A quick method...
for estimating the percentage of viable cells in a yeast population, using
methylene blue staining
K. Painting and B. Kirsop
National Collection of Yeast Cultures, AFRC Institute of Food Research, Norwich
Laboratory, Colney Lane, Norwich NR4 7UA, United Kingdom
World Federation for Culture Collections
Technical information sheet No 2
Published by: UNESCO / WFCC - Education Committee 1989
Introduction
It is sometimes necessary to obtain a quick estimate of the percentage of viable
cells in a yeast sample. In traditional plate count methods results are not
available until 3 or 4 days after inoculation A quick staining method can
provide an estimate of viability in a few minutes.
Principle
Yeast cells that are viable contain an enzyme -that decolourises methylene blue,
whereas dead cells do not. When cells from a yeast sample are suspended in the
dye, it penetrates into all the cells, but is reduced only by the living cells.
It is very simple, therefore, to distinguish between living and dead cells by
examining them microscopically: dead cells are stained blue and living cells are
unstained.
Preparation of methylene blue solution
Dissolve 0.01 g methylene blue in 10 ml distilled water. Add 2 g sodium citrate
dihydrate and stir until dissolved. Filter through filter paper, making the
volume up to 100 ml with distilled water.
Procedure
Mix equal volumes of yeast sample and methylene blue solution on a microscope
slide. (It may be convenient to mix equal quantities of each, using a wire
inoculation loop.) The cell concentration should be adjusted so that about 50
yeast cells are present in a microscope field, using a 40 x objective and 10 x
or 12.5 x eyepieces.
Approximately 1000 cells should be examined and the percentage of unstained
cells of the total recorded. This figure represents the percentage viability of
the sample. When counting cells, buds that are less in size than half the parent
cell are ignored.
Note: It should be remembered that this method measures the presence of an
enzyme in a cell, rather than the ability of the cell to divide. It is possible
that the enzyme is present in cells incapable of dividing and the method is thus
less accurate than methods, such as plate count and slide count methods, which
measure the ability of cells to produce daughter cells. It nevertheless provides
a very useful rapid indication of the viability of yeasts samples.