BACTERIA FIXING ATMOSPHERIC NITROGEN 129 



second and a fourth tube from the third; the plates are poured and incubated 

 at 20° to 25°C. The organism is isolated upon sterile agar slants or liquid 

 media from a typical colony upon the plate, using the third and fourth plates 

 and discarding the first two. The lens-shaped and pin-head colonies should be 

 selected rather than the giant colonies. In case of questionable plates, replating 

 may be necessary from the culture isolated. To keep the cultures in stock, 

 one of the above agar media (ash or mannite agar) may be used. 



Isolation from soil. The Bad. radicicola can readily spread through 

 the soil 127 and persist there for a long period of time. The bacteria 

 move in the soil at a definite rate. 128 Bad. radicicola can also be cul- 

 tivated from the soil, although the specificity of the forms isolated by 

 Nobbe and Hiltner and Gage 129 has not been sufficiently demon- 

 strated. The results of Greig-Smith concerning the great abundance of 

 Bad. radicicola in the soil were not confirmed. The numbers of each 

 strain in the soil depend upon the reaction of the soil, an acidity 

 greater than pH 5.4 being detrimental to the development of most 

 strains; at a favorable reaction (pH 5.4-6.8, depending on strain), as 

 many as 100,000 to 1,000,000 cells of different strains may be found 

 per gram of soil. 130 Kellermann and Leonard 131 could isolate the 

 organism only from soils sterilized and previously inoculated. Lip- 

 man and Fowler 132 isolated Bad. radicicola from soil, in which le- 

 gumes have previously been grown, and demonstrated its ability to 

 cause the formation of nodules on the roots of leguminous plants. 

 Two media were employed: (1) 1000 grams of water, 10 grams 

 maltose, 1 gram K 2 HP0 4 , 1 gram MgS0 4 , 2 to 3 drops each of 10 per 

 cent solution of NaCl, FeCl 3 , MnS0 4 , and CaCl 2 and 15 grams of 

 agar. (2) Soil extract, obtained by boiling one part of soil with 

 three parts of water for one hour, then filtering and adding 15 grams of 

 agar and 10 grams of maltose to 1 liter of the extract. A soil in which 



127 Ball, O. M. A contribution to the life history of Bacillus (Ps.) radicicola 

 Beij. Centrbl. Bakt. II, 23: 47-59. 1909. 



128 Kellerman, K. F., and Fawcett, E. H. Movements of certain bacteria in 

 soils. Science, 25: 806. 1907. 



129 Nobbe, Hiltner and Schmid, 1895 (p. 134); Gage, G. E. Biological and 

 chemical studies on nitrosobacteria. Centrbl. Bakt. II, 27: 7-48. 1910. 



130 wn S on, J- K. Legume bacteria population of the soil. Jour. Amer. Soc. 

 Agron. 18:911-919. 1926. 



131 Kellerman, K. F., and Leonard, L. T. The prevalence of Bacillus radici- 

 cola in soil. Science, n. s. 38:95-98. 1913. 



132 Lipman, C. B., and Fowler, L. W. Isolation of Bacillus radicola from 

 soil. Science, N. S. 41: 256-259, 725. 1915. 



