38 THE ROYAL SOCIETY OF CANADA 



Gelatin Stab Cultures 



A gelatin medium was prepared by adding 12 per cent of gelatin 

 to the broth medium. It was made just alkaline to phenolphthalein. 

 Stab cultures were made from each of the agar cultures into this 

 medium and kept at 18°C. Three degrees of liquefying power were 

 noted in the various cultures. These are designated by 1, 2 and 3 

 in the following table: 1 indicates that the medium was almost 

 entirely liquefied after 2 weeks, 2 indicates a slight liquefaction, and 3 

 that no liquefaction occurred at all. In the last cases the organisms, 

 though they all grew both at the surface and along the track of the 

 needle, developed no particularly characteristic manner of growth. 



Denitrification Media 



Each of the agar cultures was subcultured into each of the two 

 denitrification media (Media B and C respectively) and incubated at 

 25°C. All the cultures in Medium B, except X, XII and XIV, 

 showed growth accompanied by varying degrees of precipitation of 

 calcium carbonate after 4 days. After 17 days this precipitation had 

 increased considerably in some of the cultures, particularly at the sur- 

 face and particularly in the cases of Cultures lA, III and XXII. 

 Each of the cultures was tested at this stage for alkalinity to phenol- 

 phthalein, for ammonia and for nitrite. None of them gave any 

 reaction for ammonia. All the cultures in Medium C showed growth 

 and precipitation after 2 days; they had developed a strong turbidity 

 and heavy precipitate, accompanied in some cases by a scum and a 

 ring of calcium carbonate on the walls of the tube at the surface of 

 the culture, after 10 days. They were tested at this stage for alkalinity 

 to phenolphthalein, for ammonia and for nitrite. All of them showed 

 the presence of ammonia. 



The results of the tests for alkalinity to phenolphthalein and for 

 nitrite are summarized in the following table, + indicating a positive 

 reaction, — a negative one. The uninoculated tubes incubated with 

 the cultures showed no deposition of calcium carbonate and no alkalin- 

 ity to phenolphthalein. The definitely alkaline reaction which some 

 of the cultures produced in Medium B may be due to a limited denitri- 

 fication having taken place or to the production of normal calcium 

 carbonate in the solution by oxidation of the calcium acetate. The 

 latter explanation seems most probable since the strongest alkaline 

 reaction was set up in some cases where no trace of nitrite was found 

 and there was not the faintest indication of bubbling in any instance. 

 The precipitation of calcium carbonate to some extent in all these 

 cultures, and in some cases quite heavily, brings out very definitely 



