Nitrogen Metabolism of Bacteria 31 



a method by which all interfering substances were excluded. The 

 Wiechowski" method, for which great accuracy is claimed in the deter- 

 mination of allantoin in urine, was employed repeatedly, but without 

 success. 



This method consists in precipitation by phosphotungstic acid, the removal of 

 the excess of this reagent by means of lead oxid and acetic acid, the elimination 

 of the chlorid with silver acetate, and the final precipitation of all heavy metals 

 with hydrogen sulfid. After removal of the latter by aeration the solution is 

 made alkaline with magnesium oxid and the allantoin precipitated by means of 

 a solution consisting of 20% sodium acetate and 1% mercuric acetate. 



It was found that in all bacterial cultures on peptone media a precipitate was 

 obtained at this point which had a varying, in some cases high, total-nitrogen 

 content. Moreover, the sterile control usually gave a precipitate here also, 

 which varied from a slight opalescence to an appreciable deposit. It was not 

 possible to prepare the characteristic crystals of allantoin from any of these 

 precipitates. It is probable that the latter consisted, in the most part, of amino- 

 acids which escaped precipitation by the phosphotungstic acid. Levene and 

 Beatty" showed that many of these acids are precipitated completely by this 

 reagent only when it is present in very high concentration. Such concentrations 

 would be entirely impractical in the present connection. 



Attempts to show that uric acid is decomposed by bacteria with the forma- 

 tion of allantoin were unsuccessful for the same reasons as have just been 

 outlined. A medium having asparagin as the nitrogenous base was also used 

 in these experiments. The Wiechowski method was inapplicable here also, since 

 asparagin, not being precipitated by phosphotungstic acid, is brought down with 

 the allantoin by the sodium-acetate mercuric-acetate reagent. If allantoin was 

 ever present in this precipitate, it was there in such small quantities that it could 

 not be detected in the presence of asparagin. 



This inability to separate allantoin from asparagin would seem to render 

 the Wiechowski method inapplicable also to the determination of allantoin in 

 plants. In fact, it has been shown recently in this laboratory that a very large 

 portion of the nitrogen determined as allantoin in plant tissue by this method 

 is in reality asparagin, where this substance is present in considerable amounts.* 



The problem of the formation of allantoin by bacteria seems to 

 depend for its solution on the perfection of the methods for the deter- 

 mination of this compound and its separation from interfering sub- 

 stances. 



Creatin and Creatinin. — Most of the peptone cultures or peptone 

 solutions reported in the first part of this paper were tested for creatin 

 and creatinin. In general, the results were in close agreement with 

 those of Fitzgerald and Schmidt. ^^ That is, only B. proteus was found 

 to form appreciable amounts of creatinin on solutions of peptone alone. 

 It was found however that in those cultures containing glucose, a test 

 for this substance and for creatin gave positive results in a large num- 



" Neubauer's Analyse des Harns, 1913, 2, p. 1076. 



"« Levene and Beatty, Ztschr. f. physiol. ' Chem., 1906, 47, p. 149. 



* Yet unpublished thesis of G. C. Swan, Stanford University, May, 1915. 



