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If once a pellicle has formed, transfers into the said culture liquid, prepared 

 either with distilled or with tap-water, come easily and without exception to develop- 

 ment. 



2. Source of nitrogen required. 



In the above mentioned nutrient liquid we have chosen kaliumnitrate as source 

 of nitrogen. As well, however, kaliumnitrite or some anorganic ammonium salt may be 

 used. Very good results were obtained with: 



Distilled water 100 



Ammonium sulphate (or NH4 Cl) o.oi o.i 



Dikaliumphosphate 0.02 



Mineral solution* i drop 



and with: 



Distilled water 100 



Kaliumnitrite o.oi o.r 



Dikaliumphosphate 0.02 



Mineral solution* i drop. 



As both these liquids answer to the conditions of life of the microbes of nitrifica- 

 tion, the formation of nitrite or nitrate is actually to be observed when using them, 

 and when inoculating with garden-soil or with crude cultures. With the easily pro- 

 duced pure cultures of B. oligocarbophilus, of which more below, a good development 

 of the film is possible, by which experiment it can at the same time be proved, that 

 this microbe itself does not nitrify. Hence, ammonium salts or nitrites, added to excess 

 can, even for a year or longer, continue unchanged under the luxuriantly growing 

 pellicle of B. oligocarbophilus, whereas, in the presence of nitrifying ferments, they 

 completely disappear in a few weeks, being then found back as nitrates. If the fer- 

 ments of nitrification alone are present, there is no question of film-formation and the 

 nutrient solutions remain perfectly clear. 



Not only the nature of the nitrogen-furnishing substances, but also their quantity 

 can in these experiments, as already inferred in the recipes, vary between fairly wide 

 limits, and the same may be said concerning the conditions for the water culture of 

 higher and lower green plants. The limits allowable for B. oligocarbophilus, have not 

 yet been precisely fixed, but they certainly have a broader range for this organism 

 (circa o.i 10 pro mille) than for the higher plants (0.5 5 pro mille). 



By many experiments it was established, that in absence of kalium, phosphor, and 

 magnesium, a still slighter growth occurs, than when no nitrogen compounds are 

 given. Evidently B. oligocarbophilus finds in the atmosphere, in a condition fit for 

 nutrition, a quantity of nitrogen, which, although insufficient, should not be overlooked. 

 If the distilled water in the artificial solution is replaced by tap-water, a some- 

 what higher rate of organic substance is produced. As in tap-water a small quantity of 

 nitrogen compounds occur, -- here, at Delft, about 0.4 milligrams of combined nitrogen 

 per litre, - - whilst it contains the other necessary elements (phosphor and kalium, of 

 course, excepted) in an obviously favorable form for the nutrition of our mikrobe, one 

 can simply use for its culture: 



Tap-water 100 



Dikaliumphosphate 0.02 



