30 MICRO-ORGANISMS AND FERMENTATION. 



graved, and this is then connected with a moist chamber (Fig. 

 2); 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 1 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.cm. of sterilised water. This flask, then, will in all likeli- 

 hood contain about 10 cells. If it is now vigorously shaken 

 for some time until the cells are equally distributed in the 

 water, and then 1 c.cm. of the liquid introduced into each of 20 

 flasks containing nutritive liquid, it is probable that half of these 

 20 flasks have received one cell each. But, here again, as in 

 LISTER'S experiments, it is entirely a calculation of probabilities. 

 If the flasks are allowed to stand for further development of 

 micro-organisms, there will be a chance of getting a pure culture 

 in some of them. But no certain inferences can be drawn. 

 HANSEN succeeded, however, in adding a new factor, which 

 first gave certainty to this experiment. Thus, if the freshly 

 inoculated flasks are vigorously shaken, and then left in repose, 

 the individual cells will sink to the bottom, and be deposited 

 on the wall of the flask. It is self-evident that if a flask 

 contains, for instance, three cells, these cells will always, or at 

 least in the great majority of cases, be deposited in three dis- 

 tinct 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 be found, we have obtained a pure culture. 



It is evident that by means of this method we are also able 

 to introduce a single cell directly into the flask with nutritive 

 solution, or the individual cell is allowed to develop in a drop 

 suspended in a moist chamber, and the growth which forms is 

 then transferred to the flask. 



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 pre- 

 paration of pure cultures for use in physiological investigations. 

 The foundation of such methods was laid by SCHROETER (1872), 

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



