1、BRITISH STANDARD BS EN 13783:2002 The European Standard EN 13783:2001 has the status of a British Standard ICS 67.050 NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBS EN 13783:2002 This British Standard, having been prepared under the direction of the Consumer Products and Se
2、rvices Sector Policy and Strategy Committee, was published under the authority of the Standards Policy and Strategy Committee on 24 January 2002 BSI 24 January 2002 ISBN 0 580 38936 7 National foreword This British Standard is the official English language version of EN 13783:2001. The UK participat
3、ion in its preparation was entrusted to Technical Committee AW/-/3, Food analysis Horizontal methods, which has the responsibility to: A list of organizations represented on this committee can be obtained on request to its secretary. Cross-references The British Standards which implement internation
4、al or European publications referred to 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 al
5、l the necessary provisions of a contract. Users of British Standards are responsible for their correct application. Compliance with a British Standard does not of itself confer immunity from legal obligations. aid enquirers to understand the text; present to the responsible European committee any en
6、quiries 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. Summary of pages This document comprises a front cover, an inside front cover, the EN title page, pages 2 to 15 and a bac
7、k cover. The BSI copyright date displayed in this document indicates when the document was last issued. Amendments issued since publication Amd. No. Date CommentsEUROPEANSTANDARD NORMEEUROPENNE EUROPISCHENORM EN13783 November2001 ICS07.100.30;67.050 Englishversion FoodstuffsDetectionofirradiatedfood
8、usingDirect EpifluorescentFilterTechnique/AerobicPlateCount (DEFT/APC)Screeningmethod ProduitsalimentairesDtectiondalimentsionissen utilisantlatechniquedpifluorescenceaprsfiltrationet dnombrementdelaflorearobiesurmilieuglos (DEFT/APC)Mthodeparcriblage LebensmittelNachweisderBestrahlungvon Lebensmitt
9、elnmitEpifluoreszenzFiltertechnik/aerober mesophilerKeimzahl(DEFT/APC)Screeningverfahren ThisEuropeanStandardwasapprovedbyCENon29September2001. CENmembersareboundtocomplywiththeCEN/CENELECInternalRegulationswhichstipulatetheconditionsforgivingthisEurope an Standardthestatusofanationalstandardwithout
10、anyalteration.Uptodatelistsandbibliographicalreferencesconcernings uchnational standardsmaybeobtainedonapplicationtotheManagementCentreortoanyCENmember. ThisEuropeanStandardexistsinthreeofficialversions(English,French,German).Aversioninanyotherlanguagemadebytra nslation undertheresponsibilityofaCENm
11、emberintoitsownlanguageandnotifiedtotheManagementCentrehasthesamestatusasthe official versions. CENmembersarethenationalstandardsbodiesofAustria,Belgium,CzechRepublic,Denmark,Finland,France,Germany,Greece, Iceland,Ireland,Italy,Luxembourg,Netherlands,Norway,Portugal,Spain,Sweden,SwitzerlandandUnited
12、Kingdom. EUROPEANCOMMITTEEFORSTANDARDIZATION COMITEUROPENDENORMALISATION EUROPISCHESKOMITEEFRNORMUNG ManagementCentre:ruedeStassart,36B1050Brussels 2001CEN Allrightsofexploitationinanyformandbyanymeansreserved worldwideforCENnationalMembers. Ref.No.EN13783:2001EEN13783:2001(E) 2 Contents page Forewo
