APPLIED MICROBIOLOGY, Mar., 1967, p. 266-276Copyright © 1967 American Society for Microbiology

Vol. 15, No.2Printed in U.S.A.

Recovery Patterns of Spores of Putrefactive Anaerobe3679 in Various Subculture Media After

Heat Treatment,JORG A. L. AUGUSTIN2 AND I. J. PFLUG

Department ofFood Science, Michigan State University, East Lansing, Michigan

Received for publication 22 August 1966

A comparative study was made of the heat resistance of spores of putrefactiveanaerobe 3679 grown in two different sporulation media and of the recovery pat-tern of these spores in several subculturing media after treatment with moist anddry heat. The heat resistance of the spores was characterized in the form of D andz values. The D values were determined by the modified Schmidt method. The zvalues were established by the graphic method. The results revealed significantdifferences in D and z values, depending on the type of heat and sporulation andsubculture media. Spores grown in beef heart infusion showed higher heat resist-ance than those grown in Trypticase. Among the seven subculture media used,the largest number of spores was recovered in beef infusion. The magnitude of theD values at 121.1 C obtained with spores heated in moist heat decreased, dependingon the subculture medium used, in the following order: beef infusion, pea infusion,yeast extract, liver infusion, Eugonbroth, Trypticase, synthetic medium. Withspores subjected to dry heat, D values at 148.9 C decreased with the subculturemedium in the following order: beef infusion, yeast extract, pea infusion and liverinfusion, Trypticase, Eugonbroth, synthetic medium. The z values obtained withspores subjected to dry heat were approximately double those obtained with moistheat. Their relative magnitude varied slightly, depending on the type of subculturemedium used. However, the relative magnitudes of the D values and z values withreference to the subculture media used were different with moist heat from thoseobtained with dry heat. Two theories are discussed as possible explanations for thelogarithmic order of death of bacterial spores. The results obtained in these experi-ments, together with the findings of other workers, are most compatible with thetheory that heat treatment of spores results in an increased rate of random injuryto the genetic material of the spores.

A number of workers have reported differencesin the numbers of spores of putrefactive anaerobe(PA) 3679 surviving in various subculture mediaafter exposure to moist heat. Frank and Campbell(2), in an effort to find a subculture mediumwhich was more convenient to prepare thanYesair's pork infusion medium, found that thetype of subculture medium used made a con-siderable difference in the decimal reductiontimes (D values) at 121 C (250 F). Wheaton andPratt (10) reported similar findings: survivorcounts were considerably higher in most infusion-type media, as well as in yeast extract, than inthe formulated and dehydrated media. These

1 Journal paper 3911, Michigan Agricultural Experi-ment Station, East Lansing.

2Present address: Armour and Co., Oak Brook, Ill.

studies were limited to moist-heat treatment atonly one temperature.

It was the purpose of this investigation toobtain information regarding the resistance ofspores of PA 3679 to moist and dry heat over awide temperature range. Of specific interest wereanswers to questions regarding the nutritionalrequirements of spores for germination and out-growth after heat treatment, the nature of deathof bacterial spores, and the mechanism that leadsto death of bacterial spores.

MATERIALS AND METHODSPreparation ofspore suspension. Two spore suspen-

sions were prepared from a strain ofPA 3679 obtainedfrom C. F. Schmidt (Continental Can Co., Chicago,Ill.). One suspension was prepared with beef heartinfusion as described by Wheaton and Pratt (10),

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SPORE RECOVERY AFTER HEAT TREATMENT

and the other with a medium recommended by C.Vinton (personal communication), containing 3%Trypticase (BBL), 0.1% yeast extract (BBL), and0.1% ammonium sulfate. After maximal sporulation,as determined by microscopic examination of thespore suspension, the beef heart infusion spores wereseparated from the solid portion of the infusion bypassing the mixture through two layers of cheesecloth. The spores were separated from the broth bycentrifugation in an International centrifuge at 1,500rev/min and were washed in distilled water until thesupernatant fluid appeared relatively clear. To rid thespores of their vegetative sporangia, the spores weresubjected to a treatment in lysozyme and trypsin at45 C as described by Grecz, Anellis, and Schneider(3), but without the application of sonic oscillation.Finally the spores were subjected to further washingsin distilled water and centrifugation until microscopicexamination revealed the spore suspension to be freefrom vegetative cells and debris. The clean sporesuspension was stored in 0.066 M phosphate buffer at4 C. The same cleaning procedure was applied to thespores grown in Trypticase except that the filtrationthrough cheese cloth was omitted.

Subculture media. The following media were usedfor subculturing the heat-treated spores as well as forenumeration of the initial spore concentration.

Beef infusion. Beef infusion, 1,000 ml; peptone(Difco), 5.0 g; Tryptone (Difco), 1.6 g; starch (BBL),1.0 g; K2HPO4, 1.25 g; pH, 7.4.

Pea infusion. Pea infusion, 1,000 ml; Tryptone, 10.0g; K2HPO4, 1.0 g; pH, 7.4.

