by

Marcus Bryan Sellers

This exercise will enable the students to observe the development of a biofilm in real time under constant microscopic examination. This demonstration models the accumulation of biofilms in pipelines and water mains, a significant industrial problem.

• Build the circuit board as shown in the diagram. The materials required are listed in the appendix. The circuit board is an Emitter Follower Regulator. This device first steps down the voltage received from the AC/DC converter from 5 V to 1.5 V. Then, through adjusting the potentiometer, it permits the voltage to the pump to vary from 0 V to 1.5 V.

• Drill four holes in a plastic refrigerator container as follows:

• Connect the tubes as shown in the drawing. It is important that the outflow tube from the pump to the 3-way connector and the overflow tube be made of nylon tubing and that the outflow tube from the 3-way be flexible tubing. An overflow tube leading from the pump outlet back to the container reservoir was found to be necessary because without it the pressure in the capillary tube became excessive.

• To build the capillary tube assembly: Insert each end of the hollow rectangular capillary tube into two separate 8 cm lengths of rubber tubing and glue each end with the space-filling Super Glue®. Be careful not to occlude the lumen of the capillary tube with Super Glue® and make sure the connections are completely sealed. Do one side at a time and allow time for the glue to dry before gluing the other end. This is the capillary tube in which the biofilm will form and be observed.

• Connect this capillary tube assembly to the end of the flexible rubber tube coming from the pump with one of the 2-way connectors. Connect the other end of the capillary tube assembly to the nylon return flow tube with another 2-way connector.

• Mount the capillary tube assembly on a 1 x 3-inch microscope slide. This will require building a base, which will support the capillary tube and permit clearance for the plastic tubing to which it is connected. In our experience, two layers of microscope slide glass are just about right.

• Attach the mounted capillary tube assembly to the base just constructed with plumber's putty. Place the base plate onto the stage of a bright field or phase contrast microscope. Secure the free ends of the flexible tubing to the base plate mount with plumber's putty to keep the tubing from twisting.

• Once the glue has dried completely test the integrity of the system by placing about
  3-4 cm water into the container, connecting the circuit board to a 5 V AC/DC transformer and turn it on. There must be no leakage around the capillary tube connections.

• Once the system is watertight establish a microbial culture in the container (50 ml of a hay infusion works well).

• Do not leave the microscope light on when not in use. The heat will kill the microbes.

• After about a week, a rich biofilm will be seen growing on the inner surface of the capillary tube. The top and bottom layers of the biofilm are readily observed through the capillary wall. The flow rate of the liquid through the capillary tubes can be observed and is clearly non-laminar. The flow rate near the capillary wall is considerably slower than that closer to the center of the capillary lumen.

• Keep the output line from the pump at about the same level as the capillary tube. Both the Overflow and Return line should be above the level of liquid in the reservoir and the pressure in the lines, when not in use, should be enough to have a good rate of flow out of both lines.

• Have Fun.

• We have found that the thickness of the capillary wall is greater than the focal length of our oil immersion lenses. We DO NOT recommend the use of standard oil immersion lenses for this reason. All of our work was done at high dry magnification. Thinner flat capillary tubes are available from Friedrich & Dimmock, but we have not yet tried them.
• The pump will last for approximately 2 weeks of continuous use at less than 1.5 V. The pumps lifetime is inversely proportional to voltage over time, and to the extent of bioflim accumulation within the pump.
• The circuit board will typically last for about 2 weeks in continuous use, or about 6 weeks in intermittent use.
• If nothing can be seen through the microscope while the pump is running, try slowing down the flow by adjusting the potentiometer on the circuit board. Slow moving organisms are easier to see than faster ones. If still nothing can be seen, use bigger organisms.