Studies in Fat Metabolism of Timothy Grass Bacillus. 265 



Estimation of Sunthesised Protein. 



The contents of one Eoux bottle were washed quantitatively into a beaker 

 and made slightly acid with acetic acid ; 20 c.c. of " dialysed iron " were 

 added and the whole heated on a water-bath, filtered whilst hot, and washed 

 with hot water till free from phosphate. The precipitate and paper were 

 then transferred to a Kjeldahl incinerating flask and the nitrogen estimated 

 in the usual manner. The results are calculated to represent grm. of 

 nitrogen in 100 c.c. medium, i.e., two-thirds of one Eoux bottle. 



Estimation of Lipoids of Bacillus. 



(1) " Fat." — The contents of one Roux bottle (150 c.c.) were transferred 

 quantitatively to a 250-c.c. measuring flask, and made up to volume. They 

 were then filtered through a dry pleated filter into a dry flask, and the filtrate 

 was set aside for estimation of the constituents of the medium. The 

 precipitate, consisting of the insoluble constituents of the medium together 

 with the agglutinated bacteria, was transferred on the filter paper to a dish 

 and dried in an atmosphere of nitrogen at about 97°. The final stages of the 

 drying were carried out in vacuo. The filter paper and dried contents were 

 then transferred to a Soxhlet extractor and extracted with ether (purified 

 from alcohol and aldehydic substances). The extraction flasks employed, of 

 about 150 c.c. capacity and about 35 grm. in weight, were fitted to the 

 extractor with ground glass joints, in order to get good quantitative results. 

 After about 8 hours' extraction, the ether was distilled off and the flask dried 

 in nitrogen and later in vacuo at 97°. The extract thus obtained represents 

 the " fat " fraction described above. 



(2) "Phosphatide." — The Soxhlet thimble and contents were then trans- 

 ferred to a wide-mouthed extraction flask fitted with a condenser. To this 

 about 30 c.c. of alcohol (previously distilled from caustic soda) were added ; 

 the alcohol was boiled on a water-bath for 20 minutes and then distilled off 

 and the flask and contents dried in an atmosphere of nitrogen. The small 

 amount of material which had dissolved out of the thimble was then washed 

 back with ether and the thimble and contents were returned to the Soxhlet 

 extractor; a second extraction was then made with ether and the flask dried 

 and weighed as before. This extract is the "phosphatide " fraction referred 

 to above. 



No detailed chemical examination of these fractions has been made, but it 

 has been shown that the " fat " fraction contains no phosphorus, whilst the 

 I phosphatide " fraction contains a small but fairly constant quantity 

 (estimated by Neuman's method). Both fractions were tested for nitrogen by 

 Kjeldahl's method : traces (below 1 per cent.) were found in both fractions. 



vol. xcni. — B. u 



