50 Wager and Peniston.—Cytological Observations: on 
disappeared in most of the cells (Fig. 13). Its presence is, however, 
indicated by the position of the refractive granules, and it is immediately 
visible on the addition of iodine solution (Fig. 16). In the course of two 
hours the cytoplasm becomes perfectly hyaline, and the cell appears to 
contain nothing but a number of refractive granules suggesting the possible 
limits of the vacuole (Figs. 14, 15), which is brought out clearly by iodine 
(Fig. 16). 
If some of these cells are placed in water, the vacuole becomes visible 
in from thirty minutes to an hour. It then continues to increase in size 
for about four hours, at the end of which time the cytoplasm has appa¬ 
rently become vacuolated ; in some of the cells two, three, or four large 
vacuoles occur, and there is some difficulty in determining which is the 
nuclear vacuole (Figs. 23-6). All these vacuoles except one (the nuclear 
vacuole) disappear on fixing. They must not be confused with the 
vacuoles which occur in connexion with the formation of glycogen during 
fermentation. After from 5-8 hours in Pasteur’s solution the vacuole is 
again visible in many of the cells, but it is not clearly defined in the 
majority of the cells until some hours later (Figs. 17, 18). 
The apparent disappearance of the vacuole is possibly a plasmolytic 
phenomenon brought about by transferring the yeast from the beer wort 
to the more concentrated Pasteur’s solution. The refractive index of the 
cytoplasm is raised by the abstraction of water to much the same as that 
of the vacuole, so that the contents of the cell appear homogeneous. In all 
such cases, however, the vacuole is rendered visible in iodine solution. 
Fermentation begins slowly ; its rate of progress during the first few 
hours depends upon the condition of the cell when the culture is started. 
Yeast in the condition described, containing little glycogen, becomes 
distributed throughout the fermenting medium almost at once, none of the 
cells settling to the bottom, and a slight foam makes its appearance in the 
course of 1-2 hours. After about five hours the fermentation increases 
markedly up to 15-20 hours; during this period the cells bud rapidly, and 
form a thick foam with small air-bubbles. Fermentation continues vigorously, 
and at a normal temperature reaches its maximum in 30-45 hours. The 
budding activity is much reduced after 15-20 hours, and from that stage 
onwards the foam becomes frothy owing to the appearance in it of large 
air-bubbles. Shortly after the height of fermentation is reached, cells settle 
to the bottom in large quantities, fermentation rapidly decreases, and the 
foam disappears. After fifteen hours’ fermentation, the central vacuole in¬ 
creases in size ; it remains very large during the most active period, and 
shortly before the height of fermentation almost fills the cell. It then 
decreases in size (Figs. 20, 21), and when the cells sink is frequently smaller 
than in the early stages of fermentation. In many cells it occupies a very 
small proportion of the cell space, and in some has apparently completely 
