34 THE ROYAL SOCIETY OF CANADA 



Medium D 



Glucose 2*0 grams 



Potassium phosphate (K2HPO4) -2 gram 



Sea-water 1000 cc. 



Medium E. 



Same as Medium D with Mannose substituting Glucose. 



Medium F 



Same as Medium D with Glycerine substituting Glucose. The 

 media were boiled, made just alkaline to phenolphthalein, and filtered 

 before sterilization. As in the previous- instances each sample of 

 sea-water was inoculated with each medium and a set of each kept at 

 28°C and air temperature respectively. 



The media were all perfectly clear after 3 months; no bacterial 

 development appeared to have taken place and it is exceedingly 

 unlikely that any nitrogen-fixation had occurred. Facilities were not 

 available for confirmation of this by chemical means, but, in the absence 

 of any sign of growth in the culture, this seemed hardly necessary. 



So far as they go then these experiments point to the absence of 

 any free-living aerobic nitrogen-fixing bacteria in the samples examined. 

 It would, however, be interesting to try other carbon compounds in 

 the media, since those used in these experiments may not be sufficiently 

 near in composition to those which result from the break down of 

 marine plants and animals and are used naturally by nitrogen-fixing 

 bacteria in the sea if such exist. It would also be particularly inter- 

 esting to look for them in waters where denitrification occurs, since 

 here, not only does nitrogen-fixation become a process of considerable 

 significance inasmuch as it may modify the apparent loss of nitrogen 

 occurring, but also, since there would be an abnormally large amount 

 of free nitrogen dissolved in the water, conditions would seem to be 

 good for the development of organisms capable of its assimilation. 



Characterization of the Bacteria Isolated 



Broth Cultures 



In order to more closely characterize the bacteria isolated in the 

 course of this work the cultures obtained were inoculated in the first 

 place into broth made up with : — 

 (a) Tap-water. 



{h) Tap-water with sufficient sodium chloride added (about 

 3 per cent) to make it up to the density of the sea-water used 

 during the experiments. 