13、rd2 1 Scope 3 2 Normativereferences 3 3 Principle3 4 Reagents.3 5 Apparatus .6 6 Samplingtechnique .7 7 Procedure .8 8 Evaluation.9 9 Limitations10 10 Validation10 11 Testreport 11 AnnexA (informative) Furtherinformationontheapplicability.12 AnnexB (informative) Practicalexampleforcalculationofthemi
14、croscopefactor.13 AnnexC (informative) Flowdiagramoftheprocedure14 Bibliography 15 Foreword ThisEuropeanStandardhasbeenpreparedbyTechnicalCommitteeCEN/TC275“FoodAnalysisHorizontal Methods“,thesecretariatofwhichisheldbyDIN. ThisEuropeanStandardshallbegiventhestatusofanationalstandard,eitherbypublicat
15、ionofanidenticaltextor byendorsement,atthelatestbyMay2002,andconflictingnationalstandardsshallbewithdrawnatthelatestby May2002. ThisEuropeanStandardwaselaboratedonthebasisofaprotocoldevelopedfollowingaconcertedaction supportedbytheCommissionofEuropeanUnion(XIIC.)(BCR),andascreeninginvestigationcarri
16、edoutbythe DanishNationalFoodAgencyandthelocalEnvironmentandfoodAgency,MLKFYN,DKOdense.Expertsand laboratoriesfromE.U.andEFTAcountries,contributedjointlytothedevelopmentoftheconcertedactionprotocol. WARNING:Theuseofthisstandardmayinvolvehazardousmaterials,operationsandequipment.Thisstandard doesnotp
17、urporttoaddressallthesafetyproblemsassociatedwithitsuse.Itistheresponsibilityoftheuserofthis standardtoestablishappropriatesafetyandhealthpracticesanddeterminetheapplicabilityofregulatory limitationspriortouse. TheannexesA,BandCareinformative. ThisstandardincludesaBibliography. AccordingtotheCEN/CEN
18、ELECInternalRegulations,thenationalstandardsorganizationsofthefollowing countriesareboundtoimplementthisEuropeanStandard:Austria,Belgium,CzechRepublic,Denmark,Finland, France,Germany,Greece,Iceland,Ireland,Italy,Luxembourg,Netherlands,Norway,Portugal,Spain,Sweden, SwitzerlandandtheUnitedKingdom.EN13
19、783:2001(E) 3 1Scope ThisEuropeanStandardspecifiesamicrobiologicalscreeningmethodforthedetectionofirradiationtreatmentof herbsandspices,usingthecombineddirectepifluorescentfiltertechnique(DEFT)andaerobicplatecount(APC). TheDEFT/APCtechniqueisnotradiationspecific,therefore,itisrecommendedtoconfirmpos
20、itiveresultsusinga standardisedmethod(e.g.EN1788,prEN13751)tospecificallyproveanirradiationtreatmentofthesuspected food. Themethodhasbeensuccessfullytestedininterlaboratorytestswithherbsandspices1to5. 2 Normativereferences ThisEuropeanStandardincorporatesbydatedorundatedreference,provisionsfromother
21、publications.These normativereferencesarecitedattheappropriateplacesinthetext,andthepublicationsarelistedhereafter.For datedreferences,subsequentamendmentstoorrevisionsofanyofthesepublicationsapplytothisEuropean Standardonlywhenincorporatedinitbyamendmentorrevision.Forundatedreferencesthelatestediti
22、onofthe publicationreferredtoapplies(includingamendments). ISO4833,MicrobiologyGeneralguidancefortheenumerationofmicroorganismsColonycounttechniqueat 30C. ISO7218, MicrobiologyoffoodandanimalfeedingstuffsGeneralrulesformicrobiologicalexaminations. 3Principle ThemethodisbasedonthecomparisonoftheAPCwi
