HEAT ACTIVATION INDUCING GERMINATION IN THE SPORES OF THERMOTOLERANT AND THERMOPHILIC AEROBIC BACTERIA HAROLD R. CURRAN AND FRED R. EVANS Dairy Research Laboratories, Bureau of Dairy Industry, Agricultural Research Administration, U. S. Department of Agriculture Received for publication October 27, 1944 Relatively mild heating of the spores of mesophilic aerobes has been shown to hasten their subsequent germination (Evans and Curran, 1943). Although effective in bringing about a more rapid germination, with one exception, such heating had no measurable influence upon the number of spores that germinated. This tangible evidence of pregermination stimulation led us to extend our obser- vations to a group of thermotolerant aerobes isolated from commercially canned evaporated milk that had spoiled. With nearly all the latter types, preheating was found to exert a determining influence upon the number of spores that germinated. This phenomenon, which must be regarded as true heat activation, has not been reported previously for bacterial forms, although evidence of its operation may be found in the publications of Mudge and Thorwaldsen (1930) and Christian (1931), both of which dealt with obscure milk defects in which heat affected the development of sporeforming organisms. Seeking to explain observed qualitative and quantitative changes in the flora of pasteurized milk, Mudge and Thorwaldsen formulated an interesting hypothesis which assumes for certain thermophiles a complex life cycle involving both visible and invisible spore forms. The latter, normally dormant, might be induced to germinate by physical and chemical agencies, including heat. Christian believed that heat- ing promoted the development of a "coconut" or "carbolic" taint in commercial sterilized milk by destroying a product of vegetative activity inhibitory to the germination of the spores. Some light is shed upon the heat activation reaction by experiments reported in this paper. The relationship between the amount of pregermination heat and the degree of activation, and certain factors exclusive of pregermination heat which affect the heat response are given especial consideration. METHODS AND MATERIALS Observations were made on the following organisms: 15u, 4149, 6 (American Can Company); CON ("A," Continental Can Company); 9499 (National Can- ners Association); G1 and H2 (Whitehouse Evaporated Milk Company); LB (Bureau of Dairy Industry); Bacillus coagulans (Iowa State College), a thermo- tolerant aerobe isolated from spoilage in commercially processed evaporated milk; Bacillus calidolactis, 2 strains, one from Iowa State College and 1518 from the National Canners Association, both obligate thermophilic aerobes recovered from spoiled evaporated milk and corn, respectively; Bacillus subtilis 6051 and 6634 (American Type Culture Collection); and 3679 (American Can Company), 335 on March 25, 2020 by guest http://jb.asm.org/ Downloaded from
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HEAT ACTIVATION INDUCING GERMINATION IN THE SPORES OFTHERMOTOLERANT AND THERMOPHILIC AEROBIC BACTERIA
HAROLD R. CURRAN AND FRED R. EVANS
Dairy Research Laboratories, Bureau of Dairy Industry, Agricultural ResearchAdministration, U. S. Department of Agriculture
Received for publication October 27, 1944
Relatively mild heating of the spores of mesophilic aerobes has been shownto hasten their subsequent germination (Evans and Curran, 1943). Althougheffective in bringing about a more rapid germination, with one exception, suchheating had no measurable influence upon the number of spores that germinated.This tangible evidence of pregermination stimulation led us to extend our obser-vations to a group of thermotolerant aerobes isolated from commercially cannedevaporated milk that had spoiled. With nearly all the latter types, preheatingwas found to exert a determining influence upon the number of spores thatgerminated. This phenomenon, which must be regarded as true heat activation,has not been reported previously for bacterial forms, although evidence of itsoperation may be found in the publications of Mudge and Thorwaldsen (1930)and Christian (1931), both of which dealt with obscure milk defects in whichheat affected the development of sporeforming organisms. Seeking to explainobserved qualitative and quantitative changes in the flora of pasteurized milk,Mudge and Thorwaldsen formulated an interesting hypothesis which assumesfor certain thermophiles a complex life cycle involving both visible and invisiblespore forms. The latter, normally dormant, might be induced to germinate byphysical and chemical agencies, including heat. Christian believed that heat-ing promoted the development of a "coconut" or "carbolic" taint in commercialsterilized milk by destroying a product of vegetative activity inhibitory to thegermination of the spores.Some light is shed upon the heat activation reaction by experiments reported
in this paper. The relationship between the amount of pregermination heatand the degree of activation, and certain factors exclusive of pregerminationheat which affect the heat response are given especial consideration.
