606 SELMAN A. WAKSMAN 



it did not grow in bouillon, on nutrient agar, or other common 

 organic and inorganic media, favorable for the cultivation of 

 bacteria and fungi; it gave a uniform microscopic picture, both 

 in unstained and stained preparations. The culture was kept on 

 liquid media, for over one and one-half years by transferring it to 

 fresh lots of media every one to four weeks. All attempts to 

 develop a solid medium for the cultivation of the organism failed 

 until December of last year. 



A detailed study of the media commonly used for the cultiva- 

 tion of colorless sulfur bacteria not accumulating sulfur within 

 their cells is given elsewhere (Waksman, 1922). The two 

 liquid media best adapted for the isolation and cultivation of 

 Thiobacillus thiooxidans have the following composition: 



I II 



(NH4)jS0, 0.2 gram (NH4)jS04 0.2 gram 



MgSOi-THjO 0.5 gram MgSOi-TH.0 0.5gram 



KHoPO, 3.0 grams KH2PO4 l.Ogram 



CaCl2 0.25 gram Re-precipitated Ca3(PO«)2 2.5 grams 



Elementary, powdered Sulfur 10.0 grams 



sulfur 10 grams /m\ 



Distilled water 1000 cc. HjPO, I— j to adjust reaction to 



pH = 3.0 

 Distilled water 1000 cc. 



The sulfur in both media and the Ca3P04 in medium II are 

 weighed out separately in the indi\'idual flasks into which the 

 media are distributed (100-cc. portions are usually placed in 

 250-cc. flasks). The media are sterihzed for tliirty minutes, in 

 flowing steam, on three consecutive days. 



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

 a fresh vigorous culture (seven to fourteen days old) of the Th. 

 thiooxidans growth will be manifested 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 same phenomenon is observed when the medium is inoculated 

 with a little soil containing the organisms, onlj^ the length of 

 time required for development is sometimes a little longer, 

 depending upon the abundance of the organism in the soil, 

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



