Name: Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells (Video) | JoVE Journal
Uploaded: 2011-01-15T17:00:00.000+00:00
Duration: 17 min 30 s
Description: Learn how to set up a flow cell system for microbial biofilm analysis using Pseudomonas aeruginosa and Saccharomyces cerevisiae. This protocol demonstrates proper assembly, sealing, and visualization with confocal microscopy.

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5 minutes

1. Assembly of the Flow Cell System with All Components
The assembled flow system includes: autoclavable tubing, bubble traps, medium/waste bottle and flow cells as shown in Figure 1. All these parts can be reused between experiments.
<img src="/files/ftp_upload/2383/2383fig1.jpg" alt="Continuous flow system diagram with pump, tubing, bubble traps, and channels for media transfer." data-altupdatedat="1788235205000" data-alt="Figure 1"> 
Figure 1. The flow cell system setup (essential components of the setup). The flow cell system consists of several components: a medium bottle, a peristaltic pump, bubble traps, the flow cell, a waste bottle, and diverse sections of tubing interconnected by various connectors. Figure kindly provided by Rune Lyngklip.
2. Assembly of the Flow Cell
<ol>
<li>The flow cell (Figure 2a) is treated with thin lanes of silicone glue, using a syringe (Figure 3).</li>
<li>Place a cover slip on top of the silicone lines (Figure 3). Glass cover slips are used as substratum for P. aeruginosa while PVC cover slips are applied for S. cerevisiae biofilm.</li>
</ol>
<img src="/files/ftp_upload/2383/2383fig2.jpg" alt="Microfluidic chip design diagrams with dimensions, illustrating experimental setup for fluid control." data-altupdatedat="1788235205000" data-alt="Figure 2"> 
Figure 2. Schematic drawing of flow cell and bubble trap2. Detailed description of the dimensions used for the production of a) flow cell b) bubble trap, DTU Systems Biology. Reprinted with permission of John Wiley &amp; Sons, Inc. (DTU Systems Biology was formerly entitled Biocentrum, as depicted in the figure)
<img src="/files/ftp_upload/2383/2383fig3.jpg" alt="Battery assembly diagram showing layered structure with cell arrangement process." data-altupdatedat="1788235205000" data-alt="Figure 3"> 
Figure 3. Illustration of the silicone glue application lines for attachment of the glass substratum. The indicated cover glass is placed over the silicone glue to attach it to the flow cell.
<ol start="3">
<li>Turn the flow cell over and place it on a flat surface with the cover slip side down. Press gently on the back of the flow cell to push the cover slip onto the base of the flow cell. Turn the flow cell over and inspect for areas that are not sealed by the silicone. The handle (piston) part of a syringe can be used as a tool to gently press the glass and flow cell together.</li>
</ol>
3. Medium Bottle
<ol>
<li>Place a feeding silicone tube (2 mm inner diameter) in a medium bottle and insert a straight connector at the other end.</li>
<li>Add medium to the bottle (for medium content see "media" paragraph) 
Cover connector and bottle with metal foil and autoclave the medium. Make sure that the end of the supply tube is fixed above the liquid level in the medium bottle or a siphon effect may empty the medium bottle when autoclaving.</li>
</ol>
4. Connecting the Bubble Trap, Flow Cell and Pump
Assemble all tubing according to the outline in Figure 1. Use silicone tubing except for the part that goes through the peristaltic pump where Marprene tubing is applied.
<ol>
<li>In order to connect the tube from the medium bottle to all individual flow chambers, split a tube into the required number of inlets applied in the experiment. Use T-connectors to make the desirable number of connection tubes from the feed tube to the Marprene tubes in the pump (see Figure 1, T-connector). It is generally a good idea to keep the same sequence order of tubes and flow cells throughout the system, to facilitate identification of components in case of faults in the system.</li>
<li>Connect each individual feeding tube (2 mm in diameter) to Marprene tubes in the pump using straight connectors. Connect the Marprene tubing to a bubble trap (Figure 2b) via intermediate silicone tubing (1 mm in diameter). Make sure that the pump is connected to the inlet at the tallest part of the bubble trap.</li>
<li>Connect the resulting outlet tube of the bubble traps to the flow cell inlet (1 mm in diameter), make sure that the length of these tubings allows the flow cell to be moved to the stage of the confocal microscope (typically 1 m).</li>
<li>Place 5 mL syringes on top of the bubble traps. Close the tops with suitable caps.</li>
<li>On the flow cell outlet connect a short, approximately 40 mm piece of (1 mm in diameter), tubing and use a "reducing" straight connector to attach a waste tube (2 mm in diameter) of needed length. Place waste tubes in the waste container.</li>
<li>Importantly, the waste container must always be placed at the same level as the flow cells, never below flow-cell level. Also, make sure that the end of the waste tubing is fixed above the expected level of waste liquid to avoid flush-back due to a siphon effect when handling the flow cells.</li>
</ol>
5. STERILIZING AND WASHING THE FLOW SYSTEM
<ol>
<li>Remove bubble trap caps and place them in 70% ethanol to keep them sterile.</li>
<li>Run at highest pump speed to fill the system with 0.5% (v/v) sodium hypochlorite in water.</li>
<li>Place the bubble trap caps back on when the bubble traps are completely filled.</li>
<li>Tap the flow cells to remove bubbles in the flow chamber. Take care not to damage the fragile cover glass.</li>
<li>Allow the system to sterilize for 3-4 h at a flow rate of 3 mL/h/channel (0.2 mm/s linear flow rate).</li>
<li>Wash the system 2-3 times to wash out all the hypochlorite. Fill and empty the system with sterile water. Bubble traps must be emptied completely between each wash. This can be done by pumping in air until bubble traps have been emptied. After emptying, remove caps from the bubble traps before refilling the system. Replace caps after the bubble traps have been completely filled with liquid. Repeat as required.</li>
<li>Run sterile water through the system at a low flow rate (1-3 mL/h/channel) over night or proceed to the next step.</li>
<li>Connect the medium bottle to the inlet and flush the system with medium over night at low flow rate (3 mL/h/channel) at the temperature where the experiment will be performed. Note: bubble traps must be emptied for water before the system is filled with medium.</li>
</ol>
6. INOCULATION OF THE FLOW CELL
<ol>
<li>From an overnight culture make a dilution to a desired optical density (for P. aeruginosa e.g. 0.001 OD600nm and 0.1 OD600nm for S. cerevisiae).</li>
<li>Use a 0.5 mL syringe with a 27G needle to load enough inoculum to fill the chamber. 250 μL is sufficient for the flow chambers having the dimensions specified in this work (Figure 2., 40 mm x 4 mm x 1 mm).</li>
<li>Stop the peristaltic pump.</li>
<li>Clamp off the silicone tubing leading to the flow cell to prevent back flow into the system.</li>
<li>Sterilize the inoculation site on the silicone tubing by wiping it with 70% ethanol.</li>
<li>Insert the needle into the silicone tube and introduce the tip into the inlet of the flow cell. Slowly inject the inoculum into the chamber (be careful not to inject air bubbles).</li>
<li>Remove the needle and wipe the injection site with 70% ethanol followed by immediate sealing of the hole using silicone glue over the injection site.</li>
<li>Turn over the flow cell and let the micro-organisms adhere to the substratum for 1 hour without flow through the flow cell.</li>
<li>Turn the flow cell, start the medium pump (3 mL/h/channel) and take the clamp off the silicone tube.</li>
<li>The system is placed for incubation, at 37°C in the case of P. aeruginosa and 30°C in the case of S. cerevisiae.</li>
<li>Biofilm in the flow chambers can now be visualized by CLSM.</li>
</ol>
7. STAINING OF BIOFILM FOR MICROSCOPY
<ol>
<li>Make a dilution of the appropriate staining (e.g. 1:1000 Syto 9 live stain for S. cerevisiae)</li>
<li>Stop the peristaltic pump.</li>
<li>Clamp of the silicone tubing leading to the flow cell.</li>
<li>Sterilize inoculation site on the silicone tubing by wiping it with 70% ethanol.</li>
<li>Use a 0.5 mL syringe with a 27G needle to load enough staining solution to fill the chamber. 250 μL is sufficient for the flow chambers used here.</li>
<li>Insert the needle into the silicone tube and introduce the tip into the inlet of the flow cell. Slowly inject the staining solution into the chamber (be careful not to inject bubbles).</li>
<li>Remove the needle and wipe the injection site with 70% ethanol followed by immediate sealing of the injection site.</li>
<li>Leave the flow cell without flow for 15 minutes.</li>
<li>Take off the clamp and start the flow (3 mL/h/channel)</li>
<li>Acquire data with the CLSM</li>
</ol>

