Name: The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis (Video) | JoVE Journal
Uploaded: 2010-12-27T17:00:00.000+00:00
Duration: 8 min 8 s
Description: Learn to grow bacterial biofilms using drip flow and rotating disk reactors for Staphylococcus aureus analysis. This method enables high biomass production and controlled shear force studies.

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1. The Drip Flow Biofilm Reactor
<ol>
<li>The drip flow biofilm reactor (available from Biosurface Technologies or custom designed versions can usually be made by university machine shops, see Figure 1) is assembled and autoclaved. Assembly involves affixing coupons in chambers and securing chamber lids. The chamber, along with biofilm medium (tryptic soy broth 2 grams/L and glucose 2 grams/L), and influent nutrient tubing are sterilized by autoclaving.</li>
<li>Inoculation of the drip flow reactor is preformed by placing the reactor on a flat surface, clamping the effluent tubing lines, filling each chamber with 10 mL tryptic soy broth and adding 10 μL of a S. aureus culture grown overnight in tryptic soy broth. The inoculated reactor is then place in a 37°C incubator for 18 hours.</li>
<li>After 18 hours of incubation, the effluent tubing is unclamped and the reactor is placed on a wooden block cut to a 10° angle.</li>
<li>Aseptically connect the influent nutrient tubing to the bottle containing the continuous flow nutrient broth. Feed the tubing line through the pump and prime the tubes by running the pump at a maximum speed (will vary depending on pump model).</li>
<li>Once the influent tubing is primed stop the pump and attach connect needles (22 gauge, 1 inch) to the end of each tube. Wipe the chamber inlet stopper with an ethanol wipe and aseptically insert the needles through the inlet stopper.</li>
<li>Turn on the pump and allow the media to slowly drip (flow rate ~125 μL/minute) over the coupons. The media should flow downward along the coupon from the inlet stopper port to the effluent port. Operate the reactor in continuous flow for 2-5 days (depending on the application), occasionally checking the reactor for proper drainage.</li>
<li>To harvest the drip flow reactor biofilms, stop the pump and carefully remove the needles from the reactor. The reactor can then be placed on a flat surface and the coupons can be aseptically removed using sterile forceps. If microscopy is desired, the coupons can now be processed accordingly (Figure 2B is a scanning electron micrograph of a S. aureus biofilm grown in a drip biofilm reactor). If quantification of the biofilm biomass or physiology studies are the goal of the study, the biofilm can be removed from the coupon using a cell scraper. While holding the coupons with forceps, gently scrape the biofilm off the coupon into a conical tube containing phosphate buffered saline using a cell scraper. Note: to quantify the colony forming units in the biofilms, it is necessary to homogenize the harvested biofilms with a tissue homogenizer to disaggregate clumps and form a homogenous suspension. Various models of tissue homogenizers are suitable for this application. We utilize a Fisher Scientific Tissuemiser Homogenizer (product # 15-338-420) at full speed for 1 minute to homogenize biofilm samples. Failure to homogenize the biofilm will result in an underestimation of the colony forming units present in the sample.</li>
</ol>
2. The Rotating Disk Biofilm Reactor
<ol>
<li>The rotating disk biofilm reactor (available from available from Biosurface Technologies or can be custom made, see Figure 3) is assembled and autoclaved. Assemble the reactor by first place the spinning disk coupons into the slots of the spinning disc and placing it into a 1-liter glass beaker with an overflow port. A number 15-rubber stopper with holes drilled in it to allow media flow and aeration is used as the reactor cap. The reactor, biofilm media (tryptic soy broth 2 g/L and glucose 2 g/L), and inlet tubing are then sterilized by autoclaving.</li>
