||high school biology, undergraduate microbiology, college microbiology, advanced college level microbiology|
|Revision Date||January 5, 2005|
This exercise describes the construction of a flow cell using materials that are both economical and readily available. The resulting reactor will be suitable for producing bacterial biofilms that can be continuously observed during growth.
Students should be able to define a biofilm, describe the differences between biofilm (surface-attached) and planktonic (suspended bacterial cells) bacteria, and describe how bacteria tend to grow on surfaces.
Given readily accessible materials, detailed instructions and figures, and a finished flow cell system to observe, a student will be able to construct a simple flow cell system that is suitable for growing biofilms in square capillary tubing on the stage of a microscope.
In practice, the student should observe adhesion of cells to the inside of the glass wall, the growth of complex biofilm structures, the sloughing of biofilm masses and re-growth of biofilm in these denuded areas.
Four components of a basic flow cell reactor system are:
This exercise will describe the function and construction of the access port and the flow cell. Suggestions for nutrient sources and waste disposal will also be made.
J. Lennox, Penn State Altoona
Figure 1. A flow cell reactor system
|1||1 x 3 x 1/8 inch Plexiglass® acrylic base. Many schools will have shops able to fabricate these. See student instructions.|
|7 inches||1/4 x 1/4 inch clear acrylic rod stock (Part # 104015). This material costs approximately $ 0.20/foot. Available from Laird Plastics, www.lairdplastics.com. E-mail or call (800 610-1016) for a local distributor|
|1||2.5-3 inch metal washer purchased at a local hardware store.|
|1||square capillary tubing, 2mm I.D. or 0.9mm I.D. The Borosilicate square tubing is available from Friedrich and Dimmock, Inc. PO Box 230, Millville, NJ 08332. The 2 mm I.D. is Catalog Number BST-2-30 and the 0.9 mm I.D. tubing is Catalog Number BST-090-15.|
|1||T-barbed fitting connector, Cole-Parmer EW-30624-28, $8.50/25|
|1||sleeve stopper Fisher Sci #03-215-5, $51.62/100|
|1||polycarbonate tensioning tool, Cole-Parmer U-06830-07, $40.00/ea|
|1||4" strap or cable tie Cole-Parmer U-06830-52, $17.00/1000|
|1||silicone tubing, cut into 3 pieces. (The length of these 3 pieces varies as needed for your application). This tubing is available from Cole-Parmer, use UA-96410-14 1/16" I.D. to fit the 2mm I.D. square capillary tubing ($40.00/25 ft) and UA-96410-16 1/8" I.D. ($45.00/25 ft) to fit the 0.9 mm I.D. capillary tubing.|
|1||Peristaltic Pump Fisherbrand, Variable Flow
Model 13-876-1 Low flow, 0.03-8.2 mL/min $ 186.48
Modes 13-876-2 Medium flow, 4-85 mL/min $ 186.48
A flow cell is a device that permits the microscopic observation of the initiation and growth of a biofilm in real time. It is constructed simply from a piece of square capillary tubing mounted on a base. The flow cell is placed on the stage of the microscope and a source of microorganisms and nutrient is permitted to pass through the tube. Observation over time will permit the student to follow the various stages in biofilm development.
J. Lennox, Penn State Altoona
The tube that forms the reactor vessel itself is a square borosilicate capillary tube obtained from Friedrich and Dimmock, Inc. PO Box 230, Millville, NJ 08332. This tubing comes in a wide variety of sizes. The sizes found most useful are the 2mm inside diameter (ID) and the 0.9 ID. The 2mm ID is fairly sturdy and easy to handle but has a wall diameter too great to use the oil immersion lens of most microscopes. The 0.9mm ID tubing has a thinner wall thickness and can be used with oil immersion but it is very fragile and recommended only for students with some experience in this procedure.
Caution: Wear goggles while cutting the tubing, it can shatter. Use all appropriate precautions when using cyano-acrylic cement. It binds to fingers as well as to plastic and glass.
