1G2 KITA.TIMA. 



IMMUNIZATION AND IMMUNIZED SERUM. 



Immunization with the venom of the family Crotalidce is as a rule 

 difficult to produce, and so it was found to be with the habu venom. 

 I employed horses, cattle, goats, etc., for this purpose, and found that 

 cattle could be made immune with comparative ease, the horse only with 

 the utmost difficulty, several of the latter falling victims to the experi- 

 ment. The venom was heated or treated with calcium hypochlorite or 

 iodine trichloride in order to diminish the toxicity before introduction 

 into the animal body, but simple dilution with water was also highly 

 satisfactory. The diluted toxin was injected subcutaneously, intravenous 

 inoculation being dangerous and often rapidly fatal to the animal. In 

 most cases we began the inoculation with 0.00001 cubic centimeter of 

 a 1 per cent solution, which was doubled during the first few injections 

 and then increased by small degrees, till at last the dose reached 500 

 cubic centimeters. It took two years to effect immunization, for the 

 snake venom tends to develop necroses and consequently the interval 

 between injection must be made much wider than is usual with other 

 toxins. 



Calmette employs the following method to determine the efficacy 

 of the serum : 



He first determines the dose of venom, which, when given intravenously, will 

 kill a rabbit in from fifteen to twenty minutes, and this is inoculated intra- 

 venously fifteen minutes after the animal receives a certain amount of serum 

 intravenously. If 1 cubic centimeter of the serum were needed to protect a rabbit 

 of 2 kilograms' weight, the serum is said to contain 2,000 immunization units. 

 However, I preferred to test the serum after the method of Erhlich and von 

 Behring, mixing the toxin and serum before inoculation. 



In order to determine the protective value of the serum, various 

 amounts of the latter are added to tubes containing a 1 per cent solution 

 of the venom. The mixture is allowed to stand for thirty minutes before 

 inoculation. The dose contains ten times the minimum lethal dose for 

 the animal. For instance, if a mouse is used, 0.05 cubic centimeter 

 of a 1 per cent solution of venom should be injected in order to give 

 the animal 0.0005 gram of venom, for the minimum lethal dose for the 

 mouse is 0.00005 gram. This dose is first mixed with a certain amount 

 of serum and then diluted with water so that the whole volume comes 

 to 0.4 cubic centimeter before it is inoculated. The proportions used 

 are as follows per 15 grams of mouse: 0.05 cubic centimeter of a 1 per 

 cent solution of venom, 0.1 cubic centimeter serum, 0.25 cubic centi- 

 meter water. 



For practical purposes, 0.5 cubic centimeter of a 1 per cent solution 

 of venom, 1 cubic centimeter of serum and 2.5 cubic centimeters of 

 water are mixed, the mixture is allowed to stand for thirty minutes, 

 and 0.4 cubic centimeter of it is inoculated subcutaneously. 



A certain interval of time is necessary to effect a complete neutral- 



