68 PRINCIPLES OF SOIL MICROBIOLOGY 



of oxidation, when the ammonium salt is still abundant, the nitrate 

 former may be entirely eliminated in the process of several consecutive 

 transfers. If nitrite is substituted in the medium, in place of the 

 ammonium salt, and the culture is inoculated with soil or from a pre- 

 vious culture during the process of nitrate formation, and transfers are 

 constantly made upon the nitrite medium, the nitrite forming organism 

 may be entirely eliminated. The two bacteria can thus be separated 

 by means of their characteristic metabolism. 



Solid media for the isolation and cultivation of the nitrite forming 

 organism. Silicic acid media were used first 20 for the isolation of the 

 nitrite forming bacterium. 



Equal volumes of clear sodium or potassium silicate (specific gravity 1.05 to 

 1.06) andHCl (specific gravity 1.10) are mixed by pouring the first into the second; 

 the mixture is then dialyzed in parchment paper dialyzers, for several days in 

 distilled water, which is repeatedly changed. 21 When the dialyzate gives no 

 further reaction, other than mere turbidity, with AgN0 3 , the dialysis is com- 

 pleted. The clear solution contains about 2 per cent silicic acid and can be 

 preserved for three months in clean glass-stoppered bottles and readily sterilized 

 at 115° to 120°C. In addition to the silicic acid, four liquid solutions are pre- 

 pared : 



1. (NH 4 ) 2 S04 3 grams 2. 2 per cent solution of ferrous 



K 2 HPO 4 1 gram sulfate 



MgS0 4 -7H 2 0.5 gram 3. Saturated NaCl solution 



Distilled water 100 cc. 4. Milk of magnesia, i.e., a thick 



suspension of finely powdered 

 magnesium carbonate in dis- 

 tilled water 

 Fifty cubic centimeters of the silicic acid solution is placed in a flask, then 2.5 cc. 

 of solution 1 are added and 1 cc. of solution 2. Enough milk of magnesia is added 

 to give the mixture a milky appearance (0.1 per cent sodium carbonate solution 

 may be used in place of the magnesia). The mixture is then poured, with con- 

 tinued stirring, into sterile, small thin-walled Petri dishes. Finally, one drop 

 of solution 3 is placed in the center of each plate. When allowed to rest in an 

 horizontal position, the liquid solidifies in about an hour. To get a more solid 

 medium, it is better to allow the dishes to rest 24 hours, then dry them out in the 

 thermostat. 



The medium is inoculated either directly into the flask before the plates are 

 poured, or by placing a drop on the surface of the plate. The drop is spread over 

 the surface of the medium with the tip of a sterile glass rod and the same rod is 

 used for the inoculation of a second and third plate, so as to obtain a series of di- 

 lutions. 



20 Winogradsky, 1891 (p. 65). 



21 Omeliansky, 1899 (p. 66). 



