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Simplified heavy metal staining techniques demonstrated with Fast Plant leaf tissue

By Eva Rice,2014-09-22 19:13
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Simplified heavy metal staining techniques demonstrated with Fast Plant leaf tissueheavy,metal,with,Fast,Plant,leaf,fast,plant,Heavy,Metal

Simplified heavy metal staining techniques

    demonstrated with Fast Plant leaf tissue

Simplified

    ;FastPlant

    ;CellRe~arzh(1989.9g

    ;heavymeta1stainingtechniquesdemonstratedwith ;leaftissue

    ;INTRoDUCTION

    ;tIARRISJOSEPHB..TEOMASG.GUILLAMSANDJEFFERY

    ;A.8aHULTZ

    ;BiologyDepart,ment,UniversityofWisconsinStevens

    ;Point,StevensPoint,WI54481U8A.

    ;ABSTRACT

    ;Fa8LbPlant(srapa,Gruciferae)leaftjssue

    ;fixedinglutaraldehydeacroleinandpostfixedino8

    ;mium,wasexaminedforrespons3toseveralOaS_ily- ;preparedheavymetalstains.Lcadanduranium, ;separatelyandincombinat]on,gavetypicalresults ;acrossthespectrumofcellorganelles.Asasinglestain ;followingosn]ium,bJsmuthproducedimagesseemingly

;equivalenttoleadanduranium.Phosphotungshieacid

    ;producedverygoodmembranedelineationbutproduced

    ;awashed——outbackgroundimagosimilartothatfromlead

    ;staining.Carbohydratecompoundswereespecially

    ;responsivetoruthenium;thecytoplasmandthematrix

    ;ofallorganellesworealsosSainedvery..

    ;wellThe

    ;procedureswerenomoredemandJngthantraditional

    ;stainingmethodsandmaybeeasilyusedinresearchand

    ;teaching.FastPlantmaterialsarearelJable.quiekand

    ;easySOUrCeoflivingmateria1.

    ;Keywords:FastPta~tBt6ssuo,sheavy伽此

    ;sta6n6ngteoh~z$que.

    ;AlternativestainingmethodsintransmissionelectronmicroscopyWhieh ;providedifierentjnformationareofwidevalueinbothre.searchandteaching. ;Methodsdevelopmen~inelectronmloroscopyhave,inmostlaboratories,invoived

    ;variationsspecificallySUitedforthesubject.Thisreportdescribesthjresultsof

    ;usingseveralstaJningmethodsonultra~hinsootisnswhjohhadboonpreparedby

    ;theapplicationoftraditionalfixa$ionandembeddingprocedures.EaSOof

;preparationanduseofsta;nSwasanobjective.

    ;FastPlantgeneticstockwasorginallysoleetedfrompopulationsofBrasg~a ;.rapa(Oruelferae,mustardfamily).Theadvantageoftheselectionwastheshort

    ;

    ;8imphfiedheavyme~alshuiningtcehniques

    ;lifocyclewhichwasfiveweeksfromseedtoseed[1].Tissueffomsthe8ep

    alIt8

    ;havebeenshowntobeusedomsilyforultrastructuralstudioswithtraditjona1 ;methods[2].ThisreportdescribestheresponseofFastPlantleaftissue88evera1

    ;simplestainingprocedures.

    ;Itwasconsideredimportanttousestandardfixationproceduressoastobeth ;introduceminimummethodologicalencumbranceandallowforeaseincomparison

    ;withfamilJarimages.

    ;MATERIALSANDMETH0DS

    ;Fization,Embedding,Sectioning.

    ;FastPlantseedlingsweregrownonfilterpaperinfingerbowlstoausefulsizewithin60hoursatroom

    ;temperaturesandordinaryl’ghting.Wholeleafletsorotherplantparts.werecutanddroppedintovials

    ;filledto1/[5]volumewith3%glutaralJehydeand3%acroleinin0.02MsodiumeacodylateburetatpH7.4

    ;andO

    3.a(3).Specimensweresubmergedunderastainlesssteelscreenandvacuumwasappliedandreleased

    ;untilairwasremovedandinfiltrationcomplete.Fixationwascontinuedfortwohours,followedbytwohours

    ;ofrinsingin0.02Mcao]ylatebuffer(8

    lOchanges),twohourspostfixingin2%osmiumtetroxidein0.O2 ;eacodylatebuffer,andfourrinsesinglassdistilledwater.Dehydrationinan

    acetoneseriesf3rinseseach)

    ;wasfollowedbyaaditionofanequalvolumeofSpurt’sresinmix(4)tothethirdchangeof100%acetone.

