Sample Preparation: A sample is smeared on a glass slide and heat-fixed. Staining: The slide is stained with crystal violet dye, which penetrates all bacteria. Mordant Application: Iodine solution is added, forming a complex with the crystal violet. Decolorization: The slide is washed with alcohol or acetone. Gram-positive bacteria retain the crystal violet-iodine complex, while Gram-negative bacteria lose the stain. Counterstaining: A counterstain, usually safranin, is applied. Gram-negative bacteria take up the counterstain and appear red or pink, while Gram-positive bacteria remain purple.
