22 PRINCIPLES OF SOIL MICROBIOLOGY 



taking 10 cc. of the lower dilution and 90 cc. of the sterile diluting fluid. How- 

 ever, even by making higher dilutions such as 1:50 or 1:100 in each case, reliable 

 results are obtained. 



The amount of soil used in making the first dilution, and the various 

 subsequent dilutions are not of primary importance, if care is taken in 

 the technic and in the calculations. By using for the first dilution 0. 1204 

 gram of soil, Hiltner and Stormer found 9,500,000 bacteria in 1 gram 

 of moist soil and, by using 6.7841 grams of the same soil, 9,400,000 

 bacteria were found in 1 gram of the same soil. In another soil, they 

 found, by using 0.5862 and 21.1820 gram portions of soil, counts of 

 7,750,000 and 7,700,000 respectively per 1 gram of soil. The higher the 

 dilution, the greater is the probable error of the results. The final dilu- 

 tion should be such as to allow between 40 and 200 colonies to develop 

 on the plate, in the case of bacteria (and actinomyces) . 40 The number of 

 fungus colonies allowed per plate on the special acid medium should be 

 20 to 100. This will necessitate that the soil should be diluted, in the 

 counting of fungi, only to about 500 to 2000, so that the highest dilution 

 is only iiU of that employed for the total bacterial numbers for the 

 same soil, in the case of productive soils. For soils very rich in organic 

 matter or for highly acid soils, the ratio between the highest dilution for 

 fungi and bacteria should be 1 : 50 or 1 : 10. 



Sterile tap water should be used for making the dilutions. Distilled 

 water has an injurious effect (plasmolysis) . Salt solution or nutrient 

 medium have no advantage over ordinary tap water. Instead of the 

 usual shaking methods, a method has been suggested 41 for the disintegra- 

 tion and dispersion of the soil particles, by means of which plate counts 

 are supposed to give results comparable with direct counts. The use 

 of a deflocculating agent and protective colloid combined with long 

 vibration of the suspension were recommended. However, conditions 

 were made so as to favor bacterial development and the method cannot 

 be recommended without further study. 



The plates are prepared from 1 cc. of the final dilution in a regular 

 bacteriological way. The number of plates to be used for each soil 

 sample is important. The number of colonies varies greatly with 

 the different plates and results based on averages of two or three 

 plates, without allowing for variability, makes them almost worthless. 

 Hiltner and Stormer recognized this fact and used four and sometimes 



40 Breed, R. S., and Dotterer, W. D. The number of colonies allowable on 

 satisfactory agar plates. N. Y. Agr. Exp. Sta. Tech. Bui. 53. 1916. 

 "Whittles, 1923-24 (p. 7). 



