Gram Staining PDF.pdf

Microbiology

1. GRAM’SSTAINING
Mr. Osman H. Ali
B.Sc. Microbiology, MPH, HDCH

2. Introduction
TheGramstainisadifferentialstainingprocedureusedto
categorizebacteriaasGrampositiveorGramnegativebasedonthe
chemicalandphysicalpropertiesoftheircell’swalls.
Thebacteriaaredifferentiatedthroughaseriesofstainingand
decolorizationsteps.Grampositivecellswillstainpurpleand
Gram-negativecellswillstainredtopink.

3. Conti…….
TheGramstainwasdevisedbytheDanishphysicianHans
ChristianGram,whileworkinginBerlinin1883.
Helaterpublishedthisprocedurein1884.

4. Purpose
TheGramstainisfundamentaltothephenotypiccharacterizationof
bacteria.
Thestainingproceduredifferentiatesorganismsofthedomain
Bacteriaaccordingtocellwallstructure.
Gram-positivecellshaveathickpeptidoglycanlayerandstainblue
topurple.
Gram-negativecellshaveathinpeptidoglycanlayerandstainredto
pink.

5. GRAM’SSTAIN
Gram staining (or Gram's method) is a method of
differentiatingbacterialspeciesintotwolargegroups
GramPositive GramNegative
Gramstainingdifferentiatesbacteriabythe chemicaland
physicalpropertiesoftheircellwalls.

6. GRAMPOSITIVEBACTERIA
•Grampositivebacteriahaveathick
cellwallofpeptidoglycan.
•Peptidoglycanisapolymerconsisting
ofsugaraminoacidsthatformamesh
likeoutsidetheplasmamembraneof
bacteriaformingcellwall.
•InGrampositivebacteria,betweenthe
cellwallandcellmembrane,thereisa
"membraneteichoicacid".

7. GRAMNEGATIVEBACTERIA
Gramnegativebacteriahaveanouter
membraneof phospholipidsand
bacterialLipopolysaccharidesoutsideof
theirthinpeptidoglycanlayer.
Thespacebetweentheoutermembrane
andthepeptidoglycanlayeriscalledthe
periplasmicspace.

9. PrincipleofGram’sStaining
The structureof the organism ‘s cell wall determineswhether
theorganismisgrampositiveor negative.
Whenstainedwitha primarystainandfixedbya mordant,some
bacteriaareabletoretainthe primarystainbyresisting
declorizationwhileother getdecolorizedby decolorizer.
Thosebacteriawhichretainthe primarystainare calledGram
positive.
Thosebacteriawhichgetdecolorizedandthenget counterstained
arecalledGram negative.

10. 1.Crystalviolet-allbacteriatakecrystalviolet-soallappears
violet.
2.Iodine–CrystalViolet-iodine(CV-I)complexisformed.
3.Acetone-bacteriawithhighlipidcontentlooseCV-I
complex(appearcolourless)butbacteriawithlesslipidcontent
retainsCV-Icomplex(appearviolet).
4.Safranine/basicfuchsin–onlycolourlessbacteriatakes–
appearpink.

12. Preparationofaslidesmear:
1.Inoculationloopisusedtotransferadropofsuspendedculturetothe
microscopeslide.
2.Afewloopfulofwaterisaddedtofacilitateaminimalamountof
colonytransfertotheexaminationslide.
3.spreadwithaninoculationlooptoaneventhinfilmoveracircleof
15mmindiameter.
4.Theslidecanbeeitherair-driedordriedwiththehelpofheatovera
gentleflame.

14. Procedureof gram stain
•Preparation ofsmear
•Air dry and Heatfixation

17. ThankYou