METHODS AND MATERIALS
Observations were made on the following organisms: 15u, 4149, 6 (AmericanCan Company); CON ("A," Continental Can Company); 9499 (National Can-ners Association); G1 and H2 (Whitehouse Evaporated Milk Company); LB(Bureau of Dairy Industry); Bacillus coagulans (Iowa State College), a thermo-tolerant aerobe isolated from spoilage in commercially processed evaporatedmilk; Bacillus calidolactis, 2 strains, one from Iowa State College and 1518 fromthe National Canners Association, both obligate thermophilic aerobes recoveredfrom spoiled evaporated milk and corn, respectively; Bacillus subtilis 6051 and6634 (American Type Culture Collection); and 3679 (American Can Company),
a putrefactive mesophilic anaerobe. Cultures 6, 15u, 4149, and LB were subse-quently examined by Doctor Ruth Gordon of the staff of the American TypeCulture Collection and provisionally identified as Bacillus subtilis.
Plain nutrient agar slopes (pH 7.0) were used as mediums in the productionof the spores unless otherwise stated. The incubation temperature was 37 C,except for 1518 which usually was 55 C. When sporulation was complete (2 to3 weeks' incubation) the growth was washed into sterile distilled water, filteredthrough cotton, and centrifuged. The water was decanted and the washingprocess repeated 3 times. The washed spores were then filtered through thecotton plunger tube as described by Morrison and Rettger (1930), which effec-tively removed most of the clumps. The concentrated stock suspensions thusprepared, practically 100 per cent spores (except 1518), were plated to determinepurity and count, and were then held at 6 C until used.The nutrient agar (pH 7.0) used as a plating medium contained, per liter:
peptone (Difco) 5 g, beef extract (Difco) 3 g, sodium chloride 5 g, glucose 3 g,and agar 13 g.The general plan of the experiments was as follows: small aliquot portions of
the stock suspensions diluted in distilled water were seeded into 8-ml quantitiesof the test medium contained in pyrex tubes. After thorough mixing, selectedtubes were set aside to serve as controls, and the remainder were heated in a95 C bath, usually for 10 minutes. The heating was accomplished in a stirredglycerol bath equipped with a thermoregulator which maintained the desiredtemperature at 4± 0.5 C. The heated and unheated control samples were thenplated on glucose agar of the composition noted above. Routinely, plates werecounted after 48 hours of incubation, and at regular short intervals countedplates were returned to the incubator and re-examined after 4 additional daysof incubation. When low subeultivation temperatures were used, the incubationperiods were necessarily exteinded, as indicated in the tables or text.
EXPERIMENTAL
Activation of spores in relation to the anount of heat applied. The proportionof spores that respond to pregermination heating is dependent upon the amountof heat applied. This is revealed in the following experiment in which a suspen-sion of spores was subjected to different periods of heating at constant tempera-tures. WVhen the spores were uniformly dispersed in sterile skim milk platedboth immediately and after varying periods of heating at 95 C, the effect ofheating was to increase materially the number of colonies which developed.The results obtained N-ith 3 members of the spoilage group are shown in figure 1.The incubation temperature of the plates was 37 C. The colony count increasedwith the time of heating rather rapidly during the first few minutes, then moreslowly until a maximum was reached, usually betweeni 15 and 30 minutes; longerheating reduced the number of colonies. It is clear that, under these conditionsof production, treatment, and subeultivation, germination of the spores is afunction of the amount of heat to which they have been exposed. Without someprelheatiuig only a small proportion of the potentially viable spores were able to
HEAT ACTIVATING GERMINATION OF AEROBIC BACTERIA SPORES
germinate; prolonging the incubation time, varying the nutritional character ofthe plating medium, and other measures, except temperature elevation, had noappreciable influence upon the number of colonies formed. The rising slopeof the curve shows that the heat activation threshold differs with the individualspores in a culture.