    ;Specimensweremixed

    ?.el1,allowedtostandfor30minutesandasecondequalvolumeofresinwasadded.

    ;Afterthoroughra~xing,andstandingfor30minutestheresinwaspouredoff,tissuesweretransferredtO

    ;100%resin,mixedandinfiltratedovernight.Subsequentlytissuestripswerecutinto1mmsquares.

    ;transferredtoplasb:cmolds,themoldsfilledandlabeled,andtheresinpolymerizedforeighthoursina70~0

;vacuumoven.TissuesweresectionedwithaSorvallMT

    2BultramicrotomeandDuPontdiamondknife.

    ;Staining.

    ;sinceosmiumwasusedasapostfixative,itcharacterizedallimagesasastain.Thusotherstainswelean

    ;additiontoosmium.Moststaimswereapp]:edbyfloatingthegridsonadropofsolution.Uranylacetatef51

    ;wasusedasanaqueous2%solution(pH5)for20minutes.Reynold’sformulation[6]ofleadcitrate(pill2),

    ;wasappliedfor30minutes.AhotwaterrinseinsuredremovalofCar[onatecrystals.Sectionswereimmersed

    ;inthebismuthformulation[7]for10minutes.Treatmentin1%phosphotung8tic(PTA)acidin95%

    ;ethanolforfiveminutesfollowedbythoroughrinsinginwatergavegoodresults.2%PTAin8O%or100%

    ;ethanolhasbeenusedbyotherworkers,andpHvariationshavegivenslightvariationsinresults[8].

    ;Stainingfor60minutesina0.5%solutionofrErheniumredpreparedin0.1ammoniumhydroxide[9]

    ;followedbyabriefdisti]ledwaterrinseproduceddefinitiveimages. ;RESULTSANDDISCUSSION

    ;Figures14showtheeffectsoftraditionalstaJningproceduresusingosmiu

m.

    ;1eadanduranium.FastPlantleaforganellesworeselectivelyresponsivetothese

    ;stains.Sinceosmiumj8widelybelievedtobindwiththeunsaturateddoublebends

    ;ofbothfatryacidsandothermolecules,itisnotsurprisingthatthere_a8an ;absenceofstaininthecellwall(Fig.1).Thisstructureismorothan75% ;earbohydrateandverylowinproto~n.Themiddlelamolla,composedofpectina

    ;pentosepolymer),wasbarelyvisiblewithosmium.Whoroasthecellmembrane

    ;waslightlystaJned,thoseofthevacuoles,nuclei,chloroplastsandmitochondria

    ;woresignificantlydifferentiated.Thethylakoid”layers”ofthechloroplastswore

    ;ospee[allyconspicuous,probablyowingtotheirchlorophylllipoprotoincomposition.

    ;Boththenueleopla~mandcytoplasmshowedaveryfine-grainedappearance. ;

    ;HarrisJBeta1.

    ;Hetarochromatinwashardlyvisible.Thesignificantappearanceofosmiumin

;membranepatternsindicatedtha

    theleaftissuewaswellpreserved.allowing

    ;sueien~osmiumbondingObecomeeasilyvisible.

    ;LeadstainingisenhancedbOhbyfixationwithosmiumandbythehighpH

    ;of

    heReynold’sformulation[8].Thoseconditionscreatesitesfortheadditionof

    ;thecation.Further,reducedosmiumattractslead

    Omembranesiteswhiohare

    ;therebysignificantlyenhanced.Sincethecellwall(Fig.2,washeavilystained ;wihleadi

    isassumedtobeareactionthroughhydrogenbondingwiththemixed ;sugarsandpectin.Thishasbeenproposedaswellforthebondingwithliver ;glycogen.Inadditiontooverallenhanoemen~ofmembranes,thegroundsubstance,

    ;espeoiMlyofmitochondriawasimprovedbylead.Yetovtoplasmiocontentsshowed

    ;awashed-outappearance.TheRNA--containingnueleolusdisplayod8clear ;granularity,whiohistypicalofRNAnuoleoproteins.

