Light-Induced Gold Iridescence (Sunburn) of EPDM Weatherseals
Characterization of the iridescence and motility mutants ...
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Herrera, 1 Characterization of the iridescence and motility mutants in Tenacibaculum sp. strain ECSMB88. Nadia Herrera California Institute of Technology 2017 Microbial Diversity Course Miniproject report Introduction Since the establishment of the Tenacibaculum genus with the discovery of two distinct species in 2001, the number of species added to the genus has greatly increased to 23 individual species (1). The distinct Tenacibaculum species have been reported as isolates from many parts of the world—from China to Peru-- as parts of marine organisms, rocky shores, and recently, deep sea sediments (2-7). Tenacibaculum was initially identified as originating from marine sponges, but the genus was quickly populated by species which were being isolated from fish pathogens (3,4,6,7). It was not until more recently that a group started identifying a “glitter-like” iridescence in the Tenacibaculum genus, as originated by studies on Cellulophaga lytica, which later developed means for enrichment for iridescent Tenacibaculum isolates (2,8- 10). Iridescence, however, had not gone unnoticed and had been described in the literature in reports, as early as 1950 (11). A challenge in presenting iridescence instances in a bacterial isolate was accompanied by the lack of color photography which could be used in those reports. This resulted in varied means by which authors reported and described the characteristic of iridescence, and as such, it has remained a relatively understudied facet of microbiological physiology. Interestingly, aside from iridescence, bacteria which have this phenotype also utilize gliding motility. Gliding motility has been much less studied than flagellar motility, and as such remains another understudied facet of microbial physiology (12). In recent literature, both gliding and iridescence have been studied in Cellulophaga, Tenacibaculum, and Aquimarina (8). An iridescent Tenacibaculum isolate, corresponding to Tenacibaculum sp. strain ECSMB88, has been isolated by a student in the 2017 cohort of Microbial Diversity, Melissa Kardish from the Marine Biological Laboratory’s Stony Beach in Woods Hole, Massachusetts. Intriguingly enough, ECSMB88 was previously isolated from the East China Sea Biofilm by Yang, J.L., and Guo, X.P. in the Fisheries and Life Science institution in China, as stated in their deposition, KU982641, in the GenBank database. This iridescent Tenacibaculum isolate readily grows gliding colonies which show iridescence upon the first 12 hours of incubation at 30°C. As such, this particular isolate presents a model organism in which to study genes behind gliding and iridescence using observations on whole colonies. This report presents 7 mutants to which defects in gliding motility and iridescence were introduced by use of a chemical mutagenesis
Transcript of Characterization of the iridescence and motility mutants ...
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CharacterizationoftheiridescenceandmotilitymutantsinTenacibaculumsp.strainECSMB88.NadiaHerreraCaliforniaInstituteofTechnology2017MicrobialDiversityCourseMiniprojectreportIntroduction
SincetheestablishmentoftheTenacibaculumgenuswiththediscoveryoftwodistinctspeciesin2001,thenumberofspeciesaddedtothegenushasgreatlyincreasedto23individualspecies(1).ThedistinctTenacibaculumspecieshavebeenreportedasisolatesfrommanypartsoftheworld—fromChinatoPeru--aspartsofmarineorganisms,rockyshores,andrecently,deepseasediments(2-7).Tenacibaculumwasinitiallyidentifiedasoriginatingfrommarinesponges,butthegenuswasquicklypopulatedbyspecieswhichwerebeingisolatedfromfishpathogens(3,4,6,7).Itwasnotuntilmorerecentlythatagroupstartedidentifyinga“glitter-like”iridescenceintheTenacibaculumgenus,asoriginatedbystudiesonCellulophagalytica,whichlaterdevelopedmeansforenrichmentforiridescentTenacibaculumisolates(2,8-10).