Activation of spores as influenced by the nature of the heating medium and bythe temperature of subcultivation. The germination response following the heat-ing of spores in a 95 C bath for 10 minutes in each of 9 different mediums isshown in table 1. The results obtained at different temperatures of subcultiva-tion reveal the importance of this factor in determining the magnitude of theheat response. The figures within the parentheses indicate the ratio of thecount to that of the unheated control. With subcultivation at 37 C, preheatingincreased the count of the thermotolerant species (number of spores which
0
0
aI
z0J
0
'20,
00 20 30 40 45MINUTE5, IN SATH AT 95 C
FIG. 1. THE GERMINATION OF SPORES BEFORE AND AFTER VARYING PERIODS OF MILDHEATING IN SKIM MILK
germinated) in all the heating mediums; but activation was especially pro-nounced in the presence of glucose, lactose, peptone, and milk. Glucose is fer-mented by these organisms; lactose, not vigorously attacked, promotes heatactivation to about the same degree as glucose. Salt depressed heat activa-tion whereas beef extract enhanced it moderately, as did agar alone. Theresults obtained in the complete agar and broth mediums reflect the balancinginfluence of components which both enhance and depress heat activation. Sub-cultivation at 48 C increased the count over that at 37 C of both the unheatedspores and those heated in water, beef extract, and salt solution, but it had littleor no effect in the presence of glucose, lactose, or milk. Less extensive datafor the obligate thermophile (1518) show similar relationships. High tempera-tures of subcultivation serve to equalize the count at high levels by inducing asecondary activation of the spores in the subculture medium, a reaction whichdoes not occur at 37 C or below. The test substances have no measurableeffect upon the count in the absence of preheating or high temperature incuba-tion. In table 2 is shown the heat activation reaction in skim milk with sub-
HEAT ACTIVATING GERMINATION OF AEROBIC BACTERIA SPORES
cultivation of the spores at temperatures down to 14 C. When the subcultiva-tion temperatures were below 30 C, a preliminary count was regarded as finalif incubation for an additional week at the same temperature resulted in no newcolonies. These data reveal the important fact that preheating insures a highpercentage of germination at very low temperatures. In the heated samplescolonies appeared earlier and developed more rapidly than those from unheated
TABLE 2The germination of the spores of thermotolerant aerobes after sublethal heating as influenced
by the temperature of subcultivation
TREATMENT OF SPORESORGANISM TEMPERATURE OFORGANISM STBCULTIVATION Heated at 95 C for 10
Heating medium, sterile skim milk.37 C plates counted after 3 and 7 days' incubation.30 C plates counted after 3 and 7 days' incubation.25 C plates counted after 2 and 3 weeks' incubation.20 C plates counted after 3 and 4 weeks' incubation.14 C plates counted after 4 and 5 weeks' incubation.
samples; this lowering of the temperature of germination for the majority ofspores is associated with mild heating involving no measurable lethal action.Of considerable practical significance would be experimental evidence to prove
that the relatively few spores which survive drastic heating react similarly todifferent subcultivation temperatures. This evidence is provided in table 3,which shows that the overwhelming majority of spores which survive severeheating germinate at lower temperatures than do unheated spores. Graphicrepresentation of these data in terms of the percentage of germination emphasizesthis fact (figure 2). It should be noted, however, that at the lowest temperatureof subcultivation (14 C) none of the spores that survived autoclaving were able
to develop whereas a small proportion of the unheated spores could. Althougha selective elimination of spores which germinate at the higher temperatures isnot excluded, the reaction appears more likely to be a further manifestation ofheat activation. A recent paper of Williams and Reed (1942) has a bearing onthis subject. These authors recorded greater thermal death times for spores ofClostridium botulinum types A and B and of an unidentified putrefactive anaerobe(3679) when the recovery cultures were incubated at 24 and 27 C than whenincubated at 31 or 37 C, although the optimal growth temperature of theseorganisms is in the 35 to 37 C range. Working with the spores of the same putre-factive anaerobe (3679), we obtained some evidence for heat activation in thisspecies, but the results were deemed inconclusive. (The special Brewer petri
TABLE 3The germination of the spores of thermotolerant aerobes after severe heating in skim milk as
influenced by the temperature of subcultivation
TREATMENT OF SPORES
ORGANISM TEMPRCTVATUREONSUBCULTIVATNotIeated Heated at 120 C; autoclavedSIJBCULTIVATIONNot heated for 10 minutes
dish cover and B-B-L anaerobic agar were used.) The net result of preheating,provided it is adequate, is to lower the temperature at which germination of themajority of spores can occur. It would appear that these spore populationsare divisible into several classes or groups depending on their minimal tempera-tures for germination-the largest, with a relatively high temperature minimum,is susceptible to heat activation, which enables the spores to germinate at sub-minimal temperatures.
