244 THE BLASTOMYCETES 



2. To this growth a quantity of sterilized water is added, and the 

 yeast cells in a given drop counted under the microscope. 



3. Supposing 10 cells to be present, a similar-sized drop is now 

 transferred to a flask containing 20 c.c. of water, which is equivalent to 

 1 yeast cell for each 2 c.c. of water. 



4. The flask containing the 20 c.c. of water with the 10 yeast cells 

 is thoroughly shaken, and this liquid divided equally, 1 c.c. being placed 

 in each of twenty flasks containing sterilized wort. 



5. If the separation has been complete, 10 out of 20 flasks should 

 contain one organism each, but this of course cannot be absolutely 

 depended on. 



6. At this stage Han sen shakes the flasks very vigorously to separate 

 the cells as much as possible, and places the flasks in the incubator, 

 allowing them to remain perfectly still, in order that the cells may sink 

 to the bottom or become attached to the walls of the flasks. 



7. At the end of several days the flask is carefully lifted and exam- 

 ined, and it is noted whether one or more white specks have been 

 formed on the walls of the glass ; if only one such speck is found, it is a 

 pure culture. 



This method is especially useful when the yeast plants are at all 

 weakly. In mixed and vigorous species, wort gelatine plate cultures 

 should be instituted. For the methods of obtaining pure cultures on a 

 large scale, see the works of Hansen and Jorgensen. 



SACCHAROMYCES CEREVISI/E I. 



This is known as the Old English top-fermentation yeast, and is 

 used by brewers and bakers. 



Microscopical Appearances. Large round or oval cells, 11 to 4 /z, 

 most frequently 8 to 6 p in diameter. These cells give off small cells by 

 budding. In the earlier stages of film formation delicate mycelial-like 

 threads are formed, which, as the film becomes older, grow longer and 

 more regular. Nuclei can be demonstrated in the cells, especially in old 

 cultures, when stained with haemotoxilin, haematine, alum solution, or 

 osmic acid. The cells are sometimes very granular. 



Spore Formation. Ascospores develop after twenty-four hours at 10 

 to 37 C., but most rapidly at 30 C., most slowly (after ten days) at 11 to 

 12 C., and below this temperature the formation ceases. For the develop- 

 ment of the ascospores, Hansen employs plaster-of- Paris blocks, which are 

 first thoroughly sterilized by heat. A small portion of yeast is laid on 

 the upper surface, and the lower surface set in a small vessel containing 

 water, within a sterile air chamber, the whole apparatus being placed in 

 an incubator, or left at room temperature. Ascospores can also be grown 



