l8 SOIL BACTERIOLOGY 



2. After the coarse particles have settled, dilute as fol- 

 lows: 



{a) Add I c.c. of the soil suspension to 9 c.c. of sterile water; equal 



to I : 100. 

 i]b) Add I c.c. of dilution (a) to 9 c.c. of sterile water; equal to i : 1000. 

 {c) Add I c.c. of dilution {h) to 9 c.c, of sterile water; equal to 1 : 10,000. 

 id) Add I c.c. of dilution {c) to 9 c.c. of sterile water; equal to i : 100,000. 



3. Inoculate two tubes of protozoa media (hay-soil 

 extract, soil extract, or any medium adapted to protozoa) 

 with I c.c. of each of the above dilutions (see p. 97). 



4. Incubate the protozoa cultures at room temperature. 



5. At regular intervals of two days each make a micro- 

 scopic examination of the cultures. Since the protozoa 

 are usually larger than bacteria — the 16 mm. two-thirds 

 and 4 mm. one-sixth — objectives will be found desirable. 



6. By means of a large-mouthed pipet or loop transfer a 

 small portion of the protozoa culture to a slide and examine. 

 A wet or hanging-drop mount may be used. In certain 

 cases the small flagellates become so numerous that it is 

 difficult to distinguish between the bacteria and protozoa. 



Note. — An abundant growth of large protozoa may be obtained if mannit 

 solution (m. 39) is inoculated with a small amount of field soil and the 

 culture incubated for one week or longer at room temperature, A drop of 

 the culture treated with Gram's iodin solution will show the presence of 

 numerous ciliates. In certain cases the ciliates are marked by numerous 

 small, deep golden bodies within their cell wall, apparently Azotobacter 

 cells. 



Exercise 3 

 Number of Bacteria According to Plate Method 



In order to reduce the error common to determinations 

 of this character, it is well to use a large sample of soil. 

 All weighings should be made as rapidly as possible to 

 avoid errors due to loss of moisture by evaporation* Bal- 



