00374242 pdf 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[.]
BRITISH STANDARD Water quality — BS EN 26461-1:1993 BS 6068-4.8: 1993 ISO 6461-1: 1986 Detection and enumeration of the spores of sulfite-reducing anaerobes (clostridia) — Part 1: Method by enrichment in a liquid medium The European Standard EN 26461-1:1993 has the status of a British Standard Confirmed July 2008 UDC 628.1/.3:620.1:543.39:579.852.13 BS EN 26461-1:1993 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 21205 X Austria Oesterreichisches Normungsinstitut Belgium Institut belge de normalisation Denmark Dansk Standardiseringsraad Finland Suomen Standardisoimisliito, r.y France Association franỗaise de normalisation Germany Deutsches Institut für 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 26461-1:1993 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 26461-1:1993 National foreword This British Standard has been prepared under the direction of the Environment and Pollution Standards Policy Committee and is the English language version of EN 26461-1:1993 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), which endorses ISO 6461-1:1986, 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 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 26461-1 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 English version Water quality — Detection and enumeration of the spores of sulfite-reducing anaerobes (clostridia) — Part 1: Method by enrichment in a liquid medium (ISO 6461-1:1986) Qualité de l’eau — Recherche et dénombrement des spores de micro-organismes anaérobies sulfito-réducteurs (clostridia) — Partie 1: Méthode par enrichissement dans un milieu liquide (ISO 6461-1:1986) Wasserbeschaffenheit — Nachweis und Zählung der Sporen sulfitreduzierender Anaerobier (clostridien) — Teil 1: Flüssigkeitsanreicherung (ISO 6461-1:1986) 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 26461-1:1993 E EN 26461-1:1993 Foreword This European Standard is the endorsement of ISO 6461-1 Endorsement of ISO 6461-1 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 26461-1:1993 Introduction 5.1 Selection of spores 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 Selection of spores in the sample by applying heat for a period of time sufficient to destroy vegetative bacteria Scope This part of ISO 6461 specifies a method for the detection and enumeration of the spores of sulfite-reducing anaerobes (clostridia) by enrichment in a liquid medium Field of application The method is applicable to all types of water, including turbid water 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 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 Principle The detection of spores of sulfite-reducing anaerobes (clostridia) in a specified volume of a water sample requires the following steps 1) At 5.2 Enrichment culture Detection and enumeration of spores of sulfite-reducing anaerobes by inoculating volumes of the sample into liquid enrichment media, followed by incubation at 37 ± °C for 44 ± h in anaerobic conditions 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 Culture media and diluent 6.2.1 Diluent Use one of the diluents given in ISO 8199 6.2.2 Differential reinforced clostridial medium (DRCM) 6.2.2.1 Single strength basal medium Composition Peptone tryptic digest of meat Meat extract Yeast extract Starch Hydrated sodium acetate Glucose L-Cysteine-hydrochloride Water 10 g 10 g 1,5 g 1g 5g 1g 0,5 g 000 ml present at the stage of draft © BSI 09-1999 EN 26461-1:1993 Preparation Mix the peptone, meat extract, sodium acetate and yeast extract with 800 ml of water With the remaining 200 ml 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 starch 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 to 7,1 to 7,2 with mol/l sodium hydroxide Transfer 25 ml aliquots of the medium into screw-capped bottles of capacity 25 ml Sterilize in the autoclave at 121 ± °C for 15 6.2.2.2 Double strength basal medium Prepare the double strength medium as in 6.2.2.1 but reduce the volume of water by half Transfer 10 ml and 50 ml aliquots of the medium into screw-capped bottles of capacities 25 ml and 100 ml respectively 6.2.3 Sodium sulfite (Na2SO3), % (m/m) solution Dissolve g of anhydrous sodium sulfite in 100 ml of water Sterilize by filtration Store at between and °C It is advisable to prepare a fresh solution every 14 days 6.2.4 Iron(III) citrate (C6H5O7Fe), % (m/m) solution Dissolve g of iron(III) citrate in 100 ml of water Sterilize by filtration Store at between and °C It is advisable to prepare a fresh solution every 14 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 Add 0,5 ml of the mixture (6.2.5.1) to each bottle of single strength medium (6.2.2.1), which has been freshly heated and cooled 6.2.5.3 Add 0,4 ml of the mixture (6.2.5.1) to each 10 ml, and ml to each 50 ml, of double strength medium (6.2.2.2) similarly treated Apparatus and glassware Usual microbiological laboratory equipment, and 7.1 Screw-cap bottles or vials and stoppers of boron silicate glass of capacities 200, 100 and 25 ml 7.2 Volumetric pipettes, of capacities 10 and ml 7.3 Water baths, thermostatically controlled 7.4 Test tubes, 150 mm × 13 mm 7.5 Iron wire 7.6 Incubator, capable of being maintained at 37 ± °C Sampling Refer to ISO 5667-2 and ISO 8199 for sampling techniques Procedure 9.1 Treatment of samples Refer to ISO 5667-3 for guidance on the preservation and handling of samples, and to ISO 8199 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 Add 50 ml of sample (9.2) to a 100 ml screw-cap bottle containing 50 ml of the double strength complete medium (6.2.5.3) Add 10 ml of sample (9.2) to a series of five 25 ml screw-cap bottles containing 10 ml of double strength complete medium (6.2.5.3) Add ml of sample (9.2) to a series of five 25 ml screw-cap bottles containing 25 ml of single strength complete medium (6.2.5.2) If necessary, add ml of a F 10 dilution of the sample (9.2) to a series of five 25 ml screw-cap bottles containing 25 ml of single strength complete medium (6.2.5.2) In order to carry out a qualitative examination of 100 ml of drinking water or bottled water without making an MPN count, use a 200 ml vial filled with a mixture of 100 ml of double strength complete medium (6.2.5.3) and 100 ml of sample (9.2) © BSI 09-1999 EN 26461-1:1993 If necessary, top up all the bottles with the single strength complete medium (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 at 37 ± °C for 44 ± h Large volumes of culture in hermetically sealed 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) sulfide, shall be regarded as positive 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 most probable number of sulfite-reducing anaerobes (clostridia) per volume of sample It 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 © BSI 09-1999 BS EN 26461-1:1993 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 Industrial Water Society Institute of Petroleum Institution of Gas Engineers Institution of Water and Environmental Management Institution of Water Officers 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 Department of Trade and Industry (Laboratory of the Government Chemist) Department of the Environment for Northern Ireland Department of the Environment (Water Directorate) 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 26461-1:1993 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 ISO 5667-2:1991 Section 6.2:1991 Guidance on sampling techniques ISO 5667-3:1985 Section 6.3:1986 Guidance on the preservation and handling of samples Part Microbiological methods ISO 8199:1988 Section 4.2:1989 Guide to the enumeration of micro-organisms by culture © BSI 09-1999 BS EN 26461-1:1993 BS 6068-4.8: 1993 ISO 6461-1: 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 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