THE STUDY OF MICROORGANISMS IN THE SOIL 129 



(b) 1 gram of rich garden soil. 



(c) 1 cubic centimeter of sewage. 



Stopper tightly with paraffined corks and incubate at 28°C. 

 for 2 or 4 weeks. 



At the end of this time remove bottles from incubator and note 

 the change in color and odor. 



Hold over the open mouth of the bottle a small piece of filter 

 paper saturated with a solution of lead acetate. A blackening 

 of the paper indicates the presence of hydrogen sulphide. 



Remove a few cubic centimeters with a pipette to a test tube 

 or small Erlenmeyer flask and add a few drops of BaCU solution. 



Compare the amount of white precipitate in the inoculated 

 cultures with that in the uninoculated control. 



The amount of hydrogen sulphide may be determined quanti- 

 tatively by titrating with iodine and sodium thiosulphate. 



Exercise 46 

 Isolation of Hydrogen Sulphide-forming Microorganisms 



Prepare five deep tubes of Medium 62. 



Make shake cultures, deep tubes, from varying dilutions of the 

 enrichment cultures of Exercise 45 (reduction of sulphates with 

 the formation of H2S). 



After the inoculated tubes have hardened, cover with glycerol 

 agar to a depth of about 2.5 centimeters. Incubate at 28°C. 

 and note the formation of colonies. After 5 to 10 days these 

 colonies become brown to black in color. Pure cultures may be 

 obtained from these deep colonies by cutting the tubes and 

 transferring the black colonies to fresh tubes of Medium 62. 



Glycerol Agar^ 



Agar, washed 20 . gm. 



Glycerol (C3H5(OH)3) 500.0 cc. 



Distilled water 500 . cc. 



^ Dissolve the agar in the water by heating in a steamer, add the glycerol 

 and filter. 



