Exercise 21. Free Nitrogen Fixation 
Symbiotic Type 
42 
MATERIALS: 
Preserved specimens of legume nodules 
Young leguminous plant with nodules 
3 sterile Petri dishes 
3 tubes nitrogen-free agar 
3 tubes sterile water 
Sterile pipettes 
Pair of forceps 
Sterile knife 
Pure culture of Rhizobium radicicolum 
Leguminous plants, such as clovers, peas, beans, lupines, and vetches, 
when grown in moist soils bear numerous nodules or tubercles upon their 
roots. At certain times in the plant’s growth these nodules contain myriads 
of bacteria. These nodule-forming organims can fix the free nitrogen of the 
air, in which state it becomes available to the plant. Such a relation existing 
between the bacteria and the plant is one of co-operation. 
1. Remove a root from a fresh, green, leguminous plant wdth a nodule at¬ 
tached. Select as large a nodule as possible. 
2. Carefully wash the nodule in three changes of sterile water. 
3. Treat the nodule in 70 per cent alcohol for 3 to 5 minutes. Pour off the 
alcohol, and wash the nodule in three changes of sterile water. 
4. Sterilize a clean slide by heating thoroughly in the flame. 
5. When the slide is cool, place a large drop of water on it; and crush the 
nodule, with a sterile knife or scalpel in the water. 
6. Label three Petri dishes 1, 2, 3 and by means of sterile pipette, place a 
large drop of sterile water in each. 
7. By means of the sterile loop transfer one loopful of the milky material 
on the slide to the first Petri dish. Mix well. Sterilize the loop and transfer 
two loopfuls from the first Petri dish to the second. Sterilize the loop, and 
transfer two loopfuls from the second dish to the third. Mix well. 
8. Prepare a slide for staining with a loopful of the material on the sterile 
slide. 
9. Melt three tubes of legume agar, cool to 45°C. and pour the three plates. 
When the agar is solid, place the plates inverted, in moist chambers at 28° C. 
for ten days or two weeks. The nitrogen-free medium known as legume agar 
has the following composition: 
Mannit (C 6 H 8 (OH 6 )_ 15 grams 
Dibasic potassium phosphate(K 2 HP0 4 )_ 0.20 “ 
Magnesium sulphate (MgS0 4 )_ 2.20 “ 
Sodium chloride (NaCl)_ 0.20 “ 
Calcium sulphate (CaS0 4 )_ 0.10 
Calcium carbonate (CaC0 3 )_ 5.00 
Water__1000.00 
Agar_ 15.00 