Liver infusion. Liver infusion, 1,000 ml; peptone,10.0 g; K2HP04, 1.0 g; pH, 7.4.

Yeast extract. Yeast extract (BBL), 10.0 g; starch,soluble (BBL), 1.0 g; K2HPO4, 2.0 g; distilled water,1,000 ml; pH, 7.2.

Trypticase. Trypticase (BBL), 50.0 g; K2HPO4, 2.0g; distilled water, 1,000 ml; pH, 7.2.

Eugonbroth. Eugonbroth (BBL), 15.0 g; distilledwater, 1,000 ml; pH, 7.2.

Synthetic medium. Amino acids (grams per milli-liter): L-arginine, 3.00; L-leucine, 1.50; L-aspartic acid,0.45; L-lysine hydrochloride, 0.85; L-Cysteine, 0.25;DL-methionine, 0.60; glycine, 0.20; DL-phenylalanine,2.00; L-glutamic acid, 0.50; L-proline, 0.45; L-histidine,0.20; DL-threonine, 1.00; DL-serine, 0.25; L-trypto-phan, 0.10; DL-alanine, 0.42; L-tyrosine, 0.40; DL-isoleucine, 0.50; DL-valine, 2.00. Vitamins (milligramsper milliliter): biotin, 0.05; riboflavine, 1.00; folic acid,0.20; pyridoxal * H20, 0.50; niacin, 4.00; thiamine * H20,1.00; DL-calcium pantothenate, 1.00; p-aminobenzoicacid (potassium salt), 0.24. Mineral salts: K2HPO4,1.00 g; NaCl, 2.0 mg; KH2PO4, 1.00 g; FeSO4-2H2O,2.0 mg; MgSO4-7H20, 40.00 mg; MnSO4-H20, 2.0mg. The medium contained 1,000 ml of distilled water,and the pH was 7.2.

Media which were to be used to study the recoveryof heat-treated spores were placed in disposableculture tubes (16 by 125 mm) in amounts of 8 ml pertube. After the tubes were filled and stoppered, theywere sterilized according to the following procedure:infusion-type media, 25 min at 121 C; formulated me-dia, 15 min at 121 C; Eugonbroth, 15 min at 118 C;

synthetic medium, 12 min at 116 C. Immediately aftersterilization, all media were cooled by immersion incold water. Inoculation of the media with sporesoccurred within 12 hr following the preparation of themedia.

Immediately prior to inoculation, the followingadditions were aseptically made to each tube: 0.1 mlof a 10% sodium thioglycolate solution, 0.1 ml of a40% dextrose solution, 0.2 ml of a 4% sodium bicar-bonate solution. Dextrose was sterilized by filtration,and the other two compounds were sterilized by heat.Dextrose was not added to Eugonbroth medium.After inoculation with heat-treated spores, the tubeswere stratified with a 1:1 mixture of presterilizedparaffin-mineral oil.

Media used for the determination of initial sporecounts were prepared by adding 15 g of agar per 1,000ml of the medium. All media were sterilized inamounts of 300 ml in 500-ml Erlenmeyer flasks. Priorto being poured into Prickett tubes containing thespore suspension, 4 ml of a 10% sodium thioglycolatesolution and 8 ml of a 4% sodium bicarbonate solutionwere added to each flask. After solidification of theagar, a small overlayer of the agar medium was addedto each tube.The criteria for growth were colony formation in

the case of agar counts, and gas formation or turbiditydevelopment, or both, with the broth-type media usedfor end point determinations.

All inoculated tubes were stored at 37 C. Thestorage times ranged from 24 to 48 hr for the agarsamples, and 2 weeks after no more positive tubesdeveloped, i.e., approximately a total of 6 weeks, forthe samples containing the broth media.

Enumeration of initial spore count. Comparativespore counts were made to determine the initial sporeconcentration with each subculturing medium. Also,periodic determinations of the initial spore countwere made throughout the period of experimentationwith the yeast extract medium only. These periodicchecks served to note any possible changes in initialspore concentration during storage.

Prior to inoculation, the spore samples were heat-shocked in a boiling-water bath for 8 min and cooledin ice water. After this treatment, the samples werediluted in a 0.85% aqueous saline solution and werethen pipetted into the Prickett tubes in amounts whichwould yield a spore count of approximately 20 to 80per tube.The spores grown in Trypticase-yeast extract

germinated spontaneously in the various culturemedia. Therefore no heat shock treatment was appliedto them prior to inoculation.

Determination of thermal resistance. All sporessubjected to heating were added in amounts of 0.01ml to standard tinplate sample cups (11-mm outsidediameter, 8 mm deep, and 0.2 mm thick) and weredried under vacuum at room temperature.

Moist-heat resistance studies were carried out inthe thermoresistometer described by Pflug (6) forexposure times of less than 3 min, and in miniatureretorts for longer exposure times. All dry-heat studieswere carried out in miniature retorts. A dry-heatatmosphere was achieved by enclosing the cups con-

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TABLE 1. Lag correction factors

U (min)z value -_________________

140.6 C 148.9 C 157.3 C 160.0 C

F

33 0.18 0.1736 0.19 0.15 0.17 0.1739 - - 0.15 0.16

taining the dried spores in thermal death time cans(TDT cans, 208 X 006) and subjecting these cans tosaturated steam.

