微生物限度检查法英文.docx

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