1、January 2008DEUTSCHE NORM English price group 17No part of this standard may be reproduced without prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).ICS 07.100.20; 13.060.70!$
2、KcJ“1406439www.din.deDDIN EN ISO 8199Water quality General guidance on the enumeration of micro-organisms by culture(ISO 8199:2005)English version of DIN EN ISO 8199:2008-01Wasserbeschaffenheit Allgemeine Anleitung zur Zhlung von Mikroorganismen durch Kulturverfahren(ISO 8199:2005)Englische Fassung
3、DIN EN ISO 8199:2008-01www.beuth.deDocument comprises 46 pagesDIN EN ISO 8199:2008-01 2 This standard is part of the series Deutsche Einheitsverfahren zur Wasser-, Abwasser- und Schlamm-untersuchung Mikrobiologische Verfahren (Gruppe K) (German standard methods for the examination of water, waste wa
4、ter and sludge Microbiological methods (group K) and describes method K 20. National foreword This standard has been prepared by Technical Committee ISO/TC 147 “Water quality” in collaboration with Technical Committee CEN/TC 230 “Water analysis” (Secretariat: DIN, Germany). The responsible German bo
5、dy involved in its preparation was the Normenausschuss Wasserwesen (Water Practice Standards Committee), Technical Committees NA 119-01-03-03 UA Mikrobiologie and NA 119-01-03 AA Wasseruntersuchung. Designation of method K 20 General guidance on the enumeration of micro-organisms by culture: Method
6、DIN EN ISO 8199 K 20 Expert assistance and specialized laboratories will be required to perform the analysis described in this standard. Existing safety requirements are to be observed. Depending on the objective of the analysis, a check shall be made on a case-by-case basis as to whether and to wha
7、t extent additional conditions will have to be specified. Standard methods published as DIN Standards are obtainable from Beuth Verlag GmbH, either individually or grouped in volumes. The standard methods included in the loose-leaf publication entitled Deutsche Einheits-verfahren zur Wasser-, Abwass
8、er- und Schlammuntersuchung will continue to be published by Wiley-VCH Verlag and Beuth Verlag GmbH. All standard methods relevant to the Abwasserverordnung (Waste Water Regulation) (AbwV) included in the new Regulation on Section 7a of the Gesetz zur Ordnung des Wasserhaushalts (German Water Manage
9、ment Act) concerning Anforderungen an das Einleiten von Abwasser in Gewsser together with the Abwasser-verordnung and the Gesetz zur Ordnung des Wasserhaushalts have been published by Beuth-Verlag GmbH as a loose-leaf collection Analysenverfahren in der Abwasserverordnung Rechtsvorschriften und Norm
10、en (Supplements). Standards or draft standards bearing the group title “German standard methods for the examination of water, waste water and sludge” are classified under the following categories (main titles): General information (group A) (DIN 38402) Sensory analysis (group B) (DIN 38403) Physical
11、 and physicochemical parameters (group C) (DIN 38404) Anions (group D) (DIN 38405) Cations (group E) (DIN 38406) Substance group analysis (group F) (DIN 38407) Gaseous constituents (group G) (DIN 38408) Parameters characterizing effects and substances (group H) (DIN 38409) Biological-ecological meth
12、ods of analysis (group M) (DIN 38410) Microbiological methods (group K) (DIN 38411) Test methods using water organisms (group L) (DIN 38412) Individual constituents (group P) (DIN 38413) Sludge and sediments (group S) (DIN 38414) Bio-assays with microorganisms (group T) (DIN 38415) DIN EN ISO 8199:2
13、008-01 3 In addition to the methods described in the DIN 38402 to DIN 38415 series of standards, there are a number of European and International Standards available as DIN EN, DIN EN ISO and DIN ISO Standards which also form part of the collection of German standard methods. Information on Parts of
14、 these series of standards that have already been published can be obtained from the offices of the Normenausschuss Wasserwesen, telephone +49 (30) 2601-2448, or from Beuth Verlag GmbH, Burggrafenstrae 6, 10787 Berlin, Germany. The DIN Standards corresponding to the International Standards referred
15、to in the EN are as follows: ISO 3696 DIN ISO 3696 ISO 5667-2 DIN EN ISO 5667-1 ISO 5667-3 DIN EN ISO 5667-3 ISO 5725-1 DIN ISO 5725-1 ISO 6887-1 DIN EN ISO 6887-1 ISO 7218 DIN EN ISO 7218 ISO 7899-1 DIN EN ISO 7899-1 ISO 9308-3 DIN EN ISO 9308-3 ISO/TS 11133-1 DIN V ENV ISO 11133-1 ISO/TS 11133-2 D
