ISO 6222 pdf download.Water quality — Enumeration of culturable micro-organisms — Colony count by inoculation in a nutrient agar culture medium
1 Scope
This European Standard specifies a method for the enumeration of culturable micro-organisms in water by counting the colonies formed in a nutrient agar culture medium after aerobic incubation at 36 °C and 22 °C. The method is intended to measure the operational efficiency of the treatment process of public drinking water supplies and for general application to all types of water. It is particularly applicable to the examination of water intended for human consumption, including water in closed containers and to natural mineral waters.
2 Normative references
This European Standard incorporates provisions from other publications. These normative references are cited at the appropriate places in the text and the publications are listed thereafter. For dated references, subsequent amendment to or revisions of any of these publications apply to this European Standard only when incorporated in it by amendment or revision. For undated references the most recent edition of the publication referred to applies. EN ISO 3696 Water for analytical laboratory use – Specification and test methods (ISO 3696:1987) EN ISO 5667-3 Water quality – Sampling – Part 3: Guidance on the preservation and handling of samples (ISO 5667-3:1994) EN 25667-2 Water quality – Sampling – Part 2: Guidance on sampling techniques (ISO 5667-2:1991) ISO 6887 Microbiology – General guidance for the preparation of dilutions for microbiological examinations ISO 8199 Water quality – General guide to the enumeration of micro-organisms by culture
3 Definitions
For the purposes of this European Standard, the following definition applies: culturable micro-organisms: All aerobic bacteria, yeasts and moulds capable of forming colonies in the medium specified under the test conditions described herein.
4 Principle
Inoculation by mixing with a specified culture medium in Petri dishes, measured volumes of the samples or dilutions of the sample. Incubation of one set of plates at 36 °C for 44 h, and another set at 22 °C for 68 h. Calculation of the number of colony-forming units (c.f.u.) per millilitre (ml) of the sample from the number of colonies formed in the medium.
5 Apparatus and glassware
Usual microbiological laboratory equipment and, in particular: 5.1 Apparatus for sterilisation by steam (autoclave) 5.2 Incubator capable of maintaining a temperature of (36 ± 2) °C 5.3 Incubator capable of maintaining a temperature of (22 ± 2) °C 5.4 GIass or plastics Petri dishes with a diameter of 90 mm or 100 mm 5.5 Water bath or similar apparatus capable of maintaining a temperature of (45 ± 1) °C 5.6 Colony counting equipment with a method of illumination against a dark background
6 Sampling
Take the samples of water in accordance with the instructions for sampling, handling and preservation given in EN 25667-2 and EN ISO 5667-3. Examine water supplied in closed containers, including natural mineral waters, within 12 h of bottling, keeping the temperature of storage at (5 ± 3) °C during this period.
7 Culture media and diluents
7.1 Basic materials For the preparation of the medium, use ingredients of uniform quality and chemicals of analytical grade; alternatively use an equivalent dehydrated complete medium and follow the manufacturer’s instructions.For making media, use glass-distilled or deionised water prepared in accordance with EN ISO 3696 grade 3 and free from substances which might inhibit growth under the conditions of the test. NOTE: The use of chemicals of other grades is permissible providing they are shown to be of equal performance in the test.Add the ingredients, or the complete dehydrated medium, to the water and dissolve by heating. Adjust the pH if necessary so that after sterilization it will be 7,2 ± 0,2 at 25 °C. Distribute volumes of 15 ml to 20 ml in tubes, bottles or other containers. For storage in larger volumes, use containers up to 500 ml capacity. Sterilise in the autoclave (5.1) at (121 ± 3)°C for (15 ± 1) minutes. For use, melt the medium, allow to cool and maintain it at (45 ± 1) °C using the water bath (5.5). It is recommended to store the medium not longer than 4 h at 45 °C, after which time the medium shall be discarded.