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Is trypan blue toxic? The dark side of the popular stain

The gold standard dye for cell counting?

4 comments

- 3 minutes

You can’t encounter cell counting without hearing about trypan blue. Most of us associate the characteristic blue color of stained cells with the microscopy of biological samples. Trypan blue has become a fixed inventory in laboratories worldwide working with cell counting and cell viability estimation. Paul Ehrlich initially synthesized the stain in 1904 to treat trypanosomiasis in mice1. Since its invention, the non-viable cell stain has experienced huge popularity in connection to cell counting However, the excitement about this stain is wearing off due to its toxicity and possible carcinogenic effects2.

This blog post will cover:

  • Critical considerations before using trypan blue
  • Trypan blue alternatives

Why the trypan blue in your laboratory should be replaced immediately!

A web search on trypan blue reveals that it is a widely used dye to determine cell viability. Because it is commonly used, it might not cross many people’s minds that the chemical they are handling is carcinogenic and can cause genetic defects2.

With this fact in mind, perhaps it is time to move on from the use of trypan blue for staining cells. Can this change be made overnight? Of course not, but we need to start paying more attention to our consumption habits and the chemicals we use.

We will only start seeing a change when more businesses find the resources to restructure protocols and workflows to eliminate the use of trypan blue.

This doesn’t only apply to businesses. As scientists, we carry some of the responsibility too. We face decisions on whether to use one chemical or another every day.

Should we purposely expose ourselves to danger by using trypan blue? When sustainability, ecology, and health are highly valued, why don’t we pay more attention to the substances we work with daily?

Trypan blue bottle on a rolling steel table

Four alternative stains for cell viability determination

To minimize the use of trypan blue for cell viability determination, we must go for alternatives. Four cell stains are listed here, including some equivalents to trypan blue.

  1. Propidium iodide (PI) can stain the DNA or RNA of non-viable cells with damaged membranes. However, there are reports of the use of PI resulting in an underestimation of cell viability, as opposed to trypan blue, which can lead to overestimation3. PI isn’t classified as having any carcinogenic effects4.
  2. Erythrosine B stains non-viable cells because the non-intact cell membrane of these cells allows the dye to pass into the cell. It is not classified as carcinogenic and isn’t toxic, which is also why erythrosine B is commonly used as a food dye5,6.
  3. Acridine orange (AO) stains all cells through its interaction with DNA. Because of its membrane permeability, it helps determine the total number of cells in a sample. The European Chemical Agency (ECHA) states that AO isn’t carcinogenic7,8.
  4. 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) cannot permeate the cell membrane, and therefore DAPI is suitable for staining non-viable cells. These cells leave the DNA exposed, since they don’t have intact cellular and nuclear membranes. Unlike trypan blue, DAPI isn’t carcinogenic7,9.

A safe choice when performing cell viability assays is to include different types of stains that can indicate both the total amount of cells and the number of non-viable cells for a viability calculation. Here, a combination of the fluorescent stains AO and DAPI will provide an overview of the condition of the cells in the cell sample. Want to know more? Read about how the Via2-Cassette™ uses these dyes.

Learn more

Want to know more about the issues of using trypan blue in your cell counting? Watch ChemoMetec’s trypan blue webinar with a short presentation and a Q&A section addressing customer questions. Or read one of these resources:

 

References

  1. HP Morgan, IW McNae, MW Nowicki et al.: The Trypanocidal Drug Suramin and Other Trypan Blue Mimetics Are Inhibitors of Pyruvate Kinases and Bind to the Adenosine Site, Journal of Biological Chemistry, Volume 286, Issue 36, 2011, p. 31232-31240
  2. European Chemical Agency (ECHA): Substance Infocard: Tetrasodium 3,3′-[(3,3′-dimethyl[1,1′-biphenyl]-4,4′-diyl)bis(azo)]bis[5-amino-4-hydroxynaphthalene-2,7-disulphonate]
  3. C Kirchhoff, H Cypionka, Propidium ion enters viable cells with high membrane potential during live-dead staining. Journal of Microbiological Methods, Volume 142, 2017, p. 79-82
  4. European Chemical Agency (ECHA): Substance Infocard: 3,8-diamino-5-[3-(diethylmethylammonio)propyl]-6-phenylphenanthridinium diiodide
  5. European Chemical Agency (ECHA): Substance Infocard: Erythrosin B
  6. S Kamiloglu, G Sari, T Ozdal et al.: Guidelines for cell viability assays. Food Frontiers, 2020.
  7. European Chemical Agency (ECHA): Substance Infocard: N,N,N’,N’-tetramethylacridin-3,6-yldiamine hydrochloride
  8. ChemoMetec: Why working with trypan blue is not a good idea
  9. European Chemical Agency (ECHA): Substance Infocard: 2-phenylindole-4′,6-dicarboxamidine dihydrohydrochloride (hydrate)

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4 Comments

  1. Willy | 10. July 2021

    Wonderful post however , I was wondering if you could write a litte more on this topic?
    I’d be very grateful if you could elaborate a little bit
    further. Kudos!

    Reply to this message
  2. Vivat Komol | ophthalmologist | 29. August 2023

    Is it safe to inject low concentration (0.06%) Trypan blue in the eye during cataract surgery?

    Reply to this message
    • Anne Holmskov | 7. September 2023

      Hi Vivat
      This article pertains to exposure to trypan blue during cell counting procedures.
      As we are not experts in eye surgery, this question would be outside of our domain.
      But it would be the right question for a doctor or an ophthalmologist.
      We hope you find your answer soon!
      Best,
      Anne

      Reply to this message

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