Gram's Stain

Solutions required

1. Crystal violet, 0.5% aqueous solution
2. Gram's iodine (Lugol's iodine)
3. Gram decolouriser (acetone/alcohol)
4. Safranin, 0.5% aqueous solution
   

Solutions can be purchased ready to use.

Method

1. Heat fix thin smear by passing slide through Bunsen burner flame ~ 3 sec
2. Stain with crystal violet - 30 sec
3. Wash with water briefly
4. Mordant with Gram's iodine - 30 sec
5. Wash with water briefly
6. Decolourise with acetone/alcohol. Allow decolouriser to flow over slide held at 30° angle until colour just ceases to flow from the smear - a few sec
7. Wash immediately with water - 5 sec
8. Counterstain with safranin - 15 sec
9. Wash with water, blot dry and examine under oil immersion at x 1000

Results

1. Gram-positive bacteria - Blue
2. Gram-negative bacteria - Red