RECLAIMING LOST NITROGEN. 14! 



to him, but which he regarded as probably microorganisms of 

 some sort. The conclusion reached was that these little bodies 

 were parasitic microorganisms invading the root, producing a 

 pathogenic effect upon the tissues, and causing the develop- 

 ment of the tubercles. The next step was naturally the isola- 

 tion of these little bodies and their careful study by more 

 modern methods, to determine whether the conclusions reached 

 were correct. Their isolation did not prove to be difficult. It 

 was only necessary to remove a fresh tubercle at a certain 

 stage of its growth, carefully wash its surface to remove acci- 

 dental soil bacteria, and then to inoculate culture media with 

 the contents of the tubercle. The result was a growth of bac- 

 teria in the culture media which could then be studied by 

 ordinary bacteriological methods. 



The study of these bacteria proceeded rapidly and in a short 

 time the organisms in question were so thoroughly isolated 

 and carefully studied that their causal relation to the tubercles 

 in question was demonstrated. That they produce the tuber- 

 cles was proved readily enough by inoculation experiments. 

 Legumes were grown in sterilized soil and watered with bac- 

 terial infusion from these cultures ; the result was in all cases 

 the growth of abundant tubercles and the fixation of nitrogen. 

 The most striking of these experiments were made with ger- 

 minating peas. Such peas, if kept moist and warm, will grow 

 for several days, sending out their normal roots even without 

 being planted in soil. It was found by dipping the tip of a 

 needle into cultures of the microorganisms and then pricking the 

 rootlets of young legumes at various points, that the develop- 

 ment of tubercles would almost inevitably follow such slight 

 wounds. In favorable experiments the tubercles appeared in 

 six days after the inoculation and always at the point of inocu- 

 lation. All of these facts proved that these microorganisms 

 were concerned in the development of the tubercles. 



The study of these little bodies very soon proved that they 



