1、BRITISH STANDARD BS EN 26461-1:1993 BS 6068-4.8: 1993 ISO 6461-1: 1986 Water quality Detection and enumeration of the spores of sulfite-reducing anaerobes (clostridia) Part 1: Method by enrichment in a liquid medium The European Standard EN26461-1:1993 has the status of a British Standard UDC 628.1/
2、.3:620.1:543.39:579.852.13BSEN26461-1:1993 This British Standard, having been prepared under the directionof the Environment andPollution Standards Policy Committee, was published underthe authority of the Standards Board and comes intoeffect on 15April1993 BSI 09-1999 The following BSI references r
3、elate to the work on this standard: Committee reference EPC/44 Special announcement in BSINews August1991 ISBN 0 580 21205 X Cooperating organizations The European Committee for Standardization (CEN), under whose supervision this European Standard was prepared, comprises the national standards organ
4、izations of the following countries: Austria Oesterreichisches Normungsinstitut Belgium Institut belge de normalisation Denmark Dansk Standardiseringsraad Finland Suomen Standardisoimisliito, r.y. France Association franaise de normalisation Germany Deutsches Institut fr Normung e.V. Greece Hellenic
5、 Organization for Standardization Iceland Technological Institute of Iceland Ireland National Standards Authority of Ireland Italy Ente Nazionale Italiano di Unificazione Luxembourg Inspection du Travail et des Mines Netherlands Nederlands Normalisatie-instituut Norway Norges Standardiseringsforbund
6、 Portugal Instituto Portugus da Qualidade Spain Asociacin Espaola de Normalizacin y Certificacin Sweden Standardiseringskommissionen i Sverige Switzerland Association suisse de normalisation United Kingdom British Standards Institution Amendments issued since publication Amd. No. Date CommentsBSEN26
7、461-1:1993 BSI 09-1999 i Contents Page Cooperating organizations Inside front cover National foreword ii Foreword 2 0 Introduction 3 1 Scope 3 2 Field of application 3 3 References 3 4 Definition 3 5 Principle 3 6 Culture media and reagents 3 7 Apparatus and glassware 4 8 Sampling 4 9 Procedure 4 10
8、 Expression of results 5 11 Test report 5 National annex NA (informative) Committees responsible 6 National annex NB (informative) Cross-references Inside back coverBSEN26461-1:1993 ii BSI 09-1999 National foreword This British Standard has been prepared under the direction of the Environment and Po
9、llution Standards Policy Committee and is the English language version of EN26461-1:1993 Water quality Detection and enumeration of the spores of sulfite-reducing anaerobes (clostridia) Part 1: Method by enrichment in a liquid medium, published by the European Committee for Standardization (CEN), wh
10、ich endorses ISO6461-1:1986, published by the International Organization for Standardization (ISO). 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
11、does not of itself confer immunity from legal obligations. Summary of pages This document comprises a front cover, an inside front cover, pages i and ii, theEN title page, pages 2 to 6, an inside back cover and a back cover. This standard has been updated (see copyright date) and may have had amendm
12、ents incorporated. This will be indicated in the amendment table on the inside front cover.EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 26461-1 January 1993 UDC 628.1/.3:620.1:543.39:579.852.13 Descriptors: Water, quality, water tests, microbiological analysis, micro-organisms, sulfite reduc
13、ing bacteria, clostridium Englishversion Water quality Detection and enumeration of the spores ofsulfite-reducing anaerobes (clostridia) Part1:Methodby enrichment in a liquid medium (ISO6461-1:1986) Qualit de leau Recherche et dnombrementdes spores de micro-organismes anarobies sulfito-rducteurs (cl
14、ostridia) Partie1:Mthode par enrichissement dans unmilieu liquide (ISO6461-1:1986) Wasserbeschaffenheit Nachweis und Zhlung der Sporen sulfitreduzierender Anaerobier (clostridien) Teil1:Flssigkeitsanreicherung (ISO6461-1:1986) This European Standard was approved by CEN on1993-01-20. CEN members are
15、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 on application to th
16、e 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 Secretariat has the same stat
17、us as the official versions. CEN members are the national standards bodies of Austria, Belgium, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom. CEN European Committee for Standardization Com
18、it Europen de Normalisation Europisches Komitee fr Normung Central Secretariat: rue de Stassart 36, B-1050 Brussels 1993 Copyright reserved to CEN members Ref. No. EN26461-1:1993 EEN26461-1:1993 BSI 09-1999 2 Foreword This European Standard is the endorsement of ISO6461-1. Endorsement of ISO6461-1 w
