Dmi 6000 b inverse microscope

The strains to be imaged were cultivated overnight in appropriate medium and then subcultivated until reaching exponential growth phase (OD₆₀₀ of 0.2) or another desired OD₆₀₀. Three microliters of culture were spotted on an agarose pad (LM medium solidified with 1% (w/v) agarose). Fluorescence images were recorded using a Leica DMI 6000 B inverse microscope (Leica, Wetzlar, Germany) equipped with an sCMOS camera and an HCX PL APO 100×/1.4-numerical-aperture objective using VisiView software (Visitron Systems, Puchheim, Germany). The obtained images were further processed using ImageJ^{62 (link)} (https://imagej.nih.gov) to adjust contrast, to change grayscale to color, to define representative areas for display, and to add scale bars. Adobe Illustrator CS6 was used to add an outline of cells where required and to create the final assembly of panels. Cells from at least three independent cultures were imaged.

Strains were cultivated overnight in the LB medium and sub cultured the next day until they reached the exponential growth phase (OD₆₀₀ = 0.2–0.3). Then, 5 μl of culture were spotted on an agarose pad [PBS buffer solified with 1% (w/v) agarose]. Fluorescence images were acquired using a Leica DMI 6000 B inverse microscope (Leica, Wetzlar, Germany) equipped with an SCMOS camera (Visitron Systems, Puchheim, Germany) and a HC PL APO 100×/1.4 oil PH3 objective. Image processing and analysis was carried out using the ImageJ-based Fiji tool (Schindelin et al., 2012 (link)).

Cells of CN-32 strains from overnight cultures were sub inoculated in 10 ml of LB with an OD₆₀₀ of 0.05. After reaching the exponential growth phase (OD₆₀₀ = 0.2–0.3), a 100 μl aliquot was placed under a coverslip fixed by four droplets of silicone (baysilone, VWR International GmbH, Darmstadt, Germany) to generate a space of 1–2 mm width. Movies of 120 frames were taken at room temperature with a Leica DMI 6000 B inverse microscope (Leica, Wetzlar, Germany) equipped with an SCMOS camera (Visitron Systems, Puchheim, Germany) and a HCX PL APO 100×/1.4 objective. The speed of at least 500 cells per strain was quantified using the MTrackJ plugin of Fiji (Meijering et al., 2012 (link)). Significance was tested using ANOVA (p = 0.05) in R version 3.0.1.