188 



fixing microbes can be removed by tliorouglily washing of the nodules 

 with alcohol and water. In the course of many jears I have experi- 

 mented in this way with numerous species of tubercle bacteria, 

 and with many modifications in the nutrient media as well in the 

 teinpei'ature as in the source of carbon. Moreover I have, as said, 

 tried to grow pure cultm-es of the bacteria themselves in the liquid 

 culture medium as also on solid culture soils of various compositions, 

 and at tirst 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 bac- 

 teria derives from the formation of thick slime walls, that is of ni- 

 trogenfree, cellulose-like substances^ around the hardly augmented 

 original protoplasmic material. ') 



Only when cultivating the microbes in plant extracts with cane 

 sugar, wherein nitrogen com[)Ounds 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 Ï was 

 not yet acquainted with the circumstance that laboratory air 

 contains suftlcient carbon and nitrogen compounds to be made percept- 

 ible by the growth of microbes which can feed on them. This was 

 afterwards demonstrated by Ir. A. van Dklden and myself in our 

 investigation on Bacillus [ActiuohaciUus) oligocarbophUus.^-) 



There exists moreover an aerobic spore-producing bacterium''), hard 

 to kill by sterilisation of (he 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 cul- 

 tures 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 pei-formed, not with 



1) The slime formation is of importance for the explanation of the "slime 

 threads" (erroneously called "infection threads") within the nodules. See "Die 

 Nalur der Fiiden der Papilionaceënknöllchon." Centralbl fur Bakteriologie. Bd. 15, 

 pag. 928, 1894. 



') Ueber eine farblose Bakterie deren Kohlenstoffnahrung aus der atmosferischen 

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



") Bacillus daniciis, T. Westermann and F. Löhnis, Centralbl. f. Bakteriologie. 

 2te Abt. Bd. 22, pag. 250, 19Ü9). 