<li>The rotating disk biofilm reactor is inoculated by placing 250 mLs of sterile medium into the reactor, and adding 0.5 mL of an overnight S. aureus culture grown in tryptic soy broth. The reactor is then placed on a stir plate set to 250 rpm and incubated overnight at the desired temperature.</li>
<li>After 16 hours of incubation connect the inlet tubing to the port on medium reservoir and connect it to the peristaltic pump. The pump is then turn on and flow set to ~0.25 mL/min (flow can be altered depending on the growth rate desired).</li>
<li>After 24 hours stop the reactor and aseptically remove the disc without touching the biofilm coupons. Remove the coupons using sterile forceps and dip each one into phosphate buffered saline to remove any loosely attached bacteria.</li>
<li>For antimicrobial compound testing, the chips can be placed into individual wells of a 96 well plate containing compounds of interest. After incubation, chips are transferred into 1.5 mL microcentrifuge tubes containing 1 mL phosphate buffer saline and homogenized with a tissue homogenizer to disperse the intact biofilm.</li>
<li>Cells are then serial diluted and plated onto nutrient agar medium to determine viable colony forming units.</li>
<li>Note: many variations can be made on the above protocol. For example, spinning disk coupons can be coated with or made out of potential anti-biofilm compounds to test efficacy. Biofilm dispersants can also be evaluated be incubating coupons in dispersant compounds and quantifying bacteria attached versus detached.</li>
</ol>	 
3. Representative Results
An example of a set up drip flow reactor is shown if Figure 1. After three days of flow copious amounts of biofilm will accumulate on the coupon surface, Figure 2A. The total biomass will vary depending on bacterial strains and precise growth conditions. A scanning electron micrograph of a S. aureus biofilm grown in the drip flow reactor is shown in Figure 2B.
A rotating disk reactor is shown in figure 3A. The protocol described can be adapted to the specific requirements of generally any microorganism capable of forming a biofilm. Figure 3B shows the spinning disc with 18 plastic disks affixed. These disk-grown biofilms are particularly well suited for antimicrobial testing and yield highly reproducible results11.
<img src="/files/ftp_upload/2470/2470fig1.jpg" alt="Drip flow reactor setup for biofilm growth, with media bottles, pump system, waste collection." data-altupdatedat="1788235205000" data-alt="Figure 1"> 
Figure 1. The drip flow reactor setup. Important components are labeled.
<img src="/files/ftp_upload/2470/2470fig2.jpg" alt="Microbial corrosion experiment setup and SEM result; metal degradation visible in both images." data-altupdatedat="1788235205000" data-alt="Figure 2"> 
Figure 2. Example of a drip flow S. aureus biofilm. A) This biofilm was grown for three days following the protocol described. Lids from the first two chambers of the reactor are removed to show the yellow S. aureus biofilm biomass. B) Sacnning electron micrograph of a S. aureus biofilm grown in the drip flow reactor.
<img src="/files/ftp_upload/2470/2470fig3.jpg" alt="Rotating disk reactor setup for medium processing; stir plate, pump, waste management; top view coupons." data-altupdatedat="1788235205000" data-alt="Figure 3"> 
Figure 3. The rotating disk reactor. A) Example of a running spinning disk reactor. Key component are labeled. B) Close up view of a spinning disk.

