1、BSI Standards PublicationWater quality Enumerationof Escherichia coli and coliformbacteriaPart 2: Most probable number methodBS EN ISO 9308-2:2014BS EN ISO 9308-2:2014National forewordThis British Standard is the UK implementation of EN ISO 9308-2:2014. It is identical to ISO 9308-2:2012. It superse
2、des BS ISO 9308-2:2012, which is withdrawn.The UK participation in its preparation was entrusted by Technical Committee EH/3 Water quality to Subcommittee EH/3/4, Microbiological methods.A list of organizations represented on this subcommittee can be obtained on request to its secretary.This publica
3、tion does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application. The British Standards Institution 2014. Published by BSI Standards Limited 2014ISBN 978 0 580 84023 4ICS 07.100.20Compliance with a British Standard cannot confer immunity
4、fromlegal obligations.This British Standard was published under the authority of theStandards Policy and Strategy Committee on 30 June 2012.Amendments/corrigenda issued since publicationDate Text affected30 June 2014 This corrigendum renumbers BS ISO 9308-2:2012 as BS EN ISO 9308-2:2014BRITISH STAND
5、ARDEUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 9308-2 April 2014 ICS 07.100.20 English Version Water quality - Enumeration of Escherichia coli and coliform bacteria - Part 2: Most probable number method (ISO 9308-2:2012) Qualit de leau - Dnombrement des Escherichia coli et des bactries
6、coliformes - Partie 2: Mthode du nombre le plus probable (ISO 9308-2:2012) Wasserbeschaffenheit - Zhlung von Escherichia coli und coliformen Bakterien - Teil 2: Verfahren zur Bestimmung der wahrscheinlichsten Keimzahl (ISO 9308-2:2012) This European Standard was approved by CEN on 11 April 2014. CEN
7、 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 appl
8、ication to the CEN-CENELEC Management Centre 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 CEN-CENELEC Ma
9、nagement Centre has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Lat
10、via, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Management Centre: Avenue Mar
11、nix 17, B-1000 Brussels 2014 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN ISO 9308-2:2014 EForeword The text of ISO 9308-2:2012 has been prepared by Technical Committee ISO/TC 147 “Water quality” of the International Organizatio
12、n for Standardization (ISO) and has been taken over as EN ISO 9308-2:2014 by Technical Committee CEN/TC 230 “Water analysis” the secretariat of which is held by DIN. This European Standard shall be given the status of a national standard, either by publication of an identical text or by endorsement,
13、 at the latest by October 2014, and conflicting national standards shall be withdrawn at the latest by October 2014. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN and/or CENELEC shall not be held responsible for identifying
14、any or all such patent rights. According to the CEN-CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Rep
15、ublic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. Endorsement notice The text of ISO 9308-2:2012 has been
16、approved by CEN as EN ISO 9308-2:2014 without any modification. ii ISO 2012 All rights reservedBS EN ISO 9308-2:2014 EN ISO 9308-2:2014 (E) ISO 2012 All rights reserved iiiContents Page iv Introduction1 Scope 1 2 Normative references 1 3 Terms and definitions . 2 4 Principle . 2 5 Apparatus and glas
17、sware . 2 6 Culture media and reagents . 3 7 Sampling 3 8 Procedure . 3 9 Expression of results 4 10 Test report 4 11 Quality assurance 4 Annex A (informative) Further microbiological information on coliform bacteria . 5 Annex B (normative) The Quanti-Tray5)Sealer and calculation of results 6 Annex
18、C (informative) Composition of the Colilert-18 medium . 42 Annex D (informative) Validation of Colilert-18/Quanti-Tray8)for the enumeration of E.coli and coliform bacteria from water 44 Bibliography 46 BS EN ISO 9308-2:2014 ISO 9308-2:2012 (E)iv ISO 2012 All rights reservedIntroduction The presence
19、and extent of faecal pollution is an important factor in assessing the quality of a body of water and the risk to human health from infection. Examination of water samples for the presence of Escherichia coli (E. coli), which normally inhabits the bowel of man and other warm-blooded animals, provide
20、s an indication of such pollution. Examination for coliform bacteria can be more difficult to interpret because some coliform bacteria live in soil and surface fresh water and are not always intestinal. Therefore, the presence of coliform bacteria, although not a proof of faecal contamination, may i
21、ndicate a failure in treatment or ingress of water into the distribution system. The International Organization for Standardization (ISO) draws attention to the fact that it is claimed that compliance with this document may involve the use of patents concerning Colilert-18 and Quanti-Tray and Quanti
22、-Tray 2000 given in this document. ISO takes no position concerning the evidence, validity and scope of these patent rights. The holder of this patent right has assured the ISO that he/she is willing to negotiate licences either free of charge or under reasonable and non-discriminatory terms and con
23、ditions with applicants throughout the world. In this respect, the statement of the holder of this patent right is registered with ISO. Information may be obtained from: IDEXX Laboratories, Inc. One IDEXX Drive Westbrook, Maine 04092 USA Attention is drawn to the possibility that some of the element
24、s of this document may be the subject of patent rights other than those identified above. ISO shall not be held responsible for identifying any or all such patent rights. ISO (http:/www.iso.org/patents) and IEC (http:/patents.iec.ch) maintain on-line databases of patents relevant to their standards.
