Sporulation of Bacillus stearothermophilus

APPLm MICROBIOLOGY, Sept. 1967, p. 975-979 Vol. 15, No. 5 Copyright i 1967 American Society for Microbiology Printed in U.S.A. Sporulation of Bacillus stearothermophilus P. J. THOMPSON AND 0. A. THAMES, Department of Microbiology, Northwestern State College, Natchitoches, Louisiana 71457, and Gerber Research Center, Gerber Products Company, Fremont, Michigan 49412 Received for publication 3 March 1967 A broth medium containing tryptone and manganese sulfate supported heavy sporulation of Bacillus stearothermophilus ATCC 7953 (NCA 1518) and four isolates identified as B. stearothermophilus. Maximal spore yields were obtained by use of inocula grown anaerobically in a medium containing glucose with aeration of sporulation medium via bubbling. After an extended stationary period, sporulation occurred concurrently with vegetative growth between 6 and 8 hr of incubation at 60 C. Omission of glucose from the inoculum or use of a "young" (2 hr) inoculum abolished the stationary period, but decreased spore yields. A requirement of oxygen for rapid vegetative growth and sporulation was demonstrated. Manganese (15 to 30 ppm) stimulated sporulation but did not enhance cell growth. Physiological studies of sporulation in the stenothermophilic bacteria have lagged behind those of the mesophiles for lack of a broth medium supporting abundant sporulation in the usual growth temperature range of Bacillus stearothermophilus (3). Thermophile spores are usually produced on an agar medium surface incubated 4 days to 2 weeks (1). The disadvantages of this procedure are manifest; less obvious is the possibility of a heat-induced dormancy (4) during prolonged incubation at the elevated growth temperature. A reproducible method for realizing sporulation of B. stearothermophilus in a broth medium is described. MATERIALS AND METHODS Microorganisms. B. stearothermophilus ATCC 7953 (NCA 1518), smooth colony type, was used as a standard of comparison in the sporulation studies, since the spores of this strain are widely used in thermal death-time and inoculated pack studies (7). Culture isolates G-1, G-5, G-6a, and G-7 recovered from canned creamed corn, cereal, spinach, and peas, respectively, were identified as B. stearothermophilus (Bergey's Manual of Determinative Bacteriology, 7th ed.). Differences were noted among the G-series isolates in regard to motility, salt tolerance, colony morphology, and gelatin liquefaction. The cultures were maintained on 2% Trypticase (BBL) plus 0.1% Liver Infusion Broth (Difco) agar slants incubated 12 hr at 65 C and stored at 7 C. Inocula. Slant growth was transferred by loop to 100 ml of tryptone (0.5%)-glucose (0.1%)-beef extract (0.3%) broth (TGE), contained in milk dilution bottles tightly fitted with Escher stoppers. Incu- ' Present address: United Foods Co., Inc., Brownsville, Tex. 78520. bation without agitation was conducted at 60 C for 12 to 16 hr. Acid production lowered the ph of the medium to 5.0 to 5.3. Growth was restricted to ca. 106 cells/ml (plate count). Inocula from TGE broth minus glucose were similarly prepared. "Young" inocula were prepared by washing the growth from a slant culture into TGE broth in milk dilution bottles and incubating at 60 C for 2 hr. The ph ranged from 6.3 to 6.6. Sporulation medium. A 100-ml amount of inoculum was decanted into a 2-liter hypering flask containing 1.5 liters of trymin medium, which consisted of: tryptone (Difco), 30 g; 0.5 M K2HPO4, 6.1 ml; 0.5 M KH2PO4, 3.9 ml; 5.4 X 10- M MnSO4.H2O, 10 ml; and 1,480 ml of water. The ph of trymin medium after autoclaving was 6.8. Aeration assembly. Aeration of inoculated sporulation medium was controlled with the train shown in Fig. 1. Incoming air pressure of 20 psi was reduced by manipulation of the needle valve. Air flow (liters per minute) was read in the sight tube of the flowmeter. Copper tubing (inner diameter, 0.95 cm) conducted air from the flowmeter to the bottom of the sterilized