ISOLATION OF NITRIFYING FERMENTS 215 



He thoroughly removed all organic matter from the glass dishes 

 and apparatus to be used, and inoculated separate portions ot 

 the medium with the nitrobacter. The cultures developed nor- 

 mally in the dark as well as in the light. To gain an idea of the 

 extent of the assimilation of carbon, the carbon in the organic 

 matter which had been formed by the organism in its growth was 

 determined by analysis. Four cultures contained 10.2, 7.1, 4.6, 

 and 4.8 mg., respectively, of assimilated carbon, and in these 

 cultures 928, 604, and 83.5 mg., respectively, of nitric acid had 

 been formed. This seemed to leave no doubt that nitrobacter is 

 able to assimilate the carbon of carbonic acid. 



Later, in 1891, Warington, in a solution containing mineral 

 salts, obtained after repeated generation a culture which nitrified 

 vigorously. This contained no organisms which would grow on 

 gelatin and was regarded by him as containing only nitrifying 

 bacteria. The organisms thus obtained were oval in form and 

 seldom 1 micromillimeter thick and only slightly longer. 



At this time Winogradsky made a decided improvement in the 

 separation of the nitrifying organism from solutions by use of the 

 Kiihne gelatin silica medium. The nutrient basis of this medium 

 as used by Winogradsky was composed of ammonium sulphate, 0.41 

 grams; magnesium sulphate, 0.05 grams; potassium phosphate, 0.1 

 gram; sodium carbonate, 0.6-0.9 gram; calcium chlorid, a trace; 

 and water, 100 c.c. 



The inoculation of the plates took place either by mixing the 

 inoculating material with the above solution before the addition 

 of gelatinous silica, or it was made as a streak or smear culture 

 on the already hardened material. In this way the nitrifying 

 organisms developed distinct colonies from which pure cultures 

 were obtained. 



The investigations of Winogradsky and simultaneously of War- 

 ington showed the following: (1) That in the soil the nitrifying 

 process was effected by two distinct but closely related organisms, 

 the one converting ammonia into nitrous acid and nitrite and the 

 other changing the nitrites into nitrates. (2) That these two 

 processes follow one another in such rapid succession that the 

 production of nitrites is only a transitory phenomenon, so that 

 if both the nitrite and nitrate organism be added to sterilized soil 

 the process is completed in the natural way, only the merest traces 

 of nitrous acid appearing. 



If to a mineral solution containing ammonium salts, a pure cul- 

 ture of mtrosomonas be added, only nitrites will appear and these 

 will remain unchanged in the absence of the nitrobacter. If, 

 however, the two organisms be added simultaneously nitrates will 

 be rapidly formed. 



