Fluorescence-based bacterial overlay method for simultaneous in situ quantification of surface-attached bacteria

Area of Science:

• Biomaterials Science
• Microbiology
• Surface Chemistry

Background:

• Bacterial adherence to surfaces is critical in biofouling and medical device infections.
• Existing methods for quantifying bacterial adhesion on small samples are limited.
• Understanding surface properties' impact on bacterial attachment is essential for material design.

Purpose of the Study:

• To develop and validate a novel method for quantifying bacterial adherence to biomaterial surfaces.
• To investigate the influence of surface properties (wettability, roughness) on bacterial adhesion.
• To assess the effect of protein preadsorption on bacterial attachment.

Main Methods:

• Developed a new in situ method using biotinylated bacteria and fluorescence-labeled avidin-D for quantification.
• Utilized a silicon wafer model system with varying surface modifications (oxidation, PEGylation, hydrocarbon, fluorocarbon).
• Investigated effects of protein preadsorption (serum, saliva, HSA, fibronectin) and employed SEM and agar replica plating for verification.

Main Results:

• Oxidized and PEGylated silicon wafers showed reduced bacterial adhesion compared to hydrocarbon and fluorocarbon surfaces.
• Increased surface roughness generally led to higher bacterial attachment.
• Protein preadsorption modulated bacterial adherence, with some protein types (saliva, serum) masking underlying surface chemistry.

Conclusions:

• The new bacterial overlay method provides reliable quantification of surface-adherent bacteria.
• Surface chemistry and roughness are key factors influencing bacterial adhesion to biomaterials.
• Protein conditioning films play a role but do not entirely eliminate surface-specific bacterial interactions.