A Chambered Coverglass Method for In Vitro Bacterial Biofilm Formation

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 the use of a chambered coverglass to study the effect of cystic fibrosis patient-derived sputum host factors on bacterial biofilm development.

Protocol

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

1. Chambered Coverglass Method for Biofilm Formation
   1. Grow bacterial isolate of interest overnight in desired media at 37 °C with shaking (200 rpm).
      NOTE: A number of different bacteria were used, including clinical isolates Pseudomonas aeruginosa, Staphylococcus aureus, Burkholderia cepacia complex and Achromobacter xylosoxidans. Choice of media depends on strains and conditions of interest, however (Luria broth) LB media can be used for initial experiments.
   2. From overnight culture, place 40 μl of culture into 4 ml of fresh media and grow for 3-4 hr at 37 °C with shaking (200 RPM) to obtain a culture with an optical density at 600 nm (OD600) of approximately 0.5-0.6.
   3. Dilute the culture from step 1.2 to 1/5 in desired media with 10% sputum filtrate or without sputum filtrate (as control). Other concentrations of sputum filtrate can be tested (i.e., 50% or 100%).
   4. Use 200 μl of the dilution to seed wells of slide chambers.
   5. Allow bacteria to attach for 4 hr at 37 °C without shaking.
   6. After 4 hr, remove the media and gently wash the biofilm with 1x fresh media. Replace with 200 μl fresh media.
      NOTE: To study the effects of sputum on the biofilm, the fresh media should contain sputum supernatant.
   7. Allow biofilms to grow for desired amount of time at 37 °C without shaking, replacing media every 12 hr, without washing until the time for microscopy.
      NOTE: To study the effect of sputum supernatants on biofilm antibiotic susceptibility, antibiotics are added to the media following 24 hr of biofilm growth and are maintained in the media until staining and imaging of biofilms.

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
Millers LB Broth	Thermo Fisher Scientific	12780-052	Standard media for overnight growth/biofilm growth