Physiology of the Sporulation Process in Clostridium Botulinum. I. Correlation of Morphological Changes with Catabolic Activities, Synthesis of Dipicolinic Acid, and Development of Heat Resistance.

Day, Lawrence E. (Michigan State University, East Lansing), and Ralph N. Costilow. Physiology of the sporulation process in Clostridium botulinum. I. Correlation of morphological changes with catabolic activities, synthesis of dipicolinic acid, and development of heat resistance. J. Bacteriol. 88:690-694. 1964.-A reasonable degree of synchrony in the sporulation of Clostridium botulinum 62-A was attained by using a large inoculum of a young culture into a medium containing 4% Trypticase and 1 ppm of thiamine. Sporulation was complete within 24 to 36 hr. Cells harvested at various intervals were studied for their fermentative activity with l-alanine and l-proline as substrates. The Q values (microliters of gas per hour per milligram of dry cells) were maximal at the time a large percentage of the cells had initiated sporulation as indicated by swelling. They declined to a plateau at about the same level as found in vegetative cells by the time 10% of the cells had completed sporulation, and finally to a much lower level when sporulation was completed. The rates of accumulation of volatile acids (acetic, valeric, and propionic acids) corresponded closely with the catabolic potential observed. However, in the case of acetic acid, there was a significant decrease in the total acid present as the number of mature spores increased to over 50% of the final number. The total acetic acid then increased at a slow rate. The production of basic compounds during growth and sporulation more than balanced the rate of acid production, because the hydrogen ion concentration decreased exponentially throughout the period as indicated by the steady increase in pH. The synthesis of dipicolinic acid coincided closely with the development of heat resistance. Refractility developed 3 to 5 hr in advance of heat resistance.