1、BRITISH STANDARD BS EN ISO 9308-3:1999 BS 6068-4.10: 1999 Incorporating Technical Corrigendum No. 1 Water quality Detection and enumeration of Escherichia coli and coliform bacteria Part 3: Miniaturized method (Most Probable Number) for the detection and enumeration of E. coli in surface and waste w
2、ater The European Standard EN ISO 9308-3:1998 has the status of a British Standard ICS 07.100.20 NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBS EN ISO 9308-3:1999 This British Standard, having been prepared under the direction of the Health and Environment Sector Committee,
3、 was published under the authority of the Standards Committee and comes into effect on 15 June 1999 BSI 07-2001 ISBN 0 580 32006 5 National foreword This British Standard is the English language version of EN ISO 9308-3:1998, including technical corrigendum May 2000. It is identical with ISO 9308-3:
4、1998, including technical corrigendum May 2000. The UK participation in its preparation was entrusted by Technical Committee EH/3, Water quality, to Subcommittee EH/3/4, Microbiological methods, which has the re- sponsibility to: aid enquirers to understand the text; present to the responsible inter
5、national/European committee any enquiries on the interpretation, or proposals for change, and keep the UK interests informed; monitor related international and European developments and promulgate them in the UK. A list of organizations represented on this subcommittee can be obtained on request to
6、its secretary. BS EN ISO 9308-3 is one of a series of standards on water quality, others of which have been, or will be, published as sections of BS 6068. This standard has therefore been giv- en the secondary identifier BS 6068-4.10. The various sections of BS 6068 are comprised within Parts 1 to 7
7、, which, together with Part 0, are listed below. Part 0: Introduction; Part 1: Glossary; Part 2: Physical, chemical and biochemical methods; Part 3: Radiological methods; Part 4: Microbiological methods; Part 5: Biological methods; Part 6: Sampling; Part 7: Precision and accuracy. NOTE The tests des
8、cribed in this British Standard should only be carried out by suitably qualified persons with an appropriate level of microbiological expertise. Standard microbiological procedures should be followed throughout. Cross-references The British Standards which implement international or European publica
9、tions referred to in this document may be found in the BSI Standards Catalogue under the section entitled “International Standards Correspondence Index”, or by using the “Find” facility of the BSI Standards Electronic Catalogue. A British Standard does not purport to include all the necessary provis
10、ions 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, the EN ISO title page, EN ISO for
11、eword page, the ISO title page, pages ii to iv, pages 1 to 18, an inside back cover and a back cover. The BSI copyright notice displayed in this document indicates when the document was last issued. Amendments issued since publication Amd. No. Date Comments 13021 Tech Corr. No.1 July 2001 Alteration
12、 to titleEUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 9308-3 November 1998 ICS 07.100.20 Incorporating technical corrigendum No.1 Descriptors: see ISO document English version Water quality Detection and enumeration of Escherichia coli and coliform bacteria Part 3: Miniaturized method (M
13、ost Probable Number) for the detection and enumeration of E coli in surface and waste water (ISO 9308-3:1998) Qualit de leau Recherche et dnombrement des Escherichia coli et des bactries coliformes Partie 3: Mthode miniaturise (nombre le plus probable) pour la recherche et le dnombrement des E.coli
14、dans les eaux de surface et rsiduaires (ISO 9308-3:1998) Wasserbeschaffenheit Nachweis und Zhlung von Escherichia coli und coliformen Bakterien Teil 3: Miniaturisiertes Verfahren (MPN-Vesrfahren) zum Nachweis und zur Zhlung von E.coil in Oberflchenwasser und Abwasser (ISO 9308-3:1998) This European
15、Standard was approved by CEN on 15 November 1998. 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 conce
16、rning 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
17、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, Czech Republic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Swed
18、en, Switzerland and United Kingdom. CEN European Committee for Standardization Comit Europen de Normalisation Europisches Komitee fr Normung Central Secretariat: rue de Stassart 36, B-1050 Brussels 1998 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national M
