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="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="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="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 &mu;L 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&deg;C in the case of P. aeruginosa and 30&deg;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 &mu;L 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+biofilms:+a+common+cause+of+persistent+infections.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growing+and+analyzing+biofilms+in+flow+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+reproducibility+in+flow-chamber+biofilms.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+roles+of+biosurfactants+in+structural+biofilm+development+by+Pseudomonas+aeruginosa.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+and+distribution+of+colistin-+and+sodium+dodecyl+sulfate-tolerant+cells+in+Pseudomonas+aeruginosa+biofilms.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+bacterial+migration+in+the+development+of+complex+multicellular+structures+in+Pseudomonas+aeruginosa+biofilms.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insight+into+the+microbial+multicellular+lifestyle+via+flow-cell+technology+and+confocal+microscopy.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+type+IV+pili,+flagellum-mediated+motility+and+extracellular+DNA+in+the+formation+of+mature+multicellular+structures+in+Pseudomonas+aeruginosa+biofilms.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+autolysin-mediated+DNA+release+in+biofilm+formation+of+Staphylococcus+epidermidis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+characterization+of+DNA+release+in+Pseudomonas+aeruginosa+cultures+and+biofilms.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+biofilm+structures+by+the+novel+computer+program+COMSTAT.">Quantification of biofilm structures by the novel computer program COMSTAT.</a> Microbiology. 146, 2395-2407 (2000).</li><li>Palmer, R. J., Haagensen, J. A., Neu, T. R., Sternberg, C., Palmer, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of Biological Confocal Microscopy. , 882-900 (2006).</li><li>Haagensen, J. A., Regenberg, B., Sternberg, C., M&#252;ller, S., Bley, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> High Resolution Microbial Single Cell Analytics Advances in Biochemical Engineering and Biotechnology. , (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="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.

<ul>
 <li>Bubble traps, polyethylene (DTU Systems Biology, Technical University of Denmark, http://www.csm.bio.dtu.dk/Instrument%20Center/Resources/Biofilm%20Setup.aspx )</li>
 <li>Clear polypropylene plastic connectors and T-connectors (Cole Parmer, 1/8 in. (3.175 mm) and 1/16 in. (1.588 mm))</li>
 <li>Clamps</li>
 <li>Confocal microscope (Zeiss, Meta LSM510)</li>
 <li>Coverslips, glass (Knittel Gl&auml;ser) 50 x 24 mm</li>
 <li>Coverslips, PVC coverslips (Rinzl) 50 x 24 mm</li>
 <li>Flow cells, polyethylene (DTU Systems Biology, Technical University of Denmark, http://www.csm.bio.dtu.dk/Instrument%20Center/Resources/Biofilm%20Setup.aspx</li>
 <li>Medium bottles (Schott)</li>
 <li>Peristaltic Pump (Watson-Marlow, 205S)</li>
 <li>Rolling cart for flow systems and pumps </li>
 <li>Silicone glue (3M Super Silicone Sealant Clear)</li>
 <li>Syringe 5 mL (Terumo)</li>
 <li>Syto 9 (Molecular Probes)</li>
 <li>0.5 mL Syringes with needles (27G, Terumo LU-100)</li>
 <li>Waste container</li>
</ul>
Tubing:
<ul>
 <li>Silicone, 3 mm outer diameter, 1 mm inner diameter (Ole Dich)</li>
 <li>Silicone, 4 mm outer diameter, 2 mm inner diameter (Ole Dich)</li>
 <li>Marprene, 3 mm outer diameter, 1 mm inner diameter (Watson-Marlow)</li>
</ul>
Media
<table border="0">
 <tbody>
 <tr>
 <td colspan="2">P. aeruginosa medium</td>
 </tr>
 <tr>
 <td>A10</td>
 <td>g/L</td>
 </tr>
 <tr>
 <td>(NH4)2SO4</td>
 <td>2.0</td>
 </tr>
 <tr>
 <td>Na2HPO4 X 2H2O</td>
 <td>6.0</td>
 </tr>
 <tr>
 <td>KH2PO4</td>
 <td>3.0</td>
 </tr>
 <tr>
 <td>NaCl</td>
 <td>3.0</td>
 </tr>
 <tr>
 <td>Autoclave</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td colspan="2">&nbsp;</td>
 </tr>
 <tr>
 <td colspan="2">FB</td>
 </tr>
 <tr>
 <td>MgCl2 6H2O</td>
 <td>0.20</td>
 </tr>
 <tr>
 <td>1 mL 1 M CaCl2</td>
 <td>0.01</td>
 </tr>
 <tr>
 <td colspan="2">100 &mu;L/L Trace metals (for P. aeruginosa-biofilms)4</td>
 </tr>
 <tr>
 <td colspan="2">Autoclave</td>
 </tr>
 <tr>
 <td colspan="2">Mix A10 and FB in a ratio of 1:10.</td>
 </tr>
 <tr>
 <td colspan="2">Add carbon source to a desired concentration.</td>
 </tr>
 </tbody>
</table>
 
