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

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What is a hemocytometer and what is it used for?

Pipette injecting liquid into a hemocytometer slide

A hemocytometer, also known as a hemacytometer or haemocytometer, is a glass microscope slide used for manually counting cells in a cell culture sample. To help researchers count the cells methodically and accurately, the hemocytometer contains a grid of varying sized squares and perpendicular lines. Each chamber grid is composed of nine squares (subgrids), with each square being 1mm 2. The most common hemocytometer grid is the Neubauer or Improved Neubauer, though other grid styles exist as alternatives for varying cell culture applications. Cell density calculations are necessary to ensure cells in culture have adequate space to behave naturally and proliferate. To find a sample's cell density, you must know the size of your grid space and the volume of the counting chamber. Hemocytometer chambers are designed to hold a specific volume of a culture sample so you can obtain accurate and reproducible cell densities. Different grids may contain varying volumes, however the standard volume for a Neubauer grid square is 0.1 mm3.

Materials for manually counting cells in a hemocytometer

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


Cell counting protocol

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.

Why do we multiply by 10,000 when counting cells?

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.


Hemocytometer calculation technique

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)


Additional cell counting protocols

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

Next protocol:

Trypan Blue Staining Protocol

Thermo Fisher offers a vast array of products for your mammalian cell lines and experiments.

EVOS Imaging Systems display high-quality optics and intuitive interfaces for capturing and analyzing images of cells


References
  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.
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