Is Trypan Blue Toxic? The Dark Side of the Popular Stain

The Gold Standard Dye for Cell Counting?

You can’t encounter cell counting without hearing about trypan blue. The characteristic blue color of stained cells is something that most of us associate with microscopy of biological samples. Trypan blue has become fixed inventory in laboratories across the world that are working with cell counting and cell viability estimation. The stain was initially synthesized by Paul Ehrlich 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 the possible carcinogenic effects it can have on the operator2.

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 quickly reveals that it is a widely used dye to determine cell viability. Because it is commonly used, it might not cross many peoples’ 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—also regarding 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? At a time where sustainability, ecology and health is highly valued, why don’t we pay more attention to the substances that we are working with daily?

Four Alternative Stains for Cell Viability Determination

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

1. Propidium iodide
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
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
Acridine orange (AO) stains all cells by its interaction with DNA. Because of its membrane permeability, it can be used to determine the total amount of cells in a sample. According to the European Chemical Agency (ECHA), AO isn’t carcinogenic7,8.

4. 4′,6-diamidino-2-phenylindole dihydrochloride
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 presentaitons and a Q&A section addressing customer questions. Or read one of these resources:

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.

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. Christian Kirchhoff, Heribert 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)