ALCOHOLIC FERMENTATION 99 



again. The mould is then sterilised by heat, a few cells of yeast are 

 placed on its upper surface, and the whole is floated in a small 

 vessel of water and covered with a bell-jar. Under these conditions 

 of limited pabulum the cell undergoes the following changes: it 

 increases in size, loses much of its granularity, and becomes homo- 

 geneous, and about thirty hours after being sown on ths gypsum 

 there appear several refractile cells inside the parent cell. These 

 are the ascospores. In addition to the gypsum, it is necessary to 

 have a plentiful supply of oxygen, some moisture (gained from the 

 vessel of water in which the gypsum stands), a certain temperature, 

 and a young condition of the protoplasm of the parent yeast cells. 

 Hansen found that the lowest temperature at which these ascospores 

 were produced was *5 3 C., and at the other extreme up to 37 C. 

 The rapidity of formation also varies with the temperature, the 

 favourable degree of warmth being about 22 25 C. (Plate 8). 



Hansen pointed out that it was possible by means of sporulation 

 to differentiate species of yeasts. For it happens that different 

 species show slight differences in spore formation, e.g. : 



(a) The spores of Saccharomyces cerevisice expand during the first 

 stage of germination, and produce partition walls, making a com- 

 pound cell with several chambers. Budding can occur at any point 

 on the surface of the swollen spores. To this group belong S. 

 pastorianus and 8. ellipsoideus. 



(5) The spores of Saccharomyces Ludwigii fuse in the first stage, and 

 afterwards grow out into a promycelium, which produces yeast cells. 



(c) The spores of Saccharomyces anomalus are different in shape 

 from the others in that they possess a projecting rim round 

 the base. 



Another point in the cultivation of yeasts has been elucidated by 

 a number of workers, among whom is Hansen, namely, the methods 

 of obtaining pure cultures. Only by starting with one individual 

 cell can it be hoped to secure a pure culture of yeasts. For the 

 study of the morphology of yeasts under the microscope the problem 

 was not a difficult one. It was comparatively easy to keep out 

 foreign germs from a cover-glass preparation sufficiently to perceive 

 germination of spores and the growth of yeasts. But when pure 

 cultures are required for various physiological purposes then a 

 different standard and method is necessary. 



Hansen employed dilution with water in the following manner : 

 Yeast is diluted with a certain amount of sterilised water. A drop 

 is carefully examined under the microscope, a single cell of yeast is 

 taken, and a cultivation made upon wort. When it has grown 

 abundantly a quantity of sterilised water is added. From this, 

 again, a single drop is taken and added to say 20 c.c. of sterilised 

 water in a fresh flask. This flask will contain, let us suppose, ten 



