Winogradsky employs for culturing upon solid media a mineral gelatine. A solution of from 3 to 4 per cent. of silicic acid in distilled water is placed in flasks. By the addition of the following salts to such a solution gelatinisation occurs:
{ Ammonium sulphate 0.4 gram (_a_) { Magnesium sulphate 0.05 " { Calcium chloride A trace
{ Potassium phosphate 0.1 gram (_b_) { Sodium carbonate 0.6, 0.9 " { Distilled water 100 cc.
The sulphates and chloride are mixed in 50 cc. of distilled water, and the latter substance in the remaining 50 cc. in separate flasks. After sterilisation and cooling these are all mixed and added in small quantities to the silicic acid.
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.