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

The authors declare no competing interests.

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><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)&amp;nbsp;</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&amp;nbsp; (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.&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</td><td>Syneo</td><td>HS1871730P1183S</td><td>Cutting edge diameter: 4.75 mm</td></tr><tr><td>Profilometer&amp;nbsp;</td><td>Bruker</td><td></td><td>Dektak XT-stylus</td></tr><tr><td>Razor blades</td><td>H&amp;auml;berle Labortechnik</td><td>9156110</td><td></td></tr><tr><td>Refridgerator</td><td>Haden</td><td></td><td>4-6 &amp;deg;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&amp;nbsp;</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.&amp;nbsp;</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...

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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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Bioengineering

3.5K Views.  Imperial College London. Met behulp van de gepresenteerde microfluïdische apparaten kunnen we dynamische interacties tussen schimmels en andere microben onderzoeken met nauwkeurige ruimtelijke en omgevingscontrole, evenals beeldvorming met hoge resolutie op het niveau van één cel. Belangrijk is dat schimmelgroei kan worden beperkt en gericht binnen het microfluïdische kanaal. Hiermee kunnen time-lapse-afbeeldingen van hoge kwaliteit worden verkregen.Daarom kunnen de complexiteiten van schimmel-schimmel- en...

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

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

A Microfluidic Device for Studying Multiple Distinct Strains

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a comparative manner. Multiwell plates are routinely used to compare many different strains or cell lines, but allow limited control over the environment dynamics. Microfluidic devices, on the other hand, allow exquisite dynamic control over the surrounding conditions, but it is challenging to image and distinguish more than a few strains in them. Here we describe a method to easily and rapidly manufacture a microfluidic device capable of applying dynamically changing conditions to multiple distinct yeast strains in one channel. The device is designed and manufactured by simple means without the need for soft lithography. It is composed of a Y-shaped flow channel attached to a second layer harboring microwells. The strains are placed in separate microwells, and imaged under the exact same dynamic conditions. We demonstrate the use of the device for measuring protein localization responses to pulses of nutrient changes in different yeast strains.

We present a simple method to produce microfluidic devices capable of applying similar dynamic conditions to multiple distinct strains, without the need for a clean room or soft lithography.

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a ...

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described in detail. The PLBR can be utilized for the analysis of single bacteria and microcolonies to investigate biotechnological and microbiological related questions concerning, e.g.&#160;cell growth, morphology, stress response, and metabolite or protein production on single-cell level. The device features continuous media flow enabling constant environmental conditions for perturbation studies, but in addition allows fast medium changes as well as oscillating conditions to mimic any desired environmental situation. To fabricate the single use devices, a silicon wafer containing sub micrometer sized SU-8 structures served as the replication mold for rapid polydimethylsiloxane casting. Chips were cut, assembled, connected, and set up onto a high resolution and fully automated microscope suited for time-lapse imaging, a powerful tool for spatio-temporal cell analysis. Here, the biotechnological platform organism Corynebacterium glutamicum&#160;was seeded into the PLBR and cell growth and intracellular fluorescence were followed over several hours unraveling time dependent population heterogeneity on single-cell level, not possible with conventional analysis methods such as flow cytometry. Besides insights into device fabrication, furthermore, the preparation of the preculture, loading, trapping of bacteria, and the PLBR cultivation of single cells and colonies is demonstrated. These devices will add a new dimension in microbiological research to analyze time dependent phenomena of single bacteria under tight environmental control. Due to the simple and relatively short fabrication process the technology can be easily adapted at any microfluidics lab and simply tailored towards specific needs.

In this protocol the fabrication, setup and basic operation of a microfluidic picoliter bioreactor (PLBR) for single-cell analysis of prokaryotic microorganisms is introduced. Industrially relevant microorganisms were analyzed as proof of principle allowing insights into growth rate, morphology, and phenotypic heterogeneity over certain time periods, hardly possible with conventional methods.

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described ...

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this purpose, the cells are passively trapped in the middle of a microchamber. Upon activation of the control layer, the cell is isolated from the surrounding volume in a small chamber. The surrounding volume can then be exchanged without affecting the isolated cell. However, upon short opening and closing of the chamber, the solution in the chamber can be replaced within a few hundred milliseconds. Due to the reversibility of the chambers, the cells can be exposed to different solutions sequentially in a highly controllable fashion, e.g. for incubation, washing, and finally, cell lysis. The tightly sealed microchambers enable the retention of the lysate, minimize and control the dilution after cell lysis. Since lysis and analysis occur at the same location, high sensitivity is retained because no further dilution or loss of the analytes occurs during transport. The microchamber design therefore enables the reliable and reproducible analysis of very small copy numbers of intracellular molecules (attomoles, zeptomoles) released from individual cells. Furthermore, many microchambers can be arranged in an array format, allowing the analysis of many cells at once, given that suitable optical instruments are used for monitoring. We have already used the platform for proof-of-concept studies to analyze intracellular proteins, enzymes, cofactors and second messengers in either relative or absolute quantifiable manner.

