TheColiformGroupII.ReactionsinECMediumat45CE.E.GELDREICH,H.F.CLARK,P.W.KABLER,C.B.HUFF,ANDR.H.BORDNERRobertA.TaftSanitaryEngineeringCenter,PublicHealthService,DepartmentofHealth,Education,andWelfare,Cincinnati,OhioReceivedforpublicationMarch13,1958Escherichiacolihasbeensuggestedbyseveralre-searchworkersasamoreaccurateindicatoroffecalcontaminationthanthemoreinclusive"coliformgroup."MediaandproceduresfortheenumerationofE.colihavebeenproposedbyEijkman(1904),Leiter(1929),PerryandHajna(1933,1944),HajnaandPerry(1939,1943),Vaughnetal.(1951),andLevineetal.(1955).Someoftheseproceduresapparentlyyieldsatisfactoryresultswithcertainsamplesofwater,milk,orfoodproducts;however,thereisnouniversalagreementastotheircriteriaofapplicationandsig-nificanceofresultsinwaterbacteriology.TherearealsouncertaintiesregardingthesignificanceofE.coliwhenassayedbyrapidtestsascomparedtothetradi-tionalIMViCclassification.Thisinvestigationwasundertakentodeterminethereactionsof12IMViCtypesofcoliformbacteriainECmediumafter24hrincubationat45C.ItisbelievedtheresultantdatawillbeofvalueinchoosingtheprocedureandintheinterpretationoftestresultsforE.coliandthecoliformgroup.MATERIALSANDMETHODSThecoliformgroupofbacteriaforthisinvestigationwasdefinedasaerobicandfacultativeanaerobicgram-negative,nonsporeformingbacteriawhichfermentedlactosewithgasformationin48hr,orless,whenincu-batedat35C(StandardMethodsfortheExaminationofWater,Sewage,andIndustrialWastes.APHA,1955a).Allofthecultureswererecentlyisolatedfromsurfacewatersamplesobtainedfromvariouslocationsandwithvaryingdegreesofpollution(Clarketal.,1957).ProceduresdescribedastentativemethodsinStandardMethodsfortheExaminationofWater,Sewage,andIndustrialWastes(APHA,1955b)wereusedforindole,methylred,andVoges-Proskauertests,andcitrateagarofSimmons(1926)wasusedforthecitratetest.TheECmediumofHajnaandPerry(1943)wasusedasaconfirmatorytestforthevariouscoliformbacteria.Itwasreconstitutedfromthedehydratedproduct,tubedwithaninvertedvialfortheobservationofgasproductionandsterilizedinanautoclaveat121Cfor15min.Eachcultureofthecoliformbacteriawasexaminedforindoleandacetylmethylcarbinolproduction,methylredreaction,utilizationofcitrate,andgasformationinECmediumin24hrat45.0Cinaconstanttemperaturewaterbath.InreadingtheECtubes,thepresenceofgasinanyquantitywasconsideredaposi-tivereactionandabsenceofgas(in24hrincubation)wasanegativereaction.TheprocedureusedinthisstudydifferedfromtherecommendationsofPerryandHajna(1944)inthattheysuggestedairincubationat45.5Cfor48hr.RESULTSTherewere5794culturesofcoliformbacteriafromuntreatedsurfacewatersuppliesof14treatmentplantsexamined.Ofthe1358E.coliincluded,83.7percentshowedpositiveECreactions.WhentheE.colistrainsaresubdividedintovarietiesIandII(seetable1),thepercentageofpositiveresultswere92.7and21.8,respec-tively.Theremaining10IMViCtypesyielded7.8percentpositivetestsfor4436cultures.Thedatasumma-TABLE1ReactionsofcommoncoliformIMViCtypesinECmediumat45.0Cin24hrCommonlyDesignaedSource*FecalNonfecalIMViCTypePositiveECPositiveECNo.reactionsNo.reactionsculturesculturesexaminedNum-Per-examinedNum-Per-bercentagebercentage1184109892.71743821.8+++-796177.2++-+4255212.2168617910.62414.2+-++23783.4-+++485163.3++++453102.25012.0989181.882-Total...........1358113683.744363487.8*AccordingtoStandardMethodsfortheExaminationofWater,Sewage,andIndustrialWastes(10thedition,1955),page391,Table22-ColiformGroup-ReactionClassification.347GELDREICH,CLARK,KABLER,HUFF,ANDBORDNERrizedintable1alsoshowthattype+++-gave77.2percentpositivereactions.DISCUSSIONWith92.7percentoftheE.colivarietyI(++--)showingpositiveECreactions,thereisrelativelygoodagreementintheclassificationofthisorganism.How-ever,only21.8percentoftheso-calledE.colivarietyII(-+--)reactedpositivelyinECmedium,thusreducingthecorrelationforthetwoE.colivarietiescollectivelyto83.7percent.TheIMViCtype+++-comprisedonlyasmallpartoftheculturesstudied,butshowed77.2percentpositiveECtests.Thereac-tionsofE.colivarietyIIandof+±+-typetotheECtestat45Csuggesttheneedforfurtherwvorktodeterminethefecalornonfecaloriginoftheseorgan-isms.ThelatterappearstobemorecloselyrelatedtoE.colivarietyIonthebasisofgrowthwithproductionofgasat45CthandoestheindolenegativevarietyII.TheseresultsarecomparabletothosereportedbyClarketal.(1957),usingaboricacidlactosebrothreactionat43C.BoththeboricacidlactosebrothandECproceduresseemtohaveapproximatelythesamecapabilitiesindifferentiatingthecoliformgroup.Itisnotthepurposeofthisstudytoestablishthevalueofanysingletypeofcoliformbacteriaasasani-taryindicatoroffecalpollution.However,theresultsasreportedstronglysuggestthataddedeffortsshouldbemadetolocatesourcesoffecalpollutionofwaterscontainingECpositivecoliforms.Becauseotherbio-chemicalcharacteristicsmayshowbettercorrelationswithfecalornonfecaloriginsofthecoliformgroup,furtherconsiderationshouldbegivenitotheuseofothermeasurementsinadditiontotheconventionalIMViCreactionsandgrowthatelevatedtemperatures.SUMMARYColiformculturesof12IMViCtypeswereincubatedinECmediuminawaterbathfor24hrat45.0C(±t0.3).Positivetestswererecordedfor92.7percentofEscherichiacolivarietyI,21.8percentofE.colivarietyII,and83.7percentwhenconsideredtogetherascoliformbacteriacommonlydesignatedasoffecalorigin.Thenonfecaltypesofcoliformculturesshowed7.8percentpositivetests.Ahighpercentageof+++-strainsgrewat45.0C,suggestingacloserelationshiptotheE.colivarietyI.SourcesofsamplescontainingpositiveECcoliformsshouldbecarefullyinspectedforpossiblefecalcon-tamination.TheECbrothprocedureasdescribedappearssuitablefortherapidenumerationofE.coliintheinvestigationoffieldproblemsandotherkindsofcontrolwork.REFERENCESAmericanPublicHealthAssociation1955aStandardmethodsfortheexaminationofwater,sewage,andindustrialwastes,10thed.,375-387.AmericanPublicHealthAssociation1955bStandardmethodsfortheexaminationofwater,sewage,andindustrialwastes,10thed.,390-393.CLARK,H.F.,GELDREICH,E.E.,KABLER,P.W.,BORDNER,R.H.,ANDHUFF,C.B.1957Thecoliformgroup.I.TheboricacidlactosebrothreactionofcoliformIMViCtypes.Appl.Microbiol.,5,396-400.EIJKMAN,C.1904Diegarungsprobebei460alshilfsmittelbeidertrinkwasseruntersuchung.Centr.Bakteriol.Parasitenk.,Abt.I,orig.,37,742-752.HAJNA,A.A.ANDPERRY,C.A.1939OptimumtemperaturlefordifferentiationofEscherichiacolifromothercoliformbacteria.J.Bacteriol.,38,275-283.HAJNA,A.A.ANDPERRY,C.A.1943Comparativestuidyofpresumptiveandconfirmatorymediaforbacteriaofthecoliformgroupandforfecalstreptococci.Am.J.PublicHealth,33,550-556.LEITER,L.W.1929TheEijkmanfermentationtestasanaidinthedetectionoffecalorganismsinwater.Am.J.Hyg.,9,705-734.LEVINE,M.,TANI
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