Methods for Growing Spirillum lipoferum and for Counting It in Pure Culture and in Association with Plants

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Abstract
Methods are described for growing Spirillum lipoferum in quantities sufficient to serve as inoculant in field trials of its associative N 2 -fixing ability with higher plants and as a source of cells for the preparation of nitrogenase, cytochromes, respiratory enzymes, etc. A heavy inoculum of S. lipoferum grown on NH 4 + was transferred to a medium of minimal nitrogen content, and initial rapid growth at the expense of residual combined nitrogen was replaced later by slower growth on N 2 . Conversion to N 2 fixation was prompt upon exhaustion of fixed nitrogen; growth on N 2 was most rapid at a pO 2 of 0.005 to 0.007 atm. Numbers of S. lipoferum can be estimated by diluting soil, crushed roots, or other material, and inoculating diluted samples into a stagnant semisolid medium. Development of a characteristic subsurface layer of organisms and demonstration the these organisms can reduce C 2 H 2 are presumptive evidence that they are S. lipoferum. With most-probable-number tables the observations can be converted to numbers of S. lipoferum in the samples. The most-probable-number method indicated that numbers of S. lipoferum may increase 100-fold or more in roots of maize removed from the plant and incubated for 24 h at 30°C at a pO 2 initially adjusted to 0.01 atm.

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