Jan. 28, i9i8 Nitrogen-Assimilating BacteHa 203 



trast to the work of Stoklasa, both strains of Azotobacter assimilated 

 more atmospheric nitrogen in the presence of nitrates in solution than 

 in the absence of these salts. 



INFLUENCE OF NITRATES ON THE PRODUCTION OP PIGMENT BY AZOTOBACTER 



It has already been noted in the experiments dealing with the effect 

 of nitrates on the fixation of atmospheric nitrogen on agar films that 

 nitrates favor pigment production. This was true in the case of both 

 strains of the Azotobacter. 



Moreover, it has been observed by other investigators that Azotobacter 

 when grown in the presence of nitrate will produce a darker pigment 

 than when grown in its absence. Beijerinck (4, p. 575) states that 

 Azotobacter in pure culture will form a dark-brown pigment in the 

 presence of glucose and a small amount of nitrate. Sackett {43) found 

 that nitrate caused an increase in pigment production by Azotobacter. 

 In media without the nitrate the pigment formation was materially 

 decreased and in some cases practically eliminated. He also noted that 

 the amount of nitrate present has a direct influence on the intensity of 

 the pigment formation. He found that when sodium nitrate was added 

 to a suitable medium to give a content of 0.0, o.oi, 0.03, 0.05,0.08, o.i, 

 0.3, and 0.5 per cent, with glucose used as the source of energy, the 

 organisms produced pigment. Streak inoculations were made, and 

 after 14 days' incubation he found that the maximum of color was 

 obtained at 0.05 to 0.08 per cent and that greater concentrations did not 

 increase the intensity of the brown-black pigment. From his results it 

 is evident that sodium nitrate caused an increase in pigment formation 

 by azotobacter. 



In order to determine the possible effect of potassium, sodium, and cal- 

 cium nitrate on pigment formation with strains A and B, the following 

 experiment was performed. 



Under normal conditions on mannit agar free from nitrate strain A 

 produced little or no pigment even after three weeks' growth. At the 

 end of this time dirty-yellow streaks occurred throughout the growth, 

 but no brown pigment was produced. However, with strain B at the 

 end of two or three weeks a decided brown to brown-black pigment was 

 produced in the absence of nitrate. 



Agar slope cultures containing increasing amounts of potassium, so- 

 dium, and calcium nitrate, as indicated in Table XVII, were prepared. 

 These were inoculated with both strains of Azotobacter and incubated 

 at 28° C. for 10 days. Daily observations were made for first evidences 

 of pigment formation. In some of the cultures of strain A growing on 

 media containing calcium nitrate this pigmentation was observed as 

 early as 48 hours subsequent to inoculation. The following day pig- 

 mentation developed in strain B. The cultures on the potassium and 

 sodium-nitrate media began to show evidence of pigmentation in four to 

 six days. The final results, obtained after 10 days' incubation, are found 

 in Table XVII. 



