JournalofVisualizedExperimentswww.jove.comCopyright©2011JournalofVisualizedExperimentsSeptember2011|55|e2855|Page1of5VideoArticleBromodeoxyuridine(BrdU)LabelingandSubsequentFluorescenceActivatedCellSortingforCulture-independentIdentificationofDissolvedOrganicCarbon-degradingBacterioplanktonStevenRobbins*1,JishaJacob*1,XinxinLu1,MaryAnnMoran2,XiaozhenMou11BiologicalSciences,KentStateUniversity2MarineSciences,UniversityofGeorgia(UGA)*TheseauthorscontributedequallyCorrespondenceto:XiaozhenMouatxmou@kent.eduURL:http://www.jove.com/video/2855DOI:doi:10.3791/2855Keywords:MolecularBiology,Issue55,BrdUincorporation,fluorescence-activatedcellsorting,FACS,flowcytometry,microbialcommunity,culture-independent,bacterioplanktonDatePublished:9/10/2011Citation:Robbins,S.,Jacob,J.,Lu,X.,Moran,M.A.,Mou,X.Bromodeoxyuridine(BrdU)LabelingandSubsequentFluorescenceActivatedCellSortingforCulture-independentIdentificationofDissolvedOrganicCarbon-degradingBacterioplankton.J.Vis.Exp.(55),e2855,doi:10.3791/2855(2011).AbstractMicrobesaremajoragentsmediatingthedegradationofnumerousdissolvedorganiccarbon(DOC)substratesinaquaticenvironments.However,identificationofbacterialtaxathattransformspecificpoolsofDOCinnatureposesatechnicalchallenge.Herewedescribeanapproachthatcouplesbromodeoxyuridine(BrdU)incorporation,fluorescenceactivatedcellsorting(FACS),and16SrRNAgene-basedmolecularanalysisthatallowsculture-independentidentificationofbacterioplanktoncapableofdegradingaspecificDOCcompoundinaquaticenvironments.TriplicatebacterioplanktonmicrocosmsaresetuptoreceivebothBrdUandamodelDOCcompound(DOCamendments),oronlyBrdU(no-additioncontrol).BrdUsubstitutesthepositionsofthymidineinnewlysynthesizedbacterialDNAandBrdU-labeledDNAcanbereadilyimmunodetected1,2.Througha24-hrincubation,bacterioplanktonthatareabletousetheaddedDOCcompoundareexpectedtobeselectivelyactivated,andthereforehavehigherlevelsofBrdUincorporation(HIcells)thannon-responsivecellsintheDOCamendmentsandcellsinno-additioncontrols(lowBrdUincorporat...
