Bacteria that retain the primary crystal violet stain that is first applied as part of the staining process are classified as gram-positive bacteria. The stain is retained because the cell wall of a gram-positive bacteria is not as permeable as the cell wall of a gram-negative bacteria. Therefore, when the decolorizer is applied to the smear, the crystal violet will be retained if the bacteria is gram-positive and the bacteria will appear blue or purple. Prior to adding the decolorizer, iodine is applied to the smear. The iodine fixes the crystal violet stain to the cellular components of gram-positive bacteria, further preventing the decolorization of the gram-positive organisms. The bacteria indicated by the arrows in Image 1 are gram-positive bacteria.
If the bacteria is gram-negative, decolorizer will pull the crystal violet stain out of the cell walls, allowing the bacteria to be stained with the counterstain so that a gram-negative bacteria appears red or pink. The arrows in Image 2 point to gram-negative bacteria.