Fluorescent in situ hybridization in combination with filter cultivation (FISHFC) method for specific detection and enumeration of viable Clostridium perfringens.
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 degrees C, and incubation for 6h. Under these conditions, micro-colony diameters reached 100 microm, a size sufficient for hybridization. Enumeration of C. perfringens using the CLP-180-aided FISHFC method was realized in 9h 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.
Division of Marine Life Science, Faculty of Fisheries Sciences, Hokkaido University, Hakodate 041-8611, Japan.
S
S
Shimizu
M
M
Ootsubo
Y
Y
Kubosawa
I
I
Fuchizawa
Y
Y
Kawai
K
K
Yamazaki
D003016:Clostridium perfringens / Q000302:isolation & purification*; D015169:Colony Count, Microbial / Q000379:methods / Q000592:standards; D003257:Consumer Product Safety; D005506:Food Contamination / Q000032:analysis*; D017404:In Situ Hybridization, Fluorescence / Q000592:standards*; D008851:Micropore Filters; D012336:RNA, Ribosomal, 16S / Q000737:chemistry / Q000235:genetics; D012680:Sensitivity and Specificity; D013045:Species Specificity; D013997:Time Factors
D012336:RNA, Ribosomal, 16S