Calculation of results. The heat resistance of thespores was evaluated by determining their D and zvalues, which are defined as the time required for90% destruction of the original spore population andthe degrees centrigrade required to obtain a 10-foldchange in D values, respectively. All D values werecalculated by use of the method of Schmidt (8) asmodified by Pflug (personal communication). Thismodification consisted of including only those valuesfor the determination of the 95% confidence limitsfor D which lie between P = 0.16 and P = 0.84.When insufficient data were obtained to use theSchmidt method, the method described by Stumbo,Murphy, and Cochrane (9) was used. The z valueswere determined graphically from the straight lineslinking the logarithms of the D values plotted againstthe linear values of the temperature.

Corrections were made for the delay in the timefor the spore samples to reach the desired treatmenttemperature if such corrections amounted to morethan 1% of the LDO values. It was found that suchcorrections were only required in a few cases at tem-peratures of 315 F (157 C) and higher. The lag cor-rections were determined on the basis of heat pene-tration tests and were calculated by the generalmethod described by Ball and Olson (la). Table 1lists the lag corrections necessary for various z valuesand various temperatures.

REsuLTs

Resistance of spores to saturated steam. Asummary of both the D values at 121 C (250 F)and z values of beef heart-grown spores, as wellas their corresponding energies of activation andentropies, is presented in Table 2. All D valuesand corresponding 95% confidence limits ofthese spores at various temperatures are compiledin Table 3. A sample of a typical thermal re-sistance curve is shown in Fig. 1. As is evidentfrom Table 2, the z values are of the same mag-nitude for all media, namely 10.8 C (19.5 F); theonly exceptions were the z values obtained withEugonbroth and Trypticase as recovery media,i.e., 9.6 C (17.5 F) and 10.1 C (18.2 F), respec-tively.The D values obtained at 110 C (230 F) with

the various subculture media were grouped intofour classes of decreasing, distinctly differentmagnitudes. The highest D values are obtainedwith either beef infusion or pea infusion. Thesecond class included liver infusion, yeast extract,and Eugonbroth. Trypticase and syntheticmedium belonged to the third and fourth class,respectively. At 135 C (275 F), beef infusionagain gave the highest D values. Distinctly differ-ent and decreasing D values in the followingorder were obtained with pea infusion, yeastextract, and liver infusion. The lowest D valuesthat were all of the same magnitude were foundwhen Trypticase, Eugonbroth, or the syntheticmedium was used for subculturing. The fact thatthe D values obtained with the infusion-typemedia and yeast extract are changed in theirrelative magnitude at the two temperatures isexplained as being due to very slight differencesin their z values.The results obtained in general are in agreement

TABLE 2. D values at 121.1 C, z values, and corresponding heats of inactivation formoist-heat resistance studies

Beef heart spores Trypticase sporesRecovery medium

Da z E AASc Da z eb ASC

min C min C

Beef infusion. 1.4 10.8 64,700 99 0.35 8.9 82,100 144Liver infusion. .9 10.9 64,700 96 -Pea infusion. 1.35 10.8 64,700 76 - -Yeast extract. 1.2 10.8 64,700 96 0.35 9.4 76,200 130Trypticase.................. 0.52 10.1 68,200 109 -_Eugonbroth.0.71 9.6 70,500 114.5 0.18 8.7 83,200 146Synthetic. 0.48 10.8 64,700 98 -

a The D values at 121.1 C were established by graphic interpolation. They represent points on thethermal destruction rate curves which were based on the D values and their corresponding 95% confi-dence limits as listed in Tables 3 and 4. An example curve is shown in Fig. 1.

b E = energy of activation = 2.303R (T2 X T1)/(T2 - T1) log k2/k1.c AS = entropy of activation = R[2.303 log Ki - 2.303 (KTi)/(h) + (AH)/(RTi)].

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VOL. 15, 1967 SPORE RECOVERY AFTER HEAT TREATMENT 269

TABLE 3. D values and their corresponding 95% confidence limits (in minutes) for moist-heat resistance ofbeef heart infusion spores and various recovery media

Temp Beef Liver Pea infusion Yeast extract Trypticase Eugonbroth Syntheticinfusion infusion medium

110.0 13.13 10.1 13.78 11.8 6.60 9.05 4.1112.21-14.04 13.27-14.29 10.5-13.1 6.20-7.02 - 3.04-5.17

10.23 13.3 9.78 4.929.71-10.76 12.92-14.00 9.48-10.09 4.63-5.20

121.1 1.38 0.935 1.60 0.86 0.497 0.797 0.4321.31-1.43 - 1.47-1.73 - - - 0.415-0.448

1.32 0.747 - 1.09 0.437 0.693 0.4461.29-1.36 0.60-0.898 - 1.00-1.18 - 0.641-0.775 0.405-487

