Improved Medium for Sporulation of <i>Clostridium perfringens</i>

TLDR

The new medium comprises yeast extract, proteose peptone, soluble starch, sodium thioglycolate, Na₂HPO₄, and 7H₂O at defined concentrations. The medium produced 10‑ to 10,000‑fold higher spore yields than SEC, 100‑ to 1,000‑fold higher than Ellner, and 10‑ to 100‑fold higher than Kim et al., with activated carbon further increasing yield and heat resistance, and optimal heat shock (60‑80 °C for 20 min) producing a 100‑fold increase over 50 °C.

Abstract

An improved sporulation medium has been developed in which all five strains of Clostridium perfringens tested exhibited a 100- to 10,000-fold increase in numbers of spores when compared with spore yields in SEC medium under comparable conditions. In addition, three of five strains produced a 100- to 1,000-fold increase, with the remaining two strains yielding approximately the same numbers of spores, when compared with strains cultured in Ellner medium. At the 40-hr sampling time, 18 of 27 strains produced a 10- to 100-fold increase in numbers of spores in our medium, when compared to spore production obtained in a medium recently reported by Kim et al. The new medium contained yeast extract, 0.4%; proteose peptone, 1.5%; soluble starch, 0.4%; sodium thioglycolate, 0.1%; and Na 2 HPO 4 . 7H 2 O, 1.0%. In some cases, the spore yield could be increased by the addition of activated carbon to the new medium. The inclusion of activated carbon in the medium resulted in spores with slightly greater heat resistance than spores produced in the new medium without added carbon or in SEC or in Ellner medium. The major differences in heat resistance of the various strains appeared to be genetically determined rather than reflections of a particular sporulation medium. A definite heat-shock requirement was shown for four of four strains, with the optimal temperature ranging from 60 C for a heat-sensitive strain to 80 C for a heat-resistant strain. Heating for 20 min at the optimal temperature resulted in a 100-fold increase over the viable count obtained after heating for 20 min at 50 C.