Light Microscopy
Background on Microscopy:
“Intheyear1675,abouthalf-way through September, Idiscoveredlivingcreaturesinrainwhichhadstoodbutafew days in a new tub that was paintedblue....
They were incredibly small, nay sosmall, in mysight that even if 100 oftheseverywee animals laystretchedoutone against another,theycouldnotreachthe length of a single grain of sand.”
(First Reported Description of Bacteria)
On his own and in his spare time,Antony van Leeuwenhoek founded the science of microbiology. Using a simple (one lens) microscope, which he constructed fromscraps of metal andfragments of glass, Leeuwenhoek tookthe first boldsteps intheexplorationofthe microbial world. Like a curiouspuppy that sniffs (with a total lack ofdiscrimination) at everything aroundhim, Leeuwenhoekputhistinylens(whichwasnot much largerthantheheadofapin),towork.Heborrowed
ox-eyes fromthelocalbutcherandmarveled atthe crystalline structureofthe eye-lens.He carefully examined thescales of his own skin and grunted"impossible" when he first spied thestinger of a flea.Imagine his surprise ashe discovered a tiny world of "littleanimals" inside of a single drop ofrainwater!
Today you will follow in the footsteps of dear old Leeuwenhoek. You will learn how to use the light microscope toobserve living things and, in the process, hone your powers of observation. After
today you will be counted among thevery few individuals that have actually "seen" microorganisms. Enjoy!
A few years ago, when you were 15 or 16yearsold,youprobablyspentagreat dealof time sittingbehindthewheelofyourdad’scarwhileit was stillparkedin the driveway. While the neighborsthoughtyoualittleodd,youprobably werefantasizingaboutthe"bigday" when you would earn your license and hit the roadforreal.Indeed these"stationary excursions" serve animportant functionfortheyprovidedyouwith the opportunity to become familiarwiththe important parts ofthecar.Youlearnedhowtoholdthesteeringwheel, where the brake and accelerator pedalsare located and other important pointssuch as how to adjustthe seat, therearview mirror and your radiostations.As the time ofthe driver's testapproached,this informal trainingwassupplemented by formal instruction atschoolorat home duringwhichyoulearnedthecorrectwaytodriveandhow tobe comfortable and confident withthiscomplex piece of equipment.
Todayyouwillbeinstructedinthe correctuseofthelight microscope.Youwill learn whereeachpart is located,whatits function isand,hopefully,willbecome comfortableandconfidentwiththis wonderful piece of equipment.
Fromthis day forward you should be able to use your microscope withminimal assistance fromyour instructor.
Remove the microscope fromthecabinetandcarryittothebench.ALWAYS use BOTH hands whencarryingthe microscope.Useonehandtogripthe arm
andanothertosupportthebase. Once you have placed the microscope safelyon the lab bench, remove the cover andrefer to Figure 1.
We will begin with a "stationaryexcursion" whereyouwilllearn the partsofthe microscope withthehelpofadiagram(Figure1).Youshouldbeable tolocatethe microscope partswhicharein boldprint. The general idea of amicroscope is to provide an enlargedimage of a small specimen, a processwhich usually requires light. Thelightsourceis located inthebaseofthemicroscope and is simply an attachedbulbwhich emits light.Thelightisdirected onto the specimen by acondenserlensandthe amount oflightwhich enters is regulatedby theirisdiaphragm. The specimen sits like asacrificiallamb on a platformcalled thestage. If the stage has knobs, which allow you to movethe slide horizontallyand vertically, it is known as amechanicalstage. Thecoarse andfineadjustmentknobs helpyoutofocustheimage byraisingand lowering the stage(in somemicroscopesthe objectives areraised orlowered).After the lighthaspassed through the specimen, it containsvaluable information whichreachesoneof theobjective lensesthatare mountedon the revolving nosepiece.This lensgathersthevaluablelightraysandperforms an initial magnification.
Finally,the information reaches youreyes through theocular lens. The
ocularlens performs thefinalmagnification of the specimen and is thelens that you look through.Ifthereisonlyoneocularthenyouareusingamonocularmicroscope; if there are two,itisa binocular microscope.
Now that you have masteredsome important microscope parts,itistimefor your written test -- CompleteTable 1.
NOTES:
Figure1–TheLightMicroscope
Pleasenotethat this microscopeis not exactlythe same as you will be using in lab, butcontainsthesamebasiccomponents.
Table1:PartsoftheLightMicroscope
MicroscopePart / Function1. Ocular
2. Objective
3.Condenser
4. Iris Diaphragm
5.Stage
6.Coarse-adjustmentknob
7. Revolving nosepiece
8. Light Source
necessary.
During the next part of your training, the instructor will demonstratethecorrectwaytousea microscope.Itisagoodideatokeepthishandouthandy for future microscopy labs.
1)Remove the microscope fromthecabinetandcarrytothebench.
a.ALWAYS use BOTH hands when carrying the microscope.
b.Useonehandtogripthe
arm andanothertosupport thebase.
2)Wind theelectriccordaroundapipe and plug in.This will preventthe cord from ensnaring some poorunsuspecting passerby.
3)Turn on light.
