Aim: To perform gram staining of given bacterial sample
Materials required: Clean glass slides, inoculating loop, bunsen burner, distilled water (for making
smears), dropper or pipette, blotting paper, microscope and staining rack
Reagents required: Crystal violet dye, iodine, alcohol (95% ethyl alcohol), saffranin dye
Principle: Gram staining is a differential staining technique used to classify bacteria into two major
groups: Gram-positive and Gram-negative, based on the structural differences in their cell walls. The
method was developed by Hans Christian Gram in 1884. Based on the structure of cell wall, this
technique differentiates bacteria into two groups i.e., Gram positive and Gram negative bacteria. The
procedure is based on the ability of microorganisms to retain colour of the stain during Gram reaction.
Gram negative bacteria are decolourised by alcohol losing the colour of primary stain , purple. Gram
positive bacteria are not decolourised by alcohol and will remain as purple. After decolourisation stop
, a counter stain is used to impart pink colour to the gram negative microorganisms.
Gram positive bacteria have a thick mesh like cell wall which is made up of peptidoglycan
(50-90%) of cell wall, which stain purple. Gram negative bacteria have a thinner layer of
peptidoglycan (10% of cell wall) and lose the crystal violet iodine complex during decolourisation
with alcohol rinse but retain the counter stain safarin thus appearing reddish
or purple.
Stain reaction:
1. Application of crystal violet (CV) to heat fixed smear : CV dissociates in aqueous solution into CV+
and Cl- ions. These two penetrate the cell wall and cell membrane of both gram positive and gram
negative . CV+ interact with negative component of bacterial cell and stain it purple.
2. Addition of gram iodine :
Iodine acts as a mordant and a trapping agent. A mordant is a substance that increase
the affinity of cell wall for a stain by binding to primary stain , thus forming a insoluble complex that
get trapped in cell. During the reaction CV-I compex is formed and all the cells turn purple.
3. Decolourization with ethyl alcohol :
Alcohol dissolve the lipid outer membrane of gram negative bacteria, thus leaving the petidoglycan
layer exposed and increase the porosity of cell wall. The CV-I complex is then washed away from the
peptidoglycan layer leaving gram negative bacteria colourless.In gram positive bacteria , alcohol has
