GRAM STAINING

  • Important staining method in bacteriology.
  • Can demonstrate morphology of bacteria.
  • Distinguishes bacteria in 2 categories:
    1. Gram positive(purple)
    2. Gram negative (pink/light red)




      GNB
      Gram Negative Bacilli

Theories of Gram stain:

  • Magnesium ribonucleate theory
  • Iso-electric pH theory
  • Peptidoglycan theory
  • Lipid content theory




Modifications of Gram staining:

  1. Original Method of Gram Staining by Hans Christian Gram(1884)
  • Aniline-Gentian violet
  • Lugol’s iodine (Iodine1gm, KI-2gm, Water-300ml)
  • Absolute alcohol
  • Bismarck brown
  1. Kopeloff & Beerman’s Gram Method
  • Methyl violet(5min)
  • Iodine(2min)
  • Acetone(1-2 sec)
  • Basic fuchsin

    GPC
    Gram Positive Cocci arranged in cluster
  1. Jensen’s Gram Method (For gonococci, Meningococci)
  • 5% Methyl violet (30sec)
  • Lugol’s Iodine 1% (30sec)
  • 95/100% Ethanol (30sec)
  • Weak Neutral Red 0.1% (2min)
  1. Preston & Morrell’s Gram Method
  • Ammonium oxalate- Crystal violet (30sec)
  • Lugol’s Iodine 1% (30sec)
  • Iodine-Acetone (0.35% iodine) (30sec)
  • Dilute carbol fuchsin (30sec)
  1. Quick Gram Method
  • Crystal/Methyl violet (5sec)
  • Iodine (5sec)
  • Acetone (2sec)
  • Basic Fuchsin (5sec)





Examples:

  • Gram positive cocci: Staphylococci, Streptococci
  • Gram positive bacilli: Corynebacteria, Bacillus
  • Gram negative cocci: Gonococci, Meningococci
  • Gram negative bacilli: Pseudomonas, Salmonella