194 



Journal of Agricultural Research 



Vol. XII. No. 4 



present but to a less extent than did control cultures containing no 

 nitrate. 



From these results it appears that nitrates do not stimulate the nitro- 

 gen-assimilation of the nonsymbiotic nitrogen-fixing bacteria. 



Inasmuch as nitrates in small amounts caused such an increase in 

 the number of Azotobacter in sterilized soil, it was thought advisable 

 to determine just what influence these salts exert on nitrogen fixation 

 by Azotobacter. Accordingly, experiments were carried out with 

 Azotobacter on agar films, in soil cultures and in solution. 



Agar-film cultures. — In this work both strains of the Azotobacter 

 were used. One hundred c. c. of mannit agar were placed in liter 

 Erlenmeyer flasks and nitrates of potassium, sodium and calcium 

 added in varying quantities. The flasks and contents were sterilized 

 at ID pounds' pressure for 25 minutes, cooled, and inoculated with 10 

 c. c. of a suspension of the organism in sterile distilled water. The 

 flasks were incubated at 28° C. for three weeks. The weight of both 

 inoculated and uninoculated flasks was maintained throughout the 

 experiment by the addition of sterile distilled water. At the end of the 

 incubation period total nitrogen analyses were made. Because of the 

 high nitrate content dilute sulphuric-salycilic acid was added slowly 

 and carefully to prevent loss of nitrogen by the evolution of gaseous 

 oxids of nitrogen. The acid was allowed to react for a few days before 

 continuing the total nitrogen determination. The results of the experi- 

 ments are presented in Tables VIII and IX. 



Table VIII. — Influence of nitrates on the fixation of nitrogen by Azotobacter {strain A) 



on agar films 



Cul- 

 ture 

 No. 



3 

 4 

 5 

 6 



7 

 8 



9 



10 



13 

 14 

 IS 



Treatment (nitrate in looc. c. of medium). 



O 



o 



o 



50 mgm. of NO3 potassium nitrate . 



do 



100 mgm. of NO3 potassium nitrate 



. . . .do 



50 mgm. of NO3 sodium nitrate. . . 



....do 



100 mgm. of NO3 sodium nitrate. 



do 



50 mgm. of NO3 calcium nitrate. . 



....do 



100 mgm. of NO3 calcium nitrate . 

 ....do 



Nitrogen contained in loo c. c. of medium. 



Inoculated. 



Found. Average. 



Mgm. 

 13.00 



12. 70 

 12. 60 

 18.50 

 18. 40 

 27. 60 



27-75 

 18.65 

 18.30 

 27. CO 

 27.65 



13-75 

 13-70 

 18.80 



19-15 



Mgm. 

 12. 80 



} 18. 45 

 I 27. 70 

 } 18. 50 

 } 27-35 

 } 13- 75 

 } 18. 95 



Uninoculated. 



Found. Average. 



Mgm. 

 4.0 

 4.0 

 4- I 

 7. 00 



7. 20 

 16.80 

 15-70 



7-50 



7-30 



15.00 



15. 20 



8.00 



8. 50 

 14. 50 

 14-30 



Mgm. 

 4-05 



I 7.10 

 I 16.25 



}'- 



} 8-25 

 14.40 



Nitrogen 

 fixed. 



Mgm. 

 8 



II 

 II 

 II 

 12 



5 

 4 



