Gram Staining – Principle , Procedure , Function of Reagents

Gram Staining

Gram staining is basically a staining technique which is broadly used in microbial and molecular biology . This technique was developed by a Danish bacteriologist Hans Christian Gram in 1884. Gram staining is used for the identification and differentiation of different types of bacteria. It differentiates Gram positive bacteria and Gram negative bacteria on the basis of their stain retaining abilities.

Function of Crystal violet (Primary stain)

In gram staining the first stain which is used is the primary stain  or we can call it as Crystal Violet stain. Primary stain gives Blue or purple colour when it binds to the cell wall of bacteria.

Function of Iodine

 Mordant plays role in intensifying the primary stain and properly fix it onto the cells. Iodine is used for this purpose.

Function of Ethyl Alcohol

Decolourizer basically dehydrates the peptidoglycan layer of the bacterial cell wall. And as a result the cell shrinks. Usually ethyl alcohol and acetone are used as decolorizers.

Sefranin (Secondary stain)

When the cells can not retain the primary stain , secondary stain is used. Sefranin is the most commonly used secondary stain .  The cells appear red  when they are stained with sefranin.

Principle of Gram Staining:

As the cell wall of bacteria is very different from the cell wall of other organisms. Yet there are differences in the cell walls within the  species of bacteria.  Gram positive bacteria have high content of peptidoglycan in its cell wall while the lipid content is quite low. This thick layer of peptidoglycan plays its role in staining. On contrary, the gram negative bacteria has low peptidoglycan content and high lipid content in its cell wall.

In gram positive bacteria, when primary stain(crystal violet) is applied, it is fixed by iodine and a CV-I ( crystal violet-iodine) complex is formed. When decolourizer is applied to it , the cell wall of bacteria dehydrates and the cell shrinks, and as a result the pores in  the cell wall get closed because of which the primary stain is unable to exit the cell wall and thus it remains attached to the cell wall. In other words the primary stain retains and does not wash off. Now, there is no need for the secondary stain .  The primary stain gives blue/purple colour . Thus we conclude that gram positive bacteria are give Blue-purple colour by gram staining.

In case of gram positive bacteria, the cell wall has thin layer of peptidoglycan , that’s why the primary stain or we can say the CV-I complex does not retain. When decolourizer is applied, the lipids in the cell wall are dissolved which causes the CV-I to leach out of the cells. And then we apply the secondary stain ( sefranin) because of which the cells appear red  in colour. As a result gram negative bacteria  give red colour upon staining.


  • Take a clean , grease-free ,sterile microscopic slide.
  • Take the bacterial sample ,which is to be examined, at the center of the slide
  • Now , make a smear of this sample on the slide
  • Air dry and heat fix the smear.
  • Use a dropper to add primary stain on the smear and allow it to stay for 1 minute
  • Now rinse the slide  gently with water
  • Now add Gram’s Iodine to the slide and keep it for 1 minute  to fix the crystal violet.
  • Now again rinse the slide gently with water
  • Now add alcohol or aetone and allow it to stay for just 10-20 seconds and immediately rinse it with water
  • Gram positive bacteria will give blue/purple colour with the primary dye
  • While for gram negative, apply the secondary dye (sefranin) with the help of dropper and allow it to stay for 1 minute
  • Finally ,  rinse the slide with water
  • The slides can be viewed under microscope which helps to distinguish the cell shape and arrangement.
  • Gram positive bacteria appear blue/purple, while gram negative bacteria appear red
Gram Positive and Gram Negative Bacteria in Gram Staining
Fig. 01: Gram Positive and Negative Bacteria showing different colour in Gram Staining.


Gram Positive Bacteria

Nocardia, Actinomyces, Bacillus , Enterococcus , Streptococcus, Mycoplasma , Listeria, Staphlococcus, Streptomyces, Lactobacillus, Corynebacterium etc

Gram Negative Bacteria:

Legionella , Helicobacter, Salmonella, Shigella, E.coli, Moraxella, Pseudomonas, Bdellovibrio,Stenotrophomonas etc.

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