1、| | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | BRITISH STANDARD BS EN ISO 7899-2:2000 BS
2、6068-4.4:2000 The European Standard EN ISO 7899-2:2000 has the status of a British Standard ICS 07.100.20 NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAW Water quality Detection and enumeration of intestinal enterococci Part 2: Membrane filtration methodThis British Standard,
3、having been prepared under the direction of the Health and Environment Sector Committee, was published under the authority of the Standards Committee and comes into effect on 15 August 2000 BSI 08-2000 ISBN 0 580 34953 5 BS EN ISO 7899-2:2000 Amendments issued since publication Amd. No. Date Comment
4、s National foreword This British Standard is the official English language version of EN ISO 7899-2:2000. It is identical with ISO 7899-2:2000. It supersedes BS 6068-4.4:1989 which is withdrawn. The UK participation in its preparation was entrusted by Technical Committee EH/3, Water quality, to Subc
5、ommittee EH/3/4, Microbiological methods, which has the responsibility to: aid enquirers to understand the text; present to the responsible international/European committee any enquiries on the interpretation, or proposals for change, and keep the UK interests informed; monitor related international
6、 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. This is one of a series of standards on water quality, others of which have been, or will be, published as Sections of BS 6068. The various
7、Sections of BS 6068 are comprised within Parts 1 to 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 Prec
8、ision and accuracy Note The tests described in this British Standard should only be carried out by suitably qualified persons with an appropriate level of microbiological expertise. Microbilogical chemical procedures should be followed throughout. Cross-references Attention is drawn to the fact that
9、 CEN and CENELEC Standards normally include an annex which lists normative references to international publications with their corresponding European publications. The British Standards which implement these international or European publications referred to in this document may be found in the BSI
10、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. Users of British Standards are responsi
11、ble 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, the ISO title page, pages ii to v, a blank
12、page, pages 1 to 7, the annex ZA page, an inside back cover and a back cover. The BSI copyright notice displayed in this document indicates when the document was last issued.EUROPEANSTANDARD NORMEEUROPENNE EUROPISCHENORM ENISO78992 April2000 ICS07.100.00 Englishversion WaterqualityDetectionandenumer
13、ationofintestinal enterococciPart2:Membranefiltrationmethod(ISO7899 2:2000) QualitdeleauRechercheetdnombrementdes entrocoquesintestinauxPartie2:Mthodeparfiltration surmembrane(ISO78992:2000) WasserbeschaffenheitNachweisundZhlungvon intestinaleEnterokokkenTeil2: Membranfiltrationsverfahren(ISO78992:2
14、000) ThisEuropeanStandardwasapprovedbyCENon15April2000. CENmembersareboundtocomplywiththeCEN/CENELECInternalRegulationswhichstipulatetheconditionsforgivingthisEurope an Standardthestatusofanationalstandardwithoutanyalteration.Uptodatelistsandbibliographicalreferencesconcernings uchnational standards
