NITRIFICATION 31 



The discovery of Dr. Munro that organisms will grow in purely 

 inorganic solutions has been made use of for the isolation of the 

 different species. Solutions such as the following have been used : 



For the Nitrous Organisms. For the Nitric Organisms. 



Ammonium chloride, 0-5 grm. Potassium nitrite, 0-3 grin. 



Potassium phosphate, 0-1 grm. Potassium phosphate, 0-1 grm. 



Magnesium sulphate, 0-02 grm. Magnesium sulphate, 0-05 grm. 



Calcium chloride, 0-01 grm. Calcium carbonate, 5 grm. 



Calcium carbonate, 5 grm. Distilled water, 1000 c.c. 

 Distilled water, 1000 c.c. 



These are seeded with traces of earth, and by carrying on the 

 cultivation for many generations a large number of organisms are 

 eliminated. This method does not lead to a pure cultivation, for 

 several forms besides the nitrifying organisms persistently maintain 

 themselves in these mineral solutions. 



So recourse was had to gelatin plate cultivations. Although 

 several organisms were isolated in this manner, none of them 

 possessed the slightest nitrifying power. 



Frankland, and later Warington (1890), succeeded in isolating 

 nitrous organisms by the dilution method. Nitrifying solutions 

 were diluted, and traces inoculated into ammoniacal solutions ; in 

 some of these nitrification occurred, although no growth could be 

 obtained on gelatin, and they were found to contain the nitrous 

 organism only. A little later Winogradsky isolated nitrous 

 organisms, first by modified gelatin plates, and afterwards by the 

 silica jelly method. 



This is carried out as follows : Sodium carbonate is fused in the 

 blowpipe, and fine white sand is added so long as effervescence is 

 produced. The mass is allowed to cool, and is then dissolved in 

 water. The solution is poured into an excess of very dilute hydro- 

 chloric acid (silicic acid and sodium chloride being formed). The 

 solution is dialysed and sterilised. For use, some of this is placed 

 in a sterile dish and is mixed with the following solution and 

 inoculated : 



Ammonium sulphate .... 0-4 grm. 



Magnesium sulphate . . . 0-5 ,, 



Di-potassium hydrogen phosphate . . 0-1 ,, 



Calcium chloride ..... trace 



Sodium carbonate ..... 0-6-0-9 grin. 



Water 100 c.c. 



This mixture sets to a jelly in five to fifteen minutes. 

 Winogradsky has also made use of agar for plates, but this 



