Autotiopliic Bacteria 63 



to aniinonia. The process of ammonia formation was, however, 

 sharply distinguished from that of ammonia oxidation, the latter 

 being an essential part of the nitrification process, although the 

 existence of two different organisms was not suspected at first. 



The various reactions involved in nitrification of nitrogenous com- 

 pounds were clearly elucidated toward the middle of the ninth 

 decade of the last century. All efforts to isolate the specific organ- 

 isms concerned in the process failed, however. This was chiefly 

 due to a lack of recognition of the specific mode of nutrition of the 

 organisms concerned in nitrification. As long as the characteristic 

 manner of energy utilization by these organisms was not understood, 

 no suitable methods could be developed for their isolation. Although 

 numerous inxestigators asserted that certain organisms, some even 

 pathogenic in nature, were able to produce nitrates, the observations 

 were not fully confirmed. It is even doubtful whether the results 

 obtained were properly interpreted; the traces of nitrate observed 

 in these experiments may haxe come from the atmosphere, and the 

 nitrite reported may have been a result of the reduction of nitrate 

 present in the medium. Many of the investigators who approached 

 this problem were primarily chemists or agronomists, whereas the 

 bacteriologists were at that time so much under the influence of the 

 gelatin plate method of Robert Koch that the fact that an organism 

 produced no growth on this medium was sufficient to justify the 

 conclusion that such an organism did not exist. 



Winogradsky was fully prepared to undertake this study by his 

 previous investigations of sulfur and iron bacteria, carried out in 

 1885-1888, which brought out the fact that these organisms were 

 able to deri\ e their energy from inorganic compounds. He reasoned 

 by analogy that the nitrifying bacteria could probably use the am- 

 monia as a soiu^ce of energy. These organisms might, therefore, pos- 

 sess properties similar to those which have the capacity to oxidize 

 other elements or simple inorganic substances. The principle of 

 elective culture was used, with ammonium salts as the only available 

 source of energy. Conditions were thereby made unfavorable for 

 the development of all those microorganisms that are unable to 

 oxidize ammonium salts and utilize the energy thereby liberated. 



Flasks containing a salt solution free from organic carbon com- 

 pounds and with an ammonium salt were inoculated with soil. Bac- 

 terial growth took place after 4-5 days' incubation at 25-30°C; some- 

 times a longer period was requued. Manured and culti\ated soils 

 contained nitrifying organisms in greatest abundance, especially in 



