Biol160Microscopy:THECOMPOUNDMICROSCOPE

(Thestudyoftheuseofamicroscopeisidentifiedasmicroscopy)
Theentirelabheremayormaynotbeused,dependinguponyourinstructor.
Theremayormaynotaswellbeaquizattheendofthislab.

OutcomesforTodaysLab:PRELABquizprompts(inblue,flowingare
HTMLlinkedsearchanswers.
1. Identifyanddescribetheparts

ofacompoundmicroscope.
2. Identifywhatimageryfromascanningelectronmicroscopeversusa
transmissionelectronmicroscopelookslike(typicallyamuchhigherset
ofmagnificationpotentialsonthesescopesversusourowninlan
compoundlightmicroscopelabscopes
3. KnowwhoRobertHookeis,andwhathenamedinregardtocells.

1.Aftertodayslabyoushouldbefamiliarwiththeanatomyandfunctionofa
binocularcompoundlightmicroscope.
2.Demonstratepropercareanduseofthemicroscope(i.e.,carrying,cleaning
lenses,focusingcarefully).
3.Demonstratetheabilityto:preparewetmounts,stainspecimens,focus,calculate
magnification,calculatefielddiameter,estimatespecimensize,andsketch
specimensastheyappearundermagnificationusingacompoundmicroscope.
I.LabProcedure,PartOne:TheMicroscope:Beabletoidentify/labelthe
diagramofacompoundmicroscope.
Beabletoidentifythefollowingstructures:stage,objectives(andspecificnames
(scanning,low,high,oil),condenser,coarseadjustment,fineadjustment,arm,
base,turret/nosepiece,lightsource,stageadjustment.

a)HandlingtheMicroscope:
1.Cleanlensesasneededusinglenspaperonly(inyourdeskdrawer)andlens
cleaningsolution(smallbottleinlabwithgreensolution)...Normaltissueistoo
coarseandmayscratchlenses.
2.Carrythemicroscopeatalltimeswithtwohandsassuch:onehandunderthe
baseandtheotherhandgraspingthearm.Microscopesarefragileandmustbe
handledwithcare.Makesuretheelectricalcordissecuredtopreventaccidents,
wrappedaroundtheocularsatthetopofthescope.
3.Putthelowestpowerobjectivelens(theshortestone)inplaceandcoverthe
microscopewiththedustcoverwhenfinished.
4.Neverusedirectsunlightasalightsource.
b)LightAdjustment:Thediaphragmregulatestheamountoflightpassing
throughthespecimen.Toomuchlightresultsinflarecausingalackof contrastor
lostdetailwhenviewingthespecimen.
Thereare2typesofdiaphragms:
1.Thediscdiaphragmcontrolstheamountoflightwithdifferentsizedopenings.
2.Theirisdiaphragmoperatesliketheirisofacameralensallowingacontinuous
rangeoflightadjustment.
c)PreparingaWetMount:
1.Placeadropofwateronacleanslide.
2.Placeaverythinlyslicedspecimeninthewater.
3.Holdcoverslipagainstwateratanangleof45degrees,thenrelease.Thiswill
reducethenumberofairbubbles.Airbubblesmayobscureportionsofortheentire

specimen.Gentlypressingonthecoverslipwithapencilerasercaneliminatesome
airbubbles.
4.Twomethodsthatpreventorreducethemovementoflivingspecimensinclude
the:
1)useofcottonfibrestotrapthespecimen. Or/and
2)usemethylcelluloseaka"Protoslo",averyviscosefluid,toreducespecimen
movement.
d)Focusing:
1.Alwaysbeginwiththelowestpowerobjectiveinposition.Thisgivesthelargest
fieldofvisionandthegreatestdepthoffield.Italsoreducesthechanceofthelens
strikingtheslide.
2.Toavoidbreakingtheslideduringfocusing,movethelowestpowerobjective
ascloseaspossibletotheslidewhilewatchingfromthesideofthemicroscope.
Centerthespecimenandfocusbymovingtheobjectiveawayfromtheslide.
3.Turntheadjustmentdialstosharpentheimage.
4.Adjustthediaphragm("toggle"belowthestage)foroptimumcontrast.
5.Whengoingfromalowerpowerobjectivelenstoahigherpowerobjectivelens:
a.Centerthespecimeninthefieldofview.(definedas?)
b.Changetothenexthigherpowerobjectivelens(donotchange/movethe
adjustmentknob!).
c.UsetheFINEadjustmentdial(onlythefine)tosharpentheimageandagain,
adjustthediaphragmforoptimumcontrast.
e)DeterminingTotalMagnification
TotalMagnification=ocularlenspowerxobjectivelenspower(E.g.5Xx4X=
20X)

Note:theunitsaretimes(X).
1.

DeterminetheTotalMagnificationforeachcombinationofocularand
objectivelensesfoundonyourmicroscope.Completethetablebelow:

OcularLensPower(X)ObjectiveLensPower(X)=TotalMagnification(X)
Whatarethetotalmagnifyingpowersifyouaresetat:

Scanning___________ LowPower___________ HighPower___________

OilPower___________
2.Resolvingpowerorresolutionisanimportantfactorintheeffective
magnificationofamicroscope.Resolvingpoweristheabilitytodiscerntwoor
morepointsasseparateanddistinct.Goodresolutionallowsonetoseedetailin
specimens,whilepoorresolutionresultsinanunclearimageofthespecimen
despitebeingmagnified.Theunaidedeyesresolutionlimitisabout0.1mmor
100m.Theresolvingpowerofthebestlightmicroscopeisabout0.0002mmor
0.2m.
a.Howmanytimesbetteristheresolutionofthebestlightmicroscopecompared
totheunaidedeye?
b.Theelectronmicroscopehasamaximummagnificationof250000Xanda
resolvingpowerofabout7x107mm.Howmanytimesbetteristheresolutionof
theelectronmicroscopethanthebestlightmicroscope?
3.Observealettere,ornewspaperprintandthen,inthespacebelow(A),draw
theletterasitappearswiththeunaidedeyeonthestage.Totherightofthis
diagram(B),drawtheletterasitappearsinthefieldofviewunderlowpower.
A.

