An Assay to Study Bile-Salt Induced Biofilm Formation through EPS Matrix Detection
Trascrizione
Take a multiwell plate containing pathogenic enteric bacteria in a growth medium.
The medium in the selected wells contains bile salts — or amphipathic molecules with detergent-like activity — mimicking harsh growth conditions.
Bile salts disrupt the bacterial membrane and cause damage to DNA and proteins.
As a counter mechanism, the bacteria form a biofilm — a structured bacterial community encased in a self-produced matrix of extracellular polymeric substances, or EPS, containing polysaccharides, proteins, lipids, and nucleic acids of microbial origin.
The EPS layer shields the bacteria from exposure to the bile salts.
Post-incubation, aspirate the media to remove free-floating bacteria.
Treat with a fixation buffer to preserve the biofilm's architecture, and wash to remove excess buffer.
Add fluorescently labeled Concanavalin A — a lectin that binds to EPS polysaccharides, labeling the biofilm. Wash to remove excess Concanavalin A.
Using a plate reader, measure the fluorescence. A higher fluorescence intensity in the bile salt-containing wells indicates bile salt-induced biofilm formation.
