AS 4276.17.1:2016 Water microbiology
8 TEST PROCEDURES
The procedure shall be as follows:
(a) Heat the sample to 60 ±2°C in a water bath and maintain this temperature for 15 ±1 min. The volume heated should be greater than the volume to be analysed. The temperature should be monitored by placing an appropriate thermometer in a reference bottle of the same size as the sample bottle and containing the same volume of water at the same initial temperature as the sample being treated. The time taken to reach 60 ±2°C shall not exceed 15 min and can be minimized by ensuring the water in the water bath is circulated to maximize heat exchange.
(b) After heat treatment, prepare and filter the sample as described in AS 4276.1, making any necessary dilutions.
(c) Waters with low levels of competing spore forming bacteria Place the membrane face-upwards on a TSC plate ensuring no air bubbles are trapped under the filter.
Optionally, overlay with agar by carefully pouring 15–20 mL of TSC agar,pre-cooled to 45 ±1°C, over the membrane and allow to set.
The time between placing the membrane on the TSC agar and starting incubation should be as short as possible and shall not exceed 1 h.
Incubate the plates, inverted to avoid interference from condensing water,anaerobically at 44 ±1°C for 21 ±3 h.
(d) Waters with high levels of competing spore forming bacteria Place the membrane face-upwards on a OPSP agar plate ensuring no air bubbles are trapped under the filter.Optionally, overlay with agar by carefully pouring 15–20 mL of OPSP agar,pre-cooled to 45 ±1°C, over the membrane and allow to set.
The time between placing the membrane on the OPSP agar and starting incubation should be as short as possible and shall not exceed 1 h.
Incubate the plates, inverted to avoid interference from condensing water,anaerobically at 36 ±2°C for 21 ±3 h.
When interpretation of results becomes problematic due to interference from spreading microorganisms or unacceptable confirmation rates (presumptive C.perfringens colonies not confirming), or when samples have a history of problematic results, OPSP agar may be incubated at 44 ±1°C.
NOTE: Increasing the selectivity of a procedure may not recover some strains of the target microorganism.