39 
Exercise 19. Free Nitrogen Fixation 
Noil-symbiotic Type 
MATERIALS: 
2 one-fourth pint milk bottles and caps 
25 cc. graduate 
Nitrogen-free liquid medium 
Soil samples 
Pure cultue of Azotobacter 
Many soils upon which legumes are not growing, and to which nitrogen is 
not added in the form of chemical compounds or fertilizers of any kind, still 
have the power of materially increasing their nitrogen content. This increase 
is due to the presence in the soil of species of free nitrogen-fixing bacteria. 
These organisms are capable of maintaining an existence and of fixing nitro¬ 
gen independent of higher plants. 
The organisms that have the power to take the free nitrogen of the air and 
combine it in their cells can grow in a nitrogen-free culture solution. This 
culture medium contains only inorganic salts in solution and some simple 
carbohydrate or similar carbon compound which contains no nitrogen, e. g., 
the sugars, some of the alcohols, as mannite, etc. Such a solution is prepared 
as follows: 
Mannit (C 6 H 8 (OH c )_ 15.00 grams 
Magnesium sulphate (MgS0 4 )_ 0.20 “ 
Sodium chloride (NaCl)_ 0.20 “ 
Calcium sulphate (CaS0 4 )_ 0.10 “ 
Calcium carbonate (CaC0 3 )_ 5.00 “ 
Dibasic potassium phosphate (K 2 HP0 4 )_ 0.20 “ 
Water_'_1000.00 “ 
1. The above culture medium is on the desk. With the graduate place 
25 cc. of it in each of the two milk bottles. Be sure to stir the medium well 
before measuring any of it, as the insoluble substances settle to the bottom. 
2. Label the bottles. Inoculate one with two or 3 measures of soil A and 
the other with the same amount of soil B. Cap the bottles. 
3. Place the bottles in the incubator at 25° C. where they will not be dis¬ 
turbed. Watch for the development of a white, gelatinous film which is very 
conspicuous and soon turns brown or black. 
4. Make a microscopic preparation from the film on the surface of each 
solution, according to the following method: Place a drop of strong iodine 
solution upon a clean slide. Place some of the organisms from the film in the 
iodine solution. Carefully cover with a cover glass. The organisms will float 
in the solution under the cover glass. 
5. Examine the preparation with the 4 m. m. objective. The large oval or 
coccoid bodies are some species of the free nitrogen-fixing organism, Azotobac¬ 
ter. Long, rather oval or mace-shaped organisms, which have a blue appear¬ 
ance, are sometimes seen; they are the anaerobic species of free nitrogen-fix¬ 
ing organisms, Clostridium butyricum. 
6. Examine the agar slope culture of Azotobacter furnished. Draw and 
describe it according to the outline for study of species. See Appendix A. 
