PlantingScience
PowerofSunlightToolkit
BACKGROUND:
ThePlantingSciencePowerofSunlightToolkitprovidesbackground,materialslists,detailed
procedures,andsafetyconsiderationsforadditionalexperimentalmethodsrelatedtophotosynthesis
andrespiration.Thesetoolscanprovidestudentstheopportunitytoaskawiderrangeofresearch
questionsduringtheopeninquiryphaseofThePowerofSunlightthanwouldbepossibleusingonlythe
leafdiskfloatationmethod.Alternatively,teachersmayselectoneormoreofthesemethodsas
classroomdemonstrationsofphotosynthesisand/orrespirationinaction.
CONTENTS:
Page
PreparationofCitrate-PhosphateBufferforMaintainingpH ......................................................................2
MonitoringpHtoAssessPhotosynthesis&RespirationofAquaticPlants ..................................................4
MeasuringCellularRespirationUsingaRespirometer .................................................................................7
MeasuringPhotosynthesis&RespirationUsingaComputer-BasedProbe ...............................................11
Visualizing&CountingStomataUsingtheLeafImpressionMethod .........................................................13
VisualizingPlantCells&ChloroplastsUsingaMicroscope.........................................................................15
IdentifyingStarchinPlantLeavesUsinganIodineStainingMethod .........................................................17
IdentifyingChlorophyll&OtherPlantPigments ........................................................................................19
QuantifyingFresh&DryMassofPlants .....................................................................................................22
.
PlantingScience CC BY-NC-SA 3.0
|
www.plantingscience.org
|
Power of Sunlight—Toolkit Page 1 of 31
Last Updated 7/2013
PREPARATIONOFCITRATE-PHOSPHATE
BUFFERFORMAINTAININGPH
Purpose:Theadditionofincreasingamountsofsodiumbicarbonateto
watertendstoincreasetheacidityofthesolution,loweringitspH.Most
cellsthrivebestinthepHrangeof6to8;therefore,leafdisksinfiltrated
withhigh-concentrationsodiumbicarbonatesolutionsmayhavealtered
levelsofphotosynthesisandcellularrespiration.
AbufferresistspHchangeandthushelpsasolutionmaintainthesamepHunderawiderange
ofconditions.Awiderangeofpossiblebuffersareavailable,butmanycommonbuffershave
problemsinthepH6to8range.TohelpmaintainpHinleafdiskfloatationexperiments,we
suggesttheuseofaphosphate-citratebuffer,asdescribedhere.
TechnicalComplexity:Simple.
TimeRequired:20minutesforfullprocedure;5min.topreparebufferfromstocksolutions.
Materials:
Perclass:
Citricacid(anhydrous) Disodiumphosphate Water Balance,accuratetoatleast0.1g,Weighingpaper
Scoopula
Two1000mLgraduatedcylinders
Two1000mLstockbottleswithcapsorstoppers
Perteam:
100mLstockbottle
100mLgraduatedcylinder
Water
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.
PlantingScience CC BY-NC-SA 3.0
|
www.plantingscience.org
|
Power of Sunlight—Toolkit Page 2 of 31
Last Updated 7/2013
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
42.0
55.8
58.0
5.4
5.6
39.5
36.8
60.5
63.2
5.8
6.0
33.9
30.7
66.1
69.3
6.2
6.4
27.2
22.7
72.8
77.3
6.6
6.8
17.6
13.0
9.1
82.4
87.0
90.9
7.0
7.2
7.4
6.3
4.2
93.7
95.8
7.6
7.8
2.7
97.3
8.0
PlantingScience CC BY-NC-SA 3.0
|
www.plantingscience.org
|
Power of Sunlight—Toolkit Page 3 of 31
Last Updated 7/2013
MONITORINGPHTOASSESSPHOTOSYNTHESIS&
RESPIRATIONOFAQUATICPLANTS
Purpose:UsepHasanindirectmeasureoftheamountofcarbon
dioxideinanaquaticsystemcontainingalgaeoratleastone
aquaticplant.
HowtheMethodWorks:Carbondioxide(CO2)isoneofthe
productsofcellularrespirationandacriticalreactantin
photosynthesis.ThesimplestwaytomeasureCO2changesinanaqueoussolutionisto
measurepHchange.Carbondioxidedissolvesinwatertoformcarbonicacid:CO2+H2O!
H2CO3.Therefore,asCO2isreleasedbyaquaticorganismsduringrespiration,itformsaweak
acidinthesurroundingwater,loweringthepH.AsCO2isconsumedbyaquaticplantsduring
photosynthesis,thepHwilltendtoincrease.Similartotheleafdiskinfiltrationmethod,the
dissolvedcarbondioxideinanaquaticplant’senvironmentcanbesupplementedbyadding
sodiumbicarbonate.
Technically,pHisthenegativelogarithmofasolution’shydrogenionconcentration.Free
hydrogenionsareacidic,sothepHscaleisusedtoindicatehowacidicasolutionis,witha
lowernumberindicatingamorestronglyacidicsolution.Eveninpurewater(H2O),afew
moleculesarealwaysdissociatedintohydrogenions(H+)andhydroxideions(OH-)–aboutone
intenmillion(1/10,000,000)moleculesinaliterofpurewater.Inotherwords,thereare10-7
hydrogenionsperwatermolecule.ThepHofpurewateristherefore–log(10-7)=7.Thisis
consideredneutralpH,becauseeachacidicH+isaccompaniedbyonebasicOH-inpurewater.
EveryunitchangeinpH,e.g.,frompH7topH8orfrompH6topH5,indicatesaten-fold
changeinacidity.Ifmoreacidisaddedtothewatersothatoneineverythousandmoleculesis
ahydrogenion,theconcentrationis10-3andthepHis–log(10-3)=3.Thisconcentrationhas
10,000timesmorehydrogenionsthaninpurewater,sothesolutionis104timesmoreacidic
anditspHis4unitslower.
TechnicalComplexity:Simple.
TimeRequired:5minutesperreading.
Materials:
Inawater-filledvessel,algaeoraquaticplantsuchasElodea
(Optional)Oneortwosmallercontainersforeachtreatment
Oneofthefollowingsets,dependingonthemethodyoupreferorthematerialsavailable:
PlantingScience CC BY-NC-SA 3.0
|
www.plantingscience.org
|
Power of Sunlight—Toolkit Page 4 of 31
Last Updated 7/2013
METHODA
pHindicatorpaper
Color/pHkeychart
Scissors
METHODB
pHmeter
pHbufferstock
Smallcup
Squirtbottleofdistilledwater
METHODC
PhenolRedorBromphenolBluedyepowder
Scoopulaorspoon
Long-termstoragecontainer
Water
Graduatedcylinder
Electronicbalance
GeneralInstructions:
•
•
•
•
•
Ifthecontainerfortheaquaticplantisverylarge,pHwilltakealongtimetochange.Youcan
transfertheplantorapartofittoacontainerwithlessthan50mLofwatertomonitormore
rapidchangesinpH.
AlgalculturesarebettersuitedtoMethodAorMethodBthantoMethodC,becausetheirgreen
colorwillalterhowtheliquidappears.
Youmaywishtoremoveaquaticplantsfromtheircontainers,blottheexcesswater,andusean
electronicbalancetofindtheirmassforexperiments.
o Largerplantswillcarryoutmorephotosynthesisandrespiration,sotreatmentsare
moreequivalentiftheycontainthesameamountsofplantmass.
o Foralgalcultures,stirringwellandusingthesamevolumeofculturewillprovide
equivalenttreatments.
Forexperiments,repeatthepHmeasurementatregularintervals,e.g.,every5min,underthe
testconditions.
Youmayconcludetheexperimentafterapredeterminedtimeperiod(e.g.,30min,recording
thefinalpH)orafterapredeterminedamountofpHchangeoccurs(e.g.,0.5units,recordingthe
finaltime).
o MethodCisbestsuitedtorecordingthefinaltime,sinceitcanbedifficulttoassess
subtlechangesincolorofthesolutionandyoumightnothaveacolorkeyfor
intermediatecolors.
MethodA:MeasuringpHUsingIndicatorPaper
ThisisthesimplestwaytomeasurepH.IndicatorpaperissimplypaperimpregnatedwithapHindicator
dye.ThedyechangescolordependingonthepHofthesolution.
1. Tearoffabouta2cmstripofindicatorpaper(ifinrollform)orpullapiecefromitscontainer(if
instripform).
2. Dipthetipofthepaperintothewatercontainingtheaquaticplant.
3. Comparethecolorofthewetpapertothecolor/pHkeychart,usuallyfoundontheindicator
papercontainer.ThebestmatchindicatestheapproximatepH.
4. Useanewstripofindicatorpaperforeachmeasurement.
MethodB:MeasuringpHUsingapHMeter
ThisisthemostaccuratewaytomeasurepH.ThepHmeterhasanelectrodewhichmustalwaysbe
keptmoist.Whenitisnotinuse,keeptheelectrodecoveredorimmersedinabuffersolution.
PlantingScience CC BY-NC-SA 3.0
|
www.plantingscience.org
|
Power of Sunlight—Toolkit Page 5 of 31
Last Updated 7/2013
1. TurnonthepHmeterandallowitwarmupforatleastfiveminutes.Keepthecontrolknobin
standbyposition.
2. Ifthereisone,adjustthetemperatureknobtomatchthesolutiontemperatureafterwarm-upis
complete.
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:MeasuringpHUsinganIndicatorDye
IndicatordyesarepowdersthatchangecolorbasedonpHandcanbedissolvedinwatertomakea
concentratedsolution.BysomeofthedyesolutiontoaliquidofunknownpH,thecolorcanbe
monitored.PhenolRediscoloredredinsolutionsofpH>8.4.AsthepHdecreasesandthesolution
becomesmoreacidic,thecolorlightenstoorangeand,whenpH<6.8,toyellow.BromphenolBlueshifts
fromblueatpH>7.6toyellowatpH<6.0.
1. Wearalabcoatandgloves.Indicatordyescanstainyourhandsandclothing.
2. ScoopaspoonfuloftheindicatordyePhenolRed(orBromphenolBlue)toacontaineroftap
wateruntilthewaterisdistinctlyred(orblue).AstocksolutionofPhenolRedcanbestoredina
darkcabinetforseveralyearsandusedslowlyovertime,socheckwithyourteachertofindout
astockisalreadyavailable!
3. Measureanequalamountoftheindicatorsolutionintotwosmallercontainerspertreatment.
a. Dilutethesolutionineachcontainerusingequalamountsofwater,startingbyadding
thesameamountofwaterastheamountofindicatorsolutioninthecontainer.
b. Ifyoucanseethroughtotheothersideofthecontainer,youhavedilutedthesolution
enoughtoproceed.Otherwise,addmorewaterinequalamountstoallcontainers.
4. Blowbubblesintohalfofthecontainersthroughastraw.Theexhaledairwillcontainexcess
CO2andthesolutionwillbegintoturnorange(orgreen),thenyellow.
5. Foreachtreatment,placeidenticalpiecesofaquaticplanttissueintoeachred(orblue)and
yellowcoloredpairofcontainers.Labelthecontainerssoyouknowwhichpairsgotogether.
6. Observeandrecordthecolorofeachpairofsolutionsovertime.
PlantingScience CC BY-NC-SA 3.0
|
www.plantingscience.org
|
Power of Sunlight—Toolkit Page 6 of 31
Last Updated 7/2013
a. Itmaybehelpfultodevelopa“colorscale”beforebeginningtheexperiment.
b. Removingasmallvolumeofliquidfromeachcontainerandlookingatthemtogether
againstawhitebackgroundcanhelpensureunbiasedcolorjudgmentoverall.Return
thesolutiontothesamecontainerafterwards.
c. Qualitatively,iftheyellowsolutionchangescolortored(orblue),CO2isbeing
consumedmorequicklythanitisproduced.Ifthered(orblue)solutionchangesto
yellow,CO2isbeingproducedmorequicklythanitisconsumed.
d. Theamountoftimerequiredforthecolortoshiftprovidesaquantitativemeasureof
howquicklythepHchangesoverasetnumberofunits.Therefore,itindirectly
measuresphotosyntheticorrespirationrate.
PlantingScience CC BY-NC-SA 3.0
|
www.plantingscience.org
|
Power of Sunlight—Toolkit Page 7 of 31
Last Updated 7/2013
MEASURINGCELLULARRESPIRATIONUSINGA
RESPIROMETER
Purpose:Quantifyhowmuchcellularrespirationisoccurringin
mosttypesofplanttissueandsmallseedlings.Themaintypeof
materialbeingconsumed–carbohydrates,fattyacids,orproteins
andaminoacids–canalsobedetermined.
HowtheMethodWorks:Thismethodusesaninstrumentcalleda
respirometer.Therespirometerisassembledtocreateaclosed
system,sothatoncetheexperimenthasbegun,nothinggoesinor
out–notevenair.Aplantsampleorsmallseedlingisplacedintoa
chamber.Astheplantsamplerespiresinsidetheclosedsystem,it
usesoxygen(O2)andreleasescarbondioxide(CO2).Toquantifyrespiration,thesetwogases
mustbeconsideredseparately.CO2canberemovedfromthegasmixtureinthechamberbya
chemicalreactionbetweenitandsodiumorpotassiumhydroxide.Thereactionproduces
waterandasolid,K2CO3orNa2CO3,sothatnofreeCO2gasispresent.TheO2usedfor
respirationcanthenbedirectlymeasuredbythedecreasinggasvolumeintherespirometer.
Carryingoutthesameexperimentinanotherrespirometerwithoutaddingsodiumor
potassiumhydroxide,allowstheamountofCO2producedtobequantified.Athirdtube
withoutanytissueactsasacontrolforanychangesintemperatureandairpressure.
Thechemicalequationforrespirationassumesthattheorganicmoleculebeingbrokendownis
carbohydrate.Ifthisisso,thevolumeofCO2producedwillbeequaltothevolumeofO2
consumed.TheratioofCO2producedtoO2consumediscalledtheRespiratoryQuotient(RQ).
Ifonlycarbohydrateisbeingbrokendown,thevalueofRQ=1.However,thisneednotbethe
case.Whenfattyacidsarebrokendownaloneorwithcarbohydrates,thevalueofRQisless
than1.Metabolizingproteinsorotherorganicacids,aloneorwithcarbohydrates,resultsinan
RQgreaterthan1.
TechnicalComplexity:Medium,duetothecomplexityofsettingupthechambersforan
experiment.
TimeRequired:Allowatleast30minforbuildingtherespirometersandatleast45minforthe
experimentitself.
Materials:
Toassembleonerespirometer:
PlantingScience CC BY-NC-SA 3.0
•
|
www.plantingscience.org
Hotgluegun,withgluestick
|
Power of Sunlight—Toolkit Page 8 of 31
Last Updated 7/2013
•
•
•
•
•
•
•
•
1mLdisposabletuberculinsyringe
40µLplasticcapillarytube
Absorbentandnon-absorbentcotton
Clean,thinpipetteortoothpick
Manometerscaleorlinedpieceofcard
stock
Tape
Dranoor15%potassiumhydroxidesolution
Water(optional)
•
•
•
•
•
•
•
•
1-2respirometerspertreatment,plusa
control
Waterbathsettoroomtemperature(20oC)
Celsiusthermometer
Plantmaterial
Smallbeadsorbakedseeds
3-4washersperrespirometer
Manometerfluid,orsoapywaterwithred
foodcoloring
Eyedropperorpipette
Tocarryouttherespirometryexperiment:
Part1.PrepareMaterialsforMakingRespirometersandtoCarryOuttheExperiment(s).
•
•
•
Allowtimetogrowyourplantmaterialorsubjectplants/seedstotheenvironmentalconditions
youaretesting.
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.
PlantingScience CC BY-NC-SA 3.0
|
www.plantingscience.org
|
Power of Sunlight—Toolkit Page 9 of 31
Last Updated 7/2013
b. Ifitisplugged,youwillnotbeabletopulltheplungerback.Youcanunplugitby
carefullypeelingofftheglue.Startfromstep4toresealthejoint.
6. Makeasmanyrespirometerchambersasyourexperimentrequires,plusoneasacontrol.Ifyou
plantomeasurebothoxygenconsumedandcarbondioxideproduced,twoseparate
respirometerswillbeneededforeachtreatment.
7. Ifneeded,youmaystopandstoretherespirometerchambershere,finishingthemduring
anotherclassperiod.
Part3.PrepareYourRespirometersforanExperiment.
1. Carefullyinsertasmall,absorbentcottonplugintothesyringeendoftherespirometer.
a. Useaclean,thininstrument,suchasapipette,topackthecottontothe0mlor0cc
mark.
b. Takecarenottodislodgethecapillarytubeduringthisstep.
2. Tapeamanometerscaleontoeachcapillarytubeusingcleartape.
a. Double-sticktapeisbest,butfoldedsingle-sidedtapealsoworks.
b. Placethetapesothatyouareabletoreadthescalebylookingthroughthecapillary
tube.
c. Makesurethebottomofthescaleisalignedwiththesamepointonallrespirometers.
3. Ifneeded,youmaystophereandstoretherespirometersuntilanotherclassperiod.Ifyou
continue,youwillneedtocompletetheexperimentduringthesameclassperiodsothatthe
liquidaddeddoesnotdryout.
4. Putonyoursafetygogglesandlabgloves,becauseyouwillbeworkingwitheitheradilute
solutionofpotassiumorsodiumhydroxide(Drano).Botharecaustic.
5. Foreachrespirometerthatwillbeusedtomeasureonlyoxygenconsumption,putasmalldrop
ofpotassiumhydroxide(KOH)orsodiumhydroxide(NaOH)ontothecotton.
a. Avoidgettinganyonthewallsofthesyringeabovethecotton.
b. IfyouwillusesomerespirometerstocalculateCO2production,marktherespirometers
toindicatetheirtype.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. Removetheplungerandrepeatwiththerestoftheoxygen
respirometers.
PlantingScience CC BY-NC-SA 3.0
|
www.plantingscience.org
|
Power of Sunlight—Toolkit Page 10 of 31
Last Updated 7/2013
Step3.Runtheexperiment.
1. Checkthetemperatureofyourwaterbathtoseethatit’satroomtemperature,about20°C.
a. Ifit’snot,adjustthetemperaturewithwarmorcoldtapwater,makingsurethatthe
bathiswell-mixedwhenyoumeasurethetemperature.
b. Leavethethermometerinthewaterbath.
2. Put0.5mlgerminatingseedsorotherplantmaterialintotherespirometerbarrel(syringe).
Pushintheplungertothe1mlmark.Thiscreatesasealedrespirometerchamberwitha1ml
volume.Becarefulnottodamagetheplanttissues.
3. Repeatwithallrespirometersexceptthecontrol.
a. Forthecontrol,useeitherglassbeadsorseedsthathavebeenbakedinsteadofliving
plantmaterial.
b. Foreachoxygen-only/oxygenandcarbondioxiderespirometerpair,trytouseassimilar
amassofplanttissueineachaspossible.
4. Drop3-4washersaroundeachrespirometertoactasweightsinthewaterbath.
5. Putyourrespirometersintothewaterbathwiththecapillariespointingupward.
a. Keepthecapillariesabovewaterlevel,opentotheair.
b. Makesurethesyringebarrelsarecompletelysubmerged.
6. Checkthewaterbathtemperatureagain.
7. Usingadroppingpipet,putadropofredmanometerfluidtothetipofeachcapillary.Thedrop
shouldgetsuckedintothecapillary,ifeverythingisworking.Themanometerfluidwillforma
sealonthechambertocreateaclosedsystem.
a. Youmayhavetousetheplungertopulltheredmanometerfluidintothecapillary,
especiallyforthecontrol.Pullthefluidabouthalfwaydownthecapillary.
8. Usingathinpermanentmarker,makeamarkoneachcapillarytubewherethebottomofthe
redmanometerfluidispositioned.Thisisyourzero-timeorstartingdatapoint.Recordthe
temperatureinyourdatatable.
9. Atregularintervals(every2or5minutesshouldwork),markthebottomofthepositionofthe
manometerfluid.
a. Recordthetimeandtemperaturewhenyoumakethemarks.
b. Ifthemeniscusismovingoffthescale,quicklyrepositionitbygentlypushingordrawing
onthesyringeplungertomovethedropintheappropriatedirection.Besuretonote
thedrop’spositionbothbeforeandafterreadjusting,sothatyoucanlatercalculatethe
amountofadjustmentmade.
10. Continuetakingdatauntileitherthemanometerfluidhasreachedthesyringetipor25minutes
haspassed.
Step4.Recordthemanometerreadingsandcalculatethechangesingasvolume.
1. Removetherespirometersfromthewaterbath.
2. Inyourdatatable,recordthemanometerreadingsforeachmark.Dothisforeachmanometer.
PlantingScience CC BY-NC-SA 3.0
|
www.plantingscience.org
|
Power of Sunlight—Toolkit Page 11 of 31
Last Updated 7/2013
3. Calculatehowmuchthegasvolumechangedduringeachtimeinterval.
a. Ifthecontrolrespirometershowedanychange,adjustthevolumechangeforallofthe
otherrespirometersbythatamount.
b. Intheoxygenrespirometers,thevolumeshouldhavedecreased.Markthevolume
changeasnegative.
c. Intherespirometersthatdidnotremovecarbondioxidegas,thegasvolumemayhave
increasedordecreased.Markthevolumeaspositiveornegative,accordingly.
d. Tocalculatetheamountofcarbondioxidegasconsumedinsuchsamples,subtractthe
amountofoxygenproducedintheoxygen-onlyrespirometerfromtheamountof
changeobservedinthecorrespondingoxygen-carbondioxiderespirometer.
e. Graphtheresultingdataaschangeingasvolume(y-axis)overtime(x-axis).
Step5.(Optional)CalculateRespiratoryQuotient(RQ).
1. Theamountofvolumechangeintheoxygenrespirometersdescribestheamountofoxygen
produced.
2. Theamountofvolumechangeinaoxygen-carbondioxiderespirometerminustheamountof
volumechangeinitscorrespondingoxygen-onlyrespirometerdescribestheamountofcarbon
dioxideconsumed.
3. RQmaybecalculatedforeachpairas:RQ=volumeofCO2produced/volumeofO2consumed.
4. DeterminethetypeofmaterialbeingconsumedinrespirationbasedonRQ:
a. Iftherewasnonetchangeinthecarbondioxide/oxygentube,itmusthaveproduced
exactlyasmuchCO2asitusedO2,andyouknowthisamountfromtheKOHtube:RQ=
1.
b. Ifthecarbondioxide/oxygentubeshowedadecreaseinvolume,theamountofCO2
producedwaslessthanO2consumed.TheamountofO2consumedwasequaltowhat
youmeasuredinyourKOHtube,butenoughCO2wasproducedtomovethedrop
partwaybacktowardsitsoriginalposition:RQ<1.
c. Ifthecarbondioxide/oxygentubeshowedanincreaseinvolume,theCO2producedis
equaltotheO2consumedintheKOHtubeplusanadditionalamounttogetittothe
finalposition:RQ>1.
PlantingScience CC BY-NC-SA 3.0
|
www.plantingscience.org
|
Power of Sunlight—Toolkit Page 12 of 31
Last Updated 7/2013
MEASURINGPHOTOSYNTHESIS&RESPIRATION
USINGACOMPUTER-BASEDPROBE
Purpose:Quantifyhowmuchphotosynthesisandrespiration
isoccurringinanytypeoftissue,stem,orwholeplant.This
methodcanoftenbeusedinfieldstudies,ifdesired.
HowtheMethodWorks:Themethodusesacarbondioxide
sensortomeasureCO2levelsinaclosedsystem.Aplantor
planttissuesareplacedintoasealedchamber.Thesensoris
placedintothecontainerthroughaholeinastopperorisbuiltintothechamberitself,then
sendsdatatoacomputerasitmonitorsthelevelsofCO2overtime.Thecontainercanbe
coveredtocreatedarknessorsubjectedtovariedlightintensities.
TechnicalComplexity:Medium.
TimeRequired:About15-20minutespertreatment.
Materials:
•
•
•
•
•
•
•
•
Plantsorplantpartsgrownunderexperimentalconditions,orfoundindifferentfieldconditions
Carbondioxidesensor,functionaloveratleasttherange0–5,000ppm
o Ideally,resolutionandaccuracyshouldbeassmallaspossible
o OnepossibleexampleistheVernierCO2gassensor(CO2-BTA)
Cabletoconnectsensortocomputerorinterface,ifnotbuiltintothesensor
Computerorotherappropriateinterface,suchasahandhelddatalogger
Datacollectionsoftware
Clear,sealablechamber,ifnotbuiltaspartofthesensor
(Optional)Aluminumfoil,darkcloth,orothermeansofcreatingadarkenvironment
(Forlab-basedstudies)Lightsourcethatcanbemovedorwithadjustableintensity
GeneralInstructions:SettingUptheSensorforDataCollection
Sensorpartsandset-upoftendependonthemanufacturer.Therefore,onlygeneralguidelinescanbe
providedhere:
1. Readandcarefullyfollowthemanufacturer’sinstructionsonappropriateset-upanduseof
theCO2sensor.
2. Makesuretheappropriatesoftwareisavailableonthecomputerordataloggersothatdata
canbesaved.
PlantingScience CC BY-NC-SA 3.0
|
www.plantingscience.org
|
Power of Sunlight—Toolkit Page 13 of 31
Last Updated 7/2013
Alternatively,ifusinganinstrumentthatgivesdatareadingsbutcannotsavethem,
setupadatatabletorecordthisinformationinyourlabnotebookatregulartime
points.
Linkthesensortothecomputerordataloggerwithanappropriatedatacable.
Turnonthecomputer,sensor,andsoftwareintheorderrecommendedbythe
manufacturer.
Letthesensorwarmupforthelengthoftimethemanufacturersuggests.
Ifnecessary,calibratetheinstrumentaccordingtothemanufacturer’sinstructionsbefore
collectingdata.
"
3.
4.
5.
6.
MethodA:SamplingPlantsorPlantPartsintheLab
1. Afterthesensorhaswarmedup,recordtheCO2concentrationintheemptychamberasa
baseline.
2. Selectaplantorplantparttotest,andplaceitinthechamber.
3. CloseofforsealthechambersothatthesensorisabletomonitorCO2inside.
" Themanufacturer’sinstructionsmayexplainhowtouseabuilt-inchamber.
4. Placethechambernearalightsourceassimilaraspossibletothegrowthconditionsforthe
plant.
5. RecordCO2concentrationdata,eitherusingsoftwareorbyhand.
" Thesensormayneedtimetoadjust,butitwillnotsettleonasinglevalueifthe
plantmaterialsbeingsampledarealive.
" Checkthemanufacturer’sinstructionstodeterminehowlongtowaitbeforetaking
adatapoint,ifyouarecollectingdatabyhand.
" Aftersubtractingthebaseline,thesereadingswillgivethenetCO2consumptionor
productionovertime.
6. Coverthechamberwithfoil,cloth,orothermaterialsothatitisentirelydarkened.
7. RecordCO2concentrationdataasinstep5.
" Aftersubtractingthebaseline,thesereadingswillgivetheCO2productiondueto
respirationovertime.
8. TheCO2consumptionduetophotosynthesiscanbedeterminedbysubtractingthe
respiratoryCO2productionfromthenetCO2consumption/production.Thisvalueshouldbe
negative.
9. (Optional)YoumaywishtotestthenetCO2consumptionorproductionatseverallight
intensitiestounderstandwhetherthenetCO2consumption/productionchangesbasedon
thisvariable.
10. Testtheotherplantsamplesusingthesameprocedure.
MethodB:SamplingPlantsintheField
Forfieldstudies,ahandhelddataloggerorlaptopwillbetheeasiesttouse.
1. Setupandtesttheequipmentinthelabtobesureitworksbeforegoingintothefield.
PlantingScience CC BY-NC-SA 3.0
|
www.plantingscience.org
|
Power of Sunlight—Toolkit Page 14 of 31
Last Updated 7/2013
2. Bringthesensor,datainterface,chamber,andmeanstodarkenthechambertothefield
withyou.
3. SetuptheequipmentasdescribedintheGeneralInstructions.
4. Afterthesensorhaswarmedup,recordtheCO2concentrationintheemptychamberasa
baseline.
5. Selectaplantorplantparttotest,andplaceitinthechamber.
" Certainchambersmaynotneedtheplantpartremovedtotestit.Ifpossible,leave
theplantintact.
6. CloseoffthechambersothatthesensorisabletomonitorCO2inside.
" Themanufacturer’sinstructionsmayexplainhowtouseanybuilt-inchamber.
7. Placethechambersothattheplantorplantpartreceivesasclosetoitsnaturallightingas
possible.Besuretostandsothatyoudonotcastanyshadowsoverthechamber!
8. RecordCO2concentrationdata,eitherusingsoftwareorbyhand.
" Thesensormayneedtimetoadjust,butitwillnotsettleonasinglevalueifthe
plantmaterialinthechamberisalive.
" Checkthemanufacturer’sinstructionstodeterminehowlongtowaitbeforetaking
adatapoint,ifyouarecollectingdatabyhand.
" ThesereadingswillgivethenetCO2consumptionorproductionovertime,after
subtractingthebaseline.
9. Coverthechamberwithfoil,cloth,orothermaterialsothatitisentirelydarkened.
10. RecordCO2concentrationdataagain,asinstep8.
" ThesereadingswillgivetheCO2productionduetorespirationovertime,relativeto
thebaseline.
11. TheCO2consumptionduetophotosynthesiscanbedeterminedbysubtractingthe
respiratoryCO2productionfromthenetCO2consumption/production.Thisvalueshouldbe
negative.
12. Testotherplantsamplesusingthesameprocedure.
PlantingScience CC BY-NC-SA 3.0
|
www.plantingscience.org
|
Power of Sunlight—Toolkit Page 15 of 31
Last Updated 7/2013
VISUALIZING&COUNTINGSTOMATAUSINGTHELEAFIMPRESSIONMETHOD
Purpose:Todeterminestomataldensityandexaminethestate
ofstomatainleaves.
HowtheMethodWorks:Paintingnailpolishontothesurfaceof
aleafcreatesanimpressionofitsoutercellularstructure.The
impressionispeeledfromtheleaf,thenexaminedundera
microscopetoviewhowwidethestomataareopenedandhow
manyarepresentperunitarea(stomataldensity).
TechnicalComplexity:Moderate.Itcantakesomepracticeto
learnhowtobringspecimensintofocuswithoutdamagingthe
lenses.
TimeRequired:60minutesfromset-uptocompletion.
Materials:
•
•
•
•
•
•
•
•
•
•
Leaves(1-2pertreatment)
Clearnailpolish
Compoundmicroscopewith40Xobjective
Preparedleafanatomyslides,ifavailable
Blankmicroscopeslides
Dissectingprobeorotherpointedinstrument
Forceps
Distilledwater
Permanentmarker
Metricruler
Thelargedonutshapeaboveismadeupof
thetwocellsthatformastomate.Stomata
areopeningsthroughwhichgasesenter
andleavetheleaf.Thecellsaroundthe
pore,calledguardcells,openandcloseto
maketheporebiggerorsmaller.
