SPORE FORMATION BY THERMOPHILIC FLAT SOUR ORGANISMS

Transcription

1 SPORE FORMATION BY THERMOPHILIC FLAT SOUR ORGANISMS I. THE EFFECT of NUTRIENT CONCENTRATION AND THE PRESENCE OF SALTS CLARENCE F. SCHMIDT Research Department, Continental Can Company, Chicago, Illinois Received for publication April 14, 1950 The literature upon spore formation by bacteria has been reviewed on numerous occasions and most recently by Knaysi (1948). By way of introduction it may be said that despite the voluminous literature the group of organisms known as obligate thermophiles has been almost completely neglected. The "flat sour" obligate thermophiles, in the sense of the definition of Cameron and Esty (1926), represent a group of particular importance to the canning industry because of the extremely high thermal resistance of the spores that they produce. The data to be reported here are concerned with the effect of various factors upon spore formation by certain members of this group in a study conducted with the hope of obtaining some insight into the factors controlling spore formation and as a preliminary to further research upon factors that may influence the thermal resistance of the spores of these organisms as they are encountered in nature. Methods. The percentage of spores formed was determined in films prepared from agar slants and stained with malachite green and basic fuchsin. The heatfixed film was covered with 1 per cent aqueous malachite green, and the slide was passed through the flame of a bunsen burner until the staining solution definitely boiled several times. The preparation was then decolorized in running tap water for 10 to 15 seconds and counterstained for 1 to 2 minutes with 0.05 per cent basic fuchsin. Good differentiation of spores and vegetative cells was obtained by this procedure. The eyepiece qf the microscope contained a standard Howard mold-counting disk that served to divide the visible microscopic field into 20 squares. The number of spores present in the total field of 20 squares was counted. The number of vegetative cells per field was counted by estimating the number of cells in each small square in steps of five. That is, the number of vegetative cells in each square was estimated as 5, 10, 15, etc., and the estimates summed over the 20 squares to give the total number of vegetative cells per field. Several fields were counted in this way until a total of 800 to 1,000 cells plus spores was obtained. The percentage of spores was calculated from the ratio of spores to vegetative cells plus spores. This procedure gave reproducible results when several counts were made upon a single slide. The following instances of duplicate counts have been selected at random from a large number of data: 11.9 and 12.1; 5.6 and 6.1; 17.0 and 15.4; 4.8 and 3.8. Agreement of this same order was obtained from duplicate films prepared from the same agar slant. 205

2 206 CLARENCE F. SCHMIDT [VOL. 60 Cultures. The following cultures were used in this work: Code Source 1518 A culture of N.C.A. no in the writer's possession since S A subculture of the foregoing no carried as a separate strain since N A culture of N.C.A. no received from the National Canners Association in Isolated from thermophilic spoilage in chocolate milk in Isolated from thermophilic spoilage in chocolate milk in P-1 Isolated from thermophilic spoilage in black-eyed peas in P-2 Isolated from thermophilic spoilage in black-eyed peas in These cultures were all obligate thermophilic organisms in the sense of Cameron and Esty (1926), showing no growth at 37 C but growing at 45 C and higher. General Procedure. Agar slants in 20-by-125-mm screw-cap tubes containing 8 ml of medium were used for cultivation of the cultures. Incubation was conducted in an incubator room with air circulated by fans, operating at a temperature of C. Some data obtained when variations wider than this occurred in the incubator have not been included. All media were inoculated from stock cultures on nutrient agar slants incubated 3 days at 55 C. After the 3-day incubation the stock cultures could be held at room temperature for at least 10 days before being used as an inoculum without noticeably affecting the results. Spore formation was determined following 3 days' incubation with the No cultures and 605. Under the conditions used maximum growth appears to be obtained in 24 hours or less and in many cases maximum spore formation. However, better reproducibility was obtained following 3 days' incubation. No autolysis was observed with these cultures after 3 days' incubation, and only slight autolysis was observed after 6 days' incubation. With these cultures the percentage of spores did not increase between 3 and 6 days. Cultures P-1, P-2, and 304 appeared to undergo extensive autolysis between 48 and 72 hours. In order to avoid erroneously high percentages of spores due to the disappearance of vegetative cells by autolysis, the percentage of spores in these three cultures was determined after 48 hours' incubation. Difco nutrient agar and Difco peptone were used for the preparation of media. Sodium and calcium chlorides were prepared as concentrated stock solutions, and the requisite amount was added to the nutrient agar to give the desired concentration. Replicate determinations were made for each culture and each condition. At times duplicates were run at one time, but most of the replicates consist of single tests run at different times. The results have been interpreted by use of the mean and standard error of the replicate determinations for each culture and environmental condition. In this way, by comparing means and calculating t (Snedecor, 1946), one may decide whether there is a significant difference in the mean percentage spore formation of a culture under any two sets of conditions.

