BS EN ISO 7899-2:2000 pdf – Water quality Ð Detection and enumeration of intestinal enterococci Ð Part 2: Membrane filtration method.
1? Scope This part of ISO 7899 specifies a method for the detection and enumeration of intestinal enterococci in water by membrane filtration. This part of ISO 7899 is especially intended for examination of drinking water, water from swimming pools and other disinfected or clean waters. Nevertheless, the method can be applied to all types of water, except when a large amount of suspended matter or many interfering microorganisms are present. It is particularly suitable for the examination of large volumes of water containing only a few intestinal enterococci. 2? Normative references The following normative documents contain provisions which, through reference in this text, constitute provisions of this part of ISO 7899. For dated references, subsequent amendments to, or revisions of, any of these publications do not apply. However, parties to agreements based on this part of ISO 7899 are encouraged to investigate the possibility of applying the most recent editions of the normative documents indicated below. For undated references, the latest edition of the normative document referred to applies. Members of ISO and IEC maintain registers of currently valid International Standards. ISO 3696:1 987, Water for analytical laboratory use — Specification and test methods. ISO 5667-1 :1 980, Water quality — Sampling — Part 1 : Guidance on the design of samping programmes. ISO 5667-2:1 991 , Water quality — Sampling — Part 2: Guidance on sampling techniques. ISO 5667-3:1 994, Water quality — Sampling — Part 3: Guidance on the preservation and handling of samples . ISO 6887-1 :1 999, Microbiology of food and animal feeding stuffs — Preparation of test samples, initial suspension and decimal dilutions for microbiological examination — Part : General rules for the preparation of the initial suspension and decimal dilutions. ISO 81 99:1 988, Water quality — General guide to the enumeration of micro-organisms by culture. ISO/IEC Guide 2:1 996, Standardization and related activities — General vocabulary.
3? Terms and definitions For the purposes of this part of ISO 7899, the terms and definitions given in ISO/IEC Guide 2 and the following apply. 3.1 intestinal enterococci bacteria which are able to reduce 2,3,5-triphenyltetrazolium chloride to formazan and to hydrolyse aesculin at 44 °C on the media (6.3.1 and 6.3.2) specified in this part of ISO 7899 NOTE? See also annex A. 4 Principle 4.1? Filtration, incubation and enumeration The enumeration of intestinal enterococci is based on filtration of a specified volume of water sample through a membrane filter with a pore size (0,45 µm) sufficient to retain the bacteria. The filter is placed on a solid selective medium containing sodium azide (to suppress the growth of Gram-negative bacteria) and 2,3,5-triphenyltetrazolium chloride, a colourless dye, that is reduced to red formazan by intestinal enterococci. Typical colonies are raised, with a red, maroon or pink colour, either in the centre of the colony or throughout. 4.2? Confirmation If typical colonies are observed, a confirmation step is necessary, by transfer of the membrane, with all the colonies, onto bile-aesculin-azide agar, preheated at 44 °C. Intestinal enterococci hydrolyse aesculin on this medium in 2 h. The end-product, 6,7-dihydroxycoumarin, combines with iron(III) ions to give a tan-coloured to black compound which diffuses into the medium. 5? Apparatus Except for disposable glassware which is delivered sterile, glassware shall be sterilized in accordance with ISO 81 99. Usual microbiological laboratory equipment and particularly: 5.1? Membrane filtration apparatus, according to ISO 81 99. 5.2? Sterile membrane filters, with a nominal pore size of 0,45 µm. The quality of membrane filters may vary from brand to brand or even from batch to batch. It is therefore advisable to check the quality on a regular basis, in accordance with ISO 7704. 5.3? Incubator, capable of being maintained at 36 °C ? 2 °C. 5.4? Incubator, capable of being maintained at 44 °C ? 0,5 °C.
6? Culture media and reagents 6.1? Basic materials WARNING — The selective media described in this part of ISO 7899 contain sodium azide. As this substance is highly toxic and mutagenic, precautions shall be taken to avoid contact with it, especially by the inhalation of fine dust during the preparation of commercially available dehydrated complete media. Azide-containing media should not be mixed with strong inorganic acids, as toxic hydrogen azide (HN 3 ) may be produced. Solutions containing azide can also form explosive compounds when in contact with metal pipework, for example from sinks. Azides can be decomposed safely by the addition of an excess of a saturated nitrite solution. For uniformity of results, in the preparation of media, either use a dehydrated complete medium or use constituents of uniform quality and chemicals of recognized analytical grade. Sodium azide deteriorates with time so that dehydrated media have a limited shelf-life. NOTE? Use of chemicals of another quality is possible provided they are shown to be of equal performance in the test. 6.2? Distilled water or water of equivalent purity, in accordance with ISO 3696, Grade 1 .