Mechanisms of Mitosis - bio.miami.edu · The cells you count should be round or cuboidal and...
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Mechanisms of Mitosis Practicing the Experimental Procedures To discover how chromosomes move in a dividing cell, your team will examine the outcome of treating a rapidly dividing tissue (onion root tip) with a substance that either promotes (Indole-3_butyric acid) mitosis or inhibits (trifluralin) mitosis. Today you will learn and practice the techniques you must master in order to analyze mitotic cells. I. Preparation for Lab Procedures Before you begin, you must complete important preparations. Before you begin… 1. Don your Personal Protective Equipment (PPE)! • Gloves • Lab coat • Safety goggles • Other protective gear 2. Label all materials (beakers, onion plants, microscope slides) appropriately. It is critically important to label everything properly. 3. For best results and ease of counting, clean microscope slides well. • Place 1-3 drops of 95% ethanol on the slide • Wipe well with a Kimwipe. • Do this on both sides of the slide • Repeat, as necessary, until the slide is very shiny and clear. II. Chromosome Squash Procedure For our practice run, onion root tips have been incubated in plain water. To visualize chromosomes in the phases of mitosis, you will prepare and stain them in a procedure known as a chromosome squash. Because some reagents we will use may be somewhat caustic, you must wear the nitrile gloves provided and your own safety glasses while you perform the chromosome squash. Wear a lab coat or lab apron to protect your clothes from staining. Onion bulbs will sprout roots if they are placed in water for several days (Figure 1). The onions you will use today had all old roots removed approximately three days before your lab session. The bulbs were then immediately placed in plain water and allowed to sprout new roots to ensure the presence of fresh, growing root tips.
Transcript of Mechanisms of Mitosis - bio.miami.edu · The cells you count should be round or cuboidal and...
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.
