FIXATION OF NITROGEN BY FUNGI 



193 



of digestion, blown over and Nesslerized, gave the same color as 

 when used with all the other substances. 



In the above case the contamination was promptly discovered 

 as due to a bottle of acid, left by someone in the place of the 

 one which had been specially tested for this work. A new supply 

 of acid which gave no color on Nesslerization was then used and 



TABLE 10 



Experiment I. Cultures of Azotobacter in Ashby's solution, containing 2% mannite 

 and 0.05 mgm. of nitrogen as {NHi)2S0i. Each tube contained 5 cc. of culture 

 solution and 0.05 mgm. N. 



Ashby's solution is made up as follows: Mannite 12 to 20 gm., Monopotassium 

 phosphate 0.2 gm., Magnesium sulphate cryst. 0.2 gm./ Sodium chloride 0.2 gm., 

 Calcium sulphate 0.1 gm., Distilled water 1 liter. (Jour. Agric. Sci. 3: 38, 1907.) 



The above figures show approximately the average of four analyses. For 

 want of time they were not accurately measured but there was a liberal margin 

 over the above gain. These are the only cultures of the entire nine sets that 

 show a uniform and decisive gain. This gain, though relatively large (100%) 

 is small when considered in terms of milligrams; but by the colorimetric method 

 is quite decisive and beyond doubt as to the qualitative character of the result. 



It should be noted in this connection that the above experiment is especially 

 suited to show that the entire micro-method might be well adapted to this kind 

 of work in general, where unusually small margins are to be taken into consider- 

 ation. It has in it all the possibilities of speed, accuracy, and general conveni- 

 ence. 



I have not seen, except in one or two cases, any mention of the colorimetric 

 method used in this connection, and none as to such use of the micro-method. 

 Pennington states that he made use of the color method in a few cases where 

 verj^ small amounts of nitrogen were to be measured and it has been used in the 

 direct measurement of nitrites and nitrates. 



the color given by the blank spore tests was considered so small 

 as to be negUgible, as already reported. It would be very neces- 

 sary to guard against this source of error in either method. The 

 HCl used in the receiving flasks should also be guarded and 

 tested from time to time. There is less danger from the dis- 

 tilled water used, as the Nessler's solution is constantly diluted 



