14 Martin J. Prucha 



and the upper end protruding above the cotton plug. The tube was 

 plugged at its upper end with cotton. The whole was sterilized in the 

 autoclave for five hours at 15 pounds pressure. 



The seeds were sterilized by the bleaching-powder method. The steri- 

 lized seed were dropped into the pots through the glass tube, and by 

 manipulation of the tube they were buried in the soil. The soil of the 

 pots was inoculated by introducing through the tube a few cubic centi- 

 meters of water containing the nodule-forming organisms. 



PART I. ISOLATION AND IDENTIFICATION OF THE ORGANISM 

 ISOLATION OF BACILLUS RADICICOLA 



On October 10, 1910, a field pea plant 30 centimeters high, with a great 

 abundance of nodules, was procured. The whole plant was washed 

 thoroughly m running water. One of the nodules, of firm consistency, 

 was selected and cut off in such a way as to leave about 3 centimeters of 

 the root on each side of the nodule; cut off in this way the nodule is more 

 easily manipulated. The nodule was then disinfected in a solution of 

 one part of formalin to forty parts of water, for five minutes. Four 

 petri dishes were prepared, each containing a few drops of sterile water. 

 The nodule, after being disinfected, was washed in sterile water, placed 

 on a filter paper, and cut open, and with a pointed scalpel a part of the 

 central tissue was removed and placed in petri dish 1. This nodule 

 tissue was crushed and mixed with the water. Three loopfuls of this 

 infusion was transferred from plate 1 to plate 2, three loopfuls from 

 plate 2 to plate 3, and three loopfuls from plate 3 to plate 4. Ten cubic 

 centimeters of medium 331 were then poured into each petri dish, and after 

 sufficient agitation to effect equal distribution of the organisms the plates 

 were allowed to incubate at 20° C. In three days a few colonies charac- 

 teristic of the nodule organism became visible on plate 1; on the other 

 plates plenty of colonies were present, but were visible only by the micro- 

 scope. In ten days the small colonies became large enough to be con- 

 veniently transferred. 



As far as could be ascertained from the general appearance of the colo- 

 nies, all the plates contained only one organism. The large colonies that 

 developed on plate 1 appeared to be giant colonies, having started from 

 small pieces of the nodule tissue in which a large number of the organisms 

 were held. In order to obtain a pure culture of this organism, one of the 



