SACCHAROMYCETES. 251 



culture was to take place. Hansen adopted the following nnethod, 

 which is now in general use : — By the addition of a small quantity 

 of yeast to a sterilised malt infusion contained in a Pasteur flask, 

 a vigorous fermentation is started. When the process is well 

 advanced the liquid is poured off, and the yeast which is adherent 

 to the bottom of the flask is diluted with sterilised water to what 

 seems likely to be a suitable extent. This mixture is well shaken, 

 and from it a small drop is removed upon a glass point or fine 

 platinum wire. This drop is placed upon a slide, and at once 

 covered with a cover-glass ruled into squares, such as is used for 

 counting blood-discs, by the aid of which the cells present are 

 counted under the microscope. It is important that the drop of 

 liquid employed be barely sufficient to extend under the whole of 

 the cover, as if any escapes beyond the margin the experiment is 

 valueless. Let it be supposed that fifteen cells only can be 

 observed in the drop. Then the flask containing the diluted yeast 

 is again shaken, so as to thoroughly diffuse the cells through the 

 liquid, and a similar drop to that counted is transferred to a flask 

 containing 30 c.c. sterilised water. There is then a probability that 

 this flask contains about fifteen cells. A number of flasks con- 

 taining sterilised malt infusion being provided, the flask containing 

 the drop of diluted yeast is thoroughly shaken, and i c.c. of its 

 contents quickly transferred to each of the infusion-flasks, which 

 should number at least twenty. It is pretty certain that a number 

 of them will have received one single cell and no more, but it is of 

 the greatest importance to ascertain the fact by actual evidence. 

 For this purpose the flasks are at once subjected to a prolonged 

 and vigorous shaking, either by hand or by mechanical means, so 

 that if two or more cells have found their way into one flask they 

 may be separated one from the other. The flasks are then set 

 aside for some days at a suitable temperature, when they are care- 

 fully lifted and examined. If only one white speck has formed on 

 the bottom or sides, it is clear that only one cell has found its way 

 into the flask and a pure culture has been initiated. Those flasks 

 which exhibit more than one spot of vegetation or in which no 

 growth is visible, are rejected as useless. 



When, on these lines, Hansen had obtained his uncontamin- 

 ated cultures from single cells, he was in a position to compare 



