In the Gram stain procedure, the mordant is applied after the primary stain.

• A. True
• B. False
• C. It is skipped
• D. It is applied before the primary stain

Answer: (A)

The key idea is how the dye is retained in Gram staining through the mordant step. After applying the primary stain (crystal violet), the mordant (iodine) is added to form a crystal violet–iodine complex. This complex is larger and more tightly associated with the cell wall, especially in Gram-positive bacteria with a thick peptidoglycan layer, so it is not easily washed out during the subsequent decolorization step. That’s why Gram-positive cells stay purple. Gram-negative cells, with a thinner peptidoglycan layer and an outer membrane, do not retain the complex after decolorization and take up the counterstain, appearing pink. So the mordant is correctly applied after the primary stain. If it were applied before the primary stain or skipped, the differentiation would be compromised.