After the primary stain, Iodine is added to the specimen to act as a mordant, which clumps the crystal violet and traps it in thick layers of peptidoglycan in Gram-positive cell walls.

1) Crystal Violet  
2) Iodine
3) Alcohol
4) Safranin 

Gram Stain Procedure

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Crystal violet is the primary stain in gram staining, and it stains all cells in the specimen purple/blue.

Step 1: Crystal Violet 

Step 2: Iodine

Alcohol is then used as a decolorizing agent, which allows us to differentiate Gram-positive and Gram-negative by making Gram-negative cells clear. Alcohol washes away the Crystal Violet in Gram-negative cells by dissolving their outer membrane. In, Gram-positive cells, the alcohol dehydrates the thick peptidoglycan layer, trapping the Crystal Violet.

Step 3: Alcohol

Safranin is then used as a counterstain to color Gram-negative cells pink/red in order to distinguish them from the Gram-positive cells that remain purple.

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