Pore size black polycarbonate filters

Throughout the study, microcosms were sampled and observed under phase contrast microscopy. Total cell counts were performed by DAPI-staining (4′,6′-diamidino-2-phenylindole, Sigma–Aldrich) with an Olympus BX60 epifluorescence microscope equipped with a monochrome camera (12 bits, QIClick) and with a mercury light source. Formation water (18 mL) was fixed on-site with 2 mL of 10% borax-buffered formaldehyde (37%, Sigma–Aldrich) and stored at 4°C. Ten milliliters of sample were stained with 0.5 mL DAPI stock solution (200 μg.mL^-1) then filtered onto 0.2 μm pore-size black polycarbonate filters (Millipore) under vacuum. For the eighth subculture, counterstaining was done by filtering a mixture of 22 μL of culture with 1 μL of DAPI stock solution onto 0.2 μm pore-size black polycarbonate filters (Millipore) under vacuum. For each filter, 10 randomly selected fields were counted.

For the determination of the antimicrobial activity of the extracts, the agar well diffusion method was employed according to Guven et al. (2006) [68 (link)]. Briefly, 15 mL of molten agar (45 °C) were poured into sterile Petri dishes (Ø 90 mm). A volume of 50 μL of 5 h-old culture of each tested bacteria and 100 μL of 5 h-old culture of the fungus C. albicans were evenly spread onto the surface of the agar plates of LB, using agar medium for bacteria and Sabouraud agar medium for C. albicans. Once the plates had been aseptically dried, 5 mm wells were punched into the agar with a sterile cork borer. Each extract was dissolved in dimethyl sulfoxide (DMSO–water, 1–9; v/v) to a final concentration of 1 mg·mL⁻¹, filtered through 0.22 μm pore-size black polycarbonate filters (Millipore, Fontenay sous Bois, France), and finally 100 μL of each obtained extract solution was placed into the corresponding well. After staying at 4 °C for 2 h, the plates were incubated at the appropriate temperature during 24 h for bacterial strains, and during 48 h for C. albicans. The antimicrobial activity was assayed by measuring in mm the diameter of the inhibition zone formed around the wells.

The antimicrobial activity of IP6 was evaluated by means of agar-well diffusion assays, as described by Valgas et al. [44 ]. Fifteen milliliters of the molten agar (45°C) were poured into sterile petri dishes (Ø 90 mm). Working cell suspensions were prepared at 10⁶ CFU/mL, and 100 μl was evenly spreaded onto the surface of the agar plates of Luria–Bertani (LB) agar (Oxoid Ltd, UK). Once the plates had been aseptically dried, 06 mm wells were punched into the agar with a sterile Pasteur pipette. IP6 was dissolved in water to a final concentration of 50 mg/ml and then filtered through 0.22 μm pore-size black polycarbonate filters (Millipore). Thus, 50 μl were placed into the wells and the plates were incubated at 37°C for 24 h for bacterial strains. Antibacterial activity was evaluated by measuring the diameter of circular inhibition zones around the well. The un-inoculated media were also tested for inhibitory zones as a control. Tests were performed in triplicate.