74 PRINCIPLES OF SOIL MICROBIOLOGY 



Nitrosococcus also from the soil of North America (Ohio) : it forms small 

 yellowish colonies (224 by 160,u on the silica gel) surrounded by a 

 colorless halo, due to the solution of the MgC0 3 . Colonies of 1 mm. in 

 diameter have been obtained by renewing the (NH 4 ) 2 S0 4 in the plates 

 when necessary. The organism was found to be present in two stages: 

 (1) as megalococci, 1.25/* in diameter, of a slightly irregular form, oc- 

 curring in thick gelatinous masses; when the cultures are in the process 

 of active oxidation, the megalococci give rise to small cocci; (2) a form 

 which leaves the gelatinous mass and become free. The hanging drop 

 cultures showed no motility. 



The organism is stained as follows. The cover glass preparation is 

 fixed in flame, treated with mordant 1 minute in the cold with a 0.25 

 per cent solution of malachite green in distilled water, washed with 

 cold water, and stained cold with a 0.25 per cent water solution of 

 gentian violet for another minute. The stain is then rapidly washed 

 with water previously heated to 50 to 60°C. The megalococci are 

 stained deep purple and the small cocci purple-black. 



The existence of the several forms of nitrite-forming organisms in 

 the soils from different continents was explained by Winogradsky as due 

 to the probability that local conditions favored the adaptation of a 

 particular variety. 



Nitrate forming bacteria (Nitrobacter) . The organism that is able 

 to oxidize nitrite to nitrate was discovered by Winogradsky 32 in 1891 

 in the solutions where nitrate formation was taking place. 



It was finally cultivated in a sodium nitrite medium to which the ordinary 

 nutrients and magnesium carbonate or sodium carbonate have been added as 

 follows : 



NaN0 2 1.0 gram NaCl 0.5 gram 



K2HPO4 0.5 gram Na 2 C0 3 (anhydride) . . 1.0 gram 



MgS0 4 0.3gram FeS0 4 0.4gram 



Distilled water 1000 cc. 



(The ferrous sulfate may be reduced to a trace and the anhydrous Na2C0 3 to 

 0.5 gram.) 



Fifty cubic centimeter portions of the sterile solution in flasks are inoculated 

 with soil. The course of the reaction is followed by the disappearance of the 

 nitrous acid and the appearance of nitric acid (using diphenylamine and concen- 

 trated sulfuric acid). When all the nitrite has been oxidized, fresh portions of 

 the salt are added, in the form of a sterile solution as in the case of the ammonium 

 sulfate, and the culture is studied microscopically, using carbol fuchsin for 

 staining. 



32 Winogradsky, 1904 (p. 64). 



