Name: microfluidic tools for probing fungal-microbial interactions at the cellular level
Uploaded: 2022-06-23T13:20:00
Description: Watch this Scientific Journal Video about Microfluidic Tools for Probing Fungal-Microbial Interactions at the Cellular Level at JoVE.com

We acknowledge financial support from the Department of Bioengineering at Imperial College London and The Leverhulme Trust (Research Grant Reference: RPG-2020-352).

NOTE: A summary of the procedures outlined in this protocol are visually depicted in Figure 2.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63917/63917fig02.jpg" /> 
Figure 2: Schematic representation of the presented methodology consisting of five major sections detailed in this protocol. Device designs are created using computer aided design (CAD) softwar...

This article presents a protocol for the study of fungal-microbial interactions using channel microfluidics. The authors aim to demonstrate the versatility of these devices and encourage adaptation to suit the researcher&#39;s interests. Using the exemplar BFI and FFI devices, fungal-microbial interactions can be studied in more detail than previously accessible. By removing the background complexity and heterogeneity of the soil, moderating the growth of hyphae such that they are confined to a single monolayer, and tigh...

Soil is an exceptionally diverse environment containing an abundance of microorganisms that are instrumental to carbon and phosphorous cycles1,2. Filamentous fungi are a major component of numerous ecosystems as decomposers of organic and inorganic matter and can enhance the nutrition of plants through the formation of symbiotic relationships3,4. Within soil, fungi interact dynamically with a multitude of microbes such as other fungi5, bacteria6, viruses7 and nematodes8. These interactions have significant consequences for soil and plant health. Yet, owing to a lack of appropriate experimental systems capable of imaging interacting microorganisms with high-resolution, many remain undefined.
Investigations concerning bacterial-fungal interactions (BFIs) and fungal-fungal interactions (FFIs) have valuable applications in a range of fields, including antimicrobials in medicine and biological control agents in agriculture. For example, the fungus Coprinopsis cinerea produces the peptide copsin, which has been shown to exhibit antibacterial activity against the human pathogen Listeria monocytogenes9. Similarly, the fungal-derived compound, griseofulvin, is widely used as a treatment for human fungal infections and is additionally able to inhibit the growth of the plant pathogenic fungus Alternaria solani10,11. Several strains of the soil-dwelling bacterium Bacillus subtilis have also been demonstrated to be effective biocontrol agents of the fungal plant pathogen Rhizoctonia solani12,13. Nonetheless, due to limitations associated with traditional methodologies, BFIs and FFIs are poorly understood at the level of single cells.
Conventional studies typically explore BFIs and FFIs on the macroscale using agar plates with two or more species in confrontation. Their interaction is assessed by measuring growth rates and metabolite production of the confronting species14,15,16; however, this methodology is only resolved to the colony level. To study interactions at the cellular level, bacterial and fungal inoculants can be cultivated on glass microscope slides coated with agar that are then imaged under a microscope17. Nevertheless, it can be difficult to follow a single hypha using microscope slides due to a lack of confinement, meaning time-lapse images are harder to obtain. Further, the opportunity to spatially confine other microorganisms within defined regions of the fungal mycelium or create defined chemical environments that can be perturbed, for example, is not possible in such set-ups. The &#34;black box&#34; nature of soil also adds to the complexity of studying fungal-microbial interactions at the level of single cells18. By observing interacting species away from the incredible diversity of the soil microbiome, the exact manner by which individual members interact can be assessed. Thus, there is a continued need for versatile platforms that enable high-resolution, single-cell imaging of BFIs and FFIs.