In this article we present a microfluidic chip for single cell analysis. It allows the quantification of intracellular proteins, enzymes, cofactors, and second messengers by means of fluorescent assays or immunoassays.&#160;

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this ...

Window on a Microworld: Simple Microfluidic Systems for Studying Microbial Transport in Porous Media

Microbial growth and transport in porous media have important implications for the quality of groundwater and surface water, the recycling of nutrients in the environment, as well as directly for the transmission of pathogens to drinking water supplies. Natural porous media is composed of an intricate physical topology, varied surface chemistries, dynamic gradients of nutrients and electron acceptors, and a patchy distribution of microbes. These features vary substantially over a length scale of microns, making the results of macro-scale investigations of microbial transport difficult to interpret, and the validation of mechanistic models challenging. Here we demonstrate how simple microfluidic devices can be used to visualize microbial interactions with micro-structured habitats, to identify key processes influencing the observed phenomena, and to systematically validate predictive models. Simple, easy-to-use flow cells were constructed out of the transparent, biocompatible and oxygen-permeable material poly(dimethyl siloxane). Standard methods of photolithography were used to make micro-structured masters, and replica molding was used to cast micro-structured flow cells from the masters. The physical design of the flow cell chamber is adaptable to the experimental requirements: microchannels can vary from simple linear connections to complex topologies with feature sizes as small as 2 &mu;m. Our modular EcoChip flow cell array features dozens of identical chambers and flow control by a gravity-driven flow module. We demonstrate that through use of EcoChip devices, physical structures and pressure heads can be held constant or varied systematically while the influence of surface chemistry, fluid properties, or the characteristics of the microbial population is investigated. Through transport experiments using a non-pathogenic, green fluorescent protein-expressing Vibrio bacterial strain, we illustrate the importance of habitat structure, flow conditions, and inoculums size on fundamental transport phenomena, and with real-time particle-scale observations, demonstrate that microfluidics offer a compelling view of a hidden world.

Microfluidic devices can be used to visualize complex natural processes in real time and at the appropriate physical scales. We have developed a simple microfluidic device that mimics key features of natural porous media for studying growth and transport of bacteria in the subsurface.

Microbial growth and transport in porous media have important implications for the quality of groundwater and surface water, the recycling of nutrients in ...

Biology

Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial distribution and activities of community members. We have developed a microwell array platform that can be used to rapidly assemble and track thousands of bacterial communities in parallel. This protocol highlights the utility of the platform and describes its use for optically monitoring the development of simple, two-member communities within an ensemble of arrays within the platform. This demonstration uses two mutants of Pseudomonas aeruginosa, part of a series of mutants developed to study Type VI secretion pathogenicity. Chromosomal inserts of either mCherry or GFP genes facilitate the constitutive expression of fluorescent proteins with distinct emission wavelengths that can be used to monitor community member abundance and location within each microwell. This protocol describes a detailed method for assembling mixtures of bacteria into the wells of the array and using time-lapse fluorescence imaging and quantitative image analysis to measure the relative growth of each member population over time. The seeding and assembly of the microwell platform, the imaging procedures necessary for the quantitative analysis of microbial communities within the array, and the methods that can be used to reveal interactions between microbial species area all discussed.

The development of microbial communities depends on a combination of factors, including environmental architecture, member abundance, traits, and interactions. This protocol describes a synthetic, microfabricated environment for the simultaneous tracking of thousands of communities contained in femtoliter wells, where key factors such as niche size and confinement can be approximated.

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial ...

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

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic cooperation, competition, or predation. The study of biofilms is important in many biological fields, including environmental science, food production, and medicine. However, few studies have focused on such bacterial biofilms, partially due to the difficulty of investigating them. Most of the methods for qualitative and quantitative biofilm analyses described in the literature are only suitable for biofilms forming on abiotic surfaces or on homogeneous and thin biotic surfaces, such as a monolayer of epithelial cells.
While laser scanning confocal microscopy (LSCM) is often used to analyze in situ and in vivo biofilms, this technology becomes very challenging when applied to bacterial biofilms on fungal hyphae, due to the thickness and the three dimensions of the hyphal networks. To overcome this shortcoming, we developed a protocol combining microscopy with a method to limit the accumulation of hyphal layers in fungal colonies. Using this method, we were able to investigate the development of bacterial biofilms on fungal hyphae at multiple scales using both LSCM and scanning electron microscopy (SEM). This report describes the protocol, including microorganism cultures, bacterial biofilm formation conditions, biofilm staining, and LSCM and SEM visualizations.