16、IN ISO/TS 11133-2 ISO/TR 13843 DIN V ENV ISO 13843 ISO 16140 DIN EN ISO 16140 ISO 19458 DIN EN ISO 19458 National Annex NA (informative) Bibliography DIN EN ISO 3696, Water for analytical laboratory use Specification and test methods DIN EN ISO 5667-1, Water quality Sampling Part 1: Guidance on the
17、design of sampling programmes and sampling techniques DIN EN ISO 5667-3, Water quality Sampling Part 3: Guidance on the preservation and handling of water samples DIN ISO 5725-1, Accuracy (trueness and precision) of measurement methods and results Part 1: General principles and definitions DIN EN IS
18、O 6887-1, Microbiology of food and animal feeding stuffs Preparation of the test samples, of initial suspension and of decimal dilutions for microbiological examination Part 1: General rules for the preparation of the initial suspension and decimal dilutions DIN EN ISO 7218, Microbiology of food and
19、 animal feeding stuffs General requirements and guidance for microbiological examinations DIN EN ISO 7899-1, Water quality Detection and enumeration of intestinal enterococci in surface and waste water Part 1: Miniaturized method (most probable number) by inoculation in liquid medium DIN EN ISO 8199
20、:2008-01 4 DIN EN ISO 9308-3, Water quality Detection and enumeration of Escherichia coli and coliform bacteria in surface and waste water Part 3: Miniaturized method (most probable number) by inoculation in liquid medium DIN V ENV ISO 11133-1, Microbiology of food and animal feedings stuffs Guideli
21、nes on preparation and production of culture media Part 1: General guidelines on quality assurance for the preparation of culture media in the laboratory (ISO/TS 11133-1) DIN ISO/TS 11133-2, Microbiology of food and animal feeding stuffs Guidelines on preparation and production of culture media Part
22、 2: Practical guidelines on performance testing of culture media DIN V ENV ISO 13843, Water quality Guidance on validation of microbiological methods (ISO/TR 13843) DIN EN ISO 16140, Microbiology of food and animal feeding stuffs Protocol for the validation of alternative methods DIN EN ISO 19458, W
23、ater quality Sampling for microbiological analysis EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 8199 October 2007 ICS 07.100.20 English Version Water quality - General guidance on the enumeration of micro-organisms by culture (ISO 8199:2005) Qualit de leau - Lignes directrices gnrales po
24、ur le dnombrement des micro-organismes sur milieu de culture (ISO 8199:2005) Wasserbeschaffenheit - Allgemeine Anleitung zur Zhlung von Mikroorganismen durch Kulturverfahren (ISO 8199:2005) This European Standard was approved by CEN on 27 September 2007. CEN members are bound to comply with the CEN/
25、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 CEN Management Centre or to
26、 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 Management Centre has the same status as the official versio
27、ns. CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland, France,Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spai
28、n, Sweden, Switzerland and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: rue de Stassart, 36 B-1050 Brussels 2007 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national
29、Members. Ref. No. EN ISO 8199:2007: EContents Page 2 EN ISO 8199:2007 (E) Foreword . 3 Introduction 4 1 Scope . 5 2 Normative references . 5 3 Principle . 5 4 General . 5 5 Diluents and culture media 6 6 Sterilization of apparatus and glassware . 9 7 Samples 9 8 Enumeration after inoculation of test
30、 portions of the sample in (or on) solid media 10 9 Enumeration by inoculation in liquid media . 19 Annex A (informative) Criteria for the choice of enumeration technique . 26 Annex B (informative) MPN tables . 31 Bibliography 41 DIN EN ISO 8199:2008-01 Foreword The text of ISO 8199:2005 has been pr
31、epared by Technical Committee ISO/TC 147 “Water quality” of the International Organization for Standardization (ISO) and has been taken over as EN ISO 8199:2007 by Technical Committee CEN/TC 230 “Water analysis” the secretariat of which is held by DIN. This European Standard shall be given the statu
32、s of a national standard, either by publication of an identical text or by endorsement, at the latest by April 2008, and conflicting national standards shall be withdrawn at the latest by April 2008. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the follo
33、wing countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slov
34、enia, Spain, Sweden, Switzerland and the United Kingdom. Endorsement notice The text of ISO 8199:2005 has been approved by CEN as a EN ISO 8199:2007 without any modification. 3 EN ISO 8199:2007 (E) DIN EN ISO 8199:2008-01 Introduction Techniques for the isolation and enumeration of micro-organisms,