<table border="0">
 <tbody>
 <tr>
 <td colspan="2">S. cerevisiae synthetic complete (SC) medium</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>g/L</td>
 </tr>
 <tr>
 <td>Adenine sulfate</td>
 <td>0.02</td>
 </tr>
 <tr>
 <td>L-tryptophan</td>
 <td>0.02</td>
 </tr>
 <tr>
 <td>L-histidine-HCL</td>
 <td>0.02</td>
 </tr>
 <tr>
 <td>L-arginine-HCL</td>
 <td>0.04</td>
 </tr>
 <tr>
 <td>L-methionine</td>
 <td>0.02</td>
 </tr>
 <tr>
 <td>L-tyrosine</td>
 <td>0.05</td>
 </tr>
 <tr>
 <td>L-leucine</td>
 <td>0.06</td>
 </tr>
 <tr>
 <td>L-isoleucine</td>
 <td>0.06</td>
 </tr>
 <tr>
 <td>L-lysine-HCL</td>
 <td>0.05</td>
 </tr>
 <tr>
 <td>L-phenylalanine</td>
 <td>0.05</td>
 </tr>
 <tr>
 <td>L-aspartic acid</td>
 <td>0.10</td>
 </tr>
 <tr>
 <td>L-glutamic acid</td>
 <td>0.10</td>
 </tr>
 <tr>
 <td>L-valine</td>
 <td>0.15</td>
 </tr>
 <tr>
 <td>L-threonine</td>
 <td>0.20</td>
 </tr>
 <tr>
 <td>L-serine</td>
 <td>0.40</td>
 </tr>
 <tr>
 <td>Yeast Nitrogen base w/o amino acids and ammonium (Bacto)</td>
 <td>1.6</td>
 </tr>
 <tr>
 <td>Ammonium sulphate</td>
 <td>5.0</td>
 </tr>
 <tr>
 <td>NaOH</td>
 <td>6.0</td>
 </tr>
 <tr>
 <td>Succinic acid</td>
 <td>10.0</td>
 </tr>
 <tr>
 <td>Autoclave</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Glucose (autoclaved separately)</td>
 <td>0.20</td>
 </tr>
 </tbody>
</table>

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.

Watch this Scientific Journal Video about Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells at JoVE.com

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

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

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 ...

pseudomonas-aeruginosa-saccharomyces-cerevisiae-biofilm-flow

Research

JoVE Journal

Immunology and Infection

36.6K visualizações.  Danish Technical University. Protocolo que descreve a aplicação de um sistema de célula de fluxo para o cultivo e análise de biofilmes microbianos para Microscopia Confocal Laser Scanning (CLSM).

