In order to develop further a comprehensive characterization of spirilla and to devise a more satisfactory classification of the species, the type strains of 20 marine and fresh-water species of the genus Spirillum, plus additional strains of S. gracile, S. itersonii, S. serpens, and S. volutans, and 6 un-named strains of spirilla, were subjected to over 90 physiological tests and also various morphological tests using standardized inocula, media, incubation times, and testing methods. Determination of DNA base compositions was also performed.

All strains of spirilla grew aerobically except S, volutans which was an obligate microaerophile; S. giesbergeri and S. gracile appeared to be possible facultative rnicroacrophiles, S. lunatum and S, itersonii were the only species to. grow anaerobically in the presence of nitrate, The optimum temperature was 30 C for all strains examined. The cell diameters for all strains formed an almost continuous spectrum, ranging from 0.25 to 1.7 microns. All spirilla were actively motile and a number of the strains possessed flagellar tufts visible with living cells by dark field microscopy and in the case of S. volutans also by phase contrast. Coccoid bodies were present in 4 week cultures in the case of most marine strains; only 3 fresh-water species exhibited these forms, Several physiological characteristics were found to be uniformly or nearly uniformly positive (oxidase, deaminase activity with peptone, catalase, phosphatase, HzS from cysteine, growth in 1% bile) or negative (indol; amylase; sulfatase; hydrolysis of gelatin, casein, aesculin, and hippurate; urease; alkaline reaction in litmus milk; acid reaction from carbohydrates) for all or nearly all of the strains, In the few cases where acid was produced from carbohydrates, a reduced peptone concentration was necessary. Several physiological characteristics yielded varied reactions among the spirilla (reduction of selenite and nitrate; growth on EMB, MacConkey, TSI, and Seller's agars and in MRVP broth; RNAase and DNAase activity; and tolerance to 1% glycine}. Water-soluble pigments were produced by a few strains growing on aromatic amino acids. Three percent NaCl inhibited all fresh-water strains, while the marine strains could all grow in the presence of 9. 75% (w/v) NaCl. The DNA base compositions of the spirilla, determined from their Tm values, gave a broad range of values from 38 to 65 moles % G + C.

The limits of genus and species variation, the usefulness of standardized conditions in characterizing strains of spirilla, discrepancies arising from a comparison of the present data with earlier results, the division of the genus Spirillum on the basis of single, highly-weighted characters, and the division of genus Spirillum on the basis of correlated characters, were discussed. A modified description of the genus Spirillum (Ehrenberg, 1830) based on the present study was presented. Various physiological tests important for species differentiation were indicated. A modified classification scheme for the species of genus Spirillum was devised, and a temporary diagnostic key was also presented.