1、BRITISH STANDARD BS EN ISO 6222:1999 BS 6068-4.5: 1999 Water quality Enumeration of culturable micro-organisms Colony count by inoculation in a nutrient agar culture medium The European Standard ENISO6222:1999 has the status of a British Standard ICS 07.100.20BSENISO6222:1999 This British Standard,
2、having been prepared under the directionof the Health and Environment Sector Committee,was published underthe authority of the Standards Committee and comes into effect on 15August1999 BSI 03-2000 ISBN 0 580 32495 8 Amendments issued since publication Amd. No. Date CommentsBSENISO6222:1999 BSI 03-20
3、00 i Contents Page National foreword ii Foreword 2 Text of EN ISO 6222 3BSENISO6222:1999 ii BSI 03-2000 National foreword This British Standard is the English language version of ENISO6222:1999. It is identical with ISO6222:1999. It supersedes BS6068-4.5:1989 which is withdrawn. The UK participation
4、 in its preparation was entrusted by Technical Committee EH/3, Water quality, to Subcommittee EH/3/4, Microbiological methods, which has the responsibility to: aid enquirers to understand the text; present to the responsible international/European committee any enquiries on the interpretation, or pr
5、oposals for change, and keep the UK interests informed; monitor related international and European developments and promulgate them in the UK. A list of organizations represented on this subcommittee can be obtained on request to its secretary. BS ENISO6222 is one of a series of standards on water q
6、uality, others of which have been, or will be, published as Sections of BS6068. This standard has therefore been given the secondary identifier BS6068-4.5. The various Sections of BS6068 are comprised within Parts1 to7, which, together with Part0, are listed below. Part 0: Introduction; Part 1: Glos
7、sary; Part 2: Physical, chemical and biochemical methods; Part 3: Radiological methods; Part 4: Microbiological methods; Part 5: Biological methods; Part 6: Sampling; Part 7: Precision and accuracy. NOTEThe tests described in this British Standard should only be carried out by suitably qualified per
8、sons with an appropriate level of microbiological expertise. Standard microbiological procedures should be followed throughout. Cross-references Attention is drawn to the fact that CEN and CENELEC Standards normally include an annex which lists normative references to international publications with
9、 their corresponding European publications. The British Standards which implement these international or European publications may be found in the BSI Standards Catalogue under the section entitled “International Standards Correspondence Index”, or by using the “Find” facility of the BSI Standards E
10、lectronic Catalogue. A British Standard does not purport to include all the necessary provisions of a contract. Users of British Standards are responsible for their correct application. Compliance with a British Standard does not of itself confer immunity from legal obligations. Summary of pages Thi
11、s document comprises a front cover, an inside front cover, pages i and ii, the EN ISO title page, pages 2 to 4 and a back cover. This standard has been updated (see copyright date) and may have had amendments incorporated. This will be indicated in the amendment table on the inside front cover.EUROP
12、EAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 6222 May 1999 ICS 13.060.30 English version Water quality Enumeration of culturable micro-organisms Colony count by inoculation in a nutrient agar culturemedium (ISO 6222:1999) Qualit de leau Dnombrement des micro-organismes revivifiables Comptage
13、des colonies par ensemencement dans un milieu de culture nutritif glos (ISO 6222:1999) Wasserbeschaffenheit Quantitative Bestimmung der kultivierbaren Mikroorganismen Bestimmung der Koloniezahl durch Einimpfen in ein Nhragarmedium (ISO 6222:1999) This European Standard was approved by CEN on16March1
14、999. CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national standards may be obtained
15、 on application to the Central Secretariat or to any CEN member. This European Standard exists in three official versions (English, French, German). A version in any other language made by translation under the responsibility of a CEN member into its own language and notified to the Central Secretar
16、iat has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom. CEN Europ
17、ean Committee for Standardization Comit Europen de Normalisation Europisches Komitee fr Normung Central Secretariat: rue de Stassart 36, B-1050 Brussels 1999 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN ISO 6222:1999 EENISO6222:
18、1999 2 BSI 03-2000 Foreword The text of EN ISO6222:1999 has been prepared by Technical Committee CEN/TC230 “Water analysis”, the secretariat of which is held by DIN, in collaboration with Technical Committee ISO/TC147 “Water quality”. This European Standard shall be given the status of a national st
19、andard, either by publication of an identical text or by endorsement, at the latest by November1999, and conflicting national standards shall be withdrawn at the latest by November1999. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries
20、 are bound to implement this European Standard: Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and the United Kingdom. Contents Page Foreword 2 Introduction 3 1 Scope 3 2 Norm
21、ative references 3 3 Definitions 3 4 Principle 3 5 Apparatus and glassware 3 6 Sampling 3 7 Culture media and diluents 4 8 Procedure 4 9 Expression of results 4 10 Test report 4ENISO6222:1999 BSI 03-2000 3 Introduction Waters of all kinds invariably contain a variety of micro-organisms derived from
22、various sources, such as soil and vegetation, and estimation of the overall numbers provide useful information for the assessment and surveillance of water quality. Separate counts are usually made of the micro-organisms which are able to grow and form colonies on nutrient agar media at36 C and22 C.
