1、BRITISH STANDARDBS EN ISO 8199:2007Incorporating amendment no. 1 (renumbers BS ISO 8199:2005 as BS EN ISO 8199:2007)Water quality General guidance on the enumeration of micro-organisms by cultureThe European Standard EN ISO 8199:2007 has the status of a British StandardICS 07.100.20g49g50g3g38g50g51
2、g60g44g49g42g3g58g44g55g43g50g56g55g3g37g54g44g3g51g40g53g48g44g54g54g44g50g49g3g40g59g38g40g51g55g3g36g54g3g51g40g53g48g44g55g55g40g39g3g37g60g3g38g50g51g60g53g44g42g43g55g3g47g36g58BS EN ISO 8199:2007This British Standard was published under the authority of the Standards Policy and Strategy Commi
3、ttee on 5 October 2005 BSI 2008ISBN 978 0 580 59549 3National forewordThis British Standard is the UK implementation of EN ISO 8199:2007. It supersedes BS ISO 8199:2005 which is withdrawn. The UK participation in its preparation was entrusted by Technical Committee EH/3, Water quality, to Subcommitt
4、ee EH/3/4, Biological methods.A list of organizations represented on this subcommittee can be obtained on request to its secretary.This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application. Compliance with a British Sta
5、ndard cannot confer immunity from legal obligations.Amendments/corrigenda issued since publicationDate Comments31 January 2008 Renumbers BS ISO 8199:2005 as BS EN ISO 8199:2007EUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN ISO 8199October 2007ICS 07.100.20English VersionWater quality - General gu
6、idance on the enumeration of micro-organisms by culture (ISO 8199:2005)Qualit de leau - Lignes directrices gnrales pour lednombrement des micro-organismes sur milieu de culture(ISO 8199:2005)Wasserbeschaffenheit - Allgemeine Anleitung zur Zhlungvon Mikroorganismen durch Kulturverfahren (ISO8199:2005
7、)This European Standard was approved by CEN on 27 September 2007.CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibliographical re
8、ferences concerning such nationalstandards may be obtained on application to the CEN 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 translationunder the responsibility of a CEN member
9、into its own language and notified to the CEN Management Centre has the same status as theofficial versions.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
10、, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR NORMUNGManagement Centre: rue de Stassart, 36 B-1050 Brussels 20
11、07 CEN All rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN ISO 8199:2007: EForeword The text of ISO 8199:2005 has been prepared by Technical Committee ISO/TC 147 “Water quality” of the International Organization for Standardization (ISO) and
12、 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 status of a national standard, either by publication of an identical text or by endorsement, at the latest by April 2008, and
13、 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 following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmar
14、k, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. Endorsement notice The text of ISO 8199:2005 has been approved
15、by CEN as a EN ISO 8199:2007 without any modification. EN ISO 8199:2007Reference numberISO 8199:2005(E)INTERNATIONAL STANDARD ISO8199Second edition2005-06-15Water quality General guidance on the enumeration of micro-organisms by culture Qualit de leau Lignes directrices gnrales pour le dnombrement d
16、es micro-organismes sur milieu de culture EN ISO 8199:2007ii iiiContents Page Foreword iv Introduction v 1 Scope . 1 2 Normative references . 1 3 Principle. 1 4 General. 1 5 Diluents and culture media 2 6 Sterilization of apparatus and glassware . 5 7 Samples . 5 8 Enumeration after inoculation of t
17、est portions of the sample in (or on) solid media . 6 9 Enumeration by inoculation in liquid media 15 Annex A (informative) Criteria for the choice of enumeration technique. 22 Annex B (informative) MPN tables 27 Bibliography . 37 EN ISO 8199:2007iv Foreword ISO (the International Organization for S
18、tandardization) 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 ri
19、ght 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. Internationa
20、l Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2. The main task of technical committees is to prepare International Standards. Draft International Standards adopted by the technical committees are circulated to the member bodies for voting. Publication as
21、an International Standard requires approval by at least 75 % of the member bodies casting a vote. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. ISO
22、 8199 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 4, Microbiological methods. This second edition cancels and replaces the first edition (ISO 8199:1988), which has been technically revised. EN ISO 8199:2007vIntroduction Techniques for the isolation and enumeration
23、of micro-organisms, 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 a
24、void repetition of technical details in individual standards and to facilitate the choice of the technique most suitable for a particular problem. EN ISO 8199:2007blank1Water quality General guidance on the enumeration of micro-organisms by culture WARNING Persons using this International Standard s