1.47 - - 1.24 0.550 - 0.5121.43-1.51 - - 1.21-1.27 0.517-0.583 - -

126.7 0.468 0.273 0.334 0.132 0.164 0.1580.416-0.522 0.260-0.286 - 0.313-0.355 0.120-0.144 0.134-0.194 0.148-0.168

132.2 0.114 0.108 - 0.0533 _- - - 0.105-0.117 0.0515-0.057

0.1170.107-0.127 - _ _

135.0 0.080a 0.0492 0.066a 0.058a 0.0253 0.025a 0.02670.0465-0.0517 - - 0.0227-0.0280 _ 0.0238-0.0295

137.8 0.0403 0.0383 0.03330.0373-0.0432 - 0.0357-0.0400 0.0313-0.355 - _ _

0.0322 0.03350.0290-0.0353 0.0322-0.0350

a These D-value points were established by graphic interpolation from the thermal destruction rate curves, and are listed toallow comparative evaluation of the spore heat resistance at 135.0 C.

CURVE : MCCURVE b: DR

MCUNSU

CURVE C: DRSTSU

a

IHEATED SPOREJSPENSIONRY HEAT, UP TO 9 MONTHS'rORAGE OF SPOREJSPENSION

0.10-0.08-0.06-

0.040.03

110 120 130 140 150 160TEMPERATURE °C

FIG. 1. Thermal resistance curves of beef heartinfusion spores subcultured in beef infusion.

with those reported in the literature for PA 3679:infusion-type media generally yield a higherrecovery than formulated media, with the excep-tion of yeast extract which gives a better recoverythan the dehydrated media. Frank and Campbell(2) found a higher recovery of spores of PA 3679with yeast extract-starch-agar than with yeast ex-tract-starch-bicarbonate-agar. Wynne, Schmied-ing, and Daye (11) reported the opposite to bethe case. This disrepancy is believed to stem fromthe fact that the former authors used the samemedia for both initial and survivor counts,whereas Frank and Campbell derived theirinitial counts only from one medium, namelyEugonagar, regardless of the type of mediumused to count the survivors. As shown in Table5, initial counts vary with the type of mediumused. It is therefore quite conceivable that, ifFrank and Campbell would have used the samemedia for both initial and survivor counts,opposite results could have been found. Frankand Campbell reported lowest D values whenEugonagar was used; supplementation ofEugonagar with soluble starch, malt extract,yeast extract, or liver infusion resulted in only avery slight increase in the D value. Wheaton andPratt (10) obtained similar results; the bestrecovery was obtained with pork-pea infusion

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medium. Beef infusion gave someresults, as did yeast extract. Eugogave lowest recovery, and yeast extiin a higher number of survivors thasion.The Trypticase-grown spores we

siderably lower heat resistance. A.Tables 2 and 4, their D values at 12decrease, depending on the recoveused, in the following order: beef inlextract, Eugonbroth. The z values9.4 C (17.0 F) for yeast extract to 8.1

for beef infusion and 8.7 C (15.5 F)broth, and their energies of actientropies varied accordingly.

TABLE 4. D values and their corresiconfidence limits (in minutes) for,

resistance of Trypticase-yeastspores and various recovery

Temp Beef infusion Yeast extract

C

110.0

115.5

121.1

126.7

132.2

6.746.26-7.21

0.3290.298-0.361

0.08050.0748-0.0863

0.02730.0259-0.0290

5.27

5.124.75-5.37

0.3370.313-0.360

0.07920.0690-0.0893

0.02250.0707-0.0245

,what lower With reference to the relative heat resistancenagar again of spores grown in beef heart and Trypticase-ract resulted yeast extract, the results obtained are also inLn liver infu- agreement with those of Wheaton and Pratt (10).

D values in some cases have been reported to beBre of con- affected by the initial spore concentration. How-s shown F ever, according to Amaha (1), 10- or 100-foldC (250 F) differences in initial spore concentration result

fry medium in relatively small changes in D values. It wasvaried from therefore concluded that the small differences inrC (160 F) the initial spore concentrations between the twofor Eugon- types of spores (Table 5) were not large enough

ivation and to account for the extreme difference in their rela-tive heat resistance; these results are thus con-

sidered factual rather than coincidental.7onding 95% Resistance of spores to dry heat. The spores ofmoist-heat PA 3679 exhibited considerably higher resistanceextract to dry heat than to saturated steam. D valuesmedia varied (Tables 6 and 7) depending on the type of

Eugonbroth subculture medium used. At 123.9 C (255 F), theD values obtained with the various subculturemedia were lowest when the heated spores were

3.77 recovered in the synthetic medium. A slightly3.50-4.05

0.8410.809-0.878

0.1660.157-0.175

0.03780.0347-0.041

TABLE 5. Comparative initial counts in variousrecovery media

Recovery medium Beef heart spores Trypticase sporesRecoverimedium (per cup)' (per cup)