19、as recommended by Technical Committee CEN/TC230 “Water analysis” under whose competence this European Standard will henceforth fall. This European Standard shall be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest byJuly1993, and co
20、nflicting national standards shall be withdrawn at the latest byJuly1993. The standard was approved and in accordance with the CEN/CENELEC Internal Regulations, the following countries are bound to implement this European Standard: Austria, Belgium, Denmark, Finland, France, Germany, Greece, Iceland
21、, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland, United Kingdom.EN26461-1:1993 BSI 09-1999 3 0 Introduction The spores of sulfite-reducing anaerobes (clostridia) are widespread in the environment. They are present in human and animal faecal matter, in waste wa
22、ter and in soil. Unlike Escherichia coli and other coliform organisms, the spores survive in water for long periods as they are more resistant than vegetative forms to the action of chemical and physical factors. They may thus give an indication of remote or intermittent pollution. They may even be
23、resistant to chlorination at levels which are normally used for the treatment of water, and they are thus useful for control purposes. ISO 6461 consists of the following parts: Part 1: Method by enrichment in a liquid medium; Part 2: Method by membrane filtration. 1 Scope This part of ISO6461 specif
24、ies a method for the detection and enumeration of the spores of sulfite-reducing anaerobes (clostridia) by enrichment in a liquid medium. 2 Field of application The method is applicable to all types of water, including turbid water. 3 References ISO 3696, Water for laboratory use Specifications. ISO
25、 5667, Water quality Sampling Part 2: Guidance on sampling techniques Part 3: Guidance on the preservation and handling of samples. ISO 8199, Water quality General guidance for microbiological examination by enumeration of micro-organisms on culture media 1) . 4 Definition For the purpose of this pa
26、rt of ISO6461, the following definition applies. clostridia sulfite-reducing, spore-forming, anaerobic micro-organisms which belong to the Bacillaceae family and the genus Clostridium 5 Principle The detection of spores of sulfite-reducing anaerobes (clostridia) in a specified volume of a water samp
27、le requires the following steps. 5.1 Selection of spores Selection of spores in the sample by applying heat for a period of time sufficient to destroy vegetative bacteria. 5.2 Enrichment culture Detection and enumeration of spores of sulfite-reducing anaerobes by inoculating volumes of the sample in
28、to liquid enrichment media, followed by incubation at37 1 C for44 4h in anaerobic conditions. 6 Culture media and reagents 6.1 Basic materials In order to improve the reproducibility of the results, it is recommended that, for the preparation of the diluents and culture media, dehydrated basic compo
29、nents or complete dehydrated media be used. Similarly, commercially prepared reagents may also be used. The manufacturers instructions shall be rigorously followed. The chemical products used for the preparation of the culture media and the reagents shall be of recognized analytical quality. The wat
30、er used shall be distilled or deionized water, free from substances that might inhibit the growth of micro-organisms under the test conditions (seeISO3696). Measurements of pH shall be made using a pH meter, measurements being referred to a temperature of25 C. If the prepared culture media are not u
31、sed immediately, they shall, unless otherwise stated, be stored in the dark at approximately4 C, for no longer than 1 month. 6.2 Culture media and diluent 6.2.1 Diluent Use one of the diluents given in ISO8199. 6.2.2 Differential reinforced clostridial medium (DRCM) 6.2.2.1 Single strength basal med
32、ium Composition 1) At present at the stage of draft. Peptone tryptic digest of meat 10 g Meat extract 10 g Yeast extract 1,5 g Starch 1 g Hydrated sodium acetate 5 g Glucose 1 g L-Cysteine-hydrochloride 0,5 g Water 1 000 mlEN26461-1:1993 4 BSI 09-1999 Preparation Mix the peptone, meat extract, sodiu
33、m acetate and yeast extract with800ml of water. With the remaining200ml of distilled water, prepare a starch solution as follows: mix the starch in a little cold water to form a paste. Heat the rest of the water to boiling point and slowly add it to the paste with constant stirring. Then add this st
34、arch solution to the first mixture and heat to boiling point until it dissolves. Finally, add the glucose and L-cysteine hydrochloride. Dissolve. Adjust the pH to7,1 to7,2 with1 mol/l sodium hydroxide. Transfer25ml aliquots of the medium into screw-capped bottles of capacity25ml. Sterilize in the au
