NationalCommitteeforClinicalLaboratoryStandards.2003.Methodsfordilutionantimicrobialsusceptibilitytestsforbacteriathatgrowaerobically,5thed.ApprovedstandardM7–A6.NationalCommitteeforClinicalLaboratoryStandards,Wayne,Pa.MICDETERMINATIONBYMICROTITREBROTHDILUTIONMETHOD(NB.ForcationicantimicrobialpeptidesusethemodifiedMICmethod)REFERENCE:Amsterdam,D.1996.Susceptibilitytestingofantimicrobialsinliquidmedia,p.52-111.InLoman,V.,ed.Antibioticsinlaboratorymedicine,4thed.WilliamsandWilkins,Baltimore,MD.MATERIALS:•liquidculturesofbacteriaatsuitablegrowthphase•sterilepetridishes•sterile96-wellmicrotitreplates(round-bottomwellsarebest)•filtersterilizedantibiotics•sterilediluents•testtubes.METHOD:1.GrowtheteststrainsinthechosenmediumtotherightA600.Haveantibioticsolutionsandplatesreadybeforetheculturesreachthedesiredgrowthphase.2.Thawandweightheantibiotics.Takeanoteofthepurityatthisstage,e.g.gentamicin,577µg/mgsolid.Dissolvetheantibiotics(solventdependsonthecompound),thendiluteinthetestmediumto2xthetopconcentrationdesiredinthetest,e.g.ifhighestdesiredconcentrationis128µg/ml,diluteto256µg/ml.Keeponiceuntiluse.3.Usingthemultipipettor,dispense100µlofmediumintoallwellsofamicrotitreplate.Labeltheplateandlid.4.Pipette100µlofappropriate2xantibioticsolutionsintothewellsincolumn1(farleftofplate).5.Usingthemultipipettorsetat100µl,mixtheantibioticsintothewellsincolumn1bysuckingupanddown6-8times.Donotsplash.6.Withdraw100ulfromcolumn1andaddthistocolumn2.Thismakescolumn2atwofolddilutionofcolumn1,e.g.fortheexampleabovethiswouldbe64µg/ml.Mixupanddown6-8times.Transfer100µltocolumn3.Repeattheproceduredowntocolumn10only.Thesamesetoftipscanbeusedfortheentiredilutionseries.7.Discard100µlfromcolumn10ratherthanputtingitincolumn11.8.PourbacteriaoftherightA600intoasterilepetridish.Thebacteriamaybedilutedfirstdependingonthedesiredinoculum.TheappropriateinoculumsizeforstandardMICis2x104to105CFU/ml.9.Withthesmallermultipipettorsetto5µl,dispensebacteriaintowellsincolumns11to1inthatorder.Donotaddbacteriatocolumn12(sterilitycontrolandblankfortheplatescanner).10.Incubatetheplatesat37oCorotherdesiredtemperature.11.Streakthebacterialculturesonplatestochecktheirpurity.12.Whensatisfactorygrowthisobtained(18-36hours)scantheplateswithanELISAreader(orreadbyeye).Usecolumn12astheblank(thismeansputtingtheplateinback-to-front).13.MICcanbetakenasthelowestconcentrationofdrugthatreduces,bymorethan50%or90%forMIC50orMIC90respectively.NOTE:Putdifferentdrugsondifferentrowsofthesameplate,buttrytoavoidputtingbacteriatogether,topreventcross-contamination