PlantingScience Power of Sunlight Toolkit

PlantingScience CC BY-NC-SA 3.0 | www.plantingscience.org | Power of Sunlight—Toolkit of 31 Last Updated 7/2013

BACKGROUND:

ThePlantingSciencePowerofSunlightToolkitprovidesbackground,materialslists,detailedprocedures,andsafetyconsiderationsforadditionalexperimentalmethodsrelatedtophotosynthesis

andrespiration.ThesetoolscanprovidestudentstheopportunitytoaskawiderrangeofresearchquestionsduringtheopeninquiryphaseofThePowerofSunlightthanwouldbepossibleusingonlythe

leafdiskfloatationmethod.Alternatively,teachersmayselectoneormoreofthesemethodsasclassroomdemonstrationsofphotosynthesisand/orrespirationinaction.

CONTENTS:

PagePreparationofCitrate-PhosphateBufferforMaintainingpH ......................................................................2MonitoringpHtoAssessPhotosynthesis&RespirationofAquaticPlants ..................................................4

MeasuringCellularRespirationUsingaRespirometer .................................................................................7MeasuringPhotosynthesis&RespirationUsingaComputer-BasedProbe ...............................................11Visualizing&CountingStomataUsingtheLeafImpressionMethod .........................................................13

VisualizingPlantCells&ChloroplastsUsingaMicroscope.........................................................................15IdentifyingStarchinPlantLeavesUsinganIodineStainingMethod .........................................................17

IdentifyingChlorophyll&OtherPlantPigments ........................................................................................19QuantifyingFresh&DryMassofPlants .....................................................................................................22

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PlantingScience

PowerofSunlightToolkit


PREPARATIONOFCITRATE-PHOSPHATEBUFFERFORMAINTAININGPH

Purpose:Theadditionofincreasingamountsofsodiumbicarbonatetowatertendstoincreasetheacidityofthesolution,loweringitspH.MostcellsthrivebestinthepHrangeof6to8;therefore,leafdisksinfiltratedwithhigh-concentrationsodiumbicarbonatesolutionsmayhavealtered

levelsofphotosynthesisandcellularrespiration.

AbufferresistspHchangeandthushelpsasolutionmaintainthesamepHunderawiderangeofconditions.Awiderangeofpossiblebuffersareavailable,butmanycommonbuffershaveproblemsinthepH6to8range.TohelpmaintainpHinleafdiskfloatationexperiments,wesuggesttheuseofaphosphate-citratebuffer,asdescribedhere.

TechnicalComplexity:Simple.

TimeRequired:20minutesforfullprocedure;5min.topreparebufferfromstocksolutions.

Materials:Perclass: Perteam:Citricacid(anhydrous) 100mLstockbottle

Disodiumphosphate 100mLgraduatedcylinderWater WaterBalance,accuratetoatleast0.1g,Weighingpaper

ScoopulaTwo1000mLgraduatedcylindersTwo1000mLstockbottleswithcapsorstoppers

Procedure:1. (Oneperclass)PrepareStockSolutionA:

a. Wearsafetygogglesandlabglovesincaseofsplashesandtoavoidskinandeyeirritation.b. Weighout19.2gofanhydrouscitricacidontoweighingpaperonabalance,usinga

scoopula.c. Transferthecrystalstoa1000mLstockbottle.d. Fillthestockbottletwo-thirdsofthewayfullwithwater.

e. Stirorcap/stopperthebottleandgentlyshaketodissolvethecitricacid.f. Whenthesolidiscompletelydissolved,pourthesolutionintoa1000mLgraduatedcylinder

andaddwatertoreach1000mL.g. Returnthefullliterofsolutiontothestockbottle.h. Labelthebottleas0.1Mcitricacid.


2. (Oneperclass)PrepareStockSolutionB:a. Wearsafetygogglesandlabglovesincaseofsplashesandtoavoidskinandeyeirritation.

b. Usescoopulatoweighout28.4gofdisodiumphosphateontoweighingpaperonabalance.c. Transferthecrystalstoa1000mLstockbottle.d. Fillthestockbottletwo-thirdsofthewayfullwithwater.

e. Stirorcap/stopperthebottleandshaketodissolvethedisodiumphosphate.f. Whenthesolidiscompletelydissolved,pourthesolutionintoa1000mLgraduatedcylinder

andaddwatertoreach1000mL.

g. Returnthefullliterofsolutiontothestockbottle.h. Labelthebottleas0.2Mdisodiumphosphate.

3. (Eachteam)Preparecitrate-phosphatebuffer:

a. Wearsafetygogglesincaseofsplashes.b. SelectthedesiredpHforthefinalsolutiontobeusedforleafdiskinfiltration(seeTable).c. MeasurethecorrespondingamountofStockSolutionA(0.1Mcitricacid)intoa100mL

graduatedcylinderandtransfertoa100mLstockbottle.d. MeasurethecorrespondingamountofStockSolutionB(0.2Mdisodiumphosphate)into

thesame100mLgraduatedcylinderandtransfertothesamestockbottle.e. Labelthebottleascitrate-phosphatebuffer,makingsuretoincludethedesiredpH.f. Sodiumbicarbonatecanbedirectlydissolvedintothisbufferforuseinleafdiskinfiltration

andfloatation.

Amountof0.1Mcitricacid(SolutionA)and0.2Mdisodiumphosphate(SolutionB)toachievethedesiredbufferpH:

mLofSolutionA mLofSolutionB DesiredpH

46.4 53.6 5.2

44.2 55.8 5.4

42.0 58.0 5.6

39.5 60.5 5.8

36.8 63.2 6.0

33.9 66.1 6.2

30.7 69.3 6.4

27.2 72.8 6.6

22.7 77.3 6.8

17.6 82.4 7.0

13.0 87.0 7.2

9.1 90.9 7.4

6.3 93.7 7.6

4.2 95.8 7.8

2.7 97.3 8.0


MONITORINGPHTOASSESSPHOTOSYNTHESIS&RESPIRATIONOFAQUATICPLANTS

Purpose:UsepHasanindirectmeasureoftheamountofcarbondioxideinanaquaticsystemcontainingalgaeoratleastoneaquaticplant.

HowtheMethodWorks:Carbondioxide(CO2)isoneoftheproductsofcellularrespirationandacriticalreactantin

photosynthesis.ThesimplestwaytomeasureCO2changesinanaqueoussolutionistomeasurepHchange.Carbondioxidedissolvesinwatertoformcarbonicacid:CO2+H2O!H2CO3.Therefore,asCO2isreleasedbyaquaticorganismsduringrespiration,itformsaweakacidinthesurroundingwater,loweringthepH.AsCO2isconsumedbyaquaticplantsduringphotosynthesis,thepHwilltendtoincrease.Similartotheleafdiskinfiltrationmethod,thedissolvedcarbondioxideinanaquaticplant’senvironmentcanbesupplementedbyaddingsodiumbicarbonate.

Technically,pHisthenegativelogarithmofasolution’shydrogenionconcentration.Freehydrogenionsareacidic,sothepHscaleisusedtoindicatehowacidicasolutionis,withalowernumberindicatingamorestronglyacidicsolution.Eveninpurewater(H2O),afewmoleculesarealwaysdissociatedintohydrogenions(H+)andhydroxideions(OH-)–aboutoneintenmillion(1/10,000,000)moleculesinaliterofpurewater.Inotherwords,thereare10-7hydrogenionsperwatermolecule.ThepHofpurewateristherefore–log(10-7)=7.ThisisconsideredneutralpH,becauseeachacidicH+isaccompaniedbyonebasicOH-inpurewater.EveryunitchangeinpH,e.g.,frompH7topH8orfrompH6topH5,indicatesaten-foldchangeinacidity.Ifmoreacidisaddedtothewatersothatoneineverythousandmoleculesisahydrogenion,theconcentrationis10-3andthepHis–log(10-3)=3.Thisconcentrationhas10,000timesmorehydrogenionsthaninpurewater,sothesolutionis104timesmoreacidicanditspHis4unitslower.


