NITROGEN-FIXING ORGANISMS OF LEGUMES 257 



6. Remove the nodule with flamed forceps and take up 

 the excess of the solution between folds of sterile filter 

 paper, then dip into alcohol, the last traces of alcohol 

 being removed by passing the nodule quickly through the 

 flame. 



7. Place the nodule on a flamed and cooled slide. 



8. Holding the nodule in flamed and cooled forceps, 

 cut into it, and break it open by means of a sterile scalpel 

 or a chisel-edged platinum needle. 



9. Thrust a sterile platinum needle into the nodule in 

 the middle of the newly exposed surface and gently rotate the 

 needle so that some of the crushed tissue adheres to it. 



10. Touch the needle in a drop of sterile water in a sterile 

 Petri dish. 



11. Transfer a loopful of this suspension to a second 

 drop of sterile water in a second Petri dish and a loopful 

 from this to a third drop in a third Petri dish. 



12. Pour the relates, using tubes of nitrogen-free ash 

 agar and incubate at room temperature for two or three 

 days. 



13. Make a smear on a clean slide from the freshly cut 

 surface of the nodule, stain and examine microscopically. 

 What is the morphology of Ps. radidcola found in the 

 nodule? What are bacteroids? 



14. Make a smear directly from this same nodule on 

 a clean slide, fix and stain with eosin followed by Lugol's 

 iodin solution. The iodin demonstrates the starch which 

 is usually present in nocjules. Is starch present? 



15. After a few days of incubation the colonies of Ps. 

 radidcola will be noted on the plates as round, grayish- 

 white, translucent, slime-like drops, finely granulated and 

 often with compact white centers. 



16. Examine these colonies in the hanging drop. They 

 contain the normal, short, rod forms which during the 

 first days are very motile. 



17. Isolate several pure cultures of Ps. radidcola on 



