298 PA THOGENIC BA CTERIA. The Preparation of the Toxin.—The method employed by Roux and others at the present time was first sug- gested by Fernbach, and consists in growing the most virulent bacilli obtainable in alkaline bouillon exposed in a thin layer to the passage of a current of air. The cultures are allowed to grow for three or four weeks at a temperature of 37° C., with a stream of moist air constantly passing over them. After the given time has passed, it will be found that the acidity prima- rily produced by the bacillus gives place to a much more intense alkalinity than originally existed. The acme of the toxin-production seems to keep pace with this alka- line production. When " ripe,n 0.4 per cent, of trikresol is added to the cultures, which are then filtered through porcelain. If the toxin must be kept before using, it is best to preserve it unfiltered, as it deteriorates more rap- idly after filtration. Unfiltered toxin causes too much local irritation. If the bacillus employed was virulent and the conditions of culture were favorable, the filtered culture should be so toxic that o. i c. cm. would be fatal to a 5OO-gram guinea-pig in twenty-four hours (Roux). Even under the most favorable circumstances it is difficult to obtain a toxin which will kill in less than thirty hours. The experience of the author with Fernbach's appara- tus has not been satisfactory. The passing current of air is a frequent source of contamination to the culture, and requires great care. In the end it is questionable whether the toxin thus produced is better than that obtained from an ordinary flask exposing a large surface to the air. Park and Williams did an elaborate work upon the production of diphtheria toxin.1 They found that '' toxin of sufficient strength to kill a 4oo-gram guinea- pig in three days and a half in a dose of 0.025 c.crn., developed in suitable bouillon, contained in ordinary Erlenmeyer flasks, within a period of twenty-four hours. In such bouillon the toxin reached its greatest strength in four to seven days (0.005 c.cm. killing a 5OO-gram 1 Jour, of Exper. Med.^ vol. i., No. I, Jan., 1896, p. 164.