No. 6. DEPARTMENT OF AGRICULTURE. 337 



gelatin plales was inapplicable for the isolation of the specific ni- 

 trifying agent. 



In the same year an identical principle was disco vered and pat 

 into practice by Winogradsky '^ who succeeded in separating the nitri- 

 fying ferment by using a purely inorganic medium containing: 



Water of Lake Zurich, 1000 cc. 



Ammonium sulphate, 1 grm. 



Potassium phosphate, 1 grm. 



Basic carbonate of magnesia, an excess. 



In this solution nitrification became very active, when previously 

 inoculated with a small quantity of soil. 



By a long series of fractional cultures one was finally obtained 

 which contained but fev»^ bacteria except the nitrifying organism. 

 From this somewhat impure culture, gelatin plates were made. On 

 the principle that the foreign non-nitrifying organisms grow in gela- 

 tin while the nitrifying bacteria do not, an indirect method of isola- 

 tion was utilized. 



In the portions of the gelatin between the colonies of non-nitri- 

 fying bacteria the nitrifying organisms would be liable to be present 

 in a pure state, but unable to produce colonies because of uncongenial 

 soil. By removing bits of this apparently sterile gelatin a few nitri- 

 fying organisms, unmixed with others could be transferred to a favor- 

 able solution like the one already given. In this way Winogradsky 

 was able to isolate the nitrifying organism. 



Later, in 1891, Warrington,^^ in a solution containing mineral salts, 

 obtained, after repeated generation, a culture which nitrified vigor- 

 ously, and which, by containing no organism which would grow on 

 gelatin, was regarded by him as containing onh' nitrifying bacteria. 

 The germ thus obtained was an oval form seldom one micromillimeter 

 thick and scarcely longer than broad. 



At this time Winogradsky^^ made a decided improvement in the 

 separation of the nitrifying organism from solutions containing it 

 by the use of the Kiihne gelatin silica medium.^^ The nutrient baeia 

 of this medium as used by Wirogradsky was composed of: ammo- 

 nium sulphate, 0.41 gram, magnesium sulphate, 0.05 grm., potassium 

 phosphate, 0.10 grm., sodium carbonate, 0.6-0.9 grm., calcium 

 chloride a trace, and water 100 cc. 



The inoculation of the plates took place either by mixing the in- 

 oculating material with the above solution before the addition of 

 gelatinous silica, or it w^as made as a streak or smear culture on the 

 already hardened material. In this way the nitrifying organisms de- 

 veloped distinct colonies from which pure cultures were made. 



The investigations of Winogradsky and simultaneously of War- 

 rington showed: 

 22— G— 1902 