25、 Users are encouraged to consult the databases for the most up to date information concerning patents. BS EN ISO 9308-2:2014 ISO 9308-2:2012 (E)INTERNATIONAL STANDARD ISO 2012 All rights reserved 1Water quality Enumeration of Escherichia coli and coliform bacteria Part 2: Most probable number method
26、 WARNING Persons using this part of ISO 9308 should be familiar with normal laboratory practice. This International Standard does not purport to address all of the safety problems, if any, associated with its use. It is the responsibility of the user to establish appropriate safety and health practi
27、ces and to ensure compliance with any national regulatory conditions. IMPORTANT It is absolutely essential that tests conducted in accordance with this part of ISO 9308 be carried out by suitably qualified staff. 1 Scope This part of ISO 9308 specifies a method for the enumeration of E. coli and col
28、iform bacteria in water. The method is based on the growth of target organisms in a liquid medium and calculation of the “Most Probable Number” (MPN) of organisms by reference to MPN tables. This method can be applied to all types of water, including those containing an appreciable amount of suspend
29、ed matter and high background counts of heterotrophic bacteria. However, it must not be used for the enumeration of coliform bacteria in marine water. When using for the enumeration of E. coli in marine waters, a 110 dilution in sterile water is typically required, although the method has been shown
30、 to work well with some marine waters that have a lower than normal concentration of salts. In the absence of data to support the use of the method without dilution, a 110 dilution is used. This method relies upon the detection of E. coli based upon expression of the enzyme -D-glucuronidase and cons
31、equently does not detect many of the enterohaemorhagic strains of E. coli, which do not typically express this enzyme. Additionally, there are a small number of other E. coli strains that do not express -D-glucuronidase. The choice of tests used in the detection and confirmation of the coliform grou
32、p of bacteria, including E. coli, can be regarded as part of a continuous sequence. The extent of confirmation with a particular sample depends partly on the nature of the water and partly on the reasons for the examination. The test described in this part of ISO 9308 provides a confirmed result wit
33、h no requirement for further confirmation of positive wells. NOTE While this method describes the use of an enumeration device that is commercially available, the medium described here can also be used in a standard MPN format. 2 Normative references The following documents, in whole or in part, are
34、 normatively referenced in this document and are indispensable for its application. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. ISO 8199, Water quality General guide to the enumeration
35、 of micro-organisms by culture ISO/IEC Guide 2:2004, Standardization and related activities General vocabulary BS EN ISO 9308-2:2014 ISO 9308-2:2012 (E)2 ISO 2012 All rights reservedISO 19458, Water quality Sampling for microbiological analysis 3 Terms and definitions For the purpose of this documen
36、t, the terms and definitions given in ISO/IEC Guide 2 and the following apply. 3.1 coliform bacterium member of the Enterobacteriaceae that express the enzyme -D-galactosidase 3.2 Escherichia coli member of the Enterobacteriaceae that expresses both -D-galactosidase and -D-glucuronidase enzymes 4 Pr
37、inciple A snap pack of dehydrated medium is added to a sample of water (100 ml), or a dilution of a sample made up to 100 ml. Sample plus medium is gently shaken to ensure adequate mixing and to afford dissolution of the medium. The sample plus medium is then aseptically poured into a Quanti-Tray1)o
