This study investigates the impact of biofilm matrix integrity on bacterial resistance to ethanol. By comparing untreated and inhibitor-treated biofilms, the research highlights how matrix composition influences antimicrobial efficacy.
Take pathogenic bacteria-derived biofilms grown on nutrient agar.
The untreated biofilm features densely packed bacteria within a protective extracellular matrix.
The inhibitor-treated biofilm features loosely associated cells within a sparse matrix.
Bisect each biofilm, then expose one half to buffer as a control, and the other to ethanol to impose antimicrobial stress.
In the untreated, ethanol-exposed biofilm, the dense matrix reduces ethanol diffusion and limits cell penetration, resulting in less bacterial killing.
In the inhibitor-treated, ethanol-exposed biofilm, the sparse matrix permits ethanol to penetrate more easily, leading to greater bacterial killing.
Centrifuge the tubes and remove the supernatant. Add buffer and sonicate to disperse the cells.
Plate the diluted suspensions and incubate to allow the growth of viable bacteria.
Count colony-forming units (CFUs); fewer CFUs from the inhibitor-treated, ethanol-exposed biofilm confirm that matrix integrity is essential for ethanol resistance.
Take a clean razor blade and cut the biofilm colonies into two equal parts with the help of the template. Carefully lift one half of the colony with a clean spatula, and transfer it to a 1.5-milliliter micro-centrifuge tube, containing 500 microliters of PBS.
Take the second half of the colony, and transfer it to a micro-centrifuge tube containing 500 microliters of 50% ethanol. These will be used to assess resistance to sterilizing agents. Afterwards, similarly take the first half of the D-leucine treated colony, and place it in PBS.
Then, take the second half of this colony, and transfer it to ethanol. Incubate all the tubes containing the biofilm halves for 10 minutes at room temperature. And then, centrifuge them for five minutes at 18,000 times g.
Using a pipette, carefully remove the supernatant. Add 300 microliters of PBS, and then sonicate the cells at a low setting, with a microtip sonicator. Add an additional 700 microliters of PBS for a final volume of one milliliter.
Next, perform a serial dilution of 10 to the negative seven in PBS. Take 100 microliters of one of the dilutions, and inoculate a 1.5 % Agar LB plate. Repeat this step for two other dilutions per sample.
Spread the inoculum using glass beads, and then incubate the plates overnight at 30 degrees Celsius. Examine the plates and count the colony-forming units, or CFU. After calculating the CFU per milliliter value, calculate the percentage of survivors in the PBS versus ethanol treatments.
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