pylori at the air-liquid interface. The formation of biofilms was initiated by inoculating 10 μl of pre-cultured cell suspension (approximately 5 × 105 cells in Brucella broth) into each well. The cultures were incubated under microaerobic conditions at 37°C for 1 to 6 days with shaking (80-100 rpm). After incubation, the coverslips were removed and washed with phosphate-buffered saline (PBS). The samples were then air

dried and stained with crystal violet for 30 s. After being stained, the coverslips were rinsed with distilled H2O to remove excess dye and then air LGX818 price dried for 30 min. All dye associated with the biofilms was dissolved with 1 ml of ethanol and 200 μl of the ethanol solutions were used to measure the absorbance at 594 nm with a microplate reader to determine the amount of biofilm formation. Confocal laser scanning microscopy (CLSM) and measurement of biofilm thickness For visualization, the

biofilms of H. pylori strains on the coverslips were stained with a BacLight LIVE/DEAD bacterial viability kit solution (Molecular Probes, Leiden, The Netherlands) according to the directions of the supplier. Confocal images were collected by using a Zeiss LSM 510-META confocal laser scanning microscope (Carl Zeiss, Jena, Germany). To determine biofilm thickness, a series of horizontal (xz) optical sections at 0.5 μm intervals were taken through the height of the biofilm for measurement. Each biofilm was scanned selleck chemicals at five randomly selected positions. Each sample was observed independently more than three times. Confocal images of green and red fluorescence were constructed simultaneously using a multitrack mode. Cell viability assay To determine the numbers of viable bacteria, biofilm cells on the coverslips were scrapped into PBS. The optical densities and colony-forming units (CFU) of the cell suspensions were quantitated as the mean of three independent observations. As controls, standard cell broth cultures were used. Electron microscopic studies To observe the biofilm ultrastructure, the biofilms formed on the coverslips were examined by scanning electron microscopy (SEM). The biofilms on the coverslips

were fixed with 2% glutaraldehyde for 3 h at room temperature and the samples were observed using a JSM-5600LV electron microscope (JEOL, Tokyo, Japan). To observe Cyclin-dependent kinase 3 the OMV-like structures, the biofilms of strain TK1402 on the glass slides were examined by using transmission electron microscopy (TEM). Glass slides cut in half were placed into 6-well microtiter plates and the biofilms were allowed to form as described above. The biofilms were fixed with 2% glutaraldehyde for 3 h at room temperature. The samples were then dehydrated and embedded in Epon 813 embedding solution (Chemische Werke Lowi GmbH, Waldkaraigurg, Germany). The sections were finally observed with a JEM-100 electron microscope (Jeol). Isolation of outer membrane vesicles Isolation of OMV was performed as described previously [30]. Briefly, H.