Deltavision restoration microscope system

Vibrio vulnificus cells in late log phase were harvested by centrifugation at 10,000 × g for 10 min and washed twice with artificial sea water [ASW; 60 mM NaCl, 20 mM MgSO₄⋅7H₂O, 20 mM KCl, 2 mM CaCl₂⋅2H₂O, 50 mM Tris-HCl (pH 8.0)]. Cells were diluted with ASW to a cell density of 10⁸ CFU/ml, transferred to sterile microcentrifuge tubes, and incubated at 4°C in the dark without shaking. Then the total cell number, viability, and culturability were assessed from each tube 0, 2, 4, and 11 days after incubation. Total and viable cell numbers were determined using a Live/Dead BacLight Bacterial Viability Kit (Life Technologies) according to the manufacturer’s instructions. Samples stained with SYTO 9 dye and propidium iodide were spotted on a 1% agarose pad made with Phosphate Buffered Saline (PBS) on a glass slide. Cells were visualized using a Deltavision Restoration Microscope System (GE Healthcare Life Sciences). The numbers of bacterial cells were then counted and calibrated to give total and viable counts per milliliter. To assess the culturability of V. vulnificus cells, samples were spread on LBS plates in triplicate, and incubated at 30°C for 48 h before the CFU numbers were assessed.