Cell Counting with a Hemocytometer

Hemocytometers may appear complex, but they are straightforward to use for cell counting.
The article was written by Yevgeniy Grigoryev, last updated on February 11, 2025.
An audio version of the article is available on YouTube.

Importance of Cell Counting

Many biological applications, including microbiology, cell culture, and blood work, require determining cell concentration.
Cell counting is essential for these applications.

Cell counting is performed using a counting chamber called a hemocytometer.
The hemocytometer was invented by Louis-Charles Malassez, a 19th-century French anatomist, for blood cell counts.
A hemocytometer is a thick glass microscope slide with a grid of perpendicular lines etched in the middle.
The grid has specified dimensions, allowing for the determination of cell concentration in a specific volume of solution.
The most common hemocytometer has an "H" shape with two separate mirror-like polished grid surfaces and a coverslip mounting area.

Trypan blue is a stain used to distinguish dead cells from living cells.
Dead cells are stained blue by Trypan blue, while living cells remain colorless.
A 1:1 ratio of Trypan blue to cell sample is the most common dilution.
The dilution factor must be noted for the final calculation.

Ensure the hemocytometer and coverslip are clean using lens paper.
Coverslips for hemocytometers are thicker than conventional coverslips to overcome the surface tension of the liquid.
Place the coverslip over the counting surface before loading the cell suspension.
Introduce the sample into one of the V-shaped wells using a pipette tip, allowing it to fill by capillary action.
Introduce enough liquid to cover the mirrored surface (around 10 µl) without overfilling.
Two samples can be loaded on one hemocytometer, one into each of the two grids.
Place the loaded hemocytometer on the microscope stage and focus on the counting grid at low power.
Allow the sample to settle for a couple of minutes, avoiding movement of the coverslip to prevent air bubbles.

The full grid on a hemocytometer contains nine squares, each of which is 1 \, \text{mm}^2.
The central counting area contains 25 large squares, each with 16 smaller squares.
When counting cells that overlap a line, count only those on the top or right-hand line to avoid double-counting.
Suspensions should be dilute enough to prevent cells from overlapping and should be uniformly distributed.
To perform the count, determine the magnification needed to recognize the desired cell type.
Systematically count the cells in selected squares until a total count of approximately 100 cells is reached for statistical significance.
For large cells, count the cells inside the four large corner squares and the middle square.
For a dense suspension of small cells, count the cells in the four outer and middle squares of the central square or make a more dilute suspension.
If a cell overlaps a line, count it if it overlaps the top or right-hand line and do not count it if it overlaps the bottom or left-hand line.
The area of the middle and each corner square is 1 \, \text{mm} \times 1 \, \text{mm} = 1 \, \text{mm}^2.
The depth of each square is 0.1 \, \text{mm}.
The final volume of each square at that depth is 100 \, \text{nl}.

The formula for calculating cell concentration is:
\text{Total cells/ml} = \frac{(\text{Total cells counted} \times \text{Dilution factor} \times 10,000 \, \text{cells/ml})}{\text{Number of squares counted}}
Example:
- If the sample is diluted 1:1 with Trypan blue (dilution factor = 2).
- 325 cells are counted in the four corner squares plus the central square (number of squares counted = 5).
- Then:
  \text{Total cells/ml} = \frac{(325 \, \text{cells} \times 2 \times 10,000 \, \text{cells/ml})}{5} = 1.3 \times 10^6 \, \text{cells/ml}

To find the total number of cells in the original sample, multiply the cell concentration by the total sample volume.
Example:
- If the original sample volume is 5 \, \text{ml}.
- Then:
  \text{Total cells in sample} = 1.3 \times 10^6 \, \text{cells/ml} \times 5 \, \text{ml} = 6.5 \times 10^6 \, \text{cells}

This article was modified by A.R. Beyer for BIO211 students at Richard Bland College.
Original article accessed on 03/19/2025.
Originally published 2013; updated and republished June 2021.