Iridescence,however,hadnotgoneunnoticedandhadbeendescribedintheliteratureinreports,asearlyas1950(11).Achallengeinpresentingiridescenceinstancesinabacterialisolatewasaccompaniedbythelackofcolorphotographywhichcouldbeusedinthosereports.Thisresultedinvariedmeansbywhichauthorsreportedanddescribedthecharacteristicofiridescence,andassuch,ithasremainedarelativelyunderstudiedfacetofmicrobiologicalphysiology.Interestingly,asidefromiridescence,bacteriawhichhavethisphenotypealsoutilizeglidingmotility.Glidingmotilityhasbeenmuchlessstudiedthanflagellarmotility,andassuchremainsanotherunderstudiedfacetofmicrobialphysiology(12).Inrecentliterature,bothglidingandiridescencehavebeenstudiedinCellulophaga,Tenacibaculum,andAquimarina(8).
AniridescentTenacibaculumisolate,correspondingtoTenacibaculumsp.strainECSMB88,hasbeenisolatedbyastudentinthe2017cohortofMicrobialDiversity,MelissaKardishfromtheMarineBiologicalLaboratory’sStonyBeachinWoodsHole,Massachusetts.Intriguinglyenough,ECSMB88waspreviouslyisolatedfromtheEastChinaSeaBiofilmbyYang,J.L.,andGuo,X.P.intheFisheriesandLifeScienceinstitutioninChina,asstatedintheirdeposition,KU982641,intheGenBankdatabase.ThisiridescentTenacibaculumisolatereadilygrowsglidingcolonieswhichshowiridescenceuponthefirst12hoursofincubationat30°C.Assuch,thisparticularisolatepresentsamodelorganisminwhichtostudygenesbehindglidingandiridescenceusingobservationsonwholecolonies.Thisreportpresents7mutantstowhichdefectsinglidingmotilityandiridescencewereintroducedbyuseofachemicalmutagenesis
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screenusingMethylMethanesulfonatetomodifytheDNAinTenacibaculumsp.StrainECSMB88.--MaterialsandmethodsIsolationofTenacibaculumsp.StrainECSMB88 SamplesforisolationwerecollectedbyMelissaKardishfromStonyBeachinWoodsHole,Massachusettsat41.529°E,70.674°N,at8A.M.duringthelowtide.Aseagrassrootwascollectedfromtheeastendofthebeach,nexttothejetty.Aftercollection,theseagrassrootwasstoredinseawateruntilitwastransportedtothelab.Onceinthelab,thesamplewassubjectedtothroughmixingusingthevortex.TheresultingsupernatantwasseriallydilutedandplatedontoSeawatercompletemediumplatesandincubatedat30°C.Thesequenceofthesamplewasfoundbydoing16sPCRwithprimers8Fand1391R,asdescribedbefore(13).ThePCRproductwassubmittedforsequencingandtheresultwasalignedusingNCBIBLASTn.Aphylogenytreecomparingtherelationofthetop10hitsof16sRNAsequencingresultwasmadeusingaMUSCLEalignmentfollowedbyuseofMEGA7withdefaultparameterstogenerateatreebasedonmaximumlikelihoodbasedonpreviouswork(14,15).MMSMutagenesis Methylmethanesulfonate(MMS)wasacquiredfromSigma-Aldrich,andusedatafinalconcentrationof40uMinallreaction.10uLofaliquidovernightcultureofTenacibaculumsp.StrainECSMB88wasusedtoinoculatea5mLcultureinSeawatercompletemediumandwasgrownforaround7hoursoruntilitreachedtoanOD600nm=0.5-0.8,soastoobtaintheorganisminexponentialphase.Onceexponentialphasewasreached,a1mLaliquotofcellswasremovedfromthecultureandsubjectedtoafinalconcentrationof40uMMMS(4.44uLof99%MMSinto1mLofcells)foratotalof5minutesat30°C.Thecompletedreactionwascentrifugedusingatabletopcentrifugeoperatingat12,000RPMfor2minutes.Supernatantwasdiscardedandtheremainingcolonywasre-suspendedwithseawatercompletemedium.There-suspensionwasseriallydilutedupto10-8.Atotalof80plateswereusedforspreadingthecells,todistributethenumberofcellsattainedandscreeneverydilutionfortheemergenceofindividualcolonies.Todistributethecellsspread,100uLofinoculumwasspreadineachof20platesperdilution:10-5,10-6,10-7,and10-8.AnadditionalseawatercompletemediumplatewasinoculatedwithwildtypeTenacibaculumsp.StrainECSMB88,soastoanalyzewildtypecolonies.Aftera2-dayincubationperiodatroomtemperature(~20°C)colonieswereanalyzedandpickedforfurtheranalysis.Mutantselectionandplatinganalysis Initially,colonieswerepickedonthebasisofthreevisiblephenotypes,lossoriridescence,lossofmotility,orgainofmotility.Inthelattercase,thecolonywouldappearasmorediffusethanawildtypestandard,whereaslossofmotilitywouldbemanifestedinacolonywhichlosttheirregularedgesandformedalessmotileandcompactarrangement.Lossofiridescencewouldbeanalyzedbyvisuallyinspectingcolonieswhiletiltingtheplatetoallow
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lighttoshinethroughthecoloniesunderinspection.Fromtheinitialscreen,34colonieswerepicked,andstreakedforisolationtoverifythephenotype.Onceverified,6mutantcolonieswereusedforsubsequentanalysisonseawatercompletemediumsupplementedwith1%papermateink.Eachcolonywasplatedbypipetting2-5uLofeitherre-suspendedcolonyinseawatercompletemedium,orliquidculture.Theresilienceofthemutationwasfurtherobservedbyplatingthecellson½dilutedseawatercompletemediumplateswith1%,1.5%(sameasregularplates),2%,and2.5%agarconcentrations.Stereomicroscopy StereomicroscopyimagesofindividualcoloniesonplatesweretakenusingaZeissSteREODiscoveryV.8microscopesupplementedwithanexternallightboxforangledilluminationoftheiridescentcolonies.Spectroscopy
Asalightsource,weusedanOSRAMM11Halogenlightbulb8V/20W.ThelightenergywasdirectedinaZEISSmobilefiberandfocusedbyamicroscopecondenserlenswithanapertureof1.25underanangleof38°ontothehorizontallypositionedcolonyonaplate.Iridescentlightwascollectedintoaglassfibermountedatadistanceof1cmverticallyabovetheobjectandconnectedtoaSpectralEvolutionSR-1900SpectroradiometerSN15782C0.SpectrawererecordedandanalyzedbytheDARWINsoftwareofSpectralEvolution(1CanalStreet,UnitB1,Lawrence,MA01840).Lightmicroscopy Liquidmountslideswerepreparedshortlybeforeimaging.LightmicroscopyimagesofindividualcellsfromliquidcultureweretakenonaZeissAxioImager2microscope,using100xmagnificationandaphasecontrastplateforeaseofvisualization.Scanningelectronmicroscopy Wholecolonieswhichhadbeengrowingfor12hoursweredehydratedforscanningelectronmicroscopy(SEM)studiesutilizinganosmiumtetraoxideandglutaraldehydefixationmethod,followedbydehydrationwithethanolandhexamethyldisilazane(HMDS).Dehydratedcolonieswerethenmountedontoaspecimenstubusingdoublesidedcarbontape.A10nmcoatofplatinumwasappliedusingaLeicasputtercoater.Foradetailedprotocol,pleaseseetheendofthisreport.ImagingofthesampleswasdoneonaSUPRA™40VPFieldEmissionSEM,equippedwithGEMENIcolumntechnologysupplementedwiththeThermoNoranSystemSIXEDS.
