Assessing Bacterial Biofilm Formation Using a Cationic Dye-Based Assay

Begin with a bacterial suspension in nutrient media.

Add the suspension to a multi-well plate and incubate.

The nutrients in the media facilitate bacterial growth and attachment.

The adhered bacteria secrete extracellular polymeric substances consisting of polysaccharides, proteins, and DNA to form a layer of biofilm.

Wash multiple times with buffer to remove unbound bacteria.

Invert the plate to ensure the removal of residual liquid from the well and allow the biofilm to dry.

Treat with a fixative to preserve the cellular components of the biofilm.

Add a cationic dye that binds to negatively charged extracellular polymeric components of the biofilm.

Rinse with buffer and repeat several times to remove unbound dye.

Add a solvent to release the biofilm-bound dye.

Using a plate reader, measure the color intensity of the released dye as a readout of biofilm formation by the bacteria.