DIPHTHERIA TOXINE. 77 



Of the chemical agents that destroy the living bacilli, and 

 simultaneously have a preservative influence upon the poison 

 solution, and are also added for the latter purpose alone after 

 filtration, the following are employed : 



Iodine trichloride, IC1 3 , by BEHKING and WERNICKE. 



Trier esol, in a O3 per cent, solution, by ARONSON (loc. cit.). 



Phenol, also in a 0*3 per cent, solution (SPRONCK, loo. cit.). 



Toluene is used by EHRLICH, who keeps his poisons at 15 C. 

 in the dark under a layer of that substance. According to 

 ABBA, 1 diphtheria toxine can be preserved for two years if kept 

 under toluene in the dark and in the cold. 



The best and most suitable means of preparing and preserving 

 diphtheria virus for practical immunising purposes is the fol- 

 lowing method, described by EHRLICH and WASSERMANN : The 

 bouillon cultures prepared in the manner described above are 

 filtered through double filter paper, so that the coarse bacterial 

 membranes are separated. The filtrate is then covered with a 

 layer of toluene of the depth of about two fingers'-breadth, and 

 the whole thoroughly shaken at frequent intervals during two 

 days. All living micro-organisms will then be destroyed and 

 the liquid will contain only the poison. When required for use 

 the poison is invariably withdrawn by means of a pipette, inserted 

 beneath the preservative layer of toluene. The poison must be 

 kept in a cool place, and in particular protected from light by 

 being placed in black flasks or flasks covered with paper. This 

 is the method of preparing and preserving the poisons in use 

 in the Prussian Control Station. 



For more delicate biological and chemical investigations this 

 rough filtration is insufficient, and in such cases the liquid must 

 be completely freed from the cells of the bacilli by filtration 

 through a proper bacterial filter, and then sterilised. 



Attempts to Prepare Pure Diphtheria Toxine. Attempts to 

 isolate diphtheria toxine in a pure or, at least, concentrated 

 condition were made even in the earliest period of the investi- 

 gation of diphtheria bacilli. 



LOFFLER 2 found that evaporation or shaking with ether did 

 not yield active poisons ; but, on the other hand, by extracting 

 inoculated meat broth with glycerin he obtained a slightly toxic 

 substance which could be precipitated by means of alcohol. He 

 termed this, even in this condition, an "enzyme." 



1 Abba, " Ueb. d. Dauer des toxisch. Vermogens beim D.-T. u. Antit.," 

 Centralbl.f. Bakt., xxiii., 934, 1898. 



2 Loffler, "Der gegenw. Stand d. Frage n. d. Entsteh. d. Diphtherie," 

 Deutsch. med. Woch., 1890, Nos. 5 and 6. 