This protocol describes a new method to grow and qualitatively analyze bacterial biofilms on fungal hyphae by confocal and electron microscopy.

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic ...

Immunology and Infection

High Throughput Co-culture Assays for the Investigation of Microbial Interactions

The study of interactions between microorganisms has led to numerous discoveries, from novel antimicrobials to insights in microbial ecology. Many approaches used for the study of microbial interactions require specialized equipment and are expensive and time intensive. This paper presents a protocol for co-culture interaction assays that are inexpensive, scalable to large sample numbers, and easily adaptable to numerous experimental designs. Microorganisms are cultured together, with each well representing one pairwise combination of microorganisms. A test organism is cultured on one side of each well and first incubated in monoculture. Subsequently, target organisms are simultaneously inoculated onto the opposite side of each well using a 3D-printed inoculation stamp. After co-culture, the completed assays are scored for visual phenotypes, such as growth or inhibition. These assays can be used to confirm phenotypes or identify patterns among isolates of interest. Using this simple and effective method, users can analyze combinations of microorganisms rapidly and efficiently. This co-culture approach is applicable to antibiotic discovery as well as culture-based microbiome research and has already been successfully applied to both applications.

The co-culture interaction assays presented in this protocol are inexpensive, high throughput, and simple. These assays can be used to observe microbial interactions in co-culture, identify interaction patterns, and characterize the inhibitory potential of a microbial strain of interest against human and environmental pathogens.

The study of interactions between microorganisms has led to numerous discoveries, from novel antimicrobials to insights in microbial ecology. Many ...

Live-cell Imaging of Fungal Cells to Investigate Modes of Entry and Subcellular Localization of Antifungal Plant Defensins

Small cysteine-rich defensins are one of the largest groups of host defense peptides present in all plants. Many plant defensins exhibit potent in vitro antifungal activity against a broad-spectrum of fungal pathogens and therefore have the potential to be used as antifungal agents in transgenic crops. In order to harness the full potential of plant defensins for diseases control, it is crucial to elucidate their mechanisms of action (MOA). With the advent of advanced microscopy techniques, live-cell imaging has become a powerful tool for understanding the dynamics of the antifungal MOA of plant defensins. Here, a confocal microscopy based live-cell imaging method is described using two fluorescently labeled plant defensins (MtDef4 and MtDef5) in combination with vital fluorescent dyes. This technique enables real-time visualization and analysis of the dynamic events of MtDef4 and MtDef5 internalization into fungal cells. Importantly, this assay generates a wealth of information including internalization kinetics, mode of entry and subcellular localization of these peptides. Along with other cell biological tools, these methods have provided critical insights into the dynamics and complexity of the MOA of these peptides. These tools can also be used to compare the MOA of these peptides against different fungi.

Plant defensins play an important role in plant defense against pathogens. For effective use of these antifungal peptides as antifungal agents, understanding their modes of action (MOA) is critical. Here, a live-cell imaging method is described to study critical aspects of the MOA of these peptides.

Small cysteine-rich defensins are one of the largest groups of host defense peptides present in all plants. Many plant defensins exhibit potent in vitro ...

Single-cell Microfluidic Analysis of Bacillus subtilis

Microfluidic technology overcomes many of the limitations to traditional analytical methods in microbiology. Unlike bulk-culture methods, it offers single-cell resolution and long observation times spanning hundreds of generations; unlike agarose pad-based microscopy, it has uniform growth conditions that can be tightly controlled. Because the continuous flow of growth medium isolates the cells in a microfluidic device from unpredictable variations in the local chemical environment caused by cell growth and metabolism, authentic changes in gene expression and cell growth in response to specific stimuli can be more confidently observed. Bacillus subtilis is used here as a model bacterial species to demonstrate a &#34;mother machine&#34;-type method for cellular analysis. We show how to construct and plumb a microfluidic device, load it with cells, initiate microscopic imaging, and expose cells to a stimulus by switching from one growth medium to another. A stress-responsive reporter is used as an example to reveal the type of data that may be obtained by this method. We also briefly discuss further applications of this method for other types of experiments, such as analysis of bacterial sporulation.

Single-cell Microfluidic Analysis of Bacillus subtilis

We present a method for the microfluidic analysis of individual bacterial cell lineages using Bacillus subtilis as an example. The method overcomes shortcomings of traditional analytical methods in microbiology by allowing observation of hundreds of cell generations under tightly controllable and uniform growth conditions.

Microfluidic technology overcomes many of the limitations to traditional analytical methods in microbiology. Unlike bulk-culture methods, it offers ...

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

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