BS EN ISO 7899-1-1999 Water quality Detection and enumeration of intestinal enterococci in surface and waste water Miniaturized method (Most probable number) for surface and waste .pdf

1、BRITISH STANDARD BS EN ISO 7899-1:1999 BS 6068-4.3:1999 Incorporating Technical Corrigendum No. 1 Water quality Detection and enumeration of intestinal enterococci Part 1: Miniaturized method (Most Probable Number) for surface and waste water The European Standard EN ISO 7899-1:1998 has the status o

2、f a British Standard ICS 07.100.20 NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBS EN ISO 7899-1:1999 This British Standard, having been prepared under the direction of the Health and Environment Sector Committee, was published under the authority of the Standards Committee

3、and comes into effect on 15 June 1999 BSI 06-2001 ISBN 0 580 32007 3 National foreword This British Standard is the English language version of EN ISO 7899-1:1998, including technical corrigendum May 2000. It is identical with ISO 7899-1:1998, including technical corrigendum May 2000. It supersedes

4、BS 6068-4.3:1989 which is withdrawn.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, which has the responsibility to: aid enquirers to understand the text; present to the responsible international/Euro

5、pean 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 its secretary

6、. BS EN ISO 7899-1 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 given the secondary identifier BS 6068-4.3. The various sections of BS 6068 are comprised within Parts 1 to 7, which, togethe

7、r 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 described in this B

8、ritish 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 t

9、o 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 contra

10、ct. 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, the EN ISO foreword page,

11、the ISO title page, pages ii to iv, pages 1 to 17 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 13020 T e ch.Co rr .No .1 June 2001 Alteration to titleEUROPEAN STANDARD NOR

12、ME EUROPENNE EUROPISCHE NORM EN ISO 7899-1 November 1998 ICS 07.100.20 Descriptors: see ISO document Incorporating technical corrigendum May 2000 English version Water quality Detection and enumeration of intestinal enterococci Part 1: Miniaturized method (Most Probable Number) for surface and waste

13、 water (ISO 7899-1:1998) Qualit de leau Recherche et dnombrement des entrocoques intestinaux Partie 1: Mthode miniaturise (nombre le plus probable) pour les eaux de surface et rsiduaires (ISO 7899-1:1998) Wasserbeschaffenheit Nachweis und Zhlung von intestinalen Enterokokken Teil 1: Miniaturisiertes

14、 Verfahren (MPN-Verfahren) fr Oberflchenwasser und Abwasser (ISO 7899-1:1998) This European 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 nat

15、ional 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 a

16、ny 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, Czech Republic, Denmark, Finland, France, Germ

17、any, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, 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 1998 CEN A

18、ll rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN ISO 7899-1:1998 EEN ISO 7899-1:1998 BSI 06-2001 Foreword The text of the International Standard ISO 7899-1:1998 has been prepared by Technical Committee ISO/TC 147, Water quality, in colla

19、boration with 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, at the latest by May 1999, and conflicting national standards

20、shall be withdrawn at the latest by May 1999. According to the CEN/CENELEC Internal 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, Irelan

21、d, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and the United Kingdom. Endorsement notice The text of the International Standard ISO 7899-1:1998 was approved by CEN as a European Standard without any modification. NOTE Normative references to International Standards

22、are listed in Annex ZA (normative).EN ISO 7899-1:1998 ii BSI 06-2001 Contents Page Foreword iii Introduction 1 1S c o p e 1 2 Normative references 1 3 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 diluents 2 8 Procedure 3 9 Expression of results 5 10

23、Test report 6 11 Performance data 6 Annex A (informative) Example of software for statistical analysis of MPNs 7 Annex B (informative) Example of software for computation of MPNs 11 Annex C (informative) Synthetic sea salt 12 Annex D (informative) Performance characteristics of the method 13 Annex E

24、 (normative) Quality criteria for manufacturing of the medium in microtitre plates 14 Annex F (normative) Preparation of calibration microtitre plates 15 Annex G (informative) Bibliography 16 Annex ZA (normative) Normative references to international publications with their relevant European publica

25、tions 17 Figure F.1 16 Table 1 4 Descriptors: water, surface water, sewage, quality, bacteriological quality, water pollution, tests, water tests, microbiological analysis, detection, counting, enterobacteriaceae.EN ISO 7899-1:1998 BSI 06-2001 iii Foreword ISO (the International Organization for Sta

26、ndardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Standards is normally carried out through ISO technical committees. Each member body interested in a subject for which a technical committee has been established has the righ

27、t 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 International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. Draft Internat

28、ional Standards adopted by the technical committees are circulated to the member bodies for voting. Publication as an International Standard requires approval by at least 75 % of the member bodies casting a vote. International Standard ISO 7899-1 was prepared by Technical Committee ISO/TC 147, Water

29、 quality, Subcommittee SC 4, Biological methods. This second edition cancels and replaces the first edition (ISO 7899-1:1984), which has been technically revised. ISO 7899 consists of the following parts, under the general title Water quality Detection and enumeration of intestinal enterococci in su

