The Nitrous Organism (Nitrosomonas). Prior to Koch's gelatine method the isolation of this bacterium proved an exceedingly difficult task. But even the adoption of this isolating method seemed to give no better results, and for an excellent reason: the nitrifying organisms will not grow on gelatine. To Winogradsky and Percy Frankland belongs the credit of separately isolating the nitrous organism on the surface of gelatinous silica containing the necessary inorganic food. Professor Warington, in his lectures under the Lawes Agricultural Trust, has described this important germ as follows:
"The organism as found in suspension in a freshly nitrified solution consists largely of nearly spherical corpuscles, varying extremely in size. The largest of these corpuscles barely reaches a diameter of one-thousandth of a millimetre, and some are so minute as to be hardly discernible in photographs. The larger ones are frequently not strictly circular, and are sometimes seen in the act of dividing.
"Besides the form just described, there is another, not universally present in solutions, in which the length is considerably greater than its breadth. The shape varies, being occasionally a regular oval, but sometimes largest at one end, and sometimes with the ends truncated. The circular organisms are probably the youngest.
"This organism grows in broth, diluted milk, and other solutions without producing turbidity. When acting on ammonia it produces only nitrites. It is without action on potassium nitrite. It is, in fact, the nitrous organism which, as we have previously seen, may be separated from soil by successive cultivations in ammonium carbonate solution."
The elongated forms appear to be a sign of arrested growth. Normally the organ is about 1.8 µ long, or nearly three times as long as the nitric organism. It possesses a gelatinous capsule. "The motile cells, stained by Löffler's method, are seen to have a flagellum in the form of a spiral." When grown on silica the nitrous organism appears in the same two forms—zooglea and free cells—as when cultivated in a fluid. It commences to show growth in about four days, and is at its maximum on about the tenth day. Winogradsky found that there were considerable differences in the morphology of the organism according to the soil from which it was taken. One of the Java soils he investigated contained a nitrous organism having a spiral flagellum of thirty micromillimetres; but its movement was slow.
As we have already seen, the most astonishing property of this organism is its ability to grow and perform its specific function in solutions absolutely devoid of organic matter. Some authorities hold that it acquires its necessary carbon from carbonic acid. The mode of culturing it is as follows:
To sterilised flasks add 100 cc. of a solution made of one gram of ammonium sulphate, one gram of potassium sulphate, and 1000 cc. of pure water. To this add one gram of basic magnesium carbonate which has been previously sterilised by boiling. Now inoculate the flask with a small portion of the soil under investigation, and after four or five days sub-culture on the same medium in fresh flasks, and let this be repeated half a dozen times. Now, as this inorganic medium is unfavourable to ordinary bacteria of soil, it is clear that after several sub-cultures the nitrous organism will be isolated in pure culture.
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:
| (a) |  | Ammonium sulphate | 0.4 gram |
| Magnesium sulphate | 0.05 " | ||
| Calcium chloride | A trace | ||
| (b) | | Potassium phosphate | 0.1 gram |
| 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.