DEVELOPMENT OF ANTITOXIN. 471 



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. Behring uses the term " normal diphtheria toxin of simple 

 strength" (DTN 1 ), as indicating a toxin of which .01 c.c. is the 

 minimum lethal dose under these conditions. A toxin of which 

 the minimum lethal dose is .02 will be of half normal strength 

 (DTN- 5 ) ; and so on. 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, viz., an "immunity unit" (p. 472). 



3. Development of Antitoxin. The earlier experiments on 

 tetanus and diphtheria were performed on the 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 ter- 

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

 iodine solution in the proportion of one to three (Roux and 

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

 tetanus, of using a series of toxins weakened to varying degrees 

 by being exposed to different temperatures, viz., 60, 55, and 

 50 C. The toxin is at first injected into the subcutaneous 

 tissues, the dose being gradually increased according to the 

 results of the toxin injected. As pointed out by Behring, im- 

 munisation proceeds best when each injection produces a reac- 

 tion in the form of localised inflammatory swelling ; in other 

 words, the dose should be as large as possible, so long as gen- 

 eral injurious effects are not produced. Later, when large doses 

 of toxin injected subcutaneously are well borne, the toxin is 

 injected directly into the jugular vein of the animal. Ultimately 

 300 c.c., or more, of active diphtheria toxin thus injected may 

 be borne by a horse, such a degree of resistance being developed 

 after the. treatment has been carried out for two or three months. 

 In all cases of immunising the general health of the animal 

 ought not to suffer. If the process is pushed too rapidly the 

 antitoxic power of the serum may diminish instead of increasing, 

 and a condition of marasmus may set in and may even lead to 



