The bacterial cultures in TSB+HTMS MEK162 chemical structure medium with addition of 30 % glycerol were stored at −70 °C. Before each experiment, bacterial strains were subcultured on HAEM medium and incubated overnight at 35 °C in about 5 % CO2 atmosphere. Growth assay Preliminary in vitro antibacterial activity of compounds N-ethyl-3-amino-5-oxo-4-phenyl-2,5-dihydro-1H-pyrazole-1-carbothioamide, N-(4-metoxyphenyl)-3-amino-5-oxo-4-phenyl-2,5-dihydro-1H-pyrazole-1-carbothioamide, and N-cyclohexyl-3-amino-5-oxo-4-phenyl-2,5-dihydro-1H-pyrazole-1-carbothioamide was screened by the broth microdilution method using 96-well polystyrene microplates (NUNC, TC MICROWELL 96F Nunclon D) on the basis of MIC (minimal inhibitory

concentration), usually defined as the lowest concentration of the compounds at which there was no visible growth of microorganisms. The antibacterial activity was tested according to EUCAST (2003) selleckchem procedure with some modifications. In order to assay the influence of the tested

pyrazole derivatives on the growth of haemophili rods, 198 μl of TSB+HTMS medium without (control) and with a series of twofold dilution of the tested compounds in the range of final concentration from 1,000 to 62.5 μg ml−1 was inoculated with 2 μl of the standardized microbial suspension (total volume per each well ––200 μl), and then incubated for 18 h at 35 °C in the presence of about 5 % CO2. Then in order to assay the influence of the tested N-ethyl-3-amino-5-oxo-4-phenyl-2,5-dihydro-1H-pyrazole-1-carbothioamide with highest inhibitory effect against the planktonic cells of Haemophilus spp., 198 μl of TSB+HTMS medium without (control) and with a series of twofold dilution of the tested compound in the range of final concentration from 0.12 to 31.25 μg ml−1 was inoculated with 2 μl of the standardized microbial suspension (total volume per each well––200 μl), and then incubated for 18 h at 35 °C in the presence of about 5 % CO2. ID-8 After incubation,

spectrophotometric measurements of optical density at wavelength λ = 570 (OD570) of the bacterial cultures with or without the tested compound were done by using a microplate reader (ELx800 BioTek) in order to determine MIC. The MIC values were determined by comparison to the growth of a OICR-9429 manufacturer control (compound-free) medium. Ampicillin was used as a reference antimicrobial agent on selected H. parainfluenzae (penicillinase-positive or penicillinase-negative strains) isolates at the same conditions. The blank control wells without or with twofold dilution of the tested compounds added to TSB+HTMS broth without bacterial suspension were incubated under the same conditions. The experiments were performed in triplicate. Biofilm assay In order to assay the effect of N-ethyl-3-amino-5-oxo-4-phenyl-2,5-dihydro-1H-pyrazole-1-carbothioamide on Haemophilus spp. biofilm formation, the method based on staining with 0.1 % crystal violet described previously by Kaplan and Mulks (2005) with some modifications (Kosikowska and Malm, 2009) was used.