Winogradsky prepared vessels and solutions carefully purified from organic matter, and these solutions he sowed with the nitrifying organism, and found that they flourished. Professor Warington has employed the acid carbonates of sodium and calcium with distinct success as ingredients of an ammoniacal solution undergoing nitrification. 2. The next condition of nitrification is the presence of oxygen. Without it the reverse process, denitrification, occurs, and instead of a building up we get a breaking down, with an evolution of nitrogen gas. The amount of oxygen present has an intimate proportion to the amount of nitrification, and with 16 to 21 per cent. of oxygen present the nitrates are more than four times as much as when the smallest quantity of oxygen is supplied. The use of tillage in promoting nitrification is doubtless in part due to the aëration of the soil thus obtained. 3. A third condition is the presence of a base with which nitric acid when formed may combine. Nitrification can take place only in a feebly alkaline medium, but an excess of alkalinity will retard the process. 4. The last essential requirement is a favourable temperature. The nitrifying organism can act at a temperature as low as 37° or 39° F. (3-4° C.), but at a higher temperature it becomes much more active. According to Schlösing and Müntz, at 54° F. (12° C.) nitrification becomes really active, and it increases as the temperature rises to 99° F. (37° C.), after which it falls. A high temperature or a strong light are prejudicial to the process. We are now in a position to consider shortly some of the characters of these nitrification bacteria. They may readily be divided into two chief groups, not in consideration of their form or biological characteristics, but on account of the duties which they perform. Just as we observed that there were few denitrifying organisms which could break down ammonia compounds to nitrogen gas, so is it also true that there are few nitrifying bacteria which can build up from ammonia to the nitrates. Nature has provided that this shall be accomplished in two stages, viz., a first stage from ammonia bodies to nitrites, and a second stage from nitrites to nitrates. The agent of the former is termed the _nitrous_ organism, the latter the _nitric_ organism. Both are contributing to the final production of nitrates which can be used by plant life.[39]