562 IMMUNITY 



either that a large proportion of the endotoxin does not lead to the 

 production of antitoxin or does so only with great slowness, the 

 latter alternative being on general grounds rather improbable. 

 The best examples of antitoxic sera are those of diphtheria and 

 tetanus, though similar principles and methods are involved in 

 the case of the anti-sera to ricin and abrin, and to snake poison. 

 We shall here speak of diphtheria and tetanus. The steps in the 

 process of preparation may be said to be the following : First, 

 the preparation of a powerful toxin ; second, the estimation of 

 the power of the toxin ; third, the development of antitoxin in 

 the blood of a suitable animal, by gradually increasing doses of 

 the toxin ; fourth, the estimation from time to time of the 

 antitoxic power of the serum of the animal thus treated. 



1. Preparation of the Toxin.— The mode of preparation and 

 the conditions affecting the development of diphtheria toxin 

 have already been described (p. 408). In the case of tetanus the 

 growth takes place in glucose bouillon under an atmosphere of 

 hydrogen (vide p. 429). In either case the culture is filtered 

 through a Chamberland filter when the maximum degree of 

 toxicity has been reached. The term " toxin " is usually applied 

 for convenience to the filtered (i.e., bacterium-free) culture. 



2. Estimation of the Toxin. — The power of the toxin is 

 estimated by the subcutaneous injection of varying amounts in 

 a number of guinea-pigs, and the minimum dose which will 

 produce death is thus obtained. This, of course, varies in pro- 

 portion to the weight of the animal, and is expressed accordingly. 

 In the case of diphtheria, in Ehrlich's standard, the minimum 

 lethal dose — known as M.L.D. — is the smallest amount which 

 will certainly cause death in a guinea-pig of 250 grms. within 

 four days. The testing of a toxin directly is a tedious process, 

 and in actual practice, where many toxins have to be dealt with, 

 it is found more convenient to test them by finding how much 

 will be neutralised by a certain amount of a standard antitoxic 

 serum, namely, an "immunity unit" (p. 563). 



3. Development of Antitoxin. — The earlier experiments on 

 tetanus and diphtheria were performed on small animals, such 

 as guinea-pigs, but afterwards the sheep and the goat were used, 

 and finally horses. In the case of the small animals it was 

 found advisable to use in the first stages of the process either a 

 weak toxin or a powerful toxin modified by certain methods. 

 Such methods are the addition to the toxin of terchloride of 

 iodine (Behring and Kitasato), the addition of Gram's iodine 

 solution in the proportion of one to three (Eoux and Vaillard), 

 and the plan, adopted by Vaillard in the case of tetanus, of 



