54 THE ROYAL SOCIETY OF CANADA 



test. As the cultures get older, up to several months, there action to 

 the test gets slightly stronger. This nitrate is retained almost alto- 

 gether in the bodies of the organisms. Cultures filtered through 

 Berkefeld filter gave only a trace of nitrate in the filtrate and a strong 

 reaction in the mass of organisms which did not pass through the filter. 

 The filtrate plated out showed that some of the organisms had passed 

 through the filter. But as it took about ten days to filter enough for 

 a test, it is possible that the organisms had grown through the filter in 

 that time. Probably the presence of a small number of organisms in the 

 filtrate was responsible for the trace of nitrate in the test. Mass 

 growths on Ashby's agar when mature gave a strong nitrate reaction. 

 Ammonia was occasionally found when cultures were treated with 

 Nessler's solution, but in such cultures some contaminating denitrify- 

 ing rod bacteria proved to be present. Nitrites have not been found 

 in any test made. 



Relation to the Atmosphere. 



Thorough aeration is essential for growth in any culture media. 

 In cultures sealed up after inoculation, there is no development. If 

 later such cultures are unsealed, growth takes place. If cultures are 

 allowed to develop a little and then are sealed up, growth ceases. 



Spore Formation. 



In an article in the Centralblatt fur Bakteriologie, March, 1912, 

 Prazmowski of Krakau states that spore formation is common to the 

 Azotobacter. He says that several are produced in a cell, and that 

 these are responsible for the irregular packet and sarcinae forms found 

 in mature cultures. Observations of cultures of these four varieties 

 leads to a different conclusion. The packet forms and irregular clusters 

 appear to be formed by simple fission of the cell. They develop, so 

 far as observed, only when there is a mass of growth and when the 

 nutrient material is not very accessible to the cell, and when the cell is 

 more or less hedged about by its neighbors. As already described in 

 the case of Ai and Aj, clusters of cells are commonly produced by fission 

 within a single capsule. There is a possibility that the granules of 

 the No. 2 type, already referred to as arising from a splitting-up of the 

 supposed nuclear body, act somewhat as gonidia spores, and that when 

 the cell disintegrates in actively growing cultures, these gonidia de- 

 velop into new cells, proceeding to multiply by simple fission. Possibly 

 if these are retained in the mother cell owing to failure of the mother 

 cell to disintegrate, they will later appear as the endospores of Praz- 

 mowski. 



