390 MICROBIOLOGY OF SOIL 



The first indication that nitrate production in the soil and in de- 

 caying organic matter is due to biological activities was given by 

 Pasteur in 1862. A few years later Miiller expressed his belief in the 

 biological origin of nitrates and nitrites in sewage and drinking water. 

 It was not, however, until 1877 that the true character of nitrification 

 was made clear. In that year Schloesing and Miintz demonstrated 

 that dilute solutions of ammonia could be changed into nitrate by being 

 passed slowly through long tubes filled with soil. The amounts of 

 nitrate nitrogen found in the leachings corresponded almost exactly 

 to the amount of ammonia nitrogen used up. When the soil in the 

 tubes was first sterilized by heating or by means of chloroform and other 

 germicides, the ammonia passed through unchanged. But when soils 

 sterilized by heat or chloroform were reinfected with small quantities 

 of fresh soils nitrification again proceeded in a normal manner. 



The biological nature of nitrification having been thus established 

 numerous investigators tried to isolate the specific organisms in pure 

 culture. A large amount of work in this direction was done by 

 Schloesing and Miintz, Celli and Marino-Zuco, Munro, Warington, the 

 Franklands and many others. A large number of bacteria, yeasts and 

 molds were tested with negative results. Warington, who gathered 

 a great mass of valuable information about nitrification, almost 

 succeeded in securing pure cultures of nitrifying bacteria. Finally, 

 Winogradski showed in 1890 not only that nitrification is caused by 

 specific bacteria, but explained also why the others failed in securing 

 pure cultures. He proved that these organisms do not develop colonies 

 on the ordinary gelatin and other organic media, a fact whose recog- 

 nition was largely responsible for his successful solution of the problem. 

 The medium subsequently employed by him consisted of silica jelly 

 properly supplied with inorganic nutrient salts. After him other in- 

 vestigators proved that agar, deprived of its soluble organic matter, 

 gypsum and sandstone disks, filter-paper pads, etc., could be used 

 effectively as solid media. 



Nitrous and Nitric Bacteria. Winogradski' s investigations led to 

 the conclusion, foreshadowed by the earlier work of the Franklands and 

 Warington, that the oxidation of ammonia proceeds in two stages, viz., 



(1) 2 NH 3 + 3O 2 = 2HNO 2 + 2 H 2 O 



(2) 2 HNO 2 + O 2 = 2 HNO 3 



