A Solid Phase Adherence Assay to Assess Biofilm Formation

Take a flat-bottom multi-well plate with wells containing bacteria in growth media or bacteria in growth media supplemented with bile salt.

The bile salts induce stress, causing cellular damage and releasing DNA and proteins. These biomolecules coat the well bottoms, enabling the surviving bacteria to adhere to the solid surface.

The attached bacteria multiply, forming micro-colonies and producing auto-inducers — or chemical signaling molecules, allowing bacterial communication.

This communication triggers bacteria to colonize and initiate the production of extracellular polymeric substances or EPS, generating a biofilm.

Post-incubation, remove the media to eliminate free-floating bacteria.

Introduce a crystal violet stain that interacts with the peptidoglycan of the bacterial cell wall, giving it a violet color.

Remove excess stain and dry. Overlay with ethanol to solubilize crystal violet from the bacterial cell wall, changing the solution color.

A more intense coloration in bile salt-treated wells than in untreated wells confirms biofilm formation through solid-phase adherence.