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ResultsPhylogenytreebasedon16SrRNAsequencing
Thephylogenytree(Figure1)indicatedthatourisolatedhasacloserelationwithTenacibaculumspstrainECSMB88,andTenacibaculummesophiliumstrainHMG1.Duetothesimilarityoftheseresults,wehaveoptedforusingthenameofthefirsthitintheentiretyofthisreport.Assuch,theisolatedstrainisreferredtoasTenacibaculumspstrainECSMB88.
Figure1-Phylogenytreecalculatedfromthetoptenhitsof16srRNAsequencingofourisolate.Thephylogenytreedisplaysacomparisonof10hitsfromtheNCBIBLASTnresultsof16srRNAsequencingdataobtainedfromourisolate.Thescalebarindicatesthebranchdistancescale.
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Firstroundofmutantsselected Thefirstseriesofmutantswereselectedindividuallyfroman80platearray.Coloniesthatspreadoutrelativetoawild-typecomparisonwereconsideredtobegainofglidingmutantsand,andre-struckonseawatercompletemedium,asshowninblueboxesinfigure2.Ontheotherhand,colonieswhichappearedaslossofglidingmutantshadveryroundcolonieslackinganirregularedge,thesecolonieswerepickedandareshowninorangeboxesinfigure2.Finally,colonieswhichappearedtoloseiridescenceorchangeiridescencefromthewildtypegreen/yellowhuewerepickedforfurtheranalysis,andareshownasyellowboxesinfigure2.
Figure2-Stereomicroscopyimagesofmutantsselectedforanalysisbyre-streakingofasinglemutantcolony.Instancesofgainofgliding,lossofgliding,andlossofiridescencearehighlightedinblue,orange,andgoldboxes,respectively.Analysisofselectedmutantsfromfirstround Tounderstandthephysiologicalchangesbehindthedifferentphysicalobservationsatthecolonylevel,themutantsofinterestwereplatedonseawatercompletemediumsupplementedwithink.Theblackinkshowedthestrikingdifferenceiniridescenceasthecolonygrowsontotheplate,withgreeniridescencetypicallyresidinginthecenterofthecolonyandrediridescenceresidingontheedgesofthecolony(Figure3).Inourmutationsofinterest,thisiridescencewasonlychangedinmutants4and5,whichhadbeenhighlightedaslossofiridescencemutantsinthescreenillustratedinfigure2.Onceabasalphenotypewasobserved,thecolonieswereplatedonlow-nutrientmedium,½seawatercompletewiththesameamountofagarasisusedintypicalseawatercompletemediumplates,1.5%.Theseplates
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showedthatwithlowernutrients,theedgeoftheglidingcolonieswasspreadwidelywhilemaintainingacorrugatedshape,asseeinwildtype,andmutants1,2,4,and5(Figure3).
Ontheotherhand,mutantswhichhadbeenidentifyingashavingalossofglidingmotilitywerespreadout,buthadevenlycircularedgesasseeninmutants3and6(Figure3).Inaddition,itwasclearthatcolonies3and5hadstrikinglydifferentorganizationthantherest,highlightingacircularcolonysurroundedbyathinnerdefinedringandtaperedwithathinnerlayerofiridescentcells.Thisphenotypewasmuchreducedwhenanalyzingthesamecoloniesin½seawatercompleteand1%and2.5%agar(figures5-6).Astrikingobservationwasthatin½seawatercompletemediumwith1%agar,mostofthecoloniesspreadthoroughly,andevendisplayedlossofiridescenceinmutants2and5(figure5).Intriguingly,mutant3didnotspreadthoroughly,butinsteadremainedinasmallandcircularcolonyshape(figure5).Lastly,uponincreasingtheagarconcentration.In½seawatercompletemediumto2.5%,iridescencewasverydistinctfromtheotherconditions,andagreenhuewasobservedontheoutsideofwildtype,andmutants1-3,and5-6(Figure6).Ontheotherhand,themutantwhichhadbeenselectedforalossofiridescence,mutant4,waspigmentedcompletelydifferentlyanddisplayedverylittleiridescenceamidstaredhue(figure5).Collectively,theeffectofagarconcentrationandnutrientavailabilitygreatlyimpactshowthemutationsmanifestthemselves.Thisindicatesthatitiscriticalto“makenicewithyourbug”andplateitonmultiplemediainthepursuitofinvestigatingtheeffectsofamutation.
