THE STUDY OF MICROORGANISMS IN THE SOIL 99 



After the coarse particles have settled, dilute as follows: 



(a) Add 10 cubic centimeters of soil suspension to 90 cubic 

 centimeters of sterile water; dilution 1:100. 



(6) Add 10 cubic centimeters of dilution (a) to 90 cubic centi- 

 meters of sterile water; dilution 1:1,000. 



(c) Add 10 cubic centimeters of dilution (b) to 90 cubic centi- 

 meters of sterile water; dilution 1:10,000. 



{d) Add 10 cubic centimeters of dilution (c) to 40 cubic centi- 

 meters of water; dilution 1:50,000. 



From dilutions (6), (c), and (d), of 1:1,000, 1:10,000 and 

 1:50,000, pour plates in triplicate. 



Add to each plate about 10 cubic centimeters of the peptone- 

 sucrose-agar, Medium 18, melted and cooled to 40°C. 



Incubate at 28°C. and count after 2 and 4 days. 



Exercise 6 

 Number of Bacteria according to the Plate Method (.erobic) 



Weigh 50 to 100 grams of soil on a piece of paper or scoop. 

 Transfer the soil to a 500- or 1,000-cubic centimeters sterile 

 water blank. 



Five hundred cubic centimeters of water in a 750-cubic centi- 

 meter Erlenmeyer flask allows ample space for shaking (tap. 

 water may be used). For ordinary work, provided the blanks are 

 not stored for a long time, sterilization of the water blanks for 

 30 minutes in the steamer will be sufficient for use in soil counts. 

 Some prefer sterilization in the autoclave for 15 minutes at 15 

 pounds' pressure. 



Shake the soil suspension vigorously for at least 5 minutes and 

 allow the coarse particles to settle. 



Add 10 cubic centimeters of this first dilution (equivalent to 1 

 gram of soil) to a 90-cubic centimeter sterile water blank. 



After shaking, add 1 cubic centimeter to a 99-cubic centimeter 

 sterile water blank (dilution 1:10,000). 



Transfer 1 cubic centimeter of the above to a 9-cubic centi- 

 meter sterile water blank. As a rule, this dilution, which 

 represents 1 : 100,000 of a gram of soil to each cubic centimeter, 

 is the one from which to pour plates. If the soil is very poor, 

 use a dilution of 1:10,000; if very rich, 1:1,000,000. The 

 number of dilutions will depend on the type of soil. Garden or 