<ol><li>Costerton, J. W., Lewandowski, Z., Caldwell, D. E., Korber, D. R., Lappin-Scott, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microbial+Biofilms%5BTitle%5D)+AND+%22Annu.+Rev.+Microbiol%22%5BJournal%5D)">Microbial Biofilms.</a> Annu. Rev. Microbiol. 49, 711-745 (1995).</li>
<li>Costerton, J. W., Cheng, K. J., Gessey, G. G., Ladd, T. I., Nickel, J. C., Dasgupta, M., Marrie, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bacterial+biofilms+in+nature+and+disease%5BTitle%5D)+AND+%22Ann.+Rev.+Microbiol%22%5BJournal%5D)">Bacterial biofilms in nature and disease.</a> Ann. Rev. Microbiol. 41, 435-464 (1987).</li>
<li>O'Toole, G. A., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Initiation+of+biofilm+formation+in+Pseudomonas+fluorescens+WCS365+proceeds+via+multiple%2C+convergent+signaling+pathways%3A+a+genetic+analysis%5BTitle%5D)+AND+%22Mol.+Micro%22%5BJournal%5D)">Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signaling pathways: a genetic analysis.</a> Mol. Micro. 28, 449-461 (2002).</li>
<li>Boles, B. R., Horswill, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Agr-mediated+dispersal+of+Staphylococcus+aureus+biofilms%5BTitle%5D)+AND+%22PLoS+Pathog%22%5BJournal%5D)">Agr-mediated dispersal of Staphylococcus aureus biofilms.</a> PLoS Pathog. 4, e1000052-e1000052 (2008).</li>
<li>Goeres, D. M., Haamilton, M. A., Beck, N. A., Buckingham-Meyer, K., Hilyard, J., Loetterle, L. A., Walker, D. K., Stewart, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+method+for+growing+a+biofilm+under+low+shear+at+the+air-liquid+interface+using+the+drip+flow+biofilm+reactor%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">A method for growing a biofilm under low shear at the air-liquid interface using the drip flow biofilm reactor.</a> Nature Protocols. 4, 783-788 (2009).</li>
<li>Fu, W., Forster, T., Mayer, O., Curtin, J. J., Lehman, S. M., Donlan, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bacteriophage+cocktail+for+the+prevention+of+biofilm+formation+by+Pseudomonas+aeruginosa+on+catheters+in+an+in+vitro+model+system%5BTitle%5D)+AND+%22Antimicrob+Agents+Chemother%22%5BJournal%5D)">Bacteriophage cocktail for the prevention of biofilm formation by Pseudomonas aeruginosa on catheters in an in vitro model system.</a> Antimicrob Agents Chemother. 54, 397-404 (2010).</li>
<li>Xu, K. D., McFeters, G. A., Stewart, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biofilm+resistance+to+antimicrobial+agents%5BTitle%5D)+AND+%22Microbiology%22%5BJournal%5D)">Biofilm resistance to antimicrobial agents.</a> Microbiology. 146, 547-549 (2000).</li>
<li>Xu, K. D., Stewart, P. S., Xia, F., Huang, C. T., McFeters, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spatial+physiological+heterogeneity+in+Pseudomonas+aeruginosa+biofilm+is+determined+by+oxygen+availability%5BTitle%5D)+AND+%22Appl.+Environ.+Microbiol%22%5BJournal%5D)">Spatial physiological heterogeneity in Pseudomonas aeruginosa biofilm is determined by oxygen availability.</a> Appl. Environ. Microbiol. 64, 4035-4039 (1998).</li>
<li>Boles, B. R., Thoendel, M., Singh, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Self-generated+diversity+produces+%22insurance+effects%22+in+biofilm+communities%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+USA%22%5BJournal%5D)">Self-generated diversity produces "insurance effects" in biofilm communities.</a> Proc. Natl. Acad. Sci. USA. 101, 16630-16635 (2004).</li>
<li>Pitts, B., Willse, A., McFeters, G. A., Hamilton, M. A., Zelver, N., Stewart, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+repeatable+laboratory+method+for+testing+the+efficacy+of+biocides+against+toilet+bowl+biofilms%5BTitle%5D)+AND+%22J.+Appl.+Microbiol%22%5BJournal%5D)">A repeatable laboratory method for testing the efficacy of biocides against toilet bowl biofilms.</a> J. Appl. Microbiol. 91, 117-11 (2001).</li>
<li>Boles, B. R., Thoendel, M., Singh, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rhamnolipids+mediate+detachment+of+Pseudomonas+aeruginosa+from+biofilms%5BTitle%5D)+AND+%22Mol.+Microbiol%22%5BJournal%5D)">Rhamnolipids mediate detachment of Pseudomonas aeruginosa from biofilms.</a> Mol. Microbiol. 57, 1210-1223 (2005).</li>
<li>Hentzer, M., Teitzel, G. M., Balzer, G. J., Heydorn, A., Molin, S., Givskov, M., Parsek, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Alginate+overproduction+affects+Pseudomonas+aeruginosa+biofilm+structure+and+function%5BTitle%5D)+AND+%22J.+Bacteriol%22%5BJournal%5D)">Alginate overproduction affects Pseudomonas aeruginosa biofilm structure and function.</a> J. Bacteriol. 183, 5395-5401 (2001).</li>
<li>Lin, H. Y., Chen, C. T., Huang, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Use+of+merocyanine+540+for+photodynamic+inactivation+of+Staphylococcus+aureus+planktonic+and+biofilm+cells%5BTitle%5D)+AND+%22Appl.+Environ.+Microbiol%22%5BJournal%5D)">Use of merocyanine 540 for photodynamic inactivation of Staphylococcus aureus planktonic and biofilm cells.</a> Appl. Environ. Microbiol. 70, 6453-6458 (2004).</li>
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No conflicts of interest declared.