Figure3-Colonycomparisoninseawatercompletemedium,using1.5%agar.Wildtypeisdisplayedontheleft-handpanel,whereastheselectedmutantsareontheright-handpanel.Iridescentpropertiesarehighlightedbytheuseofblackinkontheplates,demonstratingthevariabilityofcolorsindifferentregionsofthecolony.
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Figure4-Colonycomparisoninseawatercomplete,using½concertationofmediumnutrientswhilemaintainingtherigidityoftheagarfromregularseawatercompleteplates.Theseplateshighlightadditionalglidinginallofthemutantsanalyzed,withdefinedchangesincolonymorphologyinmutants3and6.
Figure5-Colonycomparisoninseawatercomplete,using½concentrationofnutrientsinmediumand1%agar.Inthismedium,colonieswhichwerecharacterizedasgainofglidingweremuchspread,whereascolony3wasfurtherestablishedasalossofmotilitycolony.Theiridescenthueinthecoloniesbecomesdiminishedinwildtype,andmutants1-2,4-5.
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Figure6-Colonycomparisoninseawatercomplete,using½concentrationofnutrientsinmediumand2.5%agar.Inthismedium,ahardersurfacepreventsmostcoloniesfromglidingfar.Inaddition,iridescenceismanifestedasagreenpigmentalongtheedgesofmostcolonies,whereasthelossofiridescenceisprominentinmutant4.Analysisofcellmorphologyinindividualcolonies Lightmicroscopyoncellsobtainedfromliquidcultureofvaryingmutantsshowedthattheyallcontainedamixedpopulationofrodswithvaryinglengths,ashighlightedinFigure7.Inthisanalysis,itwasevidentthatamixedpopulationofrodswashomogeneousamidstallcells,withwildtype,andmutants2,4and5displayingbothaninstanceofalongrodandaformationofcellularorganizationonaglassslide.Interestingly,mutants1-3,and6onlysharedoneofthosecharacteristicswithwildtype.Sincecellularorganizationappearstobeveryvariedamidstthemutants,itwasimperativetoanalyzetheirorganizationatthewholecolonylevel,soastounderstandcellularorganizationatthecolonylevel.
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Figure7-Lightmicroscopyimagesofthemutantsselectedforanalysis.Elongatedcellshapesarehighlightedusingredarrows,whereaseventsthatappeartobeearlycellularorganizationarehighlightedusingyellowarrows.Ingeneral,mostmutantsshareeitheroneorbothofthesecharacteristicswithwildtype.SEManalysisofcoloniesandmutants SEManalysisofourcoloniesallowedfortheinvestigationofsubcellularorganizationintheentiretyofacolony.InpreparationforSEM,itwasnotedthattheiridescentpropertiesofthecoloniesappearedtosurviveddehydration,asshowninfigure8.OnceanalyzedwithSEM,immediatecolonyorganizationwasnotevidentinanyportionofthecolonyforanyofthemutationsobserved.Ahomogeneousmixtureofcellsizeincolonyformationwasobservedinwildtypeandmutants1,2,4-6(Figure9).Thisresultlikelyexplainstheglitter-likeiridescenceobservedincoloniesonagar,astheiridescenceislikelytheresultofasmallgroupofcoloniesforminganorderedarrayamidstanarrayofcellsinagivencolony.Interestingly,ourlossofiridescencemutantdisplayedanorderedarrayofcellswhichseemedtohaveaperiodicstructurewithinindividualclustersandwithincellsthemselves(Figure9).Thisresultisindicativeofastructuraldifferencecorrelatedwithachangeofpigmentinacolony.Ontheotherhand,thecellsinmutant3appeartoreleaseamaterialwhichislikelyexopolysaccharideusedinbiofilmformation.Characterizationofiridescentpropertiesinthesemutants,isthereforeofinterestforfurtherdifferentiation.
