* METHOD FOR PRODUCTION OF TETANUS TOXIN 335 



expelled by heating in the Arnold for fifteen to twenty minutes, 

 and which have been cooled down to about 50°C. These tubes 

 are incubated for 24 hours and the next day, two freshly heated 

 tubes of glucose broth are inoculated with 5 cc. of the glucose 

 broth cultures planted the previous day. On the third day, 

 determine the number of flasks that are to be inoculated and 

 inoculate as many freshly heated glucose broth tubes from the 

 second glucose broth generation as there are flasks. Ander- 

 son calls for at least six or seven generations in the glucose broth 

 before the inoculation of the toxin broth, but at the Research 

 Laboratory it has been found that three generations or rven 

 two if need be, are sufficient for obtaining a toxicity of 1-25,000. 



INOCULATION OF TOXIN BROTH 



After the second sterilization in the Arnold, the flasks are 

 ready for inoculation. The broth may be cooled down to 55°- 

 60°C. by allowing the flasks to stand at room temperature, or 

 in a more rapid way by placing the hot flasks in a large sink, 

 to which cool, and then cold, water is added until the lower 

 portions of the flasks are covered. When the bottoms of the 

 flasks are cool to the hand, the portions above the water being 

 still very hot, the inoculation may be made as follows: 



The plugs are carefully removed, the necks flamed and the 

 plugs replaced. In a similar way, the mouths of the culture 

 tubes are steriUzed and then, partly removing the plug of a 

 flask, the contents of a potato tube is poured rapidly into a 

 flask. If one prefers, the broth culture may be transferred 

 by using a pipette, but the former method has been used here 

 without subsequent contamination and found very satisfactory. 

 After inoculation, the flasks are incubated for fifteen days at 

 36°-37°C., care being taken to exclude all light from them. 



The flasks at the end of 24-48 hours show a diffuse cloudi- 

 ness with the formation of gas bubbles on the surface of the 

 broth. Toward the end of two weeks, the gas bubbles usually 

 disappear, while the cloudiness persists and a light precipitate 

 forms at the bottoms of the flasks. If it is not convenient 



