EPA 9131 TOTAL COLIFORM MULTIPLE TUBE FERMENTATION TECHNIQUE

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1、METHOD9131TOTALCOLIFORM:MULTIPLETUBEFERMENTATIONTECHNIQUE1.0SCOPEANDAPPLICATION1.1Thismethodisusedtodeterminethepresenceofamemberofthecoliformgroupingroundwaterandsurfacewater.1.2Thecoliformgroup,asanalyzedforinthisprocedure,isdefinedasallaerobicandfacultativea

2、naerobic,gram-negative,non-spore-forming,rod-shapedbacteriathatfermentlactosewithgasformationwithin48hrat35EC.2.0SUMMARYOFMETHOD2.1Themultiple-tubefermentationtechniqueisathree-stageprocedureinwhichtheresultsarestatisticallyexpressedintermsoftheMostProbableNumb

3、er(MPN).Thesestages--thepresumptivestage,confirmedstage,andcompletedtest--arebrieflysummarizedbelow.(Fortheanalysistobeaccurate,afive-tubetestisrequired.)2.1.1PresumptiveStage:Aseriesoflauryltryptosebrothprimaryfermentationtubesareinoculatedwithgraduatedquantit

4、iesofthesampletobetested.Theinoculatedtubesareincubatedat35+0.5ECfor24+2hr,atwhichtimethetubesareexaminedforgasformation.Forthetubesinwhichnogasisformed,continueincubationandexamineforgasformationattheendof48+3hr.Formationofgasinanyamountwithin48+3hrisapositive

5、presumptivetest.2.1.2ConfirmedStage:Theconfirmedstageisusedonallprimaryfermentationtubesshowinggasformationduringthe24-hrand48-hrperiods.Fermentationtubescontainingbrilliantgreenlactosebilebrothareinoculatedwithmediumfromthetubesshowingapositiveresultinthepresu

6、mptivetest.Inoculationshouldbeperformedassoonaspossibleaftergasformationoccurs.Theinoculatedtubesareincubatedfor48+3hrat35+0.5EC.Formationofgasatanytimeinthetubeindicatesapositiveconfirmedtest.2.1.3CompletedTest:Thecompletedtestisperformedonallsamplesshowingapo

7、sitiveresultintheconfirmedtest.Itcanalsobeusedasaqualitycontrolmeasureon20%ofallsamplesanalyzed.Oneormoreplatesofeosinmethylenebluearestreakedwithsampletobeanalyzed.Thestreakedplatesareincubatedfor24+2hrat35+0.5EC.Afterincubation,transferoneormoretypicalcolonie

8、s(nucleated,withorwithoutmetallicsheen)toalauryltryptosebrothfermentationtubeandanutrientagarslant.Thefermentationtubesandagarslantsareincubatedat35+0.5ECfor24+2hr,orfor48+3