Ruby red mica

Washed and concentrated bacterial cultures were placed on a freshly cleaved ruby red mica (Goodfellow Cambridge Ltd, Cambridge), incubated for 5 min at room temperature and blotted dry before placement in a dessicator for at least 2 h. Images were collected within a Nanoscope V atomic force microscope (Bruker software) using ScanAsyst in air with ScanAsyst cantilevers, at a scanrate of ~0.9–1 Hz. The final images were flattened and/or planefitted in both axes using Bruker software and presented in amplitude (error) mode.

Bacterial cultures grown overnight were diluted 25-times in LB media and allowed to grow for 2 hours at 26°C to OD₆₀₀ = 0.2. The growth conditions were then changed to T3SS-inducing conditions at 37°C. One milliliter was taken from the bacterial cultures every hour for analysis with atomic force microscopy. Each sample was centrifuged for 4 min at 1500 rpm, washed once with 2 mM MgCl₂, and re-suspended in 50–200 μl of the same solution. Ten microliters of each sample was placed on freshly cleaved ruby red mica (Goodfellow Cambridge Ltd, Cambridge), incubated 5 min at room temperature, and blotted dry before being placed into a desiccator for a minimum of 2 hours. Images were collected by a Nanoscope V AFM (Bruker software) using ScanAsyst in air with ScanAsyst cantilevers at a scan rate of approximately 0.9–1 Hz. The final images were flattened and/or plane-fitted in both axes using Bruker software and presented in amplitude (error) mode.