78 TOXINES AND ANTITOXINES. 



Subsequently Roux and YERSIN, MADSEN, and others obtained, 

 by means of precipitation with alcohol, saturation with ammonium 

 sulphate, and precipitation with calcium phosphate, active dry 

 preparations, which, of course, made no pretensions to purity. 

 The fundamental idea underlying these experiments is that the 

 diphtheria poison is carried down mechanically by the voluminous 

 precipitate e.g., of calcium phosphate produced in its solution. 



BEIEGER was the first to undertake these investigations in a 

 systematic and thorough manner. At first, in collaboration with 

 C. FRANKEL/ he looked for ptomaines in the diphtheria cultures, 

 but soon had to admit that there were absolutely no volatile bases 

 present. On this account BRIEGER has considerably modified his 

 views on the significance of ptomaines. He now obtained from 

 the cultivations, not only of diphtheria bacilli, but also of many 

 other bacteria, poisonous proteid substances which he termed 

 toxalbumins. 



The method by which BRIEGER and FRANKEL isolated their 

 diphtheria toxalbumins is as follows : 



The globulins are first separated from the bouillon by saturating it with 

 magnesium sulphate at 30 C. This gives a slight precipitate which is 

 completely non-poisonous. 



The poisonous proteids are next precipitated from the bouillon thus 

 treated, or, equally well, from the fresh bouillon, by means of ammonium 

 sulphate or sodium sulphate, or also by the addition of a large excess of 

 alcohol. Since the ammonium sulphate precipitate must be redissolved 

 and dialysed (not without loss), it is best to employ precipitation with 

 alcohol. 



The bouillon is evaporated to a third of its volume at 30 C. , and treated 

 with ten times its quantity of absolute alcohol, preferably with the addition 

 of a few drops of acetic acid. After standing for twelve hours in an ice -chest 

 the liquid is filtered, and the precipitate taken up with water and again 

 precipitated with alcohol, this treatment being repeated six or eight times 

 until the substances dissolve to a completely clear solution in water. 

 Finally, the preparation is dialysed and dried in vacuo at 40 C. 



This treatment yields a snow-white powder, the aqueous 

 solution of which does not coagulate on boiling. It gives no 

 precipitate with sodium sulphate, sodium chloride, magnesium 

 sulphate, nitric acid, or lead acetate, but is precipitated by 

 saturated solutions of carbon dioxide and by all the usual 

 reagents for proteids. The substance is thus allied to the 

 albumoses; it yields a benzoyl derivative, but not a phenyl- 

 hydrazine compound. In the dry state it can be heated to 

 70 0. without injury. It is poisonous, a dose of 2 '5 mgrms. 



1 Brieger and Frankel, "Ueber Bakteriengifte," Berl. klin. Woch., 1890, 

 241. 



