THE STUDY OF MICROORGANISMS IN THE SOIL 101 



One-cubic centimeter portions of the final dilutions are added 

 to 15 cubic centimeters of a sterile specific agar medium {e.g., 

 glucose agar for Clostridium pasteurianum, Medium 11 or 12). 



To remove oxygen from the medium steam for about 30 minutes 

 just before it is to be used. Now cool to 45°C. and add the 

 dilutions of soil, mixing well the soil suspension with the agar by 

 rotating the tube. The deep agar tube will produce anaerobic 

 conditions favorable for the development of the specific 

 organisms. 



The colonies may be counted in the tube, after a definite 

 period of incubation (7 to 14 days). 



Liquid media, incubated under anaerobic conditions may be 

 employed. 



If it is desired to determine the number of anaerobic bacteria 

 present in the form of spores and vegetative cells, one portion of 

 soil is suspended in water and heated at 80°C. for 10 minutes, 

 which results in the destruction of the vegetative cells; the 

 further dilutions are then prepared from the first suspension. 



If the colonies are to be isolated for pure culture study, allow 

 the agar medium to solidify and then pour on the surface of the 

 agar in the tubes or plates a layer of agar containing 0.05 per 

 cent of HgCl2, about 3^ centimeter in thickness. 



Exercise 8 

 Number of Spore-forming Bacteria in the Soil 



Prepare water suspensions of the soil as described in Exercise 

 6, making dilutions of 1 : 1,000, 1 : 10,000, and 1 : 100,000. 



Transfer 10 cubic centimeters of each suspension to a sterile 

 test tube. Heat these inoculated tubes for 10 minutes at 80°C. 

 Cool quickly under the tap. 



Place in Petri dishes 1 cubic centimeter portions of each of the 

 unheated and heated dilutions. Use three plates for each dilution. 



Pour plates of sodium caseinat^ or nutrose agar (Medium 4) 

 and incubate at 28°C. 



After 7 to 10 days count the number of colonies. 



Exercise 9 

 Number of Thermophilic Bacteria (Optional) 

 Incubate several samples of soil and some fresh stable manure 

 at 60°C. 



