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 ? +T    &|G%2`HelveticaG 55GallonDrum " &zG%2`HelveticaG (stainlesssteel)+x xf+T@@|||||}~ :/G%2`HelveticaG StrainerMade 8-G%2`HelveticaG from1"Mesh 7 ,G%2`HelveticaG (stainlesssteel)+ f  +XP |P ~~}||||1`  6 6 1$                   &                   6 5 4 3 3 2 1 1 1 1 1 1 1 2 3 3 4 5 6 &1 1 1 1 2 3 3 4 5 6 7 8 9 9 : ; ; ; ; ; ; ; ; : 9 9 8 7 6 5 4 3 3 2 1 1 1 1 & 6 5 4 3 3 2 1 1 1 1 1 1 1 2 3 3 4 5 6 AFIG1A.WPG ; #  e[XV hp x (#%'0*,.8135@8:<H?Ae@d..PXXXO<6X9`("Courier 10cpiOFigure1A.IncineratorAshSamplingSifter#|m '  `2CG Times 12.00ptDT TDXXm PzGXP<6X9`("Courier 10cpiDT TDXx6X@8;X@( U$  EE  E  {$ U!   X+XXX    7+XXdd7@$ $ MICROBIALSURVIVABILITYTESTFORMEDICALWASTE@hh'INCINERATORASH  cCondition alTest  M   Methods( CTM025 ) 1. @ ApplicabilityandPrinciple    @ 1.1 @` Applicability.Twotestmethodswillbeperformedtodeterminethesurvivabilityofmicroorganismsintheashduringthenormaloperationofthemedicalwasteincinerator.Thequantificationofsurvivingindicatororganismsisutilizedasanindicationoftheeffectivenessofincinerationasamedicalwastetreatmenttechnology.Thistestprocedureisintendedtorecover,identifyandquantifytheindicatororganismsusedtodeterminetheefficiencyoftheincinerator.Bacillus  stearothermophilus,asporeformingbacterium,isusedbecause & thistypeoforganismistypicallythemostresistanttothermalinactivation,therebyensuringthatmorefragileorganismswillbedestroyed.Thefollowingproceduresweredevelopedtorecoveronlytheindicatororganism. @ 1.2 @` Principle.Withtheincineratoroperatingunderrecommendedconditions,thewastestreamischargedwithknownquantitiesofBacillusstearothermophilussporesinitemsnormallyfoundinthe ( medicalwastestreamandintheinsulatedpipes.Thesamplesareaddedtotheincineratorwithtypicalmedicalwastesandarerecoveredattheendoftheburncyclewhenashesarebatchremoved.Thedestructionefficiencyoftheincineratorisdeterminedbyestablishingthesurvivabilityoftheindicatormicroorganism.2. @ Apparatus :&!6  @  @` Note:Mentionoftradenamesorspecificproductinthismethod x'"8 doesnotconstituteendorsementbytheU.S.EnvironmentalProtectionAgency. @ 2.1 @` Sterilization.Autoclavecapableofsteamsterilizationconditionsof121$Cfor15minutesat15psi.Specificapparatus p,'@ andreagentsthatcontacttherecoveredsampleshallbesterilizedundertheseconditionsbyautoclavingorotherequivalentmethod...2.2` ..` Sampling.Thedirectashsamplesarecollectedfromtheashaftercooling.Athiefsamplerisusedforcollection.Thesampleiscollectedintosterilecontainersandstoredat4$Cpriortotransportationtothelaboratory.ThepipesampleisshowninFigure1andiscomposedofthefollowingcomponents:] L<8x , '`'3ffff@@](#..(#2.2.1` ..` OuterContainer.Steelpipehavingatwoinchdiameterand6in.longwithendcapstocontainthesample.