NUMBERS OF MICROORGANISMS 23 



eight plates. The average error obtained was 8 to 10 per cent, in some 

 cases even 15 per cent, in other words, the difference between the ex- 

 treme variations was equivalent to ^ of the mean value. Variations 

 equal to one-third of the actual counts have been observed. 42 The 

 variability may be somewhat decreased by very careful technic, but 

 not reduced to a small error. 



Incubation of plates and counting of organisms. The plates are incubated at a 

 constant temperature, preferably 25° to 27°C. for agar media, while gelatin 

 media are incubated at 18°C. The plates for the determination of fungi are 

 incubated for 4S to 72 hours ; a shorter period of time is insufficient for the develop- 

 ment of all the colonies, while a longer period favors rapid overgrowth of some 

 fungi. The colonies are counted after 48 and again after 72 hours. The bacterial 

 plates are incubated for seven to twelve days. A shorter period was found 43 

 to allow only an insufficient development of the microorganisms. When the plates 

 are incubated only two or three days not all the organisms develop into visible 

 colonies, especially the slow growing non-spore forming bacteria and actino- 

 myces. Hiltner and Stormer and others also found an incubation period of seven 

 days to be most favorable. 



Lower temperatures require longer incubation periods, so that, at room temper- 

 ature, the plates should be incubated for fourteen days to give the maximum 

 development of microorganisms, while, at 25°, seven days are sufficient. Higher 

 temperatures, particularly above 30°C, have an injurious action upon the de- 

 velopment of certain soil organisms; the medium in the plate also dries out rather 

 rapidly. 



At the end of the incubation period, all colonies on the plates are counted. 

 Where only 40 to 200 colonies are allowed per plate, it is sufficient to make with 

 a glass pencil two or three cross lines over the plate and count all the colonies with 

 the naked eye. But if, for one reason or another (pure culture study, for ex- 

 ample), a lower dilution is used and the number of colonies exceeds 200 (a shorter 

 incubation period may then have to be employed) the counting plate has to be 

 utilized. In that case, of course, one has to depend on the manufacturer for the 

 accuracy of divisions on the plate. A large enough number of sections should be 

 counted so as to reduce the error involved in the determinations to a minimum. 

 All the colonies, except the fungi, are counted and recorded as the total number 

 of organisms developing on the plate, where no separate count of actinomyces 

 is desired. Otherwise, after all the colonies have been counted, the actinomyces 

 are determined separately. It is advisable to have recourse to the microscope for 

 the examination of all the doubtful colonies, which are then marked off with a 

 glass pencil. The plates may also be incubated a few days (4 to 7) longer and 

 actinomyces colonies counted. The number of actinomyces may then be sub- 

 tracted from the "total number of organisms" to give the number of true bacteria. 



42 Remy, Th. Bodenbakteriologische Studien. Centrbl. Bakt. II, 8: 657-662, 

 728-735. 1902. 



43 Conn, H. J. Soil flora studies. I. Methods best adapted to the study of the 

 microscopic soil flora. N. Y. Agr. Exp. Sta. Tech. Bui. 57. 1917. 



