1、BRITISH STANDARD BS EN 26461-2:1993 BS 6068-4.9: 1993 ISO 6461-2: 1986 Water quality Detection and enumeration of the spores of sulfite-reducing anaerobes (clostridia) Part 2: Method by membrane filtration The European Standard EN26461-2:1993 has the status of a British Standard UDC 628.1/.3:620.1:5
2、43.39:579.852.13BS EN26461-2:1993 This British Standard, having been prepared under the directionof the Environment andPollution Standards PolicyCommittee, was publishedunder the authority ofthe Standards Board and comesinto effect on 15 April1993 BSI 09-1999 The following BSI references relate to t
3、he work on this standard: Committee reference EPC/44 Special announcement in BSINews August1991 ISBN 0 580 21206 8 Cooperating organizations The European Committee for Standardization (CEN), under whose supervision this European Standard was prepared, comprises the national standards organizations o
4、f 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 Organizat
5、ion 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 Portugal
6、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 CommentsBS EN26461-2:199
7、3 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 Expressi
8、on of results 5 11 Test report 5 National annex NA (informative) Committees responsible 6 National annex NB (informative) Cross-references Inside back coverBS EN26461-2:1993 ii BSI 09-1999 National foreword This British Standard has been prepared under the direction of the Environment and Pollution
9、Standards Policy Committee. It is the English language version of EN26461-2:1993 Water quality Detection and enumeration of the spores of sulfite-reducing anaerobes (clostridia) Part2: Method by membrane filtration, published by the European Committee for Standardization (CEN), which endorses ISO646
10、1-2:1986, 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 does not of itself
11、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 amendments incorporated.
12、This will be indicated in the amendment table on the inside front cover.EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 26461-2 January 1993 UDC 628.1/.3:620.1:543.39:579.852.13 Descriptors: Water, quality, water tests, microbiological analysis, micro-organisms, sulfite reducing bacteria, clost
13、ridium, filtration analysis English version Water quality Detection and enumeration of the spores ofsulfite-reducing anaerobes (clostridia) Part2: Method by membrane filtration (ISO 6461-2:1986) Qualit de leau Recherche et dnombrement des spores de micro-organismes anarobies sulfito-rducteurs (clost
14、ridia) Partie2: Mthode par filtration sur membrane (ISO6461-2:1986) Wasserbeschaffenheit Nachweis und Zhlung der Sporen sulfitreduzierender Anaerobier (Clostridien) Teil2: Membranfiltrationsverfahren (ISO6461-2:1986) This European Standard was approved by CEN on 1993-01-20. CEN members are bound to
15、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 the Central
16、 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 status as the
17、 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 Comit Europe
18、n de Normalisation Europisches Komitee fr Normung Central Secretariat: rue de Stassart 36, B-1050 Brussels 1993 Copyright reserved to CEN members Ref. No. EN 26461-2:1993 EEN26461-2:1993 BSI 09-1999 2 Foreword This European Standard is the endorsement of ISO6461-2. Endorsement of ISO6461-2 was recom
19、mended 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 by July1993, and conflicti
20、ng national standards shall be withdrawn at the latest by July1993. 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, Irel
21、and, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland, United Kingdom.EN26461-2:1992 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 water an
22、d 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 resist
23、ant 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 specifies a
24、method for the detection and enumeration of the spores of sulfite-reducing anaerobes (clostridia) by membrane filtration. 2 Field of application The method can be applied to all types of water, except when a large amount of particulate material is liable to be retained by the membrane. 3 References
25、ISO 3696, Water for laboratory use Specifications. ISO 5667, Water quality Sampling Part2:Guidance on sampling techniques Part3:Guidance on the preservation and handling of samples. ISO 7704, Water quality Evaluation of membrane filters used for microbiological analyses. ISO 8199, Water quality Gene
26、ral guidance for microbiological examination by enumeration of micro-organisms on culture media 1) . 4 Definition For the purpose of this part of ISO6461, the following definition applies. clostridia sulfite-reducing, spore-forming, anaerobic micro-organisms which belong to the Bacillaceae family an
27、d the genus Clostridium 5 Principle The detection of spores of sulfite-reducing anaerobes (clostridia) in a specified volume of a water sample 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 vegetativ
28、e bacteria. 5.2 Membrane filtration and culture Filtration of the water sample through a membrane filter having a pore size such that bacterial spores(0,24m) are retained in or on the membrane filter. Placing of the filter on a specialized selective culture medium (sulfite-iron-agar), followed by in
