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.