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Counting cells using a haemocytometer

View our detailed step-by-step protocol explaining how to obtain a viable cell count from a hemocytometer.
Last edited Thu 17 Feb 2022

Protocol to obtain a viable cell count from suspension cells using a hemocytometer.

Stage 1 - Preparing hemocytometer

Steps

1

If using a glass hemocytometer and coverslip, clean with alcohol before use.

  • Moisten the coverslip with water and affix to the hemocytometer.
2

If using a disposable hemocytometer (for example, INCYTO DHC-N01), simply remove from the packet before use.

Stage 2 - Preparing cell suspension

Aseptic technique prevents contamination of cell cultures and reagents by microorganisms. Our aseptic technique video protocol shows you how to sterilize work areas and use appropriate sterile handling techniques, personal protective equipment, and good hygiene.

Table 1. The volume of DPBS and trypsin-EDTA required for trypsinization of adherent cells. T-flash (cm2) DPBS (mL) Trypsin-EDTA (mL) FBS containing media required to neutralize trypsin 25 2 2 6 80 3 3 9 175 5 5 15

Materials required



Steps

1

Gently swirl the flask to ensure the cells are evenly distributed.

2

Before the cells have a chance to settle, take out 0.5 mL of cell suspension using a 5 mL sterile pipette and place in an Eppendorf tube.

3

Take 100 µL of cells into a new Eppendorf tube.

  • Take 100 µL of cells into a new Eppendorf tube and add 400 µL 0.4% Trypan Blue (final concentration 0.32%).
  • Mix gently.

Stage 3 - Counting

Steps

1

Using a pipette, take 100 µL of Trypan Blue-treated cell suspension and apply to the hemocytometer.

  • If using a glass hemocytometer, very gently fill both chambers underneath the coverslip, allowing the cell suspension to be drawn out by capillary action. 
  • If using a disposable hemocytometer, pipette the cell suspension into the well of the counting chamber, allowing capillary action to draw it inside.
2

Using a microscope, focus on the grid lines of the hemocytometer with a 10X objective.

3

Using a hand tally counter, count the live, unstained cells (live cells do not take up Trypan Blue) in one set of 16 squares (Figure 1).

  • When counting, employ a system whereby cells are only counted when they are set within a square or on the right-hand or bottom boundary line.
  • Following the same guidelines, dead cells stained with Trypan Blue can also be counted for a viability estimate if required.

Figure 1. ​​Hemocytometer diagram indicating one of the sets of 16 squares that should be used for counting.

4

Move the hemocytometer to the next set of 16 corner squares and carry on counting until all 4 sets of 16 corners are counted.

Stage 4 - Viability

Steps

1

Take the average cell count from each of the sets of 16 corner squares.

2

Multiply

  • Multiply by 10,000 (104).
3

Multiply by 5 to correct for the 1:5 dilution from the Trypan Blue addition.