AP Biology Lab 5: Cell Respiration1 Overview Inthisexperimentyouwillworkwithseedsthatarelivingbutdormant.Aseedcontainsan embryoplantandafoodsupplysurroundedbyaseedcoat.Whenthenecessaryconditions aremet,germinationoccursandtherateofcellularrespirationgreatlyincreases.Inthislab youwillmeasureoxygenconsumptionduringgermination;measurethechangeingas volumeinrespirometerscontainingeithergerminatingornongerminatingpeaseeds;and measuretherateofrespirationofthesepeasattwodifferenttemperatures.

. Objectives Beforedoingthislabyoushouldunderstand: respiration,dormancy,andgermination; howarespirometerworksintermsofthegaslaws; thegeneralprocessesofmetabolisminlivingorganisms;and howtherateofcellularrespirationrelatestotheamountofactivityinacell. Afterdoingthislabyoushouldbeableto: calculatetherateofcellrespirationfromexperimentaldata; relategasproductiontorespirationrate; testtherateofcellularrespirationingerminatingversusnongerminatedseedsina controlledexperiments; testtheeffectoftemperatureontherateofcellrespirationingerminatingversus nongerminatedseedsinacontrolledexperiment;and calculateaQ10constantfortheeffectoftemperatureonenzymefunction. Introduction Cellularrespirationistheenginethatrunsallcellactivities,andthusthelevelofcell respirationisdirectlyrelatedtothelevelofactivitysustainedbyacell.Aerobiccellular respirationissummarizedbytheequation: C6H12O6+6O26CO2+6H2O+36ATP ThemostappropriatemeasureofcellrespirationwouldbetherateofconversionofADPto ATP.However,thisrequiressophisticatedbiochemicaltechniques.Afareasiermethodisto measuretherateofoxygenconsumptionorcarbondioxideproduced.However,thisleaves uswithadilemmathesetwogasvolumesoffsetoneanother!Therefore,wemustfinda waytoturnoneofthemintoasolidorliquidinordertominimizethevolume.Wedothisby placingcottonsoakedwithKOHinourrespirometer.TheKOHreactswithcarbondioxideto formasolidpowderthathasminimalvolumeandourgasvolumechangeisnowduealmost entirelytooxygenconsumed:CO2+2KOHK2CO3+H2O Inthislabwewillmeasurecellrespirationbymeasuringoxygenconsumption(changein gasvolume)insmallrespirometers.Thechangeinvolumeisdirectlyproportionaltothe amountofoxygenconsumedbytheorganismsintherespirometersandisanexcellent measureofcellularrespirationrate.Becauserespirationreactionsareenzymedrivensteps, wewillhaveachancetoseehowchangesintemperatureaffectthereactivityofenzymes 1BasedonAPCentralBiologylabManualaswellasDr.SnappandK.Moserslabmanual.