(Note:SincecomponentsarecommonlyavailableinEnglishunits,onlyEnglishunitsarespecified).Theendsofthepipeshallbethreadedtoattachthemetalcaps.Etchorstampeachtubeforidentification...2.2.2 .. InnerContainer.2in.lengthof3/8stainlesssteeltubingwithstainlesssteelSwagelokcaps.Otherleakfreestainlesssteelfittingsmaybeusedasalternatives...2.2.3 .. Insulation.Vermiculite,orotherthermalinsulatingmaterial.Theinsulationshallbesuchthattheinnercontainerismaintainedinthecenterportionoftheoutercontainertoprotectitfromthermalshock...2.3` ..` SampleRecovery...2.3.1 .. Gloves.Ofsufficientqualitytoprotectthetesterfrommicroorganismsthatmaybepresentintheash...2.3.2 .. SampleRetrievalInstruments.Shovel,tongs,thiefsorotherinstrumentsforremovinganincineratorashsampleandthepipesamplefromtheashdump...2.3.3 .. SampleStorageContainers.PipeSamplesareplacedinsideplasticbags.Directashsamplesareplacedinsterilecontainerstheninplasticbags.Allsamplesarestoredat4$Cuntiltransporttothelaboratory...2.4` ..` Analysis...2.4.1 .. Incubator.Airconvectiontype,capableofaerobicincubationat55to65$C...2.4.2 .. IncubationTubes(Sterile).Heatprooftubesorbottles.̴..2.4.3 .. WaterBath.Capableofmaintainingatemperatureof80$C...2.4.4 .. PetriDishes(Sterile)...2.4.5 .. FilterUnits(Sterile).NalgeneR0.2mcellulosenitratemembraneanalyticalfilterunitsorequivalent.3...Reagents..3.1` ..` SamplingandSampleRecovery...3.1.1 .. CrushedIce.Forsamplestorageinthefield...3.1.2 .. IndicatorMicroorganisms.Bacillusstearothermophilusspores.Eachpipeshouldcontainapproximately1x106drysporesandapproximately1x1012sporesperdayarebeingaddedtotheactualwastestream.DrysporesofthisquantitymaybeobtainedfromATCC,12301ParklawnDrive,Rockville,MD20857.(TelephoneNo.8006386597)...B.stearothermophilusisalsoaddedtothewastestreamatapproximately1x1012sporesperday.Directashsampleswillbetakeneachdaytodetermineactualsporesurvivabilityortheincineratordestructionefficiency...3.2` ..` Analysis.Theanalyticalreagentswillvaryaccordingtothecultureandidentificationprocedureused.Thereagentsdescribedarerequiredfortheexampletestslistedinthismethod.Otherreagentssuitableforappropriatealternativetestsareacceptable...3.2.1 .. Water.Allwaterusedshallbesteriledeionized...3.2.2 .. SporeReagent(Sterile).Bufferedphosphatesolution(2.0M)preparedbydissolving174.2gdibasicpotassiumphosphate(K2HPO4)and136.1gmonobasicpotassiumphosphate(KH2PO4)inwateranddilutingto1liter...3.2.3 .. TrypkticaseSoyAgarGrowthMedium.Preparebymixing15.0gpancreaticdigestofcasein,5.0gpapaicdigestofsoymeal,5.0gNaCl,and15.0gagarwith1literofwater.Boiltodissolvetheagar.Sterilizeat121$Cfor15minutes.Thisrecipewillprepare10petridishes.4...Procedure..4.1` ..` Sampling...4.1.1 .. SamplePreparation...4.1.1.1 .. Cleantheinnerpipeandcapsbeforeuse.Removethecapfromthesporevialandbreakupthesporecakeusingacleaninstrument.Secureoneendcaptotheinnercontainer.Carefullytransferthecrushedcakeof1x106sporesintotheinnersamplecontainer,andsealtheotherendcap.Allsamplinginstruments(i.e.samplingthief)usedtotaketheactualsamplearesterilized.Sterilesamplecontainersfortheashsamplesareobtained...4.1.1.2 .. Secureoneendcaptotheoutercontainer,andaddenoughvermiculitetoallowtheinnercontainertobepositionedintheapproximatemiddleandcenteroftheoutertube.Addadditionalvermiculite,gentlytappingtheoutercontainertoeffectsettling,untilfull.Securetheotherendcap.ThesporeinoculationtechniquesarediscussedinAppendixA.3willbeusedforinoculationofwasteitemsfordirectashsampling.Atotalof1x1012sporesareaddedtothewastestreampersamplingday...4.1.2 .. IncineratorSpike.Theincineratorspikewillvaryaccordingtoloadingpractice.Forsemicontinuousloadingoperations,addasetofthreesamplestotheincineratoratthebeginning,middle,andendofanormalday'sloadingperiod(9totalsamplesperdailyburn).Disperseeachsamplepersetindifferentsectionsofthewastestobecharged.Forsinglechargebatchoperations,placeall9samplesontheincineratorbedbeforeanywastesarecharged.Dividethebedintoagridof9equalareas.Placeasampleintheapproximatecenterofeachgrid.Loadthewasteschargeinamannerthatwillnotdisturbthesamplearrangement...4.2` ..` SampleRecovery.Thesamplerecoverywillvaryaccordingtoincineratorashremovalpractice.Recoverthesamplesduringashremovalfollowingthecooldownperiodforbatchremovaloperationsorastheyemergeintheashbinforcontinuouscleanoutoperations.NOTE:Forunitshavingwaterquenchedashbins,careshouldtakentoremovethesamplesquicklyfromthebintopreventcontamination.Removeexcessdebrisfromtherecoveredsamples,placeinaplasticbag,andpackinicetomaintainatorbelow4$C.Ensurethatthesamplesareprotectedfromcontaminationfrommeltedice...4.3` ..` Analysis...4.3.1 .. SamplePreparationofAnalysis.Thesamplesshouldbedeliveredtothelaboratorywithin96hours.ApHisperformedonthedirectashsamplesatthetimeofrecovery.Upondeliverytothelaboratorythepipesampleisopened,theoutercontainerandvermiculitearediscardedaftertheinnercontainerisrecovered.Theinnercontainershouldbecleanedsufficientlytopreventcrosscontaminationwhenopeningthesample.Thecontentsoftheinnerpipecontaineristhentransferredasepticallyintosteriletesttubes.Theinsideofthecontainerisrinsedwithsterilewaterandthisisplacedinthetesttube...Ashsamplesmustbethoroughlymixedtoensurehomogeneity.Approximatelyonegramofashisthenaddedto3100mlaliquotsofsterilewater.Asixlogserialdilutionofeachaliquotwillthenbeprepared...4.3.2 .. CultureDevelopment.Pourmoltentrypticasesoyagarinto10petridishesandallowtheagartoharden.Usingaseparatecellulosenitratefilter,foreachserialdilution,filtereachconcentrationfromeachashsuspension.Usingsteriletweezers,removethefiltersfromeachunitandplacefaceuponseparateagarplates.Aerobicallyincubatetheplatesinanairconvectionincubatorat65$Cfor18to24hours...4.3.4 .. IdentificationofIndicatorMicroorganisms.AvarietyoftestsmaybeusedtoidentifyB.stearothermophilus.Asaminimum,techniquestoestablishthatthemicroorganismsfoundaregrampositive,rodshaped,sporeproducersshouldbeused.Thismaybeperformedusingstain/morphological/biochemicaltestsorbystrip/cardtestingunitsfordeterminingbiochemicalprofile...4.4` ..` QualityControlProcedures...4.4.1 .. IndicatorOrganisms.Sporesfromavialnotsubjectedtotheincineratortestshallbedissolvedinsporereagent,appropriatelyaliquotedtoyieldafinalplatecountofbetween20and200colonies,developed,andenumeratedsimultaneouslywiththesamplesasacontroltoaidinestablishingcolonyidentity.5...Calculation..5.1` ..