Tube Biofilm

(P. aeruginosa lasB::gfp strain)

Contact: Mary Jo Kirisits

Part I - Tube Biofilm Preparation

1. The night before the experiment, prepare the strain. Autoclave 1/8 strength LB medium.  Pour 20 mL of 1/8 LB into three sterile 50-mL erlenmeyer flasks.  Add a scraping of uninduced lasB::gfp freezer stock to one flask.  (This is vial 6 in my strain box.)  Vortex thoroughly.
2. Prepare serial dilutions in the other two flasks - 1 mL culture + 20 mL 1/8 LB. Incubate with shaking at 37 ^oC overnight.
3. The following morning, examine the flasks. The first flask may contain clumps of cells.  If it does, use second or third dilution if the culture looks more homogeneous.
4. Subculture from one of the homogenous-looking cultures - 1 mL culture + 20 mL 1/8 LB. Monitor the OD_600nm.  When it reaches 0.1 in a 200-mL sample in a microtitre plate, it is ready to be used as an inoculum (step 15). 
5. Verify that the inoculum is dark. (This only needs to be done for a strain with a GFP reporter.) Take 1 mL of inoculum and centrifuge at 9,000 rpm for 6 minutes.  Remove the LB with a pipet, and wash in 1 mL of 1X PBS.  Centrifuge again at 9,000 rpm for 6 minutes.  Remove the PBS with pipet and add a fresh 1 mL of PBS.    View under the microscope to verify that GFP is not induced.  Aliquot 200 μL into a microtitre plate, and determine the RFU and OD₆₀₀. RFU/OD should be much less than 10⁵.  10⁴ is a good value to attain for lasB::gfp using a sensitivity of 120 for fluorescence.
6. Set up parallel silicone tubing for the number of experiments desired (usually 8).
7. Cut 3-in or 8-in pieces of silicone tubing (0.094-inch ID) in which the biofilms will be grown.
8. From the influent reservoir, tubing leads to the 8 parallel pump tubings. The pump tubings are connected to the bubble traps.  The bubble traps are connected to the self-sealing red inoculum tubes at a tee; use cable ties to secure the red inoculum tubes.  The inoculum tubes are connected to the biofilm tubing.  The biofilm tubing is connected to a short stretch (6-in) of waste tubing. Autoclave this entire system. 
9. Prepare 0.6% hydrogen peroxide, which should be made fresh before every use. Hydrogen peroxide is a strong oxidant and will degrade over time, especially if not covered in foil. (10 mL 30% H₂O₂ / 500 mL).
10. Flush system with hydrogen peroxide solution at 14 rpm for 2 hours. Fill the bubble traps.
11. Switch from hydrogen peroxide to sterile water, using aseptic technique in the transfer. Flush the system with sterile water at 14 rpm for 2 hours.  Fill the bubble traps.
12. Switch from sterile water to sterile LB, using aseptic technique in the transfer. Flush the system with sterile LB at 14 rpm for 2 hours. Fill the bubble traps.
13. Stop the flow. Tightly clamp the silicone tubing just upstream of the inoculation site.
14. Thoroughly clean the inoculation site with ethanol.
15. Inoculate each tube with the dark inoculum (OD_600nm = 0.100). For 3-in biofilm tubing, use a 0.5-mL inoculum; for 8-in biofilm tubing, use a 1-