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1、INTERNATIONA1.STANDARDISO4768editionFirst2O23-O7Measurementmethodofanti-biofi1.mactivityonp1.asticandothernon-poroussurfacesMethodedemesurede!activityanti-biofi1.msur1.ep1.astiqueetautressurfacesnonporeuses1S0R4to8V20B1ISO2023COPYRIGHTPROTECTEDDOCUMENTISO2023IUirhM*hedbdi1.iUedotherwiseupdhi.o啪InyM1
2、.tta0DmkfifiU81.andonnet8CH-1214Vernier,GenevaPhone：M1.227490111觥ftte:丽丽BQrgPub1.ishedinSwitzer1.andContentsForewordivIntroductionv2 Scope13 Normativereferences14 Termsanddefinitions1Materia1.s14.1 Bacteria1.straintobeusedforthetests14.2 Reagents24.3 4Oj1.tureQttMkra1.andso1.ution；4132N1.1.U1.entaga
3、r24.3.3Suspensionmedium-1/5Trypticsoybroth(15TSB)24.4.4.567三fif三三rmedjum0,1%Crysta1.vio1.etaqueousso1.ution1.0%Sodiumdodecy1.su1.fateaqueousso1.utionApparatusSteri1.izationofapparatusandstorageofstockcu1.tures6.1 Dr)r-heatsteri1.ization6.2 High-pressuresteamsteri1.izationProcedure47.1 Biofi1.mformat
4、iononthesurfaceofthetestspecimens47.1.1 Pre-cu1.tureOfbacteria.47.1.3 Preparationoftestinobe1.apedinensincubatingmedium47.1.4 Inocu1.ationoftestspecimens57.1.5 Incubationoftheinocu1.atedtestspecimens57.2 7kertionfiit三ftfWens.67.2.2 Stainingoftestspecimenswithcrysta1.vio1.et67.2.3 Secondwashingoftest
5、specimens67.2.4 Biofi1.mfromspecimens7.2.5 Absorbancerecoverymeasurement77.3 Determiningtheviab1.ebacteriacount7Expressionofresu1.ts88.1 Conditionsforva1.idtest88.2 Ca1.cu1.ationofanti-biof1.mactivityonthebasisofabsorptionspectrummethod8.3 Effectivenessof8Repeatabi1.ityandreproducibi1.ity8Testreport
6、AnnexA(informative)Repeatabi1.ityandreproducibi1.ity1()Bib1.iographyForewordISO(theInternationa1.OrganizationforStandardization)isawor1.dwidefederationofnationa1.standardsttiugh(ISOtnnhnkoft11rtnihvcekdHPfff1.PbbGgbOdymtf1.CIrdrtttd6tand)GScnoWharKecf1.tdcommitteehasbeenestab1.ishedhastherighttobere
7、presentedonthatcommittee.Internationa1.organizations,governmenta1.andnon-governmenta1.,in1.iaisonwithISO,a1.sotakepartinthework.ISOco1.1.aboratesc1.ose1.ywiththee1.ectrotechnica1.standardization.Internationa1.E1.ectrotechnica1.Commission(IEC)ona1.1.mattersofTheproceduresusedtodeve1.opthisdocumentand
8、thoseintendedforitsfurthermaintenanceare蜘硼搬OfISo1.EC(1.三te翩在他网WRenee册Fhtheeditoria1.ru1.esoftheISO/IECDirectives.Part2(secwww.iso.org/direc1.ives).而图撇nts)aWKrtakestfi嗣嬲蜘帆ncte除硼，8曲删殴ntm部阚i野棍IVedaimedpatentrightsinrespectthereof.Asofthedateofpub1.icationofthisdocument,ISOhadnorreceivednoticeof（八）paten
9、t(三)whichmayberequiredtoimp1.ementthisdocument.However,imp1.ementorsare懈怖翻琳躺/mj，wWjSOQFg/眸IentS洗蝮油那ion,扁MMSPOn赖岫油嬲9的阳躲Orasuchpatentrights.Anytradeusedinthisconstitutenameendorsenent.documentisinformationgivenfortheconvenienceofusersanddoesnotForanexp1.anationofthevo1.untarynatureofstandards,themeani
10、ngofISOspecifictermsand幽幽蛔豳酒侬遮W即辆O)assEif1.眦聚inwe帼1.吼。nBar池帆四dead朝乩CSvbu11uisobrgiso/foreword.htmI.Anyfeedbackorquestionsonthisdocumentshou1.dbedirectedtotheuser,snationa1.standardsbody.Acomp1.ete1.istingofthesebodiescanbefoundatwww4skeFgf1.embeFfUHmkIntroductionenvironmentmicroorganismssuchthes1.im
