I have done the Anti-TB test of some synthesized compounds. I would like to know the color change in details. Does it happen due to drug & strain interaction?
How & why does color change from pink to blue?
Some details are give below:
Anti-TB activity using Alamar Blue Dye
1) The anti mycobacterial activity of compounds were assessed against M. tuberculosis using microplate Alamar Blue assay (MABA).
2) This methodology is non-toxic, uses a thermally stable reagent and shows good correlation with proportional and BACTEC radiometric method.
3) Briefly, 200µl of sterile deionized water was added to all outer perimeter wells of sterile 96 wells plate to minimize evaporation of medium in the test wells during incubation.
4) The 96 wells plate received 100 µl of the Middlebrook 7H9 broth and serial dilution of compounds were made directly on plate.
5) The final drug concentrations tested were 100 to 0.2 µg/ml.
6) Plates were covered and sealed with parafilm and incubated at 37ºC for five days.
7) After this time, 25µl of freshly prepared 1:1 mixture of Alamar Blue reagent and 10% tween 80 was added to the plate and incubated for 24 hrs.
8) A blue color in the well was interpreted as no bacterial growth, and pink color was scored as growth.
9) The MIC was defined as lowest drug concentration which prevented the color change from blue to pink.