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.
The colour of the dye turns from blue to pink only when the organisms are alive and viable. If an compound has anti-mycobacterial activity, then it helps to determine at what concentration it exerts its bactericidal effect (at which point the blue remains unaffected); hence useful to determine MIC
You can read the following article
Franzblau SG, Witzig RS, McLaughlin JC, Torres P, Madico G,
Hernandez A, Degnan MT, Cook MB, Quenzer VK,
Ferguson RM, Gilman RH (1998) Rapid, low-technology
MIC determination with clinical Mycobacterium tuberculosis
isolates by using the microplate Alamar Blue assay.
J Clin Microbiol 36:362–366
I will add that the change in color is a result of redox reaction. Oxidized form is blue, reduced form is pink. Moreover, the two forms differ in fluorescence, so the antimycobacterial activity can be determined as a change in fluorescence, which is said to be more precise that spectrophotometric determinationin VIS.
The substrate in Alamar Blue is Resazurin. It changes to a pink-coloured and fluorescent compound Resorufin when reduced. It is useful for quantifying the bactericidal as well as bacteriostatic activity of compounds towards mycobacteria. Other papers describing its use in mycobacterial inhibition assays include:
Bassett IM, Lun S, Bishai WR, Guo H, Kirman JR, Altaf M, O’Toole RF (2013). Detection of inhibitors of phenotypically drug-tolerant Mycobacterium tuberculosis using an in vitro bactericidal screen. Journal of Microbiology 51(5): 651-658.
Taneja NK, Tyagi JS (2007). Resazurin reduction assays for screening of anti-tubercular compounds against dormant and actively growing Mycobacterium tuberculosis, Mycobacterium bovis BCG and Mycobacterium smegmatis. Journal of Antimicrobial Chemotherapy 60(2): 288-293.
Resazurin is also pH indicator; approximate pH range for color change: 3.8-6.4, color of acid form: orange, color of base form: violet
http://antoine.frostburg.edu/chem/senese/101/acidbase/indicators.shtml
https://en.wikipedia.org/wiki/Resazurin
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