NUMBERS OF MICROORGANISMS 9 



The application of the direct microscopic examination to the study 

 of occurrence and distribution of microorganisms in the soil gives more 

 direct evidence as to the presence and relative abundance of specific 

 groups of microorganisms. The direct microscopic examination has 

 been used 7 for counting bacteria in animal feces; it can also be used 

 for counting various microorganisms in culture. To determine the 

 numbers of microorganisms quantitatively by the use of the direct 

 microscopic method, various difficulties are encountered: 



1. Some of the microorganisms, like the protozoa, will be destroyed in the 

 process of staining. 



2. Others, like the fungi, may prove too large, for the very small quantity of 

 soil that can be used for the examination. 



3. The bacteria themselves are found in clumps upon the colloidal film and not 

 in the soil solution. Not only is it difficult to count the bacteria in the film, but 

 the variability is so great that it would take a large number of counts to obtain 

 reliable results 



The same procedure is followed as for qualitative determinations. 



The soil is diluted by means of a weak solution of gelatin (0.15 gram gelatin in 

 1000 cc. of hot water and kept sterile in a cotton plugged flask) using one part of 

 soil to 3 to 10 parts of solution depending on the soil type, heavier soils requiring 

 a higher dilution. The smear is prepared from 0.1 cc. of the infusion measured 

 out from a thin graduated pipette, to cover 1 sq. cm. on a clean slide, previously 

 rinsed in alcohol; the smears are allowed to dry over a steam bath. For staining 

 either rose bengal (1 gram in 100 cc. of 5 per cent phenol solution) or erythrosine 

 can be employed. The stain is allowed to act 1 to 3 minutes, then washed and 

 dried. With the rose bengal stain the bacteria are found deep pink or red, the 

 mineral particles uncolored, some of the dead organic matter light pink but most 

 of it yellow or unstained. The preparations are examined with an oil immersion 

 objective and a high-power eye-piece. By means of a simple equation, the 

 number of organisms can then be determined. It is advisable not to count the 

 entire field, but to mark off the central portion. 8 A disc with circles and cross 

 lines is placed in the eye-piece. Conn suggested to use a circle of such a size as to 

 cover an area on the slide either 80 or 113 microns in diameter. Every organism 

 in the area will represent two and one millions respectively per cubic centimeter, 

 using a 1.9 ( T V inch) fluorite objective CN.A. 1.32) with a 12.5 X ocular. This 

 quantity is multiplied by the dilution of the soil to give the number of bacteria 

 per gram of soil. 



However, the uneven distribution of the bacteria in the soil, causing 

 great irregularities and the difficulty of distinguishing bacterial cells from 



7 Klein, A. Die physiologische Bakteriologie des Darm-Kanals. Arch. Hyg. 

 45: 117. 1902. 



8 Breed, R. S., and Brew, J. D. Counting bacteria by means of the microscope. 

 N. Y. Agr. Exp. Sta., Tech. Bui. 49. 1916. 



