FORMATION OF NODULES BY BICILLUS 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 i| 

 to 4 mm. in diameter. 



Exercise 35 > 

 Formation of Nodules by Bacilltis Radicicola 



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

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

 chlorid (HgCy 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. 



(b) 3 and 4, alfalfa inoculated with culture isolated by student. 



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



In the case of large legumes — e. 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. 



