1 28 Immunity 



Bacillus coll, and is stood in a warm place overnight. The colon bacilli ferment 

 and remove the muscle and other sugars. The infusion is then made into 

 bouillon, titrated so that the reaction equals + i.i when tested with phenolph- 

 thalein. It then receives an addition of 0.2 per cent, of dextrose, and is steriliEed 

 in the autoclave. To secure the best toxic product, the bacilli at hand must be 

 carefully studied and that naturally possessing the strongest toxicogenic power 

 employed for the cultures. The greatest toxicity seems to develop between the 

 fifth and seventh days. If the culture is permitted to remain in the incubating 

 oven beyond this period, the toxin gradually is transformed to toxoid and its 

 activity declines. The fatal dose for a 250-300 gram guinea-pig should be about 

 0.00 1 cc. given hypodermically. 



II. The Immunization of the Animals. — All commercial manufacturers of 

 diphtheria antitoxic serums now use horses, as recommended by Roux, instead 

 of the sheep, dogs, and goats with which the earlier investigators worked. The 

 horse is readily immunized, gives an abundant supply of blood which clots readily 

 and yields a beautiful clear amber serum. 



The horse selected should be in perfect health, and should be tested with 

 mallein and tuberculin to avoid obscure glanders and tuberculosis. 



A small dose of the toxic bouillon — say o.i cc. — should be given in the begin- 

 ning, as one occasionally finds exceptionally susceptible animals that will suc- 

 cumb to larger doses. If a marked local and general reaction follows, it may be 

 better to try another animal. If no reaction is brought about, the immunization 

 is carried on as rapidly as possible. The toxin is injected hypodermatically 

 into the tissues of the neck, the skin being thoroughly cleaned and disinfected 

 before each injection. The doses are cautiously increased and may often be 

 doubled each day. If any unfavorable symptoms arise, treatment must be in- 

 terrupted for a day or two. The animal jaelds good- antitoxic serum when it 

 can endure several doses of 500 cc. of the strong toxin mentioned above. 



m. Bleeding. — When the withdrawal of a small quantity of blood by a 

 hypodermic needle introduced into the jugular vein shows that the serum con- 

 tains a maximum antitoxic strength (300 to 1000 units per cubic centimeter), 

 the horse is ready to bleed. Some horses can be bled without resistance, but 

 most of them require to be fastened in appropriate stocks. The blood is taken 

 from the jugular vein, which is superficial, of large size, and easily accessible. 

 The skin is carefully shaved over an area about 9 square inches in extent, thor- 

 oughly disinfected. A small incision is made over the center of the vein, which 

 is made prominent by pressure at the base of the neck, and the point of a small 

 sterile trocar being inserted in the incision through the skin, it is directed obliquely 

 upward into the vein. The blood is allowed to flow through a sterile tube 

 attached to the cannula into sterile bottles prepared to receive it. A large horse 

 may furnish 7 to 9 liters; small horses, s to '/ liters. 



IV. Preparation of the Serum. — The blood is stood away in a cool place until 

 the clot retracts after coagulation and the clear serum separates. The serum 

 is then withdrawn under strict aseptic precautions. It is variously prepared 

 for the market. Some manufacturers bottle it without any added preservative; 

 some add a crystal of thymol; some Pasteurize it; some add carbolic acid; some 

 add trikresol. 



The plain serum would be ideal, but the danger of subsequent contamination 

 through careless treatment makes it rather better to have an antiseptic added. 

 Trikresol is probably the most satisfactory of these, though it throws down a 

 precipitate that necessitates the filtration of the product, and leaves the serum 

 slightly opalescent. , 



V. Determining the Potency of the Serum. — The potency of the serum is 

 expressed as so many "immunizing units." Only one method of testing is 

 in use at the present time, though to understand it, it seems wise to mention 

 the original method from which it was derived. 



(A) Behring's Method. — Behring's unit was an arbitrary standard chosen in 

 consequence of certain conditions existing at the time it was devised. It is 

 diificult to understand apart from the circumstances governing its creation, but 

 may be defined as " Ten times the least quantity of antitoxin serum that will protect 

 a standard (300 gram) guinea-pig against ten times the least certainly fatal dose of 

 toxic bouillon." 



The method of determining it is not diflBcult to those skilled in laboratory 

 technic, and is as follows: 



I. Determine accurately the least certainly fatal dose of a sterile diphtheria 

 toxic bouillon for a standard guinea-pig. 



