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Determining the concentration of cells in a liquid sample is a routine yet essential task in clinical and research laboratories. Accurate cell counts inform doctors about a patient’s immune status, help diagnose infections, and guide fertility assessments. In research, they enable scientists to quantify bacteria, yeasts, and other microorganisms for ecological studies or industrial processes. The most common, reliable method for cell counting is the hemocytometer, a precise counting chamber widely taught in biology courses and used in professional labs worldwide.
When a cell suspension contains millions of cells, direct counting becomes impractical because cells overlap and cannot be distinguished. To achieve a suitable dilution, transfer 10 µL of the original solution into a sterile test tube that contains 90 µL of an appropriate diluent (e.g., phosphate‑buffered saline or culture medium). Mix thoroughly; this creates a ten‑fold dilution (dilution factor = 10⁻¹). Label the tube and repeat the procedure until the sample is dilute enough for accurate counting. Each additional ten‑fold dilution multiplies the dilution factor by 10 (e.g., a second dilution gives 10⁻², a third 10⁻³).
The hemocytometer is a small, clear chamber with a grid etched onto its bottom surface. Its depth is typically 0.1 mm, and the grid is divided into large and small squares whose areas are 1 mm² and 0.04 mm², respectively. Pipette a small volume of the diluted suspension into the chamber’s well. Capillary action spreads the liquid across the grid. Place the chamber on a microscope stage and view it at low magnification (×10–×40).
Choose a convenient pattern of squares—commonly the four corners and a central square—to count at least 100 cells. For large cells, use the larger squares; for smaller cells, count the smaller squares. Record the number of cells in each selected square.
Each large square has a volume of 0.1 mm³ (0.1 mm × 1 mm²). If you counted 103 cells across five large squares, the total counted volume is 0.5 mm³. Doubling this volume to represent a full 1 mm³ gives 206 cells. Because 1 mL equals 1 cm³ and contains 1,000 mm³, the concentration in the diluted sample is:
Formula: Volume of counted area × number of squares = total counted volume
Cells ÷ total counted volume = cells per mm³
cells per mm³ × 1,000 = cells per mL
Adjust for the dilution factor used during preparation. For a dilution factor of 10⁻² (i.e., 1:100), the original sample concentration is:
Thus, the initial undiluted suspension contained approximately 20.6 million cells per milliliter.
