What is the role of the mordant (iodine) in Gram staining?

• A. It fixes the smear
• B. It forms a crystal violet-iodine complex to trap the dye in Gram-positive cells.
• C. It decolorizes
• D. It acts as a counterstain

Answer: (B)

The mordant’s job is to form the crystal violet–iodine complex, which locks the dye in place. Iodine binds to crystal violet, creating a larger CV–I complex that is held in the thick peptidoglycan layer of Gram-positive cell walls. This makes the stain resistant to the decolorizing step, so Gram-positive cells stay purple. In Gram-negative cells, the thinner peptidoglycan and outer membrane allow the CV–I complex to be washed out during decolorization, so those cells take up the counterstain and appear pink.