66 



PRINCIPLES OF SOIL MICROBIOLOGY 



intervals, by the shaking of the flask; this hastened the process of nitri- 

 fication. Still more intensive nitrification was obtained by allowing 

 the ammoniacal solutions to flow through peat and carbon black inocu- 

 lated with nitrifying organisms. 13 Ignited soil placed in flat bottomed 

 Fernbach flasks, combined with a slow rotary movement of the culture 

 gave intensive nitrification. 14 



An alkaline reaction is essential for nitrite and nitrate formation. In 

 the absence of basic carbonates, only ammonium carbonate is oxidized ; 

 in the presence of sodium, magnesium or calcium carbonate, free ammonia 

 as well as the sulfate, phosphate and chloride of ammonium can nitrify. 15 

 This is due to the fact that the optimum reaction for the organisms is on 

 the alkaline side of neutrality (pH 7.0 to 8.0). The presence of the base 

 in the medium is essential as a neutralizing agent to prevent the reac- 

 tion of the medium from becoming too acid, due to the formation of 

 nitric and sulfuric acids from the oxidation of the ammonium salt. 



In addition to the medium given above, two other media were used: 15,18 



(NHO2SO4 



K 2 HP0 4 



MgS(V7H 2 



CaCl 2 



NaCl 



FeS0 4 



Distilled water. , 



Magnesium carbonate (MgC0 3 



MEDIUM 2 



2 . 0-2 . 5 grams 



1.0 grams 



. 5 gram 



Trace 



1000 cc. 

 Excess (0.5 gram 

 per flask) 



2.0 grams 

 1 .0 grams 

 0.5 gram 



2.0 grams 



0.4 gram 



1000 cc. 



Excess (0.5 gram 



per flask) 



The medium is distributed into flasks (about 50 cc. portions), which are then 

 plugged with cotton and sterilized at 15 pounds pressure for 15 minutes. It is 

 best to sterilize the ammonium sulfate separately, as a 5 or 10 per cent solution, 

 and then add it by means of a sterile pipette to the sterile flasks. Gibbs 17 em 

 ployed medium 3, but reduced the ammonium salt to one gram and substituted 

 a trace of ferric sulfate for the ferrous salt. 



13 Mi\ntz, A., and Laine, E. Utilisation des tourbiers dans la production 

 intensive des nitrates. Compt. Rend. Acad. Sci. 142: 1239-1244. 1906; also 

 Ann. Sci. Agron. Ser. 3, 2: 287-395. 1906. 



14 Bonazzi, A. On nitrification. Jour. Bact. 4: 43-59. 1919. 



15 Winogradsky, 1904 (p. 64). 



18 Omeliansky, V. Uber die Isolierung der Nitrifikationsmikroben aus dern 

 Erdboden. Centrbl. Bakt. II, 5: 537-549. 1899. 



17 Gibbs, M. W. The isolation and study of nitrifying bacteria. Soil Science, 

 8: 427-481. 1919. 