23、 Colony counts are useful for assessing the integrity of ground water sources and the efficiency of water treatment processes such as coagulation, filtration and disinfection and provide an indication of the cleanliness and integrity of the distribution system. They can also be used to assess the su
24、itability of a supply for the preparation of food and drink, where the water supply should contain few micro-organisms to avoid contaminating the product with spoilage organisms. The main value of colony counts lies in the detection of changes from those expected, based on frequent, long term monito
25、ring. Any sudden increase in the count can be an early warning of serious pollution and calls for immediate investigation. 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 af
26、ter aerobic incubation at36 C and22 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,
27、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 referenc
28、es, 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 Specifi
29、cation and test methods. (ISO 3696:1987) EN ISO 5667-3, Water quality Sampling Part 3: Guidance on the preservation and handling of samples. (ISO5667-3:1994) EN 25667-2, Water quality Sampling Part 2: Guidance on sampling techniques. (ISO5667-2:1991) ISO 6887, Microbiology General guidance for the p
30、reparation 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 mo
31、ulds 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 at36 C for44h, and anothe
32、r set at22 C for68h. 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 (autoc
33、lave) 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 Glass or plastics Petri dishes with a diameter of90mm or100mm 5.5 Water bath or similar apparatus capable of maintaining a temperature of (45 1) C 5.6 Colony counti
34、ng 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 EN25667-2 and ENISO5667-3. Examine water supplied in closed containers, including natural mineral waters, wit
35、hin12h of bottling, keeping the temperature of storage at(5 3) C during this period.ENISO6222:1999 4 BSI 03-2000 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 d
36、ehydrated complete medium and follow the manufacturers instructions. For making media, use glass-distilled or deionised water prepared in accordance with ENISO3696 grade3 and free from substances which might inhibit growth under the conditions of the test. NOTEThe use of chemicals of other grades is
37、 permissible providing they are shown to be of equal performance in the test. 7.2 Diluent For the dilutions, use the peptone diluent given in ISO8199. 7.3 Yeast extract agar Add the ingredients, or the complete dehydrated medium, to the water and dissolve by heating. Adjust the pH if necessary so th
38、at after sterilization it will be7,2 0,2 at25 C. Distribute volumes of15ml to20ml in tubes, bottles or other containers. For storage in larger volumes, use containers up to500ml capacity. Sterilise in the autoclave (5.1) at (121 3) C for(15 1) minutes. For use, melt the medium, allow to cool and mai
39、ntain it at (45 1) C using the water bath (5.5). It is recommended to store the medium not longer than4h at45 C, after which time the medium shall be discarded. 8 Procedure 8.1 Preparation and inoculation Carry out preparation of the sample, make dilutions and inoculate the culture media, in accorda
40、nce with ISO8199, ENISO5667-3 and ISO6887. Use the pour-plate method (ISO8199). Place a volume of the test sample (or its dilution) not exceeding2ml in the Petri dish, add15ml to20ml of the molten medium (7.3) and mix carefully by gentle rotation; allow the medium to set. Time between addition of th
41、e test sample (or its dilution) and addition of the molten medium shall not exceed15min. Inoculate at least one plate for incubation at each temperature. 8.2 Incubation and examination Invert the plates and incubate one set at (36 2) C for (44 4) h; incubate the other set at (22 2) C for (68 4) h. E
42、xamine the plates as soon as they are removed from the incubators; if this is not possible, store them at (5 3) C and examine them within48h. Reject any plate with confluent growth. 8.3 Counting of colonies For each temperature of incubation, and following the procedures described in ISO8199, count
43、the colonies present in each plate and calculate the estimated number of colony forming units present in1ml of sample. 9 Expression of results Express the results as the number of colony-forming units per millilitre (cfu/ml) of the sample for each temperature of incubation. If there are no colonies
44、in the plates inoculated with the test volumes of the undiluted sample, express the results as not detected in one millilitre. If there are more than300 colonies on the plates inoculated with the highest dilutions used, express the results as300 or as approximate only. 10 Test report The test report
45、 shall make reference to this European Standard and give all relevant information, including a) all details necessary for complete identification of the sample; b) the technique (pour plate) and medium used; c) the time and temperature of incubation; d) the results of the count expressed in accordan
46、ce with clause9; e) any particular occurrence(s) observed during the course of the analysis and any other relevant facts concerning the procedure followed. Tryptone (Peptone from Casein, pancr.) 6,0 g Dehydrated yeast extract 3,0 g Agar, powdered or in pellets 10 g to 20 g (according to gel strength
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