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 sativa, Melilotus alba, Trifolium incarnatum, T. pratense, T. repens. 
Hedysareae: Desmodium acuminatum, D. canescens, D. nudiflorum. 
Vicieae: Vicia villosa, Lathyrus sativus, Pisum sativum, 
Phaseoleae: Glycine hispida, A pios 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: 
(1) 1,000 ce. distilled water; 15 grams wood ashes. 
(2) 400 cc. above solution filtered, with 4 grams of maltose. 
(3) 200 ce. 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. ‘he 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 
15 parts; 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 
ce. 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 thecenter 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 100 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. 
*Tbe maltose used by Harrison and Barlow, was probably not pure. According to Mr. Barlow it was yellowish. 