35、toclave at121 1 C for15min. 6.2.2.2 Double strength basal medium Prepare the double strength medium as in6.2.2.1 but reduce the volume of water by half. Transfer10ml and50ml aliquots of the medium into screw-capped bottles of capacities25ml and100ml respectively. 6.2.3 Sodium sulfite (Na 2 SO 3 ), 4
36、% (m/m) solution. Dissolve4g of anhydrous sodium sulfite in100ml of water. Sterilize by filtration. Store at between2 and5 C. It is advisable to prepare a fresh solution every14 days. 6.2.4 Iron(III) citrate (C 6 H 5 O 7 Fe), 7 % (m/m) solution. Dissolve7g of iron(III) citrate in100ml of water. Ster
37、ilize by filtration. Store at between2 and5 C. It is advisable to prepare a fresh solution every14 days. 6.2.5 Complete medium 6.2.5.1 On the day of analysis, mix equal volumes of the solutions of sodium sulfite (6.2.3) and iron(III) citrate (6.2.4). 6.2.5.2 Add0,5ml of the mixture (6.2.5.1) to each
38、 bottle of single strength medium (6.2.2.1), which has been freshly heated and cooled. 6.2.5.3 Add0,4ml of the mixture (6.2.5.1) to each10ml, and2ml to each50ml, of double strength medium (6.2.2.2) similarly treated. 7 Apparatus and glassware Usual microbiological laboratory equipment, and 7.1 Screw
39、-cap bottles or vials and stoppers of boron silicate glass of capacities200, 100 and25ml. 7.2 Volumetric pipettes, of capacities10 and1ml. 7.3 Water baths, thermostatically controlled. 7.4 Test tubes, 150mm 13mm. 7.5 Iron wire 7.6 Incubator, capable of being maintained at37 1 C. 8 Sampling Refer to
40、ISO5667-2 and ISO8199 for sampling techniques. 9 Procedure 9.1 Treatment of samples Refer to ISO5667-3 for guidance on the preservation and handling of samples, and to ISO8199. 9.2 Selection of spores (technique) Before the test, the sample of water should be heated in a water bath at75 5 C for15min
41、 from the time it reaches that temperature. A similar bottle containing the same volume of water as the test sample should be used periodically as a control in order to check the heating time required. The temperature of the water in the control bottle can be constantly recorded by thermometer. 9.3
42、Inoculation and incubation Add50ml of sample (9.2) to a100ml screw-cap bottle containing50ml of the double strength complete medium (6.2.5.3). Add10ml of sample (9.2) to a series of five25ml screw-cap bottles containing10ml of double strength complete medium (6.2.5.3). Add 1 ml of sample (9.2) to a
43、series of five25ml screw-cap bottles containing25ml of single strength complete medium (6.2.5.2). If necessary, add1ml of a1 F 10 dilution of the sample (9.2) to a series of five25ml screw-cap bottles containing25ml of single strength complete medium (6.2.5.2). In order to carry out a qualitative ex
44、amination of100ml of drinking water or bottled water without making an MPN count, use a200ml vial filled with a mixture of100ml of double strength complete medium (6.2.5.3) and100ml of sample (9.2).EN26461-1:1993 BSI 09-1999 5 If necessary, top up all the bottles with the single strength complete me
45、dium (6.2.5.2) to bring the volume of liquid level with the neck and to ensure that only a very small volume of air remains, then seal the bottles hermetically, or incubate under anaerobic conditions. Incubate the inoculated bottles at37 1 C for44 4h. Large volumes of culture in hermetically sealed
46、glass bottles may explode due to gas production. The addition of iron wire, heated to redness and placed into the medium before inoculation, may aid anaerobiosis. 9.4 Interpretation Bottles in which blackening is observed, as a result of the reduction of sulfite and the precipitation of iron(II) sul
47、fide, shall be regarded as positive. 10 Expression of results Express the results in accordance with ISO8199. 11 Test report The test report shall state the method used, and express the results as the most probable number of sulfite-reducing anaerobes (clostridia) per volume of sample. It shall also
48、 mention any operating details not specified in this part of ISO6461, or regarded as optional, together with details of any incidents likely to have influenced the results. The test report shall include all the information necessary for the complete identification of the sample.BSEN26461-1:1993 6 BS
49、I 09-1999 National annex NA (informative) Committees responsible The United Kingdom participation in the preparation of this European Standard was entrusted by the Environment and Pollution Standards Policy Committee (EPC/-) to Technical Committee EPC/44, upon which the following bodies were represented: Association of Consulting Engineers British Association for Chemical Specialities British Gas plc Chemical Industries Association Industrial Water Society Institute of Petroleum Institution of Gas
