2.4. In Vitro Antimicrobial Activity Assay

The newly synthesized compounds 3a–3k were investigated in vitro for antimicrobial activities. In these studies, the broth microdilution was used. The tests were performed in accordance with the guidelines of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) [31] and Clinical and Laboratory Standards Institute (CLSI) [32]. All the used microbial cultures were first subcultured on nutrient agar (for bacteria) or Sabouraud agar (for fungi) (BioMaxima S.A., Lublin, Poland). The surface of Mueller-Hinton agar (BioMaxima S.A., Lublin, Poland) and RPMI (Roswell Park Memorial Institute) 1640 with MOPS (3-(N-Morpholino)propanesulfonic acid) (Sigma-Aldrich Chemicals, St. Louis, MO, USA) were inoculated with the suspensions of bacterial or fungal species, respectively. Microbial suspensions were prepared in sterile saline (0.85% NaCl) with an optical density of 0.5 McFarland standard scale. Samples containing examined compounds were dissolved in dimethyl sulfoxide (DMSO). The initial concentrations of these derivatives were 50 mg/mL in DMSO. In the next stage, all microbial suspensions were plated on solid media with 2 mg/mL of the tested compounds (diluted 25-fold in medium) and then incubated under specific conditions. The inhibition of bacterial and fungal growth was assessed by comparison with control cultures in media without any sample tested. Standard drugs—ciprofloxacin (antibacterial chemotherapeutic) and nystatin (antifungal antibiotic) (Sigma-Aldrich Chemicals, St. Louis, MO, USA)—were used as reference substances [31,32].

Furthermore, the MIC (minimal inhibitory concentration) of the new thiazoles was evaluated by the microdilution broth method in 96-well polystyrene plates. In this study, two-fold dilutions of the tested compounds in selective broth—Mueller-Hinton (BioMaxima S.A., Lublin, Poland) for bacteria and RPMI 1640 with MOPS (Sigma-Aldrich Chemicals, St. Louis, MO, USA) for fungi—were performed. The final concentrations of these compounds ranged from 1000 (diluted 50-fold in broth) to 0.0038 µg/mL. Bacterial and fungal suspensions were prepared in sterile NaCl with an optical density of 0.5 McFarland standard. Next, the microbial suspension was introduced into each well of the microplate containing broth and various concentrations of the tested substances. After 24 h incubation, the MIC value was assessed in the BioTek spectrophotometer (Biokom, Janki, Poland) as the minimal concentration of the samples that showed complete microbial growth inhibition. Appropriate DMSO, sterile and growth controls were performed. The media with and without tested substances/DMSO were used as controls [31,32,33,34].

Subsequently, MBC (minimal bactericidal concentration) or MFC (minimal fungicidal concentration) was determined by transferring the cultures from each MIC determination well to the appropriate solid medium. After incubation, the lowest compound concentrations with no visible growth observed were evaluated as a bactericidal or fungicidal concentration. All the experiments were repeated three times as independent assays, and representative data are presented [31,32,33,34].