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1、Designation:D6974?09(Reapproved2013)2StardPracticefEnumerationofViableBacteriaFungiinLiquidFuels—FiltrationCultureProcedures1ThisstardisissuedunderthefixeddesignationD6974thenumberimmediatelyfollowingthedesignationindica

2、testheyearofiginaladoptioninthecaseofrevisiontheyearoflastrevision.Anumberinparenthesesindicatestheyearoflastreapproval.Asuperepsilon()indicatesaneditialchangesincethelastrevisionreapproval.ε1NOTE—Subsections8.48.8.2were

3、editiallycrectedinAugust2014.ε2NOTE—ReferencesinSection9thetitleofIP385wereeditiallycrectedinJanuary2015.1.Scope1.1Thispracticecoversamembranefilter(MF)procedurefthedetectionenumerationofHeterotrophicbacteria(HPC)fungiin

4、liquidfuelswithkinematicviscosities≤24mm2s1atambienttemperature.1.2ThisquantitativepracticeisdrawnlargelyfromIPMethod385TestMethodD5259.1.3Thistestmaybeperfmedeitherinthefieldinthelabaty.1.4Theabilityofindividualmicrobes

5、tofmcoloniesonspecificgrowthmediadependsonthetaxonomyphysiologicalstateofthemicrobestobeenumeratedthechemistryofthegrowthmediumincubationconditions.Consequentlytestresultsshouldnotbeinterpretedasabsolutevalues.Ratherthey

6、shouldbeusedaspartofadiagnosticconditionmonitingefftthatincludesothertestparametersinaccdancewithGuideD6469.1.5Thispracticeoffersalternativeoptionsfdeliveringfuelsamplemicrobestothefiltermembranevolumesdilutionsfilteredg

7、rowthmediausedtocultivatefuelbnemicrobesincubationtemperatures.Thisflexibilityisofferedtofacilitatediagnosticeffts.Whenthispracticeisusedaspartofaconditionmonitingprogramasingleprocedureshouldbeusedconsistently.1.6Theval

8、uesstatedinSIunitsaretoberegardedasstard.Nootherunitsofmeasurementareincludedinthisstard.1.7Thisstarddoesnotpurpttoaddressallofthesafetyconcernsifanyassociatedwithitsuse.Itistheresponsibilityoftheuserofthisstardtoestabli

9、shappropriatesafetyhealthpracticesdeterminetheapplicabilityofregulatylimitationspritouse.2.ReferencedDocuments2.1ASTMStards:2D1129TerminologyRelatingtoWaterD1193SpecificationfReagentWaterD4175TerminologyRelatingtoPetrole

10、umPetroleumProductsLubricantsD5259TestMethodfIsolationEnumerationofEnterococcifromWaterbytheMembraneFilterProcedureD6426TestMethodfDeterminingFilterabilityofdleDistillateFuelOilsD6469GuidefMicrobialContaminationinFuelsFu

11、elSystemsD7463TestMethodfAdenosineTriphosphate(ATP)ContentofMicroganismsinFuelFuelWaterMixturesFuelAssociatedWaterD7464PracticefManualSamplingofLiquidFuelsAssociatedMaterialsFuelSystemComponentsfMicrobiologicalTestingE13

12、26GuidefEvaluatingNonconventionalMicrobiologicalTestsUsedfEnumeratingBacteriaF1094TestMethodsfMicrobiologicalMonitingofWaterUsedfProcessingElectronMicroelectronicDevicesbyDirectPressureTapSamplingValvebythePresterilizedP

13、lasticBagMethod2.2EnergyInstituteStards:3IP385DeterminationoftheViableAerobicMicrobialContentofFuelsFuelComponentsBoilingBelow390C—FiltrationCultureMethod3.Terminology3.1Definitions:3.1.1Fdefinitionoftermsusedinthismetho

14、drefertoTerminologiesD1129D4175GuideD6469.1ThispracticeisunderthejurisdictionofASTMCommitteeD02onPetroleumProductsLiquidFuelsLubricantsisthedirectresponsibilityofSubcommitteeD02.14onStabilityCleanlinessofLiquidFuels.Curr

15、enteditionapprovedMay12013.PublishedAugust2013.iginallyapprovedin2003.Lastpreviouseditionapprovedin2009asD6974–09.DOI:10.1520D697409R13E02.2FreferencedASTMstardsvisittheASTMwebsitewww.astm.gcontactASTMCustomerServiceat.F

16、AnnualBookofASTMStardsvolumeinfmationrefertothestard’sDocumentSummarypageontheASTMwebsite.3AvailablefromEnergyInstitute61NewCavendishSt.LondonWIG7ARU.K.:www.energyinst.g.uk.Copyright?ASTMInternational100BarrHarbDrivePOBo

17、xC700WestConshohockenPA194282959.UnitedStates17.10WaterBathcapableofmaintainingatemperatureof4762Creceiving500mLbottles.Waterbathcapacityshouldbesufficienttoaccommodateatleastonebottleofeachtypeofagargrowthmediumused.7.1

18、1GlassBottlesscrewcapwithgastightclosures500mLnominalcapacity.7.12CultureTubesglass16by125mmscrewcap.7.13Autoclavewithcapacitytohold500mLglassbottlesupright.NOTE4—Items7.10–7.13arenotneededifusingcommerciallypreparedPetr

19、idishesasindicatedinNote3.8.ReagentsMaterials8.1PurityofReagents—Reagentgradechemicalsshallbeusedinalltests.UnlessotherwiseindicateditisintendedthatallreagentsconfmtothespecificationsoftheCommitteeonAnalyticalReagentsoft

