University of Central Florida (UCF) MCB3020C General Microbiology Lab Midterm Practice Exam

During the Gram staining procedure, Gram-positive bacteria retain the crystal violet dye used in the staining process and appear purple under the microscope. This occurs because the thick peptidoglycan layer in the cell wall of Gram-positive bacteria traps the dye during the decolorization step with alcohol or acetone. When the crystal violet is retained, it masks any subsequent counterstaining with safranin (which would appear pink).

Thus, the appearance of Gram-positive bacteria is defined by the retention of this dye, leading to their distinctive purple color rather than pink or any other color. In contrast, Gram-negative bacteria, which have a thinner peptidoglycan layer, do not retain the crystal violet and will take up the pink coloration from the safranin counterstain after decolorization.