Biofilm Morphology Analysis by Congo Red

One microlitre loop of 20 h old biofilms grown on silicone, polystyrene (cell culture dishes) and K. pneumoniae cultures in MH broth were spotted onto LB agar plate containing 25 mg/mL Congo red (Sigma). Cultures were incubated for 72 h at 37 °C for colony morphology analysis as described before [14 (link)]. Colonies were further analysed by transmission electron microscopy (TEM) under a Morgagni 268D TEM (FEI Electron Optics, Hillsboro, OR, USA) after negative staining with 0.5% uranyl acetate (EMS, Hatfield, PA, USA) in water for 5 min at room temperature. Digital images were acquired with a CCD Mega-View camera (Olympus Soft Imaging Solutions GmbH, Munich, Germany).