Confocal Imaging of Bacterial Biofilms Induced by Sputum Filtrates from Cystic Fibrosis Patients

Incubate a chambered coverglass containing adhered bacteria in a medium supplemented with cystic fibrosis patient-derived sputum filtrate and in the medium alone.

The sputum filtrate contains host-derived components that stimulate bacterial signaling pathways, triggering matrix production and biofilm formation.

Once the biofilms have formed, discard the medium containing free-floating bacteria and residual host-derived components.

Wash with a buffer to remove loosely attached bacteria.

Add a staining mixture containing membrane-permeable and membrane-impermeable fluorescent dyes, and incubate in the dark.

The membrane-permeable dye crosses intact membranes, labeling both viable and non-viable bacteria. In contrast, the impermeable dye enters only damaged membranes, selectively labeling non-viable bacteria.

After incubation, remove the staining solution and wash with the buffer to eliminate excess dye and reduce background fluorescence.

Add fresh medium and acquire images using confocal microscopy.

Biofilms grown with sputum filtrate exhibit increased thickness, highlighting the role of host-derived components in biofilm formation.

Following growth, remove the media from the chamber wells and gently wash each chamber two times with 300 microliters of sterile PBS.

Next, to prepare the biofilm staining mixture, add 1 microliter of each dye provided in the viability kit to each milliliter of solution needed, and mix the solution.

The optimal amount of dye and staining time is determined empirically for each organism.

Add 200 microliters of the staining mixture to each well of the chambered cover glass. Incubate the cover glass.

After incubation, remove the staining mixture from the chambers and wash each well with 300 microliters of sterile PBS.

Replace the PBS with fresh media. Finally, proceed with visualization via confocal microscopy.