So, you've a lot of cells and you want to know just how many? Cell counting is a key skill and it is crucial to use the correct proceedures.

In this tutorial we assume that you have obtained your cells in a suspension within an unknown volume of some fluid. If this is not the case, it is advantageous if the cells are manipulated to this stage before commencing counting.

Resources Needed

Hemacytometer - including cover slip
Microscope - many types are suitable
Handheld counter - of the click per count type, useful, but not essential
Gilson pipette - about 200uL
Tip for gilson pipette - 1 sterile, 1 not
Trypan Blue - small quantity required

Preparing sample

It is essential that the cells are in a single cell suspension. Drawing up and expressing the suspension through a 23G needle and suitably sized syringe will generally achieve this. If the cells when viewed under the microscope still appear to be in clumps, then an incubation in pronase 10mg/mL for half an hour should achieve a single cell suspension.

At this point, remove a small aliquot using the Gilson pipette and a sterile tip. Add to this aliquot an equal amount of Trypan Blue. These should be well mixed.

Preparation of Hemacytometer

The hemacytometer consists of two parts - the one part containing the grid, and the smaller part being the cover slip. Breath on the cover slip so it becomes clouded, and then place it squarely above the grid. A small amount of pressure and a small slide will ensure a good stick. On the sides of the coverslip small rainbow-like rings can be seen - these are an indication of a proper fit, and are known as Newton's Rings. Taking a small amount of the cell and trypan blue mix, add a small drop to each side of the grid, so that it flows under the coverslip to cover the grid. Now you are ready to count cells.

Cell counting

The cell counter, when viewed under the microscope has two sides. Taking just one side, it is possible to see a central grid. This is the grid upon which cells will be counted. It is normally 1mm x 1mm, and the gap between coverslip and grid is generally 0.1mm. This gives a total volume above the grid of 0.0001mL.

Trypan Blue is excluded from viable cells, so when you view under the microscope, the blue cells are dead ones, and the whiter coloured cells are live. Count in each of the two central grids the total number of cells alive, and the total number dead. The counting completed, clean Trypan Blue from the Hemacytometer using 70% industrial methylated spirits and a paper towel.

Calculations

We recommend you try our Cell Counting Calculator for finding out your total number of cells. The principle is to calculate how many cells are in the volume you've counted (0.0001mL), then allow for the dilution with Trypan Blue, and finally scale that up to the total volume of cell suspension you have.