A hemocytometer is a specialized glass slide used for precise cell counting under a microscope. This instrument determines the concentration of cells in a liquid sample, finding wide application in cell culture, microbiology, and blood analysis. Accurate cell counts are important for experimental consistency and reliable diagnostic results, providing a standardized method for quantifying cellular suspensions.
Essential Preparations
Careful preparation of materials and the cell sample is important. Gather a hemocytometer, its coverslip, a microscope, a pipette with tips, and your cell suspension, which may need a diluent or stain like Trypan Blue. Thoroughly clean the hemocytometer and coverslip with lens paper or alcohol to remove debris.
Prepare the cell suspension, often by diluting it to achieve a countable concentration of 50-100 cells per large square. If using a stain like Trypan Blue for viability, mix it with your sample at the recommended ratio, typically 1:1, ensuring gentle mixing. Load the hemocytometer chamber by placing about 10 µL of the mixed sample under the coverslip, letting capillary action draw the fluid in without air bubbles or overfilling.
Navigating the Grid and Counting Cells
With the hemocytometer loaded, place it on the microscope stage and adjust focus to view the etched grid lines, typically with a 10x objective. The common Improved Neubauer hemocytometer features a central grid of nine large 1 mm by 1 mm squares.
Each large square has a known volume of 0.1 mm³. For routine counting, focus on the four corner squares and the central square of this grid.
Within these selected squares, you will see further subdivisions. For larger cells, count within the entire 1 mm² square; for smaller or denser suspensions, use the smaller subdivisions. Systematically move across the square, often in a serpentine pattern, to ensure all cells are counted once.
The “touching rule” prevents double-counting: count cells touching the top and left boundaries, but exclude those touching the bottom and right. Apply this rule consistently across all squares for accuracy.
Calculating Your Cell Count
After counting cells in the designated squares, convert this raw count into a concentration, usually expressed as cells per milliliter (cells/mL). The standard formula accounts for the total cells counted, number of squares observed, sample dilution factor, and the hemocytometer chamber’s known volume.
The general formula for cell concentration is: (Total cells counted / Number of squares counted) Dilution Factor 10,000. The 10,000 factor comes from each large square having a volume of 0.1 mm³, and 1 mL equaling 10,000 mm³.
For example, if you counted 200 cells across five large squares and your sample was diluted 1:2 (dilution factor of 2), the calculation is (200 / 5) 2 10,000, yielding 800,000 cells/mL. To enhance reliability, average counts from multiple squares or repeat the process with a fresh sample. Averaging mitigates uneven cell distribution.
Ensuring Accurate Results
Achieving reliable cell counts with a hemocytometer involves several key practices. Ensure a homogeneous cell suspension by gently mixing the sample before loading; cells tend to settle, so this prevents clumping and ensures even distribution. Proper dilution is also important, as a sample that is too concentrated can lead to overlapping cells and counting errors, while one that is too dilute might result in too few cells for statistically meaningful data. Aim for a concentration that yields roughly 50 to 100 cells per large square.
Care during the loading process is important to prevent air bubbles, which can obstruct the view, and to avoid overfilling the chamber, which alters the known volume. Consistency in applying the “touching rule” throughout the counting process prevents both undercounting and overcounting. Counting a sufficient number of cells, generally at least 100 cells in total, across multiple squares provides a more statistically sound result. Routinely checking microscope calibration and performing repeated counts from the same sample can help confirm reproducibility and accuracy.