Purification of cultures of bacteria by means of reverse selective bacteriostatic properties of aniline dyes.

In a recent study of the sporulation of Bacillus anthracis, this organism was recovered from the spleen and heart's blood of a mouse, dead of the experimental disease, in association with a small gram negative bacillus. The presence of the contaminating organism could not be detected on the agar transplants because of the overgrowth of B. anthracis, but it could readily be seen in smears. The observation of this gram positive spore bearing bacillus with a gram negative bacillus presented an excellent opportunity to test the validity of the idea that in such cases purification of the cultures could be effected on the principle of reverse selective bacteriostasis. If the observations previously reported on this subject were correct, it would be possible in the case of this particular mixture of B. anthracis and a gram negative bacillus, to isolate either of the two organisms, in pure culture, by simply treating the mixture with one of two dyes, whose selective bacteriostatic properties, as between organisms of these two types, were known to be opposite in character.

As has been previously reported, 1 gentian violet and acid fuchsin possess these opposite selective properties. The culture of B. anthracis contaminated with the gram negative organism (which we may call Bacillus X), was therefore stroked in a heavy suspension across a divided gentian violet plate. The upper half contained dye in a 1/100,000 dilution.

As had been expected, Bacillus × grew out in pure culture on the gentian violet half of the plate. Smears made from this culture showed practically nothing but Bacillus X, although an occasional individual B. anthracis could be seen, probably dead. An absolutely pure culture could be readily obtained by again stroking on another gentian violet plate an aqueous suspension of the growth obtained from the gentian violet half of the divided plate.