Table 4 shows the effect of different concentrations of glucose and of salt uponthe heat activation of spores; for each of the 3 cultures, glucose in 0.1 per centconcentration was most effective in promoting heat activation. The depressingaction of salt upon heat activation is evident in the higher concentrations.
The temperature at which spores are formed in relation to heat activation. Inthis experiment spores of 15u, 4149, and LB, produced at 30, 37, 45, and 52 C
HEAT ACTIVATING GERMINATION OF AEROBIC BACTERIA SPORES
were heated in milk at 95 C for 10 minutes, after which they were subcultivatedon plates at 37 and 48 C. Spores of the obligate thermophile (1518) producedat 45, 50, 55, and 65 C were similarly heated, but subcultivated at 45, 55, and61 C. The data are shown in table 5. In three of the cultures (15u and LB, and1518), activation was greatest at 45 and 55 C, respectively-temperatures whichapproximate the optimal for growth. With 4149, spores produced at suboptimaltemperatures were most responsive to heat. As in the experiment which in-
5 30 2° O20 I 5° I5Q. 45" 400 350 30* Z5o lo1*
TEMPERgATURE - C.FIG. 2. THE PERCENTAGE GERMINATION OF SPORES AT DIFFERENT TEMPERATURES BEFORE
AND AFTER AUTOCLAVING (120 C FOR 10 MINUTES) IN SKIM MILK
volved the different heating mediums (table 1), the higher temperatures of sub-cultivation effect a smoothing out of count differences, both between heated andunheated samples and among the heated samples incubated at different tem-peratures. The probable explanation for this is that heat at 48 C, but not at37 C (temperature of subcultivation), maintained for 48 hours (incubation time),is itself sufficient to activate many of the spores-a reaction which, as has beenshown, would be promoted by the peptone and glucose constituents of the medium.Similarly some activation of spores must also occur when they are formed athigh temperatures. At 52 C spore production in 15u, 4149, and LB was much
reduced over that of 37 and 45 C. Although lower in their susceptibility to heatactivation than those produced at lower temperatures, they and the spores(1518) produced at 65 C do nevertheless respond in some degree to preheating.
TABLE 4The germination of the spores of thermotolerant aerobes after sublethal heating as infduenced by
the concentration of glucose and of salt in the heating medium
TABLE 5The germination of the spores of the thermotolerant and thermophilic aerobes after sublethal
heating* as influenced by the temperature at which the spores were formedTEMPERATURE AT WHICH SPORES WERE PRODUCED
No heat
per ml
000
215521
2144
2094
30 C
Heated*
per ml 000
1,390 (6X)1,940 (3X)
314 (14X)435 (9X)
250 (12X)330 (3X)
45 C
5.4 46 (8X)18.3 55 (3X)24.4 51 (2X)
No heat
per ml
000
47170
3252
39118
37 C
Heated*
per ml 000
211 (4X)330 (1X)
4-00 (12X)457 (8X)
410 (1OX)430 (3X)
No heat
per ml
000
2187
2454
2250
45 C
Heated
per ml 000
81 (3X)97 (1X)
53 C
No heat
per ml000
0.210.34
963 (40X) 0.09964 (17X) 0.15
360 (16X) 1.88310 (6X) 2.27
50 C 55 C
4.219.827.9
* 95 C for 10 minutes in skim milk.
