STANDARD OPERATING PROCEDURES FOR GRAM STAIN

1. SPECIMEN:

1. Gram stain can be done on various samples such as sputum, pus, and tissue,etc.

2. REAGENTS:

a. Gentian violet

b. Iodine solution

c. Alcohol (95%) or Acetone-alcohol

d. Safranin

3. PROCEDURE:

1. Fix the slide by heat.

2. Primary stain :Stain the slide smear with gentian violet or crystal violet for 30-60 seconds.

3. Mordant: Wash the violet stain and flood the smear with Iodine solution for 30-60 seconds.

4. Decolorizer: Wash of the smear and flood it with alcohol ( 95 % ) or Acetone-alcohol

mixture.

5. Secondary stain : Counterstain the smear with safranin which is red dye for 1-2 minutes.

6. Observe the result.

a) Gram positive bacteria are blue purple.

b) Gram negative bacteria are pink magenta color.

7. Prepare lab report of the observations.