suction flask. Filtered air flowed through flexible plastic tubing and emerged from the gas dispersion tube in fine bubbles. Liquid was forced from the sampling port by covering the inoculation port and creating top pressure. At aeration rates of 0.7 liter per min per liter of medium, the efficiency of heat exchange was such that the temperature of the culture medium did not fall below 60 C. Bath temperature was held at 62 4 0.5 C with a circulating pump and an electrically heated tempering tank. Measurements. Plating and microscopy techniques were used to estimate spore population. A 20-ml amount of culture fluid tubed in 18-mm stoppered vials was heated in a 110 C oil bath for 5 min (4) after temperature equilibration (4 min). After cooling in flowing tap water, dilutions were prepared and 975

976 THOMPSON AND THAMES APPL. MICROBIOL.... IN ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.: M-M d * 1 :: :~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~. *........'. 4AfW 4^S IW*.UL4.OA' J.Vf * f F a.a. S s.:.~~~~~~~~.......... FIG. 1. Aeration assembly for endospore production. Incoming air at 20 psi was regulated with needle valve to give desiredflow rate reading on flowmeter gauge. Forflow rates of 0.2 to 0. 7 liter per min, the B-2S needle valve (Nuclear Products Co., Cleveland, Ohio) was used with a Bantam flowmeter (Hoke serial 996). Flow rates in excess of 0.7 liter per min required a forged needle valve (Hoke 303) and Hoke serial 990 flowmeter. The assembly was detached atflowmeter for sterilization offilterflask and culture vessel. plated in triplicate in Tryptone Glucose Extract Agar (Difco). Colonies were counted after 14 hr of incubation at 60 C. Lengthier incubation periods did not increase colony counts. Visual estimations of sporulation were made in wet mounts by use of phase microscopy. Averages of spores per total number of cells were obtained by examination of 200 organisms. Liberated spores were rarely encountered. Turbidity measurements were performed with a Coleman Universal Spectrophotometer at a wavelength of 560 m,. The Beckman model 76 ph meter was used for ph measurements. Estimations of viable vegetative cells and sporangia were obtained by plating in TGE agar. Since cells tended to clump during early log-phase growth, all samples from the sporulation medium were collected in tubes containing 2 g of sterile sand and were shaken until microscopic examination of five fields disclosed no clumps. RESULTS Growth and sporulation data for the five strains of B. stearothermophilus are presented in Fig. 2. A burst of growth followed an extended stationary phase. Sporulation paralleled growth so that the majority of spores were produced within a 2-hr interval. Observed changes in ph are typical of Bacillus species cultured in aerated media (5). The persistence of spores at 24 hr indicates germination did not occur once growth ceased. Cell and spore counts of strain 7953 are plotted in Fig. 3. Percentages of sporulation based on these data are low, whereas estimations derived from microscopic counts of cells with refractile spores range from 80 to 90% at 8 hr of incubation. This wide disagreement can only be due to inactivation of spores by the heat "shock" used to stimulate germination and kill vegetative cells, since precautions were taken to disperse clumps formed during heating. The extended stationary phase was eliminated in two ways: omission of glucose from the inoculum and use of a "young" inoculum. In either case, extensive vegetative growth and sporulation were evident at 4 hr (Fig. 4 and 5). Massive lysis of vegetative cells occurred beyond 8 hr; however, sporangia appeared intact through the phase microscope. Various peptones were substituted for tryptone in trymin medium (Table 1). Only peptone (Difco) supported heavy sporulation; however, 3% concentrations were required to achieve spore yields comparable to 2% tryptone. Sporulation was fairly constant from lot to lot of tryptone,..:...

