00297916 PDF 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[.]
BRITISH STANDARD Water quality — Detection and enumeration of the spores of sulfite-reducing anaerobes (clostridia) — Part 2: Method by membrane filtration The European Standard EN 26461-2:1993 has the status of a British Standard UDC 628.1/.3:620.1:543.39:579.852.13 BS EN 26461-2:1993 BS 6068-4.9: 1993 ISO 6461-2: 1986 BS EN 26461-2:1993 Cooperating organizations The European Committee for Standardization (CEN), under whose supervision this European Standard was prepared, comprises the national standards organizations of the following countries: This British Standard, having been prepared under the direction of the Environment and Pollution Standards Policy Committee, was published under the authority of the Standards Board and comes into effect on 15 April 1993 © BSI 09-1999 The following BSI references relate to the work on this standard: Committee reference EPC/44 Special announcement in BSI News August 1991 ISBN 580 21206 Austria Oesterreichisches Normungsinstitut Belgium Institut belge de normalisation Denmark Dansk Standardiseringsraad Finland Suomen Standardisoimisliito, r.y France Association franỗaise de normalisation Germany Deutsches Institut fỹr Normung e.V Greece Hellenic 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 Portugal Instituto Portuguès da Qualidade Spain Asociación Espola de Normalización y Certificación Sweden Standardiseringskommissionen i Sverige Switzerland Association suisse de normalisation United Kingdom British Standards Institution Amendments issued since publication Amd No Date Comments BS EN 26461-2:1993 Contents Cooperating organizations National foreword Foreword Introduction Scope Field of application References Definition Principle Culture media and reagents Apparatus and glassware Sampling Procedure 10 Expression of results 11 Test report National annex NA (informative) Committees responsible National annex NB (informative) Cross-references © BSI 09-1999 Page Inside front cover ii 3 3 3 4 5 Inside back cover i BS EN 26461-2:1993 National foreword This British Standard has been prepared under the direction of the Environment and Pollution Standards Policy Committee It is the English language version of EN 26461-2:1993 Water quality — Detection and enumeration of the spores of sulfite-reducing anaerobes (clostridia) — Part 2: Method by membrane filtration, published by the European Committee for Standardization (CEN), which endorses ISO 6461-2:1986, 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 confer immunity from legal obligations Summary of pages This document comprises a front cover, an inside front cover, pages i and ii, the EN title page, pages to 6, an inside back cover 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 ii © BSI 09-1999 EUROPEAN STANDARD EN 26461-2 NORME EUROPÉENNE January 1993 EUROPÄISCHE NORM UDC 628.1/.3:620.1:543.39:579.852.13 Descriptors: Water, quality, water tests, microbiological analysis, micro-organisms, sulfite reducing bacteria, clostridium, filtration analysis English version Water quality — Detection and enumeration of the spores of sulfite-reducing anaerobes (clostridia) — Part 2: Method by membrane filtration (ISO 6461-2:1986) Qualité de l’eau — Recherche et dénombrement des spores de micro-organismes anaérobies sulfito-réducteurs (clostridia) — Partie 2: Méthode par filtration sur membrane (ISO 6461-2:1986) Wasserbeschaffenheit — Nachweis und Zählung der Sporen sulfitreduzierender Anaerobier (Clostridien) — Teil 2: Membranfiltrationsverfahren (ISO 6461-2:1986) This European Standard was approved by CEN on 1993-01-20 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 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 Secretariat has the same status 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 Comité Européen de Normalisation Europäisches Komitee für Normung Central Secretariat: rue de Stassart 36, B-1050 Brussels © 1993 Copyright reserved to CEN members Ref No EN 26461-2:1993 E EN 26461-2:1993 Foreword This European Standard is the endorsement of ISO 6461-2 Endorsement of ISO 6461-2 was recommended by Technical Committee CEN/TC 230 “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 July 1993, and conflicting national standards shall be withdrawn at the latest by July 1993 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, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland, United Kingdom © BSI 09-1999 EN 26461-2:1992 Introduction Principle The spores of sulfite-reducing anaerobes (clostridia) are widespread in the environment They are present in human and animal faecal matter, in waste water 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 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 The detection of spores of sulfite-reducing anaerobes (clostridia) in a specified volume of a water sample requires the following steps Scope This part of ISO 6461 specifies a method for the detection and enumeration of the spores of sulfite-reducing anaerobes (clostridia) by membrane filtration 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 References ISO 3696, Water for laboratory use — Specifications ISO 5667, Water quality — Sampling — Part 2: Guidance on sampling techniques — Part 3: Guidance on the preservation and handling of samples ISO 7704, Water quality — Evaluation of membrane filters used for microbiological analyses ISO 8199, Water quality — General guidance for microbiological examination by enumeration of micro-organisms on culture media1) Definition For the purpose of this part of ISO 6461, the following definition applies clostridia sulfite-reducing, spore-forming, anaerobic micro-organisms which belong to the Bacillaceae family and the genus Clostridium 1) 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 Membrane filtration and culture Filtration of the water sample through a membrane filter having a pore size such that bacterial spores (0,2 4m) are retained in or on the membrane filter Placing of the filter on a specialized selective culture medium (sulfite-iron-agar), followed by incubation at 37 ± °C for 20 ± h and 44 ± h, and counting of any black colonies 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 dehydrated media be used Similarly, commercially prepared reagents may also be used The manufacturer’s 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 distilled or deionized water, free from substances that might inhibit the