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Figure8-SEMcolonydehydrationafterfixation.Huesofpinkandgreenonthecolonysurfaceappeartomanifestaretentionofcolonyiridescencethroughtheprocedureofdehydration.Colonysizewasreducedtoabout1/10oftheoriginalsizeandcoloniesbecamecorrugatedfromdehydrationafterpreparation.
Figure9-SEMofcolonies.SEMonindividualcoloniesshowedamesh-likestructureofcellsofvaryingsizesonthecolony,regardlessoflocationofimagingonthecolony.Mutant3showedaparticularlydensebedofextracellularmaterialaccumulationwhichislikelyexopolysaccharides.SpectroscopiccharacterizationofmutantsSpectroscopystudiesonindividualcolonies Spectroscopymeasurementsweretakenonindividualcoloniesgrownonseawatercompleteplatesinblackagar.Theseplatesprovidedabaseforreflectionmeasurementsoffoftheiridescenceofthecolony.Weobservedthatwildtypehasstrongestreflectionsat540nm,whichisrightinthegreenzoneofthelightspectrumandinaccordancewiththecolorthatweseebyeye.Thispeakisshiftedinmostmutants,butthemoststrikingchangeisinmutant4,
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wherethepeakmovestothe582nmrange,whichisclosertotheyellowspectrumanddifferentiatesthiscolonyashavingdifferentspectroscopicreflections.Ourresultssuggestthatthisprocedureofmeasuringiridescenceisawaytoprovidequantificationofdifferencesobservediniridescence,andcouldbeawaytotrulydiscernbetweendifferentiridescencetypes.
Figure10-Spectroscopyonindividualcoloniesgrownonseawatercompleteagarplatessupplementedwithink.Discussion
Wehaveisolatedcoloniesthathavedefectsinglidingabilityandiridescentcolor.Studyingthegeneticbasisbehindthiswillshedlightontwofacetsofbacterialphysiologywhicharenotwellunderstoodtodate.Thoughitisinitiallyobservedthatcoloniesformlittleorder,theyappeartoretainiridescenceafterdesiccation.Inaddition,theiriridescentpropertiesseemtoremainunchangedafterdehydration.Inordertofurtheranalyzecolonyorganizationatthecellularlevel,itwouldbenecessarytoimageagrowingcolonyusinganinvertedmicroscopesetup,orstudyingawholecolonyusingtransmissionelectronmicroscopy,whichcouldshedlightoncellularorganizationattheultrastructurallevel.
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Thisorganismhasalotofinterestingfeatureswhicharetraceable,andmanycharacteristicsandpropertieslefttoexplore.Forinstance,throughoutthistime,itisdifficulttousecentrifugationat3400RPMtopelleta50mLmassofcells,evenafterextendedspinning.Itislikelythatthesecellsarebeingkeptinsuspensionbytheirproductionofexopolysaccharideinthegrowthmediumoncetheyreachlagphase,asthisisnotobservedincellsgrowntoexponentialphase.Infact,studieshavebeendonewhereafoambeadisseenglidingalongacellinliquidmedium,soastosuggestthattheirglidingmotilityisactivewhilegrowingonliquidmedium,despitethelackofasurfacetoglideon(16).
