Studies of the Common Aerobic Spore-forming Bacilli: I. Staining for Fat with Sudan Black B-safranin.

In a classification of the aerobic spore-forming bacilli proposed by Smith and Clark (1937) one of the important differential points is the presence of reserve fat in the cells of organisms of the "B. cereus-B. megatherium group" when grown on glucose agar. In contrast, bacilli of the "B. subtilis-B. mesentericus group," and some other sub-groups, are said not to store fat. The authors do not describe their method for fat staining, but presumably they used Sudan III, or one of the related red dyes, in wet preparations. The obvious practical value of so simple a differential test led us to investigate the matter of fat storage more fully, and since our fat-staining preparations made with Sudans II, III, or IV were not satisfactory, we sought a better technique. We soon confirmed the findings of Hartman (1940), who first pointed out that Sudan black B, previously recommended for histological work (Lison, 1934; Leach, 1938), is much superior to the red Sudans for demonstrating fat in bacterial cells. Hartman used wet preparations only, suspending the bacteria in a solution of Sudan black B made in 70 per cent alcohol or in ethylene glycol. The latter was regarded as the more suitable solvent, since it does not evaporate rapidly. The fat droplets were recognized as blue-black bodies in a colorless cytoplasm. It occurred to us that the stained fat granules would probably