72 SOIL BACTERIOLOGY 



2. Plant to alfalfa as follows: 



(a) i and 2, sand uninoculated. 

 (&) 3 and 4, sand inoculated. 



(c) 5 and 6, coal-ashes uninoculated. 



(d) 7 and 8, coal-ashes inoculated. 



3. One week after seedlings begin to germinate add 

 100 c.c. per jar of plant food minus nitrogen. 



Note. Plant food: Add 10 grams of CaSO4 -\- 2HzO, 4.6 grams of 

 KH 2 PO 4 , and 2.3 grams of MgSO 4 + 7H 2 O to 1000 c.c. of water. 



4. This nutrient solution should be added at intervals 

 of every two weeks or whenever needed. A few drops of 

 iron chlorid or iron phosphate will be found beneficial. 



5. After four to six weeks examine for nodules. 



6. When mature, cut and analyze the tissue for total 

 nitrogen. 



Exercise 37 



Effect of Caffein on the Formation of Bacteroids (Optional) 



1. Inoculate in duplicate tubes of agarwith and without 

 cafTein (m. 46). 



2. Prepare caffein agar slant cultures of the following 

 organisms: Bacillus radicicola, from alfalfa, pea, and vetch. 



3. At regular two-day intervals study the morphology 

 of the organisms from the different cultures. 



Exercise 38 

 Nitrogen Fixation by Bacillus Radicicola in Solution 



1. Prepare four 5oo-c.c. Erlenmeyer flasks with 100 c.c. 

 each of mannit soil extract (m. 43). 



2. Arrange as follows: 



(a) i and 2, uninoculated. 



(6) 3 and 4, inoculated with a pure culture of alfalfa bacteria. 