Caution: Cyano-acrylic cements like Super Glue and can bind fingers and skin in seconds. Use great care not to get any of the glue on your hands.
First Aid: Should binding to fingers or skin occur, immerse the affected area in warm soapy water and gently peel or roll the skin back and forth until the affected parts separate. Repeated washes in warm soapy water will permit you to peel off adhering super glue. In the event of cyanoacrylic contact with the eyes, seek medical assistance immediately.
The plastic bases have the advantage of being able to fit into the mechanical stage of a student microscope. The metal washer has the advantage of mass that makes it durable and stable.
D. Williams, Center for Biofilm Engineering, Montana State University, Bozeman
Figure 3. Three types of bases for flow cell construction
It is often desirable to be able to introduce or remove materials into or out of the system without compromising the sterility of the flow cell. An access port placed upstream and or downstream from the flow cell will meet this need.
A simple access port can be made from a T-barbed connector and a rubber sleeve stopper sold by Fisher Scientific. The narrow end of the sleeve stopper is pushed firmly over the stem portion of the T-connector. Once this is inserted into the line between the nutrient source and the flow cell, materials such as nutrients, other organisms, antimicrobics and dyes can be inserted into the system by clamping off the medium flow (up stream from the access port) and injecting the desired material through the access port and into the T-connector. Clamping above the port ensures that the material flows in the direction of the flow cell.
Materials can be removed down stream from the flow cell by inserting another such access port. To remove material, remove the rubber sleeve and allow the material to flow into a sterile container. Disinfect the T-connector with alcohol and reinsert the sleeve stopper.
Note: For a more secure fit, the cup of the sleeve stopper can be inverted back over the stem of the T-connector and this can be held in place with a 4-inch cable tie tightened with a tensioning tool. When tightening the strap around the base hold the sleeve tight to the T-connector so that the cable tie wraps around the stem below the barb. (See Figure 6 in the student instructions.)
If desired, all of the components of the apparatus may be autoclaved except the flow cell itself. The flow cell can be disinfected with alcohol.
It is a good idea to place the flow cell in a pan that can hold the entire volume of the culture or medium supply in the event of a leakage accident.
The cell may be set up in a recirculating or flow through system.
A recirculating system is among the easiest to set up. In this system a plastic tube from a culture (e.g. a hay infusion) is passed through the peristaltic pump. The access port is attached to the outflow from the pump and then to the flow cell. The effluent from the flow cell is directed back into the hay infusion. In this pattern no waste disposal container is necessary. An aquarium, a known culture, a pond water sample, or a sample from any natural water supply may be substituted for the hay infusion. Each will produce its unique biofilm in the cell.
A flow through system requires a source of inoculum, a medium supply and a waste container. The sterile flow cell system is connected to the medium supply and the medium flow is adjusted to the desired flow rate by adjustment of the peristaltic pump. After the system is filled with medium, the pump is turned off and the tube between the pump and the access port is clamped off. The culture of interest is injected into the access port and the system is allowed to incubate for a period of time for attachment of cells to the flow cell to occur, commonly over night. At this point the clamp is removed and the pump started so that the newly attached cells will receive nutrient.
Note: Caution and instruct students about the proper use of needles and disposal of sharps.
Note: Biofilms typically prosper at low nutrient concentrations, so 1/10 nutrient broth, TSB or LB broth is likely to produce a better effect than full strength medium.
Evaluation of the flow cell is made by the instructor’s visual inspection of each student's flow cell and its performance during operation.
Detachment, surface migration, and other dynamic behavior in bacterial biofilms revealed by digital time-lapse imaging
Stoodley P, Hall-Stoodley L, Lappin-Scott HM
Methods in Enzymology, (2001) 337:306-319
Microscopy flowcells: Perfusion chambers for real-time study of biofilms
Palmer RJ Jr
This exercise was This exercise was suggested by Dr. Paul Stoodley
Supported in part by the Waksman Foundation for Microbiology
Developed in collaboration with Dr. John Lennox, Penn State University-Altoona
© 1999-2008 Center for Biofilm Engineering, http://www.biofilm.montana.edu