Detection of Clostridium tyrobutyricum using cultivation and biochemical methods and polymerase chain reaction

Abstract

Anaerobic spore-forming bacteria of the genus Clostridium are commonly present in raw milk and some milk products. Their spores can survive pasteurization and can provoke so called late blowing defect in cheese caused by butyric acid fermentation. The only species of the genus Clostridium that is able to provoke late blowing is Clostridium tyrobutyricum.In this work, two cultivation methods for detection of butyric acid producing clostridia in raw and pasteurized milk and in cheese samples were compared. The results show that tube method is suitable for route identification (in concentration 102 CFU/ml or /g) of clostridia in milk and cheese. The standard cultivation technique is suitable for more sensitive identification (10 CFU/ml or /g). All presumptive colonies grown anaerobically on selective RCM agar with polymyxine B (500 μg/ml) were classified to be of species Clostridium tyrobutyricum using PCR only. The confirmation using API tests were different in 50 % cases. The results show, that described PCR method is suitable for rapid screening of the presence of Clostridium tyrobutyricum in milk and cheese. PCR from one colony is possible to use for the analysis.

Keywords

• <i>Clostridium tyrobutyricum</i>
• raw and pasteurized milk
• cheese
• species-specific PCR