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

Step 1: Crystal Violet 

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.

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.

Step 4: Safranin

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

Gram stain is a multi-step staining procedure that differentiates bacteria by the structure of their cell wall

Gram Stain

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Microbiology covers the scope and sequence requirements for a single-semester microbiology course for non-majors. The book presents the core concepts of microbiology with a focus on applications for careers in allied health. The pedagogical features of the text make the material interesting and accessible while maintaining the career-application focus and scientific rigor inherent in the subject matter. Microbiology’s art program enhances students’ understanding of concepts through clear and effective illustrations, diagrams, and photographs.

Microbiology is produced through a collaborative publishing agreement between OpenStax and the American Society for Microbiology Press. The book aligns with the curriculum guidelines of the American Society for Microbiology.

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