A hemocytometer is a specialized glass slide with a precisely etched grid, designed for accurately counting microscopic particles like cells in a liquid sample. This counting chamber determines cell concentration, a fundamental measurement across various scientific and industrial disciplines. Applications include medical diagnostics for blood cell counts, biological research for monitoring cell growth, and yeast quantification in brewing. Accurate cell counting is important for consistency in experiments, sample preparation, and assessing cell population health. The device’s design, with its known grid area and chamber depth, provides a defined volume, enabling reliable cell density calculations.
Preparing for Cell Counting
Effective cell counting begins with careful sample preparation. Cell samples often require dilution to achieve a concentration suitable for accurate counting, typically 2.5 × 10^5 to 2.5 × 10^6 cells/mL for an Improved Neubauer chamber. Dilution is commonly performed using a diluent like phosphate-buffered saline (PBS) or a stain such as Trypan Blue. Trypan Blue is particularly useful as it selectively enters cells with compromised membranes, staining them blue, allowing differentiation between live (unstained) and dead (stained) cells for viability assessment. The dilution factor used must be carefully noted, as it directly impacts the final cell concentration calculation.
Before introducing the sample, the hemocytometer and its specialized coverslip must be thoroughly cleaned with alcohol, such as 70% ethanol, and dried to prevent debris or residue. This ensures clear visibility of the grid lines and cells. Once clean, the coverslip is carefully placed over the counting chamber, creating a precise 0.1 mm space above the grid. Setting up the microscope involves placing the loaded hemocytometer on the stage and adjusting the focus, typically starting with a 10x objective lens to visualize the grid lines and cells.
The Cell Counting Process
Loading the hemocytometer requires precision to ensure an even distribution of cells within the chamber. Using a micropipette, a small volume of the well-mixed, diluted cell suspension, typically 10 µL, is carefully introduced into the V-shaped well at the edge of the coverslip. Capillary action then draws the sample into the counting chamber, filling the space between the slide and the coverslip. Avoid overfilling the chamber or introducing air bubbles, as this can lead to inaccurate volume and uneven cell distribution.
The hemocytometer grid, particularly the Improved Neubauer chamber, features a large 3×3 mm counting grid subdivided into nine 1×1 mm squares. For most cell types, counting is performed in the four corner squares and the central square. Each of these 1×1 mm squares is further subdivided. A consistent counting rule is essential to prevent double-counting or missing cells; a common practice is to count cells that are entirely within a square and those that touch the top and left boundary lines, while excluding cells touching the bottom and right lines. This systematic approach ensures accurate, single-cell counting.
Calculating Cell Concentration
Once cells have been counted within the designated squares, the next step is to calculate the cell concentration of the original sample. This calculation uses a standard formula that incorporates the number of cells counted, the volume of the counting chamber, and any dilution factors applied. The general formula for cell concentration (cells/mL) is: (Total cells counted / Number of squares counted) × Dilution Factor × 10^4.
The factor of 10^4 is used because the volume above each 1 mm² square in a standard hemocytometer, with a chamber depth of 0.1 mm, is 0.1 mm³, equivalent to 10^-4 mL. Therefore, multiplying by 10^4 converts the cell count per 0.1 mm³ to cells per milliliter. For instance, if 325 cells were counted across 5 squares, and the sample was diluted 1:2 with Trypan Blue, the calculation would be: (325 cells / 5 squares) × 2 (dilution factor) × 10^4 = 130 × 10^4 cells/mL, or 1.3 × 10^6 cells/mL.
Ensuring Accuracy and Troubleshooting
Reliable cell counts require several best practices during hemocytometer use. Counting cells in multiple designated areas, such as the four corner squares and the central square, and averaging these counts helps account for slight variations in cell distribution. If counts between these areas vary significantly, recounting or preparing a new sample might be necessary. Maintaining consistent application of the counting rules, particularly regarding cells that touch the grid lines, is also important for reproducibility across different samples and experiments.
Several issues can affect the accuracy of cell counts:
Uneven cell distribution, often due to insufficient mixing of the cell suspension before loading, can lead to disproportionate cell numbers in different squares.
Air bubbles introduced during loading can displace the sample, altering the effective volume.
A dirty hemocytometer or coverslip can obscure cells or grid lines, leading to missed counts.
Incorrect dilution of the sample, either too concentrated or too dilute, can make counting difficult and introduce significant errors into the final concentration calculation.
Addressing these issues by proper mixing, careful loading, thorough cleaning, and appropriate dilution improves accuracy.