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