139 



In 1893 this problem attracted the attention of another leader of 

 microbiological thought, WINOGRADSKY, and thus hereby the prospects 

 for a solution might be deemed to be bright. 



In the foregoing years WINOGRADSKY had forged a new tool for 

 microbiological work, to wit, the principle of the elective or enrich- 

 ment culture, and immediately applied this principle with unpreced- 

 ented success in his researches on the sulphur bacteria and the nitri- 

 fying bacteria. 



WINOGRADSKY 1 ) very naturally decided to proceed in the same way 

 in his efforts to identify the nitrogen fixing bacteria present in soil. 

 He, therefore, prepared culture media free as far as possible from all 

 nitrogen compounds, but containing all other necessary elements, 

 with glucose as a source of carbon and energy, and, moreover, an 

 excess of calcium carbonate. The medium was poured in a thin layer 

 (8-9 mm) in conical flat-bottomed flasks, and after the medium had 

 been inoculated with some soil, a stream of purified air was passed 

 over the cultures. It will be clear that under these conditions luxuriant 

 growth in the medium, especially after a number of transfers to 

 identical media had been made, could only be due to organisms fixing 

 gaseous nitrogen. 



In his extensive memoir on the subject which appeared in 1895, 

 WINOGRADSKY indeed succeeded in identifying the organism which 

 predominated in his cultures and found it to be a strictly anaerobic, 

 spore-forming bacterium which provoked a typical butyric acid fer- 

 mentation 2 ). On the ground of its close relation with other butyric 

 acid bacteria the name Clostridium Pastorianum was given to the 

 new species. Apparently the development of this anaerobic organism 

 in the enrichment cultures had only been made possible by the simul- 

 taneous presence of other bacteria of an aerobic nature in the medium. 

 The pure culture did not develop at all under the conditions of the 

 enrichment culture, that is, in the presence of air. Its nitrogen fixing 

 power was, however, proved beyond doubt, by replacing the air by 

 pure nitrogen. After doing so, a gain in the nitrogen content of the 

 medium could be established with certainty. 



WINOGRADSKY was also able to demonstrate the wide distribution 

 of his Clostridium Pastorianum in soils of very different origin. By 

 these investigations the question of the nitrogen fixation in arable 

 soils seemed to be solved. 



It is impossible to indicate the reasons which made BEIJERINCK five 

 or six years later decide to raise the matter anew. But in a paper 3 ) which 

 was first published in 1901 BEIJERINCK opened his introduction with 

 the more or less startling remark: 



') Cf. S. WINOGRADSKY, Compt. rend. d. 1'Acad. d. Sc. 116, 1385, 1893; Ibid. 118, 

 353, 1894. 



2 ) S. WINOGRADSKY, Arch. d. sciences biol. publ. par 1'Instit. imp. d. med. exp. 

 a St. Petersbourg 3, 297, 1895. 



3) Centralbl. f. Bakt. u. Parasitenk. II, 7, 561, 1901. Later also in: Arch, neerl. d. 

 sciences exactes et naturelles Ser. II, 8, 190, 1903. 



