I am trying to develop a HTS method to screen compounds for there activity against Mycobacterium smegmatis. I have used Resazurin dye for viability detection. Its a blue dye which turns pink indicative of viability. Ideally, the dye is used for fluorescence detection. But there are are reports of it being used absorbance mode by reading at 570nm. I do not have a fluorometer and hence have gone forward with a absorbance mode. The problem I encountered is: how should I negate the absorbance of Media Blank (Media + Dye), as it is blue in colour and shows the OD reading similar to that of the reduced (pink) dye? Visually the viability can be assessed but calculation of IC50 is not possible as it is not a gradation of the same colour, but a completely different colour is formed which is leading to variable data. Can anybody suggest any other method? Can I use MTT as the viability dye?
You can use XTT (2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide), TTC, (2,3,5-Triphenyl Tetrazolium Chloride) or MTT, (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) as the viability dye.
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