1912.] PUBLIC DOCUMENT — No. 31. 127 



The soil decoctions used in our experiments were made as 

 follows : four hundred grams of soil were placed in a percolation 

 tube and lukewarm distilled water was allowed to percolate sev'- 

 eral times through the soil. This method was followed in each 

 instance. The decoctions thus made (the percolated water) were 

 then placed in flasks, each Hask containing 100 cubic centimeters 

 of percolate. Then these decoctions, composed of percolates from 

 sterilized and unsterilized soils, were placed in the autoclave and 

 subjected to steam pressure of 15 pounds for forty-five minutes 

 at a temperature of 250° F. 



Three series of experiments were carried on with each soil. In 

 series No. 1 a sterilized and unsterilized loam were used, and the 

 sterilized decoctions inoculated with ordinary soil bacteria. In 

 the second series of experiments a sterilized and unsterilized 

 loam, and in addition a sterilized and unsterilized subsoil, were 

 used, and the sterilized decoctions inoculated with ordinary soil 

 bacteria. In our third series of experiments a sterilized and 

 unsterilized loam and subsoil were used, as in our second series 

 of experiments, but with this difference, — inoculations were 

 made from a pure culture of Bacillus subtilis. In the above 

 series of experiments, where a sterilized loam or subsoil was 

 used, sterilization was done as follows : about 1 liter of soil was 

 placed in the autoclave and subjected to steam pressure of 15 

 pounds for forty-five minutes at a temperature of 250° F. 



The following method of inoculation was used in our first two 

 series of experiments, where ordinary soil bacteria were used. 

 Ten grams of loam w'ere placed in 100 cubic centimeters of 

 sterilized water, ^ and this decoction placed in an incubator for 

 three days, where a large number of bacteria developed. We 

 used these decoctions to inoculate our sterilized percolates of 

 sterilized and unsterilized soil in the two series of experiments, 

 these percolates being inoculated with 1 cubic centimeter of the 

 above culture and then incubated for twenty-four hours. The 

 decoctions were removed from the incubator and plated, and the 

 ordinary dilution methods followed. Cultures were made by 

 adding l^ cubic centimeter of the dilution to agar-agar in Petri- 

 dishes, and these were incubated for twenty-four hours, after 

 which the colonies were counted. The agar-agar was .5 per cent. 



' Distilled water was used in all cases in all the experiments. 



