NITRIFICATION. 59 
a small quantity of soil is added to a solution of nitrite the nitrite soon 
becomes converted into nitrate, under the influence of the fermenta- 
tion started by the presence of the soil. This shows that the soil 
must contain the nitrifying organisms. But the bacteria which 
are isolated from such soil by ordinary methods showed no power 
of nitrification. Evidently the nitrifying bacteria cannot be found 
by the ordinary bacteriological methods. 
The cause of the trouble as well as the secret of successful study 
was soon learned. In bacteriological studies the common method of 
isolating bacteria is to get them to grow in culture media made by 
the bacteriologist. The media commonly used contain a certain 
amount of organic compounds which serve as food for the bac- 
teria. But experiment soon showed that the presence of the smallest 
amount of organic matter is directly injurious to the nitrifying 
bacteria, so that they will not grow at all in ordinary culture media. 
It was necessary to devise some culture media that contained no 
organic matter, and as soon as this was done it was possible to 
isolate from the soil bacteria having the power, under proper con- 
ditions, of oxidizing ammonium and nitrite compounds into ni- 
trates. For a while the results of experiments were in some con- 
fusion, since in some cases nitrates appeared to be formed, while in 
others they did not. It became evident that nitrification was not a 
simple phenomenon, and further study showed that the nitrification, 
as occurring in ordinary soil, is a two-fold process. The first step in 
the process oxidizes the ammonia into nitrites. In most of the ex- 
periments the nitrogen was put into the culture fluids in the form of 
sulphate or carbonate of ammonia and this was readily oxidized into 
nitrite. The second step was the oxidation of the nitrites into 
nitrates. The two steps are not only independent, but they are 
brought about by two different species of bacteria. One organism 
has the power of producing nitrite out of ammonia, but can carry 
the oxidation no farther, failing to produce nitrates. The second 
species can act upon the nitrites, carrying their oxidation up to the 
form of nitrates, but it has no power to act upon ammonia. The 
two together can produce the complete nitrification of both am- 
monium and nitrite compounds. 
