90 PRINCIPLES OF SOIL MICROBIOLOGY 



When a flask with one of these two media is inoculated from a fresh vigorous 

 culture (seven to fourten days old) of the Th. thiooxidans, growth will be mani- 

 fested by a uniform turbidity, without any pellicle formation, within four to five 

 days, at 25 to 30°C, the culture becoming very turbid in seven to eight days; 

 the sulfur which has been floating on the surface begins to drop down. The same 

 phenomenon is observed when the medium is inoculated with a little soil con- 

 taining the bacterium, only the length of time required for development is some- 

 times a little longer, depending upon the abundance of the organism in the soil, 

 condition of soil, etc. By using the dilution method, even the approximate 

 number of the organisms in the soil can be estimated. The culture obtained on 

 the two media is practically pure, due to the fact that very few other organisms 

 would develop under these conditions. 



The bacterium is strictly aerobic and is benefited both by aeration and 

 greater surface exposure. When a particle of sulfur from the flask is 

 examined, it is found to be surrounded by the bacteria. At the same 

 time there is an intense increase in acidity of the medium. 



In the presence of calcium phosphate or carbonate, the sulfuric acid, 

 as soon as formed, interacts with the calcium salt giving crystals of 

 CaS0 4 -2H 2 0, which are seen in the culture hanging down from the 

 particles of sulfur floating on the surface, till finally the bottom of the 

 flask is covered with gypsum crystals. The organism forms no 

 spores and is destroyed at 55 to 60°C. in several minutes. The limiting 

 alkaline reaction is about pH 6.0, which is distinctly acid, while at the 

 other extreme it will grow at pH 1.0. The optimum lies at pH 2.0 to 

 4.0. It is possible, however, to accustom the organism to a neutral 

 and even an alkaline reaction, especially when transferred from one soil 

 to another before the reaction becomes too acid. 



The organism derives its carbon from the C0 2 of the atmosphere; 

 carbonates and bicarbonates affect it injuriously in so far as they tend 

 to make the reaction alkaline. The presence of organic substances is 

 not injurious. As a matter of fact, sugars, like lactose and galactose, 

 ethyl alcohol, and glycerol may even slightly stimulate growth but 

 without affecting sulfur oxidation and carbon assimilation. For 

 establishing the purity of the culture, the organism can be grown on a 

 solid medium having the following composition: 73 



Na 2 S 2 3 -5H 2 5.0 grams CaCl 2 0.25 gram 



KH 2 P0 4 3.0grams Agar 20.0 grams 



NH 4 C1 0.1 gram Distilled water 1000 cc. 



MgCl 2 0.1 gram 



71 Waksman, S. A. Microorganisms concerned in the oxidation of sulfur in the 

 soil. IV. A solid medium for the isolation and cultivation of Thiobacillus thio- 

 oxidans. Jour. Bact. 7: 605-608. 1922. 



