This article details a method for cultivating biofilm-forming bacteria using cystic fibrosis patient-derived sputum filtrates. The process involves preparing bacterial cultures, promoting biofilm formation, and analyzing the resulting biofilms.
Take a culture of biofilm-forming bacteria grown in nutrient-rich media.
Dilute the culture in fresh media and incubate with shaking to promote bacterial growth until the desired concentration is reached.
Next, add the culture to fresh media enriched with cystic fibrosis patient-derived sputum filtrates.
These filtrates contain host-derived factors that prime the bacteria for biofilm formation.
Dispense the prepared suspension into the wells of a chambered coverglass and incubate statically to allow the bacteria to attach to the glass surface.
After incubation, remove the media and gently rinse the wells with fresh media to eliminate non-adherent bacteria.
Add fresh media containing sputum filtrate into the wells and again incubate the chamber statically.
During incubation, host-derived factors in the media activate bacterial signaling pathways that trigger extracellular matrix production and promote structured biofilm development.
The biofilm is now ready for further analysis.
Place 40 microliters of previously prepared culture into 4 milliliters of fresh Luria broth, or LB media, and grow the bacteria to obtain a culture with an optical density of 0.55 at 600 nanometers.
Next, dilute 100 microliters of 0.5 OD culture into 400 microliters of prepared 10% sputum filtrates in LB. Use 200 microliters of the dilution to seed the wells of the slide chambers.
Allow the bacteria to attach for four hours at 37 degrees Celsius without shaking.
After four hours, remove the media and gently wash the biofilm with 1X fresh LB. Replace the remaining media with 200 microliters of fresh LB. Allow the biofilms to grow at 37 degrees Celsius without shaking. Replace the media every 12 hours without washing until the biofilm is ready for microscopy.
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