THE INFLUENCE OF IRON^ ETC. 51 



EXEECISE XXXIX. 



THE INFLUENCE OP IRON; OF PHOSPHATE; AND OF AERA- 

 TION ON THE FIXATION OF NITROGEN BY AZOTOBACTEE. 



1. Prepare 800 cc. of "Mannite solution." 



2. Prepare 200 cc. of the same solution not adding the 



3. Prepare similarly 200 cc. without the K2HPO4. 



4. Divide into 100 cc. portions in flasks and arrange as 

 follows : 



1 & 2 — Regular "mannite" solution in 500 cc. Erlenmeyers. 

 3 & 4 — Without FejfSOJj solution in 500 cc. Erlenmeyers. 

 5 & 6 — Without K2HPO4 solution in 500 cc. Erlenmeyers. 

 7 & 8 — Regular "mannite" solution in 2,000 cc. Erlenmeyers. 

 9 & 10 — Regular "mannite" solution in 1.000 cc. Erlenmeyers. 

 11 & 12 — Regular "mannite" solution in 200 cc. Erlenmeyers. 



5. Plug and sterilize in the autoclave as usual. 



6. Inoculate each with 1 cc. of a vigorous culture of Az. 

 vinelandii. 



7. Incubate 6-7 days at 20° C. 



8. Add 0.7 gm. Hg. and 20 cc. H2SO4 and determine the 

 total nitrogen by the regular Kjeldahl method. 



9. Determine the effect of the absence of Fe2( 80^)3 and 

 of K2HPO4, and the effect of aeration on the fixation of 

 nitrogen by Azotobacter. 



