Increased Sporulation of Clostridium perfringens in a Medium Prepared with the Prereduced Anaerobically Sterilized Technique or with Carbon Dioxide or Carbonate

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Abstract
Sporulation of ten strains of Clostridium perfringens was determined in Duncan-Strong sporulation medium prepared and stored in the presence of atmospheric oxygen (DS) or prepared as a prereduced anaerobically sterilized medium (DSPR). Numbers of heat-resistant spores were higher in DSPR compared to DS for 4 strains in fresh media and for 7 strains in stored media. For strains 8679 and 8799, spore numbers were as much as 5 logs greater in DSPR than in DS with a concomitant higher culture pH and fewer vegetative cell numbers. The effects of the addition of an antioxidant, degraders of hydrogen peroxide (H2O2), or scavengers of oxygen radicals to DS or H2O2 to DSPR do not suggest a role for toxic oxygen species in inhibiting sporulation of these two strains in DS. Increases in spore numbers and culture pH were similar in DSPR and in DS to which carbon dioxide (CO2) or sodium carbonate had been added. Modifications of sporulation media which increased the levels of CO2 or increased the culture pH improved sporulation of strains 8679 and 8799, respectively. These results suggest that improved sporulation in DSPR resulted from an improved buffering effect on culture pH or a direct effect of CO2 introduced during preparation of the prereduced medium.