VOL. 15, 1967 SPORULATION OF B. STEAROTHERMOPHILUS 977 8 ~~~~~~~~~~~~~12 6 ~~~~~A 10 w IL U) 0C 4 6 o~~ e 0D 2 0~~0 o 04j / /0/ 4AGE OF CULTURE, HR O FIG. 4. Viable count of Bacillus stearothermophilus ATCC 7953. Log N on ordinate refers to viable count 0.2 _ ds/ / of cells and sporangia (0) and to viable count of spores (a). Growth was started from a young inoculum. 2 4 6 8 24 12 AGE OF CULTURE, HR FIG. 2. Growth and sporulation of Bacillus stearothermophilus in trymin medium. Airflow was regulated lo at 0.7 liter per min per liter, and temperature was maintained at60c. Strain 7953, 0; G-1, 0; G-5, A; / G-6A, *; and G-7, E. All points are averages offive 8 independent trials. [ I0 W6,~~~~~~~~~~~~~~ / W, z z J~~~~~~~~~~~~~ 4 O 0 2 4 6 8 24 OF CULTURE, HR ~~~~~K Viable count of Bacillus stearothermophilus ATCC 7953 started from an inoculum lacking glucose. O6 Log 2N reftrs to viable count of cells and sporangia 2,# ~~~~~~~~~~AGE FIG. 5. 0 2 4 6 8 24 (0) and to viable count of spores (a). AGE OF CULTURE, HR FIG. 3.Viabkl count of Bacillus stearothermophilus ATCC 7953 cells, sporangia, and spores produced in only weak sporulation, approximately onetrymin medium. Open and closed circles represent total tenth that of the usual yield. viable numbers and spores, respectively. Manganese stimulated sporulation, but not

978 THOMPSON AND THAMES APPL. MICROBIOL. TABLE 1. Growth and sporulation of Bacillus stearothermophilus in several broth media Mediuma (OD Turbidity 560 mpa)b Sporulation (%)C Proteose Peptone (Difco).. 0.1 1 Peptone (Difco)... 0.7 80 Trypticase (BBL)... 0.3 20 Tryptose (Difco)... 0 0 a Media consisted of all ingredients of trymin medium except tryptone, which was replaced by 3% of the various peptones. The ph values of the autoclaved media ranged from 6.8 to 7.2. b Readings were made 12 hr from time of inoculation. c Estimations of sporulation were made with the phase microscope 12 hr from the time of inoculation. TABLE 2. Effects ofadded manganese on sporulation in Bacillus stearothermophilus ATCC 7953a Manganese addedb Sporulation at 24 hr" PPM 0 3.6 X 106 15 7.2 X 106 30 6.9 X 106 45 6.3 X 101 3.1 X 106 a OD at 560 m,u of all cultures was 0.8 :1-0.02 at 24 hr. The same lot of tryptone was used throughout. b Manganese (as MnSO4. H20) was added before autoclaving. e Means of three trials. Range = <10% of mean. Expressed in spores per milliliter. vegetative growth (Table 2). Concentrations of Mn in excess of 30 ppm depressed spore yields. Vegetative growth was severely decreased in nonaerated trymin medium, and no spores were formed. When air was supplied at various rates, full vegetative growth and sporulation followed (Table 3). A repression of sporulation in the presence of enhanced growth occurred at flow rates of 1.3 liters per min per liter of medium and greater. DIscussIoN Growth that results (Fig. 2) demonstrated thermophiles sporulate in trymin medium after a 4-hr stationary phase. Steps taken to minimize acid production (young inocula and glucose-free inocula) eliminated the lag in cell growth. Figures 4 and 5 differ from Fig. 3 in that they illustrate faster growth rates and more dense populations. TABLE 3. Sporulation in Bacillus stearothermophilus ATCC 7953 in response to aeration 12-hr samplesb 24-hr samplesb Aeration- rate!z560 a Sporulation 560 a Sporulation 0 0.01 <0.01 X 106 0.07 <0.01 X 106 0.13 0.15 <0.01 X 106 0.51 0.01 X 106 0.26 0.18 <0.01 X 106 0.55 0.22 X 106 0.67 0.78 8.0 X 106 0.82 9.2 X 106 1.3 0.80 3.7 X 106 0.87 4.6 X 106 1.7 0.90 1.4 X 106 0.98 4.8 X 106 2.0 0.86 3.5 X 106 0.90 4.8 X 106 2.7 0.97 5.0 X 106 1.5 1.5 X 106 a Expressed in liters per minute per liter of medium. b Means of three determinations. Range = <10% of mean. Sporulation is expressed in number of spores per milliliter. The peak and final spore populations of cultures displaying accelerated growth were 107 to 2 X 107 spores/ml