<ol><li>Costerton, J. W., Stewart, P. S., Greenberg, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bacterial+biofilms%3A+a+common+cause+of+persistent+infections%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Bacterial biofilms: a common cause of persistent infections.</a> Science. 284, 1318-1322 (1999).</li>
<li>Sternberg, C., Tolker-Nielsen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Growing+and+analyzing+biofilms+in+flow+cells%5BTitle%5D)+AND+%22Curr+Protoc+Microbiol%22%5BJournal%5D)">Growing and analyzing biofilms in flow cells.</a> Curr Protoc Microbiol. Chapter 1, 2-2 (2006).</li>
<li>Heydorn, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Experimental+reproducibility+in+flow-chamber+biofilms%5BTitle%5D)+AND+%22Microbiology%22%5BJournal%5D)">Experimental reproducibility in flow-chamber biofilms.</a> Microbiology. 146, 2409-2415 (2000).</li>
<li>Pamp, S. J., Tolker-Nielsen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multiple+roles+of+biosurfactants+in+structural+biofilm+development+by+Pseudomonas+aeruginosa%5BTitle%5D)+AND+%22J+Bacteriol%22%5BJournal%5D)">Multiple roles of biosurfactants in structural biofilm development by Pseudomonas aeruginosa.</a> J Bacteriol. 189, 2531-2539 (2007).</li>
<li>Haagensen, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differentiation+and+distribution+of+colistin-+and+sodium+dodecyl+sulfate-tolerant+cells+in+Pseudomonas+aeruginosa+biofilms%5BTitle%5D)+AND+%22J+Bacteriol%22%5BJournal%5D)">Differentiation and distribution of colistin- and sodium dodecyl sulfate-tolerant cells in Pseudomonas aeruginosa biofilms.</a> J Bacteriol. 189, 28-37 (2007).</li>
<li>Klausen, M., Aaes-Jorgensen, A., Molin, S., Tolker-Nielsen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Involvement+of+bacterial+migration+in+the+development+of+complex+multicellular+structures+in+Pseudomonas+aeruginosa+biofilms%5BTitle%5D)+AND+%22Mol+Microbiol%22%5BJournal%5D)">Involvement of bacterial migration in the development of complex multicellular structures in Pseudomonas aeruginosa biofilms.</a> Mol Microbiol. 50, 61-68 (2003).</li>
<li>Pamp, S. J., Gjermansen, M., Johansen, H. K., Tolker-Nielsen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tolerance+to+the+antimicrobial+peptide+colistin+in+Pseudomonas+aeruginosa+biofilms+is+linked+to+metabolically+active+cells%2C+and+depends+on+the+pmr+and+mexAB-oprM+genes%5BTitle%5D)+AND+%22Mol+Microbiol%22%5BJournal%5D)">Tolerance to the antimicrobial peptide colistin in Pseudomonas aeruginosa biofilms is linked to metabolically active cells, and depends on the pmr and mexAB-oprM genes.</a> Mol Microbiol. 68, 223-240 (2008).</li>
<li>Pamp, S. J., Sternberg, C., Tolker-Nielsen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Insight+into+the+microbial+multicellular+lifestyle+via+flow-cell+technology+and+confocal+microscopy%5BTitle%5D)+AND+%22Cytometry+Part+A%22%5BJournal%5D)">Insight into the microbial multicellular lifestyle via flow-cell technology and confocal microscopy.</a> Cytometry Part A. 75A, 90-103 (2009).</li>
<li>Barken, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Roles+of+type+IV+pili%2C+flagellum-mediated+motility+and+extracellular+DNA+in+the+formation+of+mature+multicellular+structures+in+Pseudomonas+aeruginosa+biofilms%5BTitle%5D)+AND+%22Environ+Microbiol%22%5BJournal%5D)">Roles of type IV pili, flagellum-mediated motility and extracellular DNA in the formation of mature multicellular structures in Pseudomonas aeruginosa biofilms.</a> Environ Microbiol. 10, 2331-2343 (2008).</li>
<li>Qin, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+autolysin-mediated+DNA+release+in+biofilm+formation+of+Staphylococcus+epidermidis%5BTitle%5D)+AND+%22Microbiology%22%5BJournal%5D)">Role of autolysin-mediated DNA release in biofilm formation of Staphylococcus epidermidis.</a> Microbiology. 153, 2083-2092 (2007).</li>
<li>Allesen-Holm, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+characterization+of+DNA+release+in+Pseudomonas+aeruginosa+cultures+and+biofilms%5BTitle%5D)+AND+%22Mol+Microbiol%22%5BJournal%5D)">A characterization of DNA release in Pseudomonas aeruginosa cultures and biofilms.</a> Mol Microbiol. 59, 1114-1128 (2006).</li>
<li>Heydorn, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantification+of+biofilm+structures+by+the+novel+computer+program+COMSTAT%5BTitle%5D)+AND+%22Microbiology%22%5BJournal%5D)">Quantification of biofilm structures by the novel computer program COMSTAT.</a> Microbiology. 146, 2395-2407 (2000).</li>
<li>COMSTAT2, a semi-automated java-based quantification program for the analysis of microbial biofilms [Internet]. , Available from: 
	 <a target="_blank" href="http://www.comstat.dk/">http://www.comstat.dk/</a> (2010).</li>
<li>Palmer, R. J., Haagensen, J. A., Neu, T. R., Sternberg, C. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aPalmer%2C+RJ+%22Handbook+of+Biological+Confocal+Microscopy%22">Handbook of Biological Confocal Microscopy</a>. Palmer, J. B. , Springer. 882-900 (2006).</li>
<li>Haagensen, J. A., Regenberg, B., Sternberg, C. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aHaagensen%2C+JA+%22High+Resolution+Microbial+Single+Cell+Analytics+Advances+in+Biochemical+Engineering+and+Biotechnology%22">High Resolution Microbial Single Cell Analytics Advances in Biochemical Engineering and Biotechnology</a>. Müller, S., Bley, T. , Springer. (2010).</li>
</ol>

