The Effect of Moisture on Gram Differentiation, and its Relation to Proposed Gram-Positive Substrates in Yeast and Other Organisms

Abstract

SUMMARYWet and dry Gram staining procedures have different characteristics in respect to their rates of decolorization. It has been proposed that this difference is due to a Gram-positive substrate which dissociates (becomes Gram-negative) in the presence of water. However, the differences between wet and dry Gram procedures can also be explained on the hypothesis that water influences the rate of solvent permeation through cell envelopes. Since no direct proof exists for the reality of the proposed Gram-positive substrate, and since evidence is presented here which cannot be explained by such a hypothesis, it is felt that the solvent permeation concept should be considered seriously as an explanation for the decolorization differences observed between wet and dry Gram procedures.

Analysis of the Mechanism of Gram Differentiation by Use of a Filter-Paper Chromatographic Technique

“…These conclusions follow from the facts that either iodine alone or dye alone could result in Gram differentiation, and that other dyes and several solvents could be used. Gram-positivity can not be due entirely to permeability characteristics requiring intact cell envelopes, since the data presented here and elsewhere (Bartholomew et al, 1964) demonstrate that ruptured cells can be properly differentiated. Gram-positivity can not be due to the amount of dye taken up, the manner of its adsorption onto the cell, or to a specific affinity of dye for cellwall material, since all of these possibilities have been shown to be inconsistent with known experimental data (Bartholomew andFinkelstein, 1954, 1958;Bartholomew, 1956, 1960).…”

“…It could control decolorization rate by affecting the permeation rate of the crystal violet molecule alone, the iodine molecule alone, the solvent molecule alone, or a combination of these factors. The question of permeation to the dye-iodine precipitate is not material, since it has been repeatedly demonstrated that this precipitate dissociates into separate dye and iodine molecules upon solution in alcohol (Stearn and Stearn, 1930;Bartholomew et al, 1964;Lamanna and Mallette, 1964). Unfortunately, with the data presented here, it is not possible to differentiate among the roles of the permeation rates for iodine, dye, or solvent molecules.…”

“…It was surprising, to find that the filter-paper chromatographic technique could so markedly differentiate cells stained with iodine alone or crystal violet alone, and cell-wall fragments from gram-positive and gram-negative organisms, when such differentiations were so difficult to obtain by use of glass-slide preparations (Bartholomew and Mlittwer, 1950; Bartholomew et al, 1964). The reasons for this become al)parent when one considers the differences in the state of the cellular miaterial in filter-paper chromatography as coml)pared to glass-slide experiments.…”