Name: visualization of biofilm formation in candida albicans using an automated microfluidic device
Uploaded: 2017-12-14T15:00:00
Description: Watch this Scientific Journal Video about Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device at JoVE.com

We thank all members of the Nobile lab for helpful discussions on biofilm assays. This study was supported by National Institutes of Health (NIH) grant R21 AI125801 (to C.J.N.). D.L.R. was supported by a doctoral fellowship from The University of California Institute for Mexico and the United States (UC-MEXUS) and Consejo Nacional de Ciencia y Technologia (CONACYT).

1. Fungal Cell Culture Preparation
NOTE: Conduct cell culture work (i.e. opening cryogenic stock tubes, cell culture tubes, and flasks) within a biosafety cabinet. Turn on the cabinet&#39;s ultraviolet (UV) germicidal lamp at least 1 h prior to work, and turn off the UV lamp while actively working in the cabinet. Wear gloves, safety glasses, and appropriate personal protective equipment, and decontaminate the surface of the bench and pipettes with 70% ethanol prior to the start of the experiment...

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The customizable microfluidic biofilm assay described here allows for the visualization of biofilm formation in real-time at a single cell level when exposed to a fixed rate laminar flow and constant temperature. It provides a powerful means to study the development of biofilms in wild-type and mutant strains, and the effects of antimicrobial agent treatments on biofilms under conditions that mimic physiological conditions observed in clinical settings. Unlike most in vitro biofilm assays, this method allows for...

Candida albicans is a commensal member of the human microbiota, however it is also an opportunistic pathogen, capable of causing superficial and severe fungal infections1,2. A major virulence trait of C. albicans is its ability to form resilient and drug resistant biofilms, communities of cells adhered to a surface and enclosed in an extracellular matrix material1,3. C. albicans biofilms are highly structured, containing several layers of multiple cell types (round budding yeast-form cells, oval pseudohyphal cells, and tubular hyphal cells)4. C. albicans biofilm development begins with the adherence of round yeast-form cells to a surface (seeding the biofilm), followed by the proliferation of these cells on the surface, and then the maturation of the immature biofilm structure into a fully-formed biofilm that is surrounded by extracellular matrix material4. The mature biofilm is predominantly composed of elongated hyphal cells that form dense and interconnecting networks, providing the architectural stability to the biofilm4. Throughout the biofilm life cycle, round budding yeast cells disperse from the mature biofilm, and may travel to other regions of the body to cause disseminated infections or seed new biofilms at other sites4,5. C. albicans can form biofilms on biotic surfaces, such as mucosal surfaces and throughout host tissue, and on abiotic surfaces, such as catheters, pacemakers, dentures, and prosthetic joints. Due to the recalcitrant properties of biofilms, they are extremely difficult to eradicate, and in many cases the only effective treatment strategy is removal of the infected device4. It is thus crucial to investigate biofilm formation under conditions similar to those observed in clinical settings.
There are several critical in vivo animal models used to study C. albicans biofilm formation6,7,8; however, these studies can be costly, time consuming, and are limited by the number of strains and antimicrobial agents that can be tested at a given time. In vitro biofilm assays, on the other hand, allow for the rapid, high-throughput assessment of antifungal compounds and mutant strains, and are much more cost-effective and ethical than biofilm assays carried out in animal models9,10,11,12,13,14. Here we describe an in vitro assay that we developed and optimized to observe biofilm formation temporally under laminar flow using a customizable microfluidic device14,15. The assay allows for the visualization of each stage of biofilm formation, including the initial adherence step, cell proliferation, biofilm maturation, and cell dispersion. The assay is also useful to visualize cell morphology changes throughout the development of a biofilm.
Microtiter plates, which are typically utilized for in vitro biofilm assays, while high throughput, do not allow for controlled flow conditions. Traditional laminar flow cell systems allow for the continuous assessment of biofilm formation in controlled flow conditions, but these are often time consuming to set up and tend to have limited dynamic range control and throughput. The microfluidic device utilized here overcomes these limitations by combining high throughput plates (containing 48 wells) with a built-in laminar flow chamber and is highly reproducible, versatile, and customizable.
Here, we describe a protocol for the use of a commercially available automated microfluidic device to assess biofilm formation of a wild-type C. albicans strain, the effects of a known antifungal agent on the development of a biofilm, and biofilm formation in two mutant strains (bcr1&#916;/&#916; and efg1 &#916;/&#916;) that were previously reported to have biofilm defects in vitro and in vivo16,17,18. The described protocol can be used to test the efficacy of antimicrobial agents in inhibiting biofilm formation throughout the development of a biofilm, and to identify genes required for normal biofilm development by screening mutant libraries.

