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

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The Gold Standard Dye for Cell Counting?

– 5 min read

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!

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 (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 dye^5,6.
3. 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 carcinogenic^7,8.
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 carcinogenic^7,9.

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:

• Webinar: Accurately Measuring Cell Viability: Are All Viability Dyes Created Equal?
• Webinar: Why is trypan blue toxic and unsafe to use?
• Mini Review: Manual vs Automated Cell counting
• Mini Review: Why Working with Trypan Blue is Not a Good Idea
• Product: Via2-Cassette™

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)

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.