A hemocytometer, also known as a hemacytometer or counting chamber, is a specialized glass slide with a grid pattern that is used for counting cells, most commonly in the context of cell culture or blood analysis. It is a traditional and widely used tool in cell biology and hematology for quantifying cell concentration, determining cell viability, and assessing cell morphology.
The hemocytometer consists of a thick glass slide with two raised counting areas called “counting chambers” and a coverslip. The counting chambers have an engraved grid pattern, usually composed of large squares that are further divided into smaller squares. The depth of the chamber is standardized, typically at 0.1 mm, allowing the volume of the chamber to be known and used for calculating cell concentration.
To use a hemocytometer for counting cells, follow these steps:
- Prepare the cell suspension: Ensure that the cells are well dispersed and that any cell aggregates are broken up to avoid inaccurate counting. If necessary, dilute the cell suspension with an appropriate buffer or medium to achieve a countable concentration.
- Load the hemocytometer: Clean the hemocytometer and coverslip with 70% ethanol and allow them to air dry. Place the coverslip on the counting chambers, ensuring that it rests evenly on the raised edges. Carefully pipette a small volume (10-20 µL) of the cell suspension into the chamber by placing the pipette tip at the edge of the coverslip. The capillary action will draw the cell suspension into the chamber. Repeat for the second chamber if desired.
- Count the cells: Place the hemocytometer on a microscope stage and focus on the grid pattern using a 10x or 20x objective. Count the cells in a defined number of large squares, usually following a specific pattern (e.g., four corner squares and the central square). Only count the cells that are inside the square or touching the top or right borderlines. Do not count cells touching the bottom or left borderlines to avoid double-counting.
- Calculate cell concentration: Use the following formula to calculate the cell concentration:Cell concentration (cells/mL) = (number of cells counted / number of squares) x (dilution factor) x (10^4)
The factor of 10^4 accounts for the volume of the counting chamber (0.1 mm depth and 1 mm x 1 mm square size).
- Assess cell viability (optional): If the cell suspension has been stained with a viability dye, such as trypan blue, count the viable (unstained) and non-viable (stained) cells separately and calculate the percentage of viable cells as follows:% viable cells = (number of viable cells / total number of cells) x 100
While hemocytometers are simple and inexpensive tools for counting cells, they do have some limitations, such as potential user variability, the need for manual counting, and the reliance on appropriate cell dilution and dispersion. Alternatives to hemocytometers include automated cell counters and flow cytometry, which can offer higher throughput, greater accuracy, and additional information about cell populations.