ANTITOXIC SERUM. 437 



2. Estimation of the Toxine. The power of the toxine 

 is estimated by the injection of varying amounts in a 

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

 produce death within forty-eight hours is thus obtained. 

 In the case of diphtheria, a powerful toxine is one of which 

 i c.c. subcutaneously injected kills a guinea-pig within forty- 

 eight hours, though toxines may be prepared of which 

 .02 c.c. may have this effect. In the case of tetanus, toxi- 

 city of such a degree may be reached that a dose of 

 Y^-Q- c.c. will kill a guinea-pig. It is to be noted that the 

 fatal dose of course varies with the body weight of the 

 animal. As a matter of convenience, guinea-pigs of about 

 250 grms. are usually employed. 



3. Immunisation by means of the Toxine. 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 toxine or a powerful 

 toxine modified by certain methods. Such methods are 

 the addition to the toxine of terchloride 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 toxines weakened to varying degrees by being 

 exposed to different temperatures, viz. 60, 55, and 50 C. 

 But in the case of large animals, such as the horse, the first 

 injections are simply made with small doses of the ordinary 

 toxine. The toxine is at first injected into the subcutaneous 

 tissues, the dose being gradually increased according to the 

 results of the toxine injected. As pointed out by Behring, 

 immunisation proceeds best when each injection produces a 

 reaction in the form of localised inflammatory swelling ; in 

 other words, the dose should be as large as possible, so long 

 as general injurious effects are not produced. It was found 

 by Roux that the number of injections also affected the 

 result, a higher degree of immunity being obtained by 

 several small doses than by the same quantity of toxine in 



