2.6. Alamar Blue Dye for Testing Anti-TB Activity

Microplate Alamar Blue Assay (MABA) was adopted to assess the susceptibility of Mycobacterium tuberculosis to MXF [37]. Briefly, 100 μL of Middlebrook 7H9 broth containing 3 × 10⁵ CFU/mL of M. tuberculosis (Vaccine strain, H37 RV strain, ATCC No—27294) was added to each well of a 96-well plate along with serial dilutions (ranging from 0.8 to 100 µg/mL) of either free MXF or MXF-PLGA-MS. Outer perimeter wells were filled with 200 μL deionized water to avoid dehydration in test wells. The plate was sealed with parafilm and incubated at 37 °C. Five days post incubation, 25 µL of Alamar Blue reagent and 25 µL of 10% Tween 80 were added to each well and the plate was further incubated at 37 °C. A change in color from blue to pink, in the wells, was observed at 24 h. Blue indicates no growth, whereas pink indicates growth.