andoverallamontofrespiration.WewillperformacalculationcalledaQ10constantto measurethetemperatureeffect.Q10valuesaresimplytherelativeenzymereactivityat 10Ctemperatureintervals. Theformulaforthiscalculationis:Q10= enzymereactionrateat10C enzymereactionrateat20C Review of Gas Laws Measuredgasvolumedependsupontemperatureandpressureaswellasnumberof gasmolecules,sowemustcontrolthesevariablesaswell.Areviewofthegaslawsis essentialtounderstandingthemethodbywhichoxygenconsumptionisdetermined.Recall thegeneralgaslaw:PV=nRT,whereP=pressure,V=volume,n=moles,R=gasconstantand T=temperatureinK. Thislawimpliesthefollowingimportantconceptsaboutgases:1)volumeofgasis proportionaltomolesunderconstanttemperatureandpressure;2)volumeisdirectly proportionaltotemperatureifallothervariablesareheldconstant;3)pressureandvolume areinverselyrelatedifallothervariablesareheldconstant.Thismeansthatgasvolumecan beaffectedbytemperatureandpressure,sowemustequilibratetherespirometerforthe effectsoftemperatureandpressurebeforewebegincollectinggasvolumedata. Procedure Eachgroupwillrecordrespirationingerminatedandungerminatedpeasatone temperature.Ifyouareina10Cgroup,setupyourbathimmediatelytoallowtimefor thetemperaturetoadjust.Addicetoattain10C. Contentsforeachvial Inonevialyouwillplaceonlygerminatingpeas.Inanothervialyouwillplacedormantpea andglassbeadsequalinvolumetothevolumeofgerminatingpeasused.Inthethirdvial, youwillplaceonlyglassbeads,againequalinvolumetothevolumeofgerminatingpeas used. 1. Determinethevolumeofgerminatingpeasbywaterdisplacementinacylinder.Use about20germinatingpeas. Recordthevolume.Tipthepeasoutandpatdry.Thesepeaswillgointothefirstvial. 2. Forthesecondvial,countoutanumberofdormantpeasequaltothenumberof germinatingpease(20).Determinethevolumeofthesepeasbywaterdisplacement andaddglassbeadstothecylinderuntilyoureachavolumeequaltothatofthe germinatingpeas.Tipthepeasandbeadsout,patdryandsaveforthesecondvial. 3. Forthethirdtrial,determinehowmanyglassbeadswilldisplaceavolumeequalto thatofthegerminatingpeasused.Tipthebeadsout,andsaveforthethirdvial.

Assemble3Respirometers

1. PlaceasmallwadofABSORBANTcottoninthebottomofeachvial.Addafewdrops ofKOHsothatthecottonbecomesmoistbutnotsoaked.Placeasmallwadof NONabsorbantcottonontopoftheabsorbantcotton. 2. Placetheappropriateamountofpeasand/orglassbeadsintothevial. 3. Placethestopper/pipettetightlyintothemouthofthevial.Makesurethemouthof thepipetteisnotobstructed.

EquilibratetheRespirometers,sothengasvolumechangesareduesolelytorespiration rate. 1. Temperatureequilibration:Placetherespirometersinthewaterbathwithpipette tipsoutofthewaterandrestingontapeslings(diagrama)toallowthetemperature inthevialstoequilibratewiththetemperatureinthewaterbath.WAIT5MINUTES. Thisallowsforgasvolumetocontractduetolowertemperature. 2. Indicator:After5minutes,addafewdropsoffoodcoloringtothetipofthe respirometer.Thiswillallowyoutoeasilyseethewaterseepingintothepipette.

a b

3.PressureEquilibration:Immerseall3respirometersentirelyinthewaterbath slipplingapieceofwhitepaperunderneaththem(mayalreadybewhite).Use

weightstokeeprespirometersfromfloating.Notethatwaterentersthepipettesfora shortdistanceandthenstops(unlessyouhavealeak).Thisistheresultof equilibrationtothepressureofthesystemandallowsthegasvolumetocontractdue topressure.WAIT5MINUTES. RuntheExperiment.CollectandReportResults 1. Afterthesecondequilibration,beginrecordingdata(i.e.0minreadingistaken afterthefullimmersionequilibration). 2. Recordtheheightofthegascolumnonthepipettesevery3minutesforatleast15 minutes.Notethatmovementoftheaircolumntowardthevialisconsidereda+ value.DeterminethecumulativemLofvolumechangebysubtractingthereadingsat 3,6,9,12,15minutesfromthereadingof0min. 3. Adjustthedata,takingintoaccountanypressurerelatedchangeswithinthecontrol (glassbeadonly)vial.Enterthesecalculationsofcumulativevolumeconsumedina table. 4. Keepwatchingthewatertemperatureandadjustitwithiceasneeded. 5. Enteryourvolumereadingsonthedatasheetprovided.Reportyourdatatoyour instructorandcleanup. 6. PrintouttheclassdatafromInClass.Reviewthedataaveragesandbringyourdatato thenextclasssession.