The Gram stain involves several key steps: 1. Fixation: The sample is fixed on a slide using heat or methanol. 2. Primary Stain: The slide is stained with crystal violet, which penetrates all bacterial cells. 3. Mordant: Iodine is added to form a complex with the crystal violet, making it less soluble. 4. Decolorization: The slide is washed with alcohol or acetone. Gram-positive bacteria retain the crystal violet-iodine complex, while Gram-negative bacteria do not. 5. Counterstain: Safranin or fuchsine is applied, staining the Gram-negative bacteria pink/red while having no effect on the already purple Gram-positive bacteria.
