The Mechanism of the Gram Reaction. II. The Function of Iodine in the Gram Stain

Fifty-five reagents were studied as to their ability to replace iodine in the Gram stain. None gave results as good as iodine. Eight gave usable Gram preparations, and forty-seven gave negative results. Omission of the counterstain resulted in increasing to thirty-three the number of reagents giving differentiation, but this, was not considered a true Gram differentiation. Many oxidizing agents were shown not to be substitutes for iodine; therefore the function of iodine must be more than to serve as an oxidizing agent. Many reagents which formed precipitates with the dye could not replace iodine; therefore factors other than precipitate formation must be involved. However, all agents which were good substitutes for iodine were both good oxidizing and dye precipitating agents. Experiments involving the study of cell membrane permeability showed that Gram-positive cells were less permeable to iodine in alcoholic solution than Gram-negative cells. This difference could not be demonstrated for iodine in aqueous solution. It was concluded that iodine served to form a dye-iodine precipitate (or complex) in the cell. Since Gram-positive cells were less permeable to iodine in alcohol than Gram-negative cells, this resulted in a slower dissolving out of this complex from Gram-positive cells during de-colorization and hence a slower decolorization time. The relative solubilities of dye precipitates in alcohol and in aqueous safranin solution were also indicated as an important factor influencing decolorization. Dyes which formed highly soluble precipitates with iodine could not be used in the Gram stain. It is not proposed that the mechanism of the Gram stain is entirely one of membrane permeability; chemical factors are undoubtedly important and will be discussed in a later paper. However, it is proposed that the chemical and physical factors are closely interrelated in the Gram stain mechanism.