What role does the mordant play in the Gram stain process?

In the Gram stain process, the mordant plays a crucial role by forming the crystal violet-iodine complex. After the initial application of crystal violet stain, iodine is added. The iodine serves as a mordant, which binds to the crystal violet dye and forms a larger complex that is more difficult to wash out of the bacterial cells. This complex is especially significant in differentiating between Gram-positive and Gram-negative bacteria.

In Gram-positive bacteria, the thick peptidoglycan layer retains the crystal violet-iodine complex even after the decolorization step, resulting in a purple color. In contrast, Gram-negative bacteria have a thinner peptidoglycan layer and will lose the complex during the decolorization step, leading to a pink appearance after counterstaining. This difference in retention of the dye complex is vital for the classification of bacteria in the Gram stain procedure, making the role of the mordant essential for accurate results.