Determination of antibacterial activity by microdilution broth

The minimum inhibitory concentration (MIC) of PdB was determined by the standard broth microdilution method using Mueller-Hinton broth (MHB) and custom- made 96-well plates. After seeding bacteria in trypticase soy agar, for each isolates a suspension of bacteria in the growth medium of MHB was prepared with an optical density equal to 0.5 McFarland (1 × 10⁸ CFU/mL). Using proper dilution, a concentration of 1 × 10⁵ CFU/mL was prepared. Afterward, 10 μl of each suspension was poured into a 96-wells microplate containing 100 μl of MHB and serial 2-fold dilution (ranging from 1:2 to 1:2048) of each PdB. For the positive control, the bacterial suspension was inoculated in the wells containing MHB without PdB. The antimicrobial activity of plasma and CaCl₂ was also measured. After incubation at 37 °C for 24 hours, the MIC values were determined as the lowest dilution in which no bacterial growth is visible. The test was performed in duplicate for each isolate and the test was repeated in case the MIC values for the two wells of each PdB differed³⁹.