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

Abstract

Source: Beaudoin, T., et al. Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model. J. Vis. Exp. (2016)

This video demonstrates an in vitro method to assess how patient-derived cystic fibrosis sputum influences bacterial biofilm formation. Using chamber slides, fluorescent staining, and confocal microscopy, it reveals increased thickness, altered architecture, and higher viable biomass in biofilms grown with sputum, highlighting the role of host components in shaping biofilm development.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

Staining Biofilms and Confocal Microscopy

1. Following desired growth time (24-48 hr works best), remove media from chamber wells and gently wash each chamber twice with 300 μl of sterile PBS.
2. Prepare staining mixture for biofilm by mixing 1 µl of each dye (provided in the viability kit) for each ml of solution needed. Make dye in water or media solution.

NOTE: Water is recommended by the manufacturer.

3. Add 200 µl of dye mixture to each well of chambered coverglass and incubate at room temperature, in the dark for 45 min.
4. Remove staining mixture from chambers and wash each well with 300 μl of sterile PBS. Remove PBS and replace it with fresh water or media.
5. Proceed with visualization of biofilms via confocal microscopy.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Lab-Tek II Chambered coverglass, #1.5 borosilicate, 8-well	Thermo Sicher Scientific	155409
Filmtracer Live/Dead Biofilm Viabilty Kit	Thermo Fisher Scientific	L10316
Millers LB Broth	Thermo Fisher Scientific	12780-052	Standard media for overnight gowth/biofilm growth
Phosphate Buffered Saline (Dulbecco A)	Oxoid	BR0014G	Washing of biofilm chambers after media removal
Zeiss AxioVert 200M	Carl Zeiss