3 1950] THERMOPHILIC FLAT SOUR ORGANISMS 207 RESUIS Table 1 shows the results of replicate determinations of the percentage of spore formation on nutrient agar. All values to the left of the heavy lines were obtained in experiments conducted between November, 1948, and May, 1949, whereas the results on the right of the heavy lines were obtained from experiments conducted between August and November, This method of presentation serves to indicate the order of agreement of the results obtained over a period of time. The last column shows the mean and standard error for spore formation on nutrient agar for each culture. The significance of differences between the mean percentages of spore formation was evaluated by use of the t test (Snedecor, 1946, Chapter 4). Differences between means are considered significant if t as calculated exceeds 2.5. This establishes the minimum degree of probability of significance at the 95 per cent level. In most cases in which a significant difference between means is obtained, the probability level is much higher than TABLE 1 Spore formation on nutrient agar: results of replicate tests uith mean and standard error CULTURE PERCENTAGE OF SPORES EAN FM AND N NO. Replicate number STANDARD ERROR it: S N :1 0.7 P-i i 1.9 P I ± 0.8 that taken as a minimum. As an instance, t calculated for the difference in mean percentage of spore formation between culture 605 and culture 1518-N on nutrient agar (table 1) is With 16 degrees of freedom the probability is greater than 99 per cent that the difference is significant. Using the t test as a basis of judgment, it may be asserted that there is a significant difference in spore formation between culture 605 and cultures 1518-N, 304, P-1, and P-2, and also between either culture 1518-N or 304 and cultures P-1 and P-2. The data in table 1, therefore, serve both as an illustration of the method of evaluating spore formation comparatively and as a basis of comparison with other nutrient conditions. Table 2 shows the results obtained by growing the cultures upon different concentrations of peptone. The results from nutrient agar are also included for comparison. Each value is the mean of four to six replicate determinations made at different times. Cultures 1518, 1518-S, 605, and 304 show significant increases in spore formation on 0.2 per cent and 0.5 per cent peptone as compared to nutrient agar. Culture 1518-N shows no significant change and cultures P-1 and

4 SCHMIDT[ 208 CLARENCE F. [VOL. 60 P-2 show a significant decrease in spore formation. Although cultures P-1 and P-2 show a difference between 0.2 and 0.5 per cent peptone, no significant difference is found with cultures 1518, 1518-S, 605, and 304. This indicates that the last-mentioned cultures respond to the removal of meat extract from the nutrient agar formula by an increase in spore formation but do not show further response when peptone concentration is decreased in this concentration range. With the exception of culture 1518-N, which showed a significant decrease, the percentage of spores formed on nutrient agar and 2 per cent peptone agar were the same. CXM.T NO. TABLE 2 Spore formation in relation to concentration of nutrients INT&O 010 IRM ATION, VIA AN STANDR ERMOR Nutrient apr 0.2% peptone 0.5S peptone 2%YO peptone : : ; S 5.9 = :1: : N i : A '-: P-i t : P : CULTURE NO. TABLE 3 The effect of NaCI and CaCL, in nutrient agar upon spore formation CONTROL PUCENTAGQ Or SPOR 701M&TON, MEA AN STANDR 31OR 0.05 x NaCI 0.25 x NaCl x CaCis 0.10 x CaCIS E : S N 10.2 i d it it i i :1: : a 1.8 P-i it L : 3.6 P : i Table 3 gives the results obtained by growing the organisms upon nutrient agar containing either NaCl or CaCl2 at two different concentration levels. The only cultures influenced by the concentrations of salts used were 1518, 1518-S, 1518-N, and 304. No effect was observed with 0.05 m NaCl; 0.25 M NaCl gave practically complete inhibition of spore formation with 1518 and 1518-S and a marked degree of inhibition with 1518-N and 304. Both concentrations of CaC12 inhibited spore formation almost completely with 1518 and 1518-S and to a very large degree with 1518-N. Table 4 shows a summay of the data in terms of comparison with spore formation on nutrient agar. In one sense this is a qualitative comparison. Taking one medium, nutrient agar, as the basis for comparison, the data in this table

