A hemocytometer is a specialized glass slide used for counting cells suspended in a liquid. This precision instrument allows researchers and medical professionals to determine the concentration of various cell types, such as blood cells, yeast, or cultured cells, within a given sample. Its primary application involves placing a small volume of a cell suspension onto a designated counting chamber and then observing the cells under a microscope. This method provides a direct and reliable way to quantify cellular components.
Understanding the Hemocytometer Grid
The hemocytometer features an etched grid pattern that defines counting areas and volumes. The Improved Neubauer chamber, a common type, contains a 3 mm x 3 mm central square encompassing nine large squares. Each of these nine large squares measures 1 mm x 1 mm, with a total area of 1 square millimeter per large square.
The central large square is further subdivided into 25 medium squares, each measuring 0.2 mm x 0.2 mm. Within each of these 25 medium squares are 16 even smaller squares, with dimensions of 0.05 mm x 0.05 mm. A raised edge on the hemocytometer holds the coverslip exactly 0.1 mm above the etched grid, creating a chamber of known depth. This design of defined areas and depth allows for the calculation of cell concentration within a specific volume.
Preparing Your Sample and Loading the Hemocytometer
Accurate cell counting begins with proper sample preparation to ensure an evenly distributed cell suspension. Samples may be too concentrated, requiring dilution with a diluent like saline solution, phosphate-buffered saline (PBS), or a specific cell culture medium. Thorough mixing of the diluted sample prevents cell clumping and ensures uniform distribution.
Once prepared, the hemocytometer and its coverslip must be clean and free of debris. To load the hemocytometer, a small volume, typically 10 to 20 microliters, of the well-mixed cell suspension is applied to the V-shaped groove at the edge of the coverslip. Capillary action then draws the liquid evenly into the counting chamber, filling the space between the coverslip and the grid. It is important to avoid overfilling the chamber or introducing air bubbles, as this can lead to inaccurate cell counts.
The Cell Counting Process
After loading the hemocytometer, allow the sample to settle for a few minutes to ensure cells are stationary before beginning the count. Using a microscope, typically at 10x or 20x magnification, bring the grid lines into focus. For most cell types, like white blood cells or yeast, counting is performed in the four large corner squares and the central large square of the hemocytometer grid. For smaller, more numerous cells such as red blood cells, counting may be focused on the 25 medium squares within the central large square.
A consistent counting strategy is important to avoid double-counting or missing cells. A common method, often called the “L-rule,” involves counting cells that are entirely within a square and those that touch the top and left boundary lines of the square. Cells touching the bottom and right lines are excluded from the count for that square to ensure each cell is counted only once. Moving systematically through each designated counting square helps maintain accuracy. A hand tally counter can be used to keep track of the cell count in each square, making the process more efficient.
Calculating Cell Concentration
After counting cells in the designated squares, calculate the cell concentration of the original sample. This calculation converts the observed cell count in a known volume into cells per milliliter (cells/mL). The standard formula for this calculation is: Cell Concentration = (Total Cells Counted / Number of Squares Counted) × Dilution Factor × Volume Factor.
The “Total Cells Counted” is the sum of all cells tallied across the selected squares. The “Number of Squares Counted” refers to the specific number of large squares (e.g., 5 for white blood cells) or medium squares used. The “Dilution Factor” accounts for any dilution made to the original sample before loading.
The “Volume Factor” is derived from the known volume of each counted square. For an Improved Neubauer chamber, a 1 mm x 1 mm square with 0.1 mm depth has a volume of 0.1 mm³, equivalent to 10⁻⁴ mL. Therefore, a common volume factor is 10,000 to convert to cells per milliliter. For instance, if 200 cells were counted in 5 large squares with a 1:1 dilution, the concentration would be (200 / 5) × 1 × 10,000 = 400,000 cells/mL.