DIPHTHERIA TOXINE. 79 



per kilo, causing certain death. Hence, it is not very poisonous, 

 a part of the toxine apparently having been destroyed. 



WASSERMANN and PROSKAUER l have modified this method. They evapo- 

 rate the bouillon (which has been neutralised by the addition of 10 to 

 12 c.c. of normal soda solution, and sterilised by filtration through 

 KITASATO'S niters, at a temperature of 27 to 30 C. in vacuo to a tenth 

 of its volume. It is then dialysed in running water at a lower tempera- 

 ture, by which means the salts and peptones are removed, and the globulins 

 (which are not poisonous) partially precipitated. The contents of the 

 dialyser are filtered until clear an important point and then poured 

 into ten times their volume of 60 to 70 per cent, alcohol acidified by the 

 addition of a few drops of acetic acid, and allowed to stand for twenty-four 

 hours. The resulting precipitate is separated by filtration and the filtrate 

 allowed to fall drop by drop into absolute alcohol. The new precipitate 

 that is formed is also filtered off, and both dissolved in a small quantity of 

 water and precipitated by the addition of twice the amount of a saturated 

 solution of ammonium sulphate. This precipitate is again dissolved, and 

 the solution dialysed until it no longer gives the sulphate reaction, after 

 which it is again introduced into absolute alcohol and the process repeated 

 until the aqueous solution is perfectly clear. The preparations are then 

 dried in vacuo at 37 C. They give all the reactions of albumoses. Only 

 preparations precipitated by alcohol of, at least, 60 per cent, strength are 

 poisonous not the others, so that in this way it is possible to effect a 

 further separation from the bulk of proteids simultaneously precipitated. 

 These products were also obtained in the same manner from extracts made 

 with glycerin and ordinary salt from the organs of human beings infected 

 with diphtheria. All these preparations, however, were only very slightly 

 poisonous. Ten mgrms. were required to kill a rabbit in three to four 

 days, and with 3 mgrms. death did not take place until after eight weeks. 



Products of greater toxicity were obtained by WASSERMANN 

 and PROSKAUER by extracting the organs of poisoned rabbits 

 with glycerin. This process yielded a , white powder which, 

 when injected in a dose of 0'2 mgrm. into the veins, killed a 

 rabbit in six to fourteen days. 



The separation of the true toxine from the associated proteid 

 impurities, and therewith our knowledge that the active principle 

 is not a protein is also due to BRIEGER, 2 who, in collaboration 

 with BOER, has prepared the toxine in a fairly pure condition. 



For this purpose, BRIEGER and BOER made use of the method of 

 precipitating the toxine in the form of a double salt of zinc. The 

 precipitation is practically quantitative, at all events in the case of 

 diphtheria poison, but the difficulty of separating the toxine from the 

 zinc is rendered the greater by the fact that hydrogen sulphide, which 



1 Wassermann and Proskauer, " Ueb. die von d. Diphtheriebacillen 

 erzeugten Toxalbumine, " Deutsch. med. Woch., 1891, 585. 



2 Brieger and Boer, "Ueb. d. Toxine d. Diphth. u. d. Tetanus," Deutsch. 

 med. Woch., 1896, 783 j Zeit.f. Hyg., xxi., 259, 1896. 



