266 



v/ater 100, agar 2, cane sugar i, starch i, bipotassium-phosphate 0,05, in which, be- 

 cause of the albuminous matter of the agar, enough fixed nitrogen is present to cause 

 a distinct growth of B. radicicola, but the colonies remain small. Later a little salt- 

 petre or ammoniumsulphate may be added locally, which makes the tubercle bacteria 

 like the other saprophytes thrive well, showing that they do not assimilate the free 

 atmospheric nitrogen. If on such a plate eventually germs of Azotobacter, which is 

 able to assimilate free atmospheric nitrogen, are present, these will grow quite well 

 if no nitrogen compounds are added. Such nitrogen-poor plates are also useful to 

 recognise the spore-bearing soil bacteria, which almost constantly appear at the 

 isolation of B. radicicola. 



I only call tubercle bacteria those species which develop mutually identic colonies 

 by thousands or hundreds of thousands from the externally well-sterilised and cau- 

 tiously crushed nodules. These bacteria derive for the greater part from within the 

 cells. I consider 'the deviating and less numerous colonies obtained at the culture ex- 

 periments as the product of germs accidentally present in the intercellular cavities of 

 the rind of the nodules 1 ). That the full-grown bactero'ids cannot develop on the plates 

 is well-known; hence bacteria may be expected from the tubercles only in the begin- 

 ning of their development. 



It is an important and until now not yet sufficiently investigated circumstance 

 that from the tubercles of the same plant not always the same bacteria are obtained. 

 So I found for Ornithopus perpusillus the bacteria I had isolated in March different 

 from those grown in October, whilst the tubercles came from plants growing side by 

 side and being in the same state of development. With the yellow lupine and serra- 

 della I had similar results. In most other cases, however, for example with 

 Pisum, Lathyrus, Vicia, and Tri folium, the similarity of the various mutually in- 

 dependently isolated stocks is so complete and the image of B. radicicola can so 

 distinctly be recognised, that the above observation requires nearer confirmation. But 

 we cannot now enter upon this point. 



When trying to isolate B. radicicola from materials other than the nodules, for 

 example from the soil and from the dying surface cell-layers of the root, it proves 

 very difficult to recognise this species amid the numerous other saprophytes, especially 

 when the number of the germs of the different species is to be determined quanti- 

 tatively. B. fluorescens liquefaciens causes much trouble by the liquefying of the ge- 

 latin plates, and yet it is necessary to use these plates as on them the colonies of all 

 the species lie free from one another, while on agar they are overgrown and rendered 

 unrecognisable by B. fluorescens, which extends strongly sideways. 



Concerning the question if only one or more species of tubercle bacteria exist 

 the following. 



Already in 1892 experiments thereabout were made by the late H e 1 1 r i e g e 1 -) 

 in the experimental station at Bernburg with pure cultures of the bacteria made by 

 myself at Delft. Of his results H e 1 1 r i e g e 1 sent me two reports. In the first, dated 

 24 July i92, he gives as Augenblickliches Hauptresultat : Es gelingt mit den 



*) Besides B. radicicola B. herbicola can also occur within the living cells. 

 ) He died 24 September 1805 of a stomach disease and was already suffering when 

 I visited him at Bernburg in 1892. 



