98 LABORATORY MANUAL OF MICROBIOLOGY 



Exercise 4 

 Number of Protozoa according to the Dilution Method 



Add 50 grams of surface soil to 500 cubic centimeters of sterile 

 water, as directed in the preceding exercise. 



After the coarse particles have settled, dilute as follows: 



(a) Add 10 cubic centimeters of the 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 90 cubic centi- 

 meters of sterile water; dilution 1:100,000. 



Inoculate duplicate tubes containing liquid media, using hay- 

 extract (Medium 38), soil extract (Medium 40), or any medium 

 adapted to protozoa. Nutrient agar placed in dishes may also 

 be used. 



Incubate the protozoan cultures at room temperature. 



At intervals of 2 days each, make a microscopic examination 

 of the cultures. Since the protozoa are usually larger than bac- 

 teria — the 16 millimeters, or two-thirds, and 4 millimeters, or 

 one-sixth objectives will be found desirable. 



By means of a large-mouthed pipette or loop, transfer a small 

 portion of the protozoan 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. 



If it is desired to differentiate between the number of vege- 

 tative cells of protozoa and number of cysts, another 50 grams of 

 soil maybe treated with 200 cubic centimeters of a 2 per cent solu- 

 tion of hydrochloric acid and allowed to stand over night. The 

 next morning 300 cubic centimeters of water are added and dilu- 

 tions made as before. The treatment with acid results in the 

 destruction of the vegetative cells and thus the number of 

 protozoan cysts may be obtained. 



Exercise 5 

 Number of Fungi according to the Plate Method 



Add 50 grams of soil to 500 cubic centimeters of sterile water. 

 Shake vigorously for 5 minutes. 



