1903.] 



On the Culture of the Nitroso-bacterium. 



359- 



Isolation of the Nitroso-hadermm. 

 Plate Cultures. 



In carrying out the work of isolation of the nitroso-bacterium I 

 made plate cultures containing silica, gelatine, and agar media 

 respective^. 



Silica Plates. 



I found that numerous species of micro-organisms grew on this 

 medium, and that, therefore, it was necessary to use what seemed to 

 be pure cultures of the nitroso-bacterium, as this species does not 

 form colonies rapidly, and is liable to be smothered by the more 

 quickly-growing bacteria if one attempts to isolate the nitroso- 

 bacterium from soil or a very impure culture. The nitroso-bacterium 

 grows well in this medium, and in one instance I was able to remove 

 a single colony which oxidised the ammonia in a solution. From this 

 culture inoculation was made into beef-broth agar, and plates poured.. 

 These plates grew large numbers of colonies in pure cultures. 



Numerous colonies were taken from silica plates and grown on beef- 

 broth agar, but such growth transferred to ammonia solutions did not 

 produce nitrification. This being the case I thought that probably 

 the micro-organism had lost its power of oxidising the ammonia, so 

 that I tried to devise a means by which this function might be re- 

 established j and for this purpose the micro-organism was placed in as 

 natural surroundings as were attainable. The following was the 

 method adopted. A single colony was taken from silica plate and 

 inoculated on to sloping beef-broth agar. After growing there it was- 

 transferred to a sterile ammonia solution ; this was allowed to filter 

 daily through sterile soil, thus allowing of aeration of the growth 

 Avhilst in its natural surroundings. This experiment succeeded, nitrite 

 being formed in 10 weeks. A control filter showed no change. 



Gelatine Plates. 



Winogradsky states that the nitroso-bacterium does not grow on 

 gelatine ; so that, in the first place, the method that he advocates to 

 obtain a pure culture of nitroso-bacteria was adopted. 



Particles of magnesia were removed from an oxidised ammonia 

 solution and sown on to gelatine plates. Now if these particles carried 

 nitroso-bacteria alone there would, if Winogradsky be correct, be no 

 growth, and such a particle showing no growth could be removed and 

 reinoculated into an ammonia solution, and thus a pure culture 

 obtained. But I found that around particles so inoculated into gelatine, 

 colonies invariably occurred. It was noted that these colonies were in 

 pure culture and were made up of an oval organism that was morpho- 

 logically similar to the nitroso-bacterium. 



