Microfluidic Tools for Probing Fungal-Microbial Interactions at the Cellular Level

Using the presented microfluidic devices, we can examine dynamic interactions between fungi and other microbes with precise spacial and environmental control, as well as high-resolution imaging at the single cell level. Importantly, fungal growth can be confined and directed within the microfluidic channel. Enabling high quality time lapse images to be obtained.

Therefore, the complexities of fungal-fungal and fungal-bacterial interactions can be explored. Understanding fungal microbial interactions is an essential step in defining and preserving the soil microbiome. This knowledge can be exploited for the development of novel biocontrol agents with sustainable agriculture.

To begin, prepare approximately 40 grams of poly(dimethylsiloxane)or PDMs, by thoroughly mixing the base and the curing agent in a 10 to 1 ratio using a spatula in a clean plastic cup. To remove all air bubbles, place the cup into a vacuum chamber for one hour. Next, using clear tape, secure the master mold into a plastic mount and use compressed filtered air to remove any dust particles, then pour the PDMs onto the center of the master mold and allow it to settle.

To prevent the settling of dust particles on the PDMs surface, loosely cover the master mold with a plastic lid, then transfer the master mold to an oven and cure overnight at 70 degrees Celsius. After removing the master mold from the oven and allowing it to cool, peel the cured PDMs away from the master mold and plastic frame without damaging the master mold in the PDMs. Then to maintain a dust-free surface, place clear tape over the microchannels embossed into the PDMs.

Next, use a mounted guillotine or a razor blade to cut the PDMs into slabs as designated by the design. When cutting the lateral opening of the PDMs slab used for the bacterial-fungal interaction device, or BFI device, ensure that the microchannels are fully open. However, for PDMs slabs used in the assembly of the fungal-fungal interaction device, or FFI, each corner should be trimmed so that it fits into a glass bottom Petri dish.

Next, use a precision cutter to punch inlet or outlet holes according to the device design. Next, submerge the PDMs slabs into a 0.5 molar sodium hydroxide solution. Rinse the slabs in sterile double distilled water before transferring them into a 70%ethanol solution.

After removing the ethanol and rinsing the slabs with double distilled sterile water again, immerse the slabs in sterile double distilled water and sonicate for five minutes, then remove the PDMs slab from the water, dry them with compressed filtered air, and place them in a sterile square Petri dish. Next, incubate the Petri dish with PDMs slab in an oven at 70 degree Celsius for one hour. Once the slabs have cooled in a dust-free environment, clear any dust from their surface using tape and compressed filtered air.

To activate the surfaces of the PDMs slabs and glass bottom Petri dishes, which will be subsequently bonded together, place them in a plasma cleaner with the surfaces to be activated facing upward. After removing the PDMs slab in the Petri dishes from the plasma cleaner, bond them gently by placing the activated surfaces in conformal contact with one another. Bond BFI and FFI PDMs slab with Petri dishes of 35 millimeter and 55 millimeter diameters, respectively for checking the successful bonding, try to pull the PDMs lab off its Petri dish with tweezers.

Visualize the device carefully with the eye or by generic microscopy to ensure no collapse of the inoculation inlets or microchannels. For water saturated or nutrient-rich conditions, pipette 100 microliters of the desired solution to fill BFI devices immediately after bonding. For FFI devices, add 30 microliters of the media into each inlet.

Finally, add 100 to 200 microliters of sterile double distilled water to the Petri dish to maintain humidity. However, for water unsaturated conditions, just add 100 to 200 microliters of sterile double distilled water to the Petri dish to maintain humidity. For fungal inoculation, inside a laminar flow hood, using a sterilized cork borer, remove an agar plug from a colony on the periphery of a three day old culture, ensuring to keep the growing hyphal front intact.

Then introduce the agar plug into the fungal inlet of the BFI and/or FFI device with the mycelium side down in the hyphal front oriented toward the microchannel openings to encourage hyphal infiltration of the channels. For the FFI device, introduce another agar plug with a second fungal species into the opposite inlet. Next, seal the Petri dish with a transparent film and incubate at 25 to 28 degree Celsius in the dark until imaging.

For bacterial inoculation, after removing the BFI device from the incubator, pipette 10 microliters of a bacterial suspension into the bacterial inlet in a sterile environment. Once the Petri dish is sealed with a transparent film, stored the device upright at 25 degree Celsius in the dark until imaging commences. Using this protocol, the interaction between the fungus Coprinopsis cinerea and the bacterium Bacillus subtilis could be successfully visualized.

It was observed that in the presence of the bacterium, the growth rate of the fungus started to decline drastically after about five hours of coinoculation. Moreover, the fungal hyphae associated with the bacteria had a thin and transparent appearance. Time lapse fluorescence microscopy also revealed fungal hyphae collapse after five hours of coinoculation with Bacillus subtilis.

However, from six hours onwards, the bacterial population also started to decline. Visualization of fungal-fungal interactions showed that Fusarium graminearum strongly inhibited the growth of Trichoderma rossicum, which was evident using fluorescence microscopy. This protocol further revealed significant morphological changes in the fungus Verticillium longisporum in the presence of bacteria Pseudomonas synxantha and Pseudomonas fluorescens.

The high resolution dynamic imaging technique used in the study also revealed septa formation in Fusarium graminearum in the presence of the fungus Clonostachys rosea. Fluorescence labeling afforded the dynamic quantification of Clonostachys rosea hyphal proliferation in the FFI device. These devices can be adapted and modified by the user to incorporate different species of interest and address their specific experimental questions concerning fungal microbial interactions.

Both the BFI and FFI devices have pushed scientific frontiers, revealing how fungi propagate defense signals and nutrients upon attack by fungi, virus, nematodes and enabling investigations on bacterial dispersal.