Smear preparation: A small sample is spread thinly on a glass slide. Fixation: The slide is passed through a flame to fix the sample. Crystal violet application: The primary stain is applied. Iodine treatment: A mordant is used to fix the dye in the cell wall. Decolorization: Alcohol or acetone is used to wash away the stain from Gram-negative bacteria. Counterstaining: Safranin or another dye is applied to stain Gram-negative bacteria.