30、rface and waste water. Part 1: Miniaturized method (Most Probable Number) by inoculation in liquid medium; Part 2: Method by membrane filtration. Annex E and Annex F form an integral part of this part of ISO 7899. Annex A, Annex B, Annex C, Annex D and Annex G are for information only.iv blankEN ISO

31、 7899-1:1998 BSI 06-2001 1 Introduction The aim of this part of ISO 7899 is to enumerate the major intestinal enterococci, namely E. faecalis, E. faecium, E. durans and E. hirae, which occur frequently in faeces of humans and homeothermic animals. Other faecal Enterococcus species, namely E. avium,

32、E. cecorum, E. columbae and E. gallinarum, and Streptococcus bovis/equinus strains may occasionally be included, but they occur rarely in the environmental samples. Their recovery tends to be low. Enterococcus casseliflavus and E. mundtii are non-faecal species which, when present in water samples (

33、e.g. because of influence of plant material and some industrial effluents), are enumerated as faecal enterococci. These species and other rare non-faecal species tend to produce yellow pigment on a non-selective medium. The possible interference of non-faecal Enterococcus species should therefore be

34、 considered in the interpretation of results. 1 Scope This part of ISO 7899 specifies a miniaturized method for the detection and enumeration of major intestinal enterococci in surface and waste water by inoculation in a liquid medium. The method is applicable to all types of surface and waste water

35、s, particularly those rich in suspended matter. This method is not suitable for drinking water and any other type of water for which the guideline count is less than 15 per 100 ml. 2 Normative references The following standards contain provisions which, through reference in this text, constitute pro

36、visions of this part of ISO 7899. At the time of publication, the editions indicated were valid. All standards are subject to revision, and parties to agreements based on this part of ISO 7899 are encouraged to investigate the possibility of applying the most recent editions of the standards indicat

37、ed below. Members of IEC and ISO maintain registers of currently valid International Standards. ISO 3951:1989, Sampling procedures and charts for inspection by variables for percent nonconforming. ISO 5667-1:1980, Water quality Sampling Part 1: Guidance on the design of sampling programmes. ISO 5667

38、-2:1991, Water quality Sampling Part 2: Guidance on sampling techniques. ISO 5667-3:1994, Water quality Sampling Part 3: Guidance on the preservation and handling of samples. ISO 8199:1988, Water quality General guide to the enumeration of microorganisms by culture. ISO/IEC Guide 2:1996, Standardiza

39、tion and related activities Vocabulary. 3 Definitions For the purposes of this part of ISO 7899, the definitions given in ISO/IEC Guide 2 and the following definition apply. 3.1 intestinal enterococci microorganisms capable of aerobic growth at 44 C and of hydrolysing the 4-methylumbelliferyl- -D-gl

40、ucoside (MUD), in the presence of thallium acetate, nalidixic acid and 2,3,5-triphenyltetrazolium chloride (TTC), in the liquid medium specified 4 Principle The diluted sample is inoculated in a row of microtitre plate wells containing dehydrated culture medium. The microtitre plates are examined un

41、der ultraviolet light at 366 nm in the dark after an incubation period of between 36 h and 72 h at 44 C0 , 5 C. The presence of enterococci is indicated by fluorescence resulting from the hydrolysis of MUD. The results are given as Most Probable Number (MPN) per 100 ml. 5 Apparatus With the exceptio

42、n of equipment supplied sterile, the glassware shall be sterilized in accordance with the instructions given in ISO 8199. Usual microbiological laboratory equipment, and in particular:EN ISO 7899-1:1998 2 BSI 06-2001 5.1 Apparatus for sterilization by dry heat (oven) or by steam (autoclave). 5.2 The

43、rmostatic incubator, regulated at 44 C 0,5 C. 5.3 Tunnel drier or vertical laminar air flow cabinet (preferably class II). 5.4 UV observation chamber (Woods Lamp 366 nm). WARNING. UV light can cause irritation of skin and eyes. Use protective gloves and glasses. 5.5 Portable refractometer (optional)

44、. 5.6 pH meter, with an accuracy of 0,1. 5.7 Test tubes, 16 mm 160 mm and 20 mm 200 mm, or flasks with similar capacity. 5.8 Adjustable or pre-set 8-channel multipipette, or any system suitable for measuring and distributing 200 l per well. 5.9 Sterile tips for multipipette. 5.10 Equipment for membr

45、ane filtration, in accordance with ISO 8199, 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 cover strips for sealing microtitre plates. 5.13 Sterile

46、Petri dishes, 90 mm in diameter. 6 Sampling 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 diluents 7.1 General instructions To ensure reproducible results, prepare culture medium and diluents, using eith

47、er constituents of uniform quality and chemicals of recognized analytical grade or a dehydrated diluent or complete medium prepared following the manufacturers instructions. Prepare them with distilled or demineralized water, free from substances capable of inhibiting or promoting growth under the t

48、est conditions. If the media are not used immediately, 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.

49、 7.2 Diluent 7.2.1 Special Diluent (SD) Sterilize in 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 100 ml of 50 % ethanol. It is used only to colour the SD blue and avoid confusing it with demineralized or distilled water. 7.2.2 Demineralized or distilled water Water used for dilution shall be demineralized or distilled water free from substances inhibiting growth under the