Studies on Polymyxin: Isolation and Identification of Bacillus polymyxa and Differentiation of Polymyxin from Certain Known Antibiotics.

Polymyxin is an antibiotic substance occurring in the culture filtrates of Bacillus polymyxa. The isolated substance is unique in its specificity for gram-negative bacteria. A summary of the more important results obtained during the course of several years, including chemotherapeutic and toxicity data, has been reported (Stansly, Shepherd, and White, 1947). The present contribution is concerned with the isolation and identification of the antibioticproducing organism and some early findings which both characterized and distinguished polymyxin from certain known antibiotics. Isolation of Bacillus polymyxa. Bacillus polymyxa was isolated from soil in the course of a program designed to find new antibiotics for the chemotherapy of gram-negative bacterial infections. The test organism used in this search was Salmonella schottmuelleri. Our method for isolating antibiotic-producing organisms with a specific type of activity involves the preparation of pour plates of soil dilutions using a variety of media and cultural conditions. The plates are subsequently sprayed with a suspension of the test organism by means of an apparatus designed for the purpose (Stansly, 1947). Identification of Bacillu-s polymyxa. The identification of Bacillus polymyxa was established by following the key to the identification of aerobic sporeforming bacteria by Smith, Gordon, and Clark (1946). In the preliminary work,' edition 5 of Bergey's Manual of Determinative Bacteriology (1939) and the galley proofs of edition 6 were found helpful. An 18-hour broth culture consisted of gram-negative rods with few or no gram-positive cells. Older cultures showed vegetative cells and oval spores either free or central to terminal in adhering and swollen sporangia. Broth cultures at 30 C were turbid and had a ropy sediment. Indole was not formed. Nitrates were reduced to nitrites. Hydrogen sulfide was not produced. Acid and gas were formed from glucose, lac'tose, and sucrose. Acid but no gas was produced from rhamnose and a slight amount of acid but no gas from sorbitol. Starch was hydrolyzed. Acid and gas were produced from litmus milk, which was coagulated and reduced. The existence of oval spores, central to terminal, and sporangia frequently adhering and swollen, plus the predominant gram-negative nature of the vegeta-