5 1950] THERMOPHILIC FLAT SOUR ORGANISMS 209 indicate whether a significant quantitative change in percentage of spore formation has been found on any other medium, and whether the change was an increase or decrease. This type of comparison appears to be of considerable value in analyzing data of the kind presented here. It may be noted immediately that the only generalization applicable to the seven cultures studied is that none are affected by 0.05 M NaCl in nutrient agar. No other nutrient condition studied permits any generalization about the behavior of the seven cultures. The parallel behavior of 1518 and 1518-S is of interest in that 1518-S is a subculture of 1518 carried as a separate culture for somewhat more than a year. The behavior of 1518-N in the presence of the salts is the same as that of 1518 and 1518-S, but the behavior with respect to peptone is different, indicating that cultures originally derived from the sate source and carried as subcultures may show differ- TABLE 4 Spore formation on different media compared to spore formation on nutrient agar Nutrient agar containing Culture No. 0.2%O 0.5%/ 2% Peptone Peptone Peptone 0.05 X 0.25 m X 0.10 X NaCI NaCl CaCl2 CaClz _ 1518-S _ 1518-N P-i P increase;-= decrease; 0 = no effect. ing behavior with respect to the effects of environmental or nutrient conditions on spore formation. DISCUSSION There are considerable data in the literature indicating that a decrease in the concentration of nutrients results in an increase in spore formation (Williams, ; Cook, 1931; Brunstetter and Magoon, 1932; Tarr, 1932; Knaysi, 1945, 1946, 1948). For the most part the work reported has been conducted with the aerobic mesophilic sporeforming species common in most laboratories, such as Bacilus anthracis, BaciUlls cereus, BaciUus megatherium, Bacillus mycoides, Bacilus ubtilits, etc. In some cases only a single strain or culture was used, but in others a number of cultures have been tested. The weight of the evidence is respectable and has led to the generalization that "starvation" is the primary cause or stimulus to spore formation (Knaysi, 1948). However, conflicting evidence is also available. Brunstetter and Magoon (1932) found a strain of Bacillus fusiformis in which the percentage of spores increased as the peptone concentration was increased. Kaplan and Williams (1941) found that concentration of peptone and meat extract had little influence

6 210 CLARENCE F. SCHEMIDT [VOL. 60 upon spore formation by Clostridium sporogenes. Wynne (1948) found that increasing a brain-heart infusion broth above the normal concentration decreased the percentage of spores formed by Clostridium botulinum but that almost all of the effect of increasing the concentration of the medium was due to the increased concentration of sodium chloride. Diluting the medium twofold and fourfold below the normal concentration did not appear to increase spore formation appreciably. The addition of potassium to an asporogenic medium resulted in greatly increased spore formation by Bacillus cereus (Foster and Heiligman, 1949a), and the addition of glucose to a glutamate salts medium resulted in greatly increased spore formation by the same organism (Foster a?nd Heiligman, 1949b). The existence of asporogenic cultures that do not produce spores under any of the nutrient conditions tested (Tarr, 1932; Knaysi, 1948) also poses a problem, since it would then appear that "starvation" must be defined in relation to the particular cultures used. Also of interest is the fact that although the percentage of spores formed may be increased or decreased by varying the medium from some condition taken as a standard, the change is still an alteration in the behavior of only some of the cells present. An interesting subject for speculation is why a given set of environmental conditions promote sporulation in a certain percentage of cells but apparently do not affect the remainder. Brunstetter and Magoon (1932) suggested the possibility of a relation between dissociation phases of a culture and sporulation. Knaysi (1933) described dissociated varieties of Bacillus megatherium that were asporogenic and seemed to show no tendency to recover the ability to form spores. However, after prolonged storage at refrigerator temperature spore formation in these variants was regained although the original variant colony morphology was still observed (Knaysi, 1935). Hayward, Marchetta, and Hutton (1946) found a culture of Bacillus globigii, giving very irreproducible results, which dissociated into several recognizable morphological types, one of which gave rapid and almost complete sporulation whereas the others were slower in the rate of spore formation. It appears very possible that cultures pure according to the usual morphological and biochemical standards may not be "pure cultures" from the standpoint of spore formation and that, even without the presence of recognizable morphological dissociation forms, the net effect of a given environmental condition may be the result of a selection from the original inoculum. The data presented in this report do not permit of any generalization relating nutrient conditions to spore formation. Some cultures would support the conclusion that spore formation increases with decrease of nutrients. Two other cultures confirm the behavior of Bacillus fusiformis as found by Brunstetter and Magoon (1932). One culture appears to be unaffected for the most part by the concentration of nutrients used in these experiments. The effect of salts added to the medium cannot be generalized also because of the varying behavior of the individual cultures studied. Four cultures show inhibition of spore formation by NaCl similar to the behavior of Clostridium botulinum noted by Wynne (1948). The remaining cultures are not affected by the concentrations of NaCl or CaCl2 tested. Although Fabian and Bryan (1933) found the monovalent cations to be stimulating to spore formation by several