No conflicts of interest declared.

We have demonstrated a flow cell system that represents a powerful tool in biofilm investigations. Combined with 3D imaging by confocal microscopy, the system has a range of advantages in comparison to other methods of analyzing microbial biofilms by means of more traditional microscopic techniques. This system allows 3D visualization of living microbial biofilm communities without disturbance of the community. Light microscopy will not provide detailed information about niches of the biofilm and while electron microscopy provides nanoscale resolution of the biofilm, it does not allow live cell imaging.
Using the described flow channel system we have previously elucidated the spatial distribution of bacterial cells sensitive to several antibiotics5-8 (Figure 4a), distribution of extracellular compounds, e.g. DNA9-11 and, the distribution of motile and non-motile cells of the same species within a bacterial community4,6,9 (Figure 4c). We envision that the flow cell system can be used to study aspects of yeast biofilms. This may be the spatio temporal distribution of yeast biofilm in response to environmental factors such as fungicides as well as identification of genes involved in yeast biofilm development. Though yeast is not known to differentiate into motile and non-motile cells, other aspects of biofilm diversification may be studies such as the morphological shift from yeast to pseudohyphal cells and the shift from haploid to diploid cells.
We have shown a system that comply with several microbial species and will work with several staining techniques. A variety of different staining probes and fluorescent proteins, such as GFP, enable specific niche investigations in the developing biofilm and is an efficient tool in analyzing the effect of antimicrobial agents or other environmental factors. The information that can be gained is very detailed (Figure 4) and features in the biofilm can be quantified with computer programs such as COMSTAT12,13.
Overall, the most critical aspect of the protocol is the fact that it is a time-consuming process. It is also a limitation that the cells need to be able to grow on a non-fluorescent, transparent surface. Since the biofilm formed is analyzed using a confocal microscope, the depth that can be investigated is limited to a few hundred micrometres14.There are further technical limitations inherent in the design: the system is not suited for high throughput screening, as an experienced researcher can handle at most about 15 channels per experiment, which in turn can take several days to prepare. However, antibiotics or mutants that are considered relevant for biofilm studies can initially be mass screened with other methods such as crystal violet staining before the most interesting candidates are transferred to the flow cell system. The cover glass sheets are very thin and break easily, and care should be taken when handling the systems. In addition the tubing should be examined daily during the run of an experiment; as considerable "back-growth" in the inlet tubes just upstream of the flow cells can occur. Such contamination can be solved by removing several centimeters of silicone tube from the inlet side of the flow cells, using sterile technique.
<img src="/files/ftp_upload/2383/2383fig4.jpg" alt="Biofilm growth comparison; 3D models and microscopy images; surface interaction study." data-altupdatedat="1788235205000" data-alt="Figure 4"> 
Figure 4. a) 4 day old PAO1 - GFP biofilm treated for 24h with Colistin and Propidium iodide for dead staining (red stain) b) 3D presentation of a three day old P. aeruginosa PAO1 (P. aeruginosa wild type) - GFP biofilm6 c) 3D picture presentation of a PAO1 - CFP pilA mutant (blue) with an PAO1 wild type YFP (yellow) d) 5 day old PAO1 - GFP biofilm presented as a 3D picture e) 26 h S. cerevisiae (PTR3 mutant in CEN.PK background) biofilm stained with Syto-915.

