A marine bacterial adhesion microplate test using the DAPI fluorescent dye: a new method to screen antifouling agents

Area of Science:

• Marine microbiology
• Biotechnology
• Surface science

Background:

• Marine bacterial adhesion is a critical initial step in biofilm formation.
• Existing screening methods often focus on bactericidal activity, not adhesion inhibition.
• Developing rapid, quantitative assays for antifouling agents is essential.

Purpose of the Study:

• To develop a sensitive, rapid, and quantitative microplate fluorescent assay for screening antifouling agents.
• To assess the method's efficacy in detecting marine bacterial adhesion inhibition and removal.
• To establish a screening tool for early-stage marine biofilm prevention.

Main Methods:

• Utilized a natural biofilm of Pseudoalteromonas sp. D41 in sterile natural seawater within a 96-well polystyrene microplate.
• Employed 4'6-diamidino-2-phenylindole dye for quantifying adhered bacteria via fluorescence.
• Validated the assay with commercial surface-active agents and chlorine to test antifouling properties.

Main Results:

• The fluorescence signal strongly correlated with the number of adhered bacteria, with a detection limit of one bacterium per 5 µm².
• Quantified bacterial adhesion ranging from 2 x 10⁷ to 2 x 10⁸ bacteria/cm².
• Demonstrated a sigmoid dose-response curve for antifouling agent concentration, indicating efficacy in preventing adhesion and promoting detachment.

Conclusions:

• The developed microplate fluorescent test is suitable for rapid, quantitative screening of antifouling agents.
• This assay is ideal for evaluating bacterial adhesion inhibition and removal activities in early marine biofilm formation.
• The method provides a valuable tool for the initial screening of antifouling products directly in seawater.