190 PUBLIC HEALTH BACTERIOLOGY 



At the first attempts at immunization, the animals died 

 of chronic poisoning. To avoid this, it is now usual to 

 begin with very small doses of the toxin weakened by the 

 addition of iodine terchloride or Lugol's solution (used in 

 Gram's method of staining, I in KI in water). A young 

 vigorous healthy horse (4 to 6 years old) is chosen, and 

 0-5 c.c. of toxin mixed with an equal quantity of Lugol's 

 solution is injected subcutaneously in front of the shoulder- 

 blade, using a large needle connected with a syringe by a 

 piece of rubber tubing. (The skin is previously shaved 

 and sterilized.) After the reaction has subsided, usually 

 in 5 to 8 days, a few days' interval is given, and the next 

 dose is administered, either 1 c.c. -j- 1 c.c. Lugol's solution 

 or 05 c.c. pure toxin. The amount given is thus gradually 

 increased by \ c.c. until finally in three to four months 

 the antitoxic value of the animal's serum is such that a 

 dose of 300 c.c. of active toxin may be borne. The 

 immunizing process must not be pushed too rapidly, other- 

 wise the health of the animal will suffer. Serum is then 

 obtained by inserting a sterile needle into the jugular vein 

 of the horse, and collecting the quantity of blood desired 

 (up to 6 litres at a time) through a rubber tube into sterile 

 Erlenmeyer flasks containing solution of citrate of soda. 

 These are allowed to stand until the corpuscles settle, and 

 the plasma is poured off into another flask, and allowed 

 to clot. Separate the serum, and filter. If filtered at 

 once, it is found to precipitate again on standing, hence 

 it is better to allow it to stand a few days before filtration. 

 Bottle or tube, after adding 05 per cent carbolic, but 

 before this it should be standardized. 



Standardization of antitoxic serum is very important, 

 so that accurate dosage may be determined, and also so 

 that one strength may be aimed at, since in the making 

 it is liable to variation. The making of such a standard is 

 not an easy task, because no two samples of toxin are of 

 exactly the same strength, nor are even two samples of the 

 same toxin tested at different times. This is another way 

 of saying that toxin is a very variable substance, but 

 fortunately antitoxin is not so variable. Hence Ehrlich 

 chose as his " immunity unit," the amount of antitoxic 

 serum which will neutralize 100 times the minimum lethal 



