1 86 Journal of Agricultural Research voi. xii, no. 4 



From a practical standpoint the relation of nitrates to the nitrogen- 

 assimilating organisms of the soil is of importance. Hence, it was 

 arranged to study the effect of nitrates on soil bacteria, especially those 

 forms concerned with the fixation of atmospheric nitrogen. The work 

 naturally falls into two rather distinct lines of investigation. First, the 

 influence of nitrates on Azotobacter was determined. Here studies were 

 made of the effect of nitrates on the growth of the organism in soil and 

 also the effect of these salts on the nitrogen-fixing property of these 

 bacteria. The action of Azotobacter on nitrates in solution, the relation 

 of nitrates to pigment production and to the formation of volutin bodies 

 were studied. Second, the influence of nitrates on the growth of Bacillus 

 radicicola in soil was studied. The action of B. radicicola on nitrates in 

 solution and the possible nitrogen-assimilating properties of the legume 

 in the presence of nitrates were investigated. Also the influence of 

 nitrates on gum production was determined. The latter part of the 

 investigations included a study of the relation of nitrates to nodule 

 formation on alfalfa. 



METHODS USED IN EXPERIMENTS 



Nitrates were determined by the reduction method with Devarda's 

 alloy and also by the phenolsulphonic acid (colorimetric) method. 



The total nitrogen content of all samples was determined by the 

 modified Kjeldahl method with sulphuric acid, salycilic acid, sodium 

 thiosulphate, and copper sulphate. Where nitrate nitrogen was present, 

 50 c. c. of concentrated sulphuric-salycilic acid (25 c. c. of concentrated 

 acid plus 25 c. c. of distilled water) were added to the cultures slowly 

 and with constant stirring. This acid was allowed to react for a few 

 days, after which the usual procedure was carried out. Digestion was 

 continued for five to six hours subsequent to the clarification of the 

 liquid. 



The amount of ammonia was determined by dist'ilation with steam 

 in the presence of magnesium oxid. 



Nitrites (qualitative test) were tested for with Trommsdorf 's reagent. 



In all distillations NI14 acid and alkali were used. 



In the preparation of agar cultures of alfalfa seedlings the seeds were 

 treated with a 0.25 per cent solution of mercuric chlorid and rinsed in 

 sterile distilled water. Three bacteria-free seeds were transferred to the 

 surface of soft mannit agar (0.7 per cent agar) in each tube. 



The nitrates were added in solution to all cultures. Gram-molecular 

 quantities of potassium, sodium, calcium, and ammoniun nitrates 

 (Merck's) were weighed into sterile distilled water. These solutions 

 were prepared in such a manner that 5 c. c. contained 450 mgm. of 

 nitrate. In all nitrate solutions the nitrate radical, or anion, was 

 present in the same quantities, while the cation, or metal, was present 

 in varying quantities, depending upon the particular salt. 



