80 TOXINES AND ANTITOXINES. 



would otherwise have been a very suitable agent, destroys the toxine. 

 After long and careful experiments they arrived at the following method 

 of separation : 



The poison-bouillon (blood serum was used with good results as the 

 culture medium) is treated with twice its volume of a 1 per cent, solution 

 of zinc chloride, and the resulting precipitate thoroughly washed with 

 water, and then vigorously shaken with a 3 to 6 per cent, solution of 

 ammonium carbonate. Ammonium phosphate solution is next added until 

 the whole of the precipitate passes into solution, and there is only a slight 

 turbidity due to separated zinc phosphate. This is allowed to subside and 

 then collected on a toughened filter and thoroughly washed with water. 

 The filtrate is saturated with solid ammonium sulphate, the precipitate 

 redissolved in water, and the solution precipitated with solid sodium 

 sulphate, by which means the peptones are left in solution. 



As thus prepared the toxine no longer gives the reaction of 

 proteids. The zinc compound also shows no proteid reactions, 

 and is optically inactive, but turns red when boiled with a 

 solution of iron chloride. 



Alcohol, ether, acetone, acids, and weak oxidising agents 

 rapidly destroy the poison, while weak alkalies and reducing 

 agents do not affect it. Diphtheria toxine can also be obtained, 

 although, of course, only in very small quantities, from dialysed 

 urine i.e., proteid-free culture medium (GUINOCHET, loc. cit.) 

 and from other similar nutrient liquids (UscniNSKY 1 ). The culture 

 medium used by USCHINSKY had the following composition : 

 Glycerin, 40 to 50 parts; sodium chloride, 5 to 7 parts; am- 

 monium lactate, 10 parts; calcium chloride, O'l part; magnesium 

 sulphate, 0'2 part ; and potassium hydrogen phosphate, 1 part ; 

 in 1,000 parts of water. The poison did not give the usual 

 proteid reactions. 



Properties of Diphtheria Toxine. The poison is not known in 

 a state of chemical purity. Hence, all that can be stated about 

 it relates to the preparations that contain it in admixture with 

 other substances. Its most important properties have already 

 been described by Koux and YERSIN. 



It is probably not a proteid^ since the purest preparations 

 (vide supra) do not give the proteid reactions. An attempt 

 made by ARRHENIUS and MADSEN 2 to determine the molecular 

 weight from the speed of diffusion into gelatin has so far only 

 led to the conclusion that toxines have at all events a much 

 smaller molecular weight than antitoxines. 



1 Uschinsky, "Ueber Diphtherieculturen auf eiweissfreier Nahrlosung, 

 Centralbl. f. BaU., xxi., 146, 1897. Id., " Les Poisons de la Diphtheric et 

 du Cholera," Arch, de Med. Exptr., 1893, 293. 



2 Arrhenius and Madsen, "On the molecular weight of Di.-T.," Fest- 

 schrift des Statens Serum Institut, Copenhagen, 1902. 



