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Gram-positive bacteria appear blue on a microscopic slide due to the retention of crystal violet dye during the Gram staining procedure. When performing a Gram stain, the bacteria are first treated with crystal violet, which penetrates the thick peptidoglycan layer in the walls of Gram-positive bacteria. After a subsequent wash, iodine is applied, forming a complex with the crystal violet that is trapped within the thick cell wall.

This characteristic structure helps to differentiate Gram-positive bacteria from Gram-negative bacteria, which have thinner peptidoglycan layers and do not retain the crystal violet after being treated with a decolorizer, usually acetone or ethanol. Instead, Gram-negative bacteria take up a counterstain (safranin or fuchsine) and appear red or pink under the microscope.

The other color choices do not accurately represent the Gram staining outcome for Gram-positive bacteria, as they would not appear in those colors due to the staining properties of the methods and the structural differences in bacterial cell walls.