Eclipse 80i microscope

Brucella strains and infected HeLa cells were observed with a Nikon 80i (objective phase contrast × 100, plan Apo) connected to a Hamamatsu ORCA-ER camera. For the observation of bacteria, 1.5 μl of an exponential phase culture was dropped on agarose pad (solution of 1% agarose in PBS) and sealed with VALAP (1/3 of vaseline, 1/3 of lanoline and 1/3 of paraffin wax).
Time-lapse experiments with B. abortus strains were performed from cultures in exponential phase in liquid Plommet–erythritol containing K₂HPO₄ (0.92 g l⁻¹), KH₂PO₄ (3 g l⁻¹), Na₂S₂O₃ (0.1 g l⁻¹), NaCl (5 g l⁻¹), Nicotinate (0.2 g l⁻¹), Thiamine (0.2 g l⁻¹), (NH₄)₂SO₄ (0.5 g l⁻¹), Pantothenate (0.07 g l⁻¹), Erythritol (2 g l⁻¹), MgSO₄·7H₂O (10 g l⁻¹), MnSO₄ (1.1 g l⁻¹), FeSO₄·7H₂O (1 g l⁻¹) and Biotine (1 g l⁻¹). Cultures were spread on a sealed Plommet–erythritol agarose 1% pad. Images were automatically taken every 20 min at 32 °C with NIS-Element software (Nikon).
Imaging of E. coli strains was performed using either a Nikon Eclipse 80i microscope equipped with a Hamamatsu Orca II-ER camera ( × 100 differential interference contrast or phase-contrast objectives, optivar × 1.5 when appropriate) or a Nikon Eclipse Ti-U with a Hamamatsu Orca-ER LCD camera ( × 100 phase-contrast objective). Images were processed with Metamorph software (Molecular Devices, Sunnyvale, CA).