76 TOXINES AND ANTITOXINES. 



3. Presence of a suitable peptone, preferably Chapoteaut's, in 

 the proportion of 2 per cent. ; 



4. A sufficient supply of air, for which reason the flask should 

 only be filled to about a third of its capacity ; 



5. Not too short and not too long a period of growth at 37 C. 

 This depends upon the culture, and tests must be made with each 

 different variety to determine when the maximum production of 

 poison has been attained. As a rule, it takes from about ten 

 days to three weeks. 



Lastly, means have also been sought to obtain a solution of 

 poison which, while effecting rapid immunisation, would yet be 

 but little poisonous, and so would not endanger the lives of 

 the animals used for the experiments even when given in large 

 doses. BRIEGER, KITASATO, and WASSERMANN l have found that 

 diphtheria cultivations grown upon thymus bouillon lose their 

 toxigenic capacity whilst their immunising power is but little 

 affected. Here there is presumably a formation of immunising 

 toxoids. Then, still more recently, MADSEN has succeeded in 

 effecting immunisation by means of the toxones of diphtheria, 

 and we shall have more to say about this when we deal with 

 them later on. 



When liquids rich in poisons have thus been obtained, all that 

 is necessary is to sterilise them. The methods employed for this 

 purpose are the ordinary ones in general use : heat, addition of 

 antiseptics, and nitration through bacterial niters. 



Roux and YERSIN found that solutions of the poison were rendered non- 

 poisonous after a few minutes at 58 C. , but that the dry poison could be 

 heated for more than an hour at 98 C. 



FRANKEL 2 endeavoured to sterilise cultivations by heating them for an 

 hour at 65 to 70 C., whilst BRIEGER and FRANKEL 3 found that small 

 quantities could be sterilised with certainty when heated from three to 

 five hours at 50 C., but that the poison was speedily destroyed above 

 60 C. 



BEHRING and WERNICKE 4 added calcium chloride to obtain a precipitate 

 of calcium phosphate in the cultivations, and sterilised the dried precipi- 

 tate by heating it at 77 C., and this did not materially injure the toxine 

 simultaneously carried down. 



1 Brieger, Kitsato, and Wassermann, " Ueb. Immunitat. u. Giftfesti- 

 gung," Zeit.f. Hyg., xii., 137, 1892. 



2 irankel, " Immunisierung Versuche bei Diphth.," Berl. klin. Woch., 

 1890, 1133. 



3 Brieger and Frankel, " Ueber Bakteriengifte," ibid., 1890, 240. 



4 Behring and Wernicke, "Ueb. Immunisier. u. Heilung von Versuchs- 

 tierenb. d^ Diphth.," Zeit.f. Hyg., xii., 10, 1892. 



