1、BRITISH STANDARD Water quality - Detection and enumeration of Escherichia coli and coliform bacteria - O Part 3: Miniaturized method (Most Probable Number) for the . detection and enumeration of E. coli in surface and waste water The European Standard EN IS0 9308-3: 1998 has the status of a British
2、Standard ICs 07.100.20 BS EN IS0 9308-3:1999 BS 6068-4.10: 1999 Incorporating Technical Corrigendum No. 1 NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAW BS EN IS0 9308-3:1999 direction of the Health and Environment Sector Committee, was published under the authority of the St
3、andards Committee and Amd. No. 13021 Tech Corr. No.1 National foreword Date Comments July 2001 Alteration to title This British Standard is the English language version of EN IS0 9308-3:1998, including technical corrigendum May 2000. It is identical with IS0 9308-3:1998, including technical corrigen
4、dum May 2000. The UK participation in its preparation was entrusted by Technical Committee EH3, Water quality, to Subcommittee EW3/4, Microbiological methods, which has the re- sponsibility to: - aid enquirers to understand the text; - present to the responsible internationallEuropean committee any
5、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. Alist of organizations represented on this subcommittee can be obtained on request to its secretary. BS EN IS0 9308-3
6、 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, which, together with Part O,
7、are listed below. - Part O: 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 described in this Bri
8、tish 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 publications referred to
9、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 provisions of a contract.
10、 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 IS0 title page, EN IS0 foreword page, the IS0
11、 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. This British Standard. having Amendments issued since publication comes into effect on 15June1999 O BSI 07-2001 I I ISBN
12、 O 580 32006 5 EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN IS0 9308-3 November 1998 ICs 07.100.20 Incorporating technical corrigendum No.1 Descriptors: see IS0 document English version Water quality - Detection and enumeration of Escherichia coli and coliform bacteria - Part 3: Miniaturized
13、 method (Most Probable Number) for the detection and enumeration of E coli in surface and waste water (IS0 9308-3:1998) Qualit de leau - Recherche et dnombrement des Escherichia coli et des bactries coliformes - Bakterien - Partie 3: Mthode miniaturise (nombre le plus probable) pour la recherche et
14、le dnombrement des E.coli dans les eaux de surface et rsiduaires Abwasser Wasserbeschaffenheit - Nachweis und Zhlung von Escherichia coli und coliformen Teil 3: Miniaturisiertes Verfahren (MPN-Vesrfahren) zum Nachweis und zur Zhlung von E.coiZ in Oberflchenwasser und (IS0 9308-31998) (IS0 9308-31998
15、) This European Standard was approved by CEN on 15 November 1998. CEN members are bound to comply with the CENICENELEC 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
16、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 memb
17、er 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, Czech Republic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portu
18、gal, Spain, Sweden, 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 0 1998 CEN All rights of exploitation in any form and by any means reserved worldwide
19、for CEN national O - Members. Ref. No. EN IS0 9308-3:1998 E EN IS0 9308-3:1998 Foreword The text of the International Standard IS0 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
20、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, at the latest by May 1999, and conflicting national standards shall be withdrawn at the latest by May 1999. According to the CENICENELEC Intern
21、al Regulations, 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 a
22、nd the United Kingdom. Endorsement notice The text of the International Standard IS0 9308-3:1998 was approved by CEN as a European Standard without any modification. NOTE listed in Annex ZA (normative). Normative references to International Standards are O BSI 07-2001 INTERNATIONAL STANDARD IS0 9308
23、-3 First edition 1998-1 1-1 5 Corrected 2000-05-01 Water quality - Detection and enumeration of Escherichia coli and coliform bacteria - Part 3: M i n at u rizec met hoc (Most Probable Number) for the detection and enumeration of E. Col in surface and waste water Qualit de Veau - Recherche et dnombr
24、ement des Escherichia coli et des bactries colifornes Partie 3: Mthode miniaturise (nombre le plus probable) pour la recherche et le dnombrement des E. coli dans les eaux de surface et rsiduaires Reference number IS0 9308-3:1998(E) EN IS0 9308-3:1998 Contents Foreword 1 Scope 2 Normative references
25、3 Terms and definitions 4 Principle 5 Apparatus 6 Sampling 7 Culture media and diluent 8 Procedure 9 Expression of results 10 Testreport 11 Performance data Annex A (informative) Example of software for statistical analysis of MPNs Annex B (informative) Example of software for computation of MPN Ann
