70 PRINCIPLES OF SOIL MICROBIOLOGY 



great a concentration of sodium. The detrimental influence of too great a con- 

 centration of the sodium and potassium salts can still further be lessened by 

 using a mixture of acids, such as equivalent solutions of HC1, H 2 S0 4 and H3PO4, 

 thus giving in the finished medium chlorides, sulfates and phosphates of both 

 bases. 



The solutions of the three acids are standardized separately against the solu- 

 tion of the silicates, so that 1 cc. of each acid would just neutralize 1 cc. of the 

 silicate solution, against methyl orange. Methyl red and brom cresol purple 

 can also be employed. Before the final standardization, 0.5 gram MgSO.j, 0.01 

 gram CaC0 3 or CaO, 0.01 gram Fe 2 (SO 4)3 or FeCl 3 , 0.01 gramMnS0 4 and 1 gram 

 of ammonium sulfate are added to the HC1 solution. 



The three acids are then mixed, taking 153.5 cc. of HC1, 77 cc. of H 2 S0 4 and 

 116 cc. of H3PO4. One cubic centimeter of this mixture will just neutralize 1 cc. 

 of the silicate solution, using phenolphthalein as an indicator. The two solutions 

 are placed in sterile bottles connected by siphons with automatic burettes, the 

 overflow caps of which are plugged with cotton to prevent contamination from 

 the air. The solutions are allowed to stand several hours, to sterilize the con- 

 tainers, then 5 cc. portions of the acid mixture followed by the same amounts 

 of silicate mixture are placed in sterile Petri dishes, which are then rotated 

 thoroughly; the jelly sets in five minutes. This medium has, however, never 

 been tested out sufficiently; it is possible that the osmotic concentration of the 

 salts may prove too excessive. 



In addition to the silica plate method, three more methods are 

 available for the isolation of the nitrite forming bacteria. 



Method 1. Agar may be prepared according to Beijerinck, 24 whereby 3 per cent 

 agar is dissolved in distilled water, filtered and allowed to solidify in thin layers 

 in glass containers. The solidified agar is then cut up into pieces, covered with 

 distilled water and incubated at 37° for two weeks; the soluble constituents of the 

 agar come into solution and are decomposed; the water is changed several times. 

 The agar is so purified that it can be used for the cultivation of the organisms. 

 It is preserved either under water or in a dried condition. Two-tenths per 

 cent NH4NaHP0 4 -4H 2 and 0.05 per cent KC1 and chalk are then added to the 

 agar and brought into solution. The plates have a milky appearance. The agar 

 may be washed in distilled water for several days, then dried at 60°C. A 2.5 per 

 cent solution of agar is then made, tubed in 10 cc. portions and sterilized in the 

 autoclave at 15 pounds pressure. Three solutions are then prepared and sterilized 

 in small portions. 



grams in 100 cc. 



1 . K 2 HPO 4 1.5 



2. (NH 4 ) 2 S0 4 15 



MgSO 4 0. 75 



Fe 2 (SO<)3 0.02 



u Beijerinck, W. M. Kulturversuche mit Amoben auf festem Substrate. Cen- 

 trbl. Bakt. I, 19: 257-267. 1896. 



