412 Micro-Urease Method for Determhiation of Urea [April-July 



extract and a few drops of toluene, and the tube placed in an 

 incubator at 37° C. The resultant ammonia was then determined 

 by the aspiration method. 



When this method was put into practice the authors were sur- 

 prised to find that the hydrolysis of the urea was complete in less 

 than 5 min. when the mixture was heated to 30-40° C. Tubes con- 

 taining the mixture were kept for various lengths of time at room 

 temperature; some also in a cold room at 0° C. The latter, after a 

 period of 24 hr., gave results that were identical with those for the 

 incubated mixtures, but yielded higher values than did those kept at 

 room temperature (for 1-2 hr.), so there must have been consider- 

 able enzyme action under even these conditions. The incubator was, 

 therefore, dispensed with and the tubes merely heated to 40-50° C. 

 by immersing them in a pan of warm water at the time of aspiration. 

 Preliminary extraction of the flour with ether was of no advantage 

 nor was anything gained by using warm water, The period of 

 soaking could not be shortened without reducing the strength of the 

 extract. 



The most troublesome feature of the work was the persistent 

 f rothing of the mixture during its aspiration. Toluene and pe- 

 troleum oil were of no avail. Alcohol prevented the foaming 

 for a time, but not permanently. The addition of a small piece 

 of camphor reduced the surface tension sufficiently to permit 

 aeration to proceed in a very satisfactory manner when tall cylin- 

 ders and a vigorous air current were employed, but in the 

 micro apparatus, this substance was sometimes carried over and 

 interfered with the determination. The heavier paraffin oils were 

 efficient, but at best rather disagreeable to use. The amyl and octyl 

 alcohols proved very satisfactory, and in the micro apparatus, 0.5 c.c. 

 of either of these is now used. 



The surprising activity of the extract naturally suggested the 

 simplification of the work by introducing the bean flour directly into 

 the Solution to be examined for its urea content. This part of the 

 work was undertaken with some misgivings and first tried on urine 

 samples in Folin's macro ammonia apparatus. The results were 

 very gratifying and repeated in the micro apparatus with entire 

 success. 



