The Mechanism of the Gram Reaction. III. Solubilities of Dye-Iodine Precipitates and Further Studies of Primary Dye Substitutes

Solubilities of dye-iodine precipitates in alcohol and in aqueous safranin solution were determined by direct solubility methods and by photocolorimetric methods. It was found that, increasing precipitate solubility in alcohol or safranin solution gave decreasing differentiation between Gram-positive and Gram-negative bacteria. Dyes which did not stain the cells well as a primary stain did not give good Gram stains, regardless of the solubilities of their precipitates. Some dyes (typified by methylene blue) which gave relatively alcohol-insoluble iodine precipitates gave inferior Gram differentiation because these precipitates were readily soluble in the safranin counterstain.

Solubilities of precipitates of crystal violet and various iodine substitutes were determined photocolorimetrically. The ability of a substance to replace iodine in the Gram stain correlated with its ability to give a precipitate which was only slightly soluble in alcohol and relatively insoluble in aqueous safranin solution.

It was concluded that the usual Gram reagents are not truly specific for the differentiation. Any dye and mordant could be used if the dye was deeply colored, stained the cells well, and if the precipitate of dye and mordant was only slightly soluble in alcohol and relatively insoluble in the counterstain. These factors, combined with those influencing differences in cell membrane permeability, constitute the most important factors in the Gram stain differentiation.

Studies were made concerning the ability of dyes to substitute for crystal violet in the Gram procedure. Of 29 dye samples reported on here for the first time none proved to be good substitutes for crystal violet.