    ;UraniumincreaseddirentiationoftheRNAcontainingribosomesand

    ;nuoleoli.Therewasalsoentrancementofhe~erochromatinrFJgs.3and7).The

    ;midd]elamellawaslightlystained(Fig.7).0therwiseuraniumimprovedthe

    ;imageonlyslightly,overosmium.Thismightbeexpeotedsinceuraniummaynot

    ;bindaallwithmembraneorotherprotein.Itmaybeusedtodifferen~iata ;betweenDNAandRNAbu七七

    hi8requiresverycarefulcontrolovers~ainingtime

    ;[8].

    ;A11~hreeoommonlyusedstainsprovidedthetypioalimageofaplan~leafcell ;(Fig.4).Theoellwallwasheavilys~ainedwithlead,membranesofallorganelles

    ;wihosmiumandlead,andribosomeswi

    hleadanduranium.Inaddition,Fig.4

    ;showsthats~arohofthechloroplastsisheavilys~aiued.Thisi8unusualbu

    has

    ;beenshownforyoungchloroplastsofditferen~genera1-10.11].Chloroplas

    ;fhylakoidsweres~ainedheavilyinoonra8twih

    hematrixwhichshowedthe

    ;washedouappearance~ypicalofleadreatmen

    .Cellularo~oplasmandtoalessar

    ;exten~nuoleoplasmshowedaleachedappearanceasfromleadalone(Fig.4). ;Bismuthasasinglestainfollowingosmium,wasequivalentinmanyrespects ;tobothleadanduranium(Figs.5and9.Itstainedatleastonelayerinthecell

    ;wallandmembranesofallorganelles.Chloroplaststarchandthylakoids,nuoleoli

    ;andallribosomesweresuperiorincontrast,andmembraneswereequivalentin

    ;contrasttoimagesfromotherstains(Fig.9.Bismuthisknowntobindbothwith ;basleaminogroups,freeorinproteins,andwithphosphates.It’ss~iningof

    ;ribosomeshasbeenclaimedtobebloekedbyglutaraldehydefixatJon[12],yetFigs.

    ;5and9showdistinotstainspooincity.Thismaybeduotobismuthenhancementof

    ;osmium[13].

    ;Phosphotungstioacid(PTA),appliedas1%i.n95%ethanol,showedgenerally

    ;verygood,butnotsuperior,cytoplasmioandorganellemembranestaining(Fig.

    ;6.Italsoheavilystainedatleastonefractionofthechloroplastmatrixaswellas ;heterochromatin,butasignifioantfractionofthebaekgroundofallorganelleswas

    ;unstained.Thisproduoedawashedoutappearance,andthismaybeofvaluein ;someresearchstudies.Theabsenceofthetypicalnegativestainappearance

    isdue

    ;toosmiumstaining,especiallyofmembranes.Therewasalmostaoomplereab

sence

    ;9

    ;i-l,i1IIl__?..?,l_1-{lIllIIlllllIllflII?lIi量霞

    ;j?藩口誊哥a鼋习i蔓温

    ;

    ;Sireplifledheavymetalstainingtechniques

    ;ofstaininbothstarchandinthecellwallwhichindicatedalackofaffinitybythe ;stainforcarbohydrates,eitherinthJsformulationorwiththeFastPlantle.af ;tissue.PTAlsreportedtobindwithalcohol,carboxylandaminogrou81.

    ;Alcoholconcentration,pHandsta[ningtimearevariableswhichshouldbe ;exploredonanindividualtJSSuebasisr81.

    ;Ruthenium,whichhasboonwidelyusedinlightmicroscopyformuoo- ;polysaeeharidesbothonthecellsurfaceandonmembranes,hasalsobeenused ;successfullyinelectronmicroscopy.Fig.8showsittobeagoodnonspeomo ;m8mbraneandbackgroundstainwithcortainpeculiarities.Mostconspicuousare

    ;theunstainedordestainedparticulatefractionsofbothmitoehondriaand ;chloroplasts.Thosefractionsappearedasirregularinoutlineandworeofaboutthe

    ;sam3thicknessasthethylakoidsofchloroplasts.Theyworelocatedatthesitesof

    ;chloroplastpunetawhichstainedwithuraniumandslightlywithbismuth(Fig

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