Hi!
Can I use MTT dye instead of resazurin for MIC determination via broth microdilution assay? If yes, what would be the best concentration to reduce interference of the MTT dye with the samples?
Sample nature: cell free supernatants from bacteria (extracted using ethyl acetate), evaporated then freeze dried.
Thanks so much!
Yes, you can use MTT dye for determining the minimum inhibitory concentration (MIC) via the broth microdilution assay, as MTT is a viable alternative to resazurin for assessing cell viability. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) is reduced by viable cells to form a formazan crystal, which can be quantified colorimetrically.
When using MTT, it is crucial to optimize the concentration of the dye to minimize interference with the sample. Typical working concentrations for MTT in microbial assays can range from 0.1 to 0.5 mg/mL; however, concentrations higher than this may result in background interference and may affect the growth of some organisms.
A good approach is to start with a concentration of MTT at around 0.2 mg/mL, which usually provides a balance between sensitivity and minimizing interference. You may need to perform preliminary experiments to determine the best concentration for your specific organism and the assay conditions, including the type of broth medium used.
Remember to include controls (without the dye) to determine the level of interference, if any, from the MTT pigment at the chosen concentration, as well as comparing with a standard that uses resazurin for validation.
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