TimeRequired:5minutesperreading.

Materials:Inawater-filledvessel,algaeoraquaticplantsuchasElodea(Optional)OneortwosmallercontainersforeachtreatmentOneofthefollowingsets,dependingonthemethodyoupreferorthematerialsavailable:


METHODA METHODB METHODCpHindicatorpaper pHmeter PhenolRedorBromphenolBluedyepowder

Color/pHkeychart pHbufferstock ScoopulaorspoonScissors Smallcup Long-termstoragecontainer

Squirtbottleofdistilledwater Water Graduatedcylinder Electronicbalance

GeneralInstructions:• Ifthecontainerfortheaquaticplantisverylarge,pHwilltakealongtimetochange.Youcan

transfertheplantorapartofittoacontainerwithlessthan50mLofwatertomonitormore

rapidchangesinpH.• AlgalculturesarebettersuitedtoMethodAorMethodBthantoMethodC,becausetheirgreen

colorwillalterhowtheliquidappears.

• Youmaywishtoremoveaquaticplantsfromtheircontainers,blottheexcesswater,anduseanelectronicbalancetofindtheirmassforexperiments.

o Largerplantswillcarryoutmorephotosynthesisandrespiration,sotreatmentsare

moreequivalentiftheycontainthesameamountsofplantmass.o Foralgalcultures,stirringwellandusingthesamevolumeofculturewillprovide

equivalenttreatments.

• Forexperiments,repeatthepHmeasurementatregularintervals,e.g.,every5min,underthetestconditions.

• Youmayconcludetheexperimentafterapredeterminedtimeperiod(e.g.,30min,recording

thefinalpH)orafterapredeterminedamountofpHchangeoccurs(e.g.,0.5units,recordingthefinaltime).

o MethodCisbestsuitedtorecordingthefinaltime,sinceitcanbedifficulttoassesssubtlechangesincolorofthesolutionandyoumightnothaveacolorkeyforintermediatecolors.

MethodA:MeasuringpHUsingIndicatorPaperThisisthesimplestwaytomeasurepH.IndicatorpaperissimplypaperimpregnatedwithapHindicatordye.ThedyechangescolordependingonthepHofthesolution.

1. Tearoffabouta2cmstripofindicatorpaper(ifinrollform)orpullapiecefromitscontainer(if

instripform).2. Dipthetipofthepaperintothewatercontainingtheaquaticplant.3. Comparethecolorofthewetpapertothecolor/pHkeychart,usuallyfoundontheindicator

papercontainer.ThebestmatchindicatestheapproximatepH.4. Useanewstripofindicatorpaperforeachmeasurement.

MethodB:MeasuringpHUsingapHMeterThisisthemostaccuratewaytomeasurepH.ThepHmeterhasanelectrodewhichmustalwaysbe

keptmoist.Whenitisnotinuse,keeptheelectrodecoveredorimmersedinabuffersolution.


1. TurnonthepHmeterandallowitwarmupforatleastfiveminutes.Keepthecontrolknobinstandbyposition.

2. Ifthereisone,adjustthetemperatureknobtomatchthesolutiontemperatureafterwarm-upiscomplete.

3. StandardizethemeteragainstabufferofknownpHclosetotherangeyouthinkyouwillneed.

a. Ifyou'reunsureoftherange,useapH7buffer.b. Pourasmallamountofbufferintoavesseljustlargeenoughtoholdtheelectrode.c. Inserttheelectrodeintothebuffer.

d. Allowthemetertoreachasteadyreading.e. IfitdoesnotreadthesameasthepHofthebufferyouused,adjustthemetertosetit

totheproperpH.Ifneeded,followthemanufacturer’sinstructionstodothis.4. Makeareadingonyourunknownsolution.

a. Taketheelectrodeoutofthebufferandrinsethetipoftheelectrodewithdistilled

water.Youcanusethebuffercontainertocollecttherinsewater.b. Holdthetipoftheelectrodeagainsttheoutsideofthecontainertoallowanylarge

dropsofwatertoflowofftheelectrode.

c. Inserttheelectrodeintotheunknownsolutionandmakeyourreading.d. Whenyoutaketheelectrodeoutofthesolution,rinseitagainwithdistilledwater

beforerecoveringthetiporplacingtheelectrodebackintothestandingbuffer.

MethodC:MeasuringpHUsinganIndicatorDyeIndicatordyesarepowdersthatchangecolorbasedonpHandcanbedissolvedinwatertomakea

concentratedsolution.BysomeofthedyesolutiontoaliquidofunknownpH,thecolorcanbemonitored.PhenolRediscoloredredinsolutionsofpH>8.4.AsthepHdecreasesandthesolutionbecomesmoreacidic,thecolorlightenstoorangeand,whenpH<6.8,toyellow.BromphenolBlueshifts

fromblueatpH>7.6toyellowatpH<6.0.1. Wearalabcoatandgloves.Indicatordyescanstainyourhandsandclothing.2. ScoopaspoonfuloftheindicatordyePhenolRed(orBromphenolBlue)toacontaineroftap

wateruntilthewaterisdistinctlyred(orblue).AstocksolutionofPhenolRedcanbestoredinadarkcabinetforseveralyearsandusedslowlyovertime,socheckwithyourteachertofindout

astockisalreadyavailable!3. Measureanequalamountoftheindicatorsolutionintotwosmallercontainerspertreatment.

a. Dilutethesolutionineachcontainerusingequalamountsofwater,startingbyadding

thesameamountofwaterastheamountofindicatorsolutioninthecontainer.b. Ifyoucanseethroughtotheothersideofthecontainer,youhavedilutedthesolution

enoughtoproceed.Otherwise,addmorewaterinequalamountstoallcontainers.

4. Blowbubblesintohalfofthecontainersthroughastraw.TheexhaledairwillcontainexcessCO2andthesolutionwillbegintoturnorange(orgreen),thenyellow.

5. Foreachtreatment,placeidenticalpiecesofaquaticplanttissueintoeachred(orblue)and

yellowcoloredpairofcontainers.Labelthecontainerssoyouknowwhichpairsgotogether.6. Observeandrecordthecolorofeachpairofsolutionsovertime.


a. Itmaybehelpfultodevelopa“colorscale”beforebeginningtheexperiment.b. Removingasmallvolumeofliquidfromeachcontainerandlookingatthemtogether

againstawhitebackgroundcanhelpensureunbiasedcolorjudgmentoverall.Returnthesolutiontothesamecontainerafterwards.

c. Qualitatively,iftheyellowsolutionchangescolortored(orblue),CO2isbeing

consumedmorequicklythanitisproduced.Ifthered(orblue)solutionchangestoyellow,CO2isbeingproducedmorequicklythanitisconsumed.

d. Theamountoftimerequiredforthecolortoshiftprovidesaquantitativemeasureof

howquicklythepHchangesoverasetnumberofunits.Therefore,itindirectlymeasuresphotosyntheticorrespirationrate.


MEASURINGCELLULARRESPIRATIONUSINGARESPIROMETER

Purpose:Quantifyhowmuchcellularrespirationisoccurringinmosttypesofplanttissueandsmallseedlings.Themaintypeofmaterialbeingconsumed–carbohydrates,fattyacids,orproteins

andaminoacids–canalsobedetermined.

