436 IMMUNITY. 



Second, the estimation of the power of the toxine. Third, 

 the gradual development of a high degree of resistance in 

 a suitable animal by gradually increasing doses of the 

 toxine. Fourth, the estimation from time to time of the 

 antitoxic power of the serum of the animal thus treated. 



i. Preparation of the Toxine. This implies selection of 

 a virulent culture, and also growing it in conditions suit- 

 able for the highest development of toxine. In the case of 

 diphtheria a virulent culture (its power having been previ- 

 ously tested by inoculation in guinea-pigs) is grown in large 

 vessels containing bouillon in a comparatively thin layer. 

 Some authorities consider that the highest development of 

 toxine takes place when a current of sterile moist air is 

 made to pass over the surface of the medium. This can be 

 readily done by having two apertures in the vessel, one of 

 which is connected with a tube leading to a water-exhaust 

 apparatus. The air, which enters by the other aperture, is 

 first made to pass through a vessel of water and then through 

 a glass tube plugged with sterile cotton wool. In this- way 

 a constant current of air may be maintained. The air 

 ought to pass slowly through the vessels, and its rate may 

 be regulated by having a screw -clip on the exit tube. 

 Some observers, however, find that an equally powerful 

 toxine is produced without this arrangement for aeration, 

 if the culture is made to grow as a pellicle on the 

 surface of the bouillon, in which case the growth should not 

 be disturbed till the toxine is fully formed. In order that 

 a powerful toxine may be formed, it is essential that the 

 bouillon be practically free from glucose. After the maxi- 

 mum toxicity is reached, usually in three or four weeks, 

 the culture is filtered through a Chamberland filter. In- 

 stead of bouillon, fluid blood serum of the ox or horse may 

 be used, in which the toxine is said to form more quickly. 

 The process of filtration of serum, however, is more diffi- 

 cult. Mixtures of bouillon and serum are also employed. 



In the case of tetanus, the growth takes place in glucose 

 bouillon under an atmosphere of hydrogen (vide p. 67), and 

 the culture is afterwards filtered in the same manner. 



