微生物限度检查法英文.docx
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微生物限度检查法英文.docx
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微生物限度检查法英文
GMPDocument
Title:
MicrobialLimitTestMethod
Drafted:
DateMonthYear
DocumentNumber:
SOP-CH-02-107-01
Reviewed:
DateMonthYear
EffectiveDate:
DateMonthYear
Approved:
DateMonthYear
Page:
1/23
Issuedby:
QualityDepartment
Dispensedby:
QualityDepartment
BackupNo.:
01
Reason(s)forrevision:
1.Purpose:
To establishamethodformicrobiallimittest,andtoensurethesmoothprogressofmicrobiallimittest.
2.ScopeofApplication:
Thisprocedureisapplicableforthemicrobiallimittestofhealthfood.
3.Responsibilities:
TheQClaboratorianareresponsiblefortheimplementationofthisprocedureandthelaboratorydirectorshallassumetheresponsibilityofsupervisionandinspectionfortheeffectiveimplementationofthisprocedure.
4.Contents:
4.1Onthebasisofthenewversionof GB4789.5-2010/2012
4.2 Briefdescription:
Microbiallimittestmethodreferstoamethodfordetectingthedegreeofcontaminationtowhichthenon-specifiedsterilepreparation,rawmaterials,excipientsaresubject,includingthetestfortheamountofbacteriumcontamination,aswellascontrolbacteriaandpathogenicbacteria.
Thetestproductsshallberandomlysampled. Samplingamountisusuallythree timesoftestingamount(morethantwominimumpackingunit). Thewholeprocessofinspectionshallstrictlycomplywithasepticoperationstopreventrecontamination.
Unlessotherwisespecified,theincubationtemperatureofbacteriaandcontrolbacteria is30~35 °Cinthistestmethod, andthatoffungiandyeastis 25~28 °C.
Thereportforthetestresultstakes 1g, 1ml or 10cm2 astheunit.
4.3 Determinationoftotalamountofbacteriaandmold
4.3.1 Briefdescription:
Countofbacteria,fungi,yeastsisdeterminingthenumberofviablebacteriagrowinginthenon-sterilepreparationswithinspecifiedbusinessunits,whichisanimportantindicatorforjudgingthedegreeofmicrobialcontaminationintheproduct,Also,itisoneofthecomprehensivebasesforhygieneevaluationofproducts,rawmaterials,excipients,equipmentsandappliances,process,productionenvironmentandthehealthstatusoftheoperator'sinthemanufacturingenterprise.
Platecountmethod,whichisoneofviablebacteriacountmethods,isusedfordetectingbacteria,fungiandyeasts,andiscurrentlyusedinmanycountries.
Thismethodtakesthevisibleandindividualcoloniesformedontheagarplatesbyeachbacterium(NutrientAgar),fungi(RoseBengalAgar),yeasts(RoseBengalAgarorYeastPeptoneDextroseAgar)asthecountingbasis. Themeasuredresultsreflectthecoloniesnumberofbacteria(agroupofmesophilic,aerobicandfacultativeanaerobicbacteriagrownonNutrientAgar),fungiandyeastgrowingunderspecifiedconditions.Bacteria,fungiandyeaststhathavespecificrequirementsfornutrients,oxygen,temperature, PH andotherfactorsareexcluded.
4.3.2 Equipments,instrumentsandappliances:
4.3.2.1 Sterileroom:
4.3.2.1.1 Sterileroomshallbewithgoodnaturallighting,preventedfromdampness,andawayfromthetoiletsandcontaminatedarea,composingofbufferroom(2rooms)andoperatingroom.
Thereshallbeasampletransferringboxbetweentheoperatingroomandthebufferingroom.
Sixsurfacesofthesterileroomshallbesmoothandcanwithstandcleaninganddisinfection. Theconnectingjointsbetweenwallsandfloors,andthatbetweenwallsandceilingshallbeconcave-curved,seamless,withnodeadcorner. Sewershallnotbeinstalledwithintheoperatingroom.
4.3.2.1.2Operatingroom:
operatingroomshallbeinstalledwithcleanbench,ofwhichthecleanclassis 100,000, andpartialis 100.
4.3.2.1.3Bufferroom:
washingbasin,sterileclothing,hats,masks,slippersandsoonshallbeplacedinthebufferroom. Incubatorsandothersundriesshallnotbeplacedwithinabufferroom.
4.3.2.2 Otherequipments:
cleanbench,constanttemperatureincubator,shaker,electrothermalblowingdrybox,refrigerator,autoclave.
4.3.2.3 Instrumentsandappliances:
Drugbalance,conicalflasks,culturedish,pipette,testtubes,sterilesamplingpaper,scissors,tweezers,medicinespoon,cottonwithethanol,sterileoveralls.
4.3.3 Diluentanditspreparation:
4.3.3.10.9% sterilesodiumchloridesolution:
take 9.0gofsodiumchlorideanddilutewithwaterto 1000mlfordissolution, distributeintoconicalflasks, sterilizeat121°C for 20minutesforuse.
4.3.3.2 Sterilephosphatebuffersolution (pH7.2):
take25.8g ofsodiumhydrogenphosphateand 4.4gofsodiumdihydrogenphosphate, dilutewithwaterto 1000ml,distributeintoconicalflasks, sterilizeat121°C for 20minutesforuse.
4.3.4 Medium:
NutrientAgarmedium,RoseBengalAgarMedium.
4.3.5 Samplingandtestingamountoftestproducts
4.3.5.1 Sampling:
4.3.5.1.1 Generallyrandomsamplingmethodisused,samplingsizeisusuallythree timesoftestingamountwhichismorethantwominimumpackingunit(toprepareforre-examination).
