NUMBERS OF MICROORGANISMS 21 



soil microorganisms when the soil is kept for a short time in the labora- 

 tory. Some investigators 36 found a strong decrease in numbers, on 

 keeping the soil sample for any length of time. An increase can be 

 expected in soils, in which the activities of microorganisms have been 

 kept in check, as in the case of dry soils moistened, soils treated 

 with gaseous disinfectants, samples from low depths, frozen soils in 

 the process of thawing off. In the case of normal soils, a decrease may 

 be expected; Hiltner and Stormer recorded a change in the numbers 

 of bacteria from 7,519,000 to 2,087,000 in sixteen days and from 

 8,280,000 to 7,016,000 in six days. 



The bacteriological analysis should, therefore, be made at once, or 

 as soon as the soils can be conveniently brought into the laboratory. 



Treatment of soil samples and preparation of plates. 



Before taking out portions of the samples for analysis, each sample is thoroughly 

 mixed by means of a clean spatula. When many small stones are present, the 

 sample is first sieved through a clean (sterile if possible) 2-mm. sieve. A definite 

 portion of soil is transferred by any convenient method into an Erlenmeyer flask 

 containing a definite quantity of sterile tap water, giving the first dilution. It is 

 preferable to have the first dilution 1:10 or 1:20, i.e., to use ten or twenty times 

 as much sterile water as soil taken. To give an initial dilution of 1:10, 10 gm. of 

 soil is added to 100 cc. of water. 37 If the soil contains considerable organic 

 matter, it may be advisable to triturate it first in a mortar with a little of the 

 diluting liquid. The weight of the soil is either included in the total volume of 

 the first dilution (10 grams of soil with sterile tap water to make 100 cc. volume) 

 or not (10 grams of soil added to 100 cc. of water). 



The mixture of soil and water is shaken for five minutes. This results in 

 maximum counts, shorter periods being insufficient to separate all the bacteria 

 from the soil: longer periods of shaking have a destructive influence upon the 

 bacterial numbers. The soil should not be allowed to be too long in contact with 

 the water of dilution, otherwise an injurious effect will take place which is prob- 

 ably due to the plasmolytic action of the liquid. This leads to a rapid decrease 

 in the number of microorganisms affecting the vegetative cells first. An injurious 

 effect will occur only after 3-4 hours. 38 



In preparing the further dilutions, the contents of the flask from which the 

 suspension is withdrawn should be kept in motion. The further dilutions can 

 be made up on the basis of 1:10 or to 1:100. It has been suggested 37,39 to limit 

 the further dilutions also to 1:10, i.e., each higher dilution should be made by 



36 Hiltner and Stormer, 1903 (p. 12). 



37 Wyant, Z. N. A comparison of the technic recommended by various authors 

 for quantitative bacteriological analysis of soil. Soil Sci. 11: 295-303. 1921. 



38 Engberding, 1909 (p. 14). 



39 Noyes, H. A., and Voigt, E. A technic for a bacteriologmarJr^^amipation of 

 soils. Proc. Ind. Acad. Sci. 1916, 272-301. /\y y ' */y\ 



L LIB R A R Y 2 J 



