142 LIVING ORGANISMS OF THE SOIL [chap. 



one or two of the flasks the carbonate of lime is omitted. 

 If such a non-nitrogenous medium is inoculated with a 

 mixture of organisms, only those which are able to draw 

 upon the atmospheric nitrogen can live and multiply, 

 because all the usual bacteria depending on a supply of 

 combined nitrogen are starved out and must remain 

 dormant until their necessary food comes along. The 

 series of prepared flasks are then inoculated with about 

 a gramme of soil each, and put away in a warm dark 

 place to incubate as usual. After a week or ten days' 

 time the flasks are examined ; wherever the Azotobacter 

 was present in the soil the liquid in the flask will be 

 covered with a dark brown skin, held up on the surface 

 by a number of bubbles of some gas which is evidently 

 being freely developed. The flasks containing no 

 carbonate of lime will often show no brown scum nor 

 formation of gas, unless the soil sample added is itself 

 rich in carbonate of lime. An analysis of the contents 

 of the flasks will show that nitrogen has been gathered 

 — often as much as 8 milligrams of nitrogen are fixed 

 for each gramme of sugar in the original solution. The 

 glucose that has been added is necessary, not merely as 

 food for the Azotobacter, but as material which can be 

 oxidised or burnt up to supply the energy required to 

 bring the nitrogen into combination. Almost any 

 carbohydrate will serve the purpose, but glucose and 

 mannite appear to be most readily oxidised. The 

 Azotobacter is really a very powerful oxidising agent ; 

 the gas which is given off" during the first growth in the 

 culture flasks is carbon dioxide produced in this way. 

 During, or by means of the rapid process of oxidation, 

 some of the nitrogen which is present is also drawn into 

 combination. Perhaps an even more satisfactory method 

 of demonstrating the fixation — more satisfactory because 

 it follows the sequence of changes taking place in the 



