44 ELEMENTARY BACTERIOLOGY LABORATORY EXERCISES 
EXERCISE 62 
NON-SYMBIOTIC NITROGEN FIXATION 
The bacteria which fix free nitrogen from the air may be grown on a 
medium which contains no nitrogen, only inorganic salts, and a simple carbo- 
hydrate from which they derive energy. The medium is composed of the follow- 
ing ingredients: 
Mannitol [C,H, (OH),] 15.0 grams 
Diabasic potassium phosphate (K,H PO,) ---------csccesss-seeo 0.2 gram 
Magnesium sulphate (Mg SO,) 0.2 gram 
Sodium chloride (NaCl) 0.2 gram 
Calcium sulphate (Ca SO,) 0.1 gram 
Calcium carbonate (Ca CO,) 5.0 grams 
Water 1000.0 grams 
Inoculate a flask containing a shallow layer of the above medium with about 
one gram of a fertile soil. Incubate at 30° C. 
Examine a mannitol agar slope (the above medium plus 1.5 per cent agar) 
of Azotobacter chroococcum and note the characteristics of the growth. Make 
microscopic preparations of the organism and stain. Note carefully its mor- 
phology and make drawing. 
When the flask containing the nitrogen-free mannitol medium shows a heavy 
growth, make stained microscopic preparations from the surface growth. Ex- 
amine for large oval or coccoid cells of Azofobacter. 
EXERCISE 63 
\ 
SYMBIOTIC NITROGEN FIXATION 
Examine the plant furnished and observe the position of the nodules on the 
roots. 
Select a nodule from your plant and carefully wash it in clean water. Grind 
the nodule with a drop of water on a clean slide. From this suspension make a 
smear preparation on another slide. Stain and examine under the oil-immersion 
objective. Look especially for irregularly shaped cells; such as, pear, club, “Y” 
and “T” shaped individuals. The bacteria when appearing in these shapes are 
called “bacteriods”. Draw the preparation. 
The legume bacteria may be grown in the laboratory on a medium similar 
to that used for the cultivation of the independent nitrogen fixing bacteria. These 
organisms when grown on artificial culture media do not appear as irregularly 
shaped bacteriods, but as ordinary rod-shaped organisms. 
Note the type of growth produced by Rhizobium leguminosarum on the agar 
slope furnished and make a stained preparation from it. The legume organism 
when taken from artificial media does not stain easily; usually the slimy growth 
material will take the stain, leaving the rods unstained. The bacteria thus ap- 
pear as colorless bodies on a stained background. 
