To visualize cells overnight cultures grown at 30°C in CH medium were diluted 1:100 into fresh medium. For sporulation, cells were grown at 37°C. For visualisation of protein fusions to GFP, cells were grown at 30°C. 0.7 µl cells were spotted onto thin ∼1.2% agar pads (containing 10% of the growth medium). Microscopic visualisation of cells was carried out as described (Murray and Koh, 2014 ; Adams et al., 2015 ). Membranes were stained with 0.4 µg.ml−1 FM5‐95 dye (Invitrogen). When necessary data were analysed using the Metamorph software (fluorescent trapping assays) and ImageJ (http://rsb.info.nih.gov/ij ) containing the ObjectJ plugin (cell length and oriC‐pole distance measurements). Standard deviations are from at least three experiments.
Two colour colocalisation imaging was performed using a Nikon Eclipse Ti microscope equipped with a Nikon CFP APO TIRF x100/1.49 oil objective, 488 nm and 561 nm solid‐state lasers, and Andor Xion X3 EMCCD camera. All image capture was conducted using Nikon NIS elements 4.0. Cells were immobilized on 1.5% agarose slides.
Two colour colocalisation imaging was performed using a Nikon Eclipse Ti microscope equipped with a Nikon CFP APO TIRF x100/1.49 oil objective, 488 nm and 561 nm solid‐state lasers, and Andor Xion X3 EMCCD camera. All image capture was conducted using Nikon NIS elements 4.0. Cells were immobilized on 1.5% agarose slides.