182 CHARLES OSCAR CHAMBERS 



the determination of the medium before growth. After growth, 

 however, the carbohydrate has been transformed into forms 

 that are more easily digested ; and practice at length gave suffi- 

 cient mastery of even the raw unseeded controls so that the 

 Kjeldahl method was abandoned and the determinations were 

 carried on by the micro-method exclusively. 



The Folin, or micro-method, combines the essential features 

 of the Kjeldahl and colorimetric methods reduced to micro- 

 dimensions but with the special feature of blowing the ammonia 

 over from the digestion tubes to the reception flasks and the 

 values, read by a DeBoscq calorimeter. This seems to afford 

 the highest degree of speed and accuracy when small amounts 

 of nitrogen are to be determined. For details the reader is 

 referred to the original sources as given by Folin. ^^ 



When sufficient accuracy had been attained by running a 

 large number of blanks and checking by Kjeldahl determinations 

 a number of Aspergillus cultures were prepared in large test- 

 tubes (25 X 200 mm.) and allowed to grow for varying periods 

 of time, from two to four weeks. They were then digested and 

 determined by the micro-method. Cultures were grown in 

 triplicates prepared as follows: 



Dox's^*' nutrient solution was prepared with 2 to 3% dextrose 

 and 5 cc. pipetted into each tube. To one set, A, was added 

 2 mg. of nitrogen in the form of (NH4)2S04; to another set, B, 

 1 mg., and to a third set, C, no nitrogen was added. 



The solution of (HN4)2S04 had been carefully prepared from 

 very pure stock and standardized; first by weight and then by 

 volumetric method, taking a standard that would give 1 mg. of 

 nitrogen per cubic centimeter of solution. This was further 

 standardized by running samples through the Kjeldahl process 

 and titrating against very accurately prepared solutions of 



1^ Folin, O. and Farmer, C. J., A new method for the determination of total 

 nitrogen in urine. Jour, of Biol. Chem. U: 493-501. 1912. 



Folin, and Denis, W., An apparatus for the absorption of fumes. Jour. Biol. 

 Chem. 11: 503-505. 1912. 



" Dox, A. W., The intracellular enzymes of Penicillium and Aspergillus. 

 U. S. Bur. An. Ind. Bui. 120: 37. 1910. 