25、hould be familiar with normal laboratory practice. This 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 practices and to ensure compliance with any national regulatory co
26、nditions. IMPORTANT It is absolutely essential that tests conducted in accordance 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
27、 examination of water, particularly the preparation of samples, culture media 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
28、aspects are also applicable to viruses and parasites. 2 Normative references 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 (includi
29、ng any amendments) applies. ISO 3696:1987, Water for analytical laboratory 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 cul
30、ture medium (solid or liquid). It is assumed that after incubation each micro-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 mi
31、cro-organisms sought, but also on the nature of the water and the reasons for 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 organ
32、ism, and unless otherwise stated in the specific standard. EN ISO 8199:20072 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 an
33、d temperatures may be specified for specific methods 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 shal
34、l be rapid. Tolerances on volumes and masses: 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. O
35、ther grades of chemical may be used provided they 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 gro
36、wth of micro-organisms under the test conditions. 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 med
37、ia. Before use, check the quality of the media, 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 temper
38、ature 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 of micro-organisms may be effected by filtration through a filter with a pore size of
39、 0,2 m, specified by the manufacturer as being suitable for “sterilization”. 5.2 Diluents The diluents given in this subclause are commonly used in water microbiology. However, the list is not exhaustive and other appropriate diluents may be used. After preparation, distribute each solution into bot
40、tles and sterilize, e.g. by autoclaving at (121 3) C for 15 min. Alternatively, the diluent can be aseptically distributed after sterilization. Store at room temperature or in a refrigerator at (5 3) C for a maximum of 6 months. If a diluent shows any change from its normal appearance, discard it. E
41、N ISO 8199:200735.2.1 Saline solution Composition Sodium chloride (NaCl) 8,5 g Water (see 5.1.1) 1 000 mlPreparation Dissolve the ingredients in the water, if necessary by heating. Adjust the pH by adding sodium hydroxide solution c(NaOH) = 1 mol/l or hydrochloric acid c(HCI) = 1 mol/l so that, afte
42、r sterilization (see 5.1.2), it will correspond to 7,0 0,5 at 25 C. 5.2.2 Peptone diluent Composition Enzymatic digest of casein (peptone) 1,0 g Water (see 5.1.1) 1 000 mlPreparation Dissolve the ingredients in the water, if necessary by heating. Adjust the pH by adding sodium hydroxide solution c(N
43、aOH) = 1 mol/l or hydrochloric acid c(HCl) = 1 mol/l so that, after sterilization (see 5.1.2), it will correspond to 7,0 0,5 at 25 C. 5.2.3 Peptone saline solution Composition Enzymatic digest of casein (peptone) 1,0 g Sodium chloride (NaCl) 8,5 g Water (see 5.1.1) 1 000 mlPreparation Dissolve the i
44、ngredients in the water, if necessary by heating. Adjust the pH by adding sodium hydroxide solution c(NaOH) = 1 mol/l or hydrochloric acid c(HCI) = 1 mol/l so that, after sterilization (see 5.1.2), it will correspond to 7,0 0,5 at 25 C. 5.2.4 Ringers solution, quarter-strength Composition Sodium chl
45、oride (NaCl) 2,25 g Potassium chloride (KCl) 0,105 g Calcium chloride (anhydrous) (CaCl2) 0,12 g Sodium hydrogen carbonate (NaHCO3) 0,05 g Water (see 5.1.1) 1 000 mlEN ISO 8199:20074 Preparation Dissolve the ingredients in the water, if necessary by heating. Adjust the pH by adding sodium hydroxide
46、solution c(NaOH) = 1 mol/l or hydrochloric acid c(HCI) = 1 mol/l so that, after sterilization (see 5.1.2), it will correspond to 7,0 0,2 at 25 C. 5.2.5 Phosphate buffer solution a) Phosphate solution Composition Potassium dihydrogen orthophosphate (KH2PO4) 34 g Water (see 5.1.1) to 1 000 mlPreparati
47、on Dissolve the potassium dihydrogen orthophosphate in 500 ml of the water. Adjust the pH to a value of 7,2 0,2 by adding sodium hydroxide solution c(NaOH) = 1 mol/l or hydrochloric acid c(HCI) = 1 mol/l. Add more water up to 1 000 ml. If the solution needs to be stored, sterilize it (see 5.1.2) bef
48、ore storage. b) Magnesium chloride solution Composition Magnesium chloride (MgCI2) 38 g Water (see 5.1.1) 1 000 mlAlternatively, an equivalent mass (99 g) of magnesium sulfate (MgSO4.7H2O) may be used. If the solution needs to be stored, sterilize it (see 5.1.2) before storage. Preparation Dissolve
49、the magnesium chloride in the water. c) Final solution Composition Phosphate solution (a) 1,25 ml Magnesium chloride solution (b) 5,0 ml Water (see 5.1.1) 1 000 mlPreparation Add the phosphate solution (a) and the magnesium chloride solution (b) to the water, dispense in convenient volumes and sterilize (see 5.1.2). The final pH should correspond to 7,0 0,2 at 25 C. 5.3 Culture media Once a bottle of dehydrated medium (chemical) is opened, date the container and indicate a max
