Types of staining techniques in microbiology

This file will tell about different Types of staining techniques in microbiology and different stains used in staining

1. STAINS & STAINING
Faculty: Dr. Rakesh Sharda

2. OBJECTIVES OF STAINING
Improvesvisibiltiybygreatercontrastbetweenthe
organismandthebackground,
differentiatevariousmorphologicaltypes(byshape,size,
arrangement,etc.),
determinethestainingcharacteristicoforganismand,at
times,directdiagnosisofdisease,and
demonstratethepurityofculture.
observecertainstructures(flagella,capsules,endospores,
etc.),

3. STAINS AND DYES
➢Adyeisageneral-purposecoloringagent,
whereasastainisusedforcoloringbiological
material.
➢Astainisanorganiccompoundcontaininga
benzeneringplusachromophore andan
auxochromegroup.
➢chromophoreisachemicalgroupthatimparts
colortobenzene.
➢auxochromegroupisachemicalcompound
thatconveysthepropertyofionizationof
chromogen(abilitytoformsalts)andbindto
fibersortissues.

4. REQUIREMENTS FOR STAINING
Stain–Majorityofthestainsusedforstainingbacteriaareofthe
basictypeasnucleicacidofbacterialcellsattractthepositive
ions,e.g.methyleneblue,crystalviolet.Acidicstainsareusedfor
backgroundstaining.
Mordant–Itisachemicalthatformsaninsolublecomplexwith
thestainandfixesitorcausesthestaintopenetratemoredeeply
intothecell.Theseareusedinindirectstaining.Forexample,
Gram’siodineinGramstainingandphenolinZiehlNeelson’s
staining.
Accentuater–Itisachemicalwhichwhenaddedtoastainto
makethereactionmoreselectiveandintense.Forexample,
potassiumhydroxideaddedinLoeffler’smethyleneblue.
Decolorizer –It is a chemical used to remove the excess stain in
indirect regressive staining. For example, ethanol in Gram’s
staining.

5. Types of staining techniques
Simplestaining Differentialstaining
(Use of of single stain)(Use of two contrasting stains)
Direct Indirect SeparationVisualization
(Positive) (Negative) into groups of structures
1. Gram stain1. Flagella stain
2. Acid fast2. Capsule stain
stain 3. Spore stain

6. SIMPLE STAINING

7. SIMPLE STAINING
➢Astainingmethodthatusesonlyasingledyethatwhich
doesnotdifferentiatebetweendifferenttypesoforganisms
➢Thereisonlyasinglestainingstepandeverythingis
stainedwiththesamecolor.
➢Simplestainsareusedtostainwholecellsortostain
specificcellularcomponents.
➢Typesofsimplestaining:
1.Direct/Positivestaining:stainobject
2.Indirect/Negativestaining:stainbackground

9. Direct staining (Positive staining)
➢Asimplestainingtechniquethatstainsthebacterialcellsin
asinglecolor.
➢Manyofthebacterialstainsarebasicchemicals;these
basicdyesreactwithnegativelychargedbacterial
cytoplasm(oppositechargesattract)andtheorganism
becomesdirectlystained
➢Examplesaremethyleneblue,crystalviolet,andbasic
fuchsin.

10. Indirect staining (Negative staining)
➢Inthisstainingprocess,insteadofellsbackgroundis
stained.
➢Here,anacidicdyelikenigrosinorIndianinkis
used.Acidicstaincarriesanegativechargeandrepelled
bythebacteria,whichalsocarryanegativechargeontheir
surface.Hence,anacidicdyedonotstainbacteria,
➢Instead,itformsadepositaroundtheorganism,leavingthe
organism itselfcolorlessortransparentupon
examination.

11. STAINING:
GENERAL TECHNIQUE
SMEAR AIR DRY HEAT FIX STAIN LOOK

12. Importance of fixing the smears
“Fixationaccomplishesthreethings:
(1)itkillstheorganisms;
(2)itcausestheorganismstoadheretotheslide;and
(3)italterstheorganismssothattheymorereadilyacceptstains
(dyes).

13. DIFFERENTIAL STAINING

14. GRAM’S STAINING

15. The Gram Stain
Inthelate1800’s,ChristianGramobservedthatsome
generaofbacteriaretainedadye-Iodinecomplexwhen
rinsedwithalcohol,whileothergenerawereeasily
decolorizedwithalcoholandcouldbethenvisualizedby
acontrastingcounterstain.
Thisstainingproceduredefinestwobacterialgroups:
thosewhichretaintheprimarydyes(“Positiveby
Gram’sMethod”or“Gram-Positive”)andthosewhich
areeasilydecolorized(“NegativebyGram’sMethod”or
“Gram-Negative”).Thisisthestartingpointfor
bacterialidentificationprocedures.

16. The Gram Stain
➢Thedifferenceindyeretentionisdependentonsuch
physicalpropertiesasthickness,density,porosity,and
integrityofthebacterialcellwall,aswellasits
chemicalcomposition.
➢Gram-Positivebacteriahavethick,dense,relatively
non-porouswalls,whileGram-Negativebacteriahave
thinwallssurroundedbylipid-richmembranes.
➢Somenon-bacterialorganismswiththickcellwalls(e.g.,
someyeasts)alsostainGram-Positive.
➢Gram-Positivebacteriawhichhavelostwallintegrity
throughagingorphysicalorchemicaldamagemay
stainGram-Negative.

