-suspending menstrua. One of the glycols, designated lowtemperature glycol, has a flash point of 163 C and a fire point of 190 C; the other glycol, referred to as high-temperature glycol, has a flash point of 260 C and a fire point of 315 C. The latter heating medium was more desirable for thermal inactivation tests, because it was not volatile at temperatures above 100 C. The lag factor (j) and the slope of heating curves (f~), presented in Table 1, were calculated according to the procedure given by Ball and Olson (1). The lag factor indicates the time before the temperature differential between the heating medium and the material being heated assume straight-line heating characteristics; whereas, the symbol f~ refers to the time for
1393
,,,P2,~4.e
l?- ===
l-
:z4z.e
i4 t'--
~,3,.,~_
i78,
~E h=
236.E
I0
°
I..226.6F-
P,Jg/
"120
30
140
of ~
156.6
,b ,~s 2~ 21s 3'0 /5 ,;o
t,oo 8O
TIME IN SECONDS
FIG. 2. tCates of heat. transfer in vial of phosphate buffer and 3:1 whole milk concentrate in low-temperature glycol, shown by open and closed circles, respectively, and in high-temperature glycol, shown by open and closed triangles (bath temperature = 319.2 C or 246.6 F). the temperature of the materiM being heated to increase tenfold. W. P. SEGNER W. C. FRAZIER AND It. E. CALBERT Departments of Dairy and Food Industries and Bacteriology University of Wisconsin, Madison REFERENCES (1) BAhia, C. O., A.~TDOLSON, F. C. W. Sterilization in Food Technology. McGraw-Hill Book Co., Inc., New York. 1957. ( 2 ) STA~q~DAR~D M]iiTHODS FO~ TI:IFJ EXA~[INATION OF DATg¥ PRODUCTS. ] l t h ed. American Public HcMth Association, New York. 1960.
TABLE i Heat penetration factors in 3:1 milk concentrate and 1:15 ~ phosphate buffer using high temperature and low temperature polyalkylene glycol (bath temperature----119.2 C) Time in seconds Lag factor (j)
Heating curve slope (fh)
Heating medium
Concentrate
Buffer
Concentrate
Buffer
Low-temp. glycol High-temp. glycol
1.59 1.97
1.29 1.73
22.0 22.9
21.2 22.3