Bacillus coll. The inoculations were made by the use of 

 a hypodermic syringe and needle. The broth culture was 

 aspirated into a sterile syringe; then, after inserting 

 the needle diagonally through the paper caps, a few drops 

 were forced into the flasks. This method has, to commend 

 it, the advantage that the flasks are never opened for 

 inoculation after having been sterilized. The inoculated 

 flasks were then incubated at 37C. for about ten days. 



In order to separate the bacteria from the fluid, 

 the contents of the flasks were run through a Sharpies 

 laboratory "supercentrifuge". This machine is very simi- 

 lar to the ordinary cream separator in construction and 

 mechanism, but is run by steam or compressed air. Suffi- 

 cient force is obtained to completely separate out all 

 particles in suspension. 



This material was washed with sterile physiological 

 sodium chloride solution and recentrifuged. The sediment 

 consists of bacteria with precipitated salts, sulphides, 

 and perhaps other amorphous material. Most of these 

 foreign substances are easily removed by filtration 

 through cotton. 



The remaining bacteria were now subjected to saponi- 

 fication in potassium alcoholate, at a temperature of 

 100C. for one hour. The resultant material was then 

 acidified with dilute hydrochloric acid. It was expected 

 that this procedure would yield a definite layer of fatty 

 acids which could be removed, but such proved not to be 

 the case. Instead, there was a mass of solid material, 

 including fatty acids, proteins and salts, in a state of 

 very fine suspension, which could not be separated by 

 gravity or by centrifugal force at three thousand revolu- 

 tions per minute. It was then decided to extract the 

 fatty acids with ether. After shaking up with ether it 

 was found that only about ten percent of the ether could 

 be recovered and that only when comparatively large 

 amounts, were used. Most of the ether became a part of 

 the suspensoid mass. The entire mass was then trans- 

 ferred to a ten- inch porcelain evaporating dish, and 

 evaporated at 37C. for several days, until the moisture 

 content was very low (too low to support the previous 

 suspension). The residual mass was then shaken up with 

 ether and the latter separated, removed and evaporated 

 at room temperature. The residue consisted of a very 

 minute amount of crystalline and amorphous material, 

 presumably fatty acids. The amount was considered too 

 small to work with. 