Biofilms grown in different reactors will often have different characteristics and each reactor has different applications. In this work, we describe the use of two biofilm reactors: a drip flow biofilm reactor and a rotating disk reactor. Drip flow reactors are useful for growing low shear biofilms at an air-liquid interface and are adaptable to a variety of conditions. We find them extremely convenient for studies where a large amount of biofilm biomass is desirable. This setup can easily be adapted for studies invo...

Erratum: The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

<table><tbody><tr><td>Drip Flow Reactors</td><td>BioSurface Technologies Corporation</td><td>DFR 110</td><td></td></tr><tr><td>Rotating Disk Reactors</td><td>BioSurface Technologies Corporation</td><td></td><td></td></tr></tbody></table>

the use of drip flow and rotating disk reactors for staphylococcus aureus biofilm analysis

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

Most microbes in nature are thought to exist as surface-associated communities in biofilms.1 Bacterial biofilms are encased within a matrix and attached to a surface.2 Biofilm formation and development are commonly studied in the laboratory using batch systems such as microtiter plates or flow systems, such as flow-cells. These methodologies are useful for screening mutant and chemical libraries (microtiter plates)3 or growing biofilms for visualization (flow cells)4. Here we present detailed protocols for growing Staphylococcus aureus in two additional types of flow system biofilms: the drip flow biofilm reactor and the rotating disk biofilm reactor.
Drip flow biofilm reactors are designed for the study of biofilms grown under low shear conditions.5 The drip flow reactor consists of four parallel test channels, each capable of holding one standard glass microscope slide sized coupon, or a length of catheter or stint. The drip flow reactor is ideal for microsensor monitoring, general biofilm studies, biofilm cryosectioning samples, high biomass production, medical material evaluations, and indwelling medical device testing.6,7,8,9
The rotating disk reactor consists of a teflon disk containing recesses for removable coupons.10 The removable coupons can by made from any machinable material. The bottom of the rotating disk contains a bar magnet to allow disk rotation to create liquid surface shear across surface-flush coupons. The entire disk containing 18 coupons is placed in a 1000 mL glass side-arm reactor vessel. A liquid growth media is circulated through the vessel while the disk is rotated by a magnetic stirrer. The coupons are removed from the reactor vessel and then scraped to collect the biofilm sample for further study or microscopy imaging. Rotating disc reactors are designed for laboratory evaluations of biocide efficacy, biofilm removal, and performance of anti-fouling materials.9,11,12,13

Protocols for utilizing open system flow biofilms with drip flow reactors and rotating disk reactors are presented in detail.

Most microbes in nature are thought to exist as surface-associated communities in biofilms.1  Bacterial biofilms are encased within a matrix and attached ...

