226 METABOLISM 



special conditions of investigation, and cannot be treated in accordance with 

 ordinary methods ? 



WINOGRADSKY started from the idea that nitro-bacteria, like sulphur- 

 bacteria, were injured by good organic nutrients, and therefore he attempted 

 to cultivate them first of all in a solution which, in addition to the necessary 

 minerals contained only potassium tartrate to serve as the source of carbon, 

 and ammonium chloride as the source of nitrogen and as nitrification material. 

 When small traces of natural soil, in which nitrification was known to occur, 

 were added to this fluid the results desired were not forthcoming, even 

 although all sorts of changes were made in the concentration of the nutrient 

 fluid. Since, however, the observations of HERAEUS (1886) had proved that 

 organic bodies had little effect, the nutritive solution previously used was 

 prepared without potassium tartrate. The result was in the highest degree 

 remarkable ; a vigorous nitrification at once began in the fluid, and hence the 

 line of further research was clearly indicated. After it had been shown that 

 a carbonate of an alkaline earth had a favourable influence, the following 

 nutritive solution was always used : 



Water (Ziirich Lake) xooog. 

 Ammonium sulphate i g. 



Potassium phosphate i g. 



Basic magnesium carbonate 5-10 g. 



If 100 cc. of this solution after sterilization be impregnated with the smallest 

 possible drop of an old culture of a similar kind, a strong potassium nitrate 

 reaction may be obtained in the course of a few days, and in a fortnight all the 

 ammonia contained in the flask is found to have been transformed, while scarcely 

 any change can be distinguished in uninoculated control solutions. In such a cul- 

 ture very many types of Bacteria and other micro-organisms occur, whose number 

 is reduced by repeated transference to similarly constituted nutritive solutions, 

 but finally it was found no longer possible to reduce in this way the number of 

 types present. Five organisms were still found in the thin scum which was 

 obvious on the surface of the fluid, and among these WINOGRADSKY sought for 

 the assumed organism which excited nitrification and absorbed oxygen greedily. 

 Each of these five organisms was investigated separately, but none of them were 

 found to be the cause of nitrification. The real agent was finally discovered in 

 another part of the culture, viz. on the sediment formed from the magnesium 

 carbonate, in the form of a bacterial zoogloea derived from motile oval Bacteria, 

 which for a short time at the beginning swarmed in the fluid. After the signifi- 

 cance of this bacterium was established, WINOGRADSKY had still the greatest 

 difficulty in obtaining a pure culture of it. Although his studies are extra- 

 ordinarily rich in lessons on methods, we will not enter into them further at 

 present, but rather confine our attention to the results he obtained with the 

 use of pure cultures, and study first of all nitrification itself, and then the 

 peculiar behaviour of the bacterium in relation to cultivation with carbon. 



In the course of his studies, WINOGRADSKY soon noted that nitrification 

 was actively advanced if ammonia was added only in small quantity, and at 

 once replaced when used up. Consequently, he invariably added to his cultures 

 only 0-04-0-1 g. of ammonium sulphate at one time, and he was able to ob- 

 tain a very observable nitrification after the second addition. One culture, for 

 example, oxidized 860 mg. of ammonium sulphate in thirty-seven days, another 

 930 mg. in thirty days, i.e. on an average 4-93 to 6-6 mg. of nitrogen were nitrified 

 per day. What was surprising in these cultures was that the whole of the nitrogen 

 was not oxidized into nitrate, but that a part, variable in quantity, was always 

 changed into nitrite. G. and P. FRANKLAND (1890) also observed the formation of 

 nitrite in their cultures, while in the soil all the ammonia is altered into nitrate. 

 WINOGRADSKY was at first disposed to regard the formation of nitrite as 



