The iodine solution works as a mordant that makes the stain more more prominent and the crystal violet dye become more tightly bound to the bacterial cell. Adding iodine solution or potassium chloride is the second step in the gram-staining process.
Gram staining is the common and most widely used technique used to differentiate between gram negative and gram positive bacteria. This process is based on the principle that bacteria reacts differently to their environment, thus also reacting (or staining) differently, helping with its identification.
The process of grain staining involves the following steps:
The bacteria is treated with the crystal violet dye, then the iodine solution.
After the staining, decolorizing agents are added to the specimen. This dehydrates, shrinks and tightens the peptidoglycan layer of the bacteria. The staining agents are trapped within the cells of gram positive bacteria because of its thicker peptidoglycan layer, while gram negative bacteria will be unable to hold on the the stain due to its thin layer.
A counterstain is added to the specimen staining it red. The red coloration does not change the color of the purple coloration of gram positive cells. Conversely, it adds a red color to gram negative bacteria.