Bacterial Spores

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Abstract
With Clostridium botulinum, and to a less definite degree with other organisms, the optimum pH for sporulation in a phosphate buffered medium is slightly acid and in a number of cases it was about pH 6.2-6.3. Fermentable carbohydrates have an inhibiting effect on sporulation, mainly due to production of acid. Whenever sufficient acid is produced to bring the pH below that at which growth can occur, no sporulation takes place. With Cl. botulinum the limit for growth is about pH 6. Increasing concentrations of inorganic salts inhibit sporulation before inhibiting growth. In Cl. botulinum the chlorides seem to be quite effective in inhibiting sporulation. Nitrates in concentration of 1% have an inhibiting effect on its sporula-tion; NH4 salts have a stimulating effect, as have also sulphates to a lesser extent. Certain elements seem to be more essential for sporulation than for growth. Cl. botulinum does not sporulate in a medium consisting of peptone only. Addition of various salts to peptone shows that NI4- and phosphate-ions induce sporulation; other ions do not. The Ca- ion seems to have an added stimulating effect when added to NI4- and phosphate-ions. Increasing concentrations of O inhibit sporulation before inhibiting growth. The optimum O concentration for sporulation of anaerobes seems to be as close to 0 as it is possible to get by ordinary means. Neither growth nor sporulation was obtained with a number of anaerobes at an O concentration of 2 cm. Hg. Except with Cl. tetani and Cl. novvi, some sporulation occurred at an 0 tension of 1 cm. Hg. In aerobes, no sporulation was obtained at an O tension of 2 cm. Hg, although fair growth was observed. A number of exps. were performed with Cl. botulinum to study the change of various properties as the organisms went through the spore cycle. The properties studied were: Morphology, cataphoretic potential, thermal resistance, 0 sensitivity, and pH and toxicity of medium. In each case, change of the different properties of the organisms was correlated with morphology. The cataphoretic potential of spores was lower than that of specific vegetative cells. Germinating spores had the highest potential. Thermal death point determinations showed that a great number of spores produced were not very resistant to high temp. and must be regarded as failures. Only 1-2% of the newly formed spores survived heating at 99[degree] C. for a few minutes, 10% survived 80[degree]; 20%, 70[degree]; and 50%, 60[degree]. Only vegetative cells were O-sensitive; these were so sensitive that most of them were killed by less than 2-3 min. exposure to air. Change from non-sensitive spore to exceedingly sensitive vegetative cell was so abrupt that only in a few cases was the sensitivity of intermediary stages obtained. Toxin production did not begin appreciably until the organisms were about to stop multiplying and sporulation had begun. During the period of sporulation, the toxin titer increased very rapidly and reached a maximum at the time sporulation was complete. It then decreased, due to destruction of the toxin, especially if the toxin were allowed to come in contact with air.

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