Quantifying Enzyme-Induced Biofilm Suppression in Pathogenic Bacteria

Take a microplate containing control and treatment wells, each inoculated with a biofilm-forming, pathogenic bacterium.

The treatment wells contain an enzyme that disrupts biofilm formation.

Incubate under micro-anaerobic conditions to create a low-oxygen environment. This promotes the secretion of bacterial signaling molecules that mediate surface adhesion. In the treatment wells, the enzyme degrades these molecules, thereby reducing bacterial attachment.

Wash the wells to remove non-adherent cells.

Introduce fresh medium and incubate. The adhered bacteria proliferate and secrete extracellular polymeric substances, which form the biofilm matrix.

Remove the medium and wash the wells.

Add a cationic dye and incubate to allow binding to negatively charged biofilm components.

Wash to remove unbound dye.

Incubate with a solvent under agitation to release the bound dye into the solution.

Measure the optical density or OD to quantify the biofilm biomass.

Lower OD in the treatment wells reflects reduced biofilm formation, resulting from impaired initial cell adhesion.

To begin, grow a 5 milliliter culture of A.baumannii S1 in LB at 30 degrees Celsius in a shaking incubator for 16 hours.

Adjust the cu