AS 4276.17.2:2016 – Water microbiology Method 17.2: Spores of Clostridium perfringens—Estimation of most probable number (MPN) using the multiple tube dilution technique.
6.2 Pipettes or equlsalen measuring devices
I mL, 10 mE, 50 mL
6.3 Anaerobic incuhatorsfjar
With appropriate equipment and materials to generate an atmosphere of approximately 90% hydrogen and 10% carbon dioxide.
6.4 incubators or water bath
Able to maintain at 36 ±2°C. 44 ±1°C and 60 ±2°C.
Sample holding and storage times shall be in accordance with AS 2031.
The procedure shall be as follows:
(a) [feat the sample to 60 ±2°C in a waterbath and maintain this temperature br 1 ±1 mm. The solume heated should he greater than the oIume to be analysed. The temperature should be monitored by placing an appropriate thermometer in a reference bottle of the same size as the sampic 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 mm and can be minimized by ensuring the water in the water bath is circulated to maximize heat exchange.
(bI Using DRCSI heated by bringing to the boil or steaming for 5 minutes to drive off oxygen and cooled immediately before use, prepare and inoculate bottles with appropriate volumes dilutions of the heated sample, as described for the MPN method in ASNZS 4276.1. Top-up with single strength medium so as to leave a minimal amount of air space.
(c) Incubate the bottles at 44 ±lC for 48±4 h. A positive reaction is shown by blackening of the nwdium
NOTE: Large volumes of culture in sealed bottles may explode. Bottles may be sealed in plastic bags in order to contain any breakage or spillage during incubation. Alternatively, the bottles may bc incubated anaerobically with the caps loosened and with sufficient hcadspacc to avoid explosions.
9.1 Wafers with low levels of competing spore forming bacteria
The procedure shall be as follows: Streak a loopful of broth from blackened DRCM tubes onto TSC agar plates to obtain single colonies and incubate anaerobically at 44 ±1CC for 21 ±3 h.
9.2 Waters with high levels of competing spore forming bacteria
The procedure shall be as follows: Streak a looplul of broth from blackened DRCM tubes onto OPSP agar plates to obtain single colonies and incubate anaerobically at 36 i2C for 21 ±3 h.
When interpretation of results becomes problematic due to interference from spreading micro-organisms 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 clcctivity of a procedure may not recover some strains of the target microorganism.
9.3 SelectIon of colonies
The procedure shall be as follows:
(a) C. perfringen.c produces black or grey to yellow-brown colonies on TS( and orsP agar. Since the black colour ci the colonies rapidly fades and finally disappears under aerobic conditions, the plates have to be counted within 30 mm after completion of the anaerobic incubation. If a number of anaerobic jars arc used, the plates should he checked jar by jar or in portions if the incubation was performed in an anaerobic incubator.
(b) The target colonies arc subculturcd onto Columbia blood agar or a suitable nutrient- rich agar with or without blood (e.g. Columbia agar base, try plone soya agar). For waters with high background counts or where oihcr sulfite reducing bacteria may make it difficult to isolate C’. perfringens (e.g. spreading colonies), the use of Columbia blood agar (or a suitable nutrient rich agar containing blood) with neomycin (0.01%) may be beneficial.
(c) Incubate anaerobically at 44 ±1°C for 21 ±3 h.
NOTE: Non-selective media arc preferred when undertaking confirmation testing. 6cncrally. thr are some strains of a target micrLrganism that are inhibited by a given selective agent.
9.4 Biochemical confirmation
9.4.1 Acid phosphatase lest
The procedure shall be as follows: Pre-warm acid phosphatase reagent to approtimatelv 36°C for 30—60 minutes prior to use. Add 2 to 3 drops of the acid phosphatasc reagent onto filter paper and spread colonies, grown anaerobically on Columbia blood or a suitable nutrient rich agar plate, onto the phosphate reagent soaked paper. A purplish colour developing within 3 to 4 mm is considered as a positive reaction.
9.4.2 ,4hernatire biochemical tests.