17. GRAM POSITIVE
GRAM NEGATIVE
Cytoplasm
Cytoplasm
Lipoteichoic acid Peptidoglycan-teichoic acid
Cytoplasmic membrane
Inner (cytoplasmic) membrane
Outer Membrane
Lipopolysaccharide
Porin
Braun lipoprotein

18. Gram staining –Requirements
•Gram-stainingisafourpartprocedure.
•Thespecimenismountedandheatfixedonaslidebefore
proceedingtostainit.
•Thereagentsrequiredare:
➢CrystalViolet(thePrimaryStain)
➢IodineSolution(theMordant)
➢Decolorizer(ethanol)
➢Safranin(theCounterstain)
➢Water(preferablyinasquirtbottle)

19. Gram staining –Procedure
1Thebacteriaarefirststainedwiththebasicdyecrystalviolet(primary
stain).Bothgram-positiveandgram-negativebacteriabecomedirectly
stainedandappearpurpleafterthisstep.
2Thebacteriaarethentreatedwithgram'siodinesolution(mordant).This
allowsthestaintoberetainedbetterbyforminganinsolublecrystal
violet-iodinecomplex,calledas‘iodinelake’.Bothgram-positiveand
gram-negativebacteriaremainpurpleafterthisstep.
3Gram'sdecolorizer,amixtureofethylalcoholandacetone,isthen
added.Thisisthedifferentialstep.Gram-positivebacteriaretainthe
crystalviolet-iodinecomplexwhilegram-negativearedecolorized.
4Finally,thecounterstainsafranin(alsoabasicdye)isapplied.Sincethe
gram-positivebacteriaarealreadystainedpurple,theyarenotaffected
bythecounterstain.Gram-negativebacteria,thatarenowcolorless,
becomedirectlystainedbythesafranin.Thus,gram-positiveappear
purple,andgram-negativeappearpink.

22. ACID FAST STAINING

23. Structure of an Acid-Fast Cell Wall

24. ➢Oncestainedtheacidfastbacterialcellsresist
decolorizationwithacidifiedorganicsolvents,e.gacid
alcoholandarethereforecalledACIDFAST.
➢Acidfaststainingpropertyofthegenus,Mycobacteria,
dependsupontheirlipid-richcellwallswhicharerelatively
impermeabletovariousbasicdyesunlessthedyesare
combinedwithphenol.
➢Theexactmethodbywhichthestainisretainedisunclear
butitisthoughtthatsomeofthestainbecomestrapped
withinthecellandsomeformsacomplexwiththemycolic
acids.Thisissupportedbythefindingthatshorterchain
mycolicacidsormycobacterialcellswithdisruptedcell
wallsstainweaklyacid-fast,e.g.Nocardia
Acid fast staining -theory

25. Acid-Fast Stain of Mycobacterium tuberculosisin
Sputum
Note the reddish acid-fast bacilli among the blue normal flora and
white blood cells in the sputum that are not acid-fast.

26. SPECIAL STAINING

27. CAPSULE STAINING

28. ➢Capsulestainingisdiagnosticallyusefulsinceitis
avirulentfactor(e.g.pneumococci).
➢Bacterialcapsulesarenon-ionic,soneitheracidic
norbasicstainswilladheretotheirsurfaces.
➢Capsulesaredemonstratedeitherbynegative
staining(NigrosinorIndiaink)orbyspecial
staining,e.g.Hiss’method,Anthony’smethod

29. Hiss Method
•Thecapsuleisnon-ionicinnaturesoitdoesn'tgetstainbya
acidicstainbutabasicstain,suchascrystalviolet,stains
thecellaswellasthecapsule.
•Thisisfollowedbytreatmentwithhypertonicsolution-
20%Coppersulphatesolution,whichservesdualroleof
boththedecolorizingagentandcounterstain.
•Coppersulphatesolution,beinghypertonic,causesdiffusion
ofstaintowardsoutersurfaceofcell.
•Afterdryingofslide,thestainwhichisnotpassedfromthe
capsularlayerduringdiffusionretainsinthecapsularlayer.
Coppersulphatethendecolorizesthecapsule.
•Capsuleappearsasafaintbluehaloaroundapurplecell.

31. ENDOSPORE STAINING

32. Endospore staining
➢Sporesarenormallyimpervioustostains.
➢Underthelightmicroscopeendosporeshaveahighlight
refractivityindicativeofhighproteincontent.
➢Endosporescanbestainedby:
➢ModifiedZeihl-Nelson'smethodusing0.25-0.5%sulphuricacid
asdecolorizingagent,
➢Barthelomew-Mittwar’smethod
➢Schaeffer-Fultonstaintechnique.

33. Schaeffer-Fulton method
•Malachitegreenisusedtostaintheendospores(primary
stain)
•Themalachitegreenisforcedtopermeatethesporewall
byheating(mordant).
•Washingwithwaterremovestainfromvegetativecells,but
notfromsporewall.
•Theendosporesthusretaintheprimarydyewhilethe
vegetativecellslosetheprimarystainandtaketheredcolor
ofsecondarystain(safranin).

34. Note green endospores within pink bacilli
Endospore stain of Bacillus megaterium