[HARRISON & BAELOw] NODULE ORGANISM OF THE LEGUMINOSAE 181 



FlasJi VII, containing '250 grams of medium RS2, was planted Nov- 

 ember 29t]i, 1905, with the germinating pea seeds. It was not inocu- 

 lated, no bacteria were present and no nodules developed. 



One mould colony developed by December 12th, 1905. This colony 

 increased in size, and by May 4th, 1906, it covered, perhaps, one-fourth 

 of the surface of the agar with a thin, zoned growth. 



One of the peas formed no roots and died later, the other grew 

 well, especially the roots. The energy of the plant seemed largely spent 

 in root formation. The roots were long and slender and smooth with- 

 out evident root hairs. The stem grew well, but after several months 

 began to decline, so that, though growth continued, (the jleaves and 

 stems remained green), it did not increase in size. This excessive root 

 formation and this decline in the growth of green parts were attributed 

 to nitrogen stan^ation. These phenomena were reversed in the case of 

 the inoculated plants, i.e., the growth of stem and leaves was progressive 

 without decline and was in excess of the growth of root. 



Stains were made May 7th, 1906, from the surface of the agar and 

 from the water of condensation in the bottom of the flask. Spores of 

 a mould were numerous, but there were no bacteria. The stem of the 

 plant that died had been invaded by the mould. Its juice was not 

 turbid and on staining its tissue the mycelium of the fungus was found 

 l.'ut no bacteria. The roots of the living plant were washed, crushed 

 on a slide and stained, and no bacteria nor fungi were found in them. 



Plate cultures in gelatin and ash-maltose-agar were made May 7th, 

 1906, from the agar in the flask and from the crushed roots of the 

 living plant. ISTumerous colonies of a mould like Pénicillium glaucum 

 developed in all the plates, but no bacterial colonies. The plates were 

 observed for twenty-four days. Photograph 40, ISTo. 1, taken February 

 20th, 1906, illustrates this flask culture. 



11. Preparation and Distribution of ^Sitro-cultures. — Preparation 

 should be made in advance of the season. The medium can be made 

 up early in the spring. It is a great convenience to have attached to 

 the steam heating system a large sized autoclave, or a retort such as is 

 used in canneries. A large sterilizer supplied with flowing steam is 

 almost a necessity. Large enamel pails can be used for making up 

 media, and a vacuum pump attached to the water pump is necessary 

 for rapid filtration. If the bottles are new, it is sutBcient to rinse 

 them in cold water before filling. With a little experience one man 

 can easily prepare the medium, fill and sterilize a gross and a half of 

 bottles in a day. 



Preparation of ash-maltose-agar for commercial cultures. — Add 10 

 parts by weight of wood ashes to 100 parts of cold tap water and stir 



Sec. IV., 1906. 12 