<table>
	<tbody>
		<tr>
			<td>BioFlux 1000z</td>
			<td>Fluxion</td>
			<td></td>
			<td>Automated microfluidic device for live cell analysis</td>
		</tr>
		<tr>
			<td>48-well plate 0-20 dyne</td>
			<td>Fluxion</td>
			<td>910-0047</td>
			<td>Microfluidic plate</td>
		</tr>
		<tr>
			<td>Montage Software</td>
			<td>Fluxion</td>
			<td>Version 7.8.4.0</td>
			<td>Visualization analysis software</td>
		</tr>
		<tr>
			<td>ImageJ Software</td>
			<td>NIH</td>
			<td>https://imagej.nih.gov/ij/</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td>Criterion</td>
			<td>C7341</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Peptone</td>
			<td>BD Biosciences</td>
			<td>211677</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextrose (D-Glucose)</td>
			<td>Fisher Scientific</td>
			<td>D163</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate Monobasic</td>
			<td>Fisher Scientific</td>
			<td>P285-500</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>Sigma-Aldrich</td>
			<td>R6504</td>
			<td></td>
		</tr>
		<tr>
			<td>MOPS</td>
			<td>Sigma-Aldrich</td>
			<td>M3183</td>
			<td></td>
		</tr>
		<tr>
			<td>Nutrient Broth</td>
			<td>Criterion</td>
			<td>C6471</td>
			<td></td>
		</tr>
		<tr>
			<td>Difco D-Mannitol</td>
			<td>BD Biosciences</td>
			<td>217020</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Criterion</td>
			<td>C5001</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Corning</td>
			<td>30-003-CF</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Inoculating Loops</td>
			<td>VWR</td>
			<td>30002-094</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dishes with Clear Lid</td>
			<td>Fisher Scientific</td>
			<td>FB0875712</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Cuvettes</td>
			<td>Fisher Scientific</td>
			<td>14-955-127</td>
			<td></td>
		</tr>
		<tr>
			<td>Lens Paper</td>
			<td>VWR</td>
			<td>52846-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate and Cuvette Spectrophotometer</td>
			<td>BioTek</td>
			<td>EPOCH2TC</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaking Incubator</td>
			<td>Eppendorf</td>
			<td>M12820004</td>
			<td></td>
		</tr>
	</tbody>
</table>

visualization of biofilm formation in candida albicans using an automated microfluidic device

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. albicans is its ability to form biofilms, structured communities of cells attached to biotic and abiotic surfaces. C. albicans biofilms can form on host tissues, such as mucosal layers, and on medical devices, such as catheters, pacemakers, dentures, and joint prostheses. Biofilms pose significant clinical challenges because they are highly resistant to physical and chemical perturbations, and can act as reservoirs to seed disseminated infections. Various in vitro assays have been utilized to study C. albicans biofilm formation, such as microtiter plate assays, dry weight measurements, cell viability assays, and confocal scanning laser microscopy. All of these assays are single end-point assays, where biofilm formation is assessed at a specific time point. Here, we describe a protocol to study biofilm formation in real-time using an automated microfluidic device under laminar flow conditions. This method allows for the observation of biofilm formation as the biofilm develops over time, using customizable conditions that mimic those of the host, such as those encountered in vascular catheters. This protocol can be used to assess the biofilm defects of genetic mutants as well as the inhibitory effects of antimicrobial agents on biofilm development in real-time.

We performed the microfluidic biofilm assay described here using a wild-type C. albicans strain under two media conditions (RPMI-1640 and Spider media), the wild-type strain in the presence of the known antifungal drug amphotericin B (16 &#181;g/mL) in RPMI, and two mutant strains previously reported to have defects in biofilm formation (bcr1&#916;/&#916; and efg1 &#916;/&#916;) in Spider media.
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Watch this Scientific Journal Video about Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device at JoVE.com

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

This protocol describes the use of a customizable automated microfluidic device to visualize biofilm formation in Candida albicans under host physiological conditions.

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. ...

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