202 
Brown. — Some Studies on Yeast. 
regarded, and no doubt correctly, as explaining the remarkable fact that 
under fixed conditions the maximal cell reproduction is independent of the 
number of cells of seed-yeast per unit volume. A satisfactory explanation 
was also afforded of the observation that if more cells are added in the 
form of seed-yeast than correspond to the maximal reproduction then no 
appreciable cell-increase takes place, the limited amount of oxygen at the 
disposal of each cell being insufficient to stimulate its reproductive functions. 
A few years ago when investigating the assimilation of nitrogen by 
yeast I had occasion to examine somewhat critically these conclusions 
of Adrian Brown, and during the course of the inquiry I carried out 
a considerable number of experiments under varying conditions, especially 
with a view to determining the quantitative relation of available free oxygen 
to cell-increase. My results were entirely confirmatory of the proposition 
that the first limiting factor is the exhaustion of the stimulus given by 
a relatively very small supply of oxygen, and they further led to an 
approximation of the absolute amount of oxygen required by a yeast-cell 
to complete its process of subdivision. 
The experiments were for the most part conducted on malt-worts of 
a sp. gr. varying from 1-050 to 1-055, which contain an excess of all the 
nutrient materials required for yeast reproduction. Before seeding with 
a definite amount of yeast the nutrient liquids were fully aerated by strong 
agitation in the presence of air at the temperature at which the experiment 
was to be conducted, and immediately after seeding the air in the flasks 
over the liquid was displaced with C 0 2 . In this manner the free oxygen 
concerned in the subsequent process was confined to that which was origin- 
ally present in solution, and the absolute values representing this oxygen 
were deducible from the known coefficient of solubility of atmospheric 
oxygen in the liquid. 1 
After seeding the yeast, a series of cell-countings was made in the haemo- 
cytometer, thus forming a datum line to which subsequent cell- countings 
could be referred. A close approximation to any required density of seeding 
can always be obtained in the first instance by adding definite weights of 
the freshly grown and washed yeast, after pressing between folds of filter 
paper. One gramme of an ordinary top-fermentation yeast prepared in this 
manner and evenly distributed in 100 c.c. of liquid will give a cell-count 
approximating very closely to 10 cells per unit volume of ^Vo c.mm. 
The actual cultivations were carried on as nearly as possible at a uniform 
temperature, and successive cell-countings were made at fixed intervals of 
time. In some cases the progression of cell-reproductions was obtained 
1 An objection might be raised to this procedure on the ground that immediately on replacing 
the air in the flask with CO a the nutrient liquid must begin to lose its free oxygen by a process of 
diffusion into the oxygen-free atmosphere above it. Practically this objection has no weight since, 
as we shall see later, the dissolved oxygen is very rapidly fixed by the seed-yeast when this amounts 
to anything like one cell per unit volume. 