35、based on their ability to grow on specified culture media, are an important and widely used means of assessing the microbiological quality of water. The purpose of this guide is to gather in a single document the information common to the various enumeration techniques so as to avoid repetition of t
36、echnical details in individual standards and to facilitate the choice of the technique most suitable for a particular problem. 4 EN ISO 8199:2007 (E) DIN EN ISO 8199:2008-01 WARNING Persons using this International Standard should be familiar with normal laboratory practice. This standard does not p
37、urport 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 practices and to ensure compliance with any national regulatory conditions. IMPORTANT It is absolutely essential that tests conducted in acco
38、rdance with this International Standard be carried out by suitably trained staff. 1 Scope This International Standard presents guidance for carrying out manipulations which are common to each technique for the microbiological examination of water, particularly the preparation of samples, culture med
39、ia and apparatus. It also describes the various enumeration techniques available and the criteria for the choice of a particular technique. This International Standard is mainly intended for bacteria, yeasts and moulds. Some aspects are also applicable to viruses and parasites. 2 Normative reference
40、s The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. ISO 3696:1987, Water for analytical laboratory
41、use Specification and test methods ISO 19458, Water quality Sampling for microbiological analysis 3 Principle The general principle of these techniques consists of inoculating a known volume of a water sample on or into a culture medium (solid or liquid). It is assumed that after incubation each mic
42、ro-organism present multiplies, giving either a colony visible directly on the solid medium, or changes in observable properties of the liquid medium. The choice of a particular method depends not only on the nature of the micro-organisms sought, but also on the nature of the water and the reasons f
43、or the examination. 4 General Uniformity of temperatures and (incubation) times: The following accepted ranges of temperatures and times during incubation or storage are applied, when appropriate for the intended target organism, and unless otherwise stated in the specific standard. 5 EN ISO 8199:20
44、07 (E) DIN EN ISO 8199:2008-01 Storage temperatures: ( 70 10) C; ( 20 5) C; (5 3) C; Incubation temperatures: (22 2) C; (36 2) C; Sterilization temperatures: (115 3) C; (121 3) C; (170 10) C; Incubation times: (21 3) h; (44 4) h; (68 4) h. Other times and temperatures may be specified for specific m
45、ethods when necessary. The upper incubation temperature limits are very strict (they can have large influences on the growth). The lower temperature limits may be exceeded for short periods, e.g. due to opening the door of an incubator, but recovery shall be rapid. Tolerances on volumes and masses:
46、Unless otherwise stated, the accepted range of any measured value is: stated value 5 %. 5 Diluents and culture media 5.1 General 5.1.1 Quality requirements Use constituents of uniform quality and analytical-grade chemicals for the preparation of media. Other grades of chemical may be used provided t
47、hey can be shown to produce the same results. Alternatively, dehydrated complete media or diluents may be used. Follow the manufacturers instructions strictly. Use glass-distilled or demineralized water which is free from substances that might affect growth of micro-organisms under the test conditio
48、ns. The water shall comply with the requirements of ISO 3696:1987, grade 3. Unless otherwise stated, the ingredients are added to the volume of water instead of making up to a certain volume, as is normal in the preparation of microbiological culture media. Before use, check the quality of the media
49、, diluents and filters, e.g. by following the procedures described in ISO 7704, ISO/TS 11133-1 or ISO/TS 11133-2. 5.1.2 Sterilization Dispense diluents and culture media in containers suitable for sterilization by autoclaving. For most purposes, a temperature of (121 3) C for 15 min is adequate. However, a different time and temperature may sometimes be required and details are given in each individual standard. Alternatively, with thermolabile substances, removal o