HowtheMethodWorks:Thismethodusesaninstrumentcalledarespirometer.Therespirometerisassembledtocreateaclosedsystem,sothatoncetheexperimenthasbegun,nothinggoesinorout–notevenair.Aplantsampleorsmallseedlingisplacedintoachamber.Astheplantsamplerespiresinsidetheclosedsystem,it

usesoxygen(O2)andreleasescarbondioxide(CO2).Toquantifyrespiration,thesetwogasesmustbeconsideredseparately.CO2canberemovedfromthegasmixtureinthechamberbyachemicalreactionbetweenitandsodiumorpotassiumhydroxide.Thereactionproduceswaterandasolid,K2CO3orNa2CO3,sothatnofreeCO2gasispresent.TheO2usedforrespirationcanthenbedirectlymeasuredbythedecreasinggasvolumeintherespirometer.Carryingoutthesameexperimentinanotherrespirometerwithoutaddingsodiumorpotassiumhydroxide,allowstheamountofCO2producedtobequantified.Athirdtubewithoutanytissueactsasacontrolforanychangesintemperatureandairpressure.

Thechemicalequationforrespirationassumesthattheorganicmoleculebeingbrokendowniscarbohydrate.Ifthisisso,thevolumeofCO2producedwillbeequaltothevolumeofO2consumed.TheratioofCO2producedtoO2consumediscalledtheRespiratoryQuotient(RQ).Ifonlycarbohydrateisbeingbrokendown,thevalueofRQ=1.However,thisneednotbethecase.Whenfattyacidsarebrokendownaloneorwithcarbohydrates,thevalueofRQislessthan1.Metabolizingproteinsorotherorganicacids,aloneorwithcarbohydrates,resultsinanRQgreaterthan1.

TechnicalComplexity:Medium,duetothecomplexityofsettingupthechambersforanexperiment.

TimeRequired:Allowatleast30minforbuildingtherespirometersandatleast45minfortheexperimentitself.

Materials:Toassembleonerespirometer: • Hotgluegun,withgluestick


• 1mLdisposabletuberculinsyringe• 40µLplasticcapillarytube

• Absorbentandnon-absorbentcotton• Clean,thinpipetteortoothpick

• Manometerscaleorlinedpieceofcardstock

• Tape• Dranoor15%potassiumhydroxidesolution

• Water(optional)

Tocarryouttherespirometryexperiment:

• 1-2respirometerspertreatment,plusacontrol

• Waterbathsettoroomtemperature(20oC)• Celsiusthermometer• Plantmaterial

• Smallbeadsorbakedseeds• 3-4washersperrespirometer• Manometerfluid,orsoapywaterwithred

foodcoloring• Eyedropperorpipette

Part1.PrepareMaterialsforMakingRespirometersandtoCarryOuttheExperiment(s).

• Allowtimetogrowyourplantmaterialorsubjectplants/seedstotheenvironmentalconditionsyouaretesting.

o Forexample,ifyouplantomeasureseedrespiration,youwillwanttosoaktheseedsfor

24hoursjustbeforetheexperiment.• Makearrangementswithyourteachertohavematerialsthatyouneedonhand.

o Respirometerscanbemadeandusedforexperimentsduring

separateclassperiods.Gatherthematerialsaccordingly.• Setupadatatable.

o Youwillberecordingtemperatureandrespirometerreading

at2-5mintimepointsforeachrespirometer.o Afteryourexperimentisdoneorbetweendatareadings,you

willalsobecalculatingthechangeinfluidlevelbetween

timepoints.

Part2.MakeRespirometerChambers.1. Pluginthehotgluegun.Makesureagluestickisinthebarrel.

2. Selectasyringe,makingsuretheplungerisallthewayintothebarrel.3. Fromthenarrowendofthesyringewheretheneedleusuallygoes,gentlyinsertacapillarytube

untilittouchestheplungertipandcan’tgoanyfurther.

4. Sealthejointbetweenthecapillaryandthesyringetip:a. Holdthesyringesothatthecapillarytubeispointingupwards.b. Putadroportwoofhotglueatthejointbetweenthecapillaryandoutersyringetipso

thatitsealsallthewayaround.c. Continuetoholdthesyringeinanuprightpositionuntilthegluecoolsandhardens,

about1-2minutes.

5. Checktoensurethegluehasn’tcloggedthecapillary.a. Pullbackontheplungertoseeifaircanpassthroughfreely.


b. Ifitisplugged,youwillnotbeabletopulltheplungerback.Youcanunplugitbycarefullypeelingofftheglue.Startfromstep4toresealthejoint.

6. Makeasmanyrespirometerchambersasyourexperimentrequires,plusoneasacontrol.Ifyouplantomeasurebothoxygenconsumedandcarbondioxideproduced,twoseparaterespirometerswillbeneededforeachtreatment.

7. Ifneeded,youmaystopandstoretherespirometerchambershere,finishingthemduringanotherclassperiod.

Part3.PrepareYourRespirometersforanExperiment.1. Carefullyinsertasmall,absorbentcottonplugintothesyringeendoftherespirometer.

a. Useaclean,thininstrument,suchasapipette,topackthecottontothe0mlor0ccmark.

b. Takecarenottodislodgethecapillarytubeduringthisstep.2. Tapeamanometerscaleontoeachcapillarytubeusingcleartape.

a. Double-sticktapeisbest,butfoldedsingle-sidedtapealsoworks.

b. Placethetapesothatyouareabletoreadthescalebylookingthroughthecapillarytube.

c. Makesurethebottomofthescaleisalignedwiththesamepointonallrespirometers.

3. Ifneeded,youmaystophereandstoretherespirometersuntilanotherclassperiod.Ifyoucontinue,youwillneedtocompletetheexperimentduringthesameclassperiodsothattheliquidaddeddoesnotdryout.

4. Putonyoursafetygogglesandlabgloves,becauseyouwillbeworkingwitheitheradilutesolutionofpotassiumorsodiumhydroxide(Drano).Botharecaustic.

5. Foreachrespirometerthatwillbeusedtomeasureonlyoxygenconsumption,putasmalldropofpotassiumhydroxide(KOH)orsodiumhydroxide(NaOH)ontothecotton.

a. Avoidgettinganyonthewallsofthesyringeabovethecotton.

b. IfyouwillusesomerespirometerstocalculateCO2production,marktherespirometerstoindicatetheirtype.DonotaddKOHorNaOHtotheCO2respirometers!

6. Addasmallplugofnon-absorbentcottonabovetheKOH/NaOH-treatedplug(oruntreated

plug,forrespirometersthatwillbeusedtocalculateCO2production).a. Pushtheplugdownwithaclean,thininstrument,asyoudidwiththeabsorbentcotton.b. ThisnewplugwillnotabsorbtheKOH/NaOH,whichwill

protectyourspecimenfromtouchingthecausticsubstance.7. Keepyourgogglesandgloveson.Pointthetipofanoxygen

respirometerintoasinkorcontainer.Gently,butfirmly,pushthe

plungerintothesyringetosqueezeouttheexcessKOH/NaOH.a. Youmaywishtorinsejustthecapillarytubebydrawingin

waterormanometerfluid,thenexpellingitagain.

8. Removetheplungerandrepeatwiththerestoftheoxygenrespirometers.


Step3.Runtheexperiment.1. Checkthetemperatureofyourwaterbathtoseethatit’satroomtemperature,about20°C.

a. Ifit’snot,adjustthetemperaturewithwarmorcoldtapwater,makingsurethatthebathiswell-mixedwhenyoumeasurethetemperature.

b. Leavethethermometerinthewaterbath.

