Why is iodine used in Gram staining?

Why is iodine used in Gram staining?

• A. It acts as a nutrient source
• B. It locks the crystal violet stain to the cell wall
• C. It speeds up decolorization
• D. It stains the background

Answer: (B)

Iodine acts as a mordant in Gram staining, forming a large crystal violet–iodine complex that becomes trapped in the cell wall. This complex increases the dye’s size and reduces its solubility, making it harder to wash out during the decolorization step. In Gram-positive bacteria, the thick peptidoglycan layer holds onto this CV–iodine complex, so the purple stain is retained. In Gram-negative bacteria, the thinner wall and outer membrane don’t retain the complex after decolorization, so the cells lose the dye and take up the counterstain. So the key role of iodine is to lock the crystal violet in place, ensuring the stain remains in Gram-positive cells while being washed out from Gram-negative cells.

Iodine acts as a mordant in Gram staining, forming a large crystal violet–iodine complex that becomes trapped in the cell wall. This complex increases the dye’s size and reduces its solubility, making it harder to wash out during the decolorization step. In Gram-positive bacteria, the thick peptidoglycan layer holds onto this CV–iodine complex, so the purple stain is retained. In Gram-negative bacteria, the thinner wall and outer membrane don’t retain the complex after decolorization, so the cells lose the dye and take up the counterstain. So the key role of iodine is to lock the crystal violet in place, ensuring the stain remains in Gram-positive cells while being washed out from Gram-negative cells.