Standard-Methods-for-the-Examination-of-Water-and-

StandardMethodsfortheExaminationofWaterandWastewater©Copyright1999byAmericanPublicHealthAssociation,AmericanWaterWorksAssociation,WaterEnvironmentFederationPart 9000MICROBIOLOGICALEXAMINATION9010INTRODUCTION*#(1)Thefollowingsectionsdescribeproceduresformakingmicrobiologicalexaminationsofwatersamplestodeterminesanitaryquality.Themethodsareintendedtoindicatethedegreeofcontaminationwithwastes.Theyarethebesttechniquescurrentlyavailable;however,theirlimitationsmustbeunderstoodthoroughly.Testsfordetectionandenumerationofindicatororganisms,ratherthanofpathogens,areused.Thecoliformgroupofbacteria,ashereindefined,istheprincipalindicatorofsuitabilityofawaterfordomestic,industrial,orotheruses.Theculturalreactionsandcharacteristicsofthisgroupofbacteriahavebeenstudiedextensively.Experiencehasestablishedthesignificanceofcoliformgroupdensityasacriterionofthedegreeofpollutionandthusofsanitaryquality.Thesignificanceofthetestsandtheinterpretationofresultsarewellauthenticatedandhavebeenusedasabasisforstandardsofbacteriologicalqualityofwatersupplies.Themembranefiltertechnique,whichinvolvesadirectplatingfordetectionandestimationofcoliformdensities,isaseffectiveasthemultiple-tubefermentationtestfordetectingbacteriaofthecoliformgroup.Modificationofproceduraldetails,particularlyoftheculturemedium,hasmadetheresultscomparablewiththosegivenbythemultiple-tubefermentationprocedure.Althoughtherearelimitationsintheapplicationofthemembranefiltertechnique,itisequivalentwhenusedwithstrictadherencetotheselimitationsandtothespecifiedtechnicaldetails.Thus,twostandardmethodsarepresentedforthedetectionandenumerationofbacteriaofthecoliformgroup.Itiscustomarytoreportresultsofthecoliformtestbythemultiple-tubefermentationprocedureasaMostProbableNumber(MPN)index.Thisisanindexofthenumberofcoliformbacteriathat,moreprobablythananyothernumber,wouldgivetheresultsshownbythelaboratoryexamination;itisnotanactualenumeration.Bycontrast,directplatingmethodssuchasthemembranefilterprocedurepermitadirectcountofcoliformcolonies.InbothprocedurescoliformdensityisreportedconventionallyastheMPNormembranefiltercountper100mL.Useofeitherprocedurepermitsappraisingthesanitaryqualityofwaterandtheeffectivenessoftreatmentprocesses.Becauseitisnotnecessarytoprovideaquantitativeassessmentofcoliformbacteriaforallsamples,aqualitative,presence-absencetestisincluded.Fecalstreptococciandenterococcialsoareindicatorsoffecalpollutionandmethodsfortheirdetectionandenumerationaregiven.Amultiple-tubedilutionandamembranefilterprocedureareincluded.StandardMethodsfortheExaminationofWaterandWastewater©Copyright1999byAmericanPublicHealthAssociation,AmericanWaterWorksAssociation,WaterEnvironmentFederationMethodsforthedifferentiationofthecoliformgroupareincluded.Suchdifferentiationgenerallyisconsideredoflimitedvalueinassessingdrinkingwaterqualitybecausethepresenceofanycoliformbacteriarendersthewaterpotentiallyunsatisfactoryandunsafe.Speciationmayprovideinformationoncolonizationofadistributionsystemandfurtherconfirmthevalidityofcoliformresults.Coliformgroupbacteriapresentinthegutandfecesofwarm-bloodedanimalsgenerallyincludeorganismscapableofproducinggasfromlactoseinasuitableculturemediumat44.5±0.2°C.Inasmuchascoliformorganismsfromothersourcesoftencannotproducegasundertheseconditions,thiscriterionisusedtodefinethefecalcomponentofthecoliformgroup.Boththemultiple-tubedilutiontechniqueandthemembranefilterprocedurehavebeenmodifiedtoincorporateincubationinconfirmatorytestsat44.5°Ctoprovideestimatesofthedensityoffecalorganisms,asdefined.ProceduresforfecalcoliformsandEscherichiacoliincludea24-hmultiple-tubetestusingA-1medium,a7-hrapidmethod,andchromogenicsubstratecoliformtests.Thisdifferentiationyieldsvaluableinformationconcerningthepossiblesourceofpollutioninwater,andespeciallyitsremoteness,becausethenonfecalmembersofthecoliformgroupmaybeexpectedtosurvivelongerthanthefecalmembersintheunfavorableenvironmentprovidedbythewater.Theheterotrophicplatecountmaybedeterminedbypourplate,spreadplate,ormembranefiltermethod.Itprovidesanapproximateenumerationoftotalnumbersofviablebacteriathatmayyieldusefulinformationaboutwaterqualityandmayprovidesupportingdataonthesignificanceofcoliformtestresults.Theheterotrophicplatecountisusefulinjudgingtheefficiencyofvarioustreatmentprocessesandmayhavesignificantapplicationasanin-plantcontroltest.Italsoisvaluableforcheckingqualityoffinishedwaterinadistributionsystemasanindicatorofmicrobialregrowthandsedimentbuildupinslow-flowsectionsanddeadends.Experienceintheshipmentofun-icedsamplesbymailindicatesthatnoticeablechangesmayoccurintypeornumbersofbacteriaduringsuchshipmentforevenlimitedperiodsoftime.Therefore,refrigerationduringtransportationisrecommendedtominimizechanges,particularlywhenambientairtemperatureexceeds13°C.Proceduresfortheisolationofcertainpathogenicbacteriaandprotozoaarepresented.Theseproceduresaretediousandcomplicatedandarenotrecommendedforroutineuse.Likewise,tentativeproceduresforentericvirusesareincludedbuttheirroutineuseisnotadvocated.Examinationofroutinebacteriologica