ISO 14189 pdf download Water quality — Enumeration of Clostridium perfringens — Method using membrane filtration
This International Standard specifies a method for the enumeration of vegetative cells and spores ofClostridium perfringens by the membrane filtration method in samples of water intended for humanconsumption. However, the method can be applied to all types of water samples provided they do notcontain particulate or colloidal matter that interferes with filtration.
2 Normative references
The following documents, in whole or in part, are normatively referenced in this document and areindispensable for its application, For dated references, only the edition cited applies. For undatedreferences, the latest edition of the referenced document (including any amendments) applies
ISO 8199, Water quality – General guidance on the enumeration ofmicro-organisms by culture
ISO/TS 11133-1, Microbiology offood and animalfeeding stuffs – Guidelines on preparation and productionof culture media — Part 1: General quidelines on quality assurance for the preparation of culture media inthe laboratory
SO 19458, Water quality — Sampling for microbiological analysisISO/IEC Guide 2:2004,Standardization and related activities – General vocabulary
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO/IEC Guide 2 and thefollowing apply:
presumptive Clostridium perfringensbacteriawhichproduceallshades ofblackor greyto vellowbrown colonies ontryptose-sulfite-cycloserineagar, even if the colour is faint, after anaerobic incubation at (44 + 1) C for (21 + 3) h
Note 1 to entry: Unlike colonies growing directly on the agar medium, colonies on the membrane do not alwaysdisplay a distinct blackening, so faint colonies are included in the count.
confirmed Clostridium perfringensbacteria that produce characteristic colonies on tryptose-sulfite-cycloserine agar and possess theenzyme acid phosphatase
A measured volume of the sample, or a dilution of it, is filtered through a membrane with a pore size of 0,45 µm sufficient to retain spores of clostridia. The membrane is incubated on a selective/differential agar (tryptose-sulfite-cycloserine agar) anaerobically at (44 ± 1) °C for (21 ± 3) h. C. perfringens usually produce black or grey to yellow brown colonies as a result of the reduction of sulfite to sulfide which reacts with a ferric salt in the medium. Characteristic colonies are counted and confirmatory tests are carried out. The result is calculated as the colony count per sample volume. If a count of spores alone is required the sample is first pre-treated at (60 ± 2) °C to inactivate vegetative bacteria.
NOTE 1 The medium contains cycloserine as a selective agent to inhibit Bacillus species.
NOTE 2 Incubation at 44 °C increases the selectivity of the test for C. perfringens.
5 Apparatus and glassware
Except for disposable glassware or plastics ware which is delivered sterile, sterilize glassware as specified in ISO 8199.
Usual microbiological equipment and particularly:
5.1 Membrane filtration apparatus, as specified in ISO 8199.
5.2 Sterile filter funnels
Use funnels either delivered sterile or sterilized as specified in ISO 8199. Alternatively flaming of funnels made of metal prior to their use is acceptable.
NOTE For this method it is insufficient to disinfect funnels by boiling.