MechanismsofMitosisPracticingtheExperimentalProcedures

Todiscoverhowchromosomesmoveinadividingcell,yourteamwillexaminetheoutcomeoftreatingarapidlydividingtissue(onionroottip)withasubstancethateitherpromotes(Indole-3_butyricacid)mitosisorinhibits(trifluralin)mitosis.Todayyouwilllearnandpracticethetechniquesyoumustmasterinordertoanalyzemitoticcells.

I.PreparationforLabProceduresBeforeyoubegin,youmustcompleteimportantpreparations.

Beforeyoubegin…1.DonyourPersonalProtectiveEquipment(PPE)!

• Gloves• Labcoat• Safetygoggles• Otherprotectivegear

2.Labelallmaterials(beakers,onionplants,microscopeslides)appropriately.Itiscriticallyimportanttolabeleverythingproperly.

3.Forbestresultsandeaseofcounting,cleanmicroscopeslideswell.

• Place1-3dropsof95%ethanolontheslide• WipewellwithaKimwipe.• Dothisonbothsidesoftheslide• Repeat,asnecessary,untiltheslideisveryshinyandclear.

II.ChromosomeSquashProcedureForourpracticerun,onionroottipshavebeenincubatedinplainwater.Tovisualizechromosomesinthephasesofmitosis,youwillprepareandstaintheminaprocedureknownasachromosomesquash.

Becausesomereagentswewillusemaybesomewhatcaustic,youmustwearthenitrileglovesprovidedandyourownsafetyglasseswhileyouperformthechromosomesquash.Wearalabcoatorlabaprontoprotectyourclothesfromstaining.

Onionbulbswillsproutrootsiftheyareplacedinwaterforseveraldays(Figure1).Theonionsyouwillusetodayhadalloldrootsremovedapproximatelythreedaysbeforeyourlabsession.Thebulbswerethenimmediatelyplacedinplainwaterandallowedtosproutnewrootstoensurethepresenceoffresh,growingroottips.

Theonionroottipcellcycleisabout24hours.Thusitmaytakeapproximately24hoursofincubationwithanyparticularreagentbeforeonecanexpecttoseeanyeffectonmitoticcells.

Figure1.Sproutinggreenonions(scallions),Alliumsp.

Plantmitosisoccursinmeristemcellsatthetipsofrootsandshoots.Thesecandifferentiateintoanyothertypeofcell.

Theapicalmeristemisaboutonemillimeterfromtheapparenttipoftheroot(therootcap,composedofdeadcells)(Figure2a).

Forsafetyreasons,studentswillnotcutroots.Yourlabinstructorwillgivethemtoyou.

1.Obtainanonionrootfromyourlabinstructor.Theroottipisdelicate,anddesiccateseasily.

• Keeptheonionrootwetatalltimes!• Donotleaveonionrootsoutofthewaterorlyingonthelabbench.

Figure 2a. Onion root tip anatomy. Only the cells at theverytipoftheroot(ZoneofCellDivision)areundergoingmitosis. These are visually distinct in a fresh root tip,appearingmoreroundor square than theelongatedcellsintheZoneofElongationaboveit.

Figure2b. Roottipofcorn(Zeamays). Notethe clear appearance of the root cap. Justabove it is theapicalmeristemandtheZoneof Cell Division. The darker, longitudinallines above the cell division zone mark thenewlyformedvascularcambium.

2.Placetherootonanappropriatelylabeledslide.

3.Usingthedissectingscope,identifytheroottip.

• Long,rectangularcellsabovetheroottiparenolongerundergoingmitosis.• Donotincludenon-mitoticcellsinyoursquashorcounts.• Withasharprazorblade,cutoffonlythemeristematicregionoftheroottip.

4.Withfine-tippedforceps,placetheroottipwithapicalmeristemintoa1.5mlmicrocentrifugetube.(Forcepstipsarefragile.Handlewithcare.)

5.Fillthemicrocentrifugetubehalfwaywith1MHCl(dropperbottleonyourlabbench)Thiswillsoftentheconnectionbetweenthecells.Usecaution:HClisastrongacid.

6.LABELTHETUBEwithaSharpiemarker.

7.Closethetubeandplaceitinahot60°Cwaterbathforexactly8minutes.(Toolonginhotacidyieldsasoggymassofcellsthatwilldisintegratewhenyourinse).

8.Carefullyremovethetubefromthehotbath.

9.Toremovethe1MHCl,fillthetubewithdeionized(DI)water,andthensuctionitoutwithaplasticsqueezepipet.Repeatthisprocedureforatotalofthreerinses.

Placeallremovedwastewaterintothecontaineratyourstationlabeled"WASTESOLUTIONS".

Nothinggoesintothesinksortrashcans!

10.Add2dropsof0.5%toluidinebluetothetube.

11.Incubateatroomtemperaturefor5minutesGentlyflickthetubewithyourfingernailaboutonceperminutetodistributethestain.Makesuretheroottipstaysinthestain.

