Cultivation of Spirillum volutans in a Bacteria-Free Environment.

Despite the early description of Spirillum volutans Ehrenberg in 1832, attempts by various investigators to grow this organism in pure culture have been unsuccessful (Williams and Rittenberg, Intern. Bull. Bacteriol. Nomencl. Taxonom. 7:49, 1957); however, by a most ingenious dialysis sac technique, Rittenberg and Rittenberg (Arch. Mikrobiol. 42:138, 1962) were able to isolate the Pringsheim strain in pure culture. Growth did not occur independently, however, and the sac had to be suspended in a culture of other bacteria which were presumed to supply a nutritional factor required by S. volutans, although no chemical supplements added were able to replace this requirement. Our strain of S. volutans was obtained from a pond water-hay infusion. After 36 serial transfers by the Rittenberg method, a pure sac culture was finally obtained. The spirillum so isolated appeared to fit the morphological description of the Pringsheim strain in all respects (Fig. 1). The cell diameter ranged from 1.5 to 1.8 ,u, the wavelength from 16 to 24 Au, and the cell length from 18 to 40 jA. Volutin granules were abundant (Fig. 2). A most striking characteristic of the sac culture was the regular formation of a ring-shaped band of spirilla when covered wet mounts were prepared and allowed to stand for 10 min (Fig. 3); this suggested a possible microaerophilic nature. The Rittenbergs noted a similar ring formation in an uncovered culture drop, but apparently the ring did not contain the enormous numbers of organisms congregated in the sharply limited zone which we have observed in covered mounts. Successful independent cultivation of the organism from our sac culture was finally accom-