FIXATION OF ATMOSPHERIC NITROGEN 593 



to 65°C, for fifteen minutes. It withstands drying and passes through 

 a thin collodium membrane; it also resists ultra-violet light eight times 

 as much as the corresponding bacteria. To obtain the bacteriophage, 

 fresh nodules, previously sterilized on the surface, are ground up and are 

 placed in a nutrient medium. After 5 days, the turbid solution is 

 filtered through a Chamberland filter and a few cubic centimeters of the 

 clear filtrate is added to a fresh medium previously inoculated with a 

 corresponding pure culture of the nodule organism. This is repeated 

 after 10 days, diminishing every time the amount of liquid used for 

 infection. This results in an accumulation of the bacteriophage in the 

 culture, and if a few cubic centimeters of such a culture are added to a 

 culture of Bad. radicicola, the turbid culture of the latter will become 

 transparent due to the dissolution of the bacteria. When some of the 

 bacteriophage is placed upon an agar slant and a culture of the nodule 

 organism is then inoculated, the latter will grow only where the bac- 

 teriophage was absent. 



It is also possible that the plant produces bacteriolytic enzymes, 

 which hydrolize the bacterial cell liberating the available nitrogen. 

 The bacterium itself seems to produce a cellulose-dissolving enzyme, 

 by means of which it enters the root hairs of the host plant, dissolving 

 the cell wall; 138 however, this still needs confirmation. The presence 

 of oxidase in the slime of various nodule bacteria has also been 

 demonstrated. 139 



Production of gum by the nodule bacteria. In artificial cultures, Bad. 

 radicicola produces a gum which goes partly into solution and is partly 

 held by the zoogleal masses of the organism. This gum is precipitated 

 by alcohol, acetone, concentrated solutions of ammonium sulfate, 

 magnesium sulfate or ammoniacal lead acetate. It does not reduce 

 Fehling's solution, but, on heating with a dilute solution of sulfuric acid 

 (2 per cent) at 120°C. for one hour, reducing sugars are formed, indicat- 

 ing that it is of a hemicellulose nature. It contains no protein or other 

 forms of nitrogen. The gum is formed with various sources of energy 

 in the medium, such as cane sugar, glycerol, or legume extract, 140 and 



138 Hiltner, L. Uber die Bakteroiden der Leguminoseknollchen und ihre 

 willkt'irliche Erzeugung ausserhalb der Wirtspflanzen. Centrbl. Bakt. II, 6: 

 273-281. 1900. 



139 Fred, E. B. A physiological study of the legume bacteria. Va. Agr. Exp. 

 Sta. Ann. Rpt. 1911-1912, 145-173. 



140 Buchanan, R. E. The gum produced by Bacillus radicicola. Centrbl. 

 Bakt. II, 22: 371-396. 1909; Greig-Smith, R. The slime or gum of Rhizobium 

 leguminosarum. Centrbl. Bakt. II, 30: 552-556. 1911; Fred, 1911-12. 