Beef infusion.. 4.35 X 104 11.0 X 103Liver infusion... 3.93 X 104Pea infusion ... 3.94 X 104Yeast extract 4.29 X 104 11.3 X 103Trypticase....... 3.10 X 104Eugonbroth ... 2.98 X 104 9.46 X 103Synthetic........ 2.54 X 104

TABLE 6. D values at 148.9 C, z values, and corresponding heats of inactivation for dry-heat resistance asshown in figures

Beef heart spores Trypticase sporesRecovery medium

a z Eb ASC Da Z Eb 'SC

min C min C

Beef infusion................. 12.0 21.7 37,000 17.0 6.4 20.0 40,600 27Liver infusion ................ 10.0 20.0 40,600 25.6Pea infusion.................. 10.0 20.0 40,600 25.6 -Yeast extract... 11.2 20.0 40,600 25.6 5.6 20.0 40,600 27Trypticase.................... 9.4 20.0 40,600 25.6Eugonbroth.. 8.2 20.0 40,600 26.0 3.2 20.0 40,600 28Synthetic... 6.4 18.3 44,000 34.5

a The D values at 148.9 C were established by graphic interpolation. They represent points on thethermal destruction rate curves which were based on the D values and their corresponding 95% con-fidence limits as listed in Tables 7 and 8. An example curve is shown in Fig. 1.

b E = energy of activation and is determined as shown in Table 2.C AS = entropy of activation and is determined as shown in Table 2.

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but distinctly higher D value was found whenEugonbroth was used as the recovery medium.With all other subculture media, substantiallyhigher D values, but of the same magnitude, wereobtained. At 160 C (320 F), the use of beefinfusion as the subculture medium gave by farthe highest D values. D values obtained withyeast extract were slightly lower. Third highestD values were obtained with liver infusion,followed by the D values obtained with peainfusion and Trypticase, which were of the samemagnitude. Again, the use of Eugonbroth resultedin the second lowest D values, and syntheticmedium, in the lowest. With the exception ofbeef infusion with a z value of 21.7 C (39 F) andthe synthetic medium with a z value of 18.3 C(33 F), z values obtained with all other subculturemedia were of the magnitude of 20 C (36 F).

Results in Table 7 show an increase in the Dvalues during storage of the spores. The first Dvalues were obtained within 90 days after sporula-tion, whereas the last values, which were sub-stantially higher, were obtained between 5 and 9months after sporulation. This change is contraryto the findings reported in the literature whichcite either no change or a decrease in heat re-sistance upon storage of spores.Table 9 shows the results of an experiment in

which the effect of the addition of vitamin B12,ribonucleic acid (RNA), and deoxyribonucleicacid (DNA) to the synthetic medium was stud-ied to find out whether the addition of certaincompounds would result in a higher recovery.The results indicate that supplementing thismedium with vitamin B12, RNA, all three ofthese compounds, or a combination of any oftwo of these compounds resulted in an increasedD value. However, experimentation was notextensive enough to allow firm conclusions.Rather, the results indicate an area in whichfurther work might be fruitful.The spores grown in Trypticase-yeast extract

again exhibited considerably lower heat resistancethan the beef heart-grown spores (Tables 4 and8). The results reveal a decrease in D values,depending on the recovery medium used, in thefollowing order: beef infusion, yeast extract,Eugonbroth.With reference to the subculturing medium,

the difference in recovery of spores prior to heattreatment follows more or less the same patternas reported for their corresponding D values,although to a considerably smaller degree (Table5). The same holds true for the Trypticase-grownspores.

Rearrangement of heat resistance data. The zvalues not only vary with the heating medium,

VOL. 15, 1967

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TABLE 8. D values and their corresponding 95% confidence limits (in minutes) for dry-heat resistance ofTrypticase-yeast extract spores and various recovery media

Temp Beef infusion Yeast extract Eugonbroth

C

123.9 101.9 (96.8-107.8) 100 (72.4) 56.5 (48.3-65.0)130 (108-151) 96.0 (84.2-107.7) 57.0 (52.4-61.7)

105 (90.5-111)109 (103-115)

140.6 17.3 (16.7-17.9) 16.6 (15.5-17.7) 9.66 (9.1-10.5)20.7 (19.2-22.2) 14.7 (13.8-15.5) 8.7418.5 (17.4-19.7) 14.0 (12.3-15.7) 7.85 (6.55-9.14)

16.3 (15.8-16.9)

148.9 6.33 (5.94-6.72) 6.82 (6.42-7.20) 4.01 (3.51-4.41)5.82 (5.44-6.19) 3.22

157.3 - 1.05 (0.84-1.26)

160.0 1.55 (1.42-1.69) 1.56 (1.39-1.72) 1.03 (0.94-1.11)1.91 (1.76-2.06) 1.53 (1.37-1.68) 1.23 (1.04-1.41)

