Those who manually count cells are likely familiar with the hemocytometer equation. But knowing the equation is one thing—do we truly understand the significance of each value within it?
Manual counting with a hemocytometer
Before diving into the equation used for manual cell counting, let’s first review the hemocytometer itself. There are different types of grids on a hemocytometer, with the most commonly used being the Improved Neubauer grid. This will be the focus of our discussion.
So the equation for manual counting is,
Total cell count (cells/mL) = (number of cells x dilution factor x 10⁴) number of squares counted
Other values are straightforward, but where does the 10⁴ value come from?
The Improved Neubauer grid consists of 9 squares in total. Each square has an area of 1 mm by 1 mm and a depth of 0.1 mm, giving it a volume of 0.1 mm³. Knowing this volume is crucial because all the cells within each square are counted, allowing us to calculate the total number of cells in the original sample. By knowing the volume, we can also convert the count to cells per mL. Here’s how it works.
We can now convert mm³ to mL.
0.1 mm³ is equal to 0.0001 mL, or 10¯⁴ mL. Since we want the unit to be per mL, this value becomes the divisor in the equation.
It’s important to note that the ‘number of cells’ in the equation refers to the total sum of cells counted across all squares, not the average. This is because the equation already accounts for the number of squares counted, so calculating an average is unnecessary.
For example,
if I use an undiluted cell sample (dilution factor = 1),
and counted 4 corner squares and got 500 cells in total,
the total cell count would be as follows:
(500 x 1 x 10⁴) / 4 = 1.25 x 10⁶ cells/mL
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