Bacterial isolates, growth conditions and biochemical identification

The milk samples obtained were centrifuged for 20 min at 3,000 rpm. The sediments of each milk sample were added to cooked meat broth (CMB) after removing the cream and supernatant. The samples were incubated anaerobically according to [18]. A loopful from each sample was streaked onto sheep blood agar plates containing 150 µg/ml neomycin sulfate and incubated anaerobically at 37 °C for a further 24 h after overnight incubation using anaerobic jars containing 95% H2 and 5% CO2 (AnaeroGen, OXOID, Ltd, England). To eliminate non-spore-forming bacteria, sterile swabs from diarrheal fecal samples were diluted in PBS (1:10) and grown in CMB at 80 °C in a water bath for 5 min. The CMB tubes were then incubated anaerobically at 37 °C the next day in a jar with gas production kits and then transferred to sheep blood agar with neomycin. C. perfringens has been distinguished from other Clostridium spp. by biochemical screening tests, including oxidase, catalase, motility nitrate reduction, blood hemolysis, indole production, urea hydrolysis, lecithinase, and sugar fermentation tests [19].