What is the most effective way to count cells under the microscope using hemocytometer. My lab is awaiting to buy a good automated cell counter but for now I have to learn this manually and applying for my experiments.
Here's a guide for effective cell counting using a hemocytometer:
Preparation:
Cleanliness: Ensure the hemocytometer and coverslip are clean and dust-free. Use ethanol or appropriate cleaning solutions recommended by the manufacturer.
Cell Suspension: Gently resuspend your cells to ensure a homogeneous distribution. Avoid clumps or uneven settling.
Hemocytometer Loading:
Micropipette Technique: Use a calibrated micropipette to load your cell suspension (typically 10 µL) onto the hemocytometer chamber.
Capillary Action: Allow the cell suspension to fill the chamber by capillary action. Avoid overfilling, which can cause inaccurate counts.
Coverslip Placement: Gently place the coverslip over the chamber at an angle, allowing it to settle slowly to avoid introducing air bubbles.
Microscope Observation:
Microscope Setup: Use a phase-contrast microscope at 10x objective for optimal cell visualization.
Counting Grid: Locate the central counting area on the hemocytometer. It typically consists of a large square subdivided into 25 smaller squares.
Counting Strategy: There are two main approaches: Absolute Count: Count all cells within the entire central square (all 25 small squares). This is more time-consuming but provides the most accurate total cell count. Relative Count: Count cells in a defined number of smaller squares (e.g., four corner squares and the center square). This is faster but less accurate for absolute cell number determination.
Counting Rules:
Cell Inclusion: Only count cells fully within a square or touching the top and right borders (not bottom or left). This avoids double counting.
Dead/Live Differentiation: If using viability dyes like Trypan Blue, count only unstained (viable) cells.