# Cell Counting with a Hemocytometer: Easy as 1, 2, 3

Many biological applications such as microbiology, cell culture, blood work and many others that use cells require that we determine cell concentration for our experiment.

Cell counting is rather straightforward and requires a counting chamber called a hemocytometer, a device invented by the 19^{th} century French anatomist Louis-Charles Malassez to perform blood cell counts. A hemocytometer consists of a thick glass microscope slide with a grid of perpendicular lines etched in the middle. The grid has specified dimensions so that the area covered by the lines is known, which makes it possible to count the number of cells in a specific volume of solution.

The most common type of hemocytometer has an “H” shape engraved in the middle that encloses two separate mirror-like polished grid surfaces and provides the cover slip mounting area (Figure 1).

## Loading the hemocytometer

Before starting ensure that both the hemocytometer and its coverslip are clean by removing any dust particles with lens paper. Coverslips that are used for mounting on hemocytometers are specially made to be thicker than the conventional microscopy coverslips because they must be able to overcome the surface tension of a drop of liquid.

Make sure to first place the coverslip over the counting surface before loading the cell suspension. Then place the pipette tip with your sample into one of the V-shaped wells, as in Figure 2, and gently expel the sample. The area under the coverslip fills by capillary action. Enough liquid should be introduced so that the mirrored surface is just covered, usually around 10 µl, but do not overfill the surface. You can load two samples on one hemocytometer, one into each of the two grids.

The loaded hemocytometer is then placed on the microscope stage and the counting grid is brought into focus at low power. Allow the sample to settle for a couple of minutes and avoid moving the coverslip as it might introduce air bubbles and make counting difficult.

## Counting cells in a hemocytometer

The full grid on a hemocytometer contains nine squares, each of which is 1 mm^{2} (Figure 3). The central counting area of the hemocytometer (Figure 3B) contains 25 large squares and each large square has 16 smaller squares. When counting, count only those cells on the lines of two sides of the large square to avoid counting cells twice (Figure 3G). Suspensions should be dilute enough so that the cells or other particles do not overlap each other on the grid, and should be uniformly distributed.

To distinguish between dead and viable cells, the sample is often diluted with a particular stain, such as Trypan blue. This staining method, also known as dye exclusion staining, uses a diazo dye that selectively penetrates cell membranes of dead cells, coloring them blue, whereas it is not absorbed by membranes of live cells, thus excluding live cells from staining. When viewed under a microscope, dead cells would appear as dark blue (Figure 4)

To perform the count, determine the magnification needed to recognize the desired cell type and systematically count the cells in selected squares so that the total count is approximately 100 cells, a minimum number of cells needed for a statistically significant count.

For large cells, you can simply count the cells inside the four large corner squares (Figure 3C-F) and the middle one (Figure 3B). For a dense suspension of small cells you may wish to count the cells in the four outer and middle squares of the central square (Figure 3B) or make a more dilute suspension.

Remember if a cell overlaps a ruling, count it as “in” if it overlaps the top or right ruling, and “out” if it overlaps the bottom or left ruling (Figure 3G).

The area of the middle (Figure 3B) and each corner square (Figure 3C-F) is 1 mm x 1 mm = 1 mm^{2}: the depth of each square is 0.1 mm. The final volume of each square at that depth is 100nl.

Once you have obtained the total cell count, cell concentration can be calculated from the following formula:

So, for example, if you diluted your sample 1:1 with Trypan blue, and you counted 325 cells in 4 corner squares plus the central big square, total cells per ml =

If you want to know how many cells you have in your original sample, just multiply the cell concentration by total sample volume. For example, if your original sample volume is 5 ml, then your sample has a total =

*Originally published on 3 July October 2013; updated and republished on 8 December 2014.*

### 23 Comments

### Leave a Comment

You must be logged in to post a comment.

You did not explain why you calculate 2x dilution factor x 10,000 cells/mL.

What is the 2? What is the dilution factor? You can’t do a calculation with a word “dilution factor” – can you explain where that part comes from? And what thing has a concentration of 10,000 cells/mL? This is not clear.

TWO THINGS

1. TO AVOID A CELL COUNTED TWICE.

2. CELLS IN THE LINE ARE NOT INSIDE THE SPECIFIC SQUARE GROOVE NEITHER NOR THEY ARE NOT OUTSIDE THE GROOVE. IF YOU INCLUDE ONE SIDE IT IS FAIR EXCLUDING THE OTHER SIDE.

I was looking for the same information and you have just posted it. That’s so wonderful and nice article. Thank you so much for sharing.

Pls am just learning for the first time how do I stain with the trypan blue is it after air drying it with slide or mixing the volume i

neede with the trypan blue

Hi,

If I use bacterial cells and I’ve got 3×10 pwr 7 cells/ml in my tube, can I consider it as 3×10 pwr 7 cfu/ml? (noting that if only viable cells are calculated).