The importance of this work in connection with soil fertility was immediately realised by Warington, who had recently come to Rothamsted.[11] He quickly confirmed the result, and made the valuable discovery that two stages were involved--the conversion of ammonia to a nitrite by one organism, and of the nitrite to nitrate by another. He made long and persistent attempts to isolate the organisms from the soil, using the best technique of his time, but though he found many bacteria none of them could nitrify ammonium salts; yet the soil did it easily. For years he continued his efforts to find the nitrifying organism, but always failed. His health was not good, his life at Rothamsted was not happy owing to disagreements with Gilbert, and although his other research work was succeeding, this investigation on which he had set his heart was not coming out; bacterial technique was not yet sufficiently far advanced. Ten bitter, disappointing years passed, and the crown of disappointment came when Winogradsky, a young bacteriologist in Paris, changed the technique and succeeded at once in isolating both the nitrite and the nitrate-forming organisms.[13] The numerous bacteria found by Warington in the soil suggested the presence of a soil population, and this idea was greatly strengthened by another line of investigation which was being followed up in France. Boussingault had shown that soils absorb oxygen and give out carbon dioxide; Schloesing extended this discovery, as also did Wollny. It was concluded that oxidation was the result of the activities of the soil organisms in decomposing the organic matter of the soil, and thus preparing the way for the nitrifying organisms. A third important function of soil bacteria was revealed by Berthelot.[1] It was known that considerable loss of nitrogen from the soil took place as the result of the conversion of nitrogen compounds into nitrates, which were subsequently washed out in the drainage water. It followed inevitably that the stock of nitrogen compounds in the soil must long ago have become exhausted had there been no addition of nitrogen compounds to the soil. Berthelot argued that there must be fixation of atmospheric nitrogen, and, following the prevailing trend of thought in France, he attributed it to bacteria. He confirmed the anticipation by exposing soil to air in such conditions that dust, rain, etc., were excluded, and he found an increase in the percentage of nitrogen. Looking back over the work, it is difficult to understand the result. The fixation of nitrogen is a process that absorbs energy, and should have necessitated some source of energy, which apparently was not supplied. But in spite of this drawback the investigation was immediately fruitful in that it gave the key to another problem which had long puzzled agriculturists.