11、es.microorganisms,andarewide1.yfoundinour1.iving“invisib1.eantivira1.testsurfaceofnon-porouseva1.uatingSimi1.ariyrsameporousantibacteria1.testandthenon-poroussurfaces.-A1.1.rightsreservedBiofi1.msareknowntobecompositionsofsubstancessuchaspo1.ysaccharides,Iipidsrandnuc1.eicacidsproducedbyvisib1.eharm
12、sandasce1.1.sofu,hercwaterpresent.ISO22196onantibacteria1.testandISO21702onantivira1.testarethetestmethodsforeva1.uatingISO18184onhygiene,*onthethetestmethodsfoqiroducts.theISOof20743onproducts.Thisdocumentdescribeshowtoeva1.uateproductsthatarctreatedtosuppressbiofi1.rnformationsonMeasurementmethodo
13、fanti-biofi1.mactivityonp1.asticandothernon-poroussurfaces1 ScopeThisdocumentspecifiesatestmethodtoeva1.uatetheanti-biofi1.mactivityofanti-biofi1.mtreatedt。PjJnt由Kan6由CihwHWiortjCffiCunfingsrtiptwiMfceraxdH)gstainteeiJi)rtdjIrt8sed,importanceofthespecies,itsstrainnumberandthespecificreasonfortheirus
14、esha1.1.beinc1.udedinthetestreport.4.2 Reagents4.3 reagentssha1.1.mediaandana1.ytica1.gradeand/orofagradeappropriateformicrobio1.ogica1.purposes.4.3.1 Genera1.instructions,commercia1.supp1.iers,whereitsha1.1.bepreparedinaccordancewiththemanufacturerscompositionofthecu1.turemediumshou1.dnotbechanged.
15、4.3. zNutrientagar(NA)Waterpowderunti1.OOOagardisso1.ved.deionizedwater.Heat.va1.uebetween7r0hotp1.ate(at25aboi)ing-preparationsha1.1.notbeused.4.3.3 Suspensionmedium-1/5Trypticsoybroth(15TSB)SrOdeionizedch1.oride,thegdipotassiumphosphatedisti1.1.edordeionizedwater000vo1.umetrica1.1.y10.1/5TSBthatha
16、sbeenkeptforoneweekor1.ongerafterthepreparationsha1.1.notbeused.4.3.4 P1.atecountagar15,0boi1.ing-wateowderinunti1.()OOthem1.agardisti1.1.eddisso1.ved.deionizedwater.pHHeat,va1.uebetweenon7,0hotp1.atemonthor1.ongerafterthepreparationsha1.1.notbeused.4.3.5 S1.antcu1.turemediumautoc1.avingand(seea1.1.
17、ow).Aftercontentssteri1.izationrso1.id.thetesttubeusedimmediate1.yafterIhcthepreparation,deionizedwater.Watersha1.1.bedisti1.1.edordeionizedandsha1.1.haveaconductivityof1Scm.A1.1.Cu1.turebeofso1.utionThecu1.turemediumspecifiedin4.3.2.4.3.3r4.3.4and4.3.5sha1.1.beused.HiemediummaybeobtainedfromThequan
18、tityofthecu1.turemediumcanbechangedaccordingtotheintendedtestvo1.ume,butthePrepareNAbydisso1.ving5,0gofmeatextract.10r0gofpeptone,Sr0gofsodiumch1.orideand15,0gofagarbath1them1.OfhasdistiIIedorAdjustthepHtowithSti1.dng,onaand7,2orin)withsodiumhydroxideorhydroch1.oricacid.Steri1.izebyautoc1.aving(see6
19、.2),Ifitisnotusedimmediate1.yafterthepreparation,storeitat5to10.NAthathasbeenkeptforonemonthor1.ongerafterthePrepare1/5TSBbydisso1.ving17,0gofapancreaticdigestofcasein,3,0gofpapaicdigestofsoybean,orgofSodiumwater.Di1.ute2,5Oftrypticasesoybrothwithand2.5gofdextrosein11:4ofdisti1.1.edandadjustthepHtoa
20、va1.uebetween7,1and7,5(at25C)withsodiumhydroxideorhydroch1.oricacid.Steri1.izebyautoc1.aving(see6.2).Ifitisnotusedimmediate1.yafterthepreparation,storeitat5toPrepareap1.atecountagarbydisso1.ving2,5gofayeastextract,5,0goftryptone,1,0gofg1.ucoseandinaofbath1ofhasorAdjustthetoWithSJirring,aand7,2or25)W
