Negative and Simple Staining
Background on Negative Staining: Asany microbiology studentwilltell you,findingbacteriaonaslideisnotaneasy task.Itdemandsagreatdealofpractice, a fairamount of patienceand a little bitof luck. Fortunately there is a simplemethod that we can use to detectmicroorganisms on a slide and it iscalled negative Staining. The processinvolves mixingmicrobes with a darkstainandspreadingthe mixture overaclean slide.Sincelivingcellsaretransparentanddonottakeupthestain, they are visible as “bright dots” against adarkly stained background as shown in Figure 1.
Cell
Figure1.Negative StainNegative staining is primarily used for:
1.revealingthepresenceof acapsule
2.providingaccuratecellsize
3.specimens that are difficult tostain
NegativeStainingProtocol
1)Clean microscope slidethoroughly.
2)Place a small drop ofnigrosinenearone edge of the slide.
3)Mix a loopful of bacterial cultureinto the drop of nigrosine as showninFigure2.Useaseptictechnique.
Figure 2
4)Placea second slide lengthwise infront of the drop of stain as shown inFigure3.Startinginthe middle ofthe slide which contains the stain, movethe other slide back at a 45oangleuntilittouchesthestain,then pushforwardtospreadthestain.
Figure 3
5)Remember thatthe slide usedtospread the stain is now contaminatedwith microbes and should be handledappropriately.
6)Airdry(5-10 min.)DO NOT heatfix!
7)Examine under oil immersion andrecordyour observations in the spaceprovided below.
Culture#1Culture#2Culture#3
Negative Staining Results
Background on Simple Staining: Nowthatyouareconvinced(Ihope)that microorganisms do in fact exist, it istime to learnhow to stain them.Wewillbegin by staining themwith a single stain,aprocesscalledsimple staining.
At some time in your distant pastyouhaveprobablybeensubjectedtoa high-schoolphysicalsciencecourse.
Duringthiscourseyoulearneda valuable lesson, which will help you tostain microorganisms. Yes, that age-oldphysical axiom“opposites attract”, the one piece ofinformation that yougleaned from this course, is actually useful! Bacteria, unbeknownst to them, carrya slightly negativecharge.Manystains,suchas methylene blue,haveapositively-chargedportion(chromophore) whichisattractedtoandsticksto the negatively charged bacterialsurface thereby “staining”the cell.
Now that you understand how simple staining works you are probablyanxious to get started, but you are not yetready.Afirststepinmost stainingtechniques (with the notable exception of negative staining) is the preparation of a bacterial smear. This techniquebasically sticks the bacteria totheslideso they won’t come off while you arestaining them
.
ProcedureforMakingaSmear
1)Clean and dry a glass slide.
2)Usingaseptic technique, transferaloopfulofabacterialbrothcultureto the center ofthe slide. You need tosmear the material outinto an areano larger than a dime. If you suspectthere are not manyorganisms in thecultureyou may needtoaddseveralloopfuls of organisms to the slidebeingsuretoplacetheminthe samespot.
3)Allowtoairdry(5-10 min.)
4)Holdingtheedgeoftheglassslide with a clothespin or forceps, pass theslide (smear sideup)throughaBunsen burner 3 or 4 times. This fixes the cells to the slide so thatsubsequent staining andwashingwill not removethem.
5)You are now ready to stain!
NOTES:
SimpleStainingProtocol
1)Cover the smear with a few drops ofmethyleneblue.Let sit for one minute.
2)Rinsewithwaterandblotdrywith bibulous paper.
3)Examine under oil-immersion andrecordyourobservationsbelow.
NOTES:
Culture #1
Culture #2
Culture #3
SimpleStainofMouthOrganisms
Thelittle critters we have been lookingatsofarare tame laboratorypetsIkeeparound for your culturing and viewing pleasure.WhatIwouldlikeforyouto do now is to do a little exploringandseewhat you can find lurking around thebase of your teeth.
1)Usingasterile swab,gently scrapearoundthebaseofyourteeth and gums for about 30 seconds.
2)Smear thematerialyoucollectedonthe swab onto a clean microscope slideandallowtoairdryfor a few minutes and then heat fix.
3)StainwithMethyleneblueas described above and observe.
4)Record your results below.