Establishing a Fungal-Bacterial Co-culture for Biofilm Formation

Treat semipermeable cellophane membranes with a chelating agent to remove metal ions that inhibit fungal growth.

Rinse with water, then sterilize to eliminate contaminating microorganisms.

Place a treated membrane onto an agar plate and inoculate it with an agar section containing a fungus.

Incubate to promote growth and colony formation.

Scrape the colony edge containing actively growing fungal hyphae. Transfer it onto another treated membrane on a low-nutrient agar medium, then incubate.

Low-nutrient medium promotes radial growth for enhanced nutrient acquisition, resulting in thin colonies.

Inoculate soil bacterial colonies into a liquid medium, and incubate with shaking to promote growth.

Centrifuge, discard the supernatant, and resuspend in a buffer. Centrifuge again, resuspend the cells, and transfer to a microplate.

Cut the cellophane-bound fungal colony, place it into the suspension, and shake to detach the cellophane from the fungus. Incubate with agitation.

Bacteria adhere to the fungal surface and form a biofilm that facilitates bacteria-fungus interaction.

To begin, prepare cellophane membranes with the same diameter as the Petri dishes, then place them in boiling EDTA for 30 minutes. Then use deionized water to rinse the sheets and autoclave them twice.

To prepare fungal pre-cultures, inoculate the fungus on agar medium, covered with an EDTA-pretreated cellophane membrane. Incubate the cultures at optimal temperature to obtain colonies approximately one centimeter in diameter. From the pre-culture, inoculate a Petri dish of appropriate agar medium, covered with the EDTA cellophane membrane by using a scalpel to gently scratch the external area of a pre-culture colony, and then transferring the hyphae to the agar plate.

Incubate the plates at optimal temperature until the colonies are approximately one centimeter in diameter. To prepare bacterial cultures such as P. fluorescens BBc6, use a sterile loop to collect two to three individual bacterial colonies from agar medium, and inoculate 25 milliliters of LB. Incubate the culture at optimal temperature overnight. After growing the bacteria overnight, centrifuge the culture at 5,000 times g for three minutes, and then suspend the pellet in 25 milliliters of sterile 0.1 molar potassium phosphate buffer.

After repeating the centrifugation, use the same buffer to adjust the final bacterial concentration to 10 to the ninth cells per milliliter. Fill a six-well microplate with 5 milliliters of the bacterial suspension.

Use a sterile razor blade to cut the cellophane membrane of the fungal culture into squares, with a single colony on each membrane. Then, with forceps, carefully remove cellophane squares containing hyphae from the solid medium and transfer them to individual wells of the bacterial suspension. Gently shake the microplate until the fungal colonies are detached from the cellophane.

Then remove the cellophane sheets, leaving the fungal colonies in the plate. Incubate the microplate with gentle agitation at 20 degrees Celsius for a time depending on the strains used and the stage to be analyzed. For P. fluorescens BBc6 RA L. bicolor, incubate the cultures for 30 minutes to get early-stage biofilms, and for up to 16 hours for mature biofilms.