GRAM STAIN

GRAM STAIN By Nimra Khan

Differential staining methods impart different colors to different organisms dividing them into Gram +ve and Gram –ve.

PRINCIPLE
Some bacteria resist decolouristaion & retain primary stain: crystal violet(gram +VE) whereas others get decolourised & take counter: stain-pink(gram-VE)

THEORIES
3 main theories explain mechanism of gram’s staining:

1)LIPID CONTENT
=>Gram +ve: protein in the cell wall on decolourisation undergo dehydration and shrinkage. The dye iodine complex stays in cell
=>Gram –ve: lipids in cell wall dissolve on decolourisation and dye iodine complex leaks out

2)Mg RIBONUCLEATE THEORY:
=>Only Gram +ve have this compound that covalently binds with dye iodine complex & imparts gram positivity.

3)IEP (Isoelectric Point) RANGE: Also called pH theory
=>Gram +Ve: unsaturated acids that get oxidized during iodine treatment lead to more acidity hence affinity towards basic dyes
=>Gram –ve: less acidity, less affinity towards basic dyes

PREPARATION OF SMEAR
1)Make a thin film of material on glass slide 2)Air dry 3) Heat fix

STAINING:

• Add crystal violet (primary stain) and wait 3-5 mins.
  
• Wash with water.
  
• Add grams iodine(mordant) wait 1 min wash with water.
  
• Decolourise with acetone.
  
• Wash with water immediately.
  
• Add safranin (counter-stain).
  Wait 1 min wash with water.
  
• Air dry. Observe under oil immersion.

RESULTS: Gram+ve appear purple. Gram-ve appear pink

Gram+ve appear purple. Gram-ve appear pink



Nimra Khan 2006MO48