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Gram negative bacteria are distinguished by their unique staining characteristics in the Gram stain process. When subjected to this type of staining, Gram negative bacteria do not retain the crystal violet dye used in the procedure and instead take up the counterstain (usually safranin), resulting in a pink appearance under a microscope. This staining difference is primarily due to the structure of their cell wall, which consists of a thin layer of peptidoglycan surrounded by an outer membrane containing lipopolysaccharides.

The ability to photosynthesize is unrelated to whether a bacterium is Gram negative or Gram positive, as photosynthesis is a metabolic process that some bacteria, regardless of Gram classification, can perform. The presence of a nucleus is another characteristic that does not apply to bacteria at all, as bacteria are prokaryotic organisms and lack a true nucleus. Lastly, while Gram negative bacteria tend to have increased resistance to certain antibiotics due to their outer membrane acting as a barrier, this characteristic is not a defining feature that distinguishes them from other types; rather, it is a consequence of their cell wall structure. Thus, the staining characteristics in the Gram stain are the key factor that distinguishes Gram negative bacteria.