214 NITRIFICATION 



The research was thus brought to the stage where it seemed 

 probable 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 a 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 indi- 

 cate the places where the gelatin had been inoculated. In some 

 of these the fungus developed. Inoculations 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. Liquid cultures inoculated with the bacteria 

 oxidized ammonia rapidly. The inference was that the bacteria 

 were the nitrifying organisms of the soil. 



Winogradsky describes the nitrite-forming organisms as of oval 

 form, about 1.1 to 1.8 n long and 0.9 to 1 ju wide, usually at rest but 

 at times capable of motion and dividing perpendicularly to the 

 longest axis. He places it in a genus by itself, which he calls 

 Nitrosomonas. 



The nitrate-forming organism, nitrobacter, is 0.3 to 0.4 /j. wide 

 and about 1 /* long. The cells occur singly or in pairs and occa- 

 sionally in threes. They are spindle-shaped, non-motile, and 

 possess a capsule which makes them difficult to stain. 



By way of comparing the activity of the nitrobacter with that 

 of the ferments as they actually occur in soil, Winogradsky made 

 a series of experiments to compare the amount of nitrification in 

 his culture liquid with that observed by Schlosing in a soil to 

 which, however, more oxygen had access than was the case with 

 Winogradsky's liquid. 



While in Schlosing's experiments by the use of 200 grams of 

 earth, 3.4, 9, and 4.1 mg. of nitrogen, respectively, were nitrified, 

 Winogradsky's pure cultures of bacteria nitrified 860 mg. of ammo- 

 nium sulphate in twenty-seven days and 930 mg. in thirty days. 

 Therefore, during the period at which nitrification was most ener- 

 getic there would be formed about 7.2 mg. of nitrogen per day. 



Winogradsky further investigated the interesting and very 

 remarkable fact previously cited, that the nitrobacter, although 

 containing no chlorophyll, grows and is able to multiply in a solu- 

 tion entirely free from organic matter. To prove this fact beyond 

 doubt he prepared a culture medium absolutely free from every 

 trace of organic matter by using twice distilled and tested water 

 and salts which had been carefully purified by recrystallization. 



