GRAM STAINING

Who could have guessed that a staining procedure devised more than a century ago would still serve as one of the most widespread methods of bacterial classification? Microbiologists of this high-tech age are still indebted to Danish physician Christian Gram, who invented the gram-staining method in 1884.

To gram stain, an investigator smears a sample of bacteria on a slide, soaks it in a violet dye and then treats it with iodine. The slide is then rinsed with alcohol and counterstained with a pink dye called safranine.

The cell walls of gram-negative bacteria have a very low affinity for the violet stain, which is rinsed out by the alcohol. Once counterstained with safranine, however, the gram-negative bacteria appear bright pink to red. Gram-positive cell walls have a high affinity for the violet stain, and retain it even through the alcohol rinse. When the process is complete, they appear dark purple to brown.

The difference between the two cell types appears to be in the amount of peptidoglycan in the cell wall. Gram positive cell walls are about five times as rich in peptidoglycan as gram-negative cell walls. In addition, gram-negatives have a second membrane (chemically different from the plasma membrane) external to the cell wall, and may also have a gelatinous sheath external to the second membrane. Cozy.

The differences in the cell wall are more than simply a classification tool. Cell wall characteristics are intimately related to the disease-causing potential of the bacterium. In fact, medical researchers have found that an extremely effective way to combat bacterial pathogens is by interfering with cell wall formation. Because the eukaryotic cell has no analog to the prokaryotic cell wall, medicines which target bacterial cell walls have little or no effect on plant or animal cells.