Microplate manager 4

The minimum inhibitory and bactericidal concentrations (MICs and MBCs) were determined using 96-well microtiter plates. The bacterial suspension was adjusted with sterile saline to a concentration of 1.0 × 10⁻⁵ cfu/mL. Compounds to be investigated were dissolved in broth LB medium (100 μL) with bacterial inocula (1.0 × 10⁻⁴ cfu per well) to achieve the wanted concentrations (1 mg/mL). The microplates were incubated for 24 h at 48 °C. The lowest concentrations without visible growth (under the binocular microscope) were defined as concentrations that completely inhibited bacterial growth (MICs). The compounds investigated were dissolved in 5% DMSO (1 mg/mL) and added in the LB medium to the inoculum. The MBCs were determined by serial sub-cultivation of 2 μL into microtiter plates containing 100 μL of broth per well and then submitted to further incubation for 72 h. The lowest concentration with no visible growth was defined as the MBC, indicating 99.5% killing of the original inoculum. The optical density of each well was measured at 655 nm by a Bio-Rad Laboratories Microplate Manager 4.0 and compared with a blank and the positive control. Streptomycin and ampicillin were used as positive controls (1 mg/mL) [58 (link),59 ]. All experiments were performed in duplicate and repeated three times.

Plasma sCD14 levels were determined using the Human sCD14 Quantikine^® ELISA (R&D Systems). Plasma samples were diluted 200-fold with sample diluent solution prior to analysis. Optical density was determined using a Microplate Reader 3550 (BioRad) and plasma sCD14 concentrations were calculated using the Microplate Manager 4.0 (BioRad).

The compounds were tested on antibacterial activity using a microdilution method [55 ,56 (link)]. Minimal inhibitory (MIC) and minimal bactericidal (MBC) concentrations of the tested samples were determined by serial dilutions of compounds dissolved in 5% DMSO-water solution in 96-well microtitre plates. Bacterial inoculum, 1.0 × 10⁴ CFU mL⁻¹, was added to LB medium and compounds dissolved in 5% DMSO solution containing 0.1% Tween 80 (v/v) (1 mg mL⁻¹). Minimal inhibitory concentration (MIC) was defined as the lowest concentration that inhibited bacterial growth, without visible growth, at the binocular microscope. The lowest concentration with no visible growth was defined as the MBC, indicating 99.5% killing of the original inoculum. All wells were measured at a wavelength of 655 nm by Microplate manager 4.0 (Bio-Rad Laboratories, Hercules, California, USA) and compared with a blank and the positive control. Antibiotics used as a positive control were Streptomycin (Sigma P 7794) and Ampicillin (Panfarma, Belgrade, Serbia) (1 mg mL⁻¹ in sterile physiological saline), while 5% DMSO was used as a negative control. All experiments were performed in duplicate and repeated three times.