12.RinsetheexcesstoluidineblueasyoudidfortheHCl.

a) FillthetubewithDIwater,thenremoveitwiththeplasticsqueezepipet.b) Repeatatotalofthreetimesc) Removealmostallofthelastrinse.d) Useadissectingprobetogentlypushtheroottipontoaclean,labeledslide.

Bythetimeyouhaveremovedthelastbitofrinsewater,youshouldbeabletoseeyourblueroottipclearly

13.AddonedropofDIwatertotheroottipontheslide.Gentlydropacoverslipoverit.

14.Placeasheetofbibulouspaper(bookletsuppliedonyourtray)overthecoverslip.

• Gentlypressstraightdownontothecoverslipwithroottipunderneath.• Becarefulnottobreakthecoverslip,oryou’llhavetostartover.

DONOTPLACEYOURSLIDEINSIDETHEBIBULOUSPAPERBOOKLET!Keepthepagescleananduncontaminatedforyourfutureslidepreps.

15.Removeanddiscardthebibulouspaper.

16.Placetheslideonyourcompoundmicroscopestage.ALWAYSBEGINMICROSCOPEOBSERVATIONSONLOWPOWER.

a) Findandfocusonyourroottipcellsintheviewingfieldonlowpower.b) Swiveltheobjectivetothenexthigherobjective,andfocusagain.c) Dothisuntilyouareproperlyfocusedwiththe40Xobjective,whichyouwillneedtousetoseenuclearmaterialclearly.

17.Examineyoursquash.Youshouldbeabletoseecellsinvariousstagesofmitosis.III.DataCollectionChooseaproperlysquashedareaandcountallofthecellsyoucansee(~50-200cells).Thecellsyoucountshouldberoundorcuboidalandflattenedintoasinglecelllayer.Donotcountlong,rectangularcells,asthesearenolongerundergoingmitosis.

SeeFigure4foranexampleofwhatyoushouldexpecttoseeinyourslides.

Figure4a.Alliumroottipcellsundergoingmitosis(acetocarminestain).http://upload.wikimedia.org/wikipedia/commons/d/d3/Onion_root_mitosis.jpg

Figure4b.Yourpreparationwillprobablylooksomethinglikethis.Yellowarrowsindicatecellsinvariousstagesofmitosis.(preparationandphotocourtesyofLindaWhite)

Countcellsinfourdifferentfieldsofviewforeachroottip.Thiswillgiveyouagoodsamplefromanindividualonion(about100-300cellsperroottip,dependingonitssizeandquality).

Record• thetotalnumberofcellsyoucanidentify• thetotalnumberofcellsinanystageofactivemitosis• thetotalnumberofcellsinEACHstageofthecellcycle

(1)interphase(2)prophase(3)metaphase(4)anaphase(5)telophase

onesample=allthecellscountedinonerootfromoneonion

AvoidPseudoreplication

• Donottakemultiplerootsfromthesameonion• Donotcountmultiplefieldsofviewasseparateexperimentalsamples.

Allcellscountedfromasingleonionplantcompriseonesample.Asingleindividualonion’sroottipsareallpartofthesameorganism.Countingthemasseparatesamplescreatesfalsereplication.IV.DataAnalysis:MitoticIndicesAMitoticIndex(M)isameasureoftheproportionofmitoticcellsinasampledcellpopulation.

M=nm/Nnm=totalnumberofmitoticcellsinthesampleN=totalnumberofcellscountedinthesample

Foreachofyoursamples,calculateandrecordaMitoticIndex,andrecordthesevaluesinatableliketheoneshown.Provideanappropriatelegendforthetable.AMitoticPhaseIndex(MP)isameasureoftheproportionofcellsinaparticularphaseofmitosisinasampledpopulationofmitoticcells.

MP=np/nm

np=#ofcellsinprophaseinthesamplenm=totalnumberofmitoticcellsinthesample

(Theequationaboveshowstheindexforprophase,butitcanbeusedforanyphase.)

Enteryourdatainatablesuchastheoneshownbelow,andprovideanappropriatelegendthatclearlydescribesthesourceofthedataandcontentsofthetable.Table1. Sample#

MitoticIndex(M)

ProphaseIndex(Mp)

MetaphaseIndex(Mm)

AnaphaseIndex(Ma)

TelophaseIndex(Mt)

Whenyourteamcollectsdatafromtreatedanduntreated(control)onionsforyourresearchproject,youwillperformessentiallythesameprotocolsyouhavepracticedhere.Youwillalsodecidewhichindicestocalculateandreporttobestreflectyourobservations.Whenyouarecompletelyfinishedwithaslidepreparation,placeitintheBrokenGlassDisposalContaineratthefrontofthelabroom.Uponcompletionofalloftoday’sexercises,notifyyourlabinstructor,whowilltheninspectyourstationforcleanliness.Ifthestationisnotproperlycleanedandrestoredtoitsoriginalcondition,youmustcorrectthatbeforeyouleavethelab.

Teamsleavinganuntidylabstation,includingundisposedslides,trash,orothermaterials,willbedocked5points.