46 PRINCIPLES OF SOIL MICROBIOLOGY 



dilutions can be made narrower and the number of protozoa determined 

 with a greater degree of accuracy. This method can also be modified so 

 as to give not only the total number of protozoa in the soil, but also the 

 number of active forms and cysts. 107 



The soil is passed through a 3 mm. sieve and two 10-gram samples are taken. 

 The total number of protozoa (active forms plus cysts) is determined, in one sam- 

 ple, while the other sample is used for the determination of cysts. Taking care to 

 agitate the flask while the liquid is being transferred, the following dilutions are 

 prepared: 



Dilutions of 25-102,400 or 50 204,800 may also be prepared. 



Nutrient agar is poured into a series of sterile Petri dishes. When the medium 

 has solidified, the dishes are inoculated in pairs with 1 cc. of each dilution. The 

 same pipette may be used when one begins with the highest dilution and goes back 

 to the lowest. The plates are incubated at 18° to 20° for 28 days and examined 

 after 7, 14, 21, and 28 days. A little sterile water is added to the plates, in case 

 they begin to dry out. The examination is made by scraping off a little of the 

 surface of the plate, placing on a slide under the microscope and examining with 

 low and high power. 



The second 10-gram sample of soil is treated with sufficient 2 per cent HC1, 

 over night, to neutralize the carbonate present in the soil and still leave an excess 

 of unchanged 2 per cent HC1. This kills all the active forms leaving the cysts 

 unharmed. The number of protozoa is then determined by the same dilution 

 method. The number of cysts subtracted from the total number of organisms, 

 obtained in the first count, gives the number of active protozoa per gram of soil. 



Fisher 108 investigated the theory of estimation of microorganisms by 

 the dilution method and found that a very high efficiency can be thus 

 obtained (87.71 per cent). By the use of a special table, the number 

 of protozoa can be calculated, when the number of negative plates is 



107 Cutler, D. W. A method for estimating the number of active protozoa in 

 the soil. Jour. Agr. Sci. 10: 135-143. 1920. 



108 Fischer, R. A. On the mathematical foundations of theoretical statistics. 

 Phil. Trans. Roy. Soc. London. Ser. A, 222: 309-368. 1922. 



