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A modification of the UK reference and ISO 14189 TSCA medium for the enumeration of Clostridium perfringens from water coupled with a membrane filter transfer technique for testing for production of acid phosphatase was evaluated. The new tryptose cycloserine agar (TCA) medium, which lacks sodium metabisulphite but contains sodium pyruvate to improve recovery, allows the isolation and confirmation of Cl. perfringens within 18–24 h of sample processing. Data from a multilaboratory study analysed according to ISO 17994 showed that TCA was equivalent to TSCA for the enumeration of Cl. perfringens. The identification of acid phosphatase-negative isolates revealed a false-negative rate for the TCA method of 0·8%. The TCA membrane filter transfer procedure provides confirmed Cl. perfringens counts in half the time of the TSCA method and is simple to undertake.The testing of drinking water for Clostridium perfringens is a regulatory parameter in Europe and the UK. Current UK and ISO methods employ membrane filtration (MF) and TSCA medium followed by subculture and confirmation of isolates by testing for acid phosphatase. This takes 48 h. We present here the results of a multilaboratory evaluation of a MF method that features a simplified isolation medium (TCA) and a membrane transfer procedure for the acid phosphatase test resulting in confirmed results being available in 18–24 h. This development significantly reduces the time to confirmed results for Cl. perfringens from water samples.