Inordertobetter-understandthesephenotypicmutations,itessentialtosequencethegenomeofthecoloniesofinterest,soastostudythegeneswhicharebeingmutatedtocreatetheeffectsseen.Outofthemutantspresentedinthisstudy,therearethreedistinctcoloniesthatshouldbestudiedtostarttounderstandpatternsofchange,andthosearewildtype,alossofmotilitymutant3,andalossofiridescentmutant5(Figure11).Onceweareabletomapthemutationstothegenome,complementationstudieswillallowustoestablishthegenesbehindthemutationsobserved.Fortunately,inthelastyearageneticsystemhasbeenestablishedforasimilartypeofbacteriawhichcanbeusedforcomplementationstudies(17).Ultimately,theseconstructscanprovetobethebeginningofaninvestigationtowardstwobacterialphenotypeswhichhaveyettobethoroughlyunderstood.
Figure11-Wholegenomesequencecandidates,colonyandSEMimages.Amidstallofthemutationsinthisstudy,thesethreecandidateswouldallowforinitialunderstandingofthemachinerybehindmotility.
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References1. Suzuki,M.,Nakagawa,Y.,Harayama,S.,andYamamoto,S.(2001)Phylogeneticanalysis
andtaxonomicstudyofmarineCytophaga-likebacteria:proposalforTenacibaculumgen.nov.withTenacibaculummaritimumcomb.nov.andTenacibaculumovolyticumcomb.nov.,anddescriptionofTenacibaculummesophilumsp.nov.andTenacibaculumamylolyticumsp.nov.IntJSystEvolMicrobiol51,1639-1652
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SupplementalinformationProtocolforSEMdehydrationandfixationofabacterialcolony
1- Usingarazorblade,cutoutthecolonyfromanagarplate.2- Scoopthecolonyontothelidofa1cmdiameterpetridish3- Add500uLoffixativeagent,Osmiumtetraoxideinwaterat1%concentrationtothe
bottomofthepetridishandspreadthroughoutplateevenly.4- Carefullyplacethecolonyovertheliquidinthebottomoftheplate,andadd300uLof
additional1%osmiumtetraoxidesoastoformameniscusaroundthecolony.Donotre-suspendcolony.
5- Allowfixationtoproceedfor1houratroomtemperature,coveryourdish.6- Pipetteoutthefixative,andreplacewith800uLof3.5%glutaraldehydein0.1M
cacodylatebuffer.7- Allowfixationtoproceedfor2hoursatroomtemperature,coveryourdish.8- Pipetteoutthefixativeandreplacewith800uLof0.1MCacodylatebuffer.9- Allowwashingtoproceedfor15minutes,andrepeatstep8foranadditional15
minutes.10- Atthispoint,yourcolonyshouldbedarkened.Pipetteoutwashingsolutionandadd800
uLof1%Osmiumtetraoxidein0.1Mcacodyltebuffer.11- Allfixationtoproceedforanhouratroomtemperature,coveryourdish.12- Removesupernatantandadd800uLofdistilledwater.13- Allowwashingtoproceedfor30minutes,andrepeatstep12foranadditional30
minutes.14- Todryyoursample,pipettegently,useanelectricpipettethatwillallowfordispensing
atlowspeed.Pipetteoutthewaterandreplacewith4mLof50%ethanol.15- Allowdryingtoproceedfor15minutes.16- Pipetteoutethanol,andreplacewith4mLof70%ethanol.17- Allowdryingtoproceedfor15minutes.18- Repeatsteps16-17with80%,90%,95%,and100%ethanol(twicefor100%).19- Afterfinalstep,removeethanolandreplacewith4mLofa1:2solutionof
Hexamethyldisilazane(HMDS):Ethanol.20- Allowdryingtocontinuefor20minutes.21- PipetteoutfirstsolutionofHMDS:Ethanolandreplacewith4mLofa2:1HMDS:Ethanol
solution.
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22- Alldryingtooccurfor20minutes.23- Repeatspets21and22with100%HMDS.24- Onceyoureachyourfinalstepof100%HMDS,pipetteoutliquidcarefully,andallow
colonytodryslowlybyleavingthecoveronthepetridishovernight.Oncedrythecolonieswillbebrittleandmuchreducedinsize.