23、ththecountobtainedusingDEFT.TheAPCgivesthe numberofviablemicroorganismsinthesampleafterapossibleirradiationandtheDEFTcountindicatesthetotal numberofmicroorganisms,includingnonviablecells,presentinthesample.ThedifferencebetweentheDEFT countandtheAPCcountinspicestreatedwithdosesof5kGyto10kGyisgenerall
24、yaboutorabove3to4log units.SimilardifferencesbetweenDEFTandAPCcountscanbeinducedbyothertreatmentsofthefoodsleading todeathofmicroorganisms,e.g.heat,thuspositiveresultsshallbeconfirmed. Aknownvolumeofsampleisfilteredthroughamembranefilteratreducedpressureinordertoconcentratethe microorganismsonthefil
25、ter.Themicroorganismsarestainedwithafluorochrome,acridineorange(AO),resulting inanorangeandorangeyellowfluorescenceunderilluminationwithbluelightat450nmto490nm.These microorganismsarecountedusinganepifluorescencemicroscopetogivetheDEFTcount.However, microorganismswhichwerenonviablebeforeirradiations
26、howgreenfluorescenceandarenotcounted.Inparallel, theAPCisdeterminedfromasecondportionofthesametestsample6to10. 4Reagents 4.1General Duringtheanalysisuseonlyreagentsofrecognizedanalyticalgrade.AllthereagentsusedintheDEFTandAPC determinationsshouldbesterilizedbymembranefiltrationthrough0,2mporesizemem
27、branefiltersorby autoclaving. 4.2Peptonesalinediluent 4.2.1Composition Sodiumchloride 8,5g Peptone 1,0g Distilledordemineralizedwater 1000mlEN13783:2001(E) 4 4.2.2Preparation Dissolvethecomponentsinthewater.AdjustthepH,ifnecessary,sothataftersterilizationthefinalpHis7,20,2 at20Cto25C.Sterilizeinthea
28、utoclave(5.13)at121C1Cfor15min.Thediluentmaybestoredinaglass bottleat4Cto6Cfornotmorethantwoweeks. 4.3Buffer, pH3,0 4.3.1 Citricacidsolution, substanceconcentration c(C 6 H 8 O 7 H 2 O)=0,1mol/l 4.3.1.1Composition Citricacidmonohydrate 21g Distilledordemineralizedwater 1000ml 4.3.1.2Preparation Diss
29、olvethecitricacidmonohydrateinthewater.Thesolutionmaybestoredinasterilizedglassbottleat4Cto6 Cfornotmorethanthreemonths. 4.3.2 Sodiumhydroxidesolution, c(NaOH)=0,1mol/l 4.3.2.1Composition Sodiumhydroxide 4,0gor Sodiumhydroxidesolution,1mol/l 100ml Distilledordemineralizedwater 900ml 4.3.2.2Preparati
30、on Dissolvethesodiumhydroxideordilutethesodiumhydroxidesolutioninthewater.Thesolutionmaybestoredina glassbottleat4Cto6Cfornotmorethanthreemonths. 4.3.3 Completebuffer, pH3,0 4.3.3.1 Composition Citricacidsolution(4.3.1) 100ml Sodiumhydroxidesolution(4.3.2) 54ml 4.3.3.2Preparation Mixthecitricacidsol
31、utionandthesodiumhydroxidesolution.AdjustpHto3,00,2withcitricacidsolutionor sodiumhydroxidesolution.Sterilizethebufferthroughamembranefilterofporesize0,2m(5.3)beforeuse.The solutionmaybestoredinaglassbottleat4Cto6Cfornotmorethanthreeweeks.EN13783:2001(E) 5 4.4Acridineorangesolution 4.4.1 Buffersolut
32、ion,pH6,6 4.4.1.1Composition Citricacidsolution(4.3.1) 35,5ml Sodiumhydroxidesolution(4.3.2) 100ml 4.4.1.2Preparation Mixthecitricacidsolutionandthesodiumhydroxidesolution.AdjustpHto6,60,2withcitricacidsolutionor sodiumhydroxidesolution.Sterilizethebufferthroughamembranefilterofporesize0,2m(5.3)befo
33、reuse.The buffersolutionmaybestoredinaglassbottleat4Cto6Cfornotmorethanthreeweeks. 4.4.2 Completeacridineorangesolution 4.4.2.1Composition Acridineorange 0,025g Buffer,pH6,6(4.4.1) 100ml 4.4.2.2Preparation Dissolvetheacridineorangeinthebuffersolution(4.4.1).Sterilizetheacridineorangesolutionthrougha
