NITROGEN-FIXING ORGANISMS OF LEGUMES 259 



Ps. radicicola forms white colonies, while Bad. tumefadens 

 absorbs the Congo red and therefore produces red or reddish 

 colonies. 



B. TEST OF THE PHYSIOLOGICAL EFFICIENCY OF 

 PS. RADICICOLA AND OBSERVATION OF NOD- 

 ULE FORMATION 



To observe nodule formation and nitrogen fixation, it 

 is necessary to have seeds germinating free from bacteria. 



Apparatus. 500 c.c. of nitrogen-free agar; six sterile 

 large est tubes with foot; sterile ordinary test tubes 

 (the agar should be distributed in all the test tubes to a 

 depth of about 5 cm.); sterile Petri dishes; clean slides; 

 mercuric chloride, 1 : 500; flask of sterile distilled water; 

 sterile pipette; seeds of some leguminous plant. (The> 

 smaller seeds are better for this experiment.) 



Culture. Ps. radidcola (specific strain). 



Method. 1. Obtain sound, mature pods of some legume 

 as pea, bean, vetch, etc. 



For testing the physiological efficiency of the pure 

 culture of Ps. radicicola just previously isolated, use seeds 

 from the same legume as that from which this particular 

 culture was isolated. 



2. Soak the pods for five minutes in mercuric chloride 

 1 : 500 and remove the excess of solution with sterile cheese- 

 cloth. 



3. Tear open the pods with flamed forceps, place the 

 seeds between folds of sterile cotton, and put the cotton in 

 a dry, warm place until the seeds are dry. 



4. Select the best of these seeds and store them in dry 

 sterile test tubes until they are to be used. 



6. Whether seeds are procured as described above, or 

 otherwise, proceed as follows: 



(a) Soak the leguminous seed in 1 : 500 mercuric chloride 

 solution for five minutes. 



