This study investigates the formation of biofilms by pathogenic enteric bacteria in the presence of bile salts, which mimic harsh growth conditions. The research highlights the protective mechanisms employed by bacteria to survive in hostile environments.
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.
For semi-quantitative detection of EPS, use a multichannel pipette to transfer the culture medium to a clear 96-well plate. Then, use 200 microliters of fixing reagent to fix the black plate for 15 minutes at room temperature. Now, record the OD600 reading by setting the control well as the blank for standardization. Then, record the reading of the culture medium while the adherent population is fixing.
Once the fixing is complete, remove the reagent and discard in the hazardous waste. Then, use 200 microliters of sterile PBS to gently wash the wells twice. After washing, aspirate the PBS. Next, add 150 microliters of 25 micrograms per milliliter of ConA-FITC. Incubate the plate at room temperature for 15 minutes. Post-incubation, gently wash the wells with 200 microliters of PBS. Then, add 150 microliters of PBS to each well, and record the fluorescence at 488 nanometers.
繁體中文