29、cubation at37 1 C for204h and444h, and counting of any black colonies. 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 components or complete dehyd
30、rated 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 water used shall be distil
31、led 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 used immediately, they s
32、hall, unless otherwise stated, be stored in the dark at approximately4 C, for no longer than1month. 6.2 Sulfite-iron-agar 6.2.1 Basal medium (nutrient agar) Composition 1) At present at the stage of draft. Meat extract 3 g Peptone 10 g Sodium chloride (NaCl) 5 g Agar 15 g Water 1 000 mlEN26461-2:199
33、2 4 BSI 09-1999 Preparation Steam to dissolve, make up to1litre with water, and adjust the pH to7,6 0,1with1mol/l sodium hydroxide solution. Sterilize at121 1 C in the autoclave for20min. Store in the refrigerator after solidifying. 6.2.2 Sodium sulfite (Na 2 SO 3 ) solution. Dissolve10g of sodium s
34、ulfite in100ml of water. It is advisable to prepare a fresh solution every two weeks. 6.2.3 Iron(II) sulfate (FeSO 4 ) solution. Dissolve8g of crystallized iron(II) sulfate in100ml of water. 6.2.4 Complete medium Immediately before use, melt the basal medium(6.2.1) and to each18ml volume add1ml of s
35、odium sulfite solution (6.2.2) and five drops of iron(II) sulfate solution (6.2.3). Add 1 ml of the sodium sulfite solution and5drops of the iron(II) sulfate solution to the agar tubes just before the embedding procedure (see9.3). 6.3 Tryptose-sulfite-agar (alternative medium) Composition Preparatio
36、n Steam to dissolve, and adjust the pH to7,6 0,1at2,5 C. Distribute into test tubes in volumes of18ml. Sterilize the medium for15min at121 1 C. Store in the refigerator at4to5 C. Discard unused medium2 weeks after preparation. 7 Apparatus and glassware Usual microbiological laboratory equipment, and
37、 7.1 Glass flasks (Erlenmeyer flask, round-bottom flask, or conical flask), of capacity2litres. 7.2 Test tubes, 160mm 16mm. 7.3 Graduated pipettes, of capacity10ml, graduated in divisions of0,1ml. 7.4 Volumetric pipettes, of capacity10ml. 7.5 Jars, of capacity1litre. 7.6 Steamer 7.7 Water bath 7.8 M
38、embrane filtration apparatus 7.9 Sterile membrane filters, of nominal pore size0,24m. NOTEThe quality of membrane filters may vary according to brand or even from batch to batch. It is therefore advisable to check the quality on a regular basis according to ISO7704. 7.10 Incubator, capable of being
39、maintained at37 1 C. 7.11 Petri dishes 8 Sampling Refer to 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
40、of water should be heated in a water bath at75 5 C for15min 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 con
41、trol bottle can be constantly recorded by thermometer. 9.3 Inoculation and incubation Refer to ISO7704 for a general description of the membrane filtration technique. According to the instructions in that document, filter a suitable volume of water. For drinking water, spring and well water, mineral
42、 waters, sea-water, and surface water, less polluted with clostridia, filter100ml; for highly polluted water or sewage use smaller volumes. Volumes of water smaller than10ml should be mixed with10to100ml of sterile water or diluent. Adjust the dilutions so that any resultant black colonies are well
43、separated and can be easily counted. Tryptose 15 g Soytone 5 g Yeast extract 5 g Sodium metabisulfite 1 g Ammonium iron(III) citrate 1 g Water 1 000 mlEN26461-2:1992 BSI 09-1999 5 After filtration, remove the membrane with sterile forceps and place it face downwards on the bottom of a Petri dish, en
44、suring that no air bubbles are trapped under the filter. Then carefully pour18ml of the liquefied complete culture medium, previously cooled to about50 C, over the membrane while holding it still with sterile forceps. After this layer of medium has set, incubate anaerobically or under other conditio
45、ns which ensure anaerobiosis at a temperature of37 1 C for20 4h and444h. If an anaerobic jar or an anaerobic incubator is used, the membrane filter may be placed on the surface of the agar face upwards. 9.4 Interpretation Count all black colonies after incubation for204h and44 4h. 10 Expression of r
46、esults Express the results in accordance with ISO8199. 11 Test report The test report shall state the method used and express the results as the number of sulfite-reducing anaerobes (clostridia) per volume of sample. The444h count should normally be reported. If this is not possible, the count at20
47、4h should be reported as approximate only. The test report shall also 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 necessar
48、y for the complete identification of the sample.BSEN26461-2:1993 6 BSI 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 Tec
49、hnical 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 Department of Trade and Industry (Laboratory of the Government Chemist) Department of the Environment for Northern Ireland Department of the Environment (Water Directorate) Industrial Water Society Institute of Petroleum Institution of Water Officers Institution of Gas Engineers Instit
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