19、embers. Ref. No. EN ISO 9308-3:1998 EEN ISO 9308-3:1998 BSI 07-2001 Foreword The text of the International Standard ISO 9308-3:1998 has been prepared by Technical Committee ISO/TC 147, Water quality, in collaboration with Technical Committee CEN/TC 230, Water analysis, the Secretariat of which is he
20、ld by DIN. 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 May 1999, and conflicting national standards shall be withdrawn at the latest by May 1999. According to the CEN/CENELEC Internal Regulati
21、ons, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and the Unit
22、ed Kingdom. Endorsement notice The text of the International Standard ISO 9308-3:1998 was approved by CEN as a European Standard without any modification. NOTE Normative references to International Standards are listed in Annex ZA (normative).EN ISO 9308-3:1998 ii BSI 07-2001 Contents Page Foreword
23、iii 1S c o p e 1 2 Normative references 1 3 Terms and definitions 1 4P r i n c i p l e 1 5A p p a r a t u s 1 6S a m p l i n g 2 7 Culture media and diluent 2 8 Procedure 3 9 Expression of results 5 10 Test report 6 11 Performance data 6 Annex A (informative) Example of software for statistical anal
24、ysis of MPNs 7 Annex B (informative) Example of software for computation of MPN 11 Annex C (informative) Synthetic sea salt 13 Annex D (informative) Performance characteristics of the method 14 Annex E (normative) Quality criteria for manufacturing of the medium in microtitre plates 14 Annex F (norm
25、ative) Preparation of calibration microtitre plates 15 Annex ZA (normative) Normative references to international publications with their relevant European publications 18 Bibliography 17 Figure F.1 16 Table 1 3 Descriptors: Water, surface water, sewage, quality, bacteriological quality, water pollu
26、tion, tests, water tests,.EN ISO 9308-3:1998 BSI 07-2001 iii Foreword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Standards is normally carried out through ISO technical commi
27、ttees. Each member body interested in a subject for which a technical committee has been established has the right to be represented on that committee. International organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the I
28、nternational Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 3. Draft International Standards adopted by the technical committees are circulated to the member
29、bodies for voting. Publication as an International Standard requires approval by at least 75 % of the member bodies casting a vote. International Standard ISO 9308-3 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 4, Microbiological methods. ISO 9308 consists of the fo
30、llowing parts, under the general title Water quality Detection and enumeration of Escherichia coli and coliform bacteria in surface and waste water: Part 1: Membrane filtration method; Part 2: Liquid enrichment method; Part 3: Miniaturized method (Most Probable Number) by inoculation in liquid media
31、. Annex E and Annex F form a normative part of this part of ISO 9308. Annex A to Annex D are for information only.iv blankEN ISO 9308-3:1998 BSI 07-2001 1 1 Scope This part of ISO 9308 specifies a miniaturized method for the detection and enumeration of Escherichia coli (E. coli) in surface and wast
32、e water by inoculation in a liquid medium. The method is applicable to all types of surface and waste waters, particularly those rich in suspended matter. This method is not suitable for drinking water and any other type of water for which the guideline is less than 15 counts per 100 ml. This method
33、 is not appropriate for enumeration and detection of coliform bacteria other than E. coli. 2 Normative references The following normative documents contain provisions which, through reference in this text, constitute provisions of this International Standard. For dated references, subsequent amendme
34、nts to, or revisions of, any of these publications do not apply. However, parties to agreements based on this International Standard are encouraged to investigate the possibility of applying the most recent editions of the normative documents indicated below. For undated references, the latest editi
35、on of the normative document referred to applies. Members of ISO and IEC maintain registers of currently valid International Standards. ISO 3951, Sampling procedures and charts for inspection by variables for percent nonconforming. ISO 5667-1, Water quality Sampling Part 1: Guidance on the design of