2. Put0.5mlgerminatingseedsorotherplantmaterialintotherespirometerbarrel(syringe).Pushintheplungertothe1mlmark.Thiscreatesasealedrespirometerchamberwitha1mlvolume.Becarefulnottodamagetheplanttissues.

3. Repeatwithallrespirometersexceptthecontrol.a. Forthecontrol,useeitherglassbeadsorseedsthathavebeenbakedinsteadofliving

plantmaterial.

b. Foreachoxygen-only/oxygenandcarbondioxiderespirometerpair,trytouseassimilaramassofplanttissueineachaspossible.

4. Drop3-4washersaroundeachrespirometertoactasweightsinthewaterbath.

5. Putyourrespirometersintothewaterbathwiththecapillariespointingupward.a. Keepthecapillariesabovewaterlevel,opentotheair.

b. Makesurethesyringebarrelsarecompletelysubmerged.6. Checkthewaterbathtemperatureagain.7. Usingadroppingpipet,putadropofredmanometerfluidtothetipofeachcapillary.Thedrop

shouldgetsuckedintothecapillary,ifeverythingisworking.Themanometerfluidwillformasealonthechambertocreateaclosedsystem.

a. Youmayhavetousetheplungertopulltheredmanometerfluidintothecapillary,

especiallyforthecontrol.Pullthefluidabouthalfwaydownthecapillary.8. Usingathinpermanentmarker,makeamarkoneachcapillarytubewherethebottomofthe

redmanometerfluidispositioned.Thisisyourzero-timeorstartingdatapoint.Recordthe

temperatureinyourdatatable.

9. Atregularintervals(every2or5minutesshouldwork),markthebottomofthepositionofthemanometerfluid.

a. Recordthetimeandtemperaturewhenyoumakethemarks.b. Ifthemeniscusismovingoffthescale,quicklyrepositionitbygentlypushingordrawing

onthesyringeplungertomovethedropintheappropriatedirection.Besuretonote

thedrop’spositionbothbeforeandafterreadjusting,sothatyoucanlatercalculatetheamountofadjustmentmade.

10. Continuetakingdatauntileitherthemanometerfluidhasreachedthesyringetipor25minutes

haspassed.

Step4.Recordthemanometerreadingsandcalculatethechangesingasvolume.1. Removetherespirometersfromthewaterbath.2. Inyourdatatable,recordthemanometerreadingsforeachmark.Dothisforeachmanometer.


3. Calculatehowmuchthegasvolumechangedduringeachtimeinterval.a. Ifthecontrolrespirometershowedanychange,adjustthevolumechangeforallofthe

otherrespirometersbythatamount.b. Intheoxygenrespirometers,thevolumeshouldhavedecreased.Markthevolume

changeasnegative.

c. Intherespirometersthatdidnotremovecarbondioxidegas,thegasvolumemayhaveincreasedordecreased.Markthevolumeaspositiveornegative,accordingly.

d. Tocalculatetheamountofcarbondioxidegasconsumedinsuchsamples,subtractthe

amountofoxygenproducedintheoxygen-onlyrespirometerfromtheamountofchangeobservedinthecorrespondingoxygen-carbondioxiderespirometer.

e. Graphtheresultingdataaschangeingasvolume(y-axis)overtime(x-axis).

Step5.(Optional)CalculateRespiratoryQuotient(RQ).1. Theamountofvolumechangeintheoxygenrespirometersdescribestheamountofoxygen

produced.

2. Theamountofvolumechangeinaoxygen-carbondioxiderespirometerminustheamountofvolumechangeinitscorrespondingoxygen-onlyrespirometerdescribestheamountofcarbondioxideconsumed.

3. RQmaybecalculatedforeachpairas:RQ=volumeofCO2produced/volumeofO2consumed.4. DeterminethetypeofmaterialbeingconsumedinrespirationbasedonRQ:

a. Iftherewasnonetchangeinthecarbondioxide/oxygentube,itmusthaveproduced

exactlyasmuchCO2asitusedO2,andyouknowthisamountfromtheKOHtube:RQ=1.

b. Ifthecarbondioxide/oxygentubeshowedadecreaseinvolume,theamountofCO2producedwaslessthanO2consumed.TheamountofO2consumedwasequaltowhatyoumeasuredinyourKOHtube,butenoughCO2wasproducedtomovethedrop

partwaybacktowardsitsoriginalposition:RQ<1.c. Ifthecarbondioxide/oxygentubeshowedanincreaseinvolume,theCO2producedis

equaltotheO2consumedintheKOHtubeplusanadditionalamounttogetittothe

finalposition:RQ>1.


MEASURINGPHOTOSYNTHESIS&RESPIRATIONUSINGACOMPUTER-BASEDPROBE

Purpose:Quantifyhowmuchphotosynthesisandrespirationisoccurringinanytypeoftissue,stem,orwholeplant.Thismethodcanoftenbeusedinfieldstudies,ifdesired.

HowtheMethodWorks:ThemethodusesacarbondioxidesensortomeasureCO2levelsinaclosedsystem.Aplantorplanttissuesareplacedintoasealedchamber.Thesensoris

placedintothecontainerthroughaholeinastopperorisbuiltintothechamberitself,thensendsdatatoacomputerasitmonitorsthelevelsofCO2overtime.Thecontainercanbecoveredtocreatedarknessorsubjectedtovariedlightintensities.

TechnicalComplexity:Medium.

TimeRequired:About15-20minutespertreatment.

Materials:• Plantsorplantpartsgrownunderexperimentalconditions,orfoundindifferentfieldconditions• Carbondioxidesensor,functionaloveratleasttherange0–5,000ppm

o Ideally,resolutionandaccuracyshouldbeassmallaspossibleo OnepossibleexampleistheVernierCO2gassensor(CO2-BTA)

• Cabletoconnectsensortocomputerorinterface,ifnotbuiltintothesensor

• Computerorotherappropriateinterface,suchasahandhelddatalogger• Datacollectionsoftware

• Clear,sealablechamber,ifnotbuiltaspartofthesensor• (Optional)Aluminumfoil,darkcloth,orothermeansofcreatingadarkenvironment• (Forlab-basedstudies)Lightsourcethatcanbemovedorwithadjustableintensity

GeneralInstructions:SettingUptheSensorforDataCollectionSensorpartsandset-upoftendependonthemanufacturer.Therefore,onlygeneralguidelinescanbeprovidedhere:

1. Readandcarefullyfollowthemanufacturer’sinstructionsonappropriateset-upanduseof

theCO2sensor.2. Makesuretheappropriatesoftwareisavailableonthecomputerordataloggersothatdata

canbesaved.


" Alternatively,ifusinganinstrumentthatgivesdatareadingsbutcannotsavethem,setupadatatabletorecordthisinformationinyourlabnotebookatregulartime

points.3. Linkthesensortothecomputerordataloggerwithanappropriatedatacable.4. Turnonthecomputer,sensor,andsoftwareintheorderrecommendedbythe

manufacturer.5. Letthesensorwarmupforthelengthoftimethemanufacturersuggests.6. Ifnecessary,calibratetheinstrumentaccordingtothemanufacturer’sinstructionsbefore

collectingdata.

MethodA:SamplingPlantsorPlantPartsintheLab1. Afterthesensorhaswarmedup,recordtheCO2concentrationintheemptychamberasa

baseline.

2. Selectaplantorplantparttotest,andplaceitinthechamber.3. CloseofforsealthechambersothatthesensorisabletomonitorCO2inside.

" Themanufacturer’sinstructionsmayexplainhowtouseabuilt-inchamber.

