96 PROTEIN POISONS 



in this menstruum than in water, it was decided to attempt 

 to disrupt the cell protein with a solution of alkali in absolute 

 alcohol. Another idea also acted as a determining factor 

 in attempting this method of hydrolysis, and in fact it 

 was at that time the dominating factor. The effect of the 

 poisonous group on animals so closely resembles that of 

 neurin that it was thought that the two might be identical, 

 or at least that the poisonous body might contain neurin. 

 Knowing that neurin can be heated without decomposition 

 in alkaline alcohol was, therefore, a reason for trying this 

 method. 



7. Previous experiments had demonstrated the advantage 

 of extracting the cell substance thoroughly with alcohol and 

 ether before submitting it to hydrolysis. This frees the 

 material from fat, wax, and other substances soluble in 

 alcohol or ether, and since it had been shown that these 

 are no part of the cell protein it is beneficial to get rid of 

 them in toto before hydrolysis is attempted. 



The following preliminary trials were made by Vaughan 

 and Wheeler (in the fall of 1903) in order to compare 

 hydrolysis with aqueous and alcoholic solutions of alkali. 



Two samples, of 10 grams each, of the cellular substance 

 of the colon bacillus were taken. This material had pre- 

 viously been thoroughly extracted with alcohol and ether. 

 One sample was mixed with 250 c.c. of a 1 per cent, aqueous 

 solution of sodium hydroxide and the other with the same 

 volume of an absolute alcohol solution of the same substance 

 in the same strength. These mixtures were heated in 

 flasks,' fitted with reflux condensers, for one hour on the 

 water-bath. Ten cubic centimeters of the clear filtrate 

 from each was evaporated, the aqueous solution to 5 c.c. 

 and the alcoholic to dryness, and then taken up in 5 c.c. 

 of water. Each was carefully neutralized with dilute 

 hydrochloric acid and injected into the abdominal cavity 

 of a guinea-pig. Both animals developed in a characteristic 

 manner the first and second stages of poisoning with the 

 split product, but neither died. This experiment showed 

 that the poison was present in both extracts, and, so far 