21、ithsodiumhydroxideorhydroch1.oricacid.Steri1.izebyautoc1.aving(sec6.2).Ifitisnotusedimmediate1.yafterthepreparation,storeitat5to10,P1.atecountagarthathasbeenkeptforoneS1.antsareprepamtusing6m1.to10m1.ofmo1.tenNApouredintoasuitab1.e-cappedtesttube.Steri1.izebydirection6.2thethebecomcp1.aceIfitisnotat
22、anang1.eofabout15tohorizonta1.storeitat5to10.S1.antcu1.turemediumkeptforonemonthor1.ongerafterthepreparationsha1.1.notbeused.Prepareacrysta1.vio1.etaqueousso1.utionbydisso1.ving1,0gofcrysta1.vio1.etin1000m1.ofdisti1.1.edorIfimwa30限PaPUrtJge1.dehNbihh(冽UIiOnabgmufi1.1.ratircmoUiyIobtaMed.mturingtheso
23、1.utionthrougha4.3.71.r0%Sodiumdodecy1.su1.fateaqueousso1.utionPrepareasodiumdodecy1.su1.fateaqueousso1.utionbydisso1.ving10,0gofsodiumdodecy1.su1.fatein1.am1.ofdisti1.1.ed5 OOOApparatUSordeionizedwater.Un1.essotherwisespecified,usethefo1.1.owingapparatusandmateria1.s.5.1Dry-heatsteri1.izer,capab1.e
24、ofmaintainingthetemperatureatava1.uebetween160and180within2ofthesetpointatequi1.ibriumconditions.5.2AUtOdaVeC叩ab1.eofmaintainingatemperatureof(1212)oCandapressureof(1035)kPa.5.3Hotp1.atewithstirrer,orhot-waterbath.54pH-meter,capab1.eofmeasuringto0r2units.5.5Ba1.ance,capab1.eofweighingto0,01g.5.6Pipe
25、tter,capab1.eofsettingap1.asticpipette.5.7P1.asticpipette,steri1.e,andcapab1.eOfmeasuringwithcapacitiesof25m1.0.25m1.5.8Freezer,capab1.eofoperatingatatemperatureof-(802)or-(202).5.9Incubator,capab1.eofmaintainingthetemperaturewithin1ofthesetpointatequi1.ibriumconditions.5.10 C1.eanbench,formicrobia1
26、.test.5.11 Spectrophotometer,capab1.eOfmeasuringat590nm.5.12 Reciproca1.!inershaker,formicrobia1.test.Swingwidth:(305)mm.5.13 Water-so1.ub1.enon-wovenfabric,apo1.yviny1.a1.coho1.non-wovenfabric.5.14 Qua1.itativefi1.terpaper.GradeNo.2.5.15 Testtubes.W5tti*(H宙ni隔aouterdiameterof63mmto65mm1.adepthof31m
27、mto35mm,an5.17 StopperedEr1.ennieyerf1.asksormediabott1.es,asrequiredforpreparationofmedia.6 Steri1.izationofapparatusandstorageofstockcu1.tures6.1 Dry-heatsteri1.izationP1.acetheobjectstobesteri1.izedinadry-heatsteri1.izer,referringtothetemperatureandtimegivenin1606.2High-pressuresteamsteri1.izatio
28、nTabIe1.TheusedtemperatureTtfIietimeJC6HHitirfU虹htU*ntrttfaationTemperatureMinimumsteri1.izationdine18030minutes170eC60minutes120minutesP1.acetheobjectstobesteri1.izedinanautoc1.aveandmaintainat(12112)forat1.east15min.6.3 Preparationofg1.asswareWaSha1.1.g1.asswarewe1.1.witha1.ka1.iorneutra1.detergen
29、t,thenrinsewe1.1.withdisti1.1.edordeionized6er.MtsOoceatcataibrortheautoc1.avepriortouse.Afckf碎W“曲y1B9Koredth最6nemA96C能海SSe躯沛部B曲密IedN卧s加f11rredcu1.tur曲制hth1.y.bediscardedandrep1.acedwithafreshcu1.ture.Theinitia1.passageremovedfromtheorigina1.masterstrainthatisobtainedfromcu1.tureco1.1.ectioniscounte
30、dasthefirsttransfer.7 Procedure7.1 BiofiImformationonthesurfaceofthetestspecimens7.1.1 Pre-cu1.tureofbacteriaUsingasteri1.einocu1.ating1.oop,transferbacteriafromthestockcu1.turetothes1.antcu1.turemediumandiincniUatmg1.3p1.)tfGtff24hctbr1.iaiiJ0nn1.fat(ittSf11SD)b(Xh.1.00ta1.iibatf1.ntiftask.AjiHWing