38、r Quanti-Tray/20001)to enumerate up to 201 organisms or 2 419 organisms per 100 ml, respectively. Trays are sealed with a Quanti-Tray1)Sealer and then incubated at (36 2) C for 18 h to 22 h. After incubation, sample wells that have a yellow colour of equal or greater intensity than that of the compa
39、rator wells are considered positive for coliform bacteria. Yellow wells that also exhibit any degree of fluorescence are considered positive for E. coli. By means of statistical tables, or a simple computer program, the most probable number (MPN) of coliform bacteria and E. coli in 100 ml of the sam
40、ple can be determined. NOTE The yellow colouration can be seen with the naked eye and results from the cleavage of ortho-nitrophenol galactoside by the enzyme -D-galactosidase. The fluorescence is demonstrable under ultraviolet light (365 nm) and originates from the cleavage of the molecule 4-methyl
41、umbelliferyl glucuronide (MUG) by the enzyme -D-glucuronidase to produce the fluorescent compound methyl umbelliferone. 5 Apparatus and glassware Use microbiological laboratory equipment and, in particular, the following: 5.1 Apparatus for sterilization by steam (autoclave) Apparatus and glassware n
42、ot supplied sterile shall be sterilized according to the instructions given in ISO 8199. 5.2 Hot air oven, for dry heat sterilization. 5.3 Incubator, thermostatically controlled at (36 2) C. 5.4 Quanti-Tray1)sealer. 5.5 Sterile wide mouthed vessels of at least 110 ml. 1) Quanti-Tray is a trademark o
43、r registered trademark of IDEXX Laboratories, Inc. or its affiliates in the United States and/or other countries. This information is given for the convenience of users of this part of ISO 9308 and does not constitute an endorsement by ISO of this product. BS EN ISO 9308-2:2014 ISO 9308-2:2012 (E) I
44、SO 2012 All rights reserved 35.6 Quanti-Tray2)comparator. 5.7 Ultraviolet lamp, 365 nm. 5.8 Quanti-Tray2)or Quanti-Tray/20002), see Annex B. 6 Culture media and reagents 6.1 Basic materials The method utilises Colilert3)-18 a medium based on the Defined Substrate Technology available for a 100 ml sa
45、mple as a ready to use powder dispensed in snap packs. Each snap pack contains sufficient medium (2,8 g) for a single test. Medium is stored under ambient conditions (2 C to 25 C) out of direct sunlight and should be used before the expiry date listed on the snap pack. The medium is composed of two
46、components to give the final concentrations as shown in Annex C. 6.2 Diluent For dilutions to be used with Colilert3)-18, use only sterile, non-inhibitory, oxidant-free water (deionized or tap). The use of buffered, saline or peptone-containing diluents interferes with the performance of the test. 6
47、.3 Antifoam B Antifoam B is a 10 % active, water soluble suspension of silicone. 7 Sampling Take the samples and deliver them to the laboratory in accordance with ISO 19458. 8 Procedure 8.1 Preparation of the sample Samples should be transported and stored at (5 3) C in accordance with ISO 19458 and
48、 analysis commenced on the day of collection or within 18 h. Under exceptional circumstances, the samples may be kept at (5 3) C for up to 24 h prior to examination. 8.2 Inoculation of media Aseptically add a single snap pack of Colilert4)-18 medium (2,8 g) to each 100 ml volume of sample or dilutio
49、n. When the medium has completely dissolved, the sample plus medium is aseptically poured into either a 2) Quanti-Tray is a trademark or registered trademark of IDEXX Laboratories, Inc. or its affiliates in the United States and/or other countries. This information is given for the convenience of users of this part of ISO 9308 and does not constitute an endorsement by ISO of this product. 3) Colilert is a trademark or registered trademark of IDEXX Laboratories, Inc. or its affiliates in the United States and/or oth
