# Yeast Cell Counting with a Neubauer Chamber

Consistent yeast performance during fermentation requires accurate cell counts to ensure correct pitching, monitor growth during fermentation, and assess viability.

A device used for determining the number of cells per unit volume of a suspension is called a counting chamber, also known by hemocytometer or haemocytometer. The counting chamber is a thick crystal slide with the size of a glass slide (30 x 70 mm and 4 mm thickness). In a simple counting chamber, the central area has a grid where cell counts are performed. Double chambers are most common than simple chamber. In this case, the chamber has two counting areas that can be loaded independently. The chamber is covered with a rectangular glass cover slip of 20 x 26 mm and the distance between the bottom of the chamber and the cover slip is 0,1 mm.

There are many type of chambers with different grids, but normally the most used is the Neubauer chamber. This chamber is engraved by laser with a grid of perpendicular lines. The grid area of the chamber consists of 9, 1 x 1 mm (1 mm2) squares. These are subdivided in 3 ways; 0.25 x 0.25 mm (0.0625 mm2), 0.25 x 0.20 mm (0.05 mm2) and 0.20 x 0.20 mm (0.04 mm2). The central, 0.20 x 0.20 mm marked, 1 x 1 mm square is further subdivided into 0.05 x 0.05 mm (0.0025 mm2) squares.

Counting yeast cells protocol

Preparing the sample
The solution containing the yeast cells must be appropriately mixed to obtain a homogenous suspension. The concentration of the yeast cells should neither be too high or too low. If the concentration is too high, then the cells overlap and are difficult to count. A low concentration of only a few cells per square results in a higher statistical error and it is then necessary to count more squares.
Suspensions that have a too high concentration should be diluted 1:10, 1:100 and 1:1000. A 1:10 dilution can be made by taking 1 part of the sample and mixing it with 9 parts water. The dilution must later be considered when calculating the final concentration.

$\text{Yeast cells/ml} = \left(\frac{\text{\# cells counted}}{\text{\# squares counted}}\right) \times 10000 \times \text{dilution factor}$