24 THE ROYAL SOCIETY OF CANADA 



The two solutions are mixed in equal volumes immediately before 

 use. This is an exceedingly delicate reagent for nitrites and gives no 

 reaction with nitrates. In use 5 drops of the reagent were added to 

 2cc. of the culture under examination. If nitrites were present a 

 distinct red colour generally developed immediately. If no red tinge 

 appeared after five minutes nitrites were certainly absent. 



For ammonia. Nessler's reagent. 



5 drops added to 2cc. of culture under examination. 



For nitrates. Either Diphenylamine or Brucine. The former 

 reagent is prepared by dissolving -5 gram of diphenylamine in lOOcc. 

 of concentrated sulphuric acid and adding 20cc. of water. The latter 

 by dissolving -2 gram of Brucine in lOOcc. of concentrated sulphuric 

 acid. 5 drops of either of these reagents were added to Ice. of culture 

 mixed with 2cc. of concentrated sulphuric acid. 



The nitrate tests could only be applied when a previous test 

 had shown nitrites absent. There is no satisfactory qualitative test 

 for nitrates in aqueous solution in the presence of nitrites. 



The samples were withdrawn from the culture flasks for testing 

 with small sterilized pipettes and all the tests were carried out under 

 like conditions. 



In no case was there any direct evidence of denitrification. 

 Nitrogen compounds were still present in quantity in the culture 

 solutions kept at 28°C after three weeks and in the cultures kept at 

 air temperature after three months. No evolution of gas was visible 

 at any time in any of the cultures. It is not possible to state definitely 

 that absolutely no denitrification took place since no quantitative 

 determinations of nitrogen were carried out. Facilities for this were 

 not available and no really reliable method for determining nitrogen 

 compounds in sea-water exists; but, judging by the intensity of the 

 qualitative reactions at the final tests, and the complete absence of 

 any bubbling in the cultures, denitrification, if it had taken place at 

 all, must have been insignificant. The nitrates and nitrites would 

 no doubt have ultimately disappeared from the medium containing 

 no peptone since bacterial growth was still active in the cultures 

 when last examined and the bacteria had in this case no other source 

 of nitrogen to draw upon. The disappearance of nitrogen in this way 

 is, however, of the same significance, from the point of view of the 

 nitrogen cycle in the sea, as the conversion of nitrate to nitrite or 

 ammonia, the nitrogen is still retained in the cycle to become available 

 on the death of the bacteria. 



