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

Abstract

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 alcm hol and relatively insoluble in aqueous safranin solution.I t 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 rrystal 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.A previous paper in this series (Mittwer, Bartholomew and Kallman, 1950) indicated that differences in cell membrane permeability to alcoholic iodine were part of the Gram stain mechanism. Since iodine forms a precipitate with crystal violet, and since the dissolution of this precipitate from the bacterial cell depends on (1) permeability, and (2) general solubility of the precipitate in alcohol and the counterstain, it is clear that a study of dye precipitate solubilities would aid greatly in interpreting the Gram stain mechanism. It is

Quantitative Analysis of the Gram Reaction

“…Clearly, the function of iodine is to lower the solubility of the absorbed dye to a critical level at which it is very susceptible to the concentration of the solvent. This supposition finds support in the demonstration that suitability of basic dyes for the Gram reaction depends partly on the solubility of their iodine complexes in ethanol (Bartholomew & Mittwer, 1951). Specific effects of solvents however are important.…”

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

“…Although no substitute for crystal violet has been generally accepted in the Gram-staining procedure, other dyes can be substituted (Bartholomew and Mittwer, , 1951. When stains such as malachite green, methylene blue, rhodamine, and safranine were used alone, the chromatographic technique gave good differentiation Each figure is the average of three readings taken from a single filter-paper strip; 95%0 ethyl alcohol was used as the solvent.The data for each pairof organismswereobtailled from experimentsset, up simultaneously.…”

Über den Farbstoff der Gramfärbung von Bakterien / On the Pigment of the Gram Staining of Bacteria

“…590 (12,0), 360 (-), 303 (3,8), 250 (2,2) nm (cm^M" 1 ) (Lit. [1]: 590 (12,1), 360 (1,4), 304 (3,9), 250 (2,2)).…”

“…Die Verbindung 1 -andere Farbstoffe der Triphenylmethylreihe, die mit KI« stabile unlösliche Triiodide bzw. Polyiodide eingehen, wären auch anwendbar[9] -wird sich auf der Oberfläche der gramnegativen Bakterien bilden[10,11] und dann leicht von dieser, samt Lipidschichten mit 96-proz. Ethanol entfernt.…”