256 GENERAL MICROBIOLOGY 



EXERCISE 9. A STUDY OF THE SYMBIOTIC NITROGEN- 

 FIXING ORGANISMS OF LEGUMES, PS. RADICI- 

 COLA 



A. ISOLATION OF PS. RADICICOLA FROM 

 NODULES OF LEGU MINOS JE 



Apparatus. Spade or trowel; sterile Petri dishes; 

 tubes of nitrogen-free ash agar; tumbler for mercuric 

 chloride solution; small piece of filter paper; small pair 

 of forceps; scalpel or chisel-edged platinum needle; platinum 

 loop; clean slides; mercuric chloride, 1-500; alcohol, 95%; 

 tubes of sterile water; aqueous-alcoholic gentian violet 

 orfuchsin; eosin; LugoPs iodin solution; saturated alcoholic 

 solution of gentian violet. 



Culture. From nodules of roots of leguminous plants. 



Method. 1. Using a spade or trowel, obtain the roots 

 of some legumes which show nodule formation in, abundance. 

 If the soil is firm, as with clay, do not forcibly pull up the 

 legume to obtain the roots as this procedure strips off the 

 nodules which develope almost without exception on the 

 young rootlets. 



Note. Heavily inoculated legumes may be stored for winter use in a 

 cool, dry, dark place. Living organisms have been found after more 

 than two years in some of the larger nodules. 



2. Thoroughly wash the roots under the tap and place 

 the plant in clean, cool water. 



3. Keep parts of the plant for identification if the 

 species is unknown. Not all plants belonging to the family 

 Leguminosce are attacked by symbiotic nitrogen-fixing 

 bacteria, only those belonging to the sub-family, Papilio- 

 nacece. 



4. Compare the size, numbers, and location of the nod- 

 ules on the roots of the different legumes. 



5. Remove a good-sized nodule from the roots, wash 

 in clean water and immerse for three minutes in mercuric 

 chloride solution, 1 : 500. 