4. Placethechambernearalightsourceassimilaraspossibletothegrowthconditionsfortheplant.

5. RecordCO2concentrationdata,eitherusingsoftwareorbyhand." Thesensormayneedtimetoadjust,butitwillnotsettleonasinglevalueifthe

plantmaterialsbeingsampledarealive.

" Checkthemanufacturer’sinstructionstodeterminehowlongtowaitbeforetakingadatapoint,ifyouarecollectingdatabyhand.

" Aftersubtractingthebaseline,thesereadingswillgivethenetCO2consumptionor

productionovertime.6. Coverthechamberwithfoil,cloth,orothermaterialsothatitisentirelydarkened.7. RecordCO2concentrationdataasinstep5.

" Aftersubtractingthebaseline,thesereadingswillgivetheCO2productionduetorespirationovertime.

8. TheCO2consumptionduetophotosynthesiscanbedeterminedbysubtractingthe

respiratoryCO2productionfromthenetCO2consumption/production.Thisvalueshouldbenegative.

9. (Optional)YoumaywishtotestthenetCO2consumptionorproductionatseverallightintensitiestounderstandwhetherthenetCO2consumption/productionchangesbasedonthisvariable.

10. Testtheotherplantsamplesusingthesameprocedure.

MethodB:SamplingPlantsintheFieldForfieldstudies,ahandhelddataloggerorlaptopwillbetheeasiesttouse.

1. Setupandtesttheequipmentinthelabtobesureitworksbeforegoingintothefield.


2. Bringthesensor,datainterface,chamber,andmeanstodarkenthechambertothefieldwithyou.

3. SetuptheequipmentasdescribedintheGeneralInstructions.4. Afterthesensorhaswarmedup,recordtheCO2concentrationintheemptychamberasa

baseline.

5. Selectaplantorplantparttotest,andplaceitinthechamber." Certainchambersmaynotneedtheplantpartremovedtotestit.Ifpossible,leave

theplantintact.

6. CloseoffthechambersothatthesensorisabletomonitorCO2inside." Themanufacturer’sinstructionsmayexplainhowtouseanybuilt-inchamber.

7. Placethechambersothattheplantorplantpartreceivesasclosetoitsnaturallightingaspossible.Besuretostandsothatyoudonotcastanyshadowsoverthechamber!

8. RecordCO2concentrationdata,eitherusingsoftwareorbyhand.

" Thesensormayneedtimetoadjust,butitwillnotsettleonasinglevalueiftheplantmaterialinthechamberisalive.

" Checkthemanufacturer’sinstructionstodeterminehowlongtowaitbeforetaking

adatapoint,ifyouarecollectingdatabyhand." ThesereadingswillgivethenetCO2consumptionorproductionovertime,after

subtractingthebaseline.

9. Coverthechamberwithfoil,cloth,orothermaterialsothatitisentirelydarkened.10. RecordCO2concentrationdataagain,asinstep8.

" ThesereadingswillgivetheCO2productionduetorespirationovertime,relativeto

thebaseline.11. TheCO2consumptionduetophotosynthesiscanbedeterminedbysubtractingthe

respiratoryCO2productionfromthenetCO2consumption/production.Thisvalueshouldbenegative.

12. Testotherplantsamplesusingthesameprocedure.


VISUALIZING&COUNTINGSTOMATAUSINGTHELEAFIMPRESSIONMETHOD

Purpose:Todeterminestomataldensityandexaminethestateofstomatainleaves.

HowtheMethodWorks:Paintingnailpolishontothesurfaceofaleafcreatesanimpressionofitsoutercellularstructure.Theimpressionispeeledfromtheleaf,thenexaminedunderamicroscopetoviewhowwidethestomataareopenedandhowmanyarepresentperunitarea(stomataldensity).

TechnicalComplexity:Moderate.Itcantakesomepracticetolearnhowtobringspecimensintofocuswithoutdamagingthelenses.

TimeRequired:60minutesfromset-uptocompletion.

Materials:• Leaves(1-2pertreatment)• Clearnailpolish

• Compoundmicroscopewith40Xobjective• Preparedleafanatomyslides,ifavailable

• Blankmicroscopeslides• Dissectingprobeorotherpointedinstrument• Forceps

• Distilledwater• Permanentmarker• Metricruler

Procedure:1. Inyournotebook,describetheleavesyouwillsample.

a. Indicatewhereandwheneachleafwasgathered(e.g.,sunorshade,timeofday,season).b. Describethespeciesortypeofplant,ifyouknow.

c. Makeaphotographorsketchoftheleaves,usinglabelsorfilenamestohelpyoukeeptrackforlater.

2. Preparetwoepidermalimpressionsfromeachleaf–onefromtheundersideandonefromthetop.

a. Painta1-cmx1-cmsquareofclearnailpolishontothesurfaceoftheleaf." Makesuretherearenogapsinthelayerofnailpolish.

Thelargedonutshapeaboveismadeupof

thetwocellsthatformastomate.Stomataareopeningsthroughwhichgasesenter

andleavetheleaf.Thecellsaroundthepore,calledguardcells,openandcloseto

maketheporebiggerorsmaller.


" Severalcoatsareokay,sinceyoudon’twantthenailpolishtotearasyoupeelitofftheleaf.

b. Allowthenailpolishtodrythoroughly.

3. Setupandpracticeusingthemicroscopewhilewaitingforthenailpolishtodry.Askyourteacherforassistance.

a. Turnthemicroscopelighton.b. Movethestagefarfromtheobjective,usingtheappropriateknob.c. Pushthelowestpowerobjectiveintoplace(usually4Xor10X).

d. Placeapreparedslideofaleafonthestage.e. Lookingatthestagefromtheside,movethestageclosetotheslidewithouttouchingthe

slideitself.

" Knowwhichwaytoturntheknobtomovethestageawayfromthesample." Becareful.Ifyoucrunchtheslideagainsttheobjective,theobjectivewillbe

permanentlydamagedandexpensivetoreplace.

f. Lookintotheocular." Onlymovethestageawayfromthesampleforroughfocus.

" Usetheotherknobformoresensitivefocus.g. Onceyoucanseethestructuresunderlowpower,youcanswitchtothe40Xobjective.

" Onlyusethesensitive-focusknobatthispower.Theobjectiveislonger,soit’s

easiertoaccidentallydamageitbybumpingintotheslide.

4. Prepareyourmicroscopeslidesoftheleafimpressions.a. Gathertwomicroscopeslidesforeachleaf–oneeachfortheimpressionfromtheupper

andlowersurface.

b. Youwillbeliftingoffeachnailpolishsquare(leafimpression)asonepiece." UseadissectingprobeorotherpointedinstrumenttoGENTLYteasetheedgeofthe

nailpolishup,liftingorpeelingthenailpolishawayfromtheleafuntilabouthalfwaylifted.

" Useforcepstofinishpeelingawaythesquare.

" Rememberwhichsideofthepeelwasfacingtheleaf.c. Placeadropofdistilledwaterontoamicroscopeslide.d. Puttheimpressionontothesurfaceofthewaterwiththesidethatwastouchingtheleaf

facingup,awayfromthewater.e. Gentlysmoothouttheimpressionusingthedissectingprobe,sothatit’sflatagainstthe

slide.

f. Withapermanentmarker,labeltheslidetoindicatetheleafsampledandwhichsideoftheleaftheimpressioncamefrom.

g. Repeatstepsb-fwiththeremainingleafimpressions.

1. Examinetheimpressionsunderthemicroscope.a. ReviewStep3forsafeuseofthemicroscope.


b. Focusontheimpression,butrememberthatyouarelookingataclearimpressionoftheleafsurface,notagreenleaf!

c. Takenotesandmakesketchesofwhatyousee.

2. Collectdataonthestomata.a. Findtheimpressionsofstomata.

" Aretheyopen,closed,inbetween,oramix?Takenotes." Makeaquicksketchofthestateofanaveragestomateintheimpressionforeach

sample.

b. Countthenumberofstomatainthe1cmx1cmimpressionandrecordthenumber." Countityourselftwice." Haveoneotherteammatedothecounttwiceforthesameslidetodoublecheck.

" Ifmanystomataarepresent,considerdoingacountusingthelowest-powerobjectiveinsteadoftheentireimpression.Calculatetheareaofthefieldofview(p.16)tofindthesamplingarea’ssize.

" Stomataldensitycanbecalculatedasthenumberofstomatespercm2.


VISUALIZINGPLANTCELLS&CHLOROPLASTSUSINGAMICROSCOPE

Purpose:Tovisualizechloroplastsandplantcells.

HowtheMethodWorks:Photosynthesisoccursinplantorganellescalledchloroplasts.Becauseoftheirgreencolor,chloroplastscanbeseeneasilyusingalightmicroscope.Awetmountslideofacell

layerteasedfromaleafispreparedandviewedat400Xmagnification.

TechnicalComplexity:Moderate.Youmayneedtomakeseveralsamplestogetathinenoughlayertoseetheindividualcellsclearly.Itcantakesomepracticetolearnhowtobringspecimensintofocuswithoutdamagingthelenses.

TimeRequired:30minutes.

Materials:• Compoundlightmicroscopewith40Xobjectiveandatleastonelower-

poweredobjective• Lenspaper• Leavesorotherorgansfromplants,oralgaecultures

• Scalpel• Microscopeslidesandcoverslips

• Eyedropperorsmallpipette• Water• (Optional)dissectingneedles

• Forceps

LabSafety:Scalpels,brokencoverslips,andbrokenslidesareverysharp.Disposeofthesematerialsinthe“sharps”containerorintheglassdisposal,NOTtheregulartrashcan.Wearingwell-fittinglabglovesis

recommendedtohelppreventaccidentalcuts.

Procedure:1. Beforeyoubegin,carefullycleantheobjectivesandeyepiecesofyourmicroscopewithlenspaper.

2. Prepareaslideofyourspecimen(s):

a. Selectaleaforotherpartoftheplant.Useascalpeltosliceaverythinfragmentfromtheleaf.


" Alternatively,placeadropofalgalcultureontoaslideusinganeyedropperorpipette.

b. Placeadropofwaterintothecenterofamicroscopeslideusinganeyedropperorpipette,thentransfertheleaffragmenttothewaterontheslide.

" Ifusinganalgalculture,thereisnoneedtoaddadditionalwater.

c. Gentlyteaseapartthetissueinthewaterdropletusingthescalpelortwodissectingneedles.

d. Placeoneendofaglasscoversliptotherightorleftofthespecimensothattherest

oftheslipisheldata45oangleoverthespecimen.e. Slowlylowerthecoverslipwithadissectingneedleorforcepssoasnottotrapair

bubbles.Thecoverslipflattensthepreparation,keepsitfromdryingout,andprotectstheobjectivelenses.

f. Pressdownonthecoverslipvery,verylightlywiththeendofthedissectingprobe

orforceps.Thisspreadsandflattensthetissue,soitiseasiertoseeonecelllayer.

3. Observeyourspecimen(s)underthemicroscope:a. Ifyouhavenotusedamicroscope(recently),askateacherforassistanceandseepp.13-14

forpracticeinstructions.b. Beginbyusingthelowest-powerobjective.c. Locateapartoftheslidethathasseemstohavethefewestlayersofcells.

d. Usethefocustohelpdistinguishindividualcells." Abouthowlargearethecells?" Wherearethecellwalls?

" Canyouseeanychloroplastsatthisscale?" Makeasketchofyourobservations,notingthetotalmagnificationinoralongside

eachdrawing.

" Providesomebriefnotesaboutthesample.Wasitpartofanexperiment?Ifso,towhattreatmentwasitsubjected?

" Alternatively,ifyouhaveadigitalcameratotakemicroscopephotos,include

informationaboutthetotalmagnificationandthesampleinthefilename.Writethefilenameinyourlabnotebook.

e. Nowusethe40Xobjectivetolookatthesample.Again,makeasketchordigitalphotoofyourobservations.Trytoanswerthefollowingquestionsinyourlabnotebook:

" Wherearethecellwalls?

" Wherearethechloroplasts,andhowarethepositionedrelativetothecellwalls?

" Abouthowmanychloroplastsareineachcell?

" Doyounoticeanymovementwithinthecell?Ifso,whatisthis?f. Repeatwithanyothersamplesyouwanttoexamine.

4. Cleanupwhenyouarefinished:


a. Washanddryallslidesandplacetheminaglasscontainernearthesink.b. Slipcoversmaybedisposedofintheglassdisposal.

c. Turnoffthemicroscope,coverit,andputitintothestoragecabinet.

Whatisthetotalmagnificationofmyspecimen?

Multiplythemagnificationoftheocularlens(usually10X)bythatoftheobjectivelens.

Example:

Supposeyouhavethe40Xobjectiveinplace.Then

thetotalmagnificationwouldbe:

10x40=400

thatis,400X.

Howbigismyspecimen?

Thefieldofviewisthefullareayoucanseeontheslidewithoutmovingit.Athighmagnification,your

fieldofviewissmall.Atlowmagnification,itislarger.Ifyouhaveaflat,clearruler,placeitonthestage.

Measurethediameteracrossthemiddleofthefieldwhenlookingintothemicroscopewitheachofthe

objectivelenses.Usingtheformulafortheareaofacircle,calculatetheareaofeachfieldofview.

Byestimatinghowmanycellsareinthefieldorhow

muchofthefieldistakenupbyonecell,youcangetaroughestimateofacell’ssize.Thestandardunitof

measureinlightmicroscopyisthemicrometer(µm),so

it’sbesttodescribethefieldofviewandspecimensizesonthisbasis:1mm=0.001µm.


IDENTIFYINGSTARCHINPLANTLEAVESUSINGANIODINESTAININGMETHOD

Purpose:Toexaminestarchgrainsasameasureofphotosyntheticand/orrespiratoryactivity.

HowtheMethodWorks:Themethodallowsyoutoseestarchinleafdisks.Starchisapolysaccharidemadeupofglucosemolecules.Plantscommonlyuseitasastoragecarbohydrate.Here,leafdisksarepreparedfromplantsgrownunderlightanddarkconditionsbeforebeingtreatedfor24hourswithenvironmentalconditionsofyourchoosing–differentlightintensities,wavelengthsoflight,light-darkcycles,temperatures,

CO2levels,orglucosesolutionsarejustafewpossibilities.Afterplacingleafdisksinanexperimentaltreatment,theyarestainedwithLugol’sSolution.Starchgranuleswillappearpurplish-blackunderamicroscope.Theirrelativedensityunderdifferenttreatmentscanbeassessed.

TechnicalComplexity:Moderate.

TimeRequired:Twoclassperiods.

Materials:PreparationPhase:

• PotassiumIodide• Iodine• Water

• Containerofabout75mL• Plantsgrowninthelightandplantsgrown

inthedarkforatleast4days.

