2.7. Determination of Minimum Inhibitory Concentration (MIC) by Serial Dilution Assay

The MIC of mucus extract was carried out via microdilution methods using MHB as described by Clinical and Laboratory Standards Institute (CLSI) with slight modifications [22]. Bacterial inoculums were prepared in MHB at 37 °C for 24 h. The mucus extract was two-fold diluted ranging from 2000 to 0.48 µg/mL (80 µL as final volume) with final phosphate buffer saline concentration <1%. Afterwards, 20 µL of bacterial suspensions (10⁸ CFU/mL) and 100 µL of MHB were loaded onto microtiter plates and the test was accomplished in 200 µL of final volume. The absorbance of each well was determined using Epoch^TM microplate spectrophotometer at 600 nm. Plates were then incubated at 37 °C for 24 h. After incubation, the absorbance was read again in the reader at the same wavelength and the obtained absorbance values were subtracted from those obtained before incubation. Assessment was carried out simultaneously for bacterial growth control (MHB + bacteria + mucus extract vehicle) and sterility control (MHB + mucus extract vehicle), as well as for the positive control gentamicin was used. MICs were recorded as the lowest concentration that inhibits the bacterial growth [23].