The Antitoxins 131 



it is employed to neutralize or "fix" the toxin circulating in the 

 blood, not to destroy the baciUi, or to effect the regeneration of the 

 tissues injuriously acted upon by the toxin. Martin is of the 

 opinion that such purely antitoxin serums are inferior to those con- 

 taining other immunity products, such as bacteriolysins, and recom- 

 mends that the whole culture instead of the filtered culture be used 

 in the immunization of the animal. If this is done, the bacteriolytic 

 effect is added' to the antitoxic effects of the serum. 



The serum may be used to prevent or to cure diphtheria. 



The antitoxin is copimercially manufactured at present by im- 

 munizing horses against increasing quantities of diphtheria toxin 

 until the proper degree of immunity has been attained, then with- 

 drawing the antitoxic blood. The details are as follows : 



I. The Preparation of the Toxin. — The toxic metabolic products of the Bacillus 

 diphtherias are for the most part freely soluble, and are therefore best prepared 

 in cultures grown in fluid media. The medium best adapted to the purpose is 

 that recommended by Theobald Smith.* 



To make it, the usual meat infusion receives the additipn of a culture of 

 Bacillus coli, 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 phenol- 

 phthalein. It then receives an addition of o, 2 per cent, of dextrose, and issterilized 

 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 2So-3oo-gram guinea-pig should be about 

 o.ooi 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 yields good antitoxic serum when it 

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



in. Bleeding. — rWhen 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 



* "Journal of Experimental Medicine," May and July, iSgg, p. 373. 