4)Cleanlenses. Itisagoodideato cleantheocularand objectivelenses before AND after use.
a.ONLY LENS PAPER ANDLENSCLEANERSHOULDBEUSED!
b.DO NOT USE Bibulouspaper-itisnotdesignedforthis purpose.
c.Gently wipe the dirty lens withacleanpieceoflenspaper.DO APPLYEXCESSIVE PRESSURE.
d.If the lens doescome clean,simplymoisten a smallpieceof lens paper with the lens cleanerandgentlywipethe lens.Thenwipewithadry piece.Theinstructorwill supplyyouwiththelens cleaner as necessary.Overuse of the lens cleaner can damage the lens so itshould be used only when
1)Place lowest objective (10x) in position (youshouldhearitclick)
2)Place slide onstage andcenter specimen.
3)Turn the coarse adjustment knob tolowertheobjective until theobjectivestops.(abuilt-in stopshould prevent the objective fromhitting theslide).
4)Lookintotheeyepiece-focuswiththecoarse adjustment knob.
5)Focus with thefine adjustment knob.
6)Adjust light withtheiris diaphragm
as needed.
7)Whilewatchingfromtheside,rotatethenosepieceto bring the next objective(10x)intoposition.
8)Focus with thecoarse(ifneeded)andfine adjustmentknobs.
Using the Oil-immersion lens
Inordertogetagoodlookat mostbacterialcells,youwillneedtousetheoil- immersion lens.First center thespecimen and focus with the high-dryobjective (43x) then follow the procedure below.
1.Rotate thenosepiecesothatthe specimen is betweenthe high-dryand oil-immersionlenses.
2.Place a SINGLE drop ofimmersion oil on theslidedirectlyoverthe specimen.
3.Watching fromthe side, rotatethe nosepieceto bring theoil-immersionlens into position.
4.Focus with thefine focusadjustmentknob ONLY.
Note:Ifthe image is too darkyou might want to open theirisdiaphragm a little.
(only about four times more powerfulthan Leeuwenhoek'smicroscope whichwas constructed over 300 years ago!)
Theocular lensmagnification of yourmicroscope is 10x.
1.Examine the smallest objective andlocate the magnifying power.
(youshouldseeanumberontheside of the objective)
2.Enterthis number inTable2below.
3.Now find and record the magnifications of each of the otherobjectives.
Table 2: Magnifying Power ofObjectives.
Magnificationreferstothe apparentincreaseinsizeofanobject whenviewedthrougha microscope.Leeuwenhoek'ssimple microscopes hada magnifying power of about 270 timesthe diameter of the specimen. Themodern compound microscopecombinesthe magnifying power of theocularlensand the objectivelens and provides amagnifying power of about 1,000 x
TheTotalMagnificationis found bymultiplying the magnifying poweroftheocularlensbythe magnifying poweroftheobjectivelensthatyouareusing.
Q:Calculate the total magnificationwhenthehigh-dryobjectiveis used.
Whenworkingwitha microscope thereare three importantconcepts that youneed to be familiar with.The first ofthese concepts is field of view. The fieldof view is the area that you can actuallysee when looking through the lens of themicroscope.Thereisarelationshipbetweenthefieldofviewandthemagnification. As the magnificationincreases, the fieldofviewdecreases.
Basicallywhatthis means isthatasyouincreasethe magnification youarelooking at less of the specimen.
Thesecondconceptyouneedtobefamiliar with is workingdistance. Asyou increase magnification by using thedifferent objective lens,youwillnoticethatasthe magnification powerofthelensincreases,sodoesthelengthofthe lens,which means thatthelensisclosertothe specimen.Therulehereisthatasthe magnification increases,theworkingdistance decreases. This why you need to be careful when using the oil immersion lens as it will be only afraction ofa millimeterfromthemicroscope slidewhenitisatits properworking distance.
Thethirdconceptyouneedtobefamiliar with is depth offield. With anylensasyoureachthe proper workingdistance, you willnoticethatthespecimen will come intofocus.Ifyouwere to continue getting closer to thespecimen there would come a pointwhen the specimen would go back out offocus. The range of distance that thespecimen is actually in focus is referredto as the depth of field. The relationship between depth of field and magnificationis as themagnificationincreases, the
depthoffielddecreases.Onceagainif youthinkaboutthisit makes sense.
With the increase in magnificationyouareactuallylookingata smaller portionof the specimen and as such the workingdistance decreases,thefieldofviewdeceases, and thedepthoffielddecreases.You literallyare gettingcloserandcloserto the portion of thespecimen you are looking at as youincreasethe magnification.
Keeping these principles in mind youwill come to the realization that thereisa limit to howsmall ofan object that canbeviewedwithatypicallightmicroscope. This limit is morecorrectlyreferred to as the limit in the resolvingpower of the microscope. The resolvingpower of the microscope istheabilityofthe microscope to allow for closelyspaced objects to be clearlydistinguished. The limit to theresolvingpowerisrelatedtothewavelengthof light used toilluminate the specimen andthe mediumthrough which the lightpasses.Generally,thelightisnotas critical as the medium. For highermagnifications oil is used and as suchincreases the resolving powerandallowsyou see smaller objects.
Inthisandmost microbiology labstheoil immersion lens is used to examinespecimens, particularly specimenscontaining bacteria. The lowermagnification lens may be helpful infinding a specimen on a microscopeslide, but willyield very little usefulinformation aboutthe specimen.
1.Obtain prepared slides of different microorganisms.
2.Inordertoclosely examine individualbacterialcells,youwillneedtousetheoil-immersion lens. (Followthe instructions provided previously)
3.Examine the slide using theoil-immersion lensandsketchyourobservationsinthespace below.