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.

Loading the chamber
The tip of the pipette is placed in the V-shaped groove on the chamber to load 10 μl of the sample into the chamber by capillary action. It is important not to overload the chamber, as doing so will give an inaccurate count. The same apply if the cover slip is moved after the sample is loaded.

Counting the cells
Carefully place Neubauer counting chamber on microscope stage and allow 1 minute for the yeast to settle. It is essential to be extremely careful with higher power objectives, since the counting chamber is much thicker than a conventional slide. The chamber or the objective lens may be damaged if the user is not careful. One entire grid on standard counting chambers with Neubauer rulings can be seen at 40x (4x objective) and every 1 x 1mm square at 400x (40x objective).

With a hand held counter count the cells in the four large corner squares and the middle one. It is advisable to count more than 100 cells to obtain an accurate cell count – 100 cells will give us an estimated error of 20%, which is the limit of what most researchers find acceptable (see Cell counting error).
Cells that are on the line of a grid require special attention. Cells that touch the top and right lines of a square should not be counted, cells on the bottom and left side should be counted. Yeast buds emerging from the mother cell are counted as separate cells if the bud is at least one-half the size of the mother

Calculating Yeast concentration
Here it is necessary to do some simple math. The following numbers are needed: number of cells counted in a square, area of the square, height of the sample, dilution factor. The objective is to find the number of cells in 1 ml of original solution.
The whole chamber has 9 squares. Each square is 1 mm2 and the chamber depth is 0.1 mm; therefore the volume overlying each square is 0.1 mm3 (or 0.0001 ml = 0.1 μl). Calculate the average number of cells per square (total cells counted/#of squares used) and multiply by 10000 and the dilution factor.

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