Cell Counting Protocols Using a Haemocytometer

Cell counting using a hemocytometer is a commonly used technique to determine the concentration of cells in a given sample. The detailed procedure explains how to obtain a viable cell count from a haemocytometer.

Preparing haemocytometer

• Ensure the haemocytometer is clean using 70% ethanol.
• Moisten the shoulders of the haemocytometer and affix the coverslip using gentle pressure and small circular motions. The phenomenon of Newton’s rings can be observed when the coverslip is correctly affixed, thus the depth of the chamber is ensured.

Preparing cell suspension

• Make sure the cell suspension to be counted is well mixed by either gentle agitation of the flask containing the cells (or other appropriate container). A serological pipette may be used if required.
• Before the cells have a chance to settle take out about 1 ml of cell suspension using a serological pipette and place in an Eppendorf tube.
• Using a 100 μl pipette, mix the cells in this sample again (gently to avoid lysing them). Take out 100 μl and place into a new Eppendorf, add 100 μl trypan blue, and mix gently again.

Counting

• Using the pipette, draw up some cell suspension containing trypan blue. Carefully fill the haemocytometer by gently resting the end of the tip at the edge of the chambers. Take care not to overfill the chamber. Allow the sample to be drawn out of the pipette by capillary action, the fluid should run to the edges of the grooves only. Re-load the pipette and fill the second chamber if required.
• Focus on the grid lines of the haemocytometer using the 10×objective of the microscope. Focus on one set of 16 corner squares as indicated by the circle.
• Using a hand tally counter, count the number of cells in this area of 16 squares. When counting, always count only live cells that look healthy (unstained by trypan blue). Count cells that are within the square and any positioned on the right-hand or bottom boundary line.
• Move the haemocytometer to another set of 16 corner squares and carry on counting until all 4 sets of 16 corner squares are counted.
• The haemocytometer is designed so that the number of cells in one set of 16 corner squares is equivalent to the number of cells×10⁴ / ml.

Creative Bioarray Relevant Recommendations

• Creative Bioarray provides cell counting kits that commonly are used for cell proliferation and viability. Additionally, we also provide a series of histological staining methods and related products to help our clients achieve dyeing purposes.

• Ensure that the hemocytometer is clean and properly calibrated before use. Use only calibrated pipettes to accurately measure and transfer the cell suspension onto the hemocytometer.
• Thoroughly mix the cell suspension to ensure uniform distribution of cells. Clumped or aggregated cells can lead to inaccurate cell counts.
• Avoid overfilling the hemocytometer chamber to prevent spillage and ensure that the chamber is evenly filled without trapping air bubbles.
• Perform cell counting in multiple replicates to ensure consistency and reliability of the results. The average count should be used for final calculations.

Cell Services

Cell Counting Kit