THE ANTHRAX PROTEIN 199 



In all our work the anthrax bacillus has been grown in 

 Roux flasks, as we have not dared try it in the large tanks, 

 consequently the amount of cellular substance obtained 

 has been small. This work was begun in 1900 and carried 

 on intermittently. We have felt it desirable to exercise 

 great care in handling quantities of the anthrax bacillus. 

 We have always opened the flasks and removed the growth 

 over a shallow tray containing some powerful germicide. 

 We will make a few extracts from our protocols. In May, 

 1900, the growth was removed from twelve Roux flasks, 

 placed in one liter of 1 per cent, sulphuric acid, and kept 

 in the incubator at 37 for twenty-four hours. At the 

 expiration of this time cultures showed the bacillus still 

 alive. The suspension was then placed in the autoclave 

 and heated at 100 for thirty minutes. Cultures now showed 

 that the bacillus had been killed. The suspension was 

 passed through a Chamberland filter with the aid of a 

 pump. The clear filtrate was poured drop by drop into 

 twice its volume of absolute alcohol. A finely flocculent, 

 white precipitate formed, was collected on a hard paper, 

 washed with alcohol until the filtrate was no longer acid, 

 and dried in vacuo over potash. This substance is not 

 colored with nitric acid and heat, but on the addition of 

 ammonia the characteristic orange of the xanthoproteic 

 test is developed beautifully. It does not give the biuret 

 and other protein tests. Later an unweighed portion of 

 this substance was dissolved in water and injected intra- 

 abdominally in a guinea-pig. Twelve hours later the 

 animal was found dead. The heart was in partial diastole 

 with clots in both auricles and ventricles. The peritoneal 

 cavity contained a few cubic centimeters of a clear fluid. 



Two days later a guinea-pig received intra-abdominally 

 60 mg. of the alcoholic precipitate. When last seen that 

 night, five hours after the injection, the animal was dying. 

 The next morning it was posted and the condition described 

 above was found. The heart's blood was found to be sterile. 

 This experiment was repeated a number of times, with 

 similar results. 



