86 PRINCIPLES OF SOIL MICROBIOLOGY 



The sulfur is oxidized to sulfuric acid which acts upon the CaC0 3 

 giving CaS0 4 and C0 2 . Beijerinck isolated, in pure culture, an organ- 

 ism, Thiobacillus denitrificans, which is a very motile, short rod, hardly- 

 distinguishable microscopically from the Thiobacillus thioparus, using 

 the following medium: 



Na 2 S 2 3 -5H 2 5.0 grams Agar 20.0 grams 



K2HPO4 0.1 gram Tap water 1000 cc. 



NaHCOs 0.2 gram 



On the plate, both organisms lose their ability to grow very rapidly, 

 long before they are dead. 



The denitrifying organism was studied in greater detail by Lieske, 64 

 who used a medium having the following composition: 



Na 2 S 2 3 -5H 2 5.0 grams MgCl 2 O.lgram 



KNO3 5.0 grams CaCl 2 Trace 



NaHCOs 1.0 gram FeCl 3 Trace 



K 2 HP0 4 0.2 gram Distilled water 1000 cc. 



This medium is placed in tall glass cylinders and is inoculated with river 

 mud containing H 2 S. In a few days, one or more opalescent zones are 

 formed in the liquid, at some distance from the surface, containing the 

 denitrifying bacteria in question. The culture is then transferred into an 

 Erlenmeyer flask filled with the medium and stoppered with a rubber 

 stopper through which a bent glass tube is passed, one end of which is 

 dipped in mercury and the rest filled with medium. The culture is 

 incubated at 25° to 30°C. and in a few days the active formation of nitro- 

 gen gas takes place. By inoculating the above medium, to which 1.5 

 per cent washed agar has been added, pure cultures are obtained. The 

 organism can also be cultivated in dilute meat extract media to which 

 thiosulfate has been added. 



Thiobacillus denitrificans Beij. as described by Lieske is a small 

 narrow rod, 1/x long, not producing any spores. It is not injured by sun- 

 shine or oxygen, although it thrives better in its absence. It is auto- 

 trophic, but is not injured by organic substances. Various carbonates 

 and bicarbonates can be used as sources of carbon, but C0 2 cannot be 

 used because of the injurious effect of the free sulfuric acid formed. 

 In the presence of nitrates, the following substances can be utilized as 

 sources of energy: hydrogen sulfide, flowers of sulfur, sodium thio- 



64 Lieske, R. Untersuchungen liber die Physiologie denitrifizierender Schwe- 

 felbakterien. Ber. deut. bot. Gesell. 30: 12-22. 1912. 



