PROTOZOA ACCORDING TO THE DILUTION METHOD 17 



2. Shake the suspension vigorously for five minutes. 



3. Allow the coarse particles to settle and dilute in the 

 following manner : 



(a) Add i c.c. of the soil extract to 99 c.c. of sterile water; equal to 



i : 1000. 

 (&) Add i c.c. of dilution (a) to 99 c.c. of sterile water; equal to 



i : 100,000. 



(c) Add 10 c.c. of dilution (b) to 90 c.c. of sterile water; equal to 



i : 1,000,000. 



(d) Add 10 c.c. of dilution (c) to 90 c.c. of sterile water; equal to 



i : 10,000,000. 



(e) Add 10 c.c. of dilution (d) to 90 c.c. of sterile water; equal to 



i : 100,000,000. 



4. Shake thoroughly between each dilution. 



5. Inoculate three tubes of bouillon with i c.c. from each 

 dilution. 



6. Incubate these at 28 C. for one week. At the end 

 of two-day periods examine the tubes for evidence of 

 growth, as shown by turbidity, pellicles, or sediment. If 

 all cultures in dilutions from (a) to (d) show growth, 

 there must be 10,000,000 or more bacteria present in i 

 gram of soil. 



Note. If it is desired to determine the numbers of specific types of bac- 

 teria present, prepare additional liquid media. Use urea bouillon for urea 

 fermenters, peptone solution for ammonifiers, etc. In this way it is possible 

 to secure an approximate idea of the number of the various groups of organ- 

 isms present in a sample of soil. 



Exercise 2 

 Number of Protozoa According to the Dilution Method 



i. Add 50 grams of soil to 500 c.c. of sterile water, as 

 given in the preceding exercise. 



