THE STUDY OF MICROORGANISMS IN THE SOIL 111 



tion allow the bottles with the melted medium to cool in a sloped 

 position. When the agar has hardened the bottles are ready for 

 inoculation with the proper bacteria. 



Select young cultures of root-nodule bacteria of alfalfa, or 

 clover and cowpea, or soybean from Exercise 13. Inoculate the 

 surface of the agar with three long streaks. Use a large loop 

 which has been bent at right angles. 



Incubate the cultures at 28°C. or room temperature, for 4 to 

 10 days. Examine the cultures microscopically (Barlow stain) 

 and pack for shipping. 



Exercise 22 



The Structure of Root Nodules 



A nodule consists mainly of a mass of large, thin-walled 

 nucleated cells often completely filled with bacteria, i.e., bac- 

 teroidal tissue. The action of the bacteria on the plant cells 

 as well as the infection threads may be seen from free hand 

 sections or better from paraffin embedded sections cut about 3 

 to 5 microns- thick. 



A. Free-hand Sections. — Cut a cross-section through a young 

 pea nodule and root. 



Mount in a drop of water and examine. 



To secure a clear section, immerse it for 2 to 5 minutes in a 

 dilute sodium hypochlorite solution and stain with safranin. 



B. Embedded Sections. — Select young nodules from the 

 desired plant grown in quartz sand or on filter-paper pads. 



Fix by immersing for 24 hours in a formol-alcohol-acetic acid 

 mixture. 



Acetic acid (glacial) 2 . 5 cc. 



Formalin 6 . 5 cc. 



Alcohol (50 per cent) 100 . cc. 



Transfer to 70 per cent alcohol and change the liquid until 

 there is no odor of acetic acid. 



Dehydrate in 80, 90, and 100 per cent alcohol, clear in chloro- 

 form, embed in paraffin, and cut microtome sections 3 to 5 microns 

 thick. In order to stain the bacteria and the infection threads 

 use method A or B. 