26、ex C (informative) Synthetic sea salt Annex D (informative) Performance characteristics of the method Annex E (normative) Quality criteria for manufacturing of the medium in microtitre plates Annex F (normative) Preparation of calibration microtitre plates Annex ZA (normative) Normative references t
27、o international publications with their relevant European publications Bibliography Figure F.l Table 1 Page 1 1 1 1 1 2 2 3 5 6 6 . 111 7 11 13 14 14 15 18 17 16 3 Descriptors: Water, surface water, sewage, quality, bacteriological quality, water pollution, tests, water tests,. 11 O BSI 07-2001 EN I
28、S0 9308-31998 Foreword IS0 (the International Organization for Standardization) is a worldwide federation of national standards bodies (IS0 member bodies). The work of preparing International Standards is normally carried out through IS0 technical committees. Each member body interested in a subject
29、 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. IS0 collaborates closely with the International Electrotechnical Commission (IEC)
30、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 bodies for voting. Publication as an Internatio
31、nal Standard requires approval by at least 75 % of the member bodies casting a vote. International Standard IS0 9308-3 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 4, Microbiological methods. IS0 9308 consists of the following parts, under the general title Water qu
32、ality - 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. Annex E and Annex F form a normative pa
33、rt of this part of IS0 9308. Annex A to Annex D are for information only. . O BSI 07-2001 111 EN IS0 9308-3:1998 1 Scope This part of IS0 9308 specifies a miniaturized method for the detection and enumeration of Escherichia coli (E. coli) in surface and waste water by inoculation in a liquid medium.
34、 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 is not appropriate for enumeration and de
35、tection 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 amendments to, or revisions of, any of these publ
36、ications 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 edition of the normative document referred to a
37、pplies. Members of IS0 and IEC maintain registers of currently valid International Standards. IS0 3951, Sampling procedures and charts for inspection by variables for percent nonconforming. IS0 5667-1, Water quality - Sampling - Part 1: Guidance on the design of samplingprogrammes. IS0 5667-2, Water
38、 quality - Sampling - Part 2: Guidance on sampling techniques. IS0 5667-3, Water quality - Sampling - Part 3: Guidance on the preservation and handling of samples. IS0 8199, Water quality - General guide to the enumeration of microorganisms by culture. ISO/IEC Guide 2, Standardization and related ac
39、tivities - Vocabulary. 3 Terms and definitions For the purposes of this part of IS0 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 of 44 “C in the specified li
40、quid 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 incubation period of 36 h mi
41、nimum and 72 h maximum at 44 “C f 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. O O 5 Apparatus With the exception of equipment supplied sterile, the glassware shall be sterilized
42、in accordance with instructions given in IS0 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 f 0,5 OC. 5.3 Tunnel drier or vertical laminar air flow cabinet (pr
43、eferably 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. O 5.5 Portable refractometer (optional). O BSI 07-2001 1 EN IS0 9308-3:1998 5.6 pH meter with an accuracy of fO,1. 5.7 Test tubes of dimension
44、s 16 mm X 160 mm and 20 mm X 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 p1 per well. 5.9 Sterile tips for multipipette. 5.10 Equipment for membrane filtration in accordance with IS0 8199,
45、including membrane filters with a nominal pore size of 0,2 pm, for sterilization of liquid media. 5.11 Sterile microtitre plates, 96-well, 350 p1, flat bottomed, nonfluorescent. 5.12 Sterile adhesive covering strips for sealing microtitre plates. 5.13 Sterile Petri dishes, 90 mm in diameter. 6 Sampl
46、ing Take the samples and deliver them to the laboratory in accordance with IS0 8199 and IS0 5667-1, IS0 5667-2 and IS0 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 ch
47、emicals 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 immediately,
48、 preserve them in the dark at (5 f 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 (SO) Synthetic s
49、ea saltl) 22,5 g Bromophenol blue solution (optional) 10 ml Demineralized or distilled water (7.2.2) 1 O00 ml Sterilize in the autoclave (5.1) at 121 “C f 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 substances inhibiting growth under the test conditions. Sterilize in the autoclave (5.1) at 121 “C f 3 “C for 15 min to 20 min. 7.3 Culture medium:
