185 



nodules only, requires reconsideration. Also oilier experiences make 

 this reconsideration necessary. But previously a few remarks on the 

 isolation of the bacteria from the nodules and from other materials, 

 and on the question of their specificity. 



A very convenient medium for isolation was already described in 

 1888, ') namely pea leaves- or clover-extract-gelatin with 2 7o ^'^^ne 

 sugar. B. radicicola grows thereon in soft, white, non-liquefying 

 colonies, while B. oriiithopod is h'oxw Ornifhoims perpusillns, (). satiuus 

 or Lupums luieus, when isolated in the autumn or in March, 

 liquefy somewhat, as does B. herhicola. ^) 



As a solid medium, poor in nitrogen compounds, I recommend a 

 plate of: Tapwater 100, agar 2, cane sugar 1, starch 1, bipotassium- 

 phosphate 0,05, in which, because 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 saltpetre 

 or ammoninmsulphate may be added locally, which makes the 

 tubercle bacteria like tiie other saprophytes thrive well, showing 

 that they do not assimilate the free atmospheric nitrogen. If on such 

 a plate eventually germs of Azotohacter, 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 extei'- 

 nally well-sterilised and cautiously 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 experiments 

 as the product of germs accidentally present in the intercellular cavities 

 of the rind of the nodules. ') That the full-grown bacteroïds cannot 

 develop on the plates is well-known ; hence bacteria may be expected 

 from the tubercles only in the beginning 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 \ found for Oimithopiis perpusillns 



5) Botan. Zeitung. i888 Pag. 764. 



3) Occasionally a great number of colonies of B. herbicola are obtained from 

 the tubercles ; whoever is unacquainted with this species may make mistakes in 

 the isolation of B. radicicola. But even with this knowledge the isolation of 

 serradella- and lupine-bacteria is difficult. Good descriptions of these forms do 

 nol -exist. 



') Besides B. radicicola B. herhicola can also occur ivithin the living cells. 



