OXIDATION OF SULFUR IN THE SOIL C07 



approximate number of the organisms in the soil can be esti- 

 mated. The culture obtained on the two media is practically 

 pure, due to the fact that very few other organisms would de- 

 velop under these conditions. However, both for the purpose of 

 establishing the purity of the culture and for the better charac- 

 terization of the organism, the development of a solid medium 

 was extremely desirable. 



Among the various solid media tried, without any success, for 

 the cultivation of Th. thiooxidans w^as Beijerinck's thiosulfate 

 medium (1904) upon which Th. thioparus grew readily. Al- 

 though Th. thiooxidans can utilize thiosulfate as a source of 

 energy, it did not grow on this medium due to the extreme alka- 

 linity of that medium and to the lack of a calcium salt. When 

 the NaHCOa is eliminated from this medium, the dibasic potas- 

 sium salt changed to the monobasic form and when calcium 

 chloride is added, a solid medium favorable for the growth of 

 Th. thiooxidans is obtained. 



The composition of the medium is as follows: 



NaaSjOa- 5H2O 5.0 grams 



ICHjPO* 3.0 grams 



NH«C1 0.1 gram 



MgCla 0.1 gram 



CaCl, 0.25 gram 



Agar 20. grams 



Distilled water 1000 cc. 



The medium is prepared as usual and sterilized at 15 pounds 

 pressure for fifteen minutes. Plates and slants are inoculated 

 from a vigorous liquid culture and incubated at 25 to 30°C. 

 Growth appears in fi\'e to six days in the form of minute straw 

 yellow to cream-colored colonies. Under the microscope, each 

 colony is surrounded with crystals of gypsum due to the action 

 of the sulfuric acid, formed from the oxidation of the thiosulfate, 

 upon the CaClj. This phenomenon is particularly prominent 

 in media containing tri-calcium phosphate in place of the chlo- 

 ride; a clear zone is formed around each colony, due to the 

 disappearance of the insoluble calcium salt. 



