Evaluating Inhibitor Effects on Pellicle Formation in Bacillus subtilis

JoVE Encyclopedia of Experiments

Microbiologie

0 weergaven • 2:57 min • February 26th, 2026

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Begin with a culture of Bacillus subtilis, a soil bacterium, grown in a nutrient-rich medium.

Centrifuge to pellet the bacteria, discard the supernatant, then resuspend the pellet in a chemically defined medium that supports biofilm formation.

Measure the optical density of the culture to determine the appropriate bacterial concentration.

Add the culture to wells containing defined medium for the control and defined medium with an inhibitor for the test.

Incubate under static conditions. The bacteria migrate upward and accumulate at the air-liquid interface, where increased oxygen availability supports bacterial growth.

In the control well, bacteria at the interface secrete extracellular matrix components and form a floating biofilm known as a pellicle, a protective layer enhancing bacterial survival.

In the test well, the inhibitor disrupts matrix synthesis, resulting in dispersed bacterial populations.

This assay shows that inhibitors block biofilm formation, which is a key feature of bacterial pathogenesis.

First, select an appropriate single colony and transfer it into 3 milliliters of LB broth. Place this starter culture into a shaking incubator for 4 hours at 37 degrees Celsius. After incubation, take 1.5 milliliters of the starter culture and centrifuge for four minutes.

Carefully remove the supernatant, and then re-suspend the pellet in 1.5 milliliters of MSgg medium. To grow pellicles, prepare a 12-well cell culture plate by adding 3 milliliters of MSgg to each well. To some of these wells, add MSgg with a small molecule inhibitor in a concentration range, distributing the location of different concentrations around the dish, to avoid edge effects.

Measure the optical density at 600 nanometers of the re-suspended starter culture. The culture should be between 0.6 and 1. This is critical for the robustness of the system.

Inoculate each well of the culture plate with 3 microliters of the re-suspended starter culture. Then, incubate the plate at 23 degrees Celsius for three days under static conditions. Afterwards, remove the plate from the incubator, and observe the pellicles.

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Laatst bijgewerkt: 1 augustus 2026