The Gram stain after more than a century.

The Gram stain, the most important stain in microbiology, was described more than a century ago. Only within the past decade, however, has an understanding of its mechanism emerged. It now seems clear that the cell wall of Gram-positive microorganisms is responsible for retention of a crystal violet:iodine complex. In Gram-negative cells, the staining procedures damage the cell surface resulting in loss of dye complexes. Gram-positive microorganisms require a relatively thick cell wall, irrespective of composition, to retain the dye. Therefore, Gram-stainability is a function of the cell wall and is not related to chemistry of cell constituents. This review provides a chronology of the Gram stain and discusses its recently discovered mechanism.