Gram staining procedure

Christian Gram, a Danish physician, developed this differential staining procedure in 1884 to sort bacteria into two classes based purely on their cell-wall chemistry. The procedure runs: prepare a bacterial smear -> stain with crystal violet (30 s) -> rinse -> apply Gram's iodine (1 min, this fixes the dye as a complex inside the cell) -> rinse -> decolourise with 95% ethanol or acetone (10-30 s) -> rinse -> counterstain with safranin (30-60 s) -> rinse and blot -> observe. Gram positive bacteria retain the crystal violet-iodine complex and stay dark violet, because their cell wall is a single, thick (0.015-0.02 um), rigid layer that is roughly 80% peptidoglycan, contains teichoic acid, and has no outer membrane or periplasmic space. Gram negative bacteria lose the crystal violet during decolourisation (their thin peptidoglycan layer, only 3-12% of the wall, can't trap it) and instead pick up the pink/red safranin counterstain; their wall is a triple-layered structure (0.0075-0.012 um) with an outer membrane carrying lipopolysaccharides, lipoproteins and porins, plus a periplasmic space, and it lacks teichoic acid. These wall differences carry real functional consequences too: Gram positive bacteria are highly susceptible to penicillin (which targets peptidoglycan synthesis) while Gram negative bacteria are much less susceptibl …