AUTOTROPHIC BACTERIA 89 



growth of a bacterium, capable of oxidizing sulfur to sulfuric acid, 

 was obtained. 71 The acid produced interacted with the tricalcium 

 phosphate and transformed it into CaS0 4 and monocalcium phos- 

 phate and finally into phosphoric acid. The bacterium was, however, 

 accompanied by other organisms, chiefly mold spores, which persisted 

 in the medium on repeated transfer. Repeated attempts to grow 

 the bacterium on agar plates failed. After all the calcium of the 

 phosphate had been transformed into calcium sulfate, the medium be- 

 came very acid, as low as pH 0.58. 



In order to make use of the fact that the organism can withstand a 

 high acid concentration, the media were prepared with an initial reac- 

 tion of pH 2.0. This allowed only the development of the sulfur- 

 oxidizing organism. The high initial acidity accompanied by the 

 use of high dilutions of the culture in making the transfers (1 : 100,000), 

 finally resulted in obtaining the culture pure. This was demonstrated 

 by the fact that no growth took place when inoculated on bouillon and 

 other media, favorable for the development of bacteria and fungi, even 

 after 10 to 14 days incubation. Microscopic examinations also demon- 

 strated the purity of the culture. This organism was described as 

 Thiohacillus thiooxidans. 72 It is a small, non-motile organism, 0.75 

 to 1.0 by 0.5 to 0.75/x, producing cloudiness throughout the medium 

 but without the formation of any pellicle. The two media best adapted 

 for the growth of this organism have the following composition : 



I II 



(NHO2SO4 0.2 gram (NH 4 ) 2 S0 4 0.2 gram 



MgS0 4 -7H 2 0.5 gram MgS0 4 -7H 2 0.5 gram 



KH 2 P0 4 3.0 grams KH 2 P0 4 1.0 gram 



CaCl 2 0.25 gram Re-precipitated 



Elementary, pow- Ca 3 (P0 4 ) 2 2.5 grams 



dered sulfur 10 grams Sulfur 10.0 grams 



Distilled water 1000 cc. H 3 P0 4 ( — ) to adjust reaction to 



pH = 3.0 



Distilled water 1000 cc. 



The sulfur in both media and the Ca 3 (P0 4 ) 2 in medium II are weighed out sepa- 

 rately in the individual flasks into which the media are distributed (100-cc. por- 

 tions are usually placed in 250-cc. flasks). The media are sterilized for thirty 

 minutes, in flowing steam, on three consecutive days. 



71 Lipman, J. G., Waksman, S. A., and Joffe, J. 8. The oxidation of sulfur by 

 soil microorganisms. Soil Sci. 12: 475-489. 1921. 



72 Waksman, S. A., and Joffe, J. S. Thiohacillus thiooxidans, a new sulfur- 

 oxidizing organism, isolated from the soil. Jour. Bact. 7: 239-256. 1922. 



