DIPHTHERIA TOXINE. 95 



duces similar injuries, although the results of my experiments contradict 

 the conclusion of LINOSSIER and LEMOiNE 1 that even minute quantities of 

 normal horse-serum produce serious illness (persistent albuminuria) in 

 rabbits. 



Concentration of the Antitoxine. Soon after the first active 

 antitoxine sera had been prepared, experiments were begun with 

 the object of isolating the antitoxine. 



At first these were concerned with the purely practical idea of 

 preparing solid substances which had not lost the specific property 

 of the antitoxine. For this purpose the usual method employed 

 was simply to precipitate the antitoxine and the proteids of the 

 serum simultaneously e.g., by means of concentrated ammonium 

 sulphate, and to dry these precipitates at a low temperature in 

 vacua. In this way fairly active, dry residual preparations of the 

 antitoxine were obtained, and these, when shaken with very weak 

 alkalies, yielded solutions of the antitoxine ranging up to several 

 hundred times the strength of the original serum. 



ARONSON, 2 for example, proceeded as follows : One hundred c.c. of the 

 serum were diluted with 100 c.c. of water, and treated with 70 c.c of a 10 

 per cent, solution of aluminium sulphate. Ammonia was then added in 

 such quantity that the solution still remained slightly acid, and the result- 

 ing precipitate was washed with 150 to 200 c. c. of cold water. It contained 

 as much as 95 per cent, of antitoxine. Similar precipitates were produced 

 by zinc sulphate and potassium ferrocyanide, and by iron chloride and 

 ammonia. These precipitates were repeatedly shaken for long periods in a 

 shaking apparatus with a hundredth part (calculated on the original serum) 

 of very dilute alkali solutions, which just turned red litmus blue. 



BuJWiD 3 freezes the serum, with the result that the water first 

 separates as ice, leaving behind a turbid solution very rich in 

 toxine. The crystals can then either be separated by "centri- 

 fuging" or slowly thawed, in which process two layers are 

 formed, so that the upper layer, containing pure water, can be 

 withdrawn. Results quite analogous to these were simultan- 

 eously obtained by ERNST, COOLIDGE, and CooK. 4 



BRIEGER and BOER S found that precipitates of calcium phos- 



1 Linossier and Lemoine, "Action nephrotoxique des inj. d. se'rums 

 normaux," Soc. BioL, lv., 515, 1903. 



2 Aronson, "Weit. Unters. iiber Diphtherie," Berl. klin. Woch., 1894, 

 425. 



3 Bujwid, " Ueb. e. Methode z. Concentr. d. Diphtherieheils. mittelst 

 Ausfrieren," Centralbl. f. Bakt., xxii., 287. 



4 Ernst, Coolidge, and Cook, "The Effect of Freezing upon the Anti- 

 diphtheritic Serum," J. Boston Med. Soc., if., 166 ; Baumgarten's Jb., 1898, 

 242 



5 Brieger and Boer, " Ueber Antitoxine und Toxine," Zeit. f. Hyg., xxi., 

 259, 



