Which statement best describes the difference between Gram-positive and Gram-negative bacteria on Gram staining?

• A. Gram-positive have outer membrane; Gram-negative do not.
• B. Gram-positive retain crystal violet and stain purple due to thick peptidoglycan; Gram-negative stain pink due to a thin peptidoglycan layer and outer membrane with lipopolysaccharide.
• C. Gram-positive stain pink; Gram-negative stain purple.
• D. Both stain purple.

Answer: (B)

Gram staining differentiates bacteria by their cell wall structure. The crystal violet-iodine complex binds strongly to the thick peptidoglycan layer found in Gram-positive cells, so these cells retain the purple stain after washing. Gram-negative bacteria have a thinner peptidoglycan layer and an outer membrane with lipopolysaccharide; during decolorization with alcohol, this structure is disrupted and the dye is washed out, leaving the cells colorless so they take up the counterstain and appear pink. That’s why the best description is that Gram-positive cells appear purple due to their thick peptidoglycan retaining the dye, while Gram-negative cells appear pink because of their thin peptidoglycan and outer membrane allowing decolorization and counterstaining. The other statements misstate the outer membrane presence or the observed colors.