4.3.5.1.2When sampling,incaseofabnormalorsuspicioussamples,suspicioussamplesshallbeselected.Thepackagewithobviousbreakagecannotbetakenassample.
4.3.5.1.3 Productswithmites,fungi,wormswhichcanbeseenfromtheappearanceofproductsandbottle(theinnersideofthelidandneck)anddeteriorationaredirectlyjudgedasunqualifiedproducts.Andthereisnoneedforsampling.
4.3.5.2 Testamount
4.3.5.2.1Testamountofalldosageformsarerequiredof morethantwo packingunits.
4.3.5.2.2 Testamountofsolidandsemi-solid(viscoustestproducts)preparationis 10g.
4.3.5.2.3 Testvolumeofliquidpreparationis 10ml.
4.3.6 Methodofoperation:
4.3.6.1 Preparationbeforeoperation
4.3.6.1.1 Thetestproductsandallthesteriledishes,conicalflasks,testtubes,pipette (1ml,10ml), measuringcylinder,diluentaremovedtoasterileroom. Alltheitemsusedineachtestmustbepreparedinadvance.Adequateamountisneededtoavoidtheinandoutoftheoperatingroom. Alloutsidepackages(kraft)areremovedaftercoded.
4.3.6.1.2 OpenUVlampandcleanbenchinthesterileroom,andmakeitworkfornolessthan 30min.
4.3.6.1.3 TheoperatorwasheshandswithsoapandclosesUVlampbeforeintothebufferroom,changeworkingshoesaftergoingintothebufferroom. Then washhandswith0.1% benzalkoniumbromidesolutionorotherdisinfectantorswabwithcottoncontainingethanol,wearsterileclothing,hats,masksandgloves.
4.3.6.1.4 Swabhandswithcottoncontainingethanolbeforeoperationatfirst,thenswabtheopeningofthetestproductbottles,boxes,bagswithcottoncontainingethanol,andunsealthetestproductswithsterilescissorsafterthealcoholvolatilized.
4.3.6.2 Preparationoftestsolution:
4.3.6.2.1 Liquidtestproducts:
take10mloftest products, addinto 90mlof sterilesodiumchloride-peptonebuffer(pH7.0), mixwell,as thetestsolution(1:
10).
4.3.6.2.2 Solid,semi-solidorviscousliquidtestproducts:
weigh10gofthetestproducts, addinto 100mlofsterilesodiumchloride-peptone buffer(pH7.0 ), heattodissolveinaconstanttemperaturewaterbath,butthetemperatureshallnotexceed 45°C, andthenshakeontheshaker,mixwell,as thetestsolution(1:
10).
4.3.6.2.3 Testproductsofenterictablet:
weigh 10gofthetestproducts, addintoflaskwith100mlofsterilizedphosphatebuffer (pH6.8),heatin waterbathat 45 °C,shaketodissolve,asthetestsolution(1:
10).
4.3.6.3 Dilutionofthetestsolution(diluted with incrementsof10times)
4.3.6.3.1 Take two smallsteriletesttubes,add sterilesodiumchloride-peptone buffer(pH7.0)withapipette, plugthetubeimmediatelyafteradditionofbuffer.
4.3.6.3.2 Takeanother 1ml sterilepipettetoadd 1mlofhomogeneoustestsolution(1:
10)into atesttubewith9ml ofsterilizedsodiumchloride-peptonebuffertube(pH7.0),miswell,namelythetestsolution(1:
100),andsoon.Each dilutionmustchangeapipette.
Whendiluted with incrementsof10times,dipthepipetteintothedilutionsolution(level1)notlowerthan 2.5cm fromliquidsurface,repeatedsuckandblowforabout 10 times,theliquidlevelshallbealittleabovetheupperscaleofthepipettewhensuction,thenliftthepipetteandsticktothetubewalltoadjusttheliquidleveltothemark,thenslowlyblowoutallthetestsolutionalongtheinnerwallofthedilutiontube(level 2) neartheliquidsurface(notouchtotheliquid)(nostickingorresidualliquidshallbeleftinthepipette).
4.3.6.4 Whilediluted with incrementsof10timesindishes,suck1mlofdilutedsolutionofvariouslevels intoeachsteriledishwiththecorrespondingpipette(whendilutefromhighleveltolowlevelthesamepipettecanbeusedduringthesuction). 2~3dishesarepreparedineachdilutionlevel(inthiscase,usuallydishinthelefthandwiththelidhalfopen,pipetteintherighthand). Duringthepouring,1ml ofthetestsolutionwillbeslowlypoured,noresidualliquidleftinthetube,topreventanti-flowingintothetipofthepipette. Atthesametimeasanegativecontrol(afterthecompletionofpouringeachlevelofdilutionfluid, suck1mlofdiluentofeachlevelwith1mlpipetteinto fourdishes,respectively,inwhich two asnegativecontrolsforthenumberofbacteriaandanother two asnegativecontrolsforfungiandyeasts).
4.3.6.5 Pouringofmedium:
heatthepre-preparedmedium(NutrientAgarforbacterialcount,RoseBengalAgarMediumusuallyforfungiandyeastscount)tomeltandcooltoabout 45°C, pour15mloftheabovedish,rotatethedishclockwiseorcounterclockwisequicklyformixing.Whenmixing,donotspillthemediumtorimandlidofthedish,andplaceontheoperatingtableforcondensation.
4.3.6.6 Culture:
Invertbacteriacountplate
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