1.80 (1.62-1.98)1.89 (1.78-1.99) 1.68 (1.56-1.80)

but also in some cases with the type of recoverymedium used. This means that the D values ob-tained with the various subculture media aresomewhat different in their temperature depend-ence. Thus, the statement made by Curran andEvans (lb) that severe heat treatment causesbacterial spores to become more exacting in theirnutritional requirements might show not only atime dependence regardless of the temperatureused, but also a temperature dependence. Todemonstrate this, the heat resistance data wererearranged. D and No values being known, theequation describing the logarithmic order ofdeath of bacterial spores was solved for N. Thetime t was selected to give an N of 1,000 with thebeef infusion medium and was held constant

TABLE 9. Effect of the addition ofRNA, DNA, andvitamin B12 to the synthetic medium on the

recovery of beef heart spores treated withmoist heat

Compounds added No. of No. D valueCompouns added replicates positive at 126.7 C

sec

Control.......... 20 0 8.65DNA (400 mg/liter).... 20 0 8.65RNA (800 mg/liter).... 20 2 8.60Vitamin B12.......... 20 2 8.60DNA, RNA, vitaminB12.20 4 9.40

DNA, RNA............ 20 4 9.40RNA, vitamin B12. 20 6 9.70DNA, vitamin B12...... 20 4 9.40

with each temperature for calculating the Nvalues for the other subculture media. The resultswere compiled in Tables 10 and 11 and are shownin Fig. 2, 3, and 4.The results showed very clearly the differences

in the numbers of surviving spores with referenceto the subculture medium. They suggested thatthe reaction of the spores in dry heat was differentfrom that in moist heat with reference to thesubculture medium. The same dependence heldwith regard to the sporulation medium.A closer look at Fig. 3 prompted the following

question: what course do the survivor lines ob-tained with the various subculture media takewhen they approach the survivor line obtainedwith beef infusion? If beef infusion mediumfulfills all the requirements for germination andoutgrowth, a break in the curve obtained withthe other medium or media would be expected at,or somewhere below, the beef infusion line. Thisline theoretically could not be crossed by thecurves obtained with the other recovery media.What will happen if the beef infusion does notfulfill all the requirements for germination andoutgrowth of the heat-treated spores becomesobvious when looking at the curves resulting withTrypticase and the synthetic medium: a crossingof the lines, as shown in Fig. 2.

Figure 4 shows that, after moist-heat treatment,the number of survivors recovered in yeastextract gradually approached and finally exceededthe number of survivors found in beef infusionas the temperature of the heat treatment in-

272 APPL. MICROBIOL.

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VOL. 15, 1967 '-lflT SPORE RECOVERY AFTER HEAT TREATMENT

TABLE 10. Number of surviving beef heart spores of PA 3679 recovered in various media after heat exposureat constant times

Moist heat Dry heatSubculture medium Initial

110.0 C 121.1 C 132.2 C 135.0 C 123.9 C 140.6 C 148.9 C 160.0 C

Beef infusion..... 100 1,000 1,000 1,000 1,000 1,000 1,000 1,000 1,000Liver infusion 90.3 159 148 166 159 871 562 407 257Pea infusion. 90.4 661 646 646 661 939 537 417 219Yeast extract. 98.4 501 458 501 479 1,150 742 603 398Trypticase ....... 71.0 7.8 1.5 0.4 0.03 646 347 240 138Eugonbroth...... 68.5 120 19.5 3.6 1.2 295 162 115 66.5Synthetic ........ 58.4 6.0 5.6 6.0 6.0 222 44.7 18.8 2.9

TABLE 11. Number of surviving trypticase spores of PA 3679 recovered in various subculture media afterheat exposure at constant times

Moist heat Dry heatSubculture medium Initial

count

110.0 C 115.5 C 121.1 C 126.7 C 123.9 C 140.6 C 148.9 C 160.0 C

Beef infusion.... 100 1,000 1,000 1,000 1,000 1,000 1,000 1,000 1,000Yeast extract.... 102.7 457 646 851 1,073 759 725 759 725Eugonbroth.. 86.0 112 91.2 74.1 52.5 83.2 75.9 79.5 91.3

creased. A closer look at Fig. 5, which shows theD values obtained with Trypticase-yeast extractspores which were subcultured in beef infusionafter moist-heat treatment at various temperatures(Table 4), indicates a possible flattening out ofthe thermal-resistance curve at temperaturesabove 129.4 C (265 F). If this were the case, thenthe beef infusion and the yeast extract curvesobtained with moist heat would run parallelstarting at, or slightly above, 129.4 C (265 F).If, however, the two lines cross each other, theonly reasonable explanation that can be given isthe same one discussed above for Trypticase andthe synthetic medium-that beef infusion doesnot fulfill all the requirements for germinationand outgrowth of Trypticase spores which havebeen subjected to moist heat.

