The distinction between Gram positive (Gram+) and negative bacteria (Gram-) has absolutely nothing to do with membrane potentials; it is all about the Gram staining procedure.
The Gram staining was named after the Danish bacteriologist Hans Christian Gram, who originally devised it in 1882 (Gram, 1884). Gram staining is a common technique used to differentiate two large groups of bacteria based on their different cell wall constituents. The Gram stain procedure distinguishes between Gram+ and Gram- groups by coloring these cells violet or red, respectively (Fig. 1).
Gram+ bacteria stain violet due to the presence of a thick layer of peptidoglycan in their cell wall, which retains the crystal violet these cells are stained with. Gram-, on the other hand stain red, which is attributed to a thinner peptidoglycan wall, which does not retain the crystal violet during the decoloring process.
Gram staining procedure
The procedure involves staining the cells with a crystal violet dye. Next, an iodine solution (iodine and potassium iodide) is added to form a complex between the crystal violet and iodine, which is insoluble in water. Then, a decolorizer, such as ethyl alcohol, is added to the sample, which dehydrates the peptidoglycan layer, shrinking and tightening it. The crystal violet-iodine complex is not able to penetrate this tightened peptidoglycan layer, and is thus trapped in the cell in Gram+ bacteria. In Gram- bacteria, however, the thinner peptidoglycan layer does not retain the crystal violet-iodine complex and the color is lost. Lastly, a counterstain, such as the weakly water soluble safranin, is added to the sample, staining it red. Since the safranin is lighter of color than crystal violet, it does not disrupt the purple coloration in Gram+ cells, but it does stain the decolorized Gram- cells red.
Fig. 1. Gram+ bacteria (left) and Gram- on the right. source: Lab Tests Online
- Gram, Fortschritte der Medizin (1884)