134 BACTERIA IN RELATION TO PLANT DISEASES. 



They isolated an organism believed to be the cause of the nodules from the following species: 



Trifolieae: Medicago saliva, Melilolus alba, Trifolium incurnatum, T. pratense, T. repens. 

 Hedysareae: Desmodium acuminatum, D. canescens. D. nudiflorum. 

 Vicieae: Vicia villosa, Lathyrus sativus, Pisum sativum. 

 Phaseoleae : Glycine hispida, Apios tuberosa, Phaseolus vulgaris. 



After experimenting with various media, they settled down to the use of a fluid medium con- 

 taining water, wood ashes, and maltose. In case a solid medium was desired, agar was added and 

 occasionally monopotassium phosphate. This medium is adapted both to the host-plant and to the 

 bacterium. They state that Bacterium leguminosarum grows well in each one of the following liquid 

 media : 



(i) i ,ooo cc. distilled water; 15 grams wood ashes. 



(2) 400 cc. above solution filtered, with 4 grams of maltose. 



(3) 200 cc. of first solution with 100 cc. distilled water, and 3 grams of maltose. 



These three solutions should be heated for half an hour in streaming steam and then boiled a 

 moment over an open flame, or heated for 10 minutes at 10 pounds pressure in the autoclave. The 

 fluids are then filtered clear, put into tubes and sterilized in streaming steam or by exposure in the 

 autoclave for 10 to 20 minutes at 10 pounds pressure. The agar media is prepared in the same 

 general way as the above with the addition of agar. They state that when 1,000 cc. of water is used 

 the sugar may vary from 4 to 20 parts; the wood ashes from 0.0 to 50 parts; the agar from 7.5 to 

 i5parts; and the acid potassium phosphate, when this is used, from 5 to 10 parts. The paper should 

 be consulted for details concerning the preparation and use of the media. 



They state that the ash-maltose-agar in Erlenmeyer flasks has many advantages over quartz or 

 water culture: one of which is that the agar contains only about 1 per cent of inert material, whereas 

 the quartz contains about 60 per cent; another is that the root-hairs and the forming nodules can 

 be seen in all parts of the agar but only when next the glass in case of quartz-sand cultures. 



Growing the plant within a glass flask affords several advantages and offers few technical diffi- 

 culties. It makes possible the most rigid pure culture methods; it requires no attention beyond the 

 initial preparation; that is, the medium does not require to be restored nor renewed, even during 

 a period of growth of eight months." 



The organism was isolated from the nodule in customary ways, the surface being sterilized after 

 washing by immersion in about 20 cc. of 1:500, or 1:1,000 mercuric chloride water, to which 2.5 

 cc. of c. p. hydrochloric acid (sp. gr. 1.20) had been added. In case the nodule floats it must be 

 held under by means of a glass rod. If it is small it may remain immersed 2 to 3 minutes, but not 

 more than 5 minutes. Large nodules may remain in the solution for half an hour. The colonies vary 

 with the plant from which the cultures are made, also with the conditions of the media, and are 

 characteristic. The deep colonies are circular, elliptical, and triangular with rounded corners. When 

 they rise to the surface they take on the form of surface colonies, except that at the center they show 

 their submerged origin. Deep colonies do not grow as well as surface colonies. They are granular, 

 white by reflected light and brownish with transmitted light. The surface colonies are raised, round, 

 wet, entire, shining and white. They appear like drops of melted paraffin. At first they are gleaming 

 and transparent, then translucent, then gradually more turbid and finally opaque. At first they are 

 watery, but they may finally become very viscid. The surface colonies may attain a diameter of 

 1 to 2 mm. in 5 days and 3 to 4 mm. in 15 days. 



They state that they have failed to detect the presence of any other organism in leguminous 

 nodules. When the interior of a nodule was inoculated into media and plates were poured, either 

 the plates remained sterile or else Bacterium leguminosarum developed. Occasionally, however, mold 

 colonies appeared and other extraneous bacteria. In stab-cultures in the ash-maltose-agar, after 2 

 or 3 days at 25 C, there was a raised, circular, transparent, wet-shining growth, spreading on the 

 surface from the point of inoculation, and a filiform growth along the needle-track which sometimes 

 had fine filaments radiating from it horizontally into the agar. These filaments were shorter toward 

 the bottom of the stab. Cultures on the ash-maltose-agar in Freudenreich flasks remain alive for 

 more than a year at room-temperature. 



In ash-maltose-water media the liquid becomes turbid and a sediment forms which is not ropy 

 but diffuses on shaking and there appears on the glass a thin wide ring of growth from the surface 

 of the fluid downward. Media consisting of distilled water ioo parts, maltose* 1 part, and ashes 0.5, 



1 and 1.5 parts, and varying from neutral to 3 , is favorable to the growth, which begins usually 

 in 3 or 4 days and increases visibly for 15 days, the liquid becoming turbid and the turbidity con- 

 tinuing with the formation of a thick white layer or precipitate. When the ashes were increased to 



2 or 2.5 parts per 100, there was a less satisfactory growth, the body of the liquid remaining clear 

 and the slimy white growth taking place in the bottom of the tubes in 15 days or more. 



*Thc maltose used by Harrison and Barlow, was probably not pure. According to Mr. Barlow it was yellowish. 



