SOIL INOCULATION 823 



As an agar medium, the following 20 may be employed: 



Mannite 10.0 grams CaC0 3 1.0 gram 



K 2 HP0 4 0.5 gram Distilled water 900 cc. 



NaCl 0.2 gram Agar 15.0 grams 



MgS0 4 -7H 2 0.2 gram Reaction pH G.8 



CaS0 4 -2H 2 0.1 gram Sterile yeast water .. . 100 cc. 



The liquefied agar is allowed to solidify in the form of slants on the 

 broad side of the flat square bottles; the solidified agar is then inoculated 

 with a few drops of a vigorous liquid culture or a suspension of a solid 

 culture in distilled water. The bottles are incubated at 28°. Each 

 culture is sufficient for the inoculation of one acre. For the preparation 

 of large quantities of medium, the following method may be employed: 21 

 175 grams of agar are dissolved in 3000 cc. of water, by placing it in the 

 autoclave at 10 to 15 pounds pressure; 2.25 pounds hard wood ashes are 

 boiled in 1000 cc. of water and filtered; 0.5 gram KH 2 P0 4 , 0.5 gram 

 MgS0 4 , 0.5 gram NaCl, 0.25 gram CaS0 4 . 2H 2 and 6.25 grams CaC0 3 

 are placed in 1000 cc. of hot water. The three solutions are mixed and 

 87.5 grams saccharose and 12.5 grams mannite are added. The medium 

 is placed in 1.5 ounce bottles and sterilized at 10 pounds. The bottles 

 are then inoculated with 2 cc. of a culture of the desired organism, incu- 

 bated seven days, and then distributed. 



The use of good fertile soil, which has been previously sterilized, for 

 the cultivation of Bad. radicicola was found 22 to give very good results 

 both for the propagation of the organism and as a culture for distribu- 

 tion. The growth of the nodule-organism on nitrogen-rich media does 

 not destroy the infecting power of the organism. 23 A sandy soil to which 

 some decomposed organic matter is added is air dried, then placed in 

 ten pots and sterilized for two hours at 100°C. Water is then added to 

 the pots to bring the soil to optimum moisture and the soil inocu- 

 lated with a suspension of the culture in Ashby's solution. A few cc. 

 are used for inoculating each pot. Soil cultures last much longer than 



20 Fred, E. B., Peterson, W. H., and Davenport, A. Fermentation charac- 

 teristics of certain pentose-destroying bacteria. Jour. Biol. Chem., 42: 175. 

 1920. 



21 Harrison, F. C. Nitro-cultures and their commercial application. Trans. 

 Roy. Soc. Canada, Ser. 3, 9: 219. 1915. 



22 Simon, J. Mitt. Okon. Gesell. Sachsen., 13: 1-27. 1908; Kuhn, A. Azoto- 

 gen, Nitragin und Impferde. Centrbl. Bakt. II, 30: 54S-552. 1911; Temple, 

 J. C. Studies of Bacillus radicicola. Ga. Agr. Exp. Bui. 120. 1916. 



23 Prucha, M. ,T. Physiological studies of Bacillus radicicola of Canada field 

 pea. Cornell Univ. Agr. Exp. Sta., Mem. 5. 1915. 