<table><tbody><tr><td colspan="4">&lt;ul&gt; &lt;li&gt;Bubble traps, polyethylene (DTU Systems Biology, Technical University of Denmark, http://www.csm.bio.dtu.dk/Instrument%20Center/Resources/Biofilm%20Setup.aspx )&lt;/li&gt; &lt;li&gt;Clear polypropylene plastic connectors and T-connectors (Cole Parmer, 1/8 in. (3.175 mm) and 1/16 in. (1.588 mm))&lt;/li&gt; &lt;li&gt;Clamps&lt;/li&gt; &lt;li&gt;Confocal microscope (Zeiss, Meta LSM510)&lt;/li&gt; &lt;li&gt;Coverslips, glass (Knittel Gl&amp;#228;ser) 50 x 24 mm&lt;/li&gt; &lt;li&gt;Coverslips, PVC coverslips (Rinzl) 50 x 24 mm&lt;/li&gt; &lt;li&gt;Flow cells, polyethylene (DTU Systems Biology, Technical University of Denmark, http://www.csm.bio.dtu.dk/Instrument%20Center/Resources/Biofilm%20Setup.aspx&lt;/li&gt; &lt;li&gt;Medium bottles (Schott)&lt;/li&gt; &lt;li&gt;Peristaltic Pump (Watson-Marlow, 205S)&lt;/li&gt; &lt;li&gt;Rolling cart for flow systems and pumps &lt;/li&gt; &lt;li&gt;Silicone glue (3M Super Silicone Sealant Clear)&lt;/li&gt; &lt;li&gt;Syringe 5 mL (Terumo)&lt;/li&gt; &lt;li&gt;Syto 9 (Molecular Probes)&lt;/li&gt; &lt;li&gt;0.5 mL Syringes with needles (27G, Terumo LU-100)&lt;/li&gt; &lt;li&gt;Waste container&lt;/li&gt;&lt;/ul&gt;&lt;p class=&quot;jove_title&quot;&gt;Tubing:&lt;/p&gt;&lt;ul&gt; &lt;li&gt;Silicone, 3 mm outer diameter, 1 mm inner diameter (Ole Dich)&lt;/li&gt; &lt;li&gt;Silicone, 4 mm outer diameter, 2 mm inner diameter (Ole Dich)&lt;/li&gt; &lt;li&gt;Marprene, 3 mm outer diameter, 1 mm inner diameter (Watson-Marlow)&lt;/li&gt;&lt;/ul&gt;&lt;p class=&quot;jove_title&quot;&gt;Media&lt;/p&gt;&lt;table border=&quot;0&quot;&gt; &lt;tbody&gt; &lt;tr&gt; &lt;td colspan=&quot;2&quot;&gt;&lt;strong&gt;&lt;em&gt;P. aeruginosa&lt;/em&gt; medium&lt;/strong&gt;&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;&lt;strong&gt;A10&lt;/strong&gt;&lt;/td&gt; &lt;td&gt;&lt;strong&gt;g/L&lt;/strong&gt;&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;(NH&lt;sub&gt;4&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;SO4&lt;/td&gt; &lt;td&gt;2.0&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; X 2H&lt;sub&gt;2&lt;/sub&gt;O&lt;/td&gt; &lt;td&gt;6.0&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;&lt;/td&gt; &lt;td&gt;3.0&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;NaCl&lt;/td&gt; &lt;td&gt;3.0&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;Autoclave&lt;/td&gt; &lt;td&gt;&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td colspan=&quot;2&quot;&gt;&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td colspan=&quot;2&quot;&gt;&lt;strong&gt;FB&lt;/strong&gt;&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;MgCl&lt;sub&gt;2&lt;/sub&gt; 6H&lt;sub&gt;2&lt;/sub&gt;O&lt;/td&gt; &lt;td&gt;0.20&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;1 mL 1 M CaCl&lt;sub&gt;2&lt;/sub&gt;&lt;/td&gt; &lt;td&gt;0.01&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td colspan=&quot;2&quot;&gt;100 &amp;#x03BC;L/L Trace metals (for &lt;em&gt;P. aeruginosa&lt;/em&gt;-biofilms)&lt;sup&gt;4&lt;/sup&gt;&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td colspan=&quot;2&quot;&gt;Autoclave&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td colspan=&quot;2&quot;&gt;Mix &lt;strong&gt;A10&lt;/strong&gt; and &lt;strong&gt;FB&lt;/strong&gt; in a ratio of 1:10.&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td colspan=&quot;2&quot;&gt;Add carbon source to a desired concentration.&lt;/td&gt; &lt;/tr&gt; &lt;/tbody&gt;&lt;/table&gt;&lt;br/&gt;&lt;table border=&quot;0&quot;&gt; &lt;tbody&gt; &lt;tr&gt; &lt;td colspan=&quot;2&quot;&gt;&lt;strong&gt;&lt;em&gt;S. cerevisiae&lt;/em&gt; synthetic complete (SC) medium&lt;/strong&gt;&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;&lt;/td&gt; &lt;td&gt;&lt;strong&gt;g/L&lt;/strong&gt;&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;Adenine sulfate&lt;/td&gt; &lt;td&gt;0.02&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;L-tryptophan&lt;/td&gt; &lt;td&gt;0.02&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;L-histidine-HCL&lt;/td&gt; &lt;td&gt;0.02&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;L-arginine-HCL&lt;/td&gt; &lt;td&gt;0.04&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;L-methionine&lt;/td&gt; &lt;td&gt;0.02&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;L-tyrosine&lt;/td&gt; &lt;td&gt;0.05&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;L-leucine&lt;/td&gt; &lt;td&gt;0.06&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;L-isoleucine&lt;/td&gt; &lt;td&gt;0.06&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;L-lysine-HCL&lt;/td&gt; &lt;td&gt;0.05&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;L-phenylalanine&lt;/td&gt; &lt;td&gt;0.05&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;L-aspartic acid&lt;/td&gt; &lt;td&gt;0.10&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;L-glutamic acid&lt;/td&gt; &lt;td&gt;0.10&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;L-valine&lt;/td&gt; &lt;td&gt;0.15&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;L-threonine&lt;/td&gt; &lt;td&gt;0.20&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;L-serine&lt;/td&gt; &lt;td&gt;0.40&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;Yeast Nitrogen base w/o amino acids and ammonium (Bacto)&lt;/td&gt; &lt;td&gt;1.6&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;Ammonium sulphate&lt;/td&gt; &lt;td&gt;5.0&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;NaOH&lt;/td&gt; &lt;td&gt;6.0&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;Succinic acid&lt;/td&gt; &lt;td&gt;10.0&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;Autoclave&lt;/td&gt; &lt;td&gt;&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;Glucose (autoclaved separately)&lt;/td&gt; &lt;td&gt;0.20&lt;/td&gt; &lt;/tr&gt; &lt;/tbody&gt;&lt;/table&gt;</td></tr></tbody></table>

