Procedure of Gram Staining Method

(Procedure of gram staining method Helps to Classify Bacteria into Two Groups)­. Gram staining method is used to distinguish and classify bacterial species into two large groups (gram positive and gram negative). The name comes from the Danish bacteriologist Hans Christian Gram who developed the technique.

Gram Staining Method


Apparatus/Chemicals for Gram Staining Method

The apparatus need to use in Gram Staining Technique are droppers, clean experimental slides and following reagents.

Reagents used in Gram Staining Method

  • Primary Stain         –     Crystal Violet
  • Mordant                  –     Grams Iodine
  • Decolorizer             –      Ethyl Alcohol
  • Secondary Stain    –      Safranin

Procedure of Gram Staining Method

1: Take a glass slide and drop some drops of crystal violet on different bacteria then you will observe some colored spot. They are bluish purple in color.

2: Drop some drops of 0.5 iodine solution on that slide. And then observe the color of bacteria which remains purple.

Significance of 0.5 % Iodine Solution: Actually, it forms complex with crystal violet dye, that is known as crystal violet iodine complex (CVI-Complex).

Fixant/Mordant: Due to this reason, there is fixation of crystal violet to the cell. That’s why iodine solution is also known as fixant/mordant. Without iodine solution there will be loss of crystal violet dye from bacterial cell.

3: Alcohol is a decolorizer that washes away stain from some bacteria. After applying alcohol, observe that some bacteria are still stained but some become colorless. The bacteria which has retained the color named as Gram positive bacteria. The colorless bacteria named as Gram negative bacteria.    

4: To increase the visibility or presence of bacteria, use the Safranin. Safranin is also called as counter stain. After applying safranin, observe that the colorless bacteria will changes its color into pink form. So, the pink color spots are Gram negative bacteria. There is appearance of two types of bacteria at the end. Therefore, this technique is also called as Differential Staining Technique.

Gram Positive Bacteria                   Gram Negative Bacteria
1.    Bacillus

2.    Clostridium

3.    Lactobacillus

1.    E. coli

2.    Agrobacterium

3.    Rhizobium (Nitrogen Fixing Bacteria)

4.    Vibrio (Comma Shaped Bacteria)

5.    Azotobacteria.



Why some of the bacteria retained color but the other loose the color?

Due to the thickness of cell wall in gram positive bacteria, there is no chance of loosening of stain.
While in gram negative bacteria.
1. The cell wall is thin due to 70-120 peptidoglycan.
2. The Lipopolysaccrides contains high amount of lipids (phospholipids) which will be dissolved by the treatment of alcohol. These are the reasons of the removal of stain in gram negative bacteria.

Bacterial Cell Walls

Effect of Antibiotics on Bacteria

Also called antibacterials. A type of antimicrobial drug used in the treatment and prevention of bacterial infections. They may either kill or inhibit the growth of bacteria. For example: Penicillin.

Effect of Penicillin on Gram + and – Bacteria

Penicillin is used to kill gram positive bacteria, but it cannot kill gram negative bacteria due to the presence of lipopolysaccharide and protein layer that surrounds the peptidoglygan layer of the cell wall, preventing penicillin from attacking.

On the other hand, the gram + bacteria do not have outer membrane Lipopolysaccrides that’s why it is easy for Penicillin to attack.

Types of Antibiotics on the Basis of Bacteria

On the basis of bacteria, the antibiotics are of two types

  1. Narrow Spectrum: Those antibiotics that kill only one type of bacteria are called narrow spectrum antibiotics.
  2. Broad Spectrum: Those antibiotics that kill both type of bacteria (Gram positive and negative bacteria) are called narrow spectrum antibiotics.
Types of Antibiotics

Types of Antibiotics

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