• Paperholepunch,plasticstraw,orNo.3corkborer

• Lightsourceforlight-grownleafdisks

• Aluminumfoilorotherlight-blockingmaterialfordark-grownleafdisks

• (Optional)Materialstoprovidean

environmentaltreatment

StainingPhase:

• Hotplateorsomeothermeanstocreateheatedliquidbath

• Beakerofwaterwithboilingchips

• Beakerofethanol• Forceps• Timerorclock/watchwithasecondhand

• Papertowels• Aluminumfoil,Petridish,orshallow

container

• Water• Compoundmicroscopewith40Xobjective


Procedure:Part1.PrepareLugol’sSolution(ifnecessary,5minutes):3. Measure60mLofdistilledwaterintoacontainer.

4. Weighout0.4gofpotassiumiodide.5. Transferthesolidintothecontaineranddissolve.6. Add0.2giodinetothesolutionandstirorswirltomix.

Part2.PrepareLeafDisks(10minutes):1. Useapaperholepunch,plasticstraw,orNo.3corkborertopunchoutyourleafdisks.

o Avoidtheheavyveins.

o Cutoutequalnumbersof leafdisksfromthelight-grownanddark-grownplants,using1-2leavesforeachenvironmentaltreatmentyouplantocarryout.

o Includeatleastonehealthygreenleafandonedark-growninadditiontoyourtestsamples.

Theseareyourcontrols,andtheywon’tbetreatedduringthe24hourperiod.o Alternatively,youcancutsquaresfromtheleavesusingscissors.

2. Dividetheleafdisksintoequivalently-sizedtreatmentgroups.o Ifyouhaveindexcardsorsmallpiecesofpaper,puttheleafdisksforeachtreatmentonto

theirowncard.Thiswillmakeiteasiertokeeptrackofthedisksforonetreatment.

3. Placethecontrolsintothesameconditionsthatwereusedduringpre-treatmentoftheleaves,lightanddark.

4. Place the treatmentgroups into their respective treatmentconditions. Treat the leafdisks for24

hours.

Part3.StaintheLeafDisksforStarch(30minutes):1. Wearsafetygoggles,alabcoat,andgloves.Youwillbeworkingwithboilingwaterandhotethanol

thatcancauseburns,andadyethatcanstainyourskinandclothing.

2. Prepareaboilingwaterbath.Askyourteachertoprepareahotethanolbath.3. Usingtheforceps,dropaleafdiskintotheboilingwaterbath.Leavefor30seconds.Thiswillkill

thecells.

4. Carefullyremovetheleaffromtheboilingwaterwithyourforcepsandplaceontoapapertowel.5. Repeatwiththeotherleafdisks,makingsuretokeeptrackofwhichtreatmenteachdiskwasgiven.6. Usingyourforceps,transferaleafdiskintothehotethanolbathandleaveittherefor2minutes.

Thiswillremovemostofthechlorophyllfromtheleaf.7. Withyourforceps,transfertheleaffromthehotethanolbathtoaroomtemperatureethanolbath

(inaglassbeaker,flask,oropenjar).Leavetheleafintheroomtemperatureethanolforone

minute.Theleafshouldbenearlywhite.8. Removetheleaffromtheroomtemperatureethanolandblotitonapapertowel.

9. RepeatSteps6-8withtheotherleafdisks,makingsuretokeeptrackofthetreatmentforeachdisk.10. Usingasquareofaluminumfoil,makeasmall“bowl,”oruseaPetridishorshallowcontainer.


11. PlaceenoughLugol’sSolutionintothebowltocoverthebottom.12. Usingtheforceps,putyourblottedleafdisksintothebowlofLugol’ssolution,eachforabout1

minute.13. Removetheleafdisksandrinsethemoffwithwater.14. Blottheleafdisksonpapertowels.

Part4.RecordData(15minutes):1. Recordthecolorintensityofyoursamplesandvariationswitheachsample.

o Starchwillbestainedadarkreddish-brown.

o Itmaybehelpfultocreateacolorguideorrankingsystemtojudgecolorintensity.o Inwhatwayarelight-grownanddark-grownsamplesdistinctfromeachother?o Aresamplesfromdifferentenvironmentalsamplesdistinctfromeachother?

2. Makeasketchofrepresentativesamplesofleafdisksfromeachtreatmentleafdisk,includingcolorvariations.

3. Visualizerepresentativesamplesfromeachtreatmentunderamicroscopeusinga40Xobjective,

andsketchwhatyousee.o Ifyouhavenotusedamicroscope(recently),askateacherforassistanceandseepp.13-14

forpracticeinstructions.


IDENTIFYINGCHLOROPHYLL&OTHERPLANTPIGMENTS

Purpose:Identifyingthepresenceorabsenceofchlorophyllandotherplantpigmentsinleaves.

HowtheMethodWorks:Allplantpigmentshaveuniquechemicalproperties,allowingustotellthemapart.Here,paperchromatographywillbeusedtoseparateplantpigments.First,fluidissqueezedoutofplantleavesontofilterpaper.Thepaperisputintoachambersothatitstiptouchesachromatography

solvent--inthiscase,acetoneorisopropylalcohol.Asthesolventisabsorbedandtravelsupthefilterpaper,theplantpigmentsarecarriedwithit.Eachpigmentwillmoveupthepaperatacharacteristicratedependingonitschemicalproperties,includingsolubilityinthesolvent,molecularmass,andamountofhydrogenbondingwiththefilterpaper.Sincethepigmentsarecolored,thesinglesamplewillseparateintopigmentbandsofdifferentcolors.


TimeRequired:60minutesfromset-uptocompletion.

Materials:• 1-2plantleavespertestsample• Jars,wide-mouthedtesttubes,400-600mLbeakers,10mLgraduatedcylinders,orothervesselof

similardepth,Coverssuitableforthevessels

• 3mmlaboratoryfilterpaper(orbleachedcoffeefilters,butthesedon’tworkaswell)• Metricruler• Pencils

• Scissors• Coin• Acetone(fingernailpolishremover)orisopropylalcohol(rubbingalcohol)

• Thindowel,straws,orpaperclips• Tape

Procedure:1. Selectoneormoreidentically-sizedvesselstouseaschromatographychambers.

o Measuretheirheight,sothatyouwillknowhowlongtocutyourpaperstrips.


o Ifyouhavemanysamplestotest,makesurethechamber(s)arelargeenoughforonevertically-placedchromatographystripperplantsamplewithoutanyofthestripstouching

anyothersorthechamber’ssides.

2. Preparethechromatographypaperstrips:a. Handlethesheet(s)offilterpaperfromtheedges,andaslittleaspossible.Thenaturaloils

fromyourhandscaninterferewithmovementofsolventupthestrip.b. Usingaruler,measureoutthesizeofthestripsontothefilterpaper.

" Allstripsshouldbeaslongasthedepthofthechromatographychamber.

" Theyshouldbeabout1.5cmwide." Measureonestripforeachsample.

c. Cutthestripswithscissors.

d. 1cmfromthebottomofeachstrip,lightlydrawapencillineasshownatright." DoNOTuseapenormarker.Pigmentsfromtheinkwillinterferewiththetest!

e. Cutthebottomintoan“arrowpoint”uptothe1cmmarkasshownatright.

3. Preparetheextractfromaleafsampledirectlyonastrip:a. Placethepaperstriponthetable.b. Positionyourleafoverthepencilmark,rememberingnottoplaceyour

fingersontothemiddleofthestrip.c. Traceoverthepencilmark,nowunderneaththeleaf,byrollingtheedgeof

acoinovertheleafabout15times.

d. Repeatthisprocesstwomoretimeswithanunusedportionofthesameleaf(oranotherfromthesameexperimentaltreatment)inthesameplaceonthestrip.Thiscompletesonestrip.

e. Repeatthisentireprocessforeachleafsampleandstrip.f. Allowtheleafextractsonthestripstodryforabout10minutes.

