267 



Keinkulturen von B. radicicola var. Pisi oder von Vicia faba, die Erbsen und Bohnen, 

 und mit denen des Bac. radic. var. Lupin, oder Ornithopodis Lupinen und Serradella 

 erfolgreich zu infiziren und zum Wachstum resp. der Assimilation des freien Stick- 

 stoffs zu bringen, und das ist was unsere anfangliche Behauptung bestatigt. Already 

 r earlier Hellriegel had arrived at the conclusion that the bacteria of Lupinits 

 and Ornithopus belong to a species different from that of Pisum and Vicia, which was 

 also my own opinion. In later years many interesting experiments were made in this 

 direction, especially by H i 1 t n e r. Yet the evidence is unsatisfactory as it proved 

 hitherto impossible in the sand cultures a ) to bring Leguminosae to complete develop- 

 ment by infection with 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 particular in B. ftuorescens liquefaciens and the nitrogen-fixing spore-for- 

 ming Granulobacter (Clostridium) pasteurianum and Helobacter cellulosae. This ob- 

 servation 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 Hell- 

 r i e g e 1'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 various other species of mi- 

 crobes, but he thought it of no consequence (1. c. p. 169). For myself I have ob- 

 served in nitrogen-free sand, besides the mentioned species, Chlorella and Cystococcus 

 and sometimes also Palmella 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 Erlenmeye r-flasks, failed as the plants 

 ceased to grow before they blossomed, although the nodules developed very well. 



T/ie tubercle bacteria do not fix the atmospheric nitrogen zvhen 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 culti- 

 vated out of the plant nor wihin 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 Asotobacter, Granulobacter and Helobacter; notwithstanding the sterilisation, the 

 soil preserves its catalytic power very little impaired. The spores of the nitrogen-fixing 

 Helobacter and Granulobacter often adhere to the nodules and, when present, fermen- 

 tation phenomena show that the experiments cannot be relied upon, B. radicicola not 

 causing fermentation. Commonly however, these fermenting and nitrogen-fixing microbes 

 can be removed by thoroughly washing of the nodules with alcohol and water. In the 



') It is a well-known fact that the Papilionaceae, when cultivated in liquids, do 

 not fix the atmospheric nitrogen indifferently whether they produce tubercles or not. 



