A Method for Sample Preparation to Visualize Biofilm Forming Bacteria on Fungal Hyphae

Introduce a cellophane-bound fungal colony into a fluorescently labeled bacterial suspension. Remove the cellophane and incubate.

The bacterial cells adhere to the fungal surface via surface molecules and receptors. The adhered bacteria secrete extracellular polymeric substances consisting of polysaccharides, proteins, and DNA, trapping the bacterial cells and forming the structural foundation of the biofilm.

Rinse the section in a strong salt solution to remove loosely bound bacteria

In a phosphate buffer, cut a section of the colony.

Transfer the section to sterile water containing wheat germ agglutinin, or WGA bound to a red fluorescent dye.

WGA, a plant protein, binds to N-acetylglucosamine and sialic acid residues, allowing the attached dye to fluoresce on the fungal cell wall.

Rinse the section in a phosphate buffer.

Partially immerse a slide in the buffer. Let the section float on it. Lift gently for the section to settle without biofilm disruption.

Place a mounting medium, followed by a coverslip for microscopic analysis.