Question

What is the purpose of the iodine in the Gram stain procedure? If this step was left out, what color would Gram negative and Gram positive cells appear after completion of the stain?

Answer 1

Iodine in the Gram stain procedure acts as a mordant and forms a complex with the crystal violet stain. This complex binds tightly with the thick peptidoglycan layer present in the cell wall of Gram positive bacteria. Thus, when the smear is rinsed with the decolorizer such as ethyl alcohol or acetone, the purple color of the primary stain (crystal violet) is retained in case of the Gran positive bacteria.

Gram negative bacteria have a thin layer of peptidoglycan and a thick outer layer formed of lipids. When they are exposed to the decolorizer, it dissolves the lipids of the cell wall and the crystal violet iodine complex leaches out of the cells. Thus, the Gram negative bacteria appears colorless and are counter stained with a secondary dye such as safranin.

If iodine is not added then both the Gram positive and Gram negative bacteria will appear pink in color as crystal violet will not be tightly binded with the peptidoglycan layer and will leach out of both the type of cells upon the addition of decolorizer. Thus, when the counter stain is added both the cells will appear pink.