The overall goal of this procedure is to grow bacterial biofilms under low or moderate sheer force using either a drip flow biofilm reactor or a rotating disc reactor. Coupons in the biofilm react as are first inoculated with bacteria and incubated at 37 degrees Celsius to allow the bacterial cells to adhere to the growth surfaces. Once the cells have attached to the coupons, a flow of growth media is started producing a sheer force that operates during biofilm development.Mature biofilms can then be harvested for analysis observation of the biofilms. Using scanning electron microscopy can be performed to determine the ultra structure of the biofilms produced. I'm Blaze Balls.The main advantage of this technique of existing methods like flow cells is that high biomass production and multiple identical biofilms are readily achieved. Demonstrating this procedure will be Rachel Stevenson and Kelly Schwartz, a technician and graduate student from my laboratory. The drip flow biofilm reactor consists of four chambers with input ports for media entry and outflow ports for waste exit.Inside each chamber is a removable coupon upon which the biofilm grows. To assemble the drip flow biofilm reactor, place the coupons in each parallel chamber and secure the chamber lids. Autoclave the assembled reactor as well as the in fluent nutrient tubing to sterilize the system before use.Also autoclave the biofilm growth medium to inoculate the sterilized drip flow reactor. Place the apparatus on a flat surface and clamp the effluent lines. Next, fill each chamber with 10 milliliters of sterile triptych soy broth based growth medium, and add 100 microliters of staphylococcus aureus.Overnight culture grown in the same medium to each chamber separately. Place the inoculated reactor in a 37 degrees Celsius incubator for 18 hours to allow the cells to adhere to the coupon surface. After the incubation, UNC clamp the effluent tubing and place the reactor on an incline wooden block cutter to 10 degree angle.Aseptically.Connect the fluent nutrient tubing to a bottle containing continuous flow nutrient broth. Feed the tubing line through the pump and prime the tubing by running the pump at full speed. Once the fluent tubing is primed, stop the pump and attach 22 gauge one inch needles to the end of each tube.Wipe the chamber inlet stopper with an ethanol wipe and aseptically. Insert the needles through the inlet stopper. Turn on the pump and allow medium to drip over the coupons.At a flow rate of roughly 125 microliters per minute, the media should flow downward from the inlet stopper port to the effluent port. Operate the reactor in continuous flow for three days. Occasionally checking the reactor for proper drainage.Drainage is checked by visual inspection of the chambers to ensure media is not pooling in the chambers. If media is pooling, pinching, the outflow tubing will usually promote proper drainage to harvest the biofilms, stop the pump and remove the needles from the reactor. Then place the reactor on a flat surface.After three days of continuous flow, staphylococcus aureus biofilms appear as yellow biomass spread along the length of the inoculated coupons. A scanning electron micrograph of the biofilm reveals staphylococcus aureus covering the coupon in a surface associated biofilm community. In order to quantify the biomass obtained, use sterile forceps to as septically, remove the coupons, harvest the bacteria using a cell scraper, then transfer them to a conical tube containing five milliliters of sterile PBS.Using a tissue, homogenizer disaggregate the bacterial clumps until a uniform suspension is obtained. Colony forming units are quantitated by plating serial dilutions onto triptych sogar plates incubating for 24 hours at 37 degrees Celsius and counting colonies. The rotating disc biofilm reactor is comprised of a chemos stat through which growth medium is pumped.A spinning disc with a star bar and 18 coupons designed to fit into the spinning disc. To assemble the reactor first, load the spinning disc coupons into the slots of the spinning disc. Next place the loaded disc inside a one liter glass beaker with an overflow port and cap the reactor with a number 15 rubber stopper with one hole for media flow and one hole for aeration.Autoclave, the assembled reactor and the inlet tubing to sterilize the system. To inoculate the reactor, fill the beaker with 250 milliliters of sterile culture medium and add 500 microliters of an overnight culture of staphylococcus aureus grown in triptych soy broth. Place the reactor on a stir plate set at 250 rotations per minute and incubate it overnight at 37 degrees Celsius to allow the cells to adhere to the coupons.Following the overnight incubation, connect the inlet tubing to the port on the medium reservoir and to the peristaltic pump. Turn on the pump and set the flow to 0.25 milliliters per minute. Allow the reactor to run for 24 hours at 37 degrees Celsius to harvest the resulting biofilms.Stop the reactor and aseptically. Remove the disc without touching the coupons. Using sterile forceps, remove the coupons and carefully dip each one in sterile PBS to remove loosely attached bacteria for antimicrobial compound testing.Aseptically, place the coupons into individual wells of a 96 well plate containing compounds of interest. Incubate the plate for four hours at 37 degrees Celsius. Then transfer the coupons into 1.5 milliliter micro centrifuge tubes containing one times PBS and disperse the biofilms using a homogenizer.Finally, plate serial dilutions of the cells on nutrient agar medium to determine the number of viable colony forming units per sample as described earlier. This graph shows the number of colony forming units in biofilms prepared using the spinning disc reactor and exposed to hydrogen peroxide, the ability to produce up to 18 identical biofilms. Using the spinning disc reactor makes this technique ideal for antimicrobial compound testing.After watching this video, you should have a good understanding of how to run drip flow and rotating disc reactor biofilms. These biofilm reactors offer alternatives to flow cell and microtiter plate biofilms, and are ideal when a high amount of biofilm biomass is needed or antimicrobial testing on established biofilms is desired.

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Learn to grow bacterial biofilms using drip flow and rotating disk reactors for Staphylococcus aureus analysis. This method enables high biomass production and controlled shear force studies.

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

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