vii.] FIXATION WITHOUT LEGUMINOUS PLANTS 187 



when grown under anaerobic conditions and supplied 

 with soluble carbohydrate, breaks the latter down with 

 the formation of butyric and other acids, and at the same 

 time draws some of the gaseous nitrogen present into com- 

 bination. This particular organism Clostridium Pastori- 

 anum is very widely diffused and can readily be isolated 

 from pond mud and similar material, where organic matter 

 is decaying under comparatively anaerobic conditions. 

 The extent of the nitrogen fixation is, however, small ; 

 in the laboratory not more than 2 to 3 mg. of nitrogen 

 are brought into combination for each gram of carbo- 

 hydrate oxidised. By far the most effective of the 

 nitrogen-fixing bacteria that are free in the soil is a large 

 organism, named by its discoverer, Beijerinck, Azotobacter 

 chroococcum. It may be easily isolated from most soils 

 by adding a small portion of soil to 50 c.c. of a culture 

 medium containing per litre 10 grams of mannite or 

 glucose, 02 gram each of potassium phosphate, mag- 

 nesium sulphate, and sodium chloride, and 01 gram of 

 calcium sulphate, half a gram of calcium carbonate being 

 also added to each flask. The solution and its flask 

 and plug of cotton wool are previously sterilised by heat. 

 After inoculation, the flask is placed in an incubator, 

 and after a week's time a considerable fermentation will 

 be observed to have taken place, attended by the 

 evolution of gas and the formation of a brown scum upon 

 the surface. By making a subculture in a similar 

 medium, inoculated with a trace of the brown scum, a 

 fairly pure growth of the Azotobacter can be obtained 

 for examination, or the amount of nitrogen fixed may 

 be determined by Kjeldahling the contents of the 

 flask. 



Azotobacter chroococcum is a large oval organism, 4 to 

 5 jul in length and 3 fj. in width, which differs from most 

 bacteria in containing glycogen, so that it stains a deep 



