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Previously we showed that Salmonella minnesota Re and Ra lipopolysaccharides (LPSs) and Escherichia coli K-12 LPS formed three-dimensional crystals, either hexagonal plates or solid columns, when they were precipitated by the addition of 2 volumes of 95% ethanol containing 375 mM MgCl2 and incubated in 70% ethanol containing 250 mM MgCl2, and stored at 4 C for 10 days. Later, Escherichia coli K-12 LPS thus treated was found to form discoid crystals as well as hexagonal plate crystals and solid column crystals. Analysis by electron diffraction of the discoid crystals from the direction perpendicular to the basal plane showed that they consisted cf hexagonal lattices with the a axis of 4.62 A. This result was quite the same as that of the hexagonal plate crystals. Electron micrographs of the edges of the discoid crystals revealed stacked sheets of the hexagonal plate crystals. From these results it was concluded that formation of the discoid crystals results from irregular overlapping of the hexagonal plate crystals.

S-form lipopolysaccharides (LPS) from Klebsiella strain LEN-1 (O3: K1 —) and from Salmonella minnesota strain 1114 were positively stained with ruthenium red, whereas R-form LPS from Klebsiella strain LEN-111 (O3— : K1 —) and Ra, Rb1, RcP+, Rd1P-, and Re LPS from the respective mutant strains of S. minnesota were not or only faintly stained by such treatment. From these results it was concluded that ruthenium red stains the O-specific polysaccharide chains of LPS. The appearance of stained preparations of S-form LPS suggested that the material responsible for this positive staining corresponded to the surface projections which were seen by the negative staining technique as attached to the ribbon-like structures and spherules of the LPS.