PREPARATION OF PURE CULTURE. 49 



to a given proportion with sterilised water, and after vigorous 

 shaking, the number of cells in a small drop of the liquid is 

 determined. The counting, in this case, is easily carried out 

 by transferring a drop to a cover-glass, on the centre of which 

 some small squares are engraved, which form a starting point 

 for the eye, and this is then attached to a moist chamber 

 (Fig. 4) ; the drop must not be allowed to extend beyond the 

 limits of the squares. The cells present in the drop are then 

 counted. Suppose, for instance, that 10 cells are found ; a 

 drop of similar size is transferred from the liquid, which must 

 first be shaken vigorously, to a flask containing a known 

 volume e.g., 20 c.c. of sterilised water. This flask, then, will 

 in all likelihood contain about 10 cells. If it is now vigorously 

 shaken for some time until the cells are equally distributed in 

 the water, and 1 c.c. of the liquid introduced into each of 

 20 flasks containing nutritive liquid, then by calculation half 

 of these 20 flasks should receive one cell each. If the infected 

 flask is strongly shaken and then allowed to stand, the single 

 cells sink and remain on the bottom. It is evident that if a 

 flask contains three cells, they will, in the great majority of 

 cases, be separated by the vigorous shaking, and be deposited 

 in three distinct places on the bottom. After some days, if 

 the flask is raised carefully, it will be observed that one or 

 more white specks have formed on the bottom of the flask. 

 If only one such speck is found, then in all probability the 

 flask has only received a single cell. 



It was by this method that Hansen prepared all his earliest 

 pure cultures, with which he carried out his fundamental 

 researches on alcoholic ferments. 



Solid nutrient media have also been employed for the 

 preparation of pure cultures by the dilution method. The 

 foundation of such methods was laid by Schroeter (1872), 

 who, in his researches on pigment-bacteria, employed slices of 

 potato as a nutrient. He had observed that when such 

 slices had been exposed for some time to the air, specks or 

 drops of different form and colour made their appearance. 

 Each of these specks usually contained one species of micro- 

 organism. 



Koch considerably developed and improved this method. 



4 



