We developed a rapid viability test for plant seeds in which yeasts metabolize organic compounds that leach from seeds (dependent on the physiological age of the seed) and thereby reduce our redox-indicator resazurin, accompanied by a color change (5). Unfortunately, seeds of some species acidify our test solution causing a pH-dependent colour change of the resazurin (abiotic reaction) which distorts our test results (6). We are looking to replace our redox indicator dye for these cases!
We already researched on several dyes from the tetrazolium-family that unfortunately also change their colour upon acidification, i.e., MTT (1), TTC (2), MTS (3) or the solubility is severely limited, e.g. MTT (3), XTT (4).
We are grateful for any advice!
1: Plumb et al. (1989) Effects of the pH Dependence of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide-Formazan Absorption on Chemosensitivity Determined by a Novel Tetrazolium-based Assay. Cancer Res 49: 4435–4440.
2: Lopez Del Egido et al. (2017) A Spectrophotometric Assay for Robust Viability Test of Seed Batches Using 2,3,5-Triphenyl Tetrazolium Chloride: Using Hordeum vulgare L. as a Model. Front. Plant Sci. 8: 747. doi: 10.3389/fpls.2017.00747
3: Riss et al. (2013) Cell Viability Assays. https://www.ncbi.nlm.nih.gov/books/NBK144065/
4: Goodwin et al. (1995) Microculture tetrazolium assays: a comparison between two new tetrazolium salts, XTT and MTS. J Immunol Methods 13: 95-103. doi: 10.1016/0022-1759(94)00277-4
5: Mohammed et al. (2019) Dead or Alive: Simple, Non-destructive, and Predictive Monitoring of Seedbanks. Trends in Plant Science 24: 783-784. DOI: 10.1016/j.tplants.2019.05.014
6: Wellmann et al. (2023) Maize Grain Germination Is Accompanied by Acidification of the Environment. Agronomy 13: 1819. doi.org/10.3390/agronomy13071819
Try fluorometric indicator like Alamar Blue, which is less sensitive to pH changes compared to some tetrazolium-based dyes.
Its fluorescence signal is dependent on redox reactions and might be suitable for your rapid viability test, minimizing interference from seed-induced pH variations. Conduct a small-scale trial to assess its compatibility with your system.
@ Zardar Khan
Dear Zardar,
thank you for your response, but Alamar Blue is a trade name of resazurin, which we are already using.
Klaus
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