bil 151 - mechanisms of mitosis chromosome squash ......bil 151 - mechanisms of mitosis chromosome...

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.