Cell Counting 101 – The Hemacytometer

The hemocytometer is a device originally designed for the counting of blood cells and is now also used to count other types as well as other microscopic particles.

The hemocytometer was invented by Louis-Charles Malassez and consists of a thick glass microscope slide with a rectangular indentation that creates a chamber. This chamber is engraved with a laser-etched grid of perpendicular lines. The device is carefully crafted so that the area bounded by the lines is known, and the depth of the chamber is also known. It is therefore possible to count the number of cells or particles in a specific volume of fluid, and thereby calculate the concentration of cells in the fluid overall.

 

 

 

In the most common design, the volume of each large square (made up from 4×4 green squares in the picture) is 0.1 mm³. The cells in four large (red in figure above) squares are counted and cells over or touching the lines on top and on the left are counted, but cells over or touching the right or bottom lines are ignored. The concentration in cells per ml = cells in four red, large squares/4 × 10,000.^[1]

Hemocytometers are often used to count blood corpuscles, organelles within cells, blood cells in cerebrospinal fluid after performing a lumbar puncture, or other cell types in suspension. Anchorage-dependent cells can also be counted if subjected to trypsinization prior to counting. Using a special hemocytometer with a depth of 0.02mm smaller particles such as sperm, yeast or bacteria can be counted. Using the ruling described above the volumes are only 1/5 compared to the standard 0.1mm deep chamber. As it is difficult to distinguish between living and dead organisms unless particular stains are used to distinguish viable from non-viable cells this results in a ‘total count’ of the bacteria. In case of bigger cells the number of dead (permeabilised) cells in the sample can be obtained by adding Trypan blue. Fluorescent dyes give better discrimination particularly when looking at bacteria.

A common mistake is to add the sample to the counting chamber before adding the cover glass. This risks that the cells could sediment / stick to the glass or some volume to evaporate before the coverslip is placed on top resulting in an overestimation of the cell concentration. Sedimentation is less of a problem with bacteria but evaporation has to be kept to a minimum

Requirements

• The original suspension must be mixed thoroughly before taking a sample. This ensures the sample is representative, and not just an artifact of the particular region of the original mixture it was drawn from.
• An appropriate dilution of the mixture with regard to the number of cells to be counted should be used. If the sample is not diluted enough, the cells will be too crowded and difficult to count. If it is too dilute, the sample size will not be enough to make strong inferences about the concentration in the original mixture.
• By performing a redundant test on a second chamber, the results can be compared. If they differ greatly, the method of taking the sample may be unreliable (e.g., the original mixture is not mixed thoroughly).