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

1. Preparation of an In Vitro Biofilm of Bacteria on the Fungal Colony

1. Centrifuge the bacterial culture at 5,000 x g for 3 min and suspend the pellet in 25 ml of sterile 0.1 M potassium phosphate buffer (25 g/L KH₂PO₄ and 2.78 g/L K₂HPO₄, pH 5.8). Repeat this step once and adjust the final bacterial concentration to 10⁹ cells/ml with the same buffer.
2. Fill a 6-well microplate with 5 ml of the bacterial suspension (or sterile 0.1 M potassium phosphate buffer for the negative control).
3. Cut the cellophane membrane of the fungal culture with a sterile razor blade to obtain squares of cellophane with a single fungal colony on each membrane. Carefully remove the cellophane squares containing hyphae from the solid medium using forceps and transfer the square of cellophane containing hyphae to a well of the microplate containing the bacterial suspension.
4. Gently shake the microplate while the fungal colonies are still attached to the cellophane; then, remove the cellophane sheets, leaving the fungal colonies in the plate.
5. Incubate the microplate with gentle agitation (~60 rpm) at a temperature adapted to the studied microorganisms.
   Note: The time of incubation depends on the speed of establishment of the biofilm and the stage to be analyzed. For P. fluorescens BBc6/L. bicolor, incubate at 20 °C for 30 min to get early-stage biofilms and up to 16 hr to get mature biofilms.

2. Laser Scanning Confocal Microscopy Analysis of the Biofilm Formation

1. Sample preparation
   1. To remove planktonic bacteria and bacteria electrostatically attached to the hyphae, rinse the fungus by transferring it to a new 6-well microplate filled with 5 ml of a strong salt solution (NaCl, 17 g/L); gently shake for 1 min.
   2. Transfer the fungus to a new 6-well microplate containing 5 ml of sterile 0.1 M potassium phosphate buffer, gently shake for 2 min, and transfer the fungus to fresh potassium phosphate buffer.
   3. Cut the part of the fungal colony to be imaged with a scalpel while keeping it in the potassium phosphate buffer, such that the obtained section contains about half of the fungal colony.
   4. To stain the sample, transfer it to a Petri dish filled with sterile water containing an appropriate fluorescent dye (depending on the aim of the analysis), and incubate it in the dark.
      1. To visualize the fungal network, use, for example, Congo Red (0.1% final concentration, 5 min of incubation), wheat germ agglutin (WGA) lectin conjugated to Alexa Fluor 633 (10 µg/ml final concentration, 10 min of incubation) or FUN1 (10 µM final concentration, 10 min of incubation).
      2. If using a non-fluorescent-tagged bacterium, counterstain the bacterial cells with a DNA-specific fluorescent probe among the numerous cell-permeant DNA dyes commercially available. For example, use DAPI (0.25 µg/ml final concentration, 10 min of incubation).
      3. To visualize the matrix proteins, use a protein stain.
   5. After staining, rinse the sample by transferring it to a Petri dish lid containing 10 ml of sterile 0.1 M potassium phosphate and gently shake for 1 min.
   6. Half submerge a slide in the Petri dish lid and delicately bring the cut section to float above the slide. Then, slowly remove the slide from the buffer solution, allowing the sample to gently settle on the slide.
      Note: For this step, it is very important to proceed gently to avoid biofilm disturbance.
   7. Finally, add 10 μl of anti-fading mounting medium to the sample and cover it with a glass coverslip.
      Note: Once the slide is prepared, the microscopic analysis must be performed as soon as possible (within at most 30 min) to avoid modification in the biofilm structure.