During the Gram stain, what happens to Gram-negative bacteria during the decolorization step with alcohol?

• A. They retain the crystal violet color and appear purple.
• B. They become colorless and must be counterstained with safranin to be seen.
• C. They lose the crystal violet stain and appear pink after counterstaining.
• D. They take up more dye and appear black.

Answer: (C)

During the alcohol decolorization step, Gram-negative bacteria lose the crystal violet–iodine complex because their outer membrane is disrupted and their thin peptidoglycan layer cannot retain the dye. They become colorless at this point, and when the counterstain is applied, they take up the pink safranin and appear pink under the microscope. This sequence—loss of the violet stain during decolorization followed by pink coloration after counterstaining—best reflects what happens to Gram-negative cells. By contrast, Gram-positive bacteria keep the purple stain due to their thick peptidoglycan layer, which is why they remain purple.