DIPHTHERIA ANTI-TOXIN. 101 



diphtheria in man began in 1891 and 1892. The first diphtheria 

 anti-toxin was put on the market in August, 1894. Since then 

 diphtheria anti-toxin has been used extensively. 



The method for making diphtheria anti-toxin has gone 

 through various stages of development. All diphtheria anti- 

 toxin used in the United States is made practically after the same 

 method, which is as follows: Young, vigorous and absolutely 

 healthy horses are immunized, although other species of animals 

 have also been used. During immunization the animal receives 

 repeated injections of diphtheria toxin. This toxin is obtained 

 by growing a virulent culture of B. diphtheria on the surface of 

 beef bouillon in an Ehrlenmeyer flask. After one week's growth 

 at a temperature of 35 to 36 C., the culture is rendered sterile 

 by adding carbolic acid in amounts to make a . .5 per cent car- 

 bolic acid solution. After 48 hours the diphtheria bacilli have 

 settled to the bottom, when the bouillon is filtered through a 

 Berkefeld filter. The germ-free bouillon contains the diphtheria 

 toxin and ought to be potent enough so that 0.01 c. c. will kill 

 a 250 gram guinea pig on or before the fourth day after 

 subcutaneous injection. The horse or animal to be immunized 

 receives enough of this toxin in bouillon to kill five thousand 

 guinea pigs of 250 grams each. At the time this amount 

 of toxin is injected the horse also receives an injection of ten 

 thousand units of diphtheria anti-toxin. After three to five days, 

 when the fever has subsided, a somewhat larger dose of toxin and 

 the same amount of anti-toxin are injected. A third injection is 

 made after another interval of from three to five days. After 

 this usually no more anti-toxin is injected with the toxin, the 

 doses of which are constantly increased and injected at intervals 

 of from five to eight days. After about two months of treatment, 

 if the immunization has been successful, the horse will usually 

 tolerate enough toxin at one injection to kill one hundred thousand 

 guinea pigs of 250 grams each. 



At the end of six weeks to two months, samples of blood are 

 drawn and tested as to protective value. If the anti-toxic value 

 is high, the horse is usually bled to death by tapping the jugular 

 vein. The withdrawal of serum is made under aseptic conditions. 

 The bottles containing the blood are slanted and after four to 

 five days the serum is drawn off. 



