Fluorescent in situ hybridization in combination with filter cultivation (FISHFC) method for specific detection and enumeration of viable Clostridium perfringens Original Research Article

To reduce time for enumeration of viable Clostridium perfringens, fluorescence in situ hybridization in combination with filter cultivation (FISHFC) was employed. The method utilized a CLP-180 probe, based on the 16S rRNA region of C. perfringens, and FISHFC fluorescence microscopy to detect C. perfringens, but not organisms from other species. Optimal cultivation requirements for micro-colony formation were TSC medium, anaerobic conditions, 37 °C, and incubation for 6 h. Under these conditions, micro-colony diameters reached 100 μm, a size sufficient for hybridization. Enumeration of C. perfringens using the CLP-180-aided FISHFC method was realized in 9 h as compared to 3–5 days required by the conventional plate count method. Moreover, viable C. perfringens counts of food samples from the two methods were not significantly different. It was concluded that the CLP-180-aided FISHFC is faster than conventional plate count methods and equally accurate (detection limit: 2 log CFU/g). The CLP-180-aided FISHFC method for rapidly evaluating C. perfringens in food samples, can contribute to ensuring food safety.