For negative staining of whole bacteria producing F-pili, 10 µl culture of bacterial cells (grown in LB until OD600nm of 1.5) was applied onto glow-discharged 300 mesh carbon-coated grids (Agar Scientific) and incubated for 2 min. Following two subsequent washes with 10 µl of water, the grids were stained with 0.2% (w/v) phosphotungstic acid for 7 s before blotting. The grids were then imaged on a Technai12 Spirit transmission electron microscope (FEI) operating at 120 kV and equipped with 2 K eagle camera (FEI). Purified F-pili were applied onto glow-discharged 400 mesh carbon-coated grids (AgarScientific) and incubated for 2 min prior two washes with 10 µl of water. The grids were stained with 2% uranyl acetate for 1 min, blotted with filter paper and imaged on a Technai12 Spirit transmission electron microscope (FEI) operating at 120 kV and equipped with 2 K eagle camera (FEI).
Patkowski J.B., Dahlberg T., Amin H., Gahlot D.K., Vijayrajratnam S., Vogel J.P., Francis M.S., Baker J.L., Andersson M, & Costa T.R. (2023). The F-pilus biomechanical adaptability accelerates conjugative dissemination of antimicrobial resistance and biofilm formation. Nature Communications, 14, 1879.
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