NITROGEN-FIXING ORGANISMS OF LEGUMES 261 



ilized seeds to each of the large test tubes. Or, place the 

 seeds between layers of moist sterile filter paper in a Petri 

 dish until they have germinated and then transfer the seeds 

 to the large test tubes. When using the larger seeds use 

 only three to six per tube, and six to ten only of the smaller 

 seeds as alfalfa, clover, etc. 



7. Put the tubes containing the ungerminated seed in a 

 warm place (30 to 35 C.) until the seeds germinate. 

 Keep the germinated seed in a well-lighted room for a few 

 days. 



8. Examine the tubes and reject all that are contaminated 

 with molds or bacteria. 



9. After a few days, inoculate four of these tubes contain- 

 ing growing leguminous plants with a pure culture of Ps. 

 radicicola, by dropping upon the seeds and surface of the 

 agar a heavy suspension of the bacteria in sterile water, by 

 means of a sterile pipette. 



10. Keep two tubes uninoculated as controls. 



Note. To imitate infection under more natural conditions, just 

 before the seeds are placed upon the agar, the agar may be melted, 

 cooled to 40 to 45 C. and inoculated with a loopful of Ps. radicicola 

 culture, mixing the organisms well through the agar with the needle. 

 After the agar has solidified the sterile seeds may be then placed on the 

 surface of the agar as before. 



11. Label the test tubes and place in some location 

 where they will be sufficiently protected from the sun, 

 heat, or cold, etc. This is very important. A piece of 

 cheese-cloth thrown over the tubes will protect them from 

 the sun. 



12. In about a month examine all test tubes and look 

 for nodules. 



13. Record the presence, number, size, and shape of 

 nodules, place of formation, etc. Show nodule-bearing 

 seedlings to the instructor. 



14. Isolate Ps. radicicola from one of these nodules. 

 This completes the cycle. 



