PRACTICAL GRAM STAINING

REAGENTS USED IN GRAM STAIN
Gram Crystal Violet 0.5%
Gram Iodine
Potassium Iodide 2%
Resublimed Iodine 1%
Gram Decolorizer
Methanol 80%
Acetone 20%
Gram Safranine 1%
REAGENTS USED IN GRAM STAIN
CRYSTAL VIOLET
Primary stain
Violet colored, stains all micro-org
GRAM IODINE
Mordant
Forms Crystal violet iodine complexes
DECOLORIZER
Acetone + Methanol
Removes Crystal violet iodine complex from thin peptidoglycan layers
Dissolves outer layer of Gram negative orgnisim

REAGENTS USED IN GRAM STAIN

GRAM SAFRANINE
Counter stain
Red colored
Stains thin walled Gram negative org.
Pus cells cytoplasm & lobes of nuclie also stain red

The Gram Stain Procedure
Step 1 - Prepare a Smear
Suspend some of the material to be stained in a drop of water on a microscope slide, spread the drop to about the size of a nickel.
Allow to air dry. Heat fix by gently warming
The Gram Stain Procedure
Step 2 - Apply the Primary Stain
Flood the Smear with Crystal Violet
Allow to stand for 1 min
Rinse with water to remove excess stain
The Gram Stain Procedure
Flood the Smear with Iodine solution
Allow to stand 2 min
Step 4 - Rinse
Rinse with water to remove excess Iodine
Step 5 - Decolorize
Drip Decolorizer (80% Methanol +20% Acetone) across the slide about 5 sec
The effluent should appear pale or clear

Step 6 - Rinse
Rinse with water to remove excess alcohol
Step 7 - Counterstain
Flood the slide with Safranin solution
Let stand for 2 minutes
Step 8 - Rinse, Dry and Observe
Rinse with water to remove excess stain
Blot dry
Observe under Oil Immersion