753 



reasonably pure and their nitrification of ammonia active, to proceed 

 to isolate the several organisms and study the characters and especially 

 the nitrifying power of each with pure cultures. All previous attempts 

 to isolate the organisms had been by means of gelatin cultures. He 

 reasoned that as no organisms had been secured capable of producing 

 nitrification in any considerable degree, that probably the organism 

 causing this phenomenon, like many others, would not grow in gelatin. 

 If this was the case other methods of culture must be adopted for its 

 isolation. He began by working with two soils, one rich in organic 

 matter, the other poor in organic matter but having large amounts of 

 carbonates. He studied the cultures in media said to be favorable to 

 the nitrification and unfavorable to reduct'on, and by continuing the 

 cultivation in these media he was able in time to exterminate those 

 forms to which the conditions of nitrification were unfavorable. When 

 no further changes were noticed in the bacteria in the solutions he iso- 

 lated the forms there present, and studied them separately with regard 

 to their ability to nitrify. He found that the presence of organic matter 

 in the culture media hindered nitrification, and he therefore used media 

 composed entirely of inorganic salts and pure water. The medium 

 finally settled upon was composed of 1,000 grams of water from Lake 

 Zurich and 1 gram each of ammonium sulphate and potassium phos- 

 phate, to which was added 0.5-1 gram of basic magnesium carbonate 

 per 100 c. c. of solution. When this culture medium was inoculated 

 with material from a culture in which nitrification had taken place, 

 the nitrification was so energetic that after 15 days every trace of 

 ammonia had disappeared ; while the same solution without inoculation 

 showed only a slight nitric acid reaction after 2 weeks. 



Cultures were continued and repeated in this medium. The number 

 of species grew less and less and after three months cultures were 

 obtained which could be regarded as containing only those species which 

 were adapted to the conditions thus provided and might be expected 

 to remain constant under these conditions. This species included the 

 nitrifying organisms and several others. 



The problem now was to isolate the nitrifying organisms. As the 

 first step, gelatin cultures were made in the ordinary way and five 

 species or organisms were found which formed colonies. Three of them 

 were bacteria, one was a small oidium, and the remaining one a peculiar 

 organism, which probably belonged to the group of sprouting fungi, 

 and is referred to beyond as a " fungus." Tests showed that neither 

 of these species of organisms caused the nitrification of the ammonia 

 salt in the liquid. 



It thus appeared that in the liquid, which contained extremely little 

 organic mattter and very small percentages of mineral salts including 

 ammonium sulphate, the ammonium was rapidly nitrified, but that when 

 portions of this culture liquid, in which nitrification was so active, were 

 put upon gelatin only a limited number of organisms developed 