Microfluidic technologies, so-called lab-on-a-chip systems, provide an ideal platform for the study of BFIs and FFIs at the level of single cells. The field of microfluidics, originating from technologies developed for chemical analysis and microelectronics, has been adopted by the biological sciences19. Microfluidic technologies regulate small volumes of fluids within a bespoke network of miniaturised channels, having at least one dimension on the micrometre scale, and their use in biological research is expanding20. In particular, microfluidic devices have been developed to examine the growth of filamentous fungi21,22,23,24,25,26,27,28,29,30. One benefit of using this technology is that the confinement of hyphae and the distribution of nutrients within microchannels more closely resembles the structure of the soil environment than conventional agar methods31. Recently, microfluidic platforms have been used to investigate interactions between human neutrophils and fungal pathogens32, bacteria and plant roots33, as well as fungi and nematodes34,35.
One of the many advantages of using microfluidics for studying microbial interactions includes the specific control of the microchannel environment. For instance, laminar flow regimes can be exploited to generate defined concentration gradients, which is especially useful when examining bacterial chemotaxis36. Another advantage is that the transparent nature of poly(dimethylsiloxane) (PDMS), an inexpensive, biocompatible elastomeric polymer commonly used in the manufacture of microfluidic devices, facilitates high-resolution imaging of single cells using brightfield and fluorescence microscopy37. Likewise, the confinement of microbes within microchannels means that time-lapse experiments tracking single cells can be performed, allowing individual cellular responses to be recorded and quantified37. Lastly, as microfluidic devices can be designed to be user-friendly, they can be easily employed by non-experts38.
Furthering knowledge of the interactions between soil-dwelling microorganisms is important for improving sustainable ecosystem management practises that maintain biodiversity and to mitigate the impact of climate change on terrestrial environments39. Thus, the development of novel microfluidic tools is fundamental to expand understanding of fungi and their interactions at the cellular level. The protocol here will focus on two microfluidic devices produced for the study of BFIs40&#160;and FFIs41&#160;as represented in Figure 1.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63917/63917fig01.jpg" /> 
Figure 1: Visual and schematic representation of the bacterial-fungal interaction (BFI) and fungal-fungal interaction (FFI) devices. (A) Image of the BFI device. A mycelial plug is placed at the entrance to one end of the microchannels to allow hyphal growth into the device. The bacterial inlet is at the opposite end. Scale bar = 5 mm. (B) Schematic overview of the BFI device, depicting the positioning of the bacterial inlets and direction of hyphal growth through the interaction microchannels. The channels are 10 &#181;m in depth, 100 &#181;m wide, and 7 mm long, with 28 observation channels in total. (C) Confrontation assay on agar plate between Coprinopsis cinerea and Bacillus subtilis NCIB 3610, scale bar = 20 mm (left). Microscopy images showing the interaction between C. cinerea and B. subtilis NCIB 3610 within the microchannel (middle and right), i.e., polar attachment of bacteria to fungal hyphae. Scale bar = 25 &#181;m (middle) and 10 &#181;m (right). (D) Image of the FFI device bonded to a glass-bottomed Petri dish, dual inoculated with mycelial plugs. Scale bar = 1 cm. (E) Schematic overview of the FFI device. Two fungal inoculant plugs are introduced into the inlets at either end of the device, permitting hyphal exploration of the microchannels. Control channels are connected to one fungal inlet only and have a dead-end channel, preventing interactions between the test fungi. Interaction channels connect both fungal inlets and permit hyphal interactions between the test subjects within the microchannel. Each interaction channel consists of 18 diamond-shaped sections, measuring a total length of 8.8 mm (490 x 430 &#181;m per diamond), 10 &#181;m deep, and having a connecting region between each diamond of 20 &#181;m. Channel types are duplicated, scale bars = 1 mm. (F) Interaction zone between two approaching hyphal fronts, growing from opposite ends of the interconnected interaction channel. Phase contrast microscopy image, scale bar = 250 &#181;m. The panels in this figure have been modified from Stanley et al., 2014 (A-C)40 and Gimeno et al., 2021 (D-F)41. <a href="https://www.jove.com/files/ftp_upload/63917/63917fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agar</td>
			<td>Difco Laboratories</td>
			<td>214010</td>