pseudomonas aeruginosa and saccharomyces cerevisiae biofilm in flow cells

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological properties compared to free living microorganisms. Biofilms in nature are often difficult to investigate and reside under poorly defined conditions1. Using a transparent substratum it is possible to device a system where simple biofilms can be examined in a non-destructive way in real-time: here we demonstrate the assembly and operation of a flow cell model system, for in vitro 3D studies of microbial biofilms generating high reproducibility under well-defined conditions2,3.
The system consists of a flow cell that serves as growth chamber for the biofilm. The flow cell is supplied with nutrients and oxygen from a medium flask via a peristaltic pump and spent medium is collected in a waste container. This construction of the flow system allows a continuous supply of nutrients and administration of e.g. antibiotics with minimal disturbance of the cells grown in the flow chamber. Moreover, the flow conditions within the flow cell allow studies of biofilm exposed to shear stress. A bubble trapping device confines air bubbles from the tubing which otherwise could disrupt the biofilm structure in the flow cell.
The flow cell system is compatible with Confocal Laser Scanning Microscopy (CLSM) and can thereby provide highly detailed 3D information about developing microbial biofilms. Cells in the biofilm can be labeled with fluorescent probes or proteins compatible with CLSM analysis. This enables online visualization and allows investigation of niches in the developing biofilm. Microbial interrelationship, investigation of antimicrobial agents or the expression of specific genes, are of the many experimental setups that can be investigated in the flow cell system.

Protocol describing the application of a flow cell system for growing and analyzing microbial biofilms for Confocal Laser Scanning Microscopy (CLSM).

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological ...

