AUTOTROPHIC BACTERIA 71 



3. NaCl 3.0 



Na 2 C0 3 1.5 



The agar is melted and cooled to 40°C. Portions (1 cc.) of the three solutions 

 are placed in sterile petri-dishes, inoculum is added, then the melted agar. All 

 the contents of the plate are properly mixed. If it is desired to use MgC0 3 , 

 it should be added to the plate directly, at the time of pouring, and Na 2 C0 3 

 omitted from solution 3. The colonies of the bacterium develop on this medium 

 only slowly and a prolonged incubation period (3 to 4 weeks) is required. Agar 

 media are also more favorable to the development of contaminating organisms 

 than silica gel. 



Method 2r. This is the magnesium carbonate — gypsum block method. 26,26 A 

 mixture is made of 300 grams gypsum (CaS0 4 -H 2 0), 30 grams MgC0 3 and 3 grams 

 MgNH 4 P04. This is carefully made into a homogeneous putty-like mass by 

 means of water or of a water extract of a fertile soil, using 250 grams per liter of 

 water, then mixing 8 parts of the powder and 3 parts of the liquid. The paste is 

 then put upon a glass plate and, by means of a knife, streaked out to a thickness 

 of 0.5 to 0.75 cm. Round portions are cut out for the plates and oblong for the 

 tubes. After the material has completely solidified, it is sterilized together with 

 the glass containers. A small amount of the sterile nutrient liquid, without the 

 ammonium sulfate and MgC0 3 , is placed at the bottom of the container, and the 

 surface of the plate is inoculated from the liquid culture. The nitrite forming bac- 

 teria develop on these blocks as yellow-brown colonies. MgC0 3 alone can be used, 

 to which the nutrient solution is added. The yellowish colonies sink into this 

 medium due to the dissolution of the MgC0 3 by the nitrous acid. The use of 

 filter paper, partly covered with the nutrient solution, in addition to some MgC0 3 , 

 has also been suggested. 27 The organism forms minute yellow dots becoming 

 gradually brown. However, even the most recent students on this subject 28 

 found that the original silica gel method of Winogradsky is still the best for the 

 isolation of the nitrite forming bacteria. 



Method 3. Winogradsky 29 recently suggested the possibility of using a 

 direct method for the isolation of nitrite forming, as well as other bacteria. This 

 new method promises to replace all the other methods, due to the rapidity with 

 which the organisms can be isolated. A series of plates containing silica gel, 

 prepared by one of the methods outlined above, and ammonium salt as the sole 

 source of energy are inoculated by placing minute particles of soil into the gel all 

 over the plates; these are then incubated. The nitrite-forming bacteria develop 



25 Omeliansky, W. L. Magnesia-Gipsplatten als neues festes Substrat fur die 

 Kultur des Nitrifikationsorganismen. Centrbl. Bakt. II, 6: 652-655. 1899. 



26 Makrinov, J. Magnesia-Gipsplatten und Magnesia-Platten mit organischer 

 Substanz als sehr geeignetes festes Substrat fur die Kultur der Nitrifikations- 

 organismen. Centrbl. Bakt., II, 24: 415-423. 1910. 



27 Omeliansky, W. L. Kleinere Mitteilungen uber Nitrifikationsmikroben. 

 Centrbl. Bakt., II, 8: 785-787. 1902. 



28 Bonazzi, A. On nitrification. III. The isolation and description of the 

 nitrate ferment. Bot. Gaz. 68: 194-207. 1919. 



29 Winogradsky, 1925 (p. 7, 11). 



