AFRANIO DO AMARAL 



1075 



BLEEDING 



Horses are bled eight to ten days after the final injection. The blood is received 

 in sodium citrate or in sodium oxalate solution to prevent coagulation. The plasma 

 thus obtained is reserved for concentration. Horses are again injected with a few 

 doses of antigen and bled two or three more times until the antitoxic activity of their 

 serum begins to show marked weakenina;. 



Fig. I, — Venom extraction. (Reprinted from the Bulletin of the Antivenin Institute of America) 



ANTIVENIN TITRATION 



Of all the methods of antitoxin titration which have been devised, three deserve 

 especial reference: 



1. Protection test. — Calmette's method' consists in determining the protective 

 power of the antivenin on 2-kg. rabbits. Antivenin to the amount of 2 cc. is given 

 subcutaneously, and two hours later i mg. of the venom is injected by the same 

 channel, 



2. Neutralization test.- — Brazil's method^ consists in determining the neutralizing 

 activity of the venom. Several test tubes are prepared, each containing i cc. of the 

 antivenin plus variable amounts of the standard venom solution, such as 0.2, 0,4, 

 0.6, 0,8, 1,0, 1,5, 2,0 cc, etc. The tubes are incubated at 37° C. during one hour to 

 bring about the complete combination of the antitoxin with the toxin (venom). The 



' Calmette, A.: op. cit., p. 258. Paris, 1907. 



2 Brazil, V.: Coll. Trahalhos Inst. Butantan, p. 221. 1909. 



