ISOLATION OF NITRIFYING FERMENTS 211 



Isolation of Nitrifying Ferments. The only definite result which 

 had been reached up to 1890 was that there must exist in soil 

 microorganisms which possess the power of nitrifying. However, 

 up to the time Winogradsky took up the subject all attempts to 

 isolate such organisms had proved futile. His previous experience 

 had been such as to confirm his belief in such organisms. He had 

 discovered microorganisms which oxidized hydrogen sulphid 

 (sulpho-bacteria) and an iron compound (ferro-bacteria). He 

 reasoned that it was extremely probable that organisms should exist 

 in water and in soil capable of availing themselves of the abundant 

 energy which would come from the oxidation of the ammonium 

 compounds contained therein. He considered that the number of 

 such species would be small and that the way to secure and study 

 such organisms would be: (1) To find a medium and condition 

 under which they would thrive and by which the growth of deni- 

 trifying organisms would be discouraged; (2) to continue the culti- 

 vation by such a method long enough to eliminate for the most 

 part other organisms; and (3) when the cultures of the oxidizing 

 organisms should have been obtained reasonably pure and their 

 nitrification of ammonia active, to proceed to isolate the various 

 organisms and study the character, and especially the nitrifying 

 power of each in pure cultures. He knew that all previous attempts 

 to isolate the organism on gelatin plates had failed and he con- 

 sidered that probably the organism would not grow on gelatin. 

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

 the other poor, but both rich in carbonate, and soon he learned that 

 organic matter hindered nitrification. After considerable work he 

 finally adopted a medium having the following composition: 



Water of Lake Zurich "... 1000 c.c. 



Ammonium sulphate 1 gm. 



Potassium phosphate 1 gm. 



Magnesium carbonate 0.5-1 gm. per 100 c.c. 



When such a medium was inoculated with suitable material, 

 nitrification was so active that after fifteen days every trace of 

 ammonia had disappeared, whereas check solution contained only 

 slight traces of nitric acid. 



Cultures were continued and repeated in this medium for three 

 months, when he considered that only the organisms suited to 

 this medium had survived. These were inoculated on to gelatin 

 plates and five species of organisms were found to form colonies 

 three bacteria, one oidium, and the fifth a peculiar organism which 

 he designated as "fungus." None, however, possessed the power 

 of converting ammonia into nitric acid. 



In the liquid cultures it was observed that a very thin film gradu- 

 ally formed on the surface of the culture and at times a slight 