34、0,2m poresizemembranefilter(5.3)beforeuse.Thesolutionmaybestoredinabrownglassbottleat4Cto6Cfor notmorethanoneweek. NOTE1Concentratedacridineorangesolutioniscommerciallyavailableandisrecommendedforsafetyreasons. NOTE2Asacridineorangeisregardedasamutagenicsubstance,disposableglovesandfacemasksshouldbe
35、usedwhen weighingthestain. 4.52Propanol 4.6TritonX100 1) ,1%cleaningsolution 4.6.1Composition TritonX100 10ml Distilledordemineralizedwater 1000ml 4.6.2Preparation MixTritonX100withwarm(80C)water.Sterilizethesolutionthrougha0,2mporesizemembranefilter(5.3). Thesolutionmaybestoredinaglassbottleat4Cto6
36、Cfornotmorethanthreeweeks. 1) TritonX100isanexampleofasuitableproductavailablecommercially.Thisinformationisonlygivenfortheconvenience ofusersofthisInternationalStandardanddoesnotconstituteanendorsementbyCENofthisproduct.EN13783:2001(E) 6 4.7TryptoneYeastExtractGlucoseAgar(Platecountagar) 4.7.1Compo
37、sition Tryptone 5,0g YeastExtract 2,5g Dextrose(Glucose) 1,0g Agar(accordingtothegelstrengthoftheagarused) 12gto18g Distilledordemineralizedwater 1000ml 4.7.2Preparation Dissolvethecomponentsordehydratedcompletemediuminthewaterwhileheatinguntilboiling.AdjustthepH,if necessary,sothataftersterilizatio
38、nthefinalpHis7,20,2at20Cto25C.Sterilizeintheautoclave(5.13)at 121C1Cfor15min. 5Apparatus UsuallaboratoryapparatusinaccordancewithISO7218and,inparticular,thefollowing: 5.1Apparatusformembranefiltrationofsamplesuspensions Filtrationequipmentmadeofstainlesssteelorglassshouldbeused.Thebottomfiltershould
39、beofsinteredglass orstainlesssteelintendedforfilterswithadiameterof25mm(5.5).Thefiltertowervolumeshouldbeatleast 10ml.Thefiltrationequipmentisplacedverticallyonasuctionflaskoramanifoldconnectedtoawaterpumpor vacuumpumpwithapressureregulator.Thevacuumduringfiltrationshouldusuallybeapproximately70kPa.
40、 NOTE Theuseofawaterpumpisnotverysuitableifthewaterpressurecannotberegulated. 5.2Filterfunnel andsuitablesuctionflasksmadeofglassforsterilefiltrationofreagentsanddiluent. 5.3Membranefilters, celluloseesterorsimilar,poresize0,2m,e.g.diameter30mmand/or47mm,forsterilefiltrationofreagents. 5.4Membranefi
41、lters, polypropylene,diameter25mm,poresize10m,forprefiltrationofsamples. 5.5Membranefilters, whitepolycarbonate,diameter25mm,poresize0,6mformembranefiltrationofsampletestsolution. 5.6Sterilefastfilterpaper, forfiltrationofspicesamples. 5.7Epifluorescencemicroscope, withsuitablelightandfiltercombinat
42、ion(450nmto490nm) 5.8Optics, 100ximmersionobjective,ocularwithmagnificationof10x.(Usingatubemagnificationof1,25atotal magnificationof1250xisachieved.) 5.9Microscopeslide, e.g.76mmx26mm.EN13783:2001(E) 7 5.10Coverslip, e.g.25mmx50mm,withathicknesscorrespondingtotherequirementsoftheobjective. 5.11 Imm
43、ersionoil, nonfluorescing.Refractiveindex1,515to1,518. 5.12Stagemicrometer, withgraduationsof0,01mm,formeasuringthediameterofthemicroscopefieldofview. 5.13Autoclave, forsterilizationofdiluentandculturemedium. 5.14Oven, fordryheatsterilizationofglassware. 5.15Whirlmixer, formixingsamplesuspensionanddiluent. 5.16Waterbath, formaintainingtheculturemediaattheappropriatetemperature. 5.17Incubator, capableofmaintainingatemperatureof30C1C. 5.18 Glassbo
