268 



course of many years I have experimented in this way with numerous species of 

 tubercle bacteria, and with many modifications in the nutrient media as well in the 

 temperature as in the source of carbon. Moreover I have, as said, tried to grow pure 

 cultures of the bacteria themselves in the liquid culture medium as also on solid 

 culture soils of various compositions, and at first I thought I had observed a rather 

 considerable increase of these organisms. This increase, however, proved to be really 

 very slight, so slight that gain of atmospheric nitrogen is not proved, whilst the obvious 

 augmentation of dry weight of the sown bacteria derives from the formation of thick 

 slime walls, that is of nitrogen-free, cellulose-like substances around the hardly aug- 

 mented original protoplasmic material 1 ). 



Only when cultivating the microbes in plant extracts with cane sugar, wherein 

 nitrogen compounds are evidently present, I could observe a very slight and by no 

 means convincing increase of the total nitrogen rate of the liquid in consequence of the 

 growth of B. radicicola. But when performing these experiments I was not yet 

 acquainted with the circumstance that laboratory air contains sufficient carbon and 

 nitrogen compounds to be made perceptible by the growth of microbes which can feed 

 on them. This was afterwards demonstrated by Ir. A. van D e 1 d e n and myself in 

 our investigation on Bacillus (Actinobacillus) oligocarbophilus 2 ). 



There exists moreover an aerobic spore-producing bacterium 3 ), hard to kill by 

 sterilisation of the nutrient liquids, which fixes free nitrogen; at that time it was still 

 quite unknown to me and even now it is very imperfectly understood. It may have 

 been present at my experiments likewise as at those of other investigators who think 

 they have observed fixation of free nitrogen out of the plant in the pure cultures of 

 B. radicicola. 



With sufficient precautions the results of such experiments are however always 

 the same: The bacteria of the nodules do in no way fix the free atmospheric nitrogen. 

 When the experiments are performed, not with nutrient liquids, but with a solid 

 medium, the results are quite the same: fixation of nitrogen does not take place then 

 Cither. Stress must be laid on the latter fact as it seems impossible to fix free 

 nitrogen by the Papilionaceae when cultivated in liquid media even under the 

 best circumstances and whether tubercles are produced or not. So it seems probable 

 that for this process a direct contact with the air is necessary, which cannot be 

 realised in the liquid culture media, but very well in solid ones. 



Further it must be observed, that the plate cultures of some of the nodule 

 organisms 4 ), for example the forms from Pisum, Vicia, and Trifolium, on glucose- 



') The slime formation is of importance for the explanation of the slime threads* 

 (erroneously called infection threads*) within the nodules. See Die Natur der Faden 

 der Papilionaceenknollchen. Centralbl. fur Bakteriologie. Bel. 15, pag. 928, 1894. 



-) Ueber eine farblose Bakterie, deren Kohlenstoffnahrung aus der atmospharischen 

 Luft herriihrt. Centralbl. f. Bakteriologie 2. Abt. Bd. 10, pag. 33, 1903. 



8 ) Bacillus danicus, T. Westermann and F. Lohnis, Centralbl. f. Bakteriologie, 

 2. Abt. Bd. 22, pag. 250, 1909. 



4 ) The wonderful experiments of Maze (Annales de 1'Institut Pasteur T. 11 

 pag. 44, 1897, T. 12, pag. i and pag. 128, 1898), who asserts that on broth gelatin plates 

 at the same time ammoniumcarbonate is produced and fixation of free nitrogen by 

 B. radicicola takes place, need not be considered, although they are taken up uncritisised 

 in the handbooks of Plantphysiology. 