Hello, I'm Matt Ma Niton from the Technical University of Denmark. Hi, and I'm Reba from the University of Copenhagen. We work on yeast and bacterial biofilms in flow cell systems And in this movie we are gonna show you how to set up a flow cell system and visualized biofilm by convertible micr.Should we do that now? Yeah, sure. Let's do it.Gluing of the flow cells. The flow cells are located between the bubble trap and waste bottle. Now we'll show you how to attach.This is stratum to the flow cell. This is done by the several components that you see on the table. A 200 microliter tip is cut into two pieces.The thin part is now inserted into a syringe, which will become the perfect tool for applying thin lines of silicon. In order to glue the glass of stratum to the flow cell, push the thin tip out of the syringe. Now, in order to make an easier application of the silicon to the base of the flow cell, cut the outmost part of the tip off, take out the piston and apply an appropriate amount of silicon into the syringe.Put the piston back into the syringe and push a little bit of silicon out in order to make sure that all the bubbles are gone. Now start applying thin lines of silicon to the flow cell. Try and make smooth and undisrupted lines of silicon to the flow cell.This will ensure that the silicon will attach evenly to the substratum When applying silicon in The opposite direction. Make sure not to scratch out the already made lines, but try and gently lift the tip as you go across, and this way you'll make sure that no openings in the silicon lines will lead to leakages between the chambers or from out the flow cell. Take a cover glass and gently place it on top of the silicon lines on the flow cell.As seen here, the cover glass will easily stick to the silicon glue. Turn the flow cell over and gently push down on the back of the flow cell with your fingers. This will ensure that the cover glass will attach in a flat manner according to the flat surface of the table.Now to make a perfect seal or to try and correct for unsealing areas, gently push down with an instrument As a piston from a syringe. Do this very carefully. As a cover glass can easily break.By doing this, you'll be able to make an even better seal and you will probably be able to correct eventually troublesome zones to avoid leakages inspecting the flow cells. In order to have a successful experiment, it's important to inspect your flow cells after the silicon has solidified. Here we show a flow cell with a non-perfect seal, which undoubtedly will lead to leakages assembly of the bubble traps and attachment of the tubings.The bubble traps are located between the pump and the flow cells. Here we show the different components for the use of the DTU bio bubble traps. They're fabricated in a way that will ensure that the bubbles are trapped in the system and only media without bubbles will enter the flow cells.Five milliliter syringes are placed on top of each of the special nozzles. On the bubble trap, a tight seal is insured by the old rings. On each nozzle, place a cap on each of the syringes.Now, as this will be the Connection for the flow cells, be sure to attach the long tubings for the flow cell to be able to reach a confocal microscope. These tubings should be attached to the bubble traps where the lower ledge of the nozzle has its outlet. As the entry ledge in the bubble trap is higher than the exit ledge, the bubbles are not able to leave the syringe and enter the flow cell attachment of the flow cells to the bubble traps.The flow cells are located between the bubble traps and the waste bottle. Here it is required to have long tubings for the flow cell to reach the confocal microscope. Again, be sure to connect the flowal to the correct side of the bubble trap.Here.Notice the Long tubings leading to the flowal from the bubble trap. This can of course vary on the construction of one setup. Now firmly attach all the tubings to the flow cell Assembly of the system Bubble traps to the pump and the T connectors.For the media feeding tubes. For the peristaltic pump, we use the RIN tubings. This specific kind of tubings are able to withstand the mechanical pressure from the pump.Set up a tube for each of the chambers in your flow cells. Here we'll now show how the tubings going from the media bottle to the pump are assembled. Cut the silicon tubing into appropriate size pieces and connect them with the T connectors.Assemble the tubings so that there will be one connection for each of the mar brain tubings in the pump. Be sure to make a tube for each of the chambers in the flow cell. Try to make your tubes as short as possible to have an easier overview of your connections.Now from the center feeding tube attached onto the T connectors short tubes that can be attached to the ING tubings in the Pump. Be sure that the silicone tubes are tightly attached to the connectors. Now with the media feeding Tubes on the left side of the pump, we'll now attach the rest of the system to the pump.Here are the tubings going to the bubble trays are attached to the rine tubings on the pump. Again, here you will lighten the overview of your experiment. If you know which tubes goes to which connection in the pump, the length of these tubings are not as crucial as the tubings on the other side of the bubble trap.Leading media to the flow cells, We have now shown You how to successfully assemble a system with one bubble trap and one flow cell On the exit point of the flow cell. We have connected tubings that are long enough to go into a waste container sterilization of the system in order to sterilize the system, make a 0.5 solution of sodium hydrochloride. Place the feeding tubes of the pump into the