56 (13X)56 (2X)66 (2X)
4.9 71 (14X)17.9 67 (3X)24.2 74 (3X)
Heated*
per ml 000
0.43 (2X)0.52 (1X)
0.62 (6X)0.66 (4X)
4.76 (2X)4.67 (2X)
65 C
5.20 17.4 (3X)18.00 22.7 (IX)26.40 33.0 (1X)
Pasteurization in relation to heat activation. Spores of 15u, 4149, and LB wereseeded into sterile skim milk, heated at 62 C for 30 minutes, then subcultivatedin the usual way. With subcultivation at 37 C (table 6) the count was increasedat least 50 per cent with the expected lower response accompanying the highertemperature of subcultivation,
HEAT ACTIVATING GERMINATION OF AEROBIC BACTERIA SPORES
Persistence of heat activation. Tests with these cultures have shown that sporesactivated by heating remain in the activated condition for a period of months.This suggests that the heat-induced change is irreversible, which would distin-guish it from a somewhat similar reaction in the ascospores of certain fungi
TABLE 6Pasteurization in relation to heat activation in spores*
TREATMENT OF SPORES
ORGANISM TEMPERATURE OFORGANISM SUTBCULTIVATION Heated at 62 C for 30Not heated minutest
C per ml per ml
15u 37 11,200 22,00015u 48 26,000 34,000
4149 37 6,800 20,0004149 48 25,000 32,000
LB 37 10,100 21,000LB 48 30,000 41,000
* Spores produced at 37 C.t Heating medium, skim milk.
TABLE 7The germination* of the sporest of two strains of Bacillus subtilis after sublethal heating in
skim milk, as influenced by the temperature of subeultivation
TREATMENT OF SPORES
ORGANISM TIEMPERATURE. OPFORGANISMI SUBCULTIVATION Heated at 85 C for 10
37 C plates counted after 3 and 7 days' incubation.20 C plates counted after 1 and 2 weeks' incubation.14 C plates counted after 3 and 4 weeks' incubation.
t Produced at 37 C.
described by Shear and Dodge (1927) and by Goddard (1935). The latter foundthat spores became deactivated by conditions which prevented germination,necessitating a second heating, following which they germinated normally.
Sublethal heating was previously shown to accelerate the germination of cer-tain mesophiles (Evans and Curran, 1943). With one exception, heat activationwas not observed. Amonig the organisms studied were 2 strains of B. subtilis,
6634 and 6051, which grew readily on agar slopes at 55 C. Because of theirtolerance to heat, the heat response of the spores of these cultures was retested,new crops being employed with subcultivation at 37 C, as formerly, and alsoat 20 and 14 C. The results are shown in table 7. Pregermination heating ofthe spores of 6051 had little influence upon the plate count at any of the threetemperatures of incubation. Subeultivation at 37 C after preheating had noappreciable influence upon the number of spores of 6634 which germinated, con-firming our previous observation. When, however, lower temperatures of sub-cultivation were used, the activating effect of heating became evident. It isnotewvorthy that at 14 C with the benefit of preheating, only 22 per cent of theviable spores were able to germinate; without preheating about 2.2 per cent wereable similarly to develop. Thus heat activation may be operative in a cultureand yet escape detection if the conditions of subeultivation are not suffi-ciently wide.
DISCUSSION
That heat activation is a factor contributing to the spoilage of canned foodsof certain types can hardly be doubted. Of 12 spore forms recovered from 10different outbreaks of spoilage in commercially processed evaporated milk, atleast 10 were subject to some degree of heat activation. These include B. coagu-lans and B. calidolactis, well-known milk-spoilage types. With broader condi-tions of observation, it is not unlikely that all could be shown to respond. Flatsour types (Bacillus stearothermophilis) are heat-activatable.Heat activation may be regarded as an adaptation by which a spore popula-
tion is able to attain a high-percentage germination at suboptimal temperatures-temperatures which, in the absence of heating, permit the germination of only asmall proportion of the total viable spores; with progressive lowering of thetemperature of subcultivation a temperature range is ultimately reached atwhich germination of part of the heated and no germination of the unheatedspores occur. Heat activation seems to be a property primarily of thermotol-erant and thermophilic species that possess unusually high thermal resistance;spores of such organisms, because of the heat treatment that they have sur-vived, are preconditioned to development at low temperatures, which greatlyincreases their spoilage potential. Where thermotolerant species are con-cerned, heat activation operates to bring the temperature of germination of themajority of the spores within the usual storage and distribution temperaturerange of canned foods.Heat activation has a bearing upon certain commercial or laboratory practices,
of which two may be mentioned. It is a favored and long-established practiceamong bacteriologists to apply sublethal heating as a means of eliminatingvegetative cells from test spore suspensions prior to their use; the attendantchange in the germinative capacity of the spores has not previously been sus-pected, and, whenever such heating has been employed prior to plating, theoperator unwittingly increased the accuracy of the count. In the manufactureof evaporated milk, it is a routine practice to forewarm the raw milk at 95 C for10 minutes prior to its condensation. This is necessary for the attainment of a
HEAT ACTIVATING GERMINATION OF AEROBIC BACTERIA SPORES
satisfactory body in the finished product. As a result of such heating, themajority of the spores of thermotolerant species become heat labile during theshort period the milk is undergoing condensation (unpublished observation),and this large proportion, in consequence, is easily killed by the subsequentprocessing treatment. This provides an example in which an established com-mercial practice quite fortuitously conduces to a desired result.