31、conditions: Temperature:352; Ra1.eofshaking:110min-and3crnwidthbyreciproca1.1.inershaker; Incubationtime:18hto24h.NOTETheincubateds1.antcu1.turecanhestoredat5to10foraweek,ifnotusedimmediate1.y.7.1.2 PreparationoftestspecimensThetestsha1.1.beperformedonat1.eastthreespecimensfromeachtreatedtestmateria
32、1.anduntreatedtestmateria1.Thesizeofthetestspecimenisexpectedtobeaf1.at.30mm2mmsquare.affec1.testsped11ensthesha11specimen.priorcxamp1.ecsi.high-prcssuresteamSterihzationseIcctedsoop1.ion.to7.1.3 Preparationoftheinocu1.atedincubatingmediumasSpectrophotometer.UsingDi1.utemicroscopesuspensioncountingC
33、hamber1.Obtainanotherappropriatemethodsuch7.1.4 Inocu1.ationoftestspecimens(square)specimenprepared,separateaccordancevesse1.withtheisattachedsurfacefacingupwards.mmFiguremmmicroorganisms.Dimensionsinmi1.1.imetresKeyg1.assp1.ate(40mm2mm.square)2Figure1-Inocu1.ationoftestspecimen7.1.5Incubationofthei
34、nocu1.atedtestspecimenstemperatureof(351)for(481)h.NOTEgrowthexamp1.e.Iftheef1.fectspecimensagentisSoe1.utionthatgrowthsubstanceetc),concentrationoftheisoneasThetheeffectoftestbesteri1.eFortotheaThesteri1.izationmethodEnsurethatthetestbacteriainpre-cu1.ture(see7.1.1)areeven1.ydispersed,andestimateth
35、enumberofbacteriadirect1.yathisandawith1/5TSBrtoorbacteria1.ce1.1.densitythatisbetween1.rO1.O3ce1.1.s/m)and1.,0104ce1.1.s/m1.Thissuspensionisusedastheinocu1.atedincubatingmedium.ThesurfacetobetestedisthesurfaceofthePrOdUCUIhedgefacesoftheproductsha1.1.notbetested.Eachtestandp1.acedintoinsteri1.ewith
36、7.1.2.treatedtog1.assp1.ateof40See*21.Itisnecessarytofixthemtotheg1.assp1.atewithanadhesivethatdoesnotaffectthegrowthofPipette20m1.oftheinocu1.atedincubatingmediumpreparedinaccordancewith7.1.3intoeachofthevesse1.scontainingthetestspecimens.Theuppersurfaceofthetestspecimensha1.1.becoveredwithincubati
37、ngmediumsha1.1.theinocu1.atedinbatingthis.medium.Ifnecessary,thevo1.umeoftheinocu1.atedtestspecimen(30mm2mm,square)Un1.essOthersvisespecified,incubatethevesse1.scontainingthetestspecimens(see7.1.4)ataTheconcentrationofthedi1.utedTSBmaybechangedfrom1/5TSBdependingontheeffectoftheagentcontainedinthetr
38、eatedtestmateria1.theFormediumabovetestofthe(duetostrongofthenoactiveofthetestthebacteriumisobservedinTSBcanbeincreasedunti1.growthisachieved(e.1/2TSBorundi1.uted).8Expressionofresu1.ts8.1 Conditionsforva1.idtest8.1.1Whenthetwoconditionsgivenin8.1.2and8.1.3aresatisfied,thetestisdeemedva1.id.Ifa1.1.c
39、onditionsarenotmet,thetestisnotconsideredva1.idandthespecimensha1.1.beretested.8.1.2co1.onyformingunits(CFU)theincubatingmediumafterincubatingfor48h(ortheperiodemp1.oyed)的mVncn.t&bthcvnhnbe说CdbmingfwdnngUBitddnt,ascdzorbccat1.seconccafttiauittin1.arat!iDtthf1.1.mactivityva1.ueswhichareusedtodefineth
40、eeffectivenesssha1.1.beagreedbya1.1.9Repeatabi1.ityandreproducibi1.ityRepeatabi1.ityandreproducibi1.ityaregiveninAnnexA.10TestreportThetestreportsha1.1.inc1.udeat1.eastthefo1.1.owinginformation:a) thenumberofthisdocumentasreference,i.e.ISO4768:2023；b) size,shapeandthicknessofthetreatedanduntreatedspecimens;speciesofthetestc) bacteriawereused;bacteriausedandtheirstrainnumbers,indicatingthereasonifothe