15、maybeobtainedonapplicationtotheCentralSecretariatortoanyCENmember. ThisEuropeanStandardexistsinthreeofficialversions(English,French,German).Aversioninanyotherlanguagemadebytra nslation undertheresponsibilityofaCENmemberintoitsownlanguageandnotifiedtotheCentralSecretariathasthesamestatusast heofficia
16、l versions. CENmembersarethenationalstandardsbodiesofAustria,Belgium,CzechRepublic,Denmark,Finland,France,Germany,Greece, Iceland,Ireland,Italy,Luxembourg,Netherlands,Norway,Portugal,Spain,Sweden,SwitzerlandandUnitedKingdom. EUROPEANCOMMITTEEFORSTANDARDIZATION COMITEUROPENDENORMALISATION EUROPISCHES
17、KOMITEEFRNORMUNG CentralSecretariat:ruedeStassart,36B1050Brussels 2000CEN Allrightsofexploitationinanyformandbyanymeansreserved worldwideforCENnationalMembers. Ref.No.ENISO78992:2000EForeword ThetextoftheI nternationalSta ndard ISO 7899 2:2000 hasb een prepared by Technical CommitteeISO/ TC14 7 “Wat
18、erqu ality“incoll aboration with TechnicalCo mmittee CEN/TC 230 “Wateranal ysis“, thesecreta riat ofwhic h ish eld by DIN. ThisEurop ean Standardshall be given thes tatusof a nationalst andard, either by publication ofani dentical textor by endorsement,att helatest byOct ober 2000,a ndcon flicting n
19、ational standardss hallbe withdrawnatthel atest byOcto ber2 000. According totheCE N/CENELECInter nalRe gulations, the national standardsorga nizations of the followingc ountriesare bound toi mplementt hisEur opean Standard:Aust ria, Belgium, Czech Republic, Denmark,Finla nd,Franc e,Ger many,Greece,
20、I celand, Ireland, Italy, Luxembourg,Ne therlands, Norway, Portugal,Sp ain, Sweden,Swi tzerlanda ndt heU nited Kingdom. Endorsementnotice Thetextof theI nternationalSta ndard ISO 7899 2:2000 wasapp roved by CEN asaEu ropean Standard withouta ny modification. NOTE:Nor mativere ferencesto Internationa
21、l Standards arelisted in annexZA (normative). EN ISO 7899-2:2000Referencenumber ISO7899-2:2000(E) INTERNATIONAL STANDARD ISO 7899-2 Secondedition 2000-04-15 WaterqualityDetectionandenumeration ofintestinalenterococci Part2: Membranefiltrationmethod Qualit de leau Recherche et dnombrement dentrocoque
22、s intestinaux Partie 2: Mthode par filtration sur membrane EN ISO 7899-2:2000ii EN ISO 7899-2:2000 iii Contents Page Foreword.iv Introduction.v 1 Scope1 2 Normativereferences1 3 Termsanddefinitions.2 4 Principle2 5 Apparatus.2 6 Culturemediaandreagents.3 7 Sampling.4 8 Procedure.4 9 Qualityassurance
23、5 10 Expressionofresults5 11 Testreport5 AnnexA(informative)Furthermicrobiologicalinformationonintestinalenterococci.6 Bibliography7 EN ISO 7899-2:2000iv Foreword ISO(theInternationalOrganizationforStandardization)isaworldwidefederationofnationalstandardsbodies(ISO memberbodies).TheworkofpreparingIn
24、ternationalStandardsisnormallycarriedoutthroughISOtechnical committees.Eachmemberbodyinterestedinasubjectforwhichatechnicalcommitteehasbeenestablishedhas therighttoberepresentedonthatcommittee.Internationalorganizations,governmentalandnon-governmental,in liaisonwithISO,alsotakepartinthework.ISOcolla
25、boratescloselywiththeInternationalElectrotechnical Commission(IEC)onallmattersofelectrotechnicalstandardization. InternationalStandardsaredraftedinaccordancewiththerulesgivenintheISO/IECDirectives,Part3. DraftInternationalStandardsadoptedbythetechnicalcommitteesarecirculatedtothememberbodiesforvotin
26、g. PublicationasanInternationalStandardrequiresapprovalbyatleast75%ofthememberbodiescastingavote. AttentionisdrawntothepossibilitythatsomeoftheelementsofthispartofISO7899maybethesubjectofpatent rights.ISOshallnotbeheldresponsibleforidentifyinganyorallsuchpatentrights. InternationalStandardISO7899-2w
