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-I The authors are indebted to Dr. Walter C. Tobie and Miss Marion H. Cook for the preliminary work which led to the conclusion that the antibiotic-producing organism had characteristics intermediate between those of Bacillus polymyza and Bacillus macerans. 549on August 3, 2020 by guest

Trypticase-soy-phosphate medium (Baltimore Biological Laboratory). 585 on July 15, 2020 by guest http://jb.asm.org/ Downloaded from SUMMARY The factors influencing Escherichia coli inhibition zones produced by the antibiotic polymyxin are considered. An agar diffusion method of assay is described and a statistical analysis presented. As customarily used, the error for a 95 per cent probability is in the neighborhood of L15 to 20 per cent. This can be further reduced, if desired, by appropriate replication.

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Isolation and Preliminary Purification of Polymyxin 3771 corresponding alcohol, tryptophol (IV), by the excellent method of Nystrom and Brown.9 An ether solution of III was treated with lithium aluminum hydride, suspended in ether, to give IV in good yield (65%). Experimental 3-Indoleacetic Acid (III).-Gramine (II) was prepared by the Mannich reaction on indole (I) according to the method of Kuhn and Stein.7 A mixture of 25.0 g. of gramine (II), 35.2 g. of sodium cyanide, 280 ml. of 95% ethyl alcohol and 70 ml. of water was boiled for eighty hours.To the cooled reaction mixture was added 350 ml. of water.The solution was treated with Norite, filtered, concentrated under reduced pressure until all the alcohol had been removed, cooled to 5°and filtered. The solid on the funnel (7.0 g., m. p. 145-150°) was recrystallized from alcohol and ether to give 5.0 g. of 3-indoleacetamide, m. p. 149-151 °. A sample of the amide, prepared for analysis by recrystallization from absolute alcohol and petroleum ether, was found to melt at 153 °. The amide has been previously reported to melt at 150-151 °.10>6

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