Febeuakt 12, 1915] 



SCIENCE 



257 



to quote Eussell/ " none of these organisms 

 (B. radicicola), however, could be found in the 

 soil, nor indeed has any one yet succeeded in 

 finding them there, although their existence 

 can not be doubted." In the literature avail- 

 able to us we have found but one instance in 

 which a claim is made of the direct isolation 

 of B. radicicola from soil not artificially inocu- 

 lated. That one is the investigation of Gage^ 

 who has himself rendered questionable the 

 value of his work by an unfortunately con- 

 fused use of terminology which has only served 

 to make more difficult than otherwise a com- 

 prehension of the present status of the subject. 

 Kellerman and Leonard^ in studying Greig- 

 Smith's claim to having discovered a specific 

 medium for B. radicicola could not find ex- 

 perimental evidence to confirm it. Inci- 

 dentally the last-named investigators tried to 

 obtain B. radicicola from different soils some 

 of which grew legumes but were unsuccessful 

 in the attempt except in the case of one soil 

 into which pure cultures of B. radicicola had 

 been introduced after its isolation from 

 alfalfa nodules. 



While not deeming the matter one of great 

 moment in any sense, since there can be no 

 doubt, as Russell remarks, that B. radicicola is 

 present in any soil in which nodules are found 

 on legumes, the writers decided to attempt the 

 isolation of that organism, and, as a matter 

 of record, submit this brief paper in evidence 

 of the success of their attempt. One of us 

 had for three or four years used as a source 

 of B. radicicola for student work in the labo- 

 ratory the nodules of a large specimen of Vicia 

 sicula growing in the Botanic Gardens on the 

 campus of the University of California, and 

 we therefore decided to attempt the isolation 

 of B. radicicola from the soil in which that 

 plant had grown. The plant had been removed 

 a year or more prior to our initiation of the 

 experiment and the soil had remained bare 

 and unused during that time. Seeds from the 

 plant in question were scattered aU over the 



1 ' ' Soil Conditions and Plant Growth, ' ' D. Van 

 Nostrand Co., 1912, p. 95. 



2 Cent, fur Bakt., 2*° Abt., Vol. 27, p. 7. 



3 Science, N. S., Vol. 38, p. 95. 



surface of the ground, and we gathered them 

 for the later tests which are described below. 

 The soil, so far as we can ascertain, had never 

 been artificially inoculated with cultures of 

 B. radicicola. 



Some of the soil just described was taken 

 from below the surface at a depth of about six 

 to eight inches, placed in a sterile container 

 and removed to the laboratory. About 30 

 grams of the soil were there placed in a sterile 

 bottle, 150 c.c. of sterile water added, and the 

 whole shaken, after being stoppered, for fifteen 

 minutes. The necessary dilutions were then 

 made for purposes of pouring plates. The 

 agar employed at first was of two kinds. The 

 first was similar to that employed by Fred 

 and was constituted as follows : 



1,000 grams water 

 10 grams maltose 

 1 gram KoHPO^ (separately neutralized) 

 1 gram MgSOj 

 2 or 3 drops each of 10 per cent, solutions of NaCl, 

 FeCls, MnSOi and CaCl^ 

 15 grams agar -agar. 



The second was a soil extract agar prepared 

 by dissolving 15 grams of agar and 10 grams 

 of maltose in an aqueous extract from the 

 soil above described. The aqueous extract was 

 obtained by boiling one part of soil with three 

 parts of water for one hour and filtering. 



In the preliminary tests the soil extract agar 

 gave by far the better results with both the 

 soil to be studied and with commercial cul- 

 tures of B. radicicola which were employed as 

 controls. By better results we mean that a 

 larger number of colonies developed on the 

 plates poured with the soil extract agar than 

 on those prepared with Fred's radicicola agar. 

 In the later work therefore the soil extract 

 agar was employed exclusively. 



From plates of the proper dilution prepared 

 as above described transfers were made to soil 

 extract maltose agar slants by means of a plati- 

 num needle from all colonies which appeared to 

 be characteristic of B. radicicola and in fact of 

 any others which appeared to be different 

 from one another. Transfers were thus made 

 from forty-four colonies. After three or four 

 days of growth on the slants, slides were pre- 



