Spreading colonies on agar medium and cultured bacterial cells were fixed with 1% paraformaldehyde (PFA) and 3.5% glutaraldehyde (GA) in 0.1 M phosphate buffer (PB) (pH 7.4) for 30 min at room temperature (RT) for heavy metal staining and charged Nanogold labeling for ASEM. Biofilms were fixed with 4% PFA for 10 min at RT for immunolabeling for optical microscopy. After labeling, colonies were further fixed with 1% GA for 10 min at RT, after which the contrast of the labels/stain was increased by Nanogold labeling enhancement and/or counterstaining with heavy metals as described previously for ASEM29. For Epon embedding and TEM, biofilms were fixed with 2.5% GA in PB at RT for 1 h and further with 1% osmic acid (OA) in the same buffer at 4 °C for 1 h.

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