Vídeo: Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

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

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living surfaces. In this paper, we describe protocols for forming Pseudomonas aeruginosa biofilms on human airway epithelial cells (CFBE cells) grown in culture. In the first method (termed the Static Co-culture Biofilm Model), P. aeruginosa is incubated with CFBE cells grown as confluent monolayers on standard tissue culture plates. Although the bacterium is quite toxic to epithelial cells, the addition of arginine delays the destruction of the monolayer long enough for biofilms to form on the CFBE cells. The second method (termed the Flow Cell Co-culture Biofilm Model), involves adaptation of a biofilm flow cell apparatus, which is often used in biofilm research, to accommodate a glass coverslip supporting a confluent monolayer of CFBE cells. This monolayer is inoculated with P. aeruginosa and a peristaltic pump then flows fresh medium across the cells. In both systems, bacterial biofilms form within 6-8 hours after inoculation. Visualization of the biofilm is enhanced by the use of P. aeruginosa strains constitutively expressing green fluorescent protein (GFP). The Static and Flow Cell Co-culture Biofilm assays are model systems for early P. aeruginosa infection of the Cystic Fibrosis (CF) lung, and these techniques allow different aspects of P. aeruginosa biofilm formation and virulence to be studied, including biofilm cytotoxicity, measurement of biofilm CFU, and staining and visualizing the biofilm.

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

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.

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living ...

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with leukemia, severe burn wounds, or organ transplants1. In patients at high-risk for developing PA bloodstream infections, the gastrointestinal (GI) tract is the main reservoir for colonization2, but the mechanisms by which PA transitions from an asymptomatic colonizing microbe to an invasive, and often deadly, pathogen are unclear. Previously, we performed in vivo transcription profiling experiments by recovering PA mRNA from bacterial cells residing in the cecums of colonized mice 3 in order to identify changes in bacterial gene expression during alterations to the host&#x2019;s immune status.
As with any transcription profiling experiment, the rate-limiting step is in the isolation of sufficient amounts of high quality mRNA. Given the abundance of enzymes, debris, food residues, and particulate matter in the GI tract, the challenge of RNA isolation is daunting. Here, we present a method for reliable and reproducible isolation of bacterial RNA recovered from the murine GI tract. This method utilizes a well-established murine model of PA GI colonization and neutropenia-induced dissemination4. Once GI colonization with PA is confirmed, mice are euthanized and cecal contents are recovered and flash frozen. RNA is then extracted using a combination of mechanical disruption, boiling, phenol/chloroform extractions, DNase treatment, and affinity chromatography. Quantity and purity are confirmed by spectrophotometry (Nanodrop Technologies) and bioanalyzer (Agilent Technologies) (Fig 1). This method of GI microbial RNA isolation can easily be adapted to other bacteria and fungi as well.

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

A reliable method for the RNA isolation of Pseudomonas aeruginosa recovered from murine cecums is described. The RNA recovered is of sufficient quantity and quality for subsequent qPCR, transcription profiling, and RNA Seq experiments. This technique can be adapted for RNA isolation of other intestinal microbes.

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with ...

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

Identification of Novel Genes Associated with Alginate Production in Pseudomonas aeruginosa Using Mini-himar1 Mariner Transposon-mediated Mutagenesis

Pseudomonas aeruginosa is a Gram-negative, environmental bacterium with versatile metabolic capabilities. P. aeruginosa is an opportunistic bacterial pathogen which establishes chronic pulmonary infections in patients with cystic fibrosis (CF). The overproduction of a capsular polysaccharide called alginate, also known as mucoidy, promotes the formation of mucoid biofilms which are more resistant than planktonic cells to antibiotic chemotherapy and host defenses. Additionally, the conversion from the nonmucoid to mucoid phenotype is a clinical marker for the onset of chronic infection in CF. Alginate overproduction by P. aeruginosa is an endergonic process which heavily taxes cellular energy. Therefore, alginate production is highly regulated in P. aeruginosa. To better understand alginate regulation, we describe a protocol using the mini-himar1 transposon mutagenesis for the identification of novel alginate regulators in a prototypic strain PAO1. The procedure consists of two basic steps. First, we transferred the mini-himar1 transposon (pFAC) from host E. coli SM10/&#955;pir into recipient P. aeruginosa PAO1 via biparental conjugation to create a high-density insertion mutant library, which were selected on Pseudomonas isolation agar plates supplemented with gentamycin. Secondly, we screened and isolated the mucoid colonies to map the insertion site through inverse PCR using DNA primers pointing outward from the gentamycin cassette and DNA sequencing. Using this protocol, we have identified two novel alginate regulators, mucE (PA4033) and kinB (PA5484), in strain PAO1 with a wild-type mucA encoding the anti-sigma factor MucA for the master alginate regulator AlgU (AlgT, &#963;22). This high-throughput mutagenesis protocol can be modified for the identification of other virulence-related genes causing change in colony morphology.

