Figure12.4Typicalbacterialshapes

Materials

• ColoniesofbacteriafromExercise12.2
• Toothpicks
• Crystalviolet
• Gram"siodine
• 95%ethanol
• Safranin
• Microscopeswithoilimmersion

Procedure

1. Beforestainingtheindividualcolonies,youshouldfirstpracticethetechniquebyobservationofthegrampositivemicro-organismsnormallyfoundinthegumliningsofyourmouth.

2. Useacleantoothpicktorubalongthegingivalcrevices(areabetweentoothsurfaceandgums)ofyourmouth.

   Rublightly!

3. Mixthescrapingswithadropofwaterpreviouslyplacedonacleanslide,spreadinathinfilmoverthecenteroftheslideandallowtoairdry.

4. Fixthesmeartotheslidebypassingtheslide(smearsideup)quicklythroughaflamethreetimes.Iftheslideishelddirectlyintheflame,itwillheatuptoorapidlyandbreak.Thetrickistogentlydrythesmearwithoutoverheatingtheslide.

5. Placetheslideonastainingrack.Applythestainsonthefixedsmearasfollows:
   • Floodtheslidewithcrystalvioletfor30sec.
   • Rinsewithwater.
   • FloodwithGram"siodinefor60sec.
   • Rinsewithwater.
   • Decolorizewith95%ethanol.
   • Rinsewithwater.
   • Counterstainwithsafraninfor60sec.
   • Rinsewithwaterandblotdry(norubbing!).
   • Examineunderoil-immersionobjectivelens.3

   Gram-positivebacteriaretaincrystalvioletafterwashingwith95%ethanol,whilegram-negativebacterialosethepurpledyeafterwashingwith95%ethanol.Thepositiveornegativereactionisameasureofthepresenceorabsenceofspecificpolysaccharidecomponentsoftheircellwalls.Safraninisusedasapinkcounterstain,sothatGram-cellscanbevisualized.Inpracticethen,thedistinctionismadebetweenpurplecells(+)andpinkcells(-).

6. Determinethebasiccellshapeofthebacteria.

7. AddtheinformationonGramstainandcellshapetotheworkdoneinExercise12.2.

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