Cell Counting with a Hemocytometer

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.

Hemocytometer

• 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.

Using a Hemocytometer: Four Simple Steps

1. Dilute Your Sample with Trypan Blue

• 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.

2. Loading the Hemocytometer

• 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.

3. Counting Cells in a Hemocytometer

• 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}.

4. Calculating Cell Concentration

• 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}

Calculating Total Cells in Original Sample

• 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}

Citation

• 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.