27、aspreparedbyTechnicalCommitteeISO/TC147, Water quality, SubcommitteeSC4, Microbiological methods. Thissecondeditioncancelsandreplacesthefirstedition(ISO7899-2:1984),whichhasbeentechnicallyrevised. ISO7899consistsofthefollowingparts,underthegeneraltitle Water quality Detection and enumeration of inte
28、stinal enterococci: Part 1: Miniaturized method (Most Probable Number) for surface and waste water Part 2: Membrane filtration method AnnexAofthispartofISO7899isforinformationonly. EN ISO 7899-2:2000 v Introduction InthispartofISO7899amethodisdescribedfortheisolationofintestinalenterococci. Enteroco
29、ccus faecalis , E. faecium, E. durans and E. hirae can be detected and enumerated with the methods described in this part of ISO7899.Inaddition,other Enterococcus species and some species of the genus Streptococcus (namely S. bovis and S. equinus ) may occasionally be detected. These Streptococcus s
30、pecies do not survive long in water and are probablynotenumeratedquantitatively.Forpurposesofwaterexamination,enterococcicanberegardedas indicatorsoffaecalpollution.Howeveritshouldbenotedthatsomeenterococcifoundinwatercanoccasionally alsooriginatefromotherhabitats. EN ISO 7899-2:2000EN ISO 7899-2:20
31、00 1 WaterqualityDetectionandenumerationofintestinal enterococci Part2: Membranefiltrationmethod 1 Scope ThispartofISO7899specifiesamethodforthedetectionandenumerationofintestinalenterococciinwaterby membranefiltration.ThispartofISO7899isespeciallyintendedforexaminationofdrinkingwater,waterfrom swim
32、mingpoolsandotherdisinfectedorcleanwaters.Nevertheless,themethodcanbeappliedtoalltypesof water,exceptwhenalargeamountofsuspendedmatterormanyinterferingmicroorganismsarepresent.Itis particularlysuitablefortheexaminationoflargevolumesofwatercontainingonlyafewintestinalenterococci. 2 Normativereference
33、s Thefollowingnormativedocumentscontainprovisionswhich,throughreferenceinthistext,constituteprovisionsof thispartofISO7899.Fordatedreferences,subsequentamendmentsto,orrevisionsof,anyofthesepublications donotapply.However,partiestoagreementsbasedonthispartofISO7899areencouragedtoinvestigatethe possib
34、ilityofapplyingthemostrecenteditionsofthenormativedocumentsindicatedbelow.Forundated references,thelatesteditionofthenormativedocumentreferredtoapplies.MembersofISOandIECmaintain registersofcurrentlyvalidInternationalStandards. ISO3696:1987, Water for analytical laboratory use Specification and test
35、 methods. ISO5667-1:1980, Water quality Sampling Part 1: Guidance on the design of samping programmes. ISO5667-2:1991, Water quality Sampling Part 2: Guidance on sampling techniques. ISO5667-3:1994, Water quality Sampling Part 3: Guidance on the preservation and handling of samples. ISO6887-1:1999,
36、Microbiology of food and animal feeding stuffs Preparation of test samples, initial suspension and decimal dilutions for microbiological examination Part : General rules for the preparation of the initial suspension and decimal dilutions. ISO8199:1988, Water quality General guide to the enumeration
37、of micro-organisms by culture. ISO/IECGuide2:1996, Standardization and related activities General vocabulary. EN ISO 7899-2:20002 3 Termsanddefinitions ForthepurposesofthispartofISO7899,thetermsanddefinitionsgiveninISO/IECGuide2andthefollowing apply. 3.1 intestinalenterococci bacteriawhichareabletor