36、 sampling programmes. ISO 5667-2, Water quality Sampling Part 2: Guidance on sampling techniques. ISO 5667-3, Water quality Sampling Part 3: Guidance on the preservation and handling of samples. ISO 8199, Water quality General guide to the enumeration of microorganisms by culture. ISO/IEC Guide 2, S
37、tandardization and related activities Vocabulary. 3 Terms and definitions For the purposes of this part of ISO 9308, the terms and definitions given in ISO/IEC Guide 2 and the following apply. 3.1 Escherichia coli E. coli -D-glucuronidase-positive microorganism growing at an incubation temperature o
38、f 44 C in the specified liquid medium containing 4-methylumbelliferyl- -D-glucuronide (MUG) 4 Principle The diluted sample is inoculated in a row of microtitre plate wells containing dehydrated culture medium. The microtitre plates are examined under ultraviolet light at 366 nm in the dark after an
39、incubation period of 36 h minimum and 72 h maximum at 44 C 0,5 C. The presence of E. coli is indicated by a blue fluorescence resulting from hydrolysis of MUG. The results are given as most probable number (MPN) per 100 ml. 5 Apparatus With the exception of equipment supplied sterile, the glassware
40、shall be sterilized in accordance with instructions given in ISO 8199 Usual microbiological laboratory equipment, and in particular: 5.1 Apparatus for sterilization by dry heat (oven) or steam (autoclave):. 5.2 Thermostatic incubator regulated at 44 C 0,5 C. 5.3 Tunnel drier or vertical laminar air
41、flow cabinet (preferably class II). 5.4 UV observation chamber (Woods Lamp, 366 nm). WARNING. UV light causes irritation of eyes and skin. Use protective glasses and gloves. 5.5 Portable refractometer (optional).EN ISO 9308-3:1998 2 BSI 07-2001 5.6 pH meter with an accuracy of 0,1. 5.7 Test tubes of
42、 dimensions 16 mm 160 mm and 20 mm 200 mm, or flasks with similar capacity. 5.8 8-channel multipipette, adjustable or preset, or any other system suitable for measuring and distributing 200 l per well. 5.9 Sterile tips for multipipette. 5.10 Equipment for membrane filtration in accordance with ISO 8
43、199, including membrane filters with a nominal pore size of 0,2 m, for sterilization of liquid media. 5.11 Sterile microtitre plates, 96-well, 350 l, flat bottomed, nonfluorescent. 5.12 Sterile adhesive covering strips for sealing microtitre plates. 5.13 Sterile Petri dishes, 90 mm in diameter. 6 Sa
44、mpling Take the samples and deliver them to the laboratory in accordance with ISO 8199 and ISO 5667-1, ISO 5667-2 and ISO 5667-3. 7 Culture media and diluent 7.1 General instructions To ensure reproducible results, prepare culture medium and diluents, using either constituents of uniform quality and
45、 chemicals of recognized analytical grade, or a dehydrated diluent or complete medium prepared following the manufacturers instructions. Prepare them with demineralized or distilled water free from substances capable of inhibiting growth under the test conditions. If the media are not used immediate
46、ly, preserve them in the dark at (5 3) C for up to one month in conditions avoiding any alterations to their composition. NOTE The use of chemicals of other grades is permissible providing they are shown to be of equivalent performance in the test. 7.2 Diluents 7.2.1 Special diluent (SD) Sterilize i
47、n the autoclave (5.1) at 121 C 3 C for 15 min to 20 min. The bromophenol blue solution is prepared by adding 0,04 g in 100ml of 50 % ethanol. It is only used to colour the SD blue and avoid confusing it with demineralized or distilled water. 7.2.2 Demineralized or distilled water, free from substanc
48、es inhibiting growth under the test conditions. Sterilize in the autoclave (5.1) at 121 C 3 C for 15 min to 20 min. 7.3 Culture medium: MUG/EC medium Composition 1) Synthetic sea salt 1) 22,5 g Bromophenol blue solution (optional) 10 ml Demineralized or distilled water (7.2.2)1 0 0 0 m l Tryptone 40
49、 g Salicin 1 g Triton X 100 1 g MUG (4-methylumbelliferyl- -D-glucuronide) 100 mg Demineralized or distilled water (7.2.2)1 0 0 0 m l 1) A typical analysis of commercially available and suitable synthetic sea salt is given in Annex C. Other diluents, such as distilled water, can be used for E.coil enumeration, unless intestinal enterococi are to be enumerated from the same dilution tubes.EN ISO 9308-3:1998 BSI 07-2001 3 Successively add tryptone, salici
