J 87 



B. radicicola only and with exclusion of all other microbes. Such 

 cultures are always at the end of the vegetation period rich in 

 various other species, in })articnlar in B. jluoreacens liquefaciens 

 and the nitrogen-tixing- spore-forming (rvanulobacter [Closiridiuin) 

 pasteur ianeni and Helobacter cellulosae. This observation holds good 

 as well for the first experiments made by myself as for those of 

 others, and this should never be lost sight of when reading the 

 descriptions of the infection experiments with the so-called "pure 

 cultures". It had not escaped Hki-lrtegel's attention, and we see it 

 in all the photographs of his above mentioned treatise at the film 

 of the glass vessels, wherein he cultivated his plants (in bright 

 daylight), which film consisted of Chlorophyceae and varions other 

 species of microbes, but he thought it of no consequence (1. c. |). 

 169). For myself I have observed in nitrogen-free sand, besides the 

 mentioned species, CliloreUa and Cystococcus and sometimes also 

 PahneUa cruenta and many Cyanophyceae. Many of my later efforts 

 to bring clover plants to complete growth on agar with nutrient 

 salts and B. radicicola in large cotton-plugged EKi.ENMKYER-flasks, 

 failed as the plants ceased to grow before they blossomed, although 

 the nodules developed very well. 



The tubercle bacteria do not Jix the atmospheric nitrogen lohen 

 cultivated in nutrient media. 



I will now call attention to my chief subject namely the want of 

 power of the tubercle bacteria to fix the free atmospheric nitrogen. 

 They do this neither when cultivated out of the plant nor within 

 the nodules. 



Regarding the first point the experiment is very simple. We have 

 but to crush the nodules and bring the thus obtained material into 

 culture soils used for the ordinary experiments to fix free nitrogen 

 and then cultivate at 20° to 30° C. ; or we use the pure cultures 

 for infection of the same media. A convenient medium is : Tapwater 

 100, Glucose 2, Dikaliumphosphate 0,05, lime 2, fresh garden soil 

 2. This liquid, to which the garden soil is added as a catalyst, 

 must previously be sterilised to kill the germs of Azotobacter, Gra- 

 nalobacter and Helobacter; notwithstanding the sterilisation, tiie 

 soil preserves its catalytic power very little im[)aired. The spores of 

 the nitrogen-fixing Helobacter and Granidobacter often adhere to 

 the nodules and, when present, fermentation phenomena show that 

 the expeiiments cannot be relied upon, Ji. radicicola not causing 

 fermentation. Commonly, iiowever, these fermenting and nitrogen- 



