If Gram-positive bacteria are over-differentiated after crystal violet staining, what color would they appear?

• A. Red
• B. Blue
• C. Purple
• D. Colorless

Answer: (A)

Gram staining relies on differences in cell wall structure to distinguish bacteria by whether they retain the crystal violet-iodine complex after the decolorization step. Gram-positive bacteria have a thick peptidoglycan layer that traps the dye and stay purple, while Gram-negative bacteria have a thinner peptidoglycan layer and an outer membrane that allows the dye to be washed out, so the counterstain makes them red.

If the decolorization step is carried too far—over-differentiation—Gram-positive cells can also lose the crystal violet and take up the counterstain. In that case, they would appear red/pink rather than purple.