I have been determining MICs for M. tuberculosis using 96 - well microtiter plates, flat well shape. After five days, I confirm positive growth by adding alamar blue to my controls.
Recently I ran into a supplier issue and I could not obtain plates with flat wells but had to revert to using V or conical shape wells. After five days of incubation, I add alamar blue. There is difference in the color change. In the flat wells, color change after five days is a distinctive pink, whereas in the v or conical shape wells, it's a purple/violet.
All conditions remain exactly the same between the two.
What could cause this issue? Is it a surface area problem? Could it be that cells sink to the bottom in v or conical shaped wells and are not in entire contact with the alamar blue?
Has anybody else observed such an issue?
Dear Shane
I have not performed alamar blue testing using 'V' bottom plates. But the possible reason may be the surface area difference between the flat and V bottom plates. In V bottom plates the cells could settle in the bottom and could produce a high intensity coloring. whereas in the flat bottom plates the cells are dispersed even the coloring would be of less intensity. I recommend you to continue with the 'Flat bottom' plates instead of V bottom which will seriously affect your results and OD values.
Best regards
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