hydrochloride solution.At this point, it is extremely important to fill the whole system with the hydrochloride solution. Allows syringes of the bubble traps to fill totally with hydrochloride when they're filled, put the caps back on, which in the meantime should have been placed in ethanol. An important step in the sterilization of the system is the removal of bubbles from the flow cells.Such bubbles can easily be seen in the flow cells shown here, but the high success of bubble removal turn the pump to its highest rounds per minute. This will lead to a higher liquid flow in the flow cells, which in time will help to get rid of the bubbles, lift the flow cell and lift the tubings leading to the flow cell and firmly tap the flow cell on the table to loosen the bubbles in order to get them flushed out. With the liquid flow, this procedure will sometimes take a long time, but you will in this way be able to get rid of the bubbles.If you're a patient, now, leave the flow cells sterilized for three to four hours at a flow of approximate 0.2 millimeters per second. Washing the system first into the system by allowing air to push the hydrochloride out of the entire System. As the system now Has been emptied up the hydrochloride, place the central feeding tube into a sterile bottle of water and cover the top.The system now needs To be washed two to three times by filling an empty in the system with sterile water, connecting the medium and saturation of tubings overnight. When connecting the media bubble to the system, the system has to be emptied of water. As with the hypo chlorate, fill the bubble trash with media before putting the caps back on.When connecting the media, be very careful to keep everything sterile. Have ethanol ready at the side at the connection point. Place the two tubes in the ethanol just before connecting the tubes.Now with the media attached to the system, it is now ready for overnight saturation or inoculation. Depending on the choice of media inoculating the flow cell. In the DTU bio flow cells, you have three inoculation sites for the three individual growth chambers.The first thing to do is to sterilize the inoculation site from an overnight culture. Make it allusion to reach your desired optical density. Use 500 microliter syringe with a 27 G needle to inoculate the sample.An inoculum of 250 microliters is sufficient for the DTU bio flow cells. Now the inoculation site is wiped off with the net ethanol swipe and the inoculation side is closed off with silicon glue. Now turn the flow cell upside down and leave it in this position for an hour without any media flow.After the flow cell has been upside down for an hour, take the clam back off and start the media flow and place a flow cell in an upside position again. Now start the incubation of the system. The incubation time and choice of temperature vary among choices, strains and experimental setups, staining of a yeast biofilm, and visualization by confocal laser scanning microscopy.In order to visualize the yeast biofilm with a confocal microscope, we stain it with the green fluorescent nucleic acid stain CY nine. Here we use a dilution of one to thousand. First, the tubings leading back to the bubble traps from the flow cells are clammed off.This will prevent backflow from the flowal during the staining procedure. Now stop the media flow to the bubble trap. Use a 500 microliter syringe as for the inoculation to inject the cyto nine stain into the flow chamber.Again, use an ethanol wipe to sterilize the inoculation site. Inject 250 microliters into the chamber here. Be very careful and gentle.When inoculating inoculation speed should be very low in order not to disrupt the biofilm. Close off the inoculation hole with silicon glue. Allow the staining to settle for 15 minutes without flow.Take off the clamp and start the flow again. You're now ready for analyzing the stained biofilm. With confocal laser scanning microscopy, the flow cells has special ledges which enable it to be placed in the holders on the microscope stage.When the flow cell is placed in the microscope stage, a drop of oil is placed on the glass of stratum. In order to use the oil immersion objective, turn on the white light and lower the objective for it to get in contact with the oil and locate the cells in the chamber. Be careful if using a glass of stratum as it can easily break.Here we show a yeast biofilm as seen with the white light in the microscope. As the fluorescent light is turned on, one can easily see the cyto nine staining of the cells. The cyto nine stain will now enable scanning of the biofilm with a confocal microscope.For further analysis with the Amer software, locate the position of interest in the flow chamber with the microscope. Remember to take into account for the different environments that is generated over time within the flow chamber. When choosing a location, now switch over the microscope from ocular visualization to scanning laser visualization.With the size LM software, now start applying the decide settings for scanning your samples. A fast scanning of your samples will give you indication of the structural composition of the biofilm in the growth chambers. Before starting the more time consuming high resolution scanning here we said What is the first and what is the last position of the scanning, and thereby how big the stack will be.We're able to scan the several frequencies due to the different lasers installed in the microscope. This will enable investigation of numerous different subpopulations. Here we see the green shadow nine stain yeast biofilm presented in the window from the specific wavelengths used.Each of the scan stacks can now be analyzed in software programs as errors.

