EXPERIMENT STATION BULLETINS. 285 



Although the beef extract used in this formula contains approximately .11% N, 

 this amount is considerably lower than that in media to which peptone has been 

 added as a nutritive element, consequently this medium comes nearer to the 

 class of nitrogen-free media than the previous one, which contains peptone. 



When a culture is desired for securing inoculations upon soil or seed, the 

 organism should be isolated from the plates at once to a nitrogen-free nutrient 

 solution, where growth takes place more slowly, but where the nitrogen assimi- 

 lating power is retained and increased, according to Moore. Among a number of 

 nitrogen-free preparations tested, solutions A and B as follows, gave the most 

 luxuriant growth: 



Solution A — 



Glucose 36.21 gr. 



MgSO, 18 gr. 



KH.PO, 3.62 gr. 



Tap water 1,000.0 cc. 



Solution B — 



Glucose 10.0 gr. 



Mg(POJ, 5 gr. 



Tap water 1,000.0 cc. 



The range of temperature at which this organism will grow is quite wide, 

 being from 7°C. to 38°C. 25°C., however, is the temperature best suited to a 

 luxuriant, characteristic development. Above 25°C. very rapid drying out of the 

 culture takes place. This is especially marked in slant agar cultures where a 

 wrinkling of the growth also ensues, which interferes with the development of 

 branched forms and also with the vitality of the organism. 



Variation of temperature, with absence of moisture, has little or no effect, how- 

 ever, upon the vitality of the organism when within the nodule itself. To establish 

 this fact, a number of sound cowpea nodules were collected in October, 1904, 

 washed sterilized with 1:500 HgCL as for isolating, and then dried under sterile 

 conditions. Three of these nodules of a uniform size were then selected and 

 placed one each in sterile tubes numbered 1, 2 and 3. 



A culture was made from No. 1, and motility, number, and cultural vitality 

 of the organism noted. The remaining tubes, Nos. 2 and 3, were placed near 

 steam connections at a temperature of 40°C. In 21 days No. 2 was tested in the 

 same manner as No. 1, and after 42 days No. 3 was tested as were Nos. 1 and 2. 

 The results of these tests showed that the constant high temperature had no 

 apparent detrimental effect on the vitality, motility, and number of organisms 

 present. 



Parallel with the above experiment, cultures were grown on solid artificial 

 media, in tubes, at a temperature of 37.5°C. for 21 and 42 days respectively. A 

 heavy growth occurred which soon became dry and shriveled, and when trans- 

 fers were made to fresh media at the end of 21 and 42 days, the organism made 

 a slow, weak growth, possessing but little vitality. This weakened condition was 

 remedied by making a series of transfers on fresh media at a temperature of 25°C. 



The effect of freezing has not been fully worked out, but the following observa- 

 tion shows that the organism, when left within the nodule as it grew in the field, 

 has considerable resistance to frost. On March 25, 1905, a nodule-laden root of 

 soy bean w^as pulled from an inoculated experimental plot at the College. Some 

 of these nodules were still intact, and contained a very active culture of the soy 

 bean organism. The hanging drop revealed marked motility and plates made from 

 the same nodule developed the characteristic tree-like colony. 



The colony first appears as a small, round spot, which soon grows up through 

 the medium to the surface. Upon reaching the surface, it spreads out at a very 

 rapid rate, and takes on a peculiar characteristic tree-like form. If colonies are 

 crowded in the plate, this characteristic growth is not so apparent and one might 

 question the source of a thick plate, while the greater dilution plate would develop 

 the colony true in character. 



Because of the rapid spreading peculiar to this colony, fewer contaminations 

 are met with if greater dilutions are made from the original tube which contains 

 the nodular material. 



Colonies from different species of legumes vary but little in general appearance. 