7 1950] THERMOPHILIC FLAT SOUR ORGANISMS 211 aerobic mesophiles, no stimulatory effect was apparent with the NaCl concentrations used in the present experiments. In consideration of the data presented in this paper and from a study of the literature the writer ventures to suggest that studies in spore formation are still in the descriptive stage and that all conclusions reached should be tagged with the qualifying phrase "with the organism or organisms studied." The formation of a spore by a bacterial cell no doubt involves or is preceded by a radical revision of cellular function and metabolism as compared to vegetative growth. That this process may be accelerated or inhibited by environmental influences is not surprising even though the ultimate outcome does not appear to be predictable. Since all cells do not react uniformly to a given environmental influence (in general the reaction obtained is an increase or decrease in the percentage of spores formed), it would seem that, in addition to environmental factors that influence metabolism or enzyme systems within the cell, there are "cellular factors" responsible for the reaction of the cell to a given environment. Therefore, spore formation under any given environmental condition is a function both of the environment and of these "cellular factors." Any stock culture used as a source of inoculum may be a mixture of varying proportions of cells containing cellular factors that, together with the given environmental conditions, will result in spore formation. Thus the absence of a 100 per cent reaction to a given environment would be explainable. If the immediate previous history of a culture (conditions of cultivation of the stock culture) could be shown to affect the degree of spore formation under given environmental conditions, the existence of such cellular factors would be strongly supported. Some evidence, as yet insufficient for publication, has been obtained in this direction. If such is the case, it may be evident why studies in spore formation are considered to be in the descriptive stage and far from the point of even minor generalizations. It is further suggested that studies of the type reported here with data statistically controlled may be the most profitable line of attack upon the problem of when, why, and how some bacteria sometimes form spores. SUMMARY AND CONCLUSIONS Spore formation by a group of seven cultures of obligate thermophilic flat sour organisms has been studied in relation to the concentration of nutrients and the presence of salts. No generalizations upon the effect of any of the tested nutrient conditions can be made since, with the exception of the absence of effect of 0.05 M NaCl in nutrient agar, no other condition studied resulted in a uniform type of behavior. From a study of these results and the literature it is suggested that "cellular factors" as well as environmental conditions exert an important influence in determining spore formation in a given environment. REFERENCES BRUNSTETTER, B. C., AND MAGOON, C. A Studies on bacterial spores. III. A contribution to the physiology of spore production in Bacillus mycoides. J. Bact., 24,

8 212 CLARENCE F. SCEMIDT [VOL. 60 CAMERON, E. J., AND ESTY, J. R The examination of spoiled canned foods. 2. Classification of flat-sour spoilage organisms from non-acid foods. J. Infectious Diseases, 39, CooK, R. P Some factors influencing spore formation in B. subtilis and the metabolism of its spores. Zentr. Bakt. Parasitenk., I, Orig., 122, FABIAN, F. W., AND BRYAN, C. S The influence of cations on aerobic sporogenesis in a liquid medium. J. Bact., 26, FosTER, J. W., AND HEIIGMAN, F. 1949a Mineral deficiencies in complex organic media as limiting factors in the sporulation of aerobic bacilli. J. Bact., 57, FOSTER, J. W., AND HEImiGMAN, F. 1949b Biochemical factors influencing sporulation in a strain of Bacillus cereus. J. Bact., 57, HAYWARD, A. E., MARCHETTA, J. A., AND HUTTON, R. S Strain variation as a factor in the sporulating properties of the so-called Bacillus globigii. J. Bact., 52, KAPLAN, I., AND WILIAMS, J. W Spore formation among the anaerobic bacteria. I. The formation of spores by Clostridium sporogenes in nutrient agar media. J. Bact., 42, KNAYsI, G Morphological and cultural studies of Bacillus megatherium with special reference to dissociation. J. Bact., 26, KNAYSI, G Further observations of certain variants of Bacillus megatherium. J. Bact., 29, KNAYSI, G A study of some environmental factors which control endospore formation by a strain of Bacillus mycoides. J. Bact., 49, KNAYSI, G On the process of sporulation in a strain of Bacillus cereus. J. Bact., 51, KNAYSI, G The endospore of bacteria. Bact. Revs., 12, SNEDECOR, GEORGE W Statistical methods. 4th ed. The Iowa State College Press, Ames, Iowa. TARR, H. L. A The relation of the composition of the culture medium to the forma. tion of the endospores of aerobic bacilli. J. Hyg., 32, WILIAMs, 0. B Bacterial endospore formation in media of varying biologic value. Proc. Soc. Exptl. Biol. Med., 28, WYNN, E. Staten 1948 Physiological studies on spore formation in Clostridium botulinum. J. Infectious Diseases,