MJG2404_LB

Time-lapse microscopy of MJG2404 grown on LB media at 37°C.Strains of interest were grown overnight in 5 mL of LB liquid broth shaking at 37°C to obtain a saturated culture. Cultures were back diluted 1,000-fold and grown to an early exponential phase (approximately 3 hours). Cells were concentrated by centrifugation, when necessary, at 5,000 <i>g </i>for 1 minute, before being resuspended in 100 μL of media. Cells were immobilized on agarose pads by spotting 0.5 μL of concentrated culture on the pad, then inverting the pad onto a glass bottom petri dish for imaging. Agarose pads for microscopy were constructed out of LB or MOPS minimal medium containing 1.5% agarose (Thermo Fisher cat. #16500500). Imaging was performed using equipment available at the University of Alabama at Birmingham High-Resolution Imaging Facility (HRIF); a Nikon Ti2 inverted fluorescencemicroscope with a tandem galvano and Nikon A1R-HD25 resonance scanner up to 30 1,024 × 1,024 images/s.Tokai Hit incubation stage chamber was used to heat samples and objectives to 37°C to facilitate the growth of bacteria for live imaging. Images were captured at 60× or 100× magnificationin both transmitted light differentialinterference contrast image and GFP or appropriate fluorescentchannels as needed. Automated time-lapse imaging was performed at 37°C, and motorized x, y, and z tracking was controlled and automated by acquisition software Nis Elements 5.0 Imaging Software available in the HRIF at UAB.