Why are there different hemocytometer grid sizes?
The hemocytometer was originally invented by Louis-Charles Malassez to count blood cells. But later on, biologists and experimental doctors discovered that it could be applied to other areas, to count yeast, sperm, and other types of human and animal cells. A hemocytometer with a different grid to account for smaller particles became necessary (as bigger squares meant more cells to count per area unit but also less guiding lines for the count – so more errors).
The current hemocytometer is composed of nine equally sized bigger squares. The central one is different from the other ones because it is divided into 25 smaller squares, while the ones in the corners are divided into 16 smaller squares. The rest of squares are not used. In addition, the smaller squares inside the central square are subdivided into 16 even smaller squares each. This allows to count very tiny cells with the same precision level as larger ones (but with a higher magnification).
What size to use with my cell type?
Let’s go through the maths – and then, if you know what the average size (diameter) of your cell is, you can tell yourself. I’ll just give the most common examples.
The main piece of information you should remember is this: each of the 9 squares in a chamber is 1mm in width (or 1mm2 in area, as it’s a square).
Corner squares – WBC
For the corner squares, each of the 16 smaller squares will be 1 mm/4 = 0.25 mm in width and 0.25 mm x 0.25 mm = 0.0625 mm2 in area (or 1 mm2/16 = 0.0625 mm2). Therefore, cells that are 10 μm or more should be counted in these corner squares (although it doesn’t hurt if you also include a count from the central square). For example, white blood cells (leukocytes) satisfy this criterion.
Central square – RBC, yeast, sperm cells…
For the central square, each of the 25 smaller squares will be 1 mm/5 = 0.2 mm in width and 0.2 mm x 0.2 mm = 0.04 mm2 in area (or 1 mm 2/25 = 0.04 mm2). In turn, each of the 25 smaller squares contains 16 even smaller squares which measure: 0.2 mm/4 = 0.05 mm in width and 0.05 mm x 0.05 mm = 0.0025 mm2 = 2500 μm2 (or 0.04 mm2/16 = 0.0025 mm2). Cells that are 10 μm or smaller should be counted in the central square – sometimes even in one of the smaller squares inside the central square. Typically you would count red blood cells, platelets, most types of yeast, and sperm cells.
So what about your cells? did you decide in which square type you0re going to count them?
Surface to volume
This step is super easy, but I’m going to make it even easier with a summary table. Just keep in mind that the vertical distance between the slide and the chamber is always 0.1 mm mul tiply your area by 0.1 mm and you will be fine.
Unit | Width | Area | Volume (mm3) | Volume (mL) | # | |
chamber | 3 mm | 9 mm2 | 0.9 mm3 | 0.0009 mL | 2 | per hemocytometer |
small sq. | 1 mm | 1 mm2 | 0.1 mm3 | 0.0001 mL | 9 | per chamber |
corner sq. | 1 mm | 1 mm2 | 0.1 mm3 | 0.0001 mL | 4 | per chamber |
smaller sq. | 0.25 mm | 0.0625 mm2 | 0.00625 mm3 | 0.00000625 mL | 16 | per corner square |
central sq. | 1 mm | 1 mm2 | 0.1 mm3 | 0.0001 mL | 1 | per chamber |
smaller sq. | 0.2 mm | 0.04 mm2 | 0.004 mm3 | 0.000004 mL | 25 | per central square |
even smaller sq. | 0.05 mm | 0.0025 mm2 | 0.00025 mm3 | 0.00000025 mL | 16 | per smaller square |
And last but not least: you should divide by the volume above in your hemocytometer calculation! That will give you the cells per mL.