754 



colonies and that none of those which were thus grown in gelatin caused 

 nitrification. The nitrifying ferment was still to be discovered. 



In the cultures in the liquid referred to it was observed that a very 

 thin film gradually formed on the surface of the culture, and a slight 

 cloudiness of the solution was noted at times. The latter disappeared 

 after a time, and was, as microscopic examination showed, caused by 

 the presence of an oval, somewhat spindle-shaped organism which 

 moved about very rapidly. Nitrification was at the same time very 

 active. It was thought that the film on the surface might contain the 

 nitrifying organisms, as the acetic acid bacteria and other oxidizing 

 ferments work at the surface where plenty of oxygen can be obtained; 

 but tests with this gave negative results. 



The plan of work was now somewhat changed: The attempt was 

 made to cultivate the nitrifying organisms more abundantly. To this 

 end a quantity of ammonium sulphate was added to the nitrified cul- 

 tures, and the process of nitrification thus continued in the culture 

 for some time. A change was noticed in the magnesium carbonate at 

 the bottom- of the solution, which gradually assumed a grayish color 

 and a gelatinous consistency. By shaking the solution vigorously this 

 mass was broken up into small flakes, which a microscopic examination 

 showed to consist of transparent particles of the salt covered with a 

 mass of oval bacteria, identical in form with those which had pre- 

 viously been noticed as the cause of the cloudiness. These bacteria 

 seemed to be on the particles exclusively and not on the walls of the 

 flask, and slowly enveloped the salt which was finally dissolved. 



A culture medium was then prepared which was free from every trace 

 of organic matter. On being inoculated nitrificatiou took jjlace in this 

 energetically, and it was found to contain large numbers of the oval 

 bacteria, as well as the fungous form previously noticed, but the other 

 forms had all disappeared. The fungus remained constant, and all 

 attempts to cultivate it out were unsuccessful. 



The research was thus brought to the stage where it seemed prob- 

 able that the oval bacteria might be the nitrifying agents. To test 

 their nature and action satisfactorily the removal of the sprouting 

 fungus was called for. To accomplish this, Winogradsky resorted to a 

 very ingenious though simple device. The fungus would develop in 

 gelatin ; the bacteria would not. Small particles of the carbonate, 

 more or less enveloped by the bacteria, were taken from the bottom of 

 the culture flask by means of a capillary tube, and placed in a large 

 flask of sterilized water; the contents of the flask were then well 

 shaken and a gelatin plate inoculated with drops of the liquid, the 

 particles of carbonate serving to indicate the places where the gelatin 

 had been inoculated. In some of these the fungus developed. Inocu- 

 lations of the culture liquid from the other spots failed to yield 

 the fungus but developed the bacteria. By this method of " inverse 

 gelatin culture" the bacteria were obtained pure. In culture liquid 



