FORMATION OF NODULES BY BACILLUS RADICICOLA 69 



dish to a second containing a few drops of water. Repeat 

 these dilutions to a third and fourth Petri dish. 



8. Pour mannit agar (m. 42) into each dish, agitate until 

 the organisms are equally distributed, and incubate at 28 C. 



9. After six to eight days examine plates. The legume 

 colonies should be characterized by a raised moist surface 

 and round entire form, at first glistening, later changing 

 to an opaque white. In size these colonies vary from ij 

 to 4 mm. in diameter. 



Exercise 35 

 Formation of Nodules by Bacillus Radicicola 



1. Wash thoroughly the seeds of several legumes (alfalfa, 

 crimson clover, red clover, etc.) and immerse in mercuric 

 chlorid (HgCfe) solution (i : 500) for three to five minutes. 

 For careful work, treat the seeds with mercuric chlorid 

 in a partial vacuum. 



2. Remove from mercuric chlorid solution and rinse 

 in-sterile water. 



3. Next drop one or two seeds of each legume into a 

 large tube containing soft mannit agar or a loose mass of 

 filter-paper pulp. 



4. Arrange as follows: 



(a) i and 2, alfalfa uninoculated. 



(6) 3 and 4, alfalfa inoculated with culture iso^Ri by student. 



(c) 5 and 6, alfalfa inoculated with culture fjmn instructor. 



In the case of large legumes ef g., soy beans it is 

 necessary to use vessels larger than ordinary test-tubes. 



5. To inoculate, use a forty-eight-hour-old culture of 

 the legume bacteria. Prepare a water suspension and from 

 this take i c.c. for each tube. 



