Studies in Fat Metabolism of Timothy Grass Bacillus. 271 



traces, but this could not be established with certainty owing to the rapid 

 darkening of the solution by easily charred matter. No solid material was 

 obtained from the Soxhlet extractor. 



These results were confirmed by two other experiments, slight variations 

 only being obtained in the amount of volatile acids distilled over. It seemed 

 therefore that, under the conditions prevailing in these experiments, inter- 

 mediate products in the breakdown of glucose accumulate only in such traces 

 that attempts at identification are hopeless. 



Experiment 3. — An Attempt to Obtain Fermentation apart from the Growth of 



the Organism. 



Six Roux bottles were filled with a medium containing the usual inorganic 

 salts and 1 per cent, glucose. From six otherwise exactly similar bottles the 

 ammonium phosphate was omitted. These twelve bottles were sown more 

 thickly than usual with the emulsion of the bacillus. At the end of fourteeen 

 days the sugar in the bottles containing no ammonia was estimated and 

 found completely untouched, whilst that in the control bottles had dis- 

 appeared in the normal manner. It thus appears that under these conditions 

 fermentation does not occur apart from growth, which in its turn is dependent 

 on a supply of nitrogen. 



Direct attempts at isolation of intermediate products having failed, 

 indirect evidence was sought by growing the organism on possible inter- 

 mediate products in the breakdown of the sugar molecule, in order to 

 ascertain : (1) whether the organism was capable of utilising and growing 

 on the intermediate products ; (2) if growth took place, how the forma- 

 tion of fat was affected. 



The formation of protein nitrogen (precipitated by colloidal iron) was 

 taken as a criterion of growth, the lipoids corresponding to the nitrogen 

 synthesised at each period being separately estimated as previously described. 

 At the point of maximum lipoid formation (e.g., point P, curve 1), the 

 growth of the organism was considered to have attained its maximum, and 

 at that point the lipoid formation was compared with the protein nitrogen 

 synthesised. 



Experiment 4.—G-roivth on, anal Utilisation of, Lactic Acid. 



A series of Eoux bottles was started in the usual way containing the 

 usual inorganic salts and also 068 per cent, lactic acid (dl) added as sodium 

 lactate. Sample bottles were withdrawn at intervals and analyses of the 

 organism carried out as in Experiment 1. Lactic acid was estimated in 