DISCUSSIONThe results of this study show clearly that,

relative to the subculture media, beef infusionprovides the most ideal conditions for germina-tion and outgrowth of the heated spores, asevidenced by the production of the highest Dvalues. The only exceptions were the Trypticase-grown spores which were subjected to moist heatat temperatures higher than 129.0 C (257 F), inwhich case higher D values were obtained whenyeast extract was used as the subculture medium.The differences in the reaction of the spores to

their subculture surroundings when subjected to

dry versus wet heat suggest that each type ofheatingmedium produces itsown stress conditions.These conditions are different for wet and dryheat and, thus, induce different changes in thespores regarding their requirements for germina-tion and outgrowth. It is quite conceivable thatsuch changes include changes in the nutritionalrequirements of the spores. If this were the case,the addition of one component to a subculturemedium in which the heated spores recoverpoorly would increase the degree of recoveryand, thus, the D value if the spores were subjectedto one type of heating medium, such as moistheat; however, the addition of this componentwould fail to improve the degree of recovery ofthe spores which were subjected to a differenttype of heating medium, such as dry heat, or viceversa.The small differences in the initial spore counts

obtained with various subculture media as com-pared with the much larger differences in survivorcounts found with the same media after exposureto heat are in agreement with the findings ofCurran and Evans (lb). However, the conclusiondrawn by these authors that spores become moreexacting in their nutritional requirements afterheat treatment cannot be confirmed on the basisof the results obtained, because the factors con-tributing to these differences in recovery are notknown. The differences could be the result ofnutritional deficiencies, or of inhibitory factors

273

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AUGUSTIN AND PFLUG

BEEF INFUSION

PEA INFUSIONa YEASTEXTRACT , i

LIVER INFUSION

APPL. MICROBIOL.

on the type of heating medium. This becomes es-pecially obvious when comparing Fig. 2 to 4.That nutritional deficiencies might be respon-

sible at least in part for the differences reportedin recovery with various media is indicated by thefact that the recovery of the spores in the syn-thetic medium can be increased if the latter issupplemented with certain components, notably,RNA, DNA, and vitamin B12. Tables on thecomposition of the various subculture mediamight provide further material to substantiate

ID3.or

2.6F

2.4

110 120 130TEMPERATURE °C

FIG. 2. Logarithms of the number of surviving beefheart spores in various subculture media after moist-heattreatment with heating times chosen to yield 1,000survivors with beef infusion as the subculture medium.

3.0-

2.0

LIVEIN-FUS C)NT EXTRAC Tr

AIVFUSIONv -

PICASE

Io2 1S0 140TEMPERATURE *C

FIG. 3. Logarithms of the number of surviving beefheart spores in various subculture media after dry-heattreatment with heating times chosen to yield 1,000survivors with beef infusion as the subculture medium.

in the form of either direct inhibitors or improperratios of some of the components of the media,such as amino acids or vitamins. For this reason,it can be concluded only that heat treatment ofPA 3679 spores results in their increased sensi-tivity to their nutritional environment. Whateverthe precise nature may be of these factors whichcontribute toward the differences in recovery ofthe surviving heat-treated spores, the requirementsof the heated spores for germination and out-growth vary at least to some degree depending

z

-C-I

BEEF INFUSION

YEAST EXTRACT_ . ~~~~v

o MOIST HEAT

X DRY HEAT

2.2

1.8

I?O

x

EUGON BROTH

TEMP a

110 c 0 130 (MOISTHEATI.

. A .4 _ , ._ . ._ ._110 120 130 (MOISTHEAT)

120 130 140 150 (DRY HEAT) 160

FIG. 4. Logarithms of the number of survivingTrypticase spores in various subculture media after heattreatment with times chosen to yield 1,000 survivorswith beef infusion as the subculture medium.

2.0

1.00.80

rC 0.60

a 040O0.30' 0.20

0.100.080.06

+

130TEMPERATURE °C

FIG. 5. Thermal-resistance curve for Trypticase-yeast extract spores subcultured in beef infusion aftermoist-heat treatment.

274

3D F

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SPORE RECOVERY AFTER HEAT TREATMENT

this conclusion. However, such data are oftenfragmentary and of insufficient accuracy to be ofany value. Furthermore, exact data on the com-position of infusion-type media do not exist at all.The change in sensitivity of the spores to their

nutritional environment manifests itself not onlyin relation to the nonheated spores versus theheat-treated spores depending on the type ofrecovery media, but also within the heat-treatedspores, where, in most cases, there is a gradualincrease in sensitivity with increasing temperature.An explanation for this phenomenon can be

derived on the basis of the findings reported byZamenhof (12), who demonstrated that dry-heattreatment of bacterial spores in vacuo resulted inmutagenic injury of these spores, and that thesite of this injury was the genetic material of thespores, i.e., the DNA molecules. This thermalinjury revealed itself in the form of alterations inthe nutritional requirements of the spores. Thedifferences in D values obtained with differentrecovery media, as well as the gradual departureof the number of survivors recovered in somemedia relative to those recovered in beef infusion,suggest that increased severeness of the heattreatment creates an increased rate of randomdenaturation of the genetic material, i.e., theDNA molecules of the spores, which is respon-sible for alterations in the nutritional require-ments of the spores. Depending on the nutrientcomposition of the subculture medium, sporescan be expected to exhibit different recoverypatterns in these media. Supplementation of themedia with the proper nutrients would then mostlikely compensate for the additional nutritionalrequirements induced into the spores as a resultof the heat treatment. If, however, a so-calledcritical gene were denatured by the heat effect,any improvement of the nutritional environmentwould fail to receive any responses from thespores.The fact that considerably higher energies of

activation and entropy values are obtained withmoist-heat than with dry-heat destruction doesnot rule out this theory. Pollard (7), who reportedsimilar differences with reference to thermalinactivation of viruses, concluded that with moistheat higher energy barriers have to be overcometo permit biological inactivation. He hypothesizedthat this is due to a tighter binding of the entirestructure, i.e., the molecules in the wet state. Hesuggested that the considerably higher entropyvalues associated with moist-heat inactivationare due to a large extent to hydration.