Identification of Novel Genes Associated with Alginate Production in Pseudomonas aeruginosa Using Mini-himar1 Mariner Transposon-mediated Mutagenesis

Here we describe a protocol using the mini-himar1 mariner transposon-mediated mutagenesis for generating a high-density insertion mutant library to screen, isolate and identify novel alginate regulators in the prototypic Pseudomonas aeruginosa strain PAO1.

Pseudomonas aeruginosa is a Gram-negative, environmental bacterium with versatile metabolic capabilities. P. aeruginosa is an opportunistic bacterial ...

Pseudomonas aeruginosa Induced Lung Injury Model

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. Here we have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa (P. aeruginosa or PA). Using this model, we were able to show lung inflammation at the early phase of injury. In addition, alveolar epithelial barrier leakiness was observed by analyzing bronchoalveolar lavage (BAL); and alveolar cell death was observed by Tunel assay using tissue prepared from injured lungs. At a later phase following injury, we observed cell proliferation required for the repair process. The injury was resolved 7 days from the initiation of P. aeruginosa injection. This model mimics the sequential course of lung inflammation, injury and repair during pneumonia. This clinically relevant animal model is suitable for studying pathology, mechanism of repair, following acute lung injury, and also can be used to test potential therapeutic agents for this disease.

Pseudomonas aeruginosa Induced Lung Injury Model

We have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa. This model mimics lung injury during pneumonia and is clinically relevant.

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. ...

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. Chronic mouse wounds models have been used to understand the underlying interactions between the microorganisms and the host. The models developed to date rely on the use of haired animals and terminal collection of wound tissue for determination of viable bacteria. While significant insight has been gained with these models, this experimental procedure requires a large number of animals and sampling is time consuming. We have developed a novel murine model that incorporates several optimal innovations to evaluate biofilm progression in chronic wounds: a) it utilizes hairless mice, eliminating the need for hair removal; b) applies pre-formed biofilms to the wounds allowing for the immediate evaluation of persistence and effect of these communities on host; c) monitors biofilm progression by quantifying light production by a genetically engineered bioluminescent strain of Pseudomonas aeruginosa, allowing real-time monitoring of the infection thus reducing the number of animals required per study. In this model, a single full-depth wound is produced on the back of STZ-induced diabetic hairless mice and inoculated with biofilms of the P. aeruginosa bioluminescent strain Xen 41. Light output from the wounds is recorded daily in an in vivo imaging system, allowing for in vivo and in situ rapid biofilm visualization and localization of biofilm bacteria within the wounds. This novel method is flexible as it can be used to study other microorganisms, including genetically engineered species and multi-species biofilms, and may be of special value in testing anti-biofilm strategies including antimicrobial occlusive dressings.

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

Here we describe a novel diabetic murine model utilizing hairless mice for real-time, non-invasive, monitoring of biofilm wound infections of bioluminescent Pseudomonas aeruginosa. This method can be adapted to evaluate infection of other bacterial species and genetically modified microorganisms, including multi-species biofilms, and test the efficacy of antibiofilm strategies.

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. ...