Procedure:
1. Inyournotebook,describetheleavesyouwillsample.
a. Indicatewhereandwheneachleafwasgathered(e.g.,sunorshade,timeofday,season).
b. Describethespeciesortypeofplant,ifyouknow.
c. Makeaphotographorsketchoftheleaves,usinglabelsorfilenamestohelpyoukeeptrack
forlater.
2. Preparetwoepidermalimpressionsfromeachleaf–onefromtheundersideandonefromthetop.
a. Painta1-cmx1-cmsquareofclearnailpolishontothesurfaceoftheleaf.
" Makesuretherearenogapsinthelayerofnailpolish.
PlantingScience CC BY-NC-SA 3.0
|
www.plantingscience.org
|
Power of Sunlight—Toolkit Page 16 of 31
Last Updated 7/2013
Severalcoatsareokay,sinceyoudon’twantthenailpolishtotearasyoupeelitoff
theleaf.
b. Allowthenailpolishtodrythoroughly.
"
3. Setupandpracticeusingthemicroscopewhilewaitingforthenailpolishtodry.Askyourteacher
forassistance.
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,liftingorpeelingthenailpolishawayfromtheleafuntilabout
halfwaylifted.
" Useforcepstofinishpeelingawaythesquare.
" Rememberwhichsideofthepeelwasfacingtheleaf.
c. Placeadropofdistilledwaterontoamicroscopeslide.
d. Puttheimpressionontothesurfaceofthewaterwiththesidethatwastouchingtheleaf
facingup,awayfromthewater.
e. Gentlysmoothouttheimpressionusingthedissectingprobe,sothatit’sflatagainstthe
slide.
f. Withapermanentmarker,labeltheslidetoindicatetheleafsampledandwhichsideofthe
leaftheimpressioncamefrom.
g. Repeatstepsb-fwiththeremainingleafimpressions.
1. Examinetheimpressionsunderthemicroscope.
a. ReviewStep3forsafeuseofthemicroscope.
PlantingScience CC BY-NC-SA 3.0
|
www.plantingscience.org
|
Power of Sunlight—Toolkit Page 17 of 31
Last Updated 7/2013
b. Focusontheimpression,butrememberthatyouarelookingataclearimpressionofthe
leafsurface,notagreenleaf!
c. Takenotesandmakesketchesofwhatyousee.
2. Collectdataonthestomata.
a. Findtheimpressionsofstomata.
" Aretheyopen,closed,inbetween,oramix?Takenotes.
" Makeaquicksketchofthestateofanaveragestomateintheimpressionforeach
sample.
b. Countthenumberofstomatainthe1cmx1cmimpressionandrecordthenumber.
" Countityourselftwice.
" Haveoneotherteammatedothecounttwiceforthesameslidetodoublecheck.
" Ifmanystomataarepresent,considerdoingacountusingthelowest-power
objectiveinsteadoftheentireimpression.Calculatetheareaofthefieldofview(p.
16)tofindthesamplingarea’ssize.
" Stomataldensitycanbecalculatedasthenumberofstomatespercm2.
PlantingScience CC BY-NC-SA 3.0
|
www.plantingscience.org
|
Power of Sunlight—Toolkit Page 18 of 31
Last Updated 7/2013
VISUALIZINGPLANTCELLS&CHLOROPLASTSUSINGA
MICROSCOPE
Purpose:Tovisualizechloroplastsandplantcells.
HowtheMethodWorks:Photosynthesisoccursinplantorganelles
calledchloroplasts.Becauseoftheirgreencolor,chloroplastscan
beseeneasilyusingalightmicroscope.Awetmountslideofacell
layerteasedfromaleafispreparedandviewedat400Xmagnification.
TechnicalComplexity:Moderate.Youmayneedtomakeseveralsamplestogetathinenough
layertoseetheindividualcellsclearly.Itcantakesomepracticetolearnhowtobring
specimensintofocuswithoutdamagingthelenses.
TimeRequired:30minutes.
Materials:
•
•
•
•
•
•
•
•
•
Compoundlightmicroscopewith40Xobjectiveandatleastonelowerpoweredobjective
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.Useascalpeltosliceaverythinfragmentfromthe
leaf.
PlantingScience CC BY-NC-SA 3.0
|
www.plantingscience.org
|
Power of Sunlight—Toolkit Page 19 of 31
Last Updated 7/2013
Alternatively,placeadropofalgalcultureontoaslideusinganeyedropperor
pipette.
Placeadropofwaterintothecenterofamicroscopeslideusinganeyedropperor
pipette,thentransfertheleaffragmenttothewaterontheslide.
" Ifusinganalgalculture,thereisnoneedtoaddadditionalwater.
Gentlyteaseapartthetissueinthewaterdropletusingthescalpelortwodissecting
needles.
Placeoneendofaglasscoversliptotherightorleftofthespecimensothattherest
oftheslipisheldata45oangleoverthespecimen.
Slowlylowerthecoverslipwithadissectingneedleorforcepssoasnottotrapair
bubbles.Thecoverslipflattensthepreparation,keepsitfromdryingout,and
protectstheobjectivelenses.
Pressdownonthecoverslipvery,verylightlywiththeendofthedissectingprobe
orforceps.Thisspreadsandflattensthetissue,soitiseasiertoseeonecelllayer.
"
b.
c.
d.
e.
f.
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,to
whattreatmentwasitsubjected?
" Alternatively,ifyouhaveadigitalcameratotakemicroscopephotos,include
informationaboutthetotalmagnificationandthesampleinthefilename.Write
thefilenameinyourlabnotebook.
e. Nowusethe40Xobjectivetolookatthesample.Again,makeasketchordigitalphotoof
yourobservations.Trytoanswerthefollowingquestionsinyourlabnotebook:
" Wherearethecellwalls?
" Wherearethechloroplasts,andhowarethepositionedrelativetothecell
walls?
" Abouthowmanychloroplastsareineachcell?
" Doyounoticeanymovementwithinthecell?Ifso,whatisthis?
f. Repeatwithanyothersamplesyouwanttoexamine.
4. Cleanupwhenyouarefinished:
PlantingScience CC BY-NC-SA 3.0
|
www.plantingscience.org
|
Power of Sunlight—Toolkit Page 20 of 31
Last Updated 7/2013
a. Washanddryallslidesandplacetheminaglasscontainernearthesink.
b. Slipcoversmaybedisposedofintheglassdisposal.
c. Turnoffthemicroscope,coverit,andputitintothestoragecabinet.
Whatisthetotalmagnificationofmyspecimen?
Multiplythemagnificationoftheocularlens(usually
10X)bythatoftheobjectivelens.