If different degrees of hydration could bepositively correlated with the size of the spores,this hypothesis could be extended as a possible

explanation for the differences in heat resistancefound among the spores which were produced intwo different sporulation media. Substantialsupport for this type of reasoning could possiblybe gained by determining the density of the sporesgrown in the two different sporulation media. Itwould be expected that the spores grown inTrypticase would be of a lower density than thosegrown in beef heart infusion. It is, therefore, notunthinkable to establish that the calcium content,the heat resistance of the spores, and sporedensities are correlated positively. If such cor-relations could be verified experimentally, itcould be possible to predict heat resistance ofspores on the basis of their density.

According to this theory, a gradual decreasefrom maximal heat of activation and entropyvalues required for the destruction of completelyhydrated spores to minimal heat of activationand entropy values needed for the destruction ofdry spores is to be expected. That this is the caseat least for the extreme degrees of hydration anddryness has been demonstrated in the results ofthis study, which clearly reveal that the values ofthe heats of activation and entropy obtained frommoist-heat destruction of the spores are close todouble those obtained with dry-heat destruction.Ingraham (5) mentioned as another possible

explanation of the logarithmic order of death thetheory of destruction of a certain structure suchas the cell membrane. The work by Hunnell andOrdal (4) offers some support to this theory. How-ever, the results obtained in this investigationappear to disprove it. If this theory would hold,then the differences in survivors recovered in thevarious subculture media after the heat treat-ments would be identical to the differences foundin the nonheated spore samples in the same media.

ACKNOWLEDGMENTThis investigation was supported by Public Health

Service grant A103708 from the National Institute ofAllergy and Infectious Diseases.

LITERATURE CITED1. AMAHA, M. 1954. Heat resistance of Cameron's

Putrefactive Anaerobe 3679 in phosphatebuffer. Food Res. 18:411.

la. BALL, C. O., AND F C. W. OLSON. 1957. Steri-lization in food technology, p. 196. McGraw-Hill Book Co., Inc., New York.

lb. CURRAN, H. R., AND F. R. EVANS. 1937. The im-portance of enrichments in the cultivation ofbacterial spores previously exposed to lethalagencies. J. Bacteriol. 34:179-189.

2. FRANK, H. A., AND L. L. CAMPBELL, JR. 1955.The influence of recovery media on thermalresistance values of spores of a putrefactiveanaerobic bacterium. Appl. Microbiol. 3:300-302.

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276 AUGUSTIN

3. GREcz, N., A. ANELLIS, AND M. D. SCHNEIDER.1962. Procedure for cleaning of Clostridiumbotulinum spores. J. Bacteriol. 84:552-558.

4. HUNNELL, J. W., AND Z. J. ORDAL. 1961. Cyto-logical and chemical changes in heat killedand germinated bacterial spores, p. 101. InH. 0. Halvorson [ed.], Spores II. BurgessPublishing Co., Minneapolis.

5. INGRAHAM, J. L. 1962. Temperature relationship,p. 265. In I. C. Gunsalus and R. Y. Stanier[ed.], The bacteria, vol. 4. Academic Press,Inc., New York.

6. PFLUG, I. J. 1960. Thermal resistance of micro-organisms to dry heat: Design of apparatus,operational problems and preliminary results.Food Technol. 14:483.

7. POLLARD, E. C. 1953. The physics of viruses.Academic Press, Inc., New York.

ALND PFLUG APPL. MICROBIOL.

8. SCHMIDT, C. F. 1957. Thermal resistance ofmicroorganisms, p. 831. In C. F. Reddish[ed.], Antiseptics, disinfectants, fungicides andsterilization, 2nd ed. Lea & Febiger, Philadel-phia.

9. STUMBO, C. R., J. R. MURPHY, AND J. COCHRAN.1950. Nature of thermal death time curves forPutrefactive Anaerobe 3679 and Clostridiumbotulinum. Food Technol. 4:321.

10. WHEATON, E., AND G. B. PRATT. 1961. Compara-tive studies on media for counting anaerobicspores. II. J. Food Sci. 26:261.

11. WYNNE, E. S., W. R. SCHMIEDING, AND G. T.DAYE, JR. 1955. A simplified medium for count-ing Clostridium spores. Food Res. 20:9.

12. ZAMENHOF, S. 1960. Effects of heating dry bac-teria and spores on their phenotype and geno-type. Proc. Natl. Acad. Sci. U.S. 46:101.

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