less than cultures displaying restricted growth. This difference was reproducible, provided the same lot of tryptone was used; however, its magnitude was not impressive. Still, spore yields obtained in trymin medium (excepting occasional lots of tryptone) were two- to threefold greater than those produced in the same volume of nutrient agar supplemented with Mn (unpublished data). B. stearothermophilus ATCC 7953 behaved differently in trymin medium than in the basamin medium described by Long and Williams (6). These investigators found sporulation was suppressed in aerated cultures at 55 C, although yields of 90 to 95% spores were realized when aerated cultures were incubated at 37 C. Since we obtained no growth of thermophiles at temperatures below 45 C in trymin medium, it was not possible to compare spore yields at 37 C. Excellent sporulation, however, was obtained at 60 C. This disagreement is not surprising in light of the demonstrated susceptibility of sporulation to nutritional influences. The comparative study of media supporting sporulation in B. stearothermophilus (Table 1) shows that peptone can substitute for tryptone, provided a higher concentration of the former is used. A factor or factors essential to sporulation may be present in these casein digests. Lessened sporulation in certain lots of tryptone may reflect a low level of the sporulation factor(s). Curran (2) cited unpublished work demonstrating the addition of Mn+4 to nutrient agar greatly increased sporulation in B. stearothermophilus.

VOL. 15, 1967 SPORULATION OF B. STEAROTHERMOPHILUS 979 In trymin medium, an increase in sporulation was engendered by added manganese, but an allor-none effect was not observed. Presumably, an absolute requirement for any metal ion would be difficult to demonstrate in so complex a medium. The requirement of oxygen for sporulation (Table 3) may be a requirement for vegetative growth, since it was not possible to separate growth from sporulation because of lack of a suitable replacement medium. Rates of air flow in excess of 1.3 liters per min per liter of medium appeared to suppress sporulation, but a rapid germination of newly formed spores could yield the same data. Efforts are currently directed towards characterizing the sporulation factor(s) in peptone and tryptone, with the aim of establishing its role in spore synthesis. Studies of heat resistance of spores produced in trymin medium are also in progress. AcKNowLEDGMNns This investigation was supported by a grant from Gerber Products Co. We gratefully acknowledge the assistance of R. Glaser and J. Canada in preparing the manuscript. LrrERATURE C1TED 1. CAMPBELL, L. L., C. M. RICHARDS, AND E. E. SNnF. 1965. Isolation of strains of Bacillus stearothermophilus with altered requirements for spore germination, p. 55-63. In L. L. Campbell and H. 0. Halvorson [ed.], Spores III. American Society for Microbiology, Ann Arbor, Mich. 2. CURRAN, H. R. 1957. The mineral requirements for sporulation, p. 1-9. In H. 0. Halvorson [ed.], Spores I. American Institute of Biological Sciences, Washington, D.C. 3. DAHL, L. G. 1955. The influence of some inorganic salts on the sporulation of a strain of Bacillus stearothermophilus. Physiol. Plantarum 8:661-668. 4. FINLEY, N., AND M. L. FIELDs. 1962. Heat activation and heat-induced dormancy of Bacillus stearothermophilus spores. Appl. Microbiol. 10:231-236. 5. HALVORSON, H. 0. 1957. Rapid and simultaneous sporulation. J. Appl. Bacteriol. 20:305-314. 6. LONG, S. K., AmN 0. B. WILL.AMs. 1960. Factors affecting growth and spore formation of Bacillus stearothermophilus. J. Bacteriol. 79:625-628. 7. TOwNSEND, C. T., I. I. SOMERS, F. C. LAMB, AND N. A. (OLN. 1954. A laboratory manual for the canning industry, p. 10-1-10-35. National Canners Association Research Laboratories, Washington, D.C. Downloaded from http://aem.asm.org/ on July 10, 2018 by guest