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SUMMARY: The effects of various reagents in the mordanting and staining of bacterial cell wab are described, The cell walls of Gram-positive bacteria were found to be much more readily stainable than those of Gram-negative organisms. In this and other respects, apart from the Gram reaction Azotobacter chroococcum resembled a Gram-positive species; some of the methods described provided an excellent illustration of its Bacillus-like morphology. The cell walls of bacteria may be stained by the use of mordants such as tannic acid (Eisenberg, 1910) or phosphomolybdic acid (Hale, 1953) followed by basic dyes, by complexes of dyes such as the fuchsin-congo-red method of Chance (1958), and certain of the methods of Yoshida et al. (1954), and by such rather exceptional dyes as Alcian blue (Tomcsik & Grace, 1955) which stain the cell wall directly without the intervention of a mordant. The dye-complex methods are open to the criticism of Girbardt & Taubeneck (1955) that they do not so much stain the wall as flocculate against it. Accordingly, in the following investigation these were avoided, and the effects of a range of compounds employed as mordants upon the cell walls of a variety of bacteria were examined and compared. In addition to Alcian blue, it was also found that Janus green would stain unmordanted cell walls, and the comparison was extended to these dyes. Since it has frequently been observed that not all bacteria react in the same manner to such staining procedures, it was considered necessary to make these tests upon a representative selection of Gram-positive and Gram-negative bacteria. And at the same time these were compared with Azotobacter, which, as has already been suggested (Bisset, 1955), although normally Gram-negative, possesses numerous characters suggestive of a relationship with the Bacillaceae. METHODS The bacteria examined were one strain each of Bacillus cereus and B. subtilis, Staphylococcus albus, Mycobaderium lacticola, a large coccus of Sarcina type, Aerobacter amogenes, Escherichia coli, Proteus vulgaris, and three of Axotobacter chroococcum. All were freshly isolated in this laboratory, with the exception of Mycobacterium lacticola which was a stock culture. The Azotobacter strains were isolated on the nitrogen-free mannitol-phos- phate medium described by Bisset & Hale (1953), on which they grew as a tough pellicle, composed of Gram-negative, almost coccal, capsulated organisms, G. Microb. xv

A Comparison of the Staining Reactions of the Cell Walls of Azotobacter chroococcum and those of Gram-positive and Gram-negative Bacteria