Int. J. Environ. Res. Public Health 2012, 9 2.7.2. Enterococci 2766 An aliquot (100 mL) of the sample collected should be filtered through a 47 mm cellulose ester membrane with filtration characteristics corresponding to a nominal pore size of 0.45 µm. Place the membrane over the Slanetz and Bartley agar medium and incubate at 36 ± 1 °C for 40–48 hours. Following incubation, colonies ranging from pink to dark red and brown (in the centre or throughout the colony) are deemed typical (presumed Enterococci). Tests should be undertaken to confirm enterococcus status: the presence of the catalase enzyme and the esculin hydrolysis test should be undertaken on all, or at least a representative number of typical colonies and biochemical identification tests performed. Tests should be carried out on colonies aged no more than 24 hours (UNI EN ISO 7899-2: 2003) [20]. 2.7.3. Pseudomonas aeruginosa An aliquot (250 mL) of the sample collected should be filtered through a 47 mm cellulose ester membrane with filtration characteristics corresponding to a nominal pore size of 0.45 µm. Place the membrane over the Pseudomonas agar/CN medium and incubate at 36 ± 1 °C for 40–48 hours After (2 ± 2) and (44 ± 4) hours assess the growth of typical colonies on isolation medium. Pseudomonas aeruginosa may develop as greenish-blue colonies that produce pyocyanin, fluorescent or reddish-brown colonies. Count all characteristic colonies (presumed to be Pseudomonsas aeruginosa). Tests should then be undertaken to confirm Pseudomonsas aeruginosa status: place under a Woods lamp and following observation on all, or at least a representative number of typical colonies, biochemical identification tests should be performed. Tests should be carried out on colonies aged no more than 24 hours (UNI EN ISO 12780:2002) [20]. 2.7.4. Clostridium perfringens An aliquot (100 mL) of the sample collected should be filtered through a 47 mm cellulose ester membrane with filtration characteristics corresponding to a nominal pore size of 0.45 µm. Place the membrane over the Tryptose Sulfite Cycloserine Agar medium, and cover the membrane completely with 5–6 mL of liquid culture medium at a temperature of 50 ± 5 °C. Leave to set and incubate at a temperature of 44 ± 1 °C for 21 ± 3 hours under anaerobic conditions. Micro-organisms belonging to the Clostridium perfringens species form black or grey/yellowish-brown colonies on the Tryptose Sulfite Cycloserine Agar medium, conveying a slightly darker shade above or below the filtrating membrane. Count all typical colonies and treat as presumed Clostridium perfringens. Isolate colonies on two parallel blood Agar plates and incubate one plate under anaerobic conditions and the other under aerobic conditions at 36 ± 1 °C for 21 ± 3 hours. Following incubation, examine plates to check the presence or absence of growth. Clostridium perfringens colonies typically produce areas of clear hemolysis on culture medium. Undertake confirmation tests only on colonies growing on blood Agar under anaerobic conditions, and subsequently perform biochemical identification tests. Tests should be carried out on colonies aged no more than 24 hours. Method ISS A 005B REV.00 (National Standard Method W 5 ISSU 3 dell’Health Protection Agency).