38、educe2,3,5-triphenyltetrazoliumchloridetoformazanandtohydrolyseaesculinat 44Conthemedia(6.3.1and6.3.2)specifiedinthispartofISO7899 NOTE SeealsoannexA. 4Principle 4.1 Filtration,incubationandenumeration Theenumerationofintestinalenterococciisbasedonfiltrationofaspecifiedvolumeofwatersamplethrougha me
39、mbranefilterwithaporesize(0,45m)sufficienttoretainthebacteria.Thefilterisplacedonasolidselective mediumcontainingsodiumazide(tosuppressthegrowthofGram-negativebacteria)and2,3,5-triphenyltetrazolium chloride,acolourlessdye,thatisreducedtoredformazanbyintestinalenterococci. Typicalcoloniesareraised,wi
40、thared,maroonorpinkcolour,eitherinthecentreofthecolonyorthroughout. 4.2 Confirmation Iftypicalcoloniesareobserved,aconfirmationstepisnecessary,bytransferofthemembrane,withallthe colonies,ontobile-aesculin-azideagar,preheatedat44C.Intestinalenterococcihydrolyseaesculinonthis mediumin2h.Theend-product
41、,6,7-dihydroxycoumarin,combineswithiron(III)ionstogiveatan-colouredtoblack compoundwhichdiffusesintothemedium. 5 Apparatus Exceptfordisposableglasswarewhichisdeliveredsterile,glasswareshallbesterilizedinaccordancewith ISO8199. Usualmicrobiologicallaboratoryequipmentandparticularly: 5.1 Membranefiltr
42、ationapparatus,accordingtoISO8199. 5.2 Sterilemembranefilters,withanominalporesizeof0,45m. Thequalityofmembranefiltersmayvaryfrombrandtobrandorevenfrombatchtobatch.Itisthereforeadvisable tocheckthequalityonaregularbasis,inaccordancewithISO7704. 5.3 Incubator,capableofbeingmaintainedat36C 2C. 5.4 Inc
43、ubator,capableofbeingmaintainedat44C 0,5C. 5.5 Autoclave,capableofbeingmaintainedat121C 3C. 5.6 Sterileforceps. 5.7 Hotplateorwaterbath,maintainedat100C. EN ISO 7899-2:2000 3 6 Culturemediaandreagents 6.1 Basicmaterials WARNINGTheselectivemediadescribedinthispartofISO7899containsodiumazide.Asthis su
44、bstanceishighlytoxicandmutagenic,precautionsshallbetakentoavoidcontactwithit,especiallyby theinhalationoffinedustduringthepreparationofcommerciallyavailabledehydratedcompletemedia. Azide-containingmediashouldnotbemixedwithstronginorganicacids,astoxichydrogenazide(HN 3 ) maybeproduced.Solutionscontai
45、ningazidecanalsoformexplosivecompoundswhenincontactwith metalpipework,forexamplefromsinks. Azidescanbedecomposedsafelybytheadditionofanexcessofasaturatednitritesolution. Foruniformityofresults,inthepreparationofmedia,eitheruseadehydratedcompletemediumoruseconstituents ofuniformqualityandchemicalsofr
46、ecognizedanalyticalgrade.Sodiumazidedeteriorateswithtimesothat dehydratedmediahavealimitedshelf-life. NOTE Useofchemicalsofanotherqualityispossibleprovidedtheyareshowntobeofequalperformanceinthetest. 6.2 Distilledwaterorwaterofequivalentpurity,inaccordancewithISO3696,Grade1. 6.3 Culturemedia 6.3.1 S
47、lanetzandBartleymedium 6.3.1.1 Basalmedium Tryptose 20,0g Yeastextract 5,0g Glucose 2,0g Dipotassiumhydrogenphosphate(K 2 HPO 4 ) 4,0g Sodiumazide(NaN 3 ) 0,4g Agar 8gto18g 1) Water 1000ml Dissolvetheingredientsinboilingwater. Oncedissolutioniscomplete,heatforanadditional5min. Coolto50Cto60C. 6.3.1.
48、2 TTCsolution 2,3,5-triphenyltetrazoliumchloride 1g Water 100ml Dissolvetheindicatorinthewaterbystirring. Sterilizebyfiltration(0,2m). Protectthesolutionagainsttheactionoflight,anddiscarditifapinktingedevelops. 1) Dependingonthegelstrengthoftheagar. EN ISO 7899-2:20004 6.3.1.3 Completemedium Basalmedium(6.3.1.1) 1000ml TTCsolution(6.3.1.2) 10ml AddtheTTCsolutiontothebasalmediumcooledto50Cto60C. AdjustthepHifnecessarysothatafterst
