Overnight cultures of the above strains were diluted to OD600 of 0.05 in 3 mL of M9 medium supplemented with 0.2% casamino acids, 0.2% maltose, and 25 μM IPTG. Cells were grown at 30°C until the OD600 reached 0.2, at which point the cells were spotted onto filter discs placed on LB agar medium supplemented with 100 μM IPTG, with or without 10 mM DTT. After 6 hours growth at 30°C, cells were suspended in liquid LB medium, fixed, and imaged using phase contrast microscopy. Scale bar, 5 μm. Where indicated, cells were fixed in 2.6% formaldehyde with 0.04% glutaraldehyde at room temperature for 1 h, followed by storage at 4°C for 24 hours. Prior to imaging, cells were immobilized on 2% agarose pads and covered with #1.5 coverslips.
Imaging was performed on a Nikon Ti inverted microscope equipped with a 100x Plan Apo 1.4 NA phase contrast objective, Andor Zyla 4.2 sCMOS camera, and Nikon motorized stage. Acquisition software was NIS Elements 4.30. The purchase of this microscope was funded in part by grant S10 RR027344–01. Microscopy was performed with the support of Microscopy Resources on the North Quad (MicRoN) at Harvard Medical School. The ImageJ plugin MicrobeJ41 was used segment cells and measure cell dimensions. Statistical significance was determined using a two-way ANOVA followed by Tukey’s Multiple Comparisons Test.
Imaging was performed on a Nikon Ti inverted microscope equipped with a 100x Plan Apo 1.4 NA phase contrast objective, Andor Zyla 4.2 sCMOS camera, and Nikon motorized stage. Acquisition software was NIS Elements 4.30. The purchase of this microscope was funded in part by grant S10 RR027344–01. Microscopy was performed with the support of Microscopy Resources on the North Quad (MicRoN) at Harvard Medical School. The ImageJ plugin MicrobeJ41 was used segment cells and measure cell dimensions. Statistical significance was determined using a two-way ANOVA followed by Tukey’s Multiple Comparisons Test.