Purpose of the hemocytometer
The hemocytometer (or haemocytometer or counting chamber) is a specimen slide which is used to determine the concentration of cells in a liquid sample. It is frequently used to determine the concentration of blood cells (hence the name “hemo-”) but also the concentration of sperm cells in a sample. The cover glass, which is placed on the sample, does not simply float on the liquid, but is held in place at a specified height (usually 0.1mm). Additionally, a grid is etched into the glass of the hemocytometer. This grid, an arrangement of squares of different sizes, allows for an easy counting of cells. This way it is possible to determine the number of cells in a specified volume.
Preparing the sample
The fluid containing the cells must be appropriately prepared before applying it to the hemocytometer.
- Proper mixing: The fluid should be a homogenous suspension. Cells that stick together in clumps are difficult to count and they are not evenly distributed.
- Appropriate concentration: The concentration of the 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 (which takes time). 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 (or better saline of correct concentration to prevent bursting of the cells). The dilution must later be considered when calculating the final concentration.
Counting the cells
- Counting cells that are on a line: 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.
- Number of squares to count: The lower the concentration, the more squares should be counted. Otherwise one introduces statistical errors. How many squares? To find out one could calculate the cell concentration per ml based on the numbers obtained from 2 different squares. If the final result is very different, then this can be an indication of sampling error.
Calculating the cell density
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 1ml of original solution.
- Step 1 – Averaging: If one did not count all of the cells in a large square (1mmx1mm) then it is necessary to average the results first before proceeding. For the purpose of this example, I use an average cell count of 123.456 cells.
- Step 2 – Computing the volume: It is necessary to determine the volume represented by the square. The width and height of the square (e.g. 0.25mm x 0.25mm) must be multiplied by the height of the sample (often printed on the hemocytometer, in this example it is 0.1mm): v = 0.25mm x 0.25mm x 0.1mm = 0.00625mm³ = 0.00625ul (where ul is microliters).
- Step 3 – Calculating the number of cells in 1 ml:if there are 123.456 cells in 0.00625ul, then how many cells are there in 1ml (=1000ul)? We do simple direct proportion:123.456cells/0.00625ul = X/1000ul
(123.456cells*1000ul)/0.00625ul = X (the ul cancel out)
X = 19 752 960 cells
- Step 4 – Correcting for dilution: If the sample was diluted before counting, then this must be taking into consideration as well. We assume that the sample was diluted 1:10. The final result is therefore 19 752 960 cells x 10 = 197 529 600 cells in 1 ml. That a lot of cells.
Things to watch out for
- Type of counting chambers: There are different types of counting chambers available, with different grid sizes. One counting chamber also has grids of different sizes. Take care that that you know the grid size and height (read the instruction manual) otherwise you’ll make calculation errors.
- Use the provided cover glasses: They are thicker than the standard 0.15mm cover glasses. They are therefore less flexible and the surface tension of the fluid will not deform them. This way the height of the fluid is standardized.
- Moving cells: Moving cells (such as sperm cells) are difficult to count. These cells must first be immobilized.
- Objective The hemocytometer is much thicker than a regular slide. Be careful that you do not crash the objective into the hemocytometer when focusing.