Fluorescence Microscopy of Bacterial Nucleoids

Fluorescence microscopy was used to screen 200 mutants for nucleoid reporter activity. All fluorescence microscopy and time-lapse fluorescence microscopy experiments were performed with a temperature controlled (Okolab Ottaviano, Italy) Ti-Eclipse inverted microscope (Nikon, Champigny-sur-Marne, France) equipped with a TI-CT-E motorized condenser, a YFP filter (Ex 500/24 nm, DM 520 nm, Em 542/27 nm), a DAPI filter (Ex 377/50 nm, DM 409 nm, Em 447/60), and a CoolSnap HQ2 FireWire CCD-camera. For imaging, cells were grown to mid-log phase and placed between LB agar pads and a cover glass, essentially as described previously [20] (link), and incubated at 37°C. Where appropriate, DAPI was added in the LB agar pad at a final concentration of 1 μg/mL, chloramphenicol at a final concentration of 2 μg/mL, nalidixic acid at a final concentration of 150 μg/mL and rifampicin at a final concentration of 100 μg/mL. Mitomycin C was added to the liquid culture 30 min prior to imaging at a final concentration of 1 μg/mL.
Images were acquired using NIS-Elements (Nikon) and resulting pictures were further handled with open source software ImageJ (downloaded from http://rsbweb.nih.gov/ij/).