Ribonucleates and the Gram stain.

Salton (J. Gen. Microbiol. 30:223, 1963) summarized the evidence which demonstrates that the gram-positive cell is able to retain the crystal violet after iodine treatment because of the characteristic nature of its cell wall. This evidence is highly convincing, yet it does not explain the experiments of Henry and Stacy (Proc. Roy. Soc. London Ser. B 133:391, 1946) or of Bartholomew and Umbreit (J. Bacteriol. 48:567, 1944), which specifically implicated magnesium ribonucleate as a chemical constituent causing the gram-positive response. We have therefore reinvestigated this problem in the light of modern knowledge. The data of Table 1 show that proteinase-free ribonuclease (colunm B) does not, by itself, render grampositive organisms gram-negative, nor does phosphodiesterase, which also hydrolyzes ribonucleic acid (RNA). However, the proteolytic enzyme, ficin (column D), does convert such cells to the gram-negative state. Other proteolytic enzymes (trypsin, pepsin, Pronase, etc.) show little activity. Lysozyme (column E) is only active on some organisms. Treatments F, G, and H were chosen because it has been shown (Salton, The Bacterial Cell Wall, Elsevier Publishing Co., Amsterdam, 1964, p. 248) that these treatments solubilize known components of the gram-positive cell wall. Therefore, one can no longer regard treatment with ribonuclease as evidence of the involvement of RNA in the Gram stain, and the earlier results were probably due to contamination of the ribonuclease by other enzymes, as suggested by Dubos (The Bacterial Cell, Harvard University Press, Cambridge, Mass., 1945, p. 81). The loss of grampositivity after the bile-salt treatment of Henry and Stacey appears to be due to the presence of enzymes in the bile salts. In all the cases in Table 1, when conversion to the gram-negative state of a gram-positive organism was achieved, it was possible to restore the gram-positive character by treatment with magnesium ribonucleate. However, this "replating" was possible only when the magnesium ribonucleate was formed in situ by precipitation on the slide. Replating did not occur when the magnesium ribonucleate, formed elsewhere, was applied to the slide. For such replating, a 1 to 5% solution of sodium ribonucleate was placed on the heat-fixed smear; 10% magnesium chloride was added dropwise, forming a precipitate; and the slide was washed and stained with the Gram stain. It appears that the magnesium ribonucleate is merely plugging up holes in the cell wall, rather than possessing any specific effect. Some cell wall is necessary, since protoplasts