Example:
Supposeyouhavethe40Xobjectiveinplace.Then
thetotalmagnificationwouldbe:
10x40=400
thatis,400X.
Howbigismyspecimen?
Thefieldofviewisthefullareayoucanseeonthe
slidewithoutmovingit.Athighmagnification,your
fieldofviewissmall.Atlowmagnification,itislarger.
Ifyouhaveaflat,clearruler,placeitonthestage.
Measurethediameteracrossthemiddleofthefield
whenlookingintothemicroscopewitheachofthe
objectivelenses.Usingtheformulafortheareaofa
circle,calculatetheareaofeachfieldofview.
Byestimatinghowmanycellsareinthefieldorhow
muchofthefieldistakenupbyonecell,youcangeta
roughestimateofacell’ssize.Thestandardunitof
measureinlightmicroscopyisthemicrometer(µm),so
it’sbesttodescribethefieldofviewandspecimen
sizesonthisbasis:1mm=0.001µm.
PlantingScience CC BY-NC-SA 3.0
|
www.plantingscience.org
|
Power of Sunlight—Toolkit Page 21 of 31
Last Updated 7/2013
IDENTIFYINGSTARCHINPLANTLEAVESUSINGAN
IODINESTAININGMETHOD
Purpose:Toexaminestarchgrainsasameasureof
photosyntheticand/orrespiratoryactivity.
HowtheMethodWorks:Themethodallowsyoutoseestarchin
leafdisks.Starchisapolysaccharidemadeupofglucose
molecules.Plantscommonlyuseitasastoragecarbohydrate.
Here,leafdisksarepreparedfromplantsgrownunderlightand
darkconditionsbeforebeingtreatedfor24hourswith
environmentalconditionsofyourchoosing–differentlight
intensities,wavelengthsoflight,light-darkcycles,temperatures,
CO2levels,orglucosesolutionsarejustafewpossibilities.Afterplacingleafdisksinan
experimentaltreatment,theyarestainedwithLugol’sSolution.Starchgranuleswillappear
purplish-blackunderamicroscope.Theirrelativedensityunderdifferenttreatmentscanbe
assessed.
TechnicalComplexity:Moderate.
TimeRequired:Twoclassperiods.
Materials:
PreparationPhase:
• PotassiumIodide
• Iodine
• Water
• Containerofabout75mL
• Plantsgrowninthelightandplantsgrown
inthedarkforatleast4days.
• Paperholepunch,plasticstraw,orNo.3
corkborer
• Lightsourceforlight-grownleafdisks
• Aluminumfoilorotherlight-blocking
materialfordark-grownleafdisks
• (Optional)Materialstoprovidean
environmentaltreatment
PlantingScience CC BY-NC-SA 3.0
|
StainingPhase:
• Hotplateorsomeothermeanstocreate
heatedliquidbath
• Beakerofwaterwithboilingchips
• Beakerofethanol
• Forceps
• Timerorclock/watchwithasecondhand
• Papertowels
• Aluminumfoil,Petridish,orshallow
container
• Water
• Compoundmicroscopewith40Xobjective
www.plantingscience.org
|
Power of Sunlight—Toolkit Page 22 of 31
Last Updated 7/2013
Procedure:
Part1.PrepareLugol’sSolution(ifnecessary,5minutes):
3.
4.
5.
6.
Measure60mLofdistilledwaterintoacontainer.
Weighout0.4gofpotassiumiodide.
Transferthesolidintothecontaineranddissolve.
Add0.2giodinetothesolutionandstirorswirltomix.
Part2.PrepareLeafDisks(10minutes):
1. Useapaperholepunch,plasticstraw,orNo.3corkborertopunchoutyourleafdisks.
o Avoidtheheavyveins.
o Cutoutequalnumbersofleafdisksfromthelight-grownanddark-grownplants,using1-2
leavesforeachenvironmentaltreatmentyouplantocarryout.
o Includeatleastonehealthygreenleafandonedark-growninadditiontoyourtestsamples.
Theseareyourcontrols,andtheywon’tbetreatedduringthe24hourperiod.
o Alternatively,youcancutsquaresfromtheleavesusingscissors.
2. Dividetheleafdisksintoequivalently-sizedtreatmentgroups.
o Ifyouhaveindexcardsorsmallpiecesofpaper,puttheleafdisksforeachtreatmentonto
theirowncard.Thiswillmakeiteasiertokeeptrackofthedisksforonetreatment.
3. Placethecontrolsintothesameconditionsthatwereusedduringpre-treatmentoftheleaves,light
anddark.
4. Place the treatment groups into their respective treatment conditions. Treat the leaf disks for 24
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.
PlantingScience CC BY-NC-SA 3.0
|
www.plantingscience.org
|
Power of Sunlight—Toolkit Page 23 of 31
Last Updated 7/2013
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,includingcolor
variations.
3. Visualizerepresentativesamplesfromeachtreatmentunderamicroscopeusinga40Xobjective,
andsketchwhatyousee.
o Ifyouhavenotusedamicroscope(recently),askateacherforassistanceandseepp.13-14
forpracticeinstructions.
PlantingScience CC BY-NC-SA 3.0
|
www.plantingscience.org
|
Power of Sunlight—Toolkit Page 24 of 31
Last Updated 7/2013
IDENTIFYINGCHLOROPHYLL&OTHERPLANTPIGMENTS
Purpose:Identifyingthepresenceorabsenceofchlorophylland
otherplantpigmentsinleaves.
HowtheMethodWorks:Allplantpigmentshaveunique
chemicalproperties,allowingustotellthemapart.Here,paper
chromatographywillbeusedtoseparateplantpigments.First,
fluidissqueezedoutofplantleavesontofilterpaper.Thepaperis
putintoachambersothatitstiptouchesachromatography
solvent--inthiscase,acetoneorisopropylalcohol.Asthesolventisabsorbedandtravelsup
thefilterpaper,theplantpigmentsarecarriedwithit.Eachpigmentwillmoveupthepaperat
acharacteristicratedependingonitschemicalproperties,includingsolubilityinthesolvent,
molecularmass,andamountofhydrogenbondingwiththefilterpaper.Sincethepigmentsare
colored,thesinglesamplewillseparateintopigmentbandsofdifferentcolors.
TechnicalComplexity:Simple.
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.