B.


b.Comparethetwodrawingsanddescribewhatyousee.(Yourdescriptionshould
includeanswerstothefollowingquestions:
Whatistheconsistency(appearance)oftheinkmagnifiedVS.nomagnification?
Describethetextureofthepaper.Fromwhatyouhaveobserved,howdoesthe
positionororientationoftheletterchange?Istheorientationthesameordifferent?
Whathappened?

c.Describethemovement(usingthebeneaththestageadjustmentdevices)ofthe
specimeninthefieldofviewwhenyoumovetheslidetotheleft.Describethe
movementofthespecimeninthefieldofviewwhenyoumovetheslideaway
fromyou.

d.Makeageneralstatementabouttheorientationandmovementofobjectsviewed
throughamicroscope.

e.Whatwouldyousaytohelpafriendwhoishavingtroublelocatingaspecimen
inthefieldofview?

4.Observetheslidemadeofmixedcoloredthreads.

a.Locatethehairsunderscanningpower.Isitpossibletohavebothhairsinfocus?

b.Locatethehairsunderlowpower.Isitpossibletohavebothhairsinfocus?
Drawwhatyousee.Indicatethetotalmagnification.

c.Locatethehairsunderhighpower.Isitpossibletohavebothhairsinfocus?

d.Whathappenstothedepthoffield(i.e.:theabilitytofocusonmorethanone
objectwhentheobjectsareatdifferentdepthsontheslide)asmagnification
increases?

f)DeterminingtheSizeoftheSpecimen:Thesizeofspecimenscanbeestimated
byknowingthediameterofthefieldatagivenmagnification.Theunitof
measurementusedthemicrometer(m).
1.Completethefollowingmetricconversions.(Recall:1mm=1000m).
600m=_____mm10mm=_____m10m=
_____mm
0.1mm=_____m.010m=_____mm0.03mm=
_____m

2.

Measurethediameterofthefieldinmmunderscanningandlowpowerby
placingtheclearplasticruler(providedinboxoncentertable)onthestage.
Convertyourmeasurementsinmmtom.Multiplythefielddiameterby
thetotalmagnificationandrecordyourresultsinthetablebelow.

FieldDiameter(mmtom)TotalMagnification(X)FDx
TM

ScanningPower

LowPower

HighPower

a.Whatdoyounoticeabouttheproduct(FDxTM)ofthefielddiameterandthe
totalmagnificationwhencompared(thethreethatis)?
b.Explainwhatyouhavediscoveredtocalculatethefielddiameterathighpower.
c.Howcouldyouestimatethesizeofthespecimenknowingthediameterofthe
field?(Hint:Ifthefielddiameteratscanningpoweris~4300m(4.3mm),andthe
specimenstretchesacrosshalfofthefield,howlongisthespecimen?)

3.

Viewtwospecimensandestimatetheirsizes.Recordyourobservations
foreachspecimenincluding:

A...specimenname

B...specimensketch

Totalmagnification:(ofA_________)and(B_________)

Estimateofspecimensize:(ofA_________)and(B_________)
Specimensmayincludepondsamples,yeastcells,onioncells,coloredmagazine
image/text,crosssectionofaplantstem,preparedtissueslides,orlower
epidermisofaElodealeafshowingguardcells.
Seestep4beforepreparingslidesofplantcells.

4.Itisoftendifficulttodistinguishstructureswithinacellfromeachotheror
theirbackgroundbecausemanystructuresarecolorlessand/orhavesimilar
textures.Usingstainsordyesthatactonspecificstructureswithinacellcanhelp
increasethecontrastbetweenstructures.Althoughmoststainskillcells,some
stains,calledvitalstainsdonot.
a.Describeashortcoming(problem)tousingstainswhenstudyingcellular
structure(s).
CAUTION:Weargloves(atthebackoftheroom)Useonlythediluteiodine
solutionandAvoidcontactwithskin,eyes,andclothing.Donotingest!.Viewing
plantcellscanbeimprovedbystainingthemwithtinctureofiodine.Iodinestains

thestarchincells.Addadropofdiluteiodinesolutioninplaceofwaterasyou
prepareyourwetmount.
ForthenextlabonBacteria(Gramstaining)comingupintwolabs:
Takeonepetridishperperson,withgrowthagarinthemandmarkoff(withawax
pencil)ontheoutsideplasticbottomofthedish(thebottomisthesidetheagaris
attachedto)intoafoursidedcrosslikepattern,labelingeachofthesections1
through4andtheplateitselfwithyourname.

TakeFOURsterileswabsandabottleofdistilledwater(toinitiallywettheswabs
with)andgoouttotakesamplesinthecollegeorgrounds,placingthemin(one
eachperswab,perspaceonthedish))thesectionsyoumarkedoffonthedishON
theagar(notintoit)withanotebook,identifyingwherespecificallythesample
camefrom(bathroomfloor,dirt,etc).Wewilllookatyourspecimensintwo
labs(theywillincubateatroomtemperaturetilthen).