P iodonitrotetrazolium violet chloride int

The determination of MIC and MBC were assayed as described by^{99 (link)}. Where the freshly prepared culture of isolated uropathogens was adjusted to OD₅₉₅ of 0.01. 100 μl of each bacterial fresh culture was put into sterilized 96-well plates. Then 20 μl of the original extract was added (serial dilutions of 10⁻¹–10⁻¹⁰ were used, 8 replicates were made for each dilution into complete raw of the 96-well plate). The plates incubated at 37 °C for 24 h. MIC was determined by the addition of 40 μl of p-iodonitrotetrazolium violet chloride (INT) (0.2 mg/ml, SIGMA-ALDRICH) to the plates and re-incubated at 37 °C for 30 min., the lowest concentration which banned color change is the MIC^{100 (link),101} . MBC was determined according to to^{99 (link),102} .

The determination of MIC and MBC were assayed as described earlier [57 (link)]. The freshly prepared culture of A. baumannii isolates was adjusted to OD₅₉₅ of 0.01. 100 μL of each isolate culture was put into sterilized 96-well plates. Then, 20 μL of the original S. aromaticum extracts (100 mg mL⁻¹) was added (serial dilutions of 10⁻¹–10⁻¹⁰ were used, eight replicates were made for each dilution into complete raw of the 96-well plate). Imipenem (10 mg mL⁻¹) and un-inoculated media were tested as the positive and negative control, respectively. After 24 h incubation at 37 °C, MIC was determined by the addition of 40μL of p-iodonitrotetrazolium violet chloride (INT) (0.2 mg/mL, Sigma-Aldrich) to the plates and re-incubated at 37 °C for 30 min. The lowest concentration which banned color change is the MIC [58 (link),59 (link)]. The MBC was determined by transferring 50 mL from each well of overnight MIC plates (and/or higher) to sterile (TSA) fresh plates. Viable colonies were counted after 24 h at 37 °C. The limit of detection for this assay was 10¹ CFU/mL.

The determination of MIC was assayed as described earlier by Salem et al.^{39 (link)} The freshly prepared cultures of both Gram-negative bacteria and/or Gram-positive bacteria were adjusted to OD₅₉₅ of 0.001. 100 μL of each isolate culture was put into sterilized 96-well plates. Then 20 μL of the screened compounds (2 mg mL⁻¹) (serial dilutions of 10⁻¹ to 10⁻¹⁰ were used, 8 replicates were made for each dilution into complete raw of the 96-well plate). In addition, levofloxacin (20 mg per disc) was used as reference drug. The un-inoculated media tested as negative control, after 24 h incubation at 37 °C. MIC was determined by the addition of 40 μL of p-iodonitrotetrazolium violet chloride (INT) (0.2 μg mL⁻¹, Sigma-Aldrich) to the plates and re-incubated at 37 °C for 30 min., the lowest concentration which banned color change is the MIC.^{40 (link)}