PlantingScience CC BY-NC-SA 3.0
|
www.plantingscience.org
|
Power of Sunlight—Toolkit Page 25 of 31
Last Updated 7/2013
o
Ifyouhavemanysamplestotest,makesurethechamber(s)arelargeenoughforone
vertically-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)inthesameplaceon
thestrip.Thiscompletesonestrip.
e. Repeatthisentireprocessforeachleafsampleandstrip.
f. Allowtheleafextractsonthestripstodryforabout10minutes.
4. Preparethechromatographychamber(s):
a. Isopropylalcoholandacetonearevolatileandflammable.Wearsafetygoggles,avoid
inhalingthefumes,anddonotusenearflames.
b. Fillthechamber(s)witheitherisopropylalcoholoracetoneto1cmindepth.
c. Coverthechamber.Allowthechambertofillwithfumeswhileyourstripsaredrying.
d. Howyoucreatethesetuptorunsampleswilldependonthechambersizeandshape.
Forexample,youcouldattach
thedriedstripstoapencil(left).
Oryoucouldhookapaperclip
throughtheflatendofastrip
andthenthrougharubber
stopper(right).
PlantingScience CC BY-NC-SA 3.0
|
www.plantingscience.org
|
Power of Sunlight—Toolkit Page 26 of 31
Last Updated 7/2013
5. Runthechromatographysamples:
a. Openthechamberandloweryourstrip(s)intoitsothetipsaretouchingthesolventandthe
plantextractlineiswellabovethesolvent.
" Iftheplantextracttouchesthesolvent,itwilldissolveintotheliquid!
" Makesurethesidesofthestripdonottouchthewallsofthechamber.
" Makesurethestripsarelevel.
b. Coverthechamberandletthesolventwickupthestrips.Donotdisturbthechamberwhile
itiswicking.
" Theleafextractwillbegintoseparateintobandsofcolorasittravelsupwards.
c. Stopthechromatographywhenthesolventreaches2cmfromthetopofthestrips,orwhen
youseeatleastfourcolorbandsclearlyseparatedonthestrips.
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=Distancepigmenttraveled
Distancesolventtraveled
7. Cleanupwhenyouarefinished:
a. Youmightnotbeallowedtopourthesolventdownthesink,soaskyourteacherhowyou
shoulddiscardit.
b. Afterdiscardingthesolvent,rinseoutthechamberswithwater.Leavenearthesinktodry.
PlantingScience CC BY-NC-SA 3.0
|
www.plantingscience.org
|
Power of Sunlight—Toolkit Page 27 of 31
Last Updated 7/2013
c. Donotthrowyourstripsawayuntilyouhaverecordedallthemeasurementsandcarried
outallthecalculations.
PlantingScience CC BY-NC-SA 3.0
|
www.plantingscience.org
|
Power of Sunlight—Toolkit Page 28 of 31
Last Updated 7/2013
QUANTIFYINGFRESH&DRYMASSOFPLANTS
Purpose:Determinechangesinplantmassovertime.
HowtheMethodWorks:Therearetwowaystomeasureplant
mass:freshorwetmassanddrymass.Inthefreshmassmethod,
thewholeplantisweighedoncethesoilisremoved.Forthedrymassmethod,plantsaredried
atlowheatbeforeweighing.Theplantsthatareweighedcannotbeusedforfurtherstudies,
butyoucandeterminethefreshmassofaplantandthendryittoalsodetermineitsdrymass.
Thisallowsyoutocalculatethewatercontentofthatplant.Youshouldusethemethod(s)best
suitedtoyourresearchquestion.Forexample,aquestionabouttheamountofcarbonfixed
duringgrowthisbettersuitedtomeasurementsofdrymass,sincethewatercontentofaplant
canvary.
TechnicalComplexity:Simple.
TimeRequired:About5minperplant.Fordryweightmeasurements,anovernightdrying
timeisrequired.
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:MeasuringFreshMass
Thismethodisthefastestwaytomeasureplantmass.Becauseaplantcanbecomposedofvarying
amountsofwaterunderdifferentenvironmentalconditionsordevelopmentalstages,itislessaccurate
thanmeasuringdrymass.
PlantingScience CC BY-NC-SA 3.0
|
www.plantingscience.org
|
Power of Sunlight—Toolkit Page 29 of 31
Last Updated 7/2013
1. Removeplantsfromsoilandwashoffanyloosesoil.
2. Blotplantsgentlywithsoftpapertoweltoremoveanyfreesurfacemoisture.
3. Weighimmediately.
o Plantshaveahighwatercomposition,sowaitingtoweighthemmayleadtosome
dryingandproduceinaccuratedata.
4. (AlternativeOption)Freshmassforrootandshootdatamaybetakenseparatelybycuttingthe
shootsatthe“crown”oftheplant,thenweighingeachpartseparately.Thecrown,orplace
whereaplant’srootsandstemmeet,isusuallyfoundatsoillevel.
MethodB:MeasuringDryMass
Thismethodremoveswaterfromtheplantsbyfirstdryingtheminanoven.Sinceplantscontainlotof
water,usingdryweightisamorereliablemeasurethanfreshweight.
1. CompletethefirsttwostepsdescribedinMethodA.
2. Ifdesired,completeStep3orStep4fromMethodAaswell.Youwillendupwithbothfresh
anddrymassdata.
3. Drytheplantsinanovensettolowheat(60oC)overnight.
o Ifyouhavemanyplantstodry,itmaybehelpfultoplacethemindividuallyinpaper
lunchbags.
o Uselabelsormakeachartofthedryingpositionstokeeptrackoftheirrespective
treatments.
4. Lettheplantscoolinadryenvironment.
o Inahumidenvironmenttheplanttissuemaytakeupwater.
o Aplasticsandwichbagwillkeepmoistureoutifyouliveinahumidclimate.
5. Oncetheplantshavecooled,weighthemonascale.
o Plantscontainmostlywater,sothedriedplantswillnotweighverymuch.
o Makesureyouhaveascalethatmeasuresmilligramstoensureaccuracy.
MethodC:CalculatingWaterContent
Watercontentcanpotentiallybeusefultoidentifyplantsthathaveadifferentresponsetotheir
environment.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)
PlantingScience CC BY-NC-SA 3.0
|
www.plantingscience.org
|
Power of Sunlight—Toolkit Page 30 of 31
Last Updated 7/2013
Freshmass(g)
PlantingScience CC BY-NC-SA 3.0
|
www.plantingscience.org
|
Power of Sunlight—Toolkit Page 31 of 31
Last Updated 7/2013