The Gram stain procedure involves four main steps: 1. Applying a primary stain (crystal violet) to a heat-fixed smear of bacterial culture. 2. Adding a mordant (iodine solution), which forms a complex with the crystal violet. 3. Decolorizing with alcohol or acetone, which dehydrates the thick peptidoglycan layer in gram-positive bacteria, trapping the crystal violet-iodine complex. 4. Counterstaining with safranin, which stains the decolorized gram-negative bacteria pink/red while gram-positive bacteria remain violet.
