In Vitro Biofilm Synthesis by Staphylococcus aureus

Bacterial biofilms are three-dimensional structures consisting of bacterial communities embedded in a self-generated extracellular matrix, which evade the host immune response.

For in vitro Staphylococcus aureus biofilm preparation, transfer an actively growing Staphylococcus aureus suspension of the desired cell density into the poly-L-lysine-coated wells of a multi-well plate.

During incubation under static conditions, the positively-charged poly-L-lysine coating facilitates electrostatic interactions with the negatively-charged glycopolymers, such as teichoic acids, on the bacterial surface, promoting bacterial attachment.

With available nutrient sources, Staphylococcus aureus divides and accumulates, forming microcolonies. Additionally, the bacteria secrete an extracellular matrix comprising polysaccharides, proteins, and extracellular DNA, which encases cells and promotes bacterial cohesion and surface adhesion. 

With continued cell division and matrix secretion, the biofilm matures into a three-dimensional structure with efficient signaling molecule distribution for cell-to-cell communication.

Upon reaching a threshold bacterial density, Staphylococcus aureus secretes proteases and nucleases, degrading the biofilm matrix and releasing a few Staphylococcus aureus into the media. Remove the media containing free-floating, planktonic bacteria.

Gently add a buffer to prevent biofilm disruption. Remove the buffer containing any remaining unattached bacteria.

The generated biofilm is ready for downstream experiments.

Begin by obtaining isolated colonies of Staphylococcus aureus from a cryopreserved stock using a streak plate technique on a nutrient-rich agar plate such as tryptic soy agar. Coat individual wells of a 96-well plate with 100 microliters of PLL diluted in sterile water, and incubate at room temperature for 30 minutes. Aspirate the PLL solution aseptically, using a vacuum-assisted aspiration trap. Allow the wells to dry overnight at room temperature.

Prepare an overnight culture by inoculating a colony of S. aureus in MEM-alpha supplemented with 2% glucose, and incubate it at 37 degrees Celsius for 16 to 18 hours at 200 rotations per minute. Dilute the overnight culture by transferring 50 microliters to 5 milliliters of fresh MEM-alpha supplemented with 2% glucose. Then, incubate it at 37 degrees Celsius at 200 rotations per minute until mid-logarithmic phase is achieved. Use MEM-alpha to normalize mid-logarithmic culture to an OD of 0.1.

Transfer 150 microliters of normalized culture to each well of the PLL-treated 96-well plate. Incubate the plate in a humidified chamber at 37 degrees Celsius for 18 to 20 hours. Aspirate the supernatant to remove the planktonic cells. Gently, wash the remaining biomass with 150 microliters of HBSS to remove the unattached cells. Repeat at least twice to remove all the planktonic cells.