Cell Counting 101 – Tip 4: The Key Factors Affecting Cell Viability

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Tip 4 of 6

– 3 min read

Catch up on how to calculate the cell concentration in Tip 3.

When you count cells using a hemocytometer, your secondary aim is often to determine cell viability. In other words, you count the number of viable cells compared to the number of non-viable cells in the sample so you know how your cell population is doing overall. Viability is used in everyday cell culture maintenance and in multiple experiments as a readout, for example in toxicity assays, to understand how cells thrive (or not) in their given environment1.

When you know the total number of cells as well as the number of dead cells in a sample, you can calculate cell viability using the formula:

cell viability determination formula

The lower the dead cell count, the higher the cell viability. If you’re not sure how to do it, visit our blog post on cell counting strategies.

Dead Stains Affect Viability Determination

One of the most widely used dyes to stain dead and non-viable cells is trypan blue. Being a membrane impermeable dye, it only stains non-viable cells due to their damaged cell membranes. You count these non-viable cells using a hemocytometer and a standard brightfield microscope. Trypan blue doesn’t stain these cells uniformly, rather, you will see the cells stained in shades of blue. In many cases, this causes inconsistencies in cell counts because it can be challenging to differentiate lightly stained non-viable cells from viable ones.

Another concern with dyes like trypan blue is that they can prove toxic to viable cells over time, so there’s also a time factor to consider in your procedure. This webinar tells you more about how trypan blue can affect cells, and in this blog post, we present some alternative stains.

Finally, a source of error you should consider is that you may not be able to differentiate between cells and cellular debris2, which can lead to your dead cell count becoming artificially high.

To overcome some of these challenges and to ensure that the cell viability percentage is reliable, you should validate the data by repeating the experiment multiple times. We’ll cover data validation in a future blog post.

And Beware of PBS, too

two people talking during a webinar about cell viability

It’s common to dilute or resuspend samples in PBS during handling. However, when you use this buffer, you risk inducing cell death3: Over time cell viability decreases dramatically because of shear stress4. You can reduce the effects of shear stress to some degree if you use PBS with shear-protectants but we recommend that you seek other alternatives. For example, you can dilute your cells in the medium they are grown in because you know that the cells thrive in this environment. If you want to minimize your use of PBS, you should also be aware that some staining dyes contain this buffer which could in turn affect your cell counting results.

For more information on PBS, watch our webinar.

The next blog post contains a step-by-step guide on how to determine the variance from multiple cell counts.

Further Reading


  1. MJ Stoddart: Cell viability assays: Introduction. Methods Mol Biol. 2011;740:1-6
  2. ChemoMetec: Manual vs. Automated Cell Counting
  3. G Spyrou, D Appelgren, A Rosén, B Ingelsson: Sizing Up Extracellular DNA: Instant Chromatin Discharge From Cells When Placed in Serum-Free Conditions. Front Cell Dev Biol. 2020 Jul 22;8:634
  4. A Chen, M Leith, R Tu, G Tahim, A Sudra, S Bhargava: Effects of diluents on cell culture viability measured by automated cell counter. PLoS One. 2017 Mar 6;12(3):e0173375

By Christina Psaradaki, Student Assistant at ChemoMetec
Christina Psaradaki studies Human Life Science Engineering at the Technical University of Denmark. At ChemoMetec, she writes for the Cell Counting Blog.


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