1 82 FIXATION OR ASSIMILATION OF NITROGEN 



(ii) Run under the cover slip a drop of iodine solution, or 

 mount a small piece of the scum in the latter. Observe the 

 yellow colour of the Azotobacter cells, and look 'out for violet- 

 stained Clostridium cells, which are generally present, 

 (iii) Inoculate a plate of the following agar medium : 

 Agar . 2 gr. 



Grape sugar . . . . . . .2 gr. 



Di-potassium phosphate . . . . .2 gr. 



Water. . ... . . . 100 c.c. 



The colonies of Azotobacter are at first colourless and shining ; 

 later they become elevated and yellow, and finally, in five or six 

 days, turn a deep brown colour. 



If no Azotobacter organisms are seen on first examination of 

 the scum as above, add i c.c. of the liquid from the mixed soil 

 and culture solution to 50 c.c. of the nutrient solution, and 

 incubate for two or three days at 28 C. Another grape-sugar 

 plate can then be inoculated with a drop of this culture, and the 

 Azotobacter colonies afterwards obtained. 



3. It is hardly likely that the only organisms capable of 

 assimilating the free nitrogen of the air are those already 

 mentioned belonging to the genera Clostridium and Azoto- 

 bacter, although Winogradsky and others obtained negative 

 results with many bacteria which they specially investi- 

 gated from this point of view. There is indeed some 

 evidence that a number of species of soil organisms are 

 able to effect the synthesis of nitrogen compounds from 

 the free nitrogen of the air in slight degree, but the diffi- 

 culties connected with the investigation of the problem 

 are great. In most cases the increase in nitrogen con- 

 tent due to nitrogen fixation is necessarily very small, 

 and frequently lies within the limits of experimental 

 error, so that much caution must be exercised in accept- 

 ing or rejecting results. 



