DIPHTHERIA TOXINE. 97 



The milk collected with aseptic precautions is treated with about 20 c.c. 

 of N. -hydrochloric acid per litre, coagulated as rapidly as possible by 

 means of rennet enzyme, and filtered from the separated paracasein. The 

 whey is then thoroughly shaken with chloroform and allowed to stand, 

 after which the supernatant liquid is decanted from the layer of chloroform 

 charged with fat and bacterial cells. Each 5 litres of the whey is next 

 treated with a calculated amount (determined by a preliminary experi- 

 ment) of 30 to 33 per cent, ammonium sulphate solution, and the pre- 

 cipitate dried on porous earthenware at 35 C. in vacuo, separated as 

 completely as possible from solid ammonium sulphate and dissolved in 

 water to form a solution of the desired concentration. 



The experiments to obtain antitoxine preparations from the 

 liquid serum have hitherto proved nearly worthless from the 

 practical point of view, since at present it is preferred to 

 immunise animals to such an extent that the serum of the 

 animal itself contains so many antitoxic units per c.c. as to 

 render concentration superfluous. 



On the other hand, the scientific side of the question has been 

 followed with equal interest, and attempts have been made to 

 isolate the antitoxine as such as completely as possible and to 

 determine whether it is to be considered as an individual sub- 

 stance, and with which proteid of the serum it combines. In 

 addition to this, experiments have been made to discover points 

 of difference between ordinary serum and that containing anti- 

 toxine. 



SZONTAGH and WELLMANN* made a comparative examination 

 of normal and antitoxic horse-serum. In neither did they detect 

 any nucleo-albumin, while the proportions of globulin and of ash 

 and chlorine, and the specific gravity were the same in each. 

 The only difference observed was that the antitoxic serum 

 contained, on the average, about 0-25 per cent, more nitrogen 

 than the normal serum ; they rightly attribute, however, no 

 special importance to this difference. On the other hand, they 

 claim to have found that the therapeutic serum shows a decrease 

 in the lowering of freezing point and in the electrical conduc- 

 tivity, which are apparently proportional to the amount of 

 antitoxine. 



FREUND and STERNBERG 2 have endeavoured to isolate the anti- 

 toxine in as pure a state as possible by the following method : 



The therapeutic serum was treated with alum and dialysed. No 

 precipitate was produced in the dialysate by zinc salts. Nor was the 



1 Szontagh and Wellmann, " Vergleichende chem. Unters. iib. d. normale 

 Pferdeserum und das Diphtherieheilserum," Deutsch. med. Woch., 1898, 421. 



2 Freund and Sternberg, " Ueber Darstellung des Heilkorpers aus d. 

 Diphtherieheilserum," Ze.it. f. Hyg., xxxi., 429, 1899. 



