Principle

Whenlightpassesfromamaterialofonerefractiveindextomaterialofanother,asfromglasstoairorfromairtoglass,itbends.Lightofdifferentwavelengthsbendsatdifferentangles,sothatasobjectsaremagnifiedtheimagesbecomelessandlessdistinct.Thislossofresolutionbecomesveryapparentatmagnificationsofabove400xorso.Infact,asyouwillseelater,evenat400xtheimagesofverysmallobjectsarebadlydistorted.

Placingadropofoilwiththesamerefractiveindexasglassbetweenthecoverslipandobjectivelenseliminatestworefractivesurfacesandconsiderablyenhancesresolution,sothatmagnificationsof1000xorgreatercanbeachieved.

Touseanoilimmersionlens,firstfocusontheareaofspecimentobeobservedwiththehighdry(400x)lens.Paceadropofimmersionoilonthecoverslipoverthatarea,andverycarefullyswingtheoilimmersionlensintoplace.Focuscarefully,preferablybyobservingthelensitselfwhilebringingitasclosetothecoverslipaspossIBLe,thenfocusingbymovingthelensawayfromthespecimen.Wheninfocusthelensnearlytouchesthecoverslip.Thefocalplaneissonarrowthatitisveryeasytofocusrightpastit.Ifyouarefocusingtowardthespecimen,youcandrivethelensrightintoit.

Whentouseoilimmersionlenses

Useanoilimmersionlenswhenyouhaveafixed(dead-notmoving)specimenthatisnothickerthanafewmicrometers.Eventhen,useitonlywhenthestructuresyouwishtoviewarequitesmall-oneortwomicrometersindimension.Oilimmersionisessentialforviewingindividualbacteriaordetailsofthestriationsofskeletalmuscle.Itisnearlyimpossibletoviewliving,motileprotistsatamagnificationof1000x,exceptfortheverysmallestandslowest.

Adisadvantageofoilimmersionviewingisthattheoilmuststayincontact,andoilisviscous.Awetmountmustbeverysecuretouseoil.Oilimmersionlensesareusedonlywithoil,andoilcan"tbeusedwithdrylenses,suchasyour400xlens.Lensesofhighmagnificationmustbebroughtveryclosetothespecimentofocusandthefocalplaneisveryshallow,sofocusingcanbedifficult.Oildistortsimagesseenwithdrylenses,soonceyouplaceoilonaslideitmustbecleanedoffthoroughlybeforeusingthehighdrylensagain.Oilonnon-oillenseswilldistortviewingandpossiblydamagethecoatings.

Exercise:examinationofstainedbacteria

BacteriacanreADIlybeseenevenwiththescanninglens,providedsufficientcontrastcanbeprovided.Darkfieldmicroscopyisbestfordetectingmotilebacteriaatlowpower,forexample.Whenbacteriaareheat-fixedandstainedtheytendtoclumptogether.Athighdrypowerindividualcellsoftencan"tbedetectedbecausethespacebetweencellwallsiswithinthelimitsofresolutionofthemicroscopeduetodiffraction.Toproperlyviewstainedbacteriaitisnecessarytouseoilimmersionmicroscopy.

Obtainaslideofgramstainedbacteria.Eachslideisetchedonthelowersurface,withtheheat-killedandstainedbacteriaontheopposite(upper)surface.Mounttheslidewithsmearupandfocusatlowpowerontheetching.Movetheslidesothatthelensisoverthesmearitself.Nowmovetheobjectiveawayfromtheslideuntiltheuppersurfacecomesintofocus.

Abacterialsmearatlowpowerlookslikeapatchofdirt.Focusonthemess,firstat40xthenat100xandfinally400x,allinbrightfieldmode.Notethatyoucanseesomedetailat400x,buttheshapesandcolorsofthebacteriaaresomewhatdistorted.

Movethe400xlensoutoftheway,placeadropofimmersionoildirectlyonthesmearwheretheobjectivewas,andswingtheoilimmersionlensinplace.Movethefinefocusupanddownslightlytoensurethatthelensisincontactwiththeoil.Nowwatchtheendoftheobjectiveandbringitasclosetotheslidesurfaceasyoucanwithouttouchingit.Noteinwhichdirectionyoumustfocustomovetheobjectiveawayfromtheslide,lookintheeyepieces,andslowlyrotatethefinefocuscontroluntiltheimageisfocused.

Mostbacterialspeciesarerod-shapedorround(cocci),althoughsomearecurved,spiral-shaped,orirregularlyshaped.Thegramstainleavessomecelltypespink(Gramnegative)andothersdarkblue(Grampositive)dependingoncellwallcharacteristics.Gramstainresultsareamajorcriterionforidentificationofspecies.Notehowmuchmorecleartheimageisat1000xwithoilthanitwasat400xwithoutoil.