Explain the Decolourizing Agent - Stain Technique?

95% ethanol is used as a decolourizing agent. It has two functions -

(1) It acts as protein - dehydrating agent, and

(2) It is a lipid solvent. The action of decolourizing agent depends on two factors -

(i) Concentration of lipids in cell wall.

(ii) Thickness of peptidoglycan layer.

In gram negatives, lipid content is high as compared to gram positives, so ethanol action increases the porosity of cell wall by dissolving the lipid layer in outer membrane. Peptidoglycan layer being thinner and less highly cross linked as compared to gram positives, is not able to retain the CV-I complex and cell become colourless. On the other hand, in gram positives, the pores become smaller due to dehydrating effect of the alcohol, so CV-I complex is retained by the thick highly cross linked peptidoglycan layer, and the cells remain purple. This step generates the differential aspect of the gram stain and is very critical.