ZOOLOGY AND BOTANY, MICEOSOOPY, ETC. 683 



cases the sugar was omitted). (2) The temperature must be the most 

 favourable — ^37° C. (3) The cultivation must be not only pure, but 

 strong. (4) The number of individuals in the nutrient medium must 

 be accurately determined ; this number must be small. (5) At the con- 

 clusion of the experiment the number of individuals must also be calcu- 

 lated. (6) The duration of the experiment must be known, and also short 

 (2-5 hours). 



The actual procedure was as follows : — From a pure cultivation of 

 the bacillus in the meat-peptone solution a small quantity on a platinum 

 wire is transferred to 50 ccm. of a sterile • 6 per cent, salt solution. After 

 having been well shaken up, 1 ccm. is taken up with a pipette and trans- 

 ferred to 50 ccm. of meat-peptone solution. With the last solution, which 

 contains at most a couple of hundred individuals to the cubic centimetre, 

 three plate cultivations are made with 1 ccm. each of the solution. In 

 this way the bacterial contents of the solution are determined with suffi- 

 cient accuracy. These quantities having been removed, the nutrient 

 solution, which has previously to the inoculation been raised to 37° C, 

 is kept at this temperature for 2-5 hours. At the expiration of this time 

 three more (secondary) plate cultivations are inoculated with 1 ccm. each 

 of the solution. This gives the number of individuals present at the 

 conclusion of the experiment. 



The enumeration of the colonies was made by numbering those visible 

 under the field of the Microscope and striking an average from 10-30 

 such enumerations. The gelatin layer of the plate should be perfectly 

 even, and not too thick. Having obtained the average number of colonies 

 to the field of vision, and then having ascertained the size of the field, 

 the number of colonies on the whole plate was calculated. As the size 

 of the field for a given objective diminishes with 

 the strength of the eye-piece, the size of the field Fig. 119. 



for each individual eye-piece should be determined 

 once for all. The higher eye-pieces with the 

 smaller fields are more convenient for the more 

 thickly crowded plates. 



This method may be further developed by 

 adapting to the diaphragm of a high eye-piece two 

 pairs of crossed threads (fig. 119). The distance 

 between the threads should amount to about 

 iyiO-1/12 of the diameter of the diaphragm. 

 The small square in the middle of the field is 



convenient for enumerating very thickly sown colonies. The number of 

 colonies seen within the small square is ascertained at many different 

 places of the cultivation plate. Colonies which happen to lie on the 

 boundary of the square are only numbered if their larger half fall within 

 the square. From many enumerations an average is obtained which 

 serves as a basis for calculating the contents of the colonies of the whole 

 plate. In this way a plate with 5-10 millions of colonies can be 

 numbered. 



In the eye-piece used by the authors the small square had the apparent 

 size of 1 • 7 sq. cm., but the actual space with the objective used v/as 

 0"0156 sq. cm., that is, the 6410th part of a sq. cm. If, therefore, there 

 were ten colonies to the square, in the gelatin layer of 80 sq. cm. super- 

 ficies there would be a total of 5,128,800 colonies. 



