the Yeast Plant. 
5 1 
disappeared, leaving nothing but a number of refractive granules at one side 
(Fig. 22). 
Soon after the beginning of fermentation other vacuoles may make 
their appearance in the peripheral cytoplasm. This is very observable in 
the cells of a compressed yeast, D.C.L., upon which some experiments have 
been made. The nuclear vacuole remains distinct in close connexion with 
the nucleolar body. On fixing and subsequently staining the cells carefully 
with fuchsin and methyl green, or Heidenhain’s haematoxylin, it is found 
that the nuclear vacuole is the only one differentiated, the others having 
apparently become lost in the process of fixing. They may possibly be 
due to the ordinary vacuolation which takes place when active metabolism 
is set up, or they may be glycogen vacuoles. It is interesting to note that 
their appearance is coincident with the formation of patches of glycogen, 
and their disappearance coincides with the gradual fusion of these glycogen 
patches into a single large mass, which nearly fills the cell. 
Methods of Fixing. 
Since it is possible that the many conflicting results obtained may have 
arisen in part from the different methods of fixing followed by various 
observers, the action of a number of fixing fluids on the yeast cell has been 
carefully noted, and the results are here briefly summarized. The yeast used 
for this purpose was obtained from a brewery in a perfectly healthy con¬ 
dition. It had been fermenting about forty-eight hours, and presented the 
characteristic microscopic appearance already described, each cell containing 
a large well-defined vacuole with usually one large conspicuous, quickly 
moving granule towards its centre, and surrounded by numerous highly 
refractive granules (Figs. 1-3). 
1. Formalin. A i*6 per cent, solution of formaldehyde was used. 
The general appearance of the cell in this solution is much the same as in 
the living condition ; the nucleolus is invisible, and the large refractive 
granule in the vacuole still remains moving vigorously. In the case of 
every other fixing agent tried, the movement of the granule ceased as soon 
as the fluid penetrated the cell. 
2. Iodine in potassium iodide, Gram’s solution (Figs. 29-31). The 
nucleolus becomes immediately visible, and after twenty-four hours the 
vacuole remains sharply defined and is not at all contracted. The refractive 
granules are conspicuous. In a few cases where one or two small vacuoles 
are present in addition to the central vacuole, these show the characteristic 
colour associated with glycogen. 
3. Chromo-aceto-osmic Acid , Flemming’s weak solution (Figs. 32-4). 
The nucleus becomes visible in a few minutes. After complete fixation, 
the cells remain much the same size as in the living condition, the nucleolus 
shows clearly, the vacuole in the majority of cells is sharply defined, although 