Recognition of the phenomenon of heat activation and some understandingof the factors which affect it are of manifest importance to the accurate enumera-tion of viable sporing thermotolerant or thermophilic aerobes. This may alsobe true for some anaerobes but our observations on the latter are not sufficientto warrant a definite conclusion.
SUMIMARY
Sublethal heating of the spores of many thermotolerant and thermophilicaerobes has a determining influence upon the number of spores which will ger-minate subsequently.A study of 12 thermotolerant cultures isolated from 10 different outbreaks of
spoilage in commercially processed evaporated milk revealed that nearly allwould respond to pregermination heating. In the absence of such treatment alarge proportion of the potentially viable spores did not germinate. Flat sourtypes (Bacillus stearothermophilus) were found to be heat-activatable.The proportion of spores wsThich responded to preheating was found to be
dependent upon the amount of heat, the composition of the medium in whichthey were heated, the temperature at which the spores were formed, and thetemperature at which they were subcultured.The heating mediums arranged in the order of their effectiveness upon heat
activation were: glucose or lactose (0.5 per cent) > peptone (0.5 per cent) >skim milk > glucose nutrient agar > beef extract (0.3 per cent) > glucosenutrient broth > distilled water > NaCl (0.5 per cent).The concentration of glucose most favorable to heat activation was approxi-
mately 0.1 per cent. NaCl usually depressed heat activation in concentrationsof 0.5 per cent and higher.At 95 C in skim milk the heat-activating process was nearly complete in 10
minutes, entirely so in 30 minutes. Long-hold pasteurization effects materialactivation of spores.The response to preheating is greater when the temperature of subeultivation
is suboptimal than when it is at the relatively high optimal temperatures.Preheating of heat-activatable spores serves to lower the minimal temperature
at which germination can occur; this applies when the heat treatment is mildand nonlethal and also when it is so drastic that relatively few spores survive.The economic implications of this are discussed.
Recognition of the phenomenon of heat activation and some understandingof the factors which affect it are essential to the accurate enumeration of viable,sporing, thermotolerant and thermophilic aerobes. The relationship of heatactivation to certain commercial or laboratory practices is indicated.
CHRISTIAN, AM. I. 1931 A contribution to the bacteriology of commercial sterilized milk.Part II. The coconut or carbolic taint. A study of the causal organism and the factorsgoverning its spore-formation. J. Dairy Research, 3, 113-132.
EVANS, F. R., AND CURRAN, H. R. 1943 The accelerating effect of sublethal heat on sporegermination in mesophilic aerobic bacteria. J. Bact., 46, 513-523.
GODDARD, D. R. 1935 The reversible heat activation inducing germination and increasedrespiration in the ascospores of Neurospora tetrasperma. J. Gen. Physiol., 19, 45-60.
MORRISON, E. W., AND RETTGER, L. F. 1930 Bacterial spores. I. A study in heat resist-ance and dormancy. J. Bact., 20, 301.
MUDGE, C. S., AND THORWALDSEN, M. L. 1930 Tbermophilic bacteria a problem. Mo.Bull., Dept. Agr. Calif., 19, 710-718.
SHEAR, C. L., AND DODGE, B. 0. 1927 Life histories and heterothallism of the red breadmold fungi of the Monilia setophila group. J. Agr. Research, 34, 1019-1042.
WILLIAMS, 0. B., AND REED, J. M. 1942 The significance of the incubation temperatureof recovery cultures in determining spore resistance to heat. J. Infectious Diseases71, 225-227.