20、heAmericanChemicalSocietywheresuchspecificationsareavailable.48.2Theagarusedinpreparationofculturemediashallbeofmicrobiologicalgrade.Wheneverpossibleusecommercialculturemedia.8.3WaterPurity—Unlessotherwiseindicatedrefere

21、ncestowatershallbeunderstoodtomeanreagentwaterasdefinedbyTypeIIIofSpecificationD1193.8.4Chlotetracycline0.1%(wv)aqueous.Dissolve0.1gchlotetracyclineinwaterdiluteto100mL.Sterilizebypassingthrougha0.2mfilter.8.5DetergentSo

22、lution0.1%(v?v)—Dissolve10mLofpolyoxyethylene(20)sbitanmonooleate5in990mLwater.Sterilizeeitherbypassingthrougha0.2mmembranefilterintoasterilevesselautoclavingat121Cf15min.8.6HydrochlicAcid1molHClL1.8.7LacticAcid10%(wv)aq

23、ueous.Dissolve10goflacticacidinwaterdiluteto100mL.Sterilizebypassingthrougha0.2mfilter.8.8MaltExtractAgar(MEA):8.8.1CompositionLitre:MaltExtract30gMycologicalPeptone5gAgar15gWater1L8.8.2Preparation—Suspendthemaltextractm

24、ycologicalpeptoneagarin1Lofwaterboiltodissolve.AdjustthepHto5.460.2usingeither1mLL1hydrochlicacid(8.6)sodiumhydroxide10%wv(8.10).Dispense250mLptionsinto500mLglassscrewcapbottles(7.11).Sterilizebyautoclavingat12162Cf10min

25、.Coolmaintainthesterilizedagarinawaterbath(7.10)at4762C.Optionallyaftertheagarhascooledto4762Cadd1mLofa0.1%aqueoussolutionofchlotetracycline(filtersterilizedbypassingthrougha0.2mfiltersee8.4)per100mLMEAmixbyshaking.Ifthe

26、mediumisrequiredatpH3.5add10%lacticacid(filtersterilizedbypassingthrougha0.2mfiltersee8.7)toadjustpH.OnceacidifiedtheMEAshallnotbereheated.MakeagarplatesofthemediumbypouringsufficientMEAintosterilepetridishestogivealayer

27、approximately4mmthick.Allowtocoolset.NOTE5—MEAisavailablefromvariousmanufacturersindehydratedfminprepouredplateswithwithoutaddedantibioticeitherofwhichmaybeused.WhensterilizingMEApreparedfromcommercialdehydratedmediafoll

28、owthemanufacturer’sinstructionsfsterilization.Avoidoverheating.NOTE6—AlternativemediatoMEAmaybeusedprovidingtheabilityofanyalternativemediumtosupptcomparablegrowthofyeastmoldsthatarelikelytobeencounteredintestsamplescanb

29、edemonstrated.NOTE7—Alternativeantibioticsmaybeusedprovidingtheirabilitytoinhibitgrowthofbacteriabutnotyeastmoldshasbeenvalidated.8.9Ringer’sSolutionOneQuarterStrength:8.9.1CompositionLitre:Sodiumchlide2.25gPotassiumchli

30、de0.105gCalciumchlide0.12gSodiumbicarbonate0.05gWater1L8.9.2Preparation—Dissolvesaltsin1Lofwaterdispense10mLptionsintoscrewcappedculturetubes(7.12).Sterilizebyautoclavingat121Cf15min.NOTE8—OnequarterstrengthRinger’ssalts

31、areavailableintabletfmfromvariousmanufacturers.8.10SodiumHydroxide10%(wv)aqueous.Dissolve10gNaOHinwaterdiluteto100mL.8.11TryptoneSoyAgar(TSA):8.11.1CompositionLitre:Tryptone15gSoyprotein5gSodiumchlide5gAgar15gWater1L8.11

32、.2Preparation—Suspendthedryingredientsin1Lofwaterboiltodissolve.Dispense250mLptionsinto500mLglassscrewcapbottles(7.11).Sterilizebyautoclavingat12162Cf10min.Coolmaintainthesterilizedagarinawaterbath(7.10)at4762C.Drawasamp

33、letestthepH.IfthepH≠7.360.3rejectthebatchmakeafreshmixture.MakeagarplatesofthemediumbypouringsufficientTSAintosterilepetridishestogivealayerapproximately4mmthick.Allowtocoolset.NOTE9—TSAisavailablefromvariousmanufacturer

34、sindehydratedfminprepouredplates.NOTE10—AlternativemediatoTSAmaybeusedprovidingtheabilityofanyalternativemediumtosupptcomparablegrowthofbacteriathatarelikelytobeencounteredintestsamplescanbedemonstrated.9.Procedure9.1Sam

35、pling:4ReagentChemicalsAmericanChemicalSocietySpecificationsAmericanChemicalSocietyWashingtonDC.FSuggestionsonthetestingofreagentsnotlistedbytheAmericanChemicalSocietyseeAnnualStardsfLabatyChemicalsBDHLtd.PooleDsetU.K.th

36、eUnitedStatesPharmacopeiaNationalFmularyU.S.PharmacopeialConventionInc.(USPC)RockvilleMD.5ThesolesourceofsupplyofTween80knowntothecommitteeatthistimeisSigmaAldrichCo.St.LouisMO63178.Ifyouareawareofalternativesuppliersple

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