			<td>Used to solidify culture medium for bacterial and fungal cultivation within Petri dishes</td>
		</tr>
		<tr>
			<td>Aluminum foil</td>
			<td>Fisher Scientific Ltd</td>
			<td>11759408</td>
			<td></td>
		</tr>
		<tr>
			<td>AutoCAD 2021</td>
			<td>Autodesk, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclave (VX-75)</td>
			<td>Systec</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge (5810R)</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chlorotrimethysilane</td>
			<td>Merck Life Sciences</td>
			<td>386529</td>
			<td>CAUTION: Chlorotrimethylsilane is a hazardous substance. Wear appropriate PPE and handle with care. Avoid contact with skin and eyes and prevent inhalation. Keep away from sources of ignition and use in a well-ventilated area.</td>
		</tr>
		<tr>
			<td>Cork borer</td>
			<td>SLS</td>
			<td>COR1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Developer solution (mr-Dev 600)</td>
			<td>Microresist Technologies</td>
			<td></td>
			<td>CAUTION: mr-Dev 600 developer solution is flammable</td>
		</tr>
		<tr>
			<td>Erlenmeyer flasks</td>
			<td>VWR</td>
			<td>214-1108</td>
			<td>e.g. 200 mL; choose size to suit your exact needs</td>
		</tr>
		<tr>
			<td>Ethanol (70% v/v)&#160;</td>
			<td>Fisher Scientific Ltd</td>
			<td>E/0650DF/15</td>
			<td>Diluted from 99.8% (Analytical Reagent Grade)</td>
		</tr>
		<tr>
			<td>Fiji</td>
			<td>ImageJ</td>
			<td></td>
			<td>Exemplar software package for imaging processing</td>
		</tr>
		<tr>
			<td>Filtered, compressed air</td>
			<td></td>
			<td></td>
			<td>Available as standard in most labs. Altervatively, an oil-free compressor with air regulator can be used.</td>
		</tr>
		<tr>
			<td>Flat-headed wafer tweezers</td>
			<td>SLS</td>
			<td>INS5026</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fisher Scientific Ltd</td>
			<td>10008051</td>
			<td>Bent, sharp</td>
		</tr>
		<tr>
			<td>Glass bottom petri dish</td>
			<td>World Precision Instruments</td>
			<td>FD35-100</td>
			<td>35 mm</td>
		</tr>
		<tr>
			<td>Glass bottom petri dish</td>
			<td>World Precision Instruments</td>
			<td>FD5040-100</td>
			<td>50 mm</td>
		</tr>
		<tr>
			<td>Glass crystallisation dishes</td>
			<td>VWR</td>
			<td>216-1865</td>
			<td>Used for washing of PDMS slabs</td>
		</tr>
		<tr>
			<td>Glass crystallisation dishes</td>
			<td>VWR</td>
			<td>216-1866</td>
			<td>Used in the development of master moulds</td>
		</tr>
		<tr>
			<td>Glass media bottles</td>
			<td>Fisher Scientific Ltd</td>
			<td>15456113</td>
			<td>e.g. 250 mL; choose size to suit your exact needs</td>
		</tr>
		<tr>
			<td>Glass syringe (Hamilton)</td>
			<td>Fisher Scientific Ltd</td>
			<td>10625251</td>
			<td>Used for dispensing chlorotrimethylsilane</td>
		</tr>
		<tr>
			<td>Hot plate (HP 160 III BM)</td>
			<td>SAWATEC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inoculation loop</td>
			<td>VWR</td>
			<td>COPA175CS01</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl alcohol</td>
			<td>Sigma-Aldrich</td>
			<td>W292907</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar flow hood</td>
			<td>Air Science (PCR)</td>
			<td></td>
			<td>Exemplar laminar flow hood used for device fabrication</td>
		</tr>
		<tr>
			<td>LB medium</td>
			<td>Fisher Scientific Ltd</td>
			<td>BP9723-500</td>
			<td>Exemplar nutrient broth for bacterial overnight culture</td>
		</tr>
		<tr>
			<td>Light emitting diode light engine (LedHUB)</td>
			<td>Omicron-Laserage Laserprodukte GmbH</td>
			<td></td>
			<td>Exemplar light source that can be used for imaging fungal-microbial interactions (fluorescence)</td>
		</tr>
		<tr>
			<td>MA6 Ultraviolet mask aligner</td>
			<td>Suss Microtec</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Malt extract</td>
			<td>VWR</td>
			<td>84618</td>
			<td>Used to make exemplar fungal culture medium (Malt extract agar)</td>
		</tr>
		<tr>
			<td>Mask Writer</td>
			<td>Applied Materials</td>
			<td>4700DP</td>
			<td>Example of a mask writer which can be used to print photo-mask for photolithography</td>
		</tr>
		<tr>
			<td>Master mould plastic mount</td>
			<td></td>
			<td></td>
			<td>3D-printed bespoke holder manufactured in-house</td>
		</tr>
		<tr>
			<td>Microbiological safety cabinet&#160; (BioMat2)</td>
			<td>Contained Air Solutions</td>
			<td></td>
			<td>Exemplar MSC used for microbial culture and device inoculation</td>
		</tr>
		<tr>
			<td>Milli-Q purified water</td>
			<td></td>
			<td></td>
			<td>Available as standard in biology labs.&#160;</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Fisher Scientific Ltd</td>
			<td>BP359-500</td>
