Cell Counting 101 – Tip 2: What To Consider When Calculating Dilution Factor

Tip 2 of 6

The previous post in this series covers how to use a hemocytometer. Now, we’re onto the dilution factor.

When you are counting cells, you often need to dilute the sample. For example, if using a hemocytometer the recommended concentration is around 106 cells/ml to ensure that the number of cells is within the optimal range of resolution when viewed through the microscope. However, the Improved Neubauer chamber, which is the most used type of hemocytometer, has an optimum range of 2.5×105 – 2.5×106 cells/ml1. If you have samples with a cell concentration above 2.5×106 cells/ml, you need to further dilute them to lower the risk of counting errors. If you have samples with a cell concentration below 2.5×105 cells/ml, you should increase cell concentration to obtain more accurate cell counts. We will return to sources of variation in cell counting in a later blog.

How To Make a Serial Dilution

To perform a serial dilution, first, you will need to prepare an appropriate diluent for your sample. Common diluents are complete or incomplete cell culture media, or saline solutions such as phosphate buffered saline (PBS). This is an example of how to make a two-fold serial dilution2:

1. Set up a suitable number of test tubes depending on the final dilution factor you desire (the dilution factor is explained below). If not sure, do a quick and dirty cell count to know the concentration range of your sample
2. Resuspend the original sample you want to dilute to make sure that you take a representative sample
3. Add 5 µl of the diluent to all the empty test tubes with a micropipette
4. Add 5 µl of the cell sample to the first test tube. Resuspend thoroughly
5. Transfer 5 µl of mixture from the first test tube to the second test tube and resuspend
6. Repeat these steps until you obtain the desired dilution factor

This example illustrates the principles of serial dilutions and can be used for different volumes of the diluent (sample) and dilutant (medium or buffer), and with different dilution buffers.

We recommend a 1:1 dilution so you don’t need to adjust the volume of the micropipette, which ensures consistency because the pipette volume isn’t altered, and the same pipette is used every time. Also, if you frequently readjust the volume of the micropipette, you risk altering the calibration which will affect your dilutions too.

Calculating the Dilution Factor

When you dilute a sample, you need to calculate the dilution factor. You need to incorporate this factor in calculations such as the total cell count to get a result that reflects the original sample, not only the diluted sample. To calculate the dilution factor, use the following formula:

For serial dilutions, like the example described above, calculate the final dilution factor with the formula:

Where α = the number of dilutions performed in total (i.e. 6 in the example above).