pseudomonas-aeruginosa-saccharomyces-cerevisiae-biofilm-flow

Research

JoVE Journal

Immunology and Infection

This paper describes different methods of growing Pseudomonas aeruginosa biofilms on cultured human airway epithelial cells. These protocols can be adapted to study different aspects of biofilm formation, including visualization of the biofilm, staining of the biofilm, measuring the colony forming units (CFU) of the biofilm, and studying biofilm cytotoxicity.

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

The assay describes a rapid means to measure early biofilm formation in bacteria and fungi.  This method uses a microtiter plate as the substratum for microbial biofilm formation, and the biofilm is visualized using crystal violet strain.  The assay provides either a qualitative or quantitative assay for early biofilm formation.

Microtiter Dish Biofilm Formation Assay

Biofilms have complex interactions with their surrounding environment. To comprehensively investigate biofilm-environment interactions, we present here a series of methods to create heterogeneous chemical environment for biofilm development, to quantify local flow velocity, and to analyze mass transport in and around biofilm colonies.

Methods for Characterizing the Co-development of Biofilm and Habitat Heterogeneity

Particle-tracking microrheology investigates the viscoelasticity of materials. Here, the technique is used to determine the viscoelasticity, creep compliance and effective crosslinking roles of different matrix components of a bacterial biofilm. The matrix consists of polymeric substances secreted by the bacteria and its components determine biofilm structure and mechanical properties.

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

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

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.

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

Here, we describe the use of a higher-throughput microfluidic bioreactor coupled with a fluorescent microscope for the analysis of shear stress effects on Pseudomonas aeruginosa biofilms expressing green fluorescent proteins, including instrument set up, the determination of biofilm coverage, growth rate, and morphological properties.

Live Cell Analysis of Shear Stress on Pseudomonas aeruginosa Using an Automated Higher-Throughput Microfluidic System

This protocol presents methodology to perform biofilm growth and biomass measurements using self-assembled deep well PCR-plate devices for high-throughput 96-well pegged lid static biofilm screening.

Generation of Greater Bacterial Biofilm Biomass using PCR-Plate Deep Well Microplate Devices

This methodology allows for the establishment of a polymicrobial biofilm model in cystic fibrosis for antimicrobial sensitivity testing in research and clinical laboratories. This model provides accurate and reliable results over a range of outputs.

Development of a Polymicrobial Colony Biofilm Model to Test Antimicrobials in Cystic Fibrosis

This study outlines a framework for developing an aggregate polymicrobial biofilm model and optimizing an RNA extraction protocol to assess Pseudomonas aeruginosa gene expression reflective of the cystic fibrosis lung environment. Applications include evaluating antimicrobial effects and studying antibiotic alternatives under conditions relevant to cystic fibrosis.

Cystic Fibrosis Aggregate Biofilm Model to Study Infection-relevant Gene Expression

Learn how to set up a flow cell system for microbial biofilm analysis using Pseudomonas aeruginosa and Saccharomyces cerevisiae. This protocol demonstrates proper assembly, sealing, and visualization with confocal microscopy.

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

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