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Short answer. 2,3,5-Triphenyltetrazolium chloride (TTC) is a water-soluble, colourless salt that living tissue reduces to an insoluble red compound, formazan. Red marks tissue with active dehydrogenase enzymes; unstained white tissue is non-viable. It is used to assess seed viability and to outline infarcts in experimental stroke models. This page is general laboratory information, not medical advice.
Definition and scope
TTC is a tetrazolium salt, identified in chemical databases as CAS 298-96-4 with molecular formula C19H15ClN4 (PubChem compound record CID 9283). The “2,3,5-” in its full name marks the positions of the three phenyl groups on the tetrazolium ring.
How does TTC staining work?
TTC itself is colourless. Living cells reduce it, and the reduced product is formazan, a red compound that does not dissolve in water. The UF/IFAS seed-technology guide states that “living tissue changes the tetrazolium to an insoluble red compound called formazan”, and describes the test as positive for the dehydrogenase enzymes involved in respiration. Sanchez-Bezanilla and colleagues (2019) describe the same chemistry in brain tissue: TTC is reduced by the mitochondrial enzyme succinate dehydrogenase of living cells, so healthy tissue turns deep red while dead tissue stays white.
What is the principle behind the tetrazolium test?
The principle is that enzyme activity stands in for viability. Dehydrogenase activity in living cells converts the colourless tetrazolium into coloured formazan. Where that activity is lost, no colour forms. The stain therefore reports metabolic activity at the moment of staining, and the colour pattern (red, pink, white) is read by eye or by later quantification.
Uses compared: seeds and brain tissue
The two settings below use the same chemistry but differ in tissue, reading and conditions. Conditions are those the source states for its own setting and are not interchangeable.
| Setting | What is stained | Conditions stated by the source | How the result is read |
|---|---|---|---|
| Seed viability testing (UF/IFAS seed technology) | Cut seeds or isolated embryos | Solution usually 0.1 to 1.0%, applied for about 24 hours (as stated by the source) | Bright red = viable; pink = losing viability; white = non-viable |
| Experimental stroke models (Sanchez-Bezanilla et al., 2019) | Brain sections from animal models | Set by the authors’ own laboratory protocol; see the paper for its conditions | Deep red = healthy tissue; white = infarcted tissue; infarct volume corrected for edema |
Limitations to check before relying on a TTC result
- It measures dehydrogenase activity, not a direct cell count. A colour change shows that reducing enzymes were active; it is not the same as counting live cells. For assays that count or measure cells, see cell proliferation assay methods compared and hemocytometer cell counting and viability.
- Edema can distort infarct size. Sanchez-Bezanilla et al. report infarct volume corrected for edema, because swelling changes the apparent size of stained and unstained regions.
- Downstream analysis can be affected. The same authors note that the TTC protocol is considered to interfere with downstream analyses; they suggest proteolysis may occur for some proteins when sections are incubated and that formazan pigment may interfere with the BCA protein assay.
- Seed results depend on reading the colour categories correctly. The UF/IFAS guide separates bright red, pink and unstained tissue, so partial staining is an interpretation, not a yes/no signal.
Related tetrazolium assays
TTC is one of several tetrazolium salts. Cell-culture assays use others, with different solubility of the coloured product. Formazan from TTC is insoluble in water, as the UF/IFAS guide states. For cultured-cell readouts see the CASRAI guides on the MTT assay, XTT assay and CCK-8 assay, and on ATP assays, which measure a different signal.
Common misunderstandings
- TTC is not triphenylmethyl chloride. Triphenylmethyl chloride is trityl chloride, a different compound used in organic synthesis. The two names look similar, but they are unrelated to the viability stain.
- Red does not mean “healthy for every purpose”. Red shows reducing enzyme activity in that tissue at the time of staining.
- The same protocol does not transfer between settings. The table above shows that seed testing and brain-section staining use different conditions.
Frequently asked questions
How does TTC staining work?
Living tissue reduces colourless TTC to insoluble red formazan through dehydrogenase activity; dead tissue lacks that activity and stays white.
What is the CAS number for 2,3,5-triphenyltetrazolium chloride?
CAS 298-96-4, formula C19H15ClN4, listed in the PubChem compound record (CID 9283).
What is the principle behind the tetrazolium test?
Dehydrogenase enzymes in living cells reduce a colourless tetrazolium salt to a coloured formazan, so colour indicates metabolic activity.
What is triphenylmethyl chloride?
Triphenylmethyl chloride is trityl chloride, a different chemical from triphenyltetrazolium chloride and not a viability stain.
Sources
All links below were checked on 2026-11-07.
- UF/IFAS Seed Technology, tetrazolium test: https://propg.ifas.ufl.edu/04-seeds/02-technology/10-seedstesting-tetrazolium.html (checked 2026-11-07).
- Sanchez-Bezanilla S, Nilsson M, Walker FR, Ong LK. Front Mol Neurosci 2019;12:181: https://www.frontiersin.org/journals/molecular-neuroscience/articles/10.3389/fnmol.2019.00181/full (checked 2026-11-07).
- PubChem, triphenyltetrazolium chloride, CID 9283: https://pubchem.ncbi.nlm.nih.gov/compound/9283 (identifiers, checked 2026-11-07).
