Manually Counting Cells in a Hemocytomer

Learn about using a hemocytometer to count cells and how to count cells manually.

In addition to the materials below, hemocytometers may be obtained from Fisher Scientific.

The procedure below provides some general directions on how to count cells manually using a hemocytometer. Dissociating adherent cells and collecting suspension cells for counting and analysis requires additional steps.

See Adherent Cell Culture Protocols
See Suspension Cell Culture Protocols

If you wish to measure the concentration of viable cells, a trypan blue stain can be added.

See the Trypan Blue Exclusion Protocol

1. Clean the chamber and cover slip with alcohol. Dry the coverslip and fix it in place.
2. Harvest the cells and gently, but thoroughly, resuspend the cell pellet (if needed). Using a micropipette, add 10 μL of the cell solution to the hemocytometer. Do not overfill.
3. Secure the chamber in the inverted microscope under a 10X objective. Use phase-contrast to distinguish the cells.
4. To calculate cell count, count the number of viable cells in the large, central gridded square (1 mm²) moving in a zig-zag pattern down each row. The gridded square is circled in the graphic below. Multiply by 10⁴ to estimate the number of cells per mL (concentration of cells in your sample). Prepare duplicate samples and average the count to find the cell concentration.

10,000, or 10⁴, is a correction factor used to convert the number of viable cells counted in one Neubauer grid square to the number of cells per one milliliter (mL).

As mentioned above, the Neubauer grid has a volume of 0.1 mm³. The volume of 0.1 mm³ = 1x10^-4 mL = 1/10,000 mL. If we want to find the concentration (cells/1 mL) of one Neubauer grid, we need to multiply the number of counted cells by 10,000 to convert the volume of one grid square to 1 mL.

Therefore, we multiply by 10,000 (10⁴) to find the concentration of cells/mL.

Using a hemocytometer requires the researcher to perform manual calculations to find the concentration of cells in the sample. In a basic hemocytometer calculation, such as the one described in the protocol above, researchers find the concentration of cells by multiplying the count of cells in the central grid by 10⁴.

However, if you are looking for the density of viable cells in your sample, you can use the formula: C = N x D x 10³.
C = count of viable cells/mL (concentration)
N = number of viable cells counted in 10 subgrids (1.0 mm³)
D = dilution factor
10³ = correction factor

You may also want to know how many cells of your total cell concentration were viable. To find this use the formula V = N/T x 100%.
V = percentage of viable cells
N = number of viable cells counted in 10 subgrids (1 mm³)
T = number of total cells counted in 10 subgrids (1 mm³)

Hemocytometers were originally developed to count blood cells, though they have since been used to count cells for various cell types and species. While counting cells in a hemocytometer is the traditional method for cell counting, it is not necessarily the most efficient or accurate. Many hemocytometer users opt for this method of cell counting because it is commonly referenced in various existing protocols and research. However, manually counting cells is often time consuming and leaves room for human error. Automatic cell counters are becoming the new standard for cell counting protocols. Compare the difference between counting cells with a hemocytometer vs an automated cell counter.

View the library of Cell Counting Information

1. Absher, Marlene (1973) Chapter 1 – Hemocytometer Counting. Tissue Culture. Academic Press. pp 395–397.
2. Hoffman, Tracy L. (2006) Counting Cells. Cell Biology (Third Edition). Academic Press. pp 21–24.
3. Camacho-Fernández, C., Hervás, D., Rivas-Sendra, A. et al. (2018) Comparison of six different methods to calculate cell densities. Plant Methods 14, 30. 
4. Fuentes, Maria. (2024) Hemocytometer - cell counting with a hemocytometer 
5. Hemocytometer image https://commons.wikimedia.org/wiki/File:Hemocytometer.jpg . License: https://creativecommons.org/licenses/by-sa/2.0/ . This image has not been edited from its original form.