196 Journal of Agricultural Research voi. xii, no. 4 



salt. Since strain B normally produces a good pigment, the influence 

 of nitrate on this strain was not very marked. The relation of nitrates 

 to pigment formation will be taken up later (pp. 203-205). 



From the results of the experiments with agar films containing various 

 amounts of nitrate, it seems apparent that potassium and sodium nitrates 

 in amounts of 50 and 100 mgm. of nitrate in 100 c. c. of medium cause a 

 small increase in the amount of nitrogen fixed. However, this increase 

 in fixation is not at all parallel with the increase in number of Azoto- 

 bacter caused by nitrates in sterilized soil. 



It may be concluded that an increase in the number of Azotobacter in 

 sterilized soil as a result of nitrate stimulation does not mean a corre- 

 sponding increase in nitrogen fixation on agar films. 



Soil cultures. — ^The conditions obtaining in these experiments were 

 strictly comparable with those heretofore cited dealing with the influence 

 of nitrates on Azotobacter in sterilized soil (pp. 187-193). 



The fixation of nitrogen was studied in pure culture in sterilized soil 

 and in unsterilized soil. One hundred and fifty gm. of soil (dry weight) 

 were weighed into i -liter Erlenmeyer flasks, nitrates were added in vary- 

 ing amounts from 10 to 200 mgm., and i per cent of mannit was also 

 added. Triplicate flasks were prepared for each amount of nitrate studied. 

 The moisture content was raised to approximately 18 per cent and the 

 flasks allowed to remain at room temperature for one day. The con- 

 tents were then thoroughly mixed and a fine crumb structure produced. 

 The flasks for the experiments with pure cultures in sterilized soil were 

 immediately sterilized at 15 pounds' pressure for three hours. After 

 cooling, two of each set were inoculated with 5 c. c. of a suspension of 

 Azotobacter (strain A) in sterile distilled water. The remaining flask of 

 each set was not inoculated, but was incubated at 28° C. with the inocu- 

 lated flasks. The moisture lost by evaporation was replaced from time 

 to time by the addition of sterile distilled water. At the end of the incu- 

 bation period the soil was removed and spread out in thin layers and 

 allowed to dry. It was then thoroughly ground in a porcelain-ball mill 

 for one hour. At the end of this time all of the soil passed through a 

 loo-mesh sieve. 



Soil cultures used in the study of the effect of nitrates on nitrogen 

 fixation in unsterilized soil were prepared in a similar manner, except 

 that the flasks were not sterilized. Previous to incubation a small 

 inoculum of Azotobacter (strain A) was added to insure the presence of 

 the nitrogen-fixing organism in the soil cultures. The proper moisture 

 content was maintained in the same manner as in the case of the pure 

 cultures in sterilized soil and the incubation period was the same for both. 

 The results are given in Tables X, XI, XII, and XIII. 



