156 DIPHTHERIA AND PSEUDODIPHTHERIA. 



of tuberculin and mallein, and free from all rheumatic and chronic 

 disease, is gradually immunized to the diphtheritic poison by 

 being injected with very small doses of the virulent toxins 

 from a diphtheria bouillon culture filtrated through porcelain. 

 The initial dose consists of 0.10 c.c. mixed with an equal 

 quantity of Gram's iodine solution ; this should produce 

 little or no constitutional disturbance, and very little if any 

 local effect. Four or five days after this first injection a 

 second injection, consisting of pure toxin 0.10 c.c., is used, 

 and every four or five days thereafter injections are re- 

 peated in progressively larger doses until the animal is 

 able to withstand doses of from 400 to 500 c.c. of toxin. 

 During those injections the animal may show decided local 

 effects, such as swelling and oedema at the point of inocula- 

 tion, but no very marked constitutional disturbances. During 

 the progress of this immunization, at intervals, by punctur- 

 ing of the jugular vein with a sterilized trocar, some blood 

 is withdrawn from the animal and its serum tested as to its 

 antitoxic value, and when the same is found sufficient the 

 toxin injections are repeated at longer intervals to maintain 

 the antitoxic property of the animal's serum, and the next 

 process is begun. 



Standardization. A large quantity of blood, 4 or 5 liters, 

 is extracted from the immunized horse at one time, collected 

 in well-sterilized vessels, and allowed to clot in an ice-chest 

 for two or three days, after which the clear serum is pipetted 

 off and stored in sterilized flasks, the antiseptic strength of 

 the serum being properly labelled on each flask. This anti- 

 toxin power, called units, is estimated as follows : 



Ten times a fatal dose of a toxin, that is known to kill a 

 250-gram guinea-pig within three days, is mixed with differ- 

 ent quantities of the serum to be tested, say, 0.10, 0.01, 

 0.001 c.c., and these mixtures injected into different guinea- 

 pigs, Nos. 1, 2, and 3, respectively. Should guinea-pig No. 1 

 survive the mixed injection, and guinea-pigs Nos. 2 and 3 

 die. the antitoxin is said to contain 10 times 10 units in 1 c.c. ; 

 that is, it is an antitoxin of 100-unit power. Should guinea- 



