Gram Staining Procedure

It is a type of differential staining in which two or more stains are used to differentiate between types of bacteria. Hans Christian Gram discovered this staining technique in 1884.

Importance:

• It is an important test for rapid diagnosis of infectious agents.

• It differentiates types of bacteria whether Gram positive or Gram negative.

• It helps in the study of the morphology of bacteria.

• It helps find evidence of capsules, spores, pus cells, epithelial cells, Yeast cells, etc.

• To understand how the Gram stain reaction affects Gram positive and Gram negative bacteria based on their cell walls' biochemical and structural differences.

Gram Staining Procedure:

1. Crystal violet: It is the primary stain which is used as a simple stain because it dyes the cell wall of bacteria cells.

2. Gram’s Iodine: It acts as a mordant which helps to fix the primary dye to the cell wall.

3. Decolourizer: It is used to remove the stain of primary dye (crystal violet) from the Gram negative bacterial cell wall. Decolourizer is composed of organic solvents like acetone, ethanol, or in combination.

4. Safranin: Finally, a counter stain is applied to stain those cells that have lost their primary stain (especially Gram negative bacteria) as a result of decolorization.

The procedure is based on the ability of microorganisms to retain the color of the stains used during the gram stain reaction. Gram-negative bacteria are decolorized by the alcohol, losing the color of the primary stain, purple.

Gram-positive bacteria are not decolorized by alcohol and will remain purple. After the decolorization step, a counterstain is used to impart a pink color to the decolorized gram negative organisms.

Gram positive bacteria: Stain dark purple due to retaining the primary dye called Crystal violet in the cell wall. Example: Staphylococcus aureus.

Gram negative bacteria: Stain red or pink due to retaining the counter-staining dye called Safranin. Example: Escherichia coli.

There are some differences between Gram positive and Gram negative bacteria which are listed below.

Differences between Gram Positive and Gram negative bacteria

Gram Positive bacteria	Gram Negative bacteria
1. They retain crystal violet dye and stain dark violet or purple, they remain colored blue or purple with gram stain when washed with absolute alcohol and water.	1. They are decolourized to accept counter stains such as Safranin or Fuchsine. They stain red or pink and they do not retain the Gram stain when washed with absolute alcohol and acetone.
2. Peptidoglycan is multilayered thick.	2. Peptidoglycan is single-layered thin.
3. Outer membrane is absent.	3. Outer membrane is present.
4. Periplasmic space is absent.	4. Periplasmic space is present.
5. Teicholic acids are present.	5. Teicholic acids are absent.
6. Lipid and lipoprotein content is low.	6. Lipid and lipoprotein content is high.
7. They have high resistance to physical disruption and drying.	7. They have a low resistance to physical disruption and drying.
8. Cell wall is 100-120 Å thick; single layered. The lipid content of the cell wall is low, whereas the Murein content is 70-80%.	8. Cell wall is 70-120 Å thick; two layered. Lipid content is 20-30% (high), Murein content is 10-20%.
9. More susceptible to antibiotics.	9. More resistant to antibiotics.