The Gram stain differentiates bacteria based on the composition of their cell walls, specifically the thickness of the peptidoglycan layer. Gram-positive bacteria retain the crystal violet dye, appearing purple due to their thick peptidoglycan layer. In contrast, Gram-negative bacteria do not retain the crystal violet and take up the counterstain (safranin), appearing pink/red because of their thinner peptidoglycan layer and outer membrane. This key differentiation guides antibiotic choice and infection management.

Reference: Jawetz, Melnick & Adelberg's Medical Microbiology, Carroll, Edition 28.