Using a haemocytometer

I've been getting some questions on how I do cell counting, so here's a quick documentation. It also serves as a quick reference for me to go back to, if I forget my calculations. My cell counts are done using a microscope at 400X magnification, and using a haemocytometer - i.e. manual cell counting. A good hemocytometer can set you back by hundreds of dollars, but I managed to get quite a decent one off eBay, even if it's made in China.

This is the central grid of the haemocytometer.

400X magnification of each small square.

A hemocytometer is essentially a glass slide that has been laser-etched with markings, and comes with a special (heavier) cover slip. This ensures that if done properly, there is always a constant volume of 0.0009ml in the chamber under the cover slip. I use the central grid which consists of a total of 25 large squares. Each of these large squares are further divided into 16 smaller squares. The entire volume of this central grid works out to be 0.0001ml. Each of the 25 large squares has a volume of 0.000004ml - you can do the calculations from here! So basically, the number of cells are counted in each large square and based on the known volume of this large square, you can calculate the cell density in terms of cells/ml.

A closer look at each of the 25 large squares, each being further sub-divided into 16 small squares.

Of course, this takes quite a bit of time with all the prep work etc, and very rarely I do have access to a flow cytometer. However, flow cytometers are unable to count yeast cells properly at our usual pitching densities, and it is also not possible to do a viability count. (That'll come in another post - how to do a viability test using a haemocytometer)

So here's my usual protocol:

Dilution
For most propagations the straight slurry is too dense to do a proper cell count. As a result, I dilute a sample by a factor of 10 before loading it into the haemocytometer. I use a variable-volume micropipette for this so the sampling is pretty accurate (once again, from sources in China due to cost but it's surprisingly accurate!) This is shaken to homogenise the sample, and 1 microlitre of this sample is loaded into the cell count chamber of the haemocytometer.

Counting
Next, I count 5 of the large squares (one from each corner, then the centre one) and divide by 5 to get an average cell count / big square. The haemocytometer I got came with a nice hand-counter, so I don't have to overload my rather tiny brain with counting.

Calculation formula 
Cells/ml = average cell count / big square x dilution factor x 10,000

So, for example I prepare a sample by diluting it 0.01ml of yeast slurry in 0.09ml of filtered water (10X dilution). I count a total of 100 cells in the 5 large squares, which equates to an average of 20 cells per large square. The cell count in this case would be = 20 cells x 10 (dilution factor) x 10,000 = 200,000 cells / ml.

Of course, there is always the chance of sampling error (eg non-homogenised sample) and observer error (eg I get distracted and lose count, or double count), so I usually do a cell count at least a couple of times to minimise error.