A New Method for Qualitative Multi-scale Analysis of Bacterial Biofilms on Filamentous Fungal Colonies Using Confocal and Electron Microscopy

The overall goal of this protocol is to use microscopy to analyze the formation of bacterial biofilms on filamentous hyphae. This method can help answer key question in the microbiology field, such as how bacterial biofilm are formed on hyphae, how specific they are, and what are they made of. The main advantage of this technique is that it permits the investigation of bacterial biofilm formation on fungal hyphae on multiple scales, from the nanometer to centimeter ranges.

Demonstrating the procedure will be done by Cora Miquel Guennoc from my laboratory, and Christophe Rose, the electron microscopy engineer from INRA PTEF Platform. 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 five 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.

To remove planktonic bacteria and bacteria electrostatically attached to the hyphae, rinse the fungus by transferring it to a new six-well microplate filled with strong salt solution. Gently shake the plate for one minute. Transfer the fungus to a new six-well microplate containing five milliliters of sterile 0.1 molar potassium phosphate buffer.

Gently shake the plate for two minutes, and then transfer the fungus to fresh buffer. While keeping the fungal colony in buffer, use a scalpel to cut the colony approximately in half. Transfer half of the colony to be stained to a Petri dish containing one milliliter of sterile water, supplemented with an appropriate fluorescent dye to visualize the fungal network, such as wheat germ agglutinin conjugated to Alexa Fluor 633.

Then incubate the sample in the dark. After staining, rinse the sample by transferring it to a Petri dish lid containing 10 milliliters of sterile 0.1 molar potassium phosphate buffer, and gently shake for one minute. Half submerge a slide in the Petri dish lid, and delicately position the cut section to float above the slide.

Then slowly lift the slide from the buffer solution, allowing the sample to gently settle on the slide. The most delicate aspect is the positioning of the sample on the slide. To avoid biofilm description, the sample and the slide must be immersed while positioning the sample, and the sample must not be moved any more before imaging.

Finally, add 10 microliters of anti-fading mounting medium to the sample, and cover it with a glass cover slip. Examine the slides under a confocal laser microscope with a 10x or 40x objective. Use a combination of tile scan and the Z-stack functions to obtain global, 3D views of the fungal colony and the biofilms.

To carry out electron microscopy, after rinsing the fungus in strong salt solution as before, transfer the fungus to sterile water rather than buffer to prevent the formation of salt crystals during the dehydration step. Transfer the biofilms to a sample holder, and remove excess water. Then, transfer the samples to the chamber of a variable pressure scanning electron microscope.

On a Peltier cooling stage, freeze the sample to negative 50 degrees Celsius, allowing it to slowly freeze dry directly in the SEM chamber, with 100 Pascals of variable pressure set for 15 hours. Retrieve the sample and transfer it into a high vacuum, film deposition system. Then coat the sample with two nanometers of platinum under argon plasma.

Observe the coated sample with a scanning electron microscope, with a field emission gun using the high resolution in-lens detector, and an electronic high tension of one kilovolt. This meso-scale analysis of biofilms demonstrates the heterogenic distribution of the biofilm of P.fluorescens BBc6 in green on the hyphae of L.bicolor S238N, shown in red. The analysis also made possible the tracking of biofilm formation over time, from the early steps in which only some GFP tagged bacteria were attached to the hyphae, shown in red, to the formation of a thick, mature biofilm.

As shown here, the high resolution meso-scale images were also used to carry out micro-scale analysis, to obtain colony architectures. In addition, in this high resolution, 2D maximum intensity projection of a 3D confocal image, specific labeling with SYPRO Ruby, shown in red, indicates the presence of proteins in the matrix, with GFP-labeled BBc6 bacteria and FUN-1 labeled fungus in dark green. After dehydration and coding of the same sample, further details of the biofilm structure were obtained by SEM imaging.

The green arrowheads point to bacterial cells in the biofilm, and the yellow arrowheads point to fungal hyphae. Once mastered, this technique can be done in about two and a half hours for one sample, not counting the incubation time for the biofilm formation and the culture of microorganisms. While attempting this procedure, it's important to remember that the success of the method highly depends on the ability to obtain very thin fungal colonies, made of only a few layers of hyphae.

Following this procedure, other methods, such as mutagenesis or in situ hybridization, can be used to answer additional questions, like the functional or chemical characterization of the biofilm, or to study multi-species biofilms. After its development, this technique paved the way for researchers from multiple fields like medicine, environmental science, the food industry, and so on, to explore biofilm formation during fungal bacterial interactions. After watching this video, you should have a good understanding of how to grow and analyze bacterial biofilm formation on fungal hyphae.