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Abstract

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Protocol

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Materials

References

Immunology and Infection

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

Published: October 18th, 2018

DOI:

10.3791/58457

1Department of Oral Biology, University at Buffalo

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. 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.

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)....

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1. Assemble the Flow Apparatus

  1. 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).
    1. Ensure that all of the flow apparatus is air tight to prevent leak.......

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Representative images of a normal overnight time-lapse experiment using wild-type C. albicans cells at 37 °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.......

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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 Köhler 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.......

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

....

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

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