DISEASE, INFECTION, AND EESISTANCE 285 



diphtheria . bacteria grow as a film over the surface and 

 within a few days produce the maximum amount of toxin. 

 Some substance, such as cresol, which will kill the diph- 

 theria bacilli without injuring the toxin, is added and the 

 broth filtered through a suitable porcelain filter. This broth 

 containing the toxin is now ready for standardization. In- 

 asmuch as no chemical test has been devised for the detec- 

 tion or estimation of a toxin it is necessary to standardize 

 by injection of the material into a suitable animal. The 

 animal of choice is the guinea pig. The unit of toxicity is 

 determined by injecting varying amounts of the toxic broth 

 into a series of guinea pigs weighing about 250 grams each. 

 Those animals which die in just three days have received a 

 minimum lethal (fatal) dose of the toxin. This unit of 

 toxicity is usually abbreviated as the M. L. D. dose. A 

 broth having a satisfactory content of toxin frequently will 

 show one thousand or more M. L. D. per cubic centimeter, 

 that is, each c.c. will contain enough poison to kill one 

 thousand guinea pigs weighing 250 grams each. 



Injection of the Toxin. Several species of animals may 

 be used in the production of diphtheria antitoxin. The 

 horse has proved to be the most suitable from the commer- 

 cial standpoint because it will yield a large quantity of 

 serum at one bleeding and usually will develop an antitoxin 

 of relatively high potency. The horses used must first be 

 examined to make sure that they are free from disease and 

 in good physical condition. They are then injected sub- 

 cutaneously with a small amount of diphtheria toxin. This 

 may cause some local swelling and fever. This soon disap- 

 pears, however, and the animal may then receive a second 

 dose of the toxin. At intervals larger and larger doses of 

 toxin are injected until it is evident that the animal has 

 reached a high degree of immunity and the blood contains 

 considerable quantities of antitoxin. A small sample of 

 blood is then withdrawn from the horse and tested to deter- 



