1 M phosphate buffer (pH 7.2) for 2 h at 4°C, and then post-fixed in 1% osmium tetroxide at 4°C for 2 h. The specimens were dehydrated with a series of ethanol solutions (30%-100%) and treated with hexamethyldisilazane LEE011 price twice for 15 min. The specimens were mounted on metal stubs, coated with a thin layer Akt inhibitor platinum under argon using a sputter-coater (SCD 005; BAL-TEC, Bannockburn, IL, USA), and then visualized by field emission-scanning electron microscopy (FE-SEM) (Supra 55VP; Carl Zeiss, Oberkochen, Germany) at the accelerating voltage of 2 kV at the National Instrumentation Center for Environmental Management (NICEM; Seoul, Korea). Images were captured
in TIFF format. Confocal microscopy To determine membrane
integrity, bacterial cells were stained with membrane-permeant and -impermeant fluorescent selleck products dyes according to the manufacturer’s instructions (Live/Dead BacLight Bacterial Viability Kit; Molecular Probes, Eugene, OR, USA) followed by confocal microscopy. Hp cells from BB agar plates were inoculated (OD600, 0.01 or 0.1) into BB-NBCS media and grown under various gas conditions. Aliquots were taken at 12 or 36 h, stained with SYTO 9 and propidium iodide (PI) for 15 min, and washed twice with phosphate buffered saline (PBS). Cells were then spread on slide glasses, covered with mounting medium and cover slips, and visualized by confocal microscopy (Leica TCS SP5; Leica Microsystems GmbH, Wetzlar, Germany). SYTO 9 is a green fluorescent membrane-permeant dye that labels all bacteria by staining nucleic acid, whereas PI is a Ribose-5-phosphate isomerase red fluorescent membrane-impermeant dye that labels only bacteria with damaged membranes. High performance liquid chromatography analysis of organic acid metabolites The concentrations of fermentation products in the Hp culture media were determined by high
performance liquid chromatography (HPLC) using the HP1100 system (Hewlett Packard, Palo Alto, CA, USA) at NICEM. Hp cells grown on agar plates were collected, washed, and inoculated into 20 ml of fresh media (OD600, 0.1). Cells were cultured under various gas conditions for 36 h, and the culture medium was collected and divided into two aliquots (one of which was spiked with 15 mM pyruvate as internal control for quantification), which were processed simultaneously. The culture medium was extracted twice with phenol/chloroform to remove proteins and then passed through a 0.45-μm syringe filter. The samples were injected into an ion exchange column (Aminex HPX-87H, 300 × 7.8 mm; Bio-Rad, Richmond, CA, USA), and eluted at 40°C with 0.01 N H2SO4 at a flow rate of 0.5 ml/min. Organic acids were analyzed with a refractive index detector HP1100 (Hewlett Packard). Solutions containing glucose and organic acids including acetate, formate, propionate, lactate, pyruvate, succinate, and butyrate were used as standards.