Upon this medium it is possible to sub-culture a pure growth from the film at the bottom of the flasks in which the nitrous organism is first isolated.
The Nitric Organism. It was soon learned that the nitrous organism, even when obtainable in large quantities and in pure culture, was not able entirely to complete the nitrifying process. As early as 1881 Professor Warington had observed that some of his cultures, though capable of changing nitrites into nitrates, had no power of oxidising ammonia. These he had obtained from advanced sub-cultures of the nitrous organism, and somewhat later Winogradsky isolated and described this companion of the nitrous organism. It develops freely in solutions to which no organic matter has been added; indeed, much organic matter will prevent its growing. He isolated it from soils from various parts of the world on the following media:
| Water | 1000.0 |
| Potassium phosphate | 1.0 |
| Magnesium sulphate | 0.5 |
| Calcium chloride | A trace |
| Sodium chloride | 2.0 |
About 20 cc. of this solution is placed in a flat-bottom flask, and a little freshly washed magnesium carbonate is added. The flask is closed with cotton wool, and the whole is sterilised. To each flask 2 cc. of a 2 per cent. solution of ammonium sulphate is subsequently added. The temperature for incubation is 30° C. Winogradsky concluded that the oxidation of nitrites to nitrates was brought about by a specific organism independently of the nitrous organism. He successfully isolated it in silica jelly. He believes the organism, like its companion, derives its nutriment solely from inorganic matter, but this is not finally established.
The form of the nitric organism (or nitromonas, as it was once termed) is allied to the nitrous organism. The cells are elongated, rarely oval, but sometimes pear-shaped. They are more than half a micromillimetre in length, and somewhat less in thickness. The cells have a gelatinous membrane. Like the other nitrifying bacteria, its development and action are favoured by the presence of the acid carbonates of calcium and sodium. Of the latter, six grams per litre or even a smaller quantity gives good results. The sulphate of calcium can be used, but the organism prefers the carbonates. The differences between these two bacteria are small, with the exception of their chemical action. The nitric organism has no action upon ammonia, and the presence of any considerable amount of ammonium carbonate hinders its development and prevents its action on a nitrite.[40]
We may here summarise the general facts respecting nitrification. Winogradsky proposes to term the group nitro-bacteria, and to classify thus:
| Nitrous organisms | = |  | Nitrosomonas, containing at least two species, viz., the European and the Java. |
| Nitrosococcus. | |||
| Nitric organism | = | Nitrobacter. |
Nitrification occurs in two stages, each stage performed by a distinct organism. By one (nitrosomonas) ammonia is converted into nitrite; by the other (nitrobacter) the nitrite is converted into nitrate.[41] Both organisms are widely and abundantly distributed in the superficial soils. They act together and in conjunction, and for one common purpose. They are separable by employing favourable media.
"If we employ a suitable inorganic solution containing potassium nitrite, but no ammonia, we shall presently obtain the nitric organism alone, the nitrous organism feeding on ammonia being excluded. If, on the other hand, we employ an ammonium carbonate solution of sufficient strength, we have selected conditions very unfavourable to the growth of the nitric organism, and a few cultivations leave the nitrous organism alone in possession of the field" (Warington).