			<td></td>
		</tr>
		<tr>
			<td>NIS-Elements Advanced Research imaging software</td>
			<td>Nikon</td>
			<td></td>
			<td>Exemplar software package for image acquisition</td>
		</tr>
		<tr>
			<td>NIS-Elements Free Viewer</td>
			<td>Nikon</td>
			<td></td>
			<td>Exemplar software package for viewing acquired images</td>
		</tr>
		<tr>
			<td>Oven (Binder BD115)</td>
			<td>Fisher Scientific Ltd</td>
			<td>15602126</td>
			<td>Used for curing poly(dimethylsiloxane)(PDMS)</td>
		</tr>
		<tr>
			<td>Oven (CLO-2AH-S)</td>
			<td>KOYO</td>
			<td></td>
			<td>Used for preparing silicon wafers</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Bemis</td>
			<td>HS234526B</td>
			<td>transparent film</td>
		</tr>
		<tr>
			<td>Petri dishes, square sterile</td>
			<td>Fisher Scientific Ltd</td>
			<td>11708573</td>
			<td>120.5 mm</td>
		</tr>
		<tr>
			<td>Petri dishes, sterile</td>
			<td>Fisher Scientific Ltd</td>
			<td>15370366</td>
			<td>90 mm&#160;</td>
		</tr>
		<tr>
			<td>Photolithography mask</td>
			<td>Micro Lithography Services Ltd. UK</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma cleaner (Zepto)</td>
			<td>Diener Electronic</td>
			<td>100012601</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic cup</td>
			<td>Semadeni</td>
			<td>8323</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic spatula</td>
			<td>Semadeni</td>
			<td>3340</td>
			<td></td>
		</tr>
		<tr>
			<td>Portable precision balance (OHAUS Scout)</td>
			<td>Fisher Scientific Ltd</td>
			<td>15519631</td>
			<td>Used for weighing PDMS, media components etc.</td>
		</tr>
		<tr>
			<td>Precision cutter</td>
			<td>Syneo</td>
			<td>HS1251135P1183</td>
			<td>Cutting edge diameter: 3.18 mm</td>
		</tr>
		<tr>
			<td>Precision cutter&#160;</td>
			<td>Syneo</td>
			<td>HS1871730P1183S</td>
			<td>Cutting edge diameter: 4.75 mm</td>
		</tr>
		<tr>
			<td>Profilometer&#160;</td>
			<td>Bruker</td>
			<td></td>
			<td>Dektak XT-stylus</td>
		</tr>
		<tr>
			<td>Razor blades</td>
			<td>H&#228;berle Labortechnik</td>
			<td>9156110</td>
			<td></td>
		</tr>
		<tr>
			<td>Refridgerator</td>
			<td>Haden</td>
			<td></td>
			<td>4-6 &#176;C</td>
		</tr>
		<tr>
			<td>Retiga R1 CCD camera</td>
			<td>Qimaging</td>
			<td></td>
			<td>Exemplar camera that can be used for imaging fungal-microbial interactions</td>
		</tr>
		<tr>
			<td>Scotch magic tape</td>
			<td>Office Depot</td>
			<td>3969954</td>
			<td>19 mm invisible tape; clear tape</td>
		</tr>
		<tr>
			<td>Shaking incubator (Cole-Parmer SI500)</td>
			<td>Fisher Scientific Ltd</td>
			<td>10257954</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon wafer</td>
			<td>Inseto</td>
			<td></td>
			<td>100 mm</td>
		</tr>
		<tr>
			<td>Soda lime glass plate</td>
			<td>Inseto</td>
			<td></td>
			<td>125 mm x 125 mm x 2 mm. Used to hold photolithography mask in mask aligner</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>Spincoater&#160;</td>
			<td>SAWATEC</td>
			<td>SM-180-BM</td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 2010 photoresist</td>
			<td>MicroChem</td>
			<td></td>
			<td>CAUTION: SU-8 photoresist is hazardous, take care when handling and prevent inhalation and contact with skin. Flammable, potentially carcinogenic and toxic to the environment.&#160;</td>
		</tr>
		<tr>
			<td>Sylgard 184 elastomer kit</td>
			<td>VWR</td>
			<td>634165S</td>
			<td>Used for the preparation of poly(dimethylsiloxane)(PDMS) devices</td>
		</tr>
		<tr>
			<td>Temperature controlled incubator</td>
			<td>Okolab</td>
			<td></td>
			<td>Exemplar incubator that can be used for imaging fungal-microbial interactions</td>
		</tr>
		<tr>
			<td>Ti2-E inverted epifluorescence microscope</td>
			<td>Nikon</td>
			<td>MEA54000</td>
			<td>Exemplar microscope that can be used for imaging fungal-microbial interactions</td>
		</tr>
		<tr>
			<td>Ultrasonic cleaner S-Line</td>
			<td>Fisher Scientific Ltd</td>
			<td>FB15050</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum desiccator</td>
			<td>Fisher Scientific Ltd</td>
			<td>10528861</td>
			<td>Silianisation and PDMS degassing should be conducted in separate desiccators</td>
		</tr>
		<tr>
			<td>x10/0.3 NA CFI Plan Fluor DL objective lens</td>
			<td>Nikon</td>
			<td>MRH20105</td>
			<td>Exemplar objective lens that can be used for imaging fungal-microbial interactions</td>
		</tr>
		<tr>
			<td>x20/0.45 NA CFI Plan Fluor DL objective lens</td>
			<td>Nikon</td>
			<td>MRH48230</td>
			<td>Exemplar objective lens that can be used for imaging fungal-microbial interactions</td>
		</tr>
	</tbody>
</table>

