19 



are essential for sensitization. Either the fats may 

 split off a molecule that is toxic, thus giving rise to 

 the reaction according to Vaughn 1 s idea, or the reaction 

 may be due to variations in surface energy and molecular 

 attraction. 



So far, then, we have prepared an antigen from the 

 fats of Bacillus coli and have demonstrated the ability 

 of this antigen to cause the production of various sero- 

 logical reactions when injected into suitable animals. 

 Our interest now turns toward the chemical composition of 

 the antigen and the attempt is made to analyse the fatty 

 mass . 



In the previous preparation of the fatty antigen 

 about 0.4 gram of fat was obtained from thirty litres of 

 broth culture. In order to obtain a larger amount of 

 material two hundred litres of broth were made and inocu- 

 lated as before. The bacilli were collected in the same 

 manner. The bacteria mass was then transferred to a 

 litre flask and covered with ether. The flask was then 

 fitted with a reflux condenser and heated to 50C on an 

 automatic electric water-bath. The heat was applied con- 

 tinuously for a period of forty-eight hours. At the end 

 of this time the flask and contents were cooled and the 

 ether was pipetted off into another flask. This latter 

 flask was tightly stopped and placed in the icebox. The 

 bacterial residue was extracted again in the same manner 

 and the ether extract was added to the first lot. A 

 total of five extractions was made. The mixed ether from 

 the five extractions was then evaporated under reduced 

 pressure (obtained by attaching an ordinary filter pump), 

 at a temperature of 25C. The remaining fatty mass was 

 then treated with potassium alcoholate. The flask was 

 fitted with a reflux condenser and the fats were saponi- 

 fied at a temperature of 90C. The flask and contents 

 were cooled and acidified with hydrochloric acid. The 

 flask was then attached to the reflux condenser and the 

 fluid was evaporated at 70C under reduced pressure. The 

 concentrated mass of fatty acids was then taken up with 

 ether. The ethereal solution, containing the fatty 

 acids, was pipetted off and the ether evaporated at room 

 temperature under reduced pressure. The mass of residual 

 fatty acids weighed 2.873 grams. A portion of these 

 acids was fluid at 30C, but the remainder were solid up 

 to about 65C. The acids were then subjected to distil- 

 lation in steam. The distillate was treated with solid 

 sodium chloride ,and then shaken up with ether. The ether 

 layer was removed and evaporated in vacuo at room temper- 

 ature. There was a very slight trace of residue, too 



