The use of egg yolk-free tryptose-sulphite-cycloserine (TSC) agar for the membrane filtration enumeration of Clostridium perfringens was evaluated using domestic sewage effluents and polluted surface waters. The medium recovered higher numbers of C. perfringens than a medium containing polymixin (mCP). Overlaying the membrane with TSC or cycloserine-free TSC did not improve recoveries. Supplementation of the medium with 50 mg l−1 neomycin sulphate resulted in significantly lower recoveries of C. perfringens. Comparison of two commercial formulations of TSC showed no difference in recoveries. A two tube confirmation procedure based on motility, nitrate reduction, lactose fermentation and gelatin liquefaction was found to be reliable to confirm C. perfringens from TSC plates. Although not as specific as mCP for the enumeration of C. perfringens TSC aappeared to be selective for sulphite reducing clostridia of predominantly faecal or urino-genital origin. A concept of faecal clostridia and C. perfringens (based on enumeration on TSC agar) analogous to that of faecal coliforms and E. coli is briefly discussed.

We first determined the analytical specificity and ubiquity (i.e., the ability to detect all or most strains) of a Clostridium perfringens-specific real-time PCR (rtPCR) assay based on the cpa gene (cpa rtPCR) by using a bacterial strain panel composed ofC.

perfringens and non-C. perfringens Clostridium strains. All non-C. perfringens Clostridium strains tested negative, whereas all C.

perfringens strains tested positive with the cpa rtPCR, for an analytical specificity and ubiquity of 100%. The cpa rtPCR assay was

then used to confirm the identity of 116 putative C. perfringens isolates recovered after filtration of water samples and culture on

mCP agar. Colonies presenting discordant results between the phenotype on mCP agar and cpa rtPCR were identified by sequencing the 16S rRNA and cpa genes. Four mCP/rtPCR colonies were identified as C. perfringens, whereas 3 mCP/rtPCR

colonies were identified as non-C. perfringens. The cpa rtPCR was negative with all 51 non-C. perfringens strains and positive

with 64 of 65 C. perfringens strains. Finally, we compared mCP agar and a CRENAME (concentration and recovery of microbial

particles, extraction ofnucleic acids, and molecular enrichment) procedure plus cpa rtPCR (CRENAME cpa rtPCR) for their

abilities to detect C. perfringens spores in drinking water. CRENAME cpa rtPCR detected as few as one C. perfringens CFU per

100 ml of drinking water sample in less than 5 h, whereas mCP agar took at least 25 h to deliver results. CRENAME cpa rtPCR

also allows the simultaneous and sensitive detection of Escherichia coli and C. perfringens from the same potable water sample.

In itself, it could be used to assess the public health risk posed by drinking water potentially contaminated with pathogens more

resistant to disinfection.

Dear Mehdi,

Thank you for the recommended article, I will consider it.

Prakit

I agree with Mehdi answer ...you can see this article i hope its useful

good luck

http://pubmedcentralcanada.ca/pmcc/articles/PMC243401/pdf/aem00205-0073.pdf 

Hi Patik,

We use mCP membrane filtration for Clostrium perfringens enumeration regularly, and we use ammonium hydroxide for confirmation. Post -incubation our colonies are yellow (straw) coloured on the mCP agar, and turn pink on exposure to the ammonium hydroxide, our filter paper remains white the mCP media remains a dark pink, we consider all pink colonies C. perfringes positive. Hope this helps.