How to Use a Hemacytometer

Classic Techniques in Cell Biology:
How to use a hemacytometer

by Heather Buschman

French physiologist Louis-Charles Malassez (1842-1909) studied a lot of things in his life. In dentistry, the residual cells of the epithelial root sheath in the periodontal ligament are now called the epithelial rests of Malassez. A genus of fungi is also named for him, which includes species that can cause dandruff and other skin infections.

Malassez also invented the hemacytometer, the thick glass microscope slide traditionally used to count the number of cells in a given volume of liquid. He made an indentation in a regular microscope slide to form a small space that could hold a few drops of cells in suspension. With etched lines and a known area and depth, the number of cells in that particular volume could be calculated. Since the invention was first used to count blood cells, it became known as the hemacytometer (hem = blood, cyto = cells), sometimes also spelled hemocytometer or haemocytometer.

Over the past few years, more than 4,000 people viewed a Scientist Solutions discussion on how to count cells with a hemacytometer. Even for people who learned cell cultures years ago, remembering the calculation can be tricky. Here’s a quick cheat sheet:

1. Transfer a small sample of cell suspension (~100 μl) to a microfuge tube.

2. Dilute 1:2 with Trypan blue or other cell stain.

3. Carefully transfer approximately 10 μl of cells to one of the semi-reflective panels on a hemacytometer covered with a cover slip. Allow the capillary action of the cover slip to gently draw in just enough liquid to fill the chamber liquid. Do not overload.

4. Under the microscope, you should see a grid of 9 squares. Focus the microscope on one of the 4 outer squares in the grid. The square should contain 16 smaller squares.

5. Count the number of live cells in at least 2 of the outer squares. (Dead cells turn dark blue with Trypan staining.)

6. Use the following equation to calculate the number of cells in the original volume:

Some tips for best results:
• Gently pipette the cell suspension up and down to mix well and reduce aggregation before removing a small sample.

• Remove any bubbles in the hemacytometer before counting.

• Count cells touching the top and right borders of each of the 4 squares, but not the cells touching the bottom and left borders.

• If there are less than 100 cells total in all 4 squares, count more squares or start again with a more concentrated cell suspension. If there are too many cells to count accurately, start again by diluting the sample 1:2 or 1:10 before adding Trypan blue.


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