growth of micro-organisms under the test conditions (see ISO 3696) Measurements of pH shall be made using a pH meter, measurements being referred to a temperature of 25 °C If the prepared culture media are not used immediately, they shall, unless otherwise stated, be stored in the dark at approximately °C, for no longer than month 6.2 Sulfite-iron-agar 6.2.1 Basal medium (nutrient agar) Composition Meat extract Peptone Sodium chloride (NaCl) Agar Water 3g 10 g 5g 15 g 000 ml At present at the stage of draft © BSI 09-1999 EN 26461-2:1992 Preparation Steam to dissolve, make up to litre with water, and adjust the pH to 7,6 ± 0,1 with mol/l sodium hydroxide solution Sterilize at 121 ± °C in the autoclave for 20 Store in the refrigerator after solidifying 6.2.2 Sodium sulfite (Na2SO3) solution Dissolve 10 g of sodium sulfite in 100 ml of water It is advisable to prepare a fresh solution every two weeks 6.2.3 Iron(II) sulfate (FeSO4) solution Dissolve g of crystallized iron(II) sulfate in 100 ml of water 7.1 Glass flasks (Erlenmeyer flask, round-bottom flask, or conical flask), of capacity litres 6.2.4 Complete medium NOTE The 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 ISO 7704 Immediately before use, melt the basal medium (6.2.1) and to each 18 ml volume add ml of sodium sulfite solution (6.2.2) and five drops of iron(II) sulfate solution (6.2.3) Add ml of the sodium sulfite solution and drops of the iron(II) sulfate solution to the agar tubes just before the embedding procedure (see 9.3) 6.3 Tryptose-sulfite-agar (alternative medium) Composition 7.3 Graduated pipettes, of capacity 10 ml, graduated in divisions of 0,1 ml 7.4 Volumetric pipettes, of capacity 10 ml 7.5 Jars, of capacity litre 7.6 Steamer 7.7 Water bath 7.8 Membrane filtration apparatus 7.9 Sterile membrane filters, of nominal pore size 0,2 4m 7.10 Incubator, capable of being maintained at 37 ± °C 7.11 Petri dishes Sampling Refer to ISO 5667-2 and ISO 8199 for sampling techniques Procedure Tryptose 15 g Soytone 5g 9.1 Treatment of samples Yeast extract 5g Sodium metabisulfite 1g Ammonium iron(III) citrate 1g Refer to ISO 5667-3 for guidance on the preservation and handling of samples, and to ISO 8199 Water 000 ml Preparation Steam to dissolve, and adjust the pH to 7,6 ± 0,1 at 2,5 °C Distribute into test tubes in volumes of 18 ml Sterilize the medium for 15 at 121 ± °C Store in the refigerator at to °C Discard unused medium weeks after preparation Apparatus and glassware Usual microbiological laboratory equipment, and 7.2 Test tubes, 160 mm × 16 mm 9.2 Selection of spores (technique) Before the test, the sample of water should be heated in a water bath at 75 ± °C for 15 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 Inoculation and incubation Refer to ISO 7704 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 waters, sea-water, and surface water, less polluted with clostridia, filter 100 ml; for highly polluted water or sewage use smaller volumes Volumes of water smaller than 10 ml should be mixed with 10 to 100 ml of sterile water or diluent Adjust the dilutions so that any resultant black colonies are well separated and can be easily counted © BSI 09-1999 EN 26461-2:1992 After filtration, remove the membrane with sterile forceps and place it face downwards on the bottom of a Petri dish, ensuring that no air bubbles are trapped under the filter Then carefully pour 18 ml of the liquefied complete culture medium, previously cooled to about 50 °C, over the membrane while holding it still with sterile forceps After this layer of medium has set, incubate anaerobically or under other conditions which ensure anaerobiosis at a temperature of 37 ± °C for 20 ± h and 44 ± h 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 for 20 ± h and 44 ± 4h © BSI 09-1999 10 Expression of results Express the results in accordance with ISO 8199 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 The 44 ± h count should normally be reported If this is not possible, the count at 20 ± h should be reported as approximate only The test report shall also mention any operating details not specified in this part of ISO 6461, 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 BS EN 26461-2:1993 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 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 Institution of Water and Environmental Management National Rivers Authority Royal Institute of Public Health and Hygiene Royal Society of Chemistry Scottish Association of Directors of Water and Sewerage Services Soap and Detergent Industry Association Water Companies Association Water Research Centre Water Services Association of England and Wales The following bodies were also represented in the drafting of the standard, through subcommittees and panels: Automatic Vending Association of Great Britain British Laboratory Ware Association British Occupational Hygiene Society British Soft Drinks Association Ltd Chartered Institution of Building Services Engineers Department of Health Institute of Hospital Engineering Ministry of Agriculture, Fisheries and Food Public Health Laboratory Service Society for Applied Bacteriology Society for General Microbiology © BSI 09-1999 BS EN 26461-2:1993 National annex NB (informative) Cross-references Publication referred to Corresponding British Standard ISO 3696:1987 BS 3978:1987 Specification for water for laboratory use BS 6068 Water quality Part Sampling Section 6.2:1991 Guidance on sampling techniques Section 6.3:1986 Guidance on the preservation and handling of samples Part Microbiological methods Section 4.2:1989 Guide to the enumeration of micro-organisms by culture ISO 5667-2:1991 ISO 5667-3:1985 ISO 8199:1988 © BSI 09-1999 BS EN 26461-2:1993 BS 6068-4.9: 1993 ISO 6461-2: 1986 BSI — British Standards Institution BSI is the independent national body responsible for preparing British Standards It presents the UK view on standards in Europe and at the international level It is incorporated by Royal Charter Revisions British Standards are updated by amendment or revision Users of British Standards should make sure that they possess the latest amendments or editions It is the constant aim of BSI to improve the quality of our products and services We would be grateful if anyone finding an inaccuracy or ambiguity while using this British Standard would inform the Secretary of the technical committee responsible, the 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