What characteristic of Gram-negative bacteria distinguishes them in staining procedures?

The distinguishing characteristic of Gram-negative bacteria in staining procedures is their presence of an outer membrane. This outer membrane is composed of a phospholipid bilayer that contains lipopolysaccharides, which contribute to the bacterial cell's overall structure and defense. During the Gram stain process, this outer membrane affects the retention of stains; when Gram-negative bacteria are subjected to decolorization, the crystal violet stain is washed out because the thin peptidoglycan layer underneath is not sufficient to retain it. Consequently, when a counterstain (usually safranin) is applied, the bacteria appear pink or red under a microscope, confirming their Gram-negative status.

In contrast, a thick peptidoglycan layer is characteristic of Gram-positive bacteria, which helps retain the crystal violet stain and appear purple after the staining procedure. The retention of the crystal violet stain itself is typical of Gram-positive bacteria, not Gram-negative. While resistance to antibiotics can be a trait of some Gram-negative bacteria due to their unique structure, it is not a defining feature of their identification during Gram staining. Thus, the outer membrane is the key feature that distinguishes Gram-negative bacteria in this context.