Staining technique which differentiates between gram positive bacteria and gram negative bacteria.

1. GRAM
STAINING
KHADIJA SAEED

2. GRAM STAINING
• GRAM STAINING IS A COMMON TECHNIQUE USED TO
DIFFERENTIATE TWO LARGE GROUPS OF BACTERIA BASED ON
THEIR CELL WALL CONSTITUENTS.
• The gram stain procedure distinguishes between :
Gram positive and gram negative groups by coloring these cells red or
violet.
• THE DYE USED ARE:
Crystal violet
Safranin

3. PRINCIPLE
• Gram positive bacteria stain violet due to the
presence of a thick layer of peptidoglycan in their
cell walls.
 Which retains the crystal violet these cells are
stained with because of presence of teichoic acid.
• Gram negative bacteria stain red, which is
attributed to a thinner peptidoglycan wall .
Which does not retain the crystal violet during the
decoloring process due to absence of teichoic acid.
Crystal violet has positive charge and teichoic acid
has negative charge, so they bind with each other.

4. PROCEDURE
1. Fix the smear of specimen either by heating or
alcohol fixation.
2. Cover the fixed smear with crystal violet stain
for 30 seconds and then wash off with water.
3. Cover the smear with iodine for 30 seconds
and wash it off with water.
4. Decolorize by pouring acetone iodine (5
seconds) and rapidly wash off.
5. Now cover the slide with safranin for 30
seconds and wash it off with water.
6. Allow the slide to air dry, observe under
microscope.

5. PROCEDURE

6. OBSERVATION