4. Preparethechromatographychamber(s):

a. Isopropylalcoholandacetonearevolatileandflammable.Wearsafetygoggles,avoidinhalingthefumes,anddonotusenearflames.

b. Fillthechamber(s)witheitherisopropylalcoholoracetoneto1cmindepth.

c. Coverthechamber.Allowthechambertofillwithfumeswhileyourstripsaredrying.d. Howyoucreatethesetuptorunsampleswilldependonthechambersizeandshape.

Forexample,youcouldattachthedriedstripstoapencil(left).

Oryoucouldhookapaperclip

throughtheflatendofastrip

andthenthrougharubberstopper(right).


5. Runthechromatographysamples:a. Openthechamberandloweryourstrip(s)intoitsothetipsaretouchingthesolventandthe

plantextractlineiswellabovethesolvent.

" Iftheplantextracttouchesthesolvent,itwilldissolveintotheliquid!" Makesurethesidesofthestripdonottouchthewallsofthechamber." Makesurethestripsarelevel.

b. Coverthechamberandletthesolventwickupthestrips.Donotdisturbthechamberwhileitiswicking.

" Theleafextractwillbegintoseparateintobandsofcolorasittravelsupwards.

c. Stopthechromatographywhenthesolventreaches2cmfromthetopofthestrips,orwhenyouseeatleastfourcolorbandsclearlyseparatedonthestrips.

d. Removethestripsfromthechromatographychamber(s).

6. Markandidentifythedistinctplantpigmentsforanalysis:a. Inpencil,drawahorizontallinewherethesolventstoppedmovingupeachstrip.b. Drawsimilarpencillinestomarkthecolorbandsoneachstrip.

c. Measurethedistancefromthelinewhereyouoriginallyputyourleafextractwiththecoin(theorigin)toeachofthelinesyouhavemarked.

" Thedistancebetweentheoriginandthefinalsolventlineisconsideredtobethe

distancethesolventtraveled." Thedistancebetweentheoriginandtheindividualpigmentlinesarethedistances

thepigmentstraveled.d. Identifythepigmentsoneachstrip.Theorder,fromtoptobottom,shouldbe:

" carotenes(yellow-orange)

" xanthophylls(yellow)" chlorophylla(brightgreentoblue-green)" chlorophyllb(yellow-greentoolivegreen)

" anthocyanin(red)" Youmaynotseeallofthesecolors.Whatyouseedependsonwhatpigmentsare

presentineachleaf.

e. Foreachpigment,calculatetheRfvalueas:Rf=DistancepigmenttraveledDistancesolventtraveled

7. Cleanupwhenyouarefinished:a. Youmightnotbeallowedtopourthesolventdownthesink,soaskyourteacherhowyou

shoulddiscardit.

b. Afterdiscardingthesolvent,rinseoutthechamberswithwater.Leavenearthesinktodry.


c. Donotthrowyourstripsawayuntilyouhaverecordedallthemeasurementsandcarriedoutallthecalculations.


QUANTIFYINGFRESH&DRYMASSOFPLANTS

Purpose:Determinechangesinplantmassovertime.

HowtheMethodWorks:Therearetwowaystomeasureplantmass:freshorwetmassanddrymass.Inthefreshmassmethod,

thewholeplantisweighedoncethesoilisremoved.Forthedrymassmethod,plantsaredriedatlowheatbeforeweighing.Theplantsthatareweighedcannotbeusedforfurtherstudies,butyoucandeterminethefreshmassofaplantandthendryittoalsodetermineitsdrymass.Thisallowsyoutocalculatethewatercontentofthatplant.Youshouldusethemethod(s)bestsuitedtoyourresearchquestion.Forexample,aquestionabouttheamountofcarbonfixedduringgrowthisbettersuitedtomeasurementsofdrymass,sincethewatercontentofaplantcanvary.


TimeRequired:About5minperplant.Fordryweightmeasurements,anovernightdryingtimeisrequired.

Materials:• Sinkwithrunningwater• Papertowels• Ascalewithmilligram(0.001g)accuracy

• Adryingoven• (Optional)Paperlunchbags• (Optional)Ziplocsandwichorgallon-sizedbags

GeneralInstructions:• Removingaplantfromitsgrowingmediumwillusuallyaffectitsgrowthrate,possiblydamaging

itintheprocess.Dryingovernightwillkillnearlyallplants.So,bothfreshanddrymass

measurementsaretypesofdestructivesampling.• Tomeasurechangesinplantmassovertime,thetotalnumberofplantsneededforthe

experimentwillbethenumberofplantssampledateachtimepoint(typicallythreeormore)

multipliedbythetotaltimepointsatwhichmeasurementsaretaken.

MethodA:MeasuringFreshMassThismethodisthefastestwaytomeasureplantmass.Becauseaplantcanbecomposedofvaryingamountsofwaterunderdifferentenvironmentalconditionsordevelopmentalstages,itislessaccurate

thanmeasuringdrymass.


1. Removeplantsfromsoilandwashoffanyloosesoil.2. Blotplantsgentlywithsoftpapertoweltoremoveanyfreesurfacemoisture.

3. Weighimmediately.o Plantshaveahighwatercomposition,sowaitingtoweighthemmayleadtosome

dryingandproduceinaccuratedata.

4. (AlternativeOption)Freshmassforrootandshootdatamaybetakenseparatelybycuttingtheshootsatthe“crown”oftheplant,thenweighingeachpartseparately.Thecrown,orplacewhereaplant’srootsandstemmeet,isusuallyfoundatsoillevel.

MethodB:MeasuringDryMassThismethodremoveswaterfromtheplantsbyfirstdryingtheminanoven.Sinceplantscontainlotof

water,usingdryweightisamorereliablemeasurethanfreshweight.1. CompletethefirsttwostepsdescribedinMethodA.2. Ifdesired,completeStep3orStep4fromMethodAaswell.Youwillendupwithbothfresh

anddrymassdata.3. Drytheplantsinanovensettolowheat(60oC)overnight.

o Ifyouhavemanyplantstodry,itmaybehelpfultoplacethemindividuallyinpaperlunchbags.

o Uselabelsormakeachartofthedryingpositionstokeeptrackoftheirrespective

treatments.4. Lettheplantscoolinadryenvironment.

o Inahumidenvironmenttheplanttissuemaytakeupwater.

o Aplasticsandwichbagwillkeepmoistureoutifyouliveinahumidclimate.5. Oncetheplantshavecooled,weighthemonascale.

o Plantscontainmostlywater,sothedriedplantswillnotweighverymuch.

o Makesureyouhaveascalethatmeasuresmilligramstoensureaccuracy.

MethodC:CalculatingWaterContentWatercontentcanpotentiallybeusefultoidentifyplantsthathaveadifferentresponsetotheirenvironment.Forexample,plantslivingunderhightemperatureorlowwaterconditionsmayhavea

lowerwatercontentthanwell-wateredplantsatamoremoderatetemperature.1. CompletethefirsttwostepsinMethodA.

2. Weighthewholeplantortherootsandshootsseparatelyasdesired.3. Placeeachplant,shoot,orrootsysteminitsownpaperlunchbag,andmarkthemtokeeptrack

ofwhichplantiswhich.

4. Drytheplantsinanovensettolowheat(60oC)overnight.5. CompletethelasttwostepsofMethodB,makingsuretorecordthedataalongsidethe

correspondingfreshmassdataforeachplantorplantpart.

6. Thewatercontentofaplantsample(%water)canbecalculatedasfollows:a. Watermass(g)=Freshmass(g)–Drymass(g)b. %water=100%xWatermass(g)


Freshmass(g)

PlantingScience Power of Sunlight Toolkit

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