Methylxanthines and related compounds were added to Duncan‐Strong (DS) medium in an effort to improve spore yields and enterotoxin production in three strains of Clostridium perfringens. Supplementation with 50–200 μg of caffeine/ml (0.26–1.03 mM) increased heat‐resistant spore yields for strain NCTC 8238 from 4.32 to 6.03 log10spores/ml after 24 h of incubation. With strains NCTC 8239 and NCTC 10240, 50–200 μg/ml caffeine (0.26–1.03 mM) or theophylline (0.28–1.11 mM) were equally effective, and increased spore yields from 4.18–4.89 to 6.00–7.13 log10spores/ml. When sporulation and enterotoxin production were evaluated in DS medium containing either starch or raffinose and supplemented with 100 μg/ml caffeine, heat‐resistant spores were first detected at 6 h, and enterotoxin at 3 h of incubation. Raffinose not only yielded higher spore numbers than starch, but also shortened the time to toxin detection and/or increased toxin production by two out of three strains. Strain NCTC 8238 produced enterotoxin (2 ng/ml) with raffinose within 3 h, while strain NCTC 10240 produced 2 ng/ml enterotoxin with starch and higher levels (4 ng/ml) with raffinose at 3 h. In contrast, strain NCTC 8239 produced enterotoxin (2 ng/ml) a
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