What characteristic distinguishes Gram-positive bacteria from Gram-negative bacteria during Gram staining?

The characteristic that distinguishes Gram-positive bacteria from Gram-negative bacteria during Gram staining is the thickness of their peptidoglycan layer. In Gram-positive bacteria, the peptidoglycan layer is significantly thicker compared to that in Gram-negative bacteria. This thicker layer retains the crystal violet stain used in the Gram staining process, resulting in a blue or purple appearance under a microscope.

In contrast, Gram-negative bacteria have a thinner peptidoglycan layer, which does not retain the crystal violet stain after the application of a decolorizer. They are subsequently stained with safranin, which causes them to appear pink or red.

Additional characteristics listed in the other choices, such as the ability to ferment sugars, the presence of pili, and the size of the bacteria, do not play a direct role in classification based on Gram staining. While some bacteria can ferment sugars, this feature does not differentiate between Gram-positive and Gram-negative types. Pili, which are hair-like structures used for attachment and conjugation, can be present in both types and do not serve as a distinguishing factor for Gram staining. Similarly, size variation exists among bacteria but is not a defining factor in the Gram staining process.