Effect of sporulation medium on heat resistance, chemical composition and germination of Bacillus megaterium spores

Journal of Bacteriology (Impact Factor: 2.81). 05/1964; 87:876-86.
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Levinson, Hillel S. (U.S. Army Natick Laboratories, Natick, Mass.), and Mildred T. Hyatt. Effect of sporulation medium on heat resistance, chemical composition, and germination of Bacillus megaterium spores. J. Bacteriol. 87:876-886. 1964.-Bacillus megaterium spores, grown on variously supplemented media, had varying concentrations of P, Ca, Mn, or dipicolinic acid. Supplementation with CaCl(2) yielded spores with increased heat resistance; addition of l-glutamate, l-proline, or increase of the phosphate concentration yielded spores with reduced heat resistance. Germination characteristics depended on both the sporulation medium and the germinant (glucose, l-alanine, l-leucine, or KNO(3)); pronounced differences were demonstrable with glucose and l-alanine, which trigger germination via different metabolic pathways. An increase in CaCl(2) during sporulation yielded spores with increased germination in glucose but not in l-alanine. Germination in l-alanine was optimal with spores produced on media containing 0.1 mm MnCl(2), but germination of such spores was minimal in glucose. An increase in the sporulation medium phosphate decreased the initial germination rate in glucose, but not in l-alanine. Spores produced in CaCl(2)-supplemented media had increased heat-activation requirements (increased dormancy) for germination induced by l-alanine, and decreased heat-shock requirements for glucose-induced germination. An increase of sporulation phosphate yielded spores with reduced dormancy for germination induced by l-alanine, but with unchanged dormancy on the other germinants. Spores produced with added l-glutamate had reduced dormancy for glucose-induced germination, and increased dormancy for germination induced by l-alanine. Addition of CaCl(2) or l-glutamate to the sporulation medium yielded spores with increased sensitivity to "ionic germination" (with KI). Spores from synthetic medium were incapacitated for full postgerminative development, as shown by repression of the changes in oxygen-uptake rate which accompany normal cell division.

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    • "It has been well known that the calcium ion contributes to heat resistance of spores (Slepecky and Foster, 1959). Since CaCl 2 has also been suggested to be effective in enhancing heat resistance of Bacillus spores (Bhothipaksa and Busta, 1978; Fleming and Ordal, 1964; Levinson and Hyatt, 1964), the fact that the addition of CaCO 3 causes the most rapid sporulation of C. sporogenes vegetative cells led us to speculate that it may function not only in inducing sporulation but also in enhancing heat resistance. To test this speculation, D and Z values of the spores that had been produced by adding CaCl 2 and CaCO 3 to a sporulation medium were determined. "
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    ABSTRACT: In this study, various mineral supplements, such as chloride salts (CaCl2, MgCl2, MnCl2, FeCl2 and KCl) supplying cations and calcium salts (CaCl2, CaCO3, CaSO4, Ca(OH)2 and CaHPO4) supplying anions, were tested if they could stimulate the sporulation of Clostridium sporogenes, a surrogate microorganism for C. botulinum. Of the cations tested, the addition of CaCl2 showed a slightly, but not significantly, greater increase in spore levels within 3 weeks of incubation, compared to that of the other cations. The optimum concentration of CaCl2 was 0.5%, which yielded nearly 10(4) CFU/ml of spores. Of the anions tested, CaCO3 promoted sporulation within one week, which was the most effective compound for promoting rapid sporulation among the minerals tested. CaSO4 produced a pattern of sporulation similar to that of CaCl2. While CaHPO4 resulted in the maximum production of spores after 4 weeks, Ca(OH)2 failed to induce sporulation. With an optimized concentration of 0.5% CaCO3, the spore yield was approximately 10(5) CFU/ml. The spores prepared in sporulation medium with CaCO3 (pH 5.0) had slightly, but not significantly, higher D values than those produced with CaCl2 (pH 5.0) at temperatures ranging from 113 to 121 degrees C. However, no significant differences were observed in Z values (both 10.76 degrees C). In a large scale spore production, D(121 degrees C) values of the spore crops prepared with CaCl2 and CaCO3 and resuspended in phosphate buffer (pH 7.0) were found to be both 0.92 min. In conclusion, our data suggest that CaCO3 is highly effective in reducing sporulation time as well as enhancing heat resistance.
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    ABSTRACT: Hyatt, Mildred T. (Pioneering Research Division. U.S. Army Natick Laboratories, Natick, Mass.), and Hillel S. Levinson. Effect of sugars and other carbon compounds on germination and postgerminative development of Bacillus megaterium spores. J. Bacteriol. 88:1403-1415. 1964.-A total of 77 carbon-containing compounds were tested for their ability to support germination and postgerminative development of Bacillus megaterium spores. The only effective germination agents were certain of the hexose sugars and their derivatives. With unheated spores, only d-glucose, d-mannose, 2-deoxy-d-glucose, d-glucosamine, and N-acetyl-d-glucosamine (all at 25 mm) supported appreciable germination (ca. 25%). Heat-shock at 60 C for 10 min increased germination and decreased the concentration of sugar required for germination, so that these compounds, at 2.5 mm, supported 40 to 60% germination. Higher concentrations (25 mm) of other compounds, d-fructose, l-sorbose, d-allose, d-altrose, 2-hydroxyethyl-d-glucose, and beta-methyl-d-glucoside, were required for appreciable germination of heated spores. Glucose or mannose contamination accounted for the germination apparently induced by certain other sugars. Ionic contamination did not appear to contribute to the germination induced by d-glucose, d-fructose, 2-deoxy-d-glucose, or l-sorbose. There was no clear-cut evidence for a multiplicity of metabolic pathways in the triggering of B. megaterium spore germination by various sugars. Postgerminative development of germinated spores was supported by a wider variety of carbon compounds, including some pentoses and hexoses, many oligosaccharides, sugar derivatives, some alcohols, and some of the tricarboxylic acid cycle intermediates. Compounds effective for germination were not necessarily utilizable for growth, and vice versa. Oxygen consumption rates reflected the progress and extent of postgerminative development on the various carbon compounds. Utilization of glucose during postgerminative development was followed, and the concentration requirements were determined.
    Journal of Bacteriology 12/1964; 88:1403-15. · 2.81 Impact Factor
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