A Cell Wall Stain Employing a Cationic Surface-active Agent as a Mordant.

For studying the morphology of bacteria in fixed smears, Knaysi (J. Bact., 49, 375) demonstrated the value of staining by a method showing the cell wall. Knaysi (J. Bact., 41, 141) developed a tannin alum fuchsin method which stains differentially the cytoplasm, cell wall, and capsule. It is the purpose of this note to report another cell wall stain in which the cell wall is positively charged by treatment with a cationic surface-active agent (Dyar and Ordal: J. Bact., 51, 149) and is then stained with an acid dye. The cytoplasm may be stained with a contrasting basic dye. By use of the following procedure, the cell wall is stained red and the cytoplasm, blue: (1) prepare and heat-fix a smear; (2) add 3 drops M/100 (0.34 per cent) cetyl pyridinium chloride; (3) add 1 drop saturated, aqueous Congo red, mixing on the slide; (4) wash; (5) counterstain for a few seconds with methylene blue; (6) wash; and (7) examine either in a drop of water under a cover slip or in oil. Side illumination is often helpful in making observations. Species of Bacilus, Micrococcus, and Escherichia and also of Saccharomyces and Schizosaccharomyces have been stained in this way. The relatively thick cell wall of yeasts is seen to be stained light red and to be accentuated by a red precipitate of dye-surface-active agent on its surface. Presumably the same thing occurs with bacteria and accounts for the clarity of the stained cell wall, which has been shown by electron photomicrographs to be actually very thin. This is further indicated by the fact that occasional bacterial cells, dislodged in the stainig procedure, leave on the slide an outline of stain corresponding in shape to the stained cell wall. When bacteria, stained by this method, are examined in water under a cover slip, the cytoplasm is sometimes seen to be shrunk away from the cell wall. When such cells of Bacillus cereus are examined dry in oil, it is frequently seen that the cell wall has contracted, now adhering closely to the shrunken cytoplasm at the sides, though not at the ends. It appears that stained cell walls maintain the rigid form of the living cells when wet but tend to shrink or collapse when dry. Although the dye-surface-active agent combination does not stain the cytoplasm, this is not considered evidence that the cationic surface-active agent has not penetrated the cell membranes, a possibility discussed by Hotchkiss (Ann. N. Y. Acad. Sci., 46, 479). Actually, the cytoplasm of vegetative cells suspended in a droplet of cetyl pyridinium chloride stains black with Sudan black B, indicating that the surface-active agent has penetrated into the cell and that its hydrocarbon portion is stained by the fat dye. 498 [VOL. 53 NOTES