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High yields of toxin have not been obtained with the nonproteolytic, and most of the proteolytic, strains of Clostridium botulinum, type B, in our culture collection. An exceptionally toxic type B culture designated as strain "okra" was received from the National Institute of Health. It is a proteolytic strain capable of producing, under certain conditions, one million minimum lethal doses of toxin for the mouse per ml of medium. From cultures of the "okra" strain a highly toxic and immunologically distinct protein has been isolated. Though this protein has not been crystallized it appears to be essentially a highly purified, single substance. The method of isolation for crystalline type A botulinum toxin of Lamanna, McElroy, and Eklund (1946) has not been applicable. This is probably a reflection of the physical and chemical differences that exist between the two serological types of toxin. Our methods and observations with the type B toxin are recorded in the following sections. Maintenance of the stock culture. The stock culture is kept in chopped-beef infusion medium consisting of meat fragments submerged in a double-strength beef infusion including 0.5 per cent sodium chloride and 1 per cent Difco proteose peptone. Once a month the culture is transferred using a 1-ml inoculum per test tube. Incubation is at 34 C for 48 hours, and storage is at room temperature. Medium and growth for toxin production. The organism is grown in 16-liter lots in 5-gallon pyrex glass carboys. The medium is composed of 1 per cent technical grade casein, 1 per cent alkaline-treated cornsteep liquor (47 to 52 per cent solids), and 0.5 per cent technical grade glucose. The cornsteep liquor is a filtrate of raw cornsteep treated by the addition of 1 part of water to 11 parts of cornsteep, alkalinizing with 40 per cent sodium hydroxide to pH 8.5, and heating at 65 C for a half hour. The casein is brought into solution by agitation at pH 9.5 or higher. The cornsteep liquor is added, and the pH adjusted to 7.2. The mixture is autoclaved at 120 C for 1 hour. Upon cooling, the proper amount of glucose solution, which has been autoclaved separately, is added. A flask with 500 ml of the same medium is preheated to remove dissolved air, cooled, and inoculated with the contents of a test tube of a 1-month-old stock culture. After overnight incubation at 34 C, 10 ml are transferred to 500 ml of medium in flasks corresponding in number to the carboys to be used for toxin production. These flasks in turn are incubated at 34 C overnight and then are used as inoculum, one per carboy. The carboys are incubated for 2 weeks at 34C. As a consequence of vigorous fermentation during the first 24 hours of growth the pH drops rapidly to values of 5.3 to 5.5. the casein comes out of solution both as a sediment and as a thick, firm pellicle floating at the surface as a result 575

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