Resazurin

The MIC test was conducted in a sterile 96-well plate, with each well containing 100 μl of potato dextrose broth (PDB) (HIMEDIA-India). Every four wells made one replication, nine different concentrations of the crude extracts were tested (1:1 dilutions) ranging from 42 to 0.16 g L⁻¹. Wells were then inoculated with one of the two tested microorganisms’ spore suspensions (A. alternata and C. gloeosporioides). The last three rows are control rows: no spores and no extract control wells, negative control with spores but no extract wells, and positive control with spores and 10 µl of the fungicidal Clatrimazole (1%) wells.
Fungal spore suspensions were adjusted to the range of 10⁴ spores L⁻¹ using a 10 day old fungal plate and sterile distilled water, the spore concentration was calculated using a heamatocytometer.
Fungal growth in each well was monitored using Resazurin (HIMEDIA-India) dye. Upon cells division, Resazurin changes its color from blue to pink and fluorescent^{27 (link)}. Results were taken within 48 h of incubation at 25 °C. MIC was recorded as the last extract concentration that shows no change in the color of Resazurin within the incubation period.

The pellicle biofilms were washed twice with distilled water and stained with resazurin (HiMedia) dye (0.337 mg/mL) for 30 min at room temperature (RT). The resazurin fluorescence was measured using a Fluoroskan Ascent (Thermo Scientific, USA) instrument at excitation (λ_ex) of 550 nm and emission (λ_em) of 600 nm.

The pellicle biofilms were washed twice with distilled water and stained with resazurin (HiMedia) dye (0.337 mg/mL) for 30 min at room temperature (RT). The resazurin fluorescence was measured using a Fluoroskan Ascent (Thermo Scientific, USA) instrument at excitation (λ_ex) of 550 nm and emission (λ_em) of 600 nm.