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa is able to form biofilms, develop antibiotic resistance, produce virulence factors, and rapidly evolve in the course of a chronic infection. Thus P. aeruginosa can cause both acute and chronic, difficult to treat infections, resulting in significant morbidity in certain patient populations. P. aeruginosa strain PA14 is a human clinical isolate with a conserved genome structure that infects a variety of mammalian and nonvertebrate hosts making PA14 an attractive strain for studying this pathogen. In 2006, a nonredundant transposon insertion mutant library containing 5,459 mutants corresponding to 4,596 predicted PA14 genes was generated. Since then, distribution of the PA14 library has allowed the research community to better understand the function of individual genes and complex pathways of P. aeruginosa. Maintenance of library integrity through the replication process requires proper handling and precise techniques. To that end, this manuscript presents protocols that describe in detail the steps involved in library replication, library quality control and proper storage of individual mutants.

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Pseudomonas aeruginosa infection causes significant morbidity in vulnerable hosts. The nonredundant transposon insertion mutant library of P. aeruginosa strain PA14, designated as PA14NR Set, facilitates analysis of gene functionality in numerous processes. Presented here is a protocol to generate high-quality copies of the PA14NR Set mutant library.

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa ...

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary tissue during pneumonia results in the production of long-lived cytotoxins that can lead to subsequent end organ failure. We have developed in vitro assays to test the hypothesis that cytotoxins are produced during pulmonary infection. Isolated rat pulmonary endothelial cells and the bacterium Pseudomonas aeruginosa are used as model systems, and the production of cytoxins following infection of the endothelial cells by the bacteria is demonstrated using cell culture followed by direct quantitation using lactate dehydrogenase assays and a novel microscopic method utilizing ImageJ technology. The amyloid nature of these cytotoxins was demonstrated by thioflavin T binding assays and by immunoblotting and immunodepletion using A11 anti-amyloid antibody. Further analyses using immunoblotting demonstrated that oligomeric tau and A&#946; were produced and released by endothelial cells following infection by P. aeruginosa. These methods should be readily adaptable to analyses of human clinical samples.

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Simple methods are described for demonstrating the production of cytotoxic amyloids following infection of pulmonary endothelium by Pseudomonas aeruginosa.

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary ...

Generation of In-Frame Gene Deletion Mutants in Pseudomonas aeruginosa and Testing for Virulence Attenuation in a Simple Mouse Model of Infection

Microorganisms are genetically versatile and diverse and have become a major source of many commercial products and biopharmaceuticals. Though some of these products are naturally produced by the organisms, other products require genetic engineering of the organism to increase the yields of production. Avirulent strains of Escherichia coli have traditionally been the preferred bacterial species for producing biopharmaceuticals; however, some products are difficult for E. coli to produce. Thus, avirulent strains of other bacterial species could provide useful alternatives for production of some commercial products. Pseudomonas aeruginosa is a common and well-studied Gram-negative bacterium that could provide a suitable alternative to E. coli. However, P. aeruginosa is an opportunistic human pathogen. Here, we detail a procedure that can be used to generate nonpathogenic strains of P. aeruginosa through sequential genomic deletions using the pEX100T-NotI plasmid. The main advantage of this method is to produce a marker-free strain. This method may be used to generate highly attenuated P. aeruginosa strains for the production of commercial products, or to design strains for other specific uses. We also describe a simple and reproducible mouse model of bacterial systemic infection via intraperitoneal injection of validated test strains to test the attenuation of the genetically engineered strain in comparison to the FDA-approved BL21 strain of E. coli.

Generation of In-Frame Gene Deletion Mutants in Pseudomonas aeruginosa and Testing for Virulence Attenuation in a Simple Mouse Model of Infection

Here, we describe a simple and reproducible protocol of mouse model of infection to evaluate the attenuation of the genetically modified strains of Pseudomonas aeruginosa in comparison to the United States Food and Drug Administration (FDA)-approved Escherichia coli for commercial applications.

Microorganisms are genetically versatile and diverse and have become a major source of many commercial products and biopharmaceuticals. Though some of ...

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Subtitles

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells