Name: real-time imaging and quantification of fungal biofilm development using a two-phase recirculating flow system
Uploaded: 2018-10-18T15:00:00
Description: Watch this Scientific Journal Video about Real-time Imaging and Quantification of Fungal Biofilm Development Using a Two-Phase Recirculating Flow System at JoVE.com

The authors would like to acknowledge Dr. Wade Sigurdson for providing valuable input in the design of the flow apparatus.

1. Assemble the Flow Apparatus
<ol>
	<li>Configure the parts listed in the Table of Materials according to the schematic in Figure 1 with the considerations discussed below. 
	NOTE: For convenience, the flow apparatus is divided into two sides, the green side (everything upstream of the slide to the media flasks), and the orange side (everything downstream of the slide to the media flasks).
	<ol>
		<li>Ensure that all of the flow apparatus is air tight to prevent leak...

<ol>
	<li>Pankhurst, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candidiasis+(oropharyngeal).">Candidiasis (oropharyngeal).</a> BMJ clinical evidence. 2012, 1304 (2012).</li><li>Ramage, G., Vandewalle, K., Wickes, B. L., L&#243;pez-Ribot, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+biofilm+formation+by+Candida+albicans.">Characteristics of biofilm formation by Candida albicans.</a> Revista iberoamericana de micolog&#237;a. 18 (4), 163-170 (2001).</li><li>Nobile, C. J., Mitchell, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+cell-surface+genes+and+biofilm+formation+by+the+C.+albicans+transcription+factor+Bcr1p.">Regulation of cell-surface genes and biofilm formation by the C. albicans transcription factor Bcr1p.</a> Current biology: CB. 15 (12), 1150-1155 (2005).</li><li>Blankenship, J. R., Mitchell, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+build+a+biofilm:+a+fungal+perspective.">How to build a biofilm: a fungal perspective.</a> Current opinion in microbiology. 9 (6), 588-594 (2006).</li><li>Ara&#250;jo, D., Henriques, M., Silva, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Portrait+of+Candida+Species+Biofilm+Regulatory+Network+Genes.">Portrait of Candida Species Biofilm Regulatory Network Genes.</a> Trends in microbiology. 25 (1), 62-75 (2017).</li><li>Lane, W. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel-plate+flow+chamber+and+continuous+flow+circuit+to+evaluate+endothelial+progenitor+cells+under+laminar+flow+shear+stress.">Parallel-plate flow chamber and continuous flow circuit to evaluate endothelial progenitor cells under laminar flow shear stress.</a> Journal of visualized experiments. (59), e3349 (2012).</li><li>Bakker, D. P., van der Plaats, A., Verkerke, G. J., Busscher, H. J., van der Mei, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+velocity+profiles+for+different+flow+chamber+designs+used+in+studies+of+microbial+adhesion+to+surfaces.">Comparison of velocity profiles for different flow chamber designs used in studies of microbial adhesion to surfaces.</a> Applied and environmental microbiology. 69 (10), 6280-6287 (2003).</li><li>Zhang, W., Sileika, T. S., Chen, C., Liu, Y., Lee, J., Packman, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+planar+flow+cell+for+studies+of+biofilm+heterogeneity+and+flow-biofilm+interactions.">A novel planar flow cell for studies of biofilm heterogeneity and flow-biofilm interactions.</a> Biotechnology and bioengineering. 108 (11), 2571-2582 (2011).</li><li>Uppuluri, P., Lopez-Ribot, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+easy+and+economical+in+vitro+method+for+the+formation+of+Candida+albicans+biofilms+under+continuous+conditions+of+flow.">An easy and economical in vitro method for the formation of Candida albicans biofilms under continuous conditions of flow.</a> Virulence. 1 (6), 483-487 (2010).</li><li>Diaz, P. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergistic+interaction+between+Candida+albicans+and+commensal+oral+streptococci+in+a+novel+in+vitro+mucosal+model.">Synergistic interaction between Candida albicans and commensal oral streptococci in a novel in vitro mucosal model.</a> Infection and immunity. 80 (2), 620-632 (2012).</li><li>McCall, A., Edgerton, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-Time+Approach+to+Flow+Cell+Imaging+of+Candida+albicans+Biofilm+Development.">Real-Time Approach to Flow Cell Imaging of Candida albicans Biofilm Development.</a> Journal of fungi. 3 (1), 13 (2017).</li><li>Zhang, B., Zerubia, J., Olivo-Marin, J. -. 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Using the flow system as outlined above allows for the generation of quantitative time-lapse videos of fungal biofilm growth and development. To allow for comparisons between experiments it is of critical importance to ensure that the imaging parameters are kept the same. This includes ensuring that the microscope is set up for K&#246;hler illumination for each experiment (many guides are available online for this process). Aside from imaging parameters, there are some important steps to keep in mind when working with th...

Candida albicans (C. albicans) is an opportunistic fungal pathogen of humans that can infect many tissue types, including oral mucosal surfaces, causing oropharyngeal candidiasis and resulting in a lower quality of life for affected individuals1. Biofilm formation is an important characteristic for the pathogenesis of C. albicans, and numerous studies have been done on the formation and function of C. albicans biofilms2,3,4,5, many of which have been conducted using static (no flow) in vitro models. However, C. albicans must adhere and grow in the presence of salivary flow in the oral cavity. Numerous flow systems have been developed to allow for live-cell imaging6,7,8,9,10. These different flow systems have been designed for different purposes, and therefore each system has different strengths and weaknesses. We found that many of the flow systems appropriate for C. albicans were costly, required complex fabricated parts, or could not be imaged during flow and had to be stopped prior to imaging. Therefore, we developed a novel flow apparatus to study C. albicans biofilm formation under flow11. During the design of our flow apparatus, we followed these major considerations. First, we wanted to be able to quantify multiple aspects of the biofilm growth and development in real-time without requiring the use of fluorescent cells (allowing us to study mutant strains and unmodified clinical isolates easily). Second, we wanted all parts to be commercially available with little to no modifications (i.e., no custom fabrication), allowing others to more easily recreate our system, and allowing for easy repairs. Third, we also wanted to allow for extended imaging times at reasonably high flow rates. Lastly, we wanted, following a period of cells attaching to the substrate under flow, to be able to monitor the biofilm growth over an extended time without introducing new cells.
These considerations led us to develop the two-flask recirculating flow system illustrated in Figure 1. The two flasks allow us to split the experiment into two phases, an attachment phase that draws from the cell-seeded attachment flask, and a growth phase that uses cell-free media to continue the biofilm growth without the addition of new cells. This system is designed to work with an incubation chamber for the microscope, with the slide and the tubing preceding it (2 to 5,&#160;Figure 1) being placed inside the incubator, and all other components placed in a large secondary container outside the microscope. Additionally, a hotplate stirrer with an attached temperature probe is used to maintain fungal cells in the attachment flask at 37 &#176;C. As it is recirculating, this system is capable of continuous imaging during flow (can be over 36 h depending on conditions), and can be used on most standard microscopes, including upright or inverted benchtop microscopes. Here, we discuss the assembly, operation, and cleaning of the flow apparatus, as well as provide some basic ImageJ quantitative algorithms to analyze the videos after an experiment.

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	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pump</td>
			<td style="width:71px;">Cole Parmer</td>
			<td style="width:119px;">07522-20</td>
			<td style="width:229px;">6</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pump head</td>
			<td style="width:71px;">Cole Parmer</td>
			<td style="width:119px;">77200-60</td>
			<td>6</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tubing</td>
			<td style="width:71px;">Cole Parmer</td>
			<td style="width:119px;">96410-14</td>
			<td>N/A</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Bubble trap adapter</td>
			<td style="width:71px;">Cole Parmer</td>
			<td style="width:119px;">30704-84</td>
			<td>3</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Bubble trap vacuum adapter for 1/4&#8221; ID vacuum line</td>
			<td style="width:71px;">Cole Parmer</td>
			<td style="width:119px;">31500-55</td>
			<td>3</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">In-line filter adapter (4 needed)</td>
			<td style="width:71px;">Cole Parmer</td>
			<td style="width:119px;">31209-40</td>
			<td>8,9</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Orange-side Y</td>
			<td style="width:71px;">Cole Parmer</td>
			<td style="width:119px;">31209-55</td>
			<td>7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Green-side Y</td>
			<td style="width:71px;">ibidi</td>
			<td style="width:119px;">10827</td>
			<td>2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">* Slides</td>
			<td style="width:71px;">ibidi</td>
			<td style="width:119px;">80196</td>
			<td>4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">* Slide luers</td>
			<td style="width:71px;">ibidi</td>
			<td style="width:119px;">10802</td>
			<td>4</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Vacuum assisted Bubble trap</td>
			<td style="width:71px;">Elveflow/Darwin microfluidics</td>
			<td style="width:119px;">KBTLarge - Microfluidic Bubble Trap Kit</td>
			<td>3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Media flasks</td>
			<td style="width:71px;">Corning</td>
			<td style="width:119px;">4980-500</td>
			<td>1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.2 &#181;m air filter</td>
			<td style="width:71px;">Corning</td>
			<td style="width:119px;">431229</td>
			<td>1</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Threaded glass bottle for PD and filter flask (2 needed)</td>
			<td style="width:71px;">Corning</td>
			<td style="width:119px;">1395-100</td>
			<td>5,10</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ported Screw cap for PD and filter flask (2 needed)</td>
			<td style="width:71px;">Wheaton</td>
			<td style="width:119px;">1129750</td>
			<td>5,10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Screwcap tubing connector</td>
			<td style="width:71px;">Wheaton</td>
			<td style="width:119px;">1129814</td>
			<td>5,10</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tubing connector beveled washer</td>
			<td style="width:71px;">Danco</td>
			<td style="width:119px;">88579</td>
			<td>5,10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tubing connector flat washer</td>
			<td style="width:71px;">Danco</td>
			<td style="width:119px;">88569</td>
			<td>5,10</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Clamps for in-line filters and downstream Y (7 needed)</td>
			<td style="width:71px;">Oetiker/MSC Industrial Supply Company</td>
			<td style="width:119px;">15100002-100</td>
			<td>7,8,9</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Clamp tool</td>
			<td style="width:71px;">Oetiker/MSC Industrial Supply Company</td>
			<td style="width:119px;">14100386</td>
			<td>N/A</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">20 micron in-line media filter</td>
			<td style="width:71px;">Analytical Scientific Instruments</td>
			<td style="width:119px;">850-1331</td>
			<td>8</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">10 micron in-line media filter</td>
			<td style="width:71px;">Analytical Scientific Instruments</td>
			<td style="width:119px;">850-1333</td>
			<td>9</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">2 micron inlet media filter</td>
			<td style="width:71px;">Supelco/Sigma-Aldrich</td>
			<td style="width:119px;">58267</td>
			<td>10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">* 0.22 &#181;m media filter</td>
			<td style="width:71px;">Millipore</td>
			<td style="width:119px;">SVGV010RS</td>
			<td>11</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">* 0.22 &#181;m media filter &#8220;adapter&#8221;</td>
			<td style="width:71px;">BD Biosciences</td>
			<td style="width:119px;">329654</td>
			<td>11</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Rubber stopper</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">14-131E</td>
			<td>1</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Hotplate stirrer with external probe port</td>
			<td style="width:71px;">ThermoFisher Scientific</td>
			<td style="width:119px;">88880006</td>
			<td>N/A</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Temperature probe</td>
			<td style="width:71px;">ThermoFisher Scientific</td>
			<td style="width:119px;">88880147</td>
			<td>N/A</td>
		</tr>
	</tbody>
</table>

real-time imaging and quantification of fungal biofilm development using a two-phase recirculating flow system

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. While models for the growth under flow have been developed, many of these systems are expensive, or do not allow imaging while the cells are under flow. We have developed a novel apparatus that allows us to image the growth and development of Candida albicans cells under flow and in real-time. Here, we detail the protocol for the assembly and use of this flow apparatus, as well as the quantification of data that are generated. We are able to quantify the rates that the cells attach to and detach from the slide, as well as to determine a measure of the biomass on the slide over time. This system is both economical and versatile, working with many types of light microscopes, including inexpensive benchtop microscopes, and is capable of extended imaging times compared to other flow systems. Overall, this is a low-throughput system that can provide highly detailed real-time information on the biofilm growth of fungal species under flow.

Representative images of a normal overnight time-lapse experiment using wild-type C. albicans cells at 37 &#176;C can be seen in Figure 2A and Supplemental Video 1. The images have been contrast enhanced to improve visibility. Quantification of the original data was performed, and representative graphs can be seen in Figure 2B. To generate these graphs, the data were fir...

Watch this Scientific Journal Video about Real-time Imaging and Quantification of Fungal Biofilm Development Using a Two-Phase Recirculating Flow System at JoVE.com

Real-time Imaging and Quantification of Fungal Biofilm Development Using a Two-Phase Recirculating Flow System

We describe the assembly, operation, and cleaning of a flow apparatus designed to image fungal biofilm formation in real time while under flow. We also provide and discuss quantitative algorithms to be used on the acquired images.

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. ...

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