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.
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Method Article
Microfluidic Tools for Probing Fungal-Microbial Interactions at the Cellular Level
In This Article
Summary
Abstract
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.
Introduction
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 nematodes
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Protocol
NOTE: A summary of the procedures outlined in this protocol are visually depicted in Figure 2.
Figure 2: Schematic representation of the presented methodology consisting of five major sections detailed in this protocol. Device designs are created using computer aide....
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Results
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.......
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Discussion
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'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.......
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Disclosures
The authors declare no competing interests.
Acknowledgements
We acknowledge financial support from the Department of Bioengineering at Imperial College London and The Leverhulme Trust (Research Grant Reference: RPG-2020-352).
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| Agar | Difco Laboratories | 214010 | Used to solidify culture medium for bacterial and fungal cultivation within Petri dishes |
| Aluminum foil | Fisher Scientific Ltd | 11759408 | |
| AutoCAD 2021 | Autodesk, USA | ||
| Autoclave (VX-75) | Systec | ||
| Centrifuge (5810R) | Eppendorf | ||
| Chlorotrimethysilane | Merck Life Sciences | 386529 | 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. |
| Cork borer | SLS | COR1000 | |
| Developer solution (mr-Dev 600) | Microresist Technologies | CAUTION: mr-Dev 600 developer solution is flammable | |
| Erlenmeyer flasks | VWR | 214-1108 | e.g. 200 mL; choose size to suit your exact needs |
| Ethanol (70% v/v) | Fisher Scientific Ltd | E/0650DF/15 | Diluted from 99.8% (Analytical Reagent Grade) |
| Fiji | ImageJ | Exemplar software package for imaging processing | |
| Filtered, compressed air | Available as standard in most labs. Altervatively, an oil-free compressor with air regulator can be used. | ||
| Flat-headed wafer tweezers | SLS | INS5026 | |
| Forceps | Fisher Scientific Ltd | 10008051 | Bent, sharp |
| Glass bottom petri dish | World Precision Instruments | FD35-100 | 35 mm |
| Glass bottom petri dish | World Precision Instruments | FD5040-100 | 50 mm |
| Glass crystallisation dishes | VWR | 216-1865 | Used for washing of PDMS slabs |
| Glass crystallisation dishes | VWR | 216-1866 | Used in the development of master moulds |
| Glass media bottles | Fisher Scientific Ltd | 15456113 | e.g. 250 mL; choose size to suit your exact needs |
| Glass syringe (Hamilton) | Fisher Scientific Ltd | 10625251 | Used for dispensing chlorotrimethylsilane |
| Hot plate (HP 160 III BM) | SAWATEC | ||
| Inoculation loop | VWR | COPA175CS01 | |
| Isopropyl alcohol | Sigma-Aldrich | W292907 | |
| Laminar flow hood | Air Science (PCR) | Exemplar laminar flow hood used for device fabrication | |
| LB medium | Fisher Scientific Ltd | BP9723-500 | Exemplar nutrient broth for bacterial overnight culture |
| Light emitting diode light engine (LedHUB) | Omicron-Laserage Laserprodukte GmbH | Exemplar light source that can be used for imaging fungal-microbial interactions (fluorescence) | |
| MA6 Ultraviolet mask aligner | Suss Microtec | ||
| Malt extract | VWR | 84618 | Used to make exemplar fungal culture medium (Malt extract agar) |
| Mask Writer | Applied Materials | 4700DP | Example of a mask writer which can be used to print photo-mask for photolithography |
| Master mould plastic mount | 3D-printed bespoke holder manufactured in-house | ||
| Microbiological safety cabinet (BioMat2) | Contained Air Solutions | Exemplar MSC used for microbial culture and device inoculation | |
| Milli-Q purified water | Available as standard in biology labs. | ||
| NaOH | Fisher Scientific Ltd | BP359-500 | |
| NIS-Elements Advanced Research imaging software | Nikon | Exemplar software package for image acquisition | |
| NIS-Elements Free Viewer | Nikon | Exemplar software package for viewing acquired images | |
| Oven (Binder BD115) | Fisher Scientific Ltd | 15602126 | Used for curing poly(dimethylsiloxane)(PDMS) |
| Oven (CLO-2AH-S) | KOYO | Used for preparing silicon wafers | |
| Parafilm | Bemis | HS234526B | transparent film |
| Petri dishes, square sterile | Fisher Scientific Ltd | 11708573 | 120.5 mm |
| Petri dishes, sterile | Fisher Scientific Ltd | 15370366 | 90 mm |
| Photolithography mask | Micro Lithography Services Ltd. UK | ||
| Plasma cleaner (Zepto) | Diener Electronic | 100012601 | |
| Plastic cup | Semadeni | 8323 | |
| Plastic spatula | Semadeni | 3340 | |
| Portable precision balance (OHAUS Scout) | Fisher Scientific Ltd | 15519631 | Used for weighing PDMS, media components etc. |
| Precision cutter | Syneo | HS1251135P1183 | Cutting edge diameter: 3.18 mm |
| Precision cutter | Syneo | HS1871730P1183S | Cutting edge diameter: 4.75 mm |
| Profilometer | Bruker | Dektak XT-stylus | |
| Razor blades | Häberle Labortechnik | 9156110 | |
| Refridgerator | Haden | 4-6 °C | |
| Retiga R1 CCD camera | Qimaging | Exemplar camera that can be used for imaging fungal-microbial interactions | |
| Scotch magic tape | Office Depot | 3969954 | 19 mm invisible tape; clear tape |
| Shaking incubator (Cole-Parmer SI500) | Fisher Scientific Ltd | 10257954 | |
| Silicon wafer | Inseto | 100 mm | |
| Soda lime glass plate | Inseto | 125 mm x 125 mm x 2 mm. Used to hold photolithography mask in mask aligner | |
| Sodium chloride | Sigma-Aldrich | S7653 | |
| Spincoater | SAWATEC | SM-180-BM | |
| SU-8 2010 photoresist | MicroChem | 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. | |
| Sylgard 184 elastomer kit | VWR | 634165S | Used for the preparation of poly(dimethylsiloxane)(PDMS) devices |
| Temperature controlled incubator | Okolab | Exemplar incubator that can be used for imaging fungal-microbial interactions | |
| Ti2-E inverted epifluorescence microscope | Nikon | MEA54000 | Exemplar microscope that can be used for imaging fungal-microbial interactions |
| Ultrasonic cleaner S-Line | Fisher Scientific Ltd | FB15050 | |
| Vacuum desiccator | Fisher Scientific Ltd | 10528861 | Silianisation and PDMS degassing should be conducted in separate desiccators |
| x10/0.3 NA CFI Plan Fluor DL objective lens | Nikon | MRH20105 | Exemplar objective lens that can be used for imaging fungal-microbial interactions |
| x20/0.45 NA CFI Plan Fluor DL objective lens | Nikon | MRH48230 | Exemplar objective lens that can be used for imaging fungal-microbial interactions |
References
- Zhu, Y. -G., Miller, R. M. Carbon cycling by arbuscular mycorrhizal fungi in soil-plant systems. Trends in Plant Science. 8 (9), 407-409 (2003).
- Dai, Z., et al. Long-term nutrient inputs shift soil microbial fun....
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