BIL151-MechanismsofMitosisChromosomeSquash:ControlSamples

Toexaminehowchromosomesmoveinadividingcell,yourteamwilldecidewhetherto(virtually)treatgrowingonionswithasubstancethateitherpromotes(Indole-3_butyricacid(IBA))mitosisorinhibits(trifluralin)mitosis.Todayyouwilllearnaboutoneofthemanystainingandvisualizationtechniquesusedtoanalyzemitoticcells.

TheroottipsdescribedintheexampletofollowwouldcompriseCONTROLsamples,astheyaregrowninplainwater,notIBAnortrifluralin.Althoughyouwillnotbephysicallyperformingthisprocedure,youshouldstillreadandbefamiliarwithitsoyouwouldbeabletofollowthesedirectionsandperformitinthefuture.WatchtheChromosomeSquashVideolinkedtoyouronlinelabmanualforalivedemonstration.

I.PreparationforLabProceduresBeforeyoubegin,youmustcompleteimportantpreparations.1.PutonyourPersonalProtectiveEquipment(PPE)!

• Gloves• Labcoat• Safetygoggles• Otherprotectivegear

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

3.Forbestresultsandeaseofcounting,cleanyourmicroscopeslidesthoroughly.

• Place1-3dropsof95%ethanolontheslide• PolishwellwithaKimwipe.• Dothistobothsidesoftheslide• Repeat,asnecessary,untiltheslideisveryshinyandclear.

II.ChromosomeSquashProcedureForourcontrolsamples,onionroottipshavebeenincubatedinplainwater.Tovisualizechromosomesinthephasesofmitosis,youwillprepareandstaintheminaprocedureknownasachromosomesquash.

Becausesomereagentswewillusemaybesomewhatcaustic,youmustwearthenitrileglovesprovidedandyourownsafetyglasseswhileyouperformthechromosomesquash.Wearalabcoatorlabaprontoprotectyourclothesfromstaining.

Onionbulbswillsproutrootsiftheyareplacedinwaterforseveraldays(Figure1).Theonionsyouwillusetodayhadalloldrootsremovedapproximatelythreedaysbeforeyourlabsession.Thebulbswerethenimmediatelyplacedinplainwaterandallowedtosproutnewrootstoensurethepresenceoffresh,growingroottips.Theonionroottipcellcycleisabout24hours.Thusitmaytakeapproximately24hoursofincubationwithanyparticularreagentbeforeonecanexpecttoseeanyeffectonmitoticcells.(Considerthiswhendesigningyourprojectmethods.)

Figure1.Sproutinggreenonions(scallions),Alliumsp.

Plantmitosisoccursinmeristemcellsatthetipsofrootsandshoots.Thesecandifferentiateintoanyothertypeofcell.

Theapicalmeristemisaboutonemillimeterfromtheapparenttipoftheroot(therootcap,composedofdeadcells)(Figure2a).

Forsafetyreasons,studentswillnotcutroots.Yourlabinstructorwillgivethemtoyou.

• 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.

1.Obtainanonionrootfromyourlabinstructor.Theroottipisdelicate,anddesiccateseasily.KEEPITWET.

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.DataCollectionEachteamwillcollectdatafrom

• CONTROLonionsincubatedinplainwater• TREATMENTonionsincubatedinthereagentyourteamchose

A.ControlSampleReplicatesChromosomesquashesandcellcountsfromonionsincubatedinplainwaterwillcompriseyourCONTROLsamples.Youwillreceivemicrographsofeightcontrolonions.

Eachofyourfourteammembersshouldcountcellsfromtwocontrolonions.Countfourfieldsofviewpercontrolonion.

Bytheendofthesession,yourteamwillhavedatafromeightcontrolsamples.B.TreatmentSampleReplicatesBased on what your team submitted in its Project Protocol Worksheet, you will beprovided with sets of prepared micrographs representing TREATMENT samples.Dependingonyourteam’sresearchproject,thesewillhavebeentreatedwitheither

• indole-3-butyricacidinwater• trifluralininwater

Yourteamwillcountmitoticcellsfromeighttreatmentsamplemicrographs.

Eachofyourfourteammembersshouldcountcellsfromtwotreatmentonions.Countfourfieldsofviewpertreatmentonion.

Bytheendofthesession,yourteamwillhavedatafromeighttreatmentsamples.Whenyourteamisinpossessionofallyourcontrolandtreatmentmicrographs,youwillmeetinZoombreakoutroomstocountmitoticcellsandtabulateyourresults.C.ProcedureforCountingMitoticCellsChooseaproperlysquashedareaandcountallofthecellsyoucansee(~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).Foreachfieldofview,record

• thetotalnumberofcellsyoucanidentify• thetotalnumberofcellsinanystageofactivemitosis• thetotalnumberofcellsinEACHstageofthecellcycle

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

onesample=allthecellscountedinonerootfromoneonion

AvoidPseudoreplication

• Multiplerootsfromthesameonionarenotreplicates• Donotcountmultiplefieldsofviewfromthesameonionasseparateexperimentalsamples.

Allcellscountedfromasingleonionplantcompriseonesample.Asingleindividualonion’sroottipsareallpartofthesameorganism.Countingthemasseparatesamplescreatesfalsereplication.

IV.DataAnalysis:MitoticIndicesRecallthataMitoticIndex(M)isameasureoftheproportionofmitoticcellsinasampledcellpopulation.

M=nm/Nnm=totalnumberofmitoticcellsinthesampleN=totalnumberofcellscountedinthesample

Foreachofyoursamples,calculateandrecordaMitoticIndex,andrecordthesevaluesinatableliketheoneshown.Provideanappropriatelegendforthetable.

AMitoticPhaseIndex(MP)isameasureoftheproportionofcellsinaparticularphaseofmitosisinasampledpopulationofmitoticcells.

MP=np/nm

np=#ofcellsinprophaseinthesamplenm=totalnumberofmitoticcellsinthesample

(Theequationaboveshowstheindexforprophase,butitcanbeusedforanyphase.)UsetheMitoticIndexWorksheetsforCONTROLonions(linkedintheonlinelabmanual)torecordyourdataandmitoticindices.D.IfThisWereInPerson:Don’tForgettheCleanup!Cleaningupafteryourselfisacriticalpartofgoodlaboratorytechnique.Whenfinishedwithaslidepreparation,placeitintheBrokenGlassDisposalContaineratthefrontofthelabroom.Uponcompletionoflabwork,notifyyourinstructor,whowilltheninspectyourstationforcleanliness.Ifthestationisnotproperlycleanedandrestoredtoitsoriginalcondition,youmustcorrectthatbeforeyouleavethelab.

Teamsleavinganuntidylabstation,including• undisposedslides• trash• slidesleftonmicroscopestage• otherinfractions• usedsurfacesorequipmentnotproperlydisinfected

…willbedocked5points.