` MicrobialSurvivabilityofIndicatorSpores.dQA=zx 0 @Xdddddddd@d(#.. Eq.1where:e[XD hp x (#%'0*,.8135@8:<H?Ae..MS..=0D ..D MicrobialSurvivability,percent(to6sig.figures.)..Sr..=0D ..D Numberofsporecoloniescountedintheanalysis...Ss..=0D ..D Numberofsporesinoriginalspikevial...100=0D ..D Conversiontopercent.e[XV hp x (#%'0*,.8135@8:<H?Ae6...Bibliography..1.AmericanSocietyforMicrobiology.ManualofMethodsforGeneralBacteriology.Washington,D.C.1981.524p...2.V ..V Buchannan,R.E.andN.E.Gibbons.Bergey'sManualofDeterminativeBacteriology.Baltimore,MD.TheWilliamsandWilkinsCompany.1974.1268p.#Xd#@..| EPAMEDICALWASTEINCINERATIONTESTS@.. PROPOSEDASHSAMPLINGPROTOCOL@..xRevision28/7/90I...PRESAMPLINGPREPARATION..A.V ..V Sifter(seeFigure1A)..V ..V 1.0 .. ScrubthesifterwithsoapywaterandcleanwithadisinfectantsuchasVirex128.Allowthesiftertoairdryinaclean,dryarea...V ..V 2.0 .. Disinfectwitha3percenthydrogenperoxidesolution,allowtoairdryinaclean,dryarea...V ..V 3.0 .. Coverthecanwithcleanplasticandsealtheliduntiluse...B.V ..V Other..V ..V 1.0 .. Wherepossible,usedisposableitemsthathavebeendisinfectedforsampling.Examplesofdisposableitemsaredisposablegloves,garments,etc.Usenewbottlestostoresamples._ F62x , h) `'3 @_II...ASHSAMPLINGPROCEDURE..A.V ..V Opentheincineratordoor...B.0V ..V Removeshovelfromthestoragecontainerandallowtoairdryinaclean,dryarea...C.0V ..V Removeashfromthechamberwithashovelanddeposititintothetopofthesifter...D.0V ..V Gentlysifttheashuntilfinesfallintothebottomofthesifter...E.0V ..V Removethesifterbasketanddepositcoursematerialintoataredtrashcan...F.0V ..V RepeatStepsCthroughEuntilthedesiredamountofashisremoved.Theamountoftheashthatisremovedwillbedeterminedbythehospital'snormalashremovalprocedures...G.0V ..V Whentheashhasbeenextracted,weighandrecordtheamountofcoursematerialremoved...H.0V ..V Placethelidtightlyonthesifter.Ǵ..I.0V ..V Rollthesifterforaminimumofoneminutetouniformlymixashfines...J.0V ..V Removethelidaftertheashhassettled...K.0V ..V Removesamplingthief(grainsampler,threefeetlongwith1/2inchslots)fromstoragecontainerandallowtoairdryinaclean,dryplace...L.0V ..V Takesamplesusingsampler.Collectfoursamplesinto950mlbottlesbyrepeatedlyinsertingsamplerintorandomlocationsintheashbedwithinthesifterdrum.Sealthebottles,wipeeachwithdisinfectantandlabelthebottlesimmediately.Storelabeledbottlesinarefrigerator(microbialsamplesonly)...M.0V ..V Thefoursampleswillbesentforanalysisasfollows:..V ..V 1. .. 950mlfordioxinanalysis.....V ..V 2.0 .. 950mlformetal/carbon/lossonignition(LOI)analysis...V ..V 3.0 .. 950mlforsporeenumerationanalysis...V ..V 4.0 .. 950mlforarchivepurposes.III.0..POSTSAMPLINGPREPARATION..A.0V ..V Cleanandpreparetheutensilsfornextuse.Storethemincontainersfilledwith3percenthydrogenperoxidesolution...B.0V ..V Cleananddisinfectthesifterforthenextuse.