Haemocytometer Cell Counts and Viability Studies

In order to ensure that cell cultures have reached the optimum level of growth before routine subculture or freezing, it is helpful to obtain an accurate cell count and a measure of the percentage viability of the cell population. 

The most common routine method for cell counting that is efficient and accurate is with the use of a haemocytometer. There are several types on the market, of which the Improved Neubauer has proved most popular. 

A thick, flat counting chamber coverslip rests on the counting chamber at a distance of 0.1 mm above the base of the slide. The base of the slide has rulings accurately engraved on it, comprising 1-mm squares, some of which are further divided into smaller squares. 

When cell suspensions are allowed to fill the chamber, they can be observed under a microscope and the cells counted in a chosen number of ruled squares. From these counts, the cell count per millilitre of suspension can be calculated. Hybridoma cells and others that grow in suspension may be counted directly. Cell lines that are attached will need to be removed from the tissue culture flask by trypsinization. Because accuracy of counting requires a minimum of approximately 10 cells ml it may be necessary to resuspend the cells in a smaller volume of medium. 

To ensure that a cell culture is growing exponentially it is useful to know the percentage viability and percentage of dead cells and hence the stage of growth of the cells. This can be estimated by their appearance under the microscope, because live healthy cells are usually round, retractile and relatively small in comparison to dead cells, which can appear larger, crenated and non-refractile when in suspension. The use of viability stains such as Trypan blue ensures a more quantitative analysis of the condition of the culture. Trypan blue is a stain that will only enter across the membranes of dead/non-viable cells. 

---