AUTOTROPHIC BACTERIA 67 



When 1 gram of soil is inoculated into the flasks containing the 

 culture medium, growth will usually take place at 25° to 30°C after 4 

 to 5 days, but sometimes only after weeks. Some soils, however, such 

 as acid peat and certain acid forest soils, 18 may not contain the organisms 

 in question. They are not very sensitive to drying, 17 but the action 

 of steam or volatile antiseptics is injurious and results in their destruc- 

 tion in the soil. Manured and cultivated soils contain the nitrifying 

 organisms in greatest abundance, especially in the upper layers of soil. 19 



The appearance of growth is accompanied by the formation of nitrous 

 acid and disappearance of ammonia. The former is demonstrated by 

 the starch-zinc iodide reagent and the latter by Nessler's reagent. 

 When all the ammonium is oxidized, a fresh portion of ammonium sul- 

 fate is added; a sterile 10 per cent solution of the salt is kept in a flask 

 with a plugged graduated pipette: 1 cc. will be equivalent to addition 

 of 0.2 per cent of the salt to the medium. W T hen a second portion of the 

 ammonium salt is added, it is oxidized much more rapidly since the 

 specific organisms have already developed abundantly. A third por- 

 tion is oxidized even more rapidly, until a certain limit of reaction is 

 attained depending on the solution of the MgC0 3 and the accumulation 

 of nitrous acid. The culture is then transferred to a fresh flask with 

 medium, using preferably a few drops from the bottom of the flask, 

 since the bacteria form a sediment on the MgC0 3 . Oxidation sets in 

 now more rapidly and goes on more regularly. After four or five 

 more transfers and repeated addition of ammonium sulfate to each 

 culture before a new transfer is made, the culture is rich enough in 

 the specific organisms and can be used for isolation purposes. 



The oxidation of nitrous to nitric acid by the nitrate-forming bac- 

 teria does not begin until the oxidation of the ammonium salt to nitrous 

 acid by the nitrite-forming bacteria is completed; this is due to the fact 

 that free ammonia, liberated from the interaction of the ammonium 

 salt and carbonate, has a toxic effect upon the nitrate organism. 6 

 The latter is present in crude culture together with the nitrite former 

 and, even when the latter reaches its maximum, the former is still 

 dormant. However, as soon as all the ammonia is transformed into 

 nitrite, the nitrate former becomes active. When transfers are made 

 from the crude culture, the stage of oxidation will account for the organ- 

 ism which will be prevalent. If the transfer is made at an early stage 



18 Migula, W. Beitrage zur Kenntnis der Nitrifikation. Centrbl. Bakt. II, 

 6: 365-370. 1900. 



19 Warington, 1878 (p. 63). 