microfluidic tools for probing fungal-microbial interactions at the cellular level

Filamentous fungi are successful inhabitants of soil and play a major role in soil ecosystems, such as in the decomposition of organic and inorganic matter, as well as regulation of nutrient levels. There they also find numerous opportunities to interact with a variety of other microbes such as bacteria or other fungi. Studying fungal interactions at the cellular level, however, can be challenging owing to the black box-like nature of soil. New microfluidic tools are being developed for the study of fungal interactions; two platforms designed to study bacterial-fungal and fungal-fungal interactions are highlighted. Within these microchannels, fungal-microbial interactions can be monitored in controlled physico-chemical environments at higher temporal and spatial resolution than previously possible. Application of these tools have yielded numerous novel biological insights, such as the observation of bacterial polar attachment to hyphae or revealing uncharacterised fungal-fungal antagonisms. A key feature of these methodologies regards the ease of use of this tool by non-experts, yielding highly translatable technologies for use in microbiology labs.

Representative results are presented from the exemplar BFI40 and FFI41 devices. Hyphal growth rate measurements can easily be obtained using these devices in combination with basic microscopy techniques. Figure 3A-B illustrates bacterial-fungal interactions between C. cinerea hyphae and B. subtilis NCIB 3610. The presence of B. subtilis halts the growth of C. cinerea after ca...

Watch this Scientific Journal Video about Microfluidic Tools for Probing Fungal-Microbial Interactions at the Cellular Level at JoVE.com

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

Owing to the opacity of soil, interactions between its constituent microbes cannot easily be visualised with cellular resolution. Here, two microfluidic tools are presented, which offer new opportunities for investigating fungal-microbial interactions. The devices are versatile and simple to use, enabling high spatiotemporal control and high-resolution imaging at the cellular level.

Filamentous fungi are successful inhabitants of soil and play a major role in soil ecosystems, such as in the decomposition of organic and inorganic ...

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