Molecular Mechanism of Gram Staining

Abstract

The passage of small molecular substances through the walls of Staphylococcus aureus is reduced in presence of 95% ethanol, as well as solutions of aniline, dimethyl aniline and nitrobenzene in 95% ethanol. But these three solvents, unlike 95% ethanol alone, can extract dye from Gram-stained Staphylococcus aureus almost completely, indicating the inadequacy of the permeability theory of Gram staining. It has been observed that, if iodine from Gram-stained Staphylococcus aureus is removed by thiosulphate, the retained dye can be extracted by 95% ethanol. However, if after thiosulphate treatment Staphylococcus aureus with the retained dye is treated with graded amounts of iodine, extractibility of the dye by 95% ethanol is progressively reduced. This supports the model proposed for the mechanism of Gram staining which involves retention of crystal violet by the cell component presumably by electrostatic bonds and formation of chargetransfer complex of crystal violet and iodine through π-electrons. Stability of this cell component-dye-iodine complex towards 95% ethanol determines the Gram character of the cell. © 1969, Applied Microbiology, Molecular and Cellular Biosciences Research Foundation. All rights reserved.