70 A MANUAL OP BACTERIOLOGY 



3. Crush a well-washed tubercle between two glass slides. 

 Transfer a drop of water from the crushed tubercle to a cover glass 

 and stain in the usual manner. Examine the preparation for the 

 rod-shaped B. radicicola and for branched rods — bacteroids. 



Exercise 100. Isolation and Culture of Bacillus radicicola 



BccHANAN, R. E. Centralbl. f. Bakt., 2te Abt., 23 : 59. 1909. 

 Prazmowski. Landw. Vers.-Stat. 37 ■ 199. 1890. 

 Harrison and Barlow. Centralbl. f. Bakt., 2te Abt., 19 : 426. 1907. 

 Also works cited under Exercise 99. 



1. Obtain several young tubercles from the roots of clover or 

 other legume. Wash thoroughly in tap water. 



2. Immerse the tubercles for several minutes in 1 : 1000 solu- 

 tion of mercuric chloride. Transfer with sterile forceps through 

 several changes of sterile distilled water. 



3. Crush the tubercles with a sterile spatula in a sterile Petri 

 dish and make plates from the contents of the nodule. Use the 

 synthetic agar described in Exercise 88. 



4. Watch the plates for small, white, moist colonies. Transfer 

 from them to ordinary laboratory media (see Exercise 108). 



5. Pour plates also from commercial cultures of B. radicicola. 



Exercise 101. The Production of Bacteroids of Bacillus radicicola upon 

 Artificial Media 



Until recently the branched rod, or bacteroid, of B. radicicola 

 has been found only in the nodule of the legume. Zipfel has 

 shown, however, that these forms may be produced in cultures 

 in the laboratory (Centralbl. f. Bakt., 2te Abt., 32: 97. 1911) 

 by the following method: 



1. Extract 100 g. of bean or pea meal with 100 cc. of normal 

 KOH and 5 liters of water for twenty-four hours. Siphon off 

 the clear liquid and neutralize with phosphoric acid. Make the 

 volume up to 5 liters. Prepare a medium by taking 



Legume-seed extract 1000 cc. 



Agar 30 g. 



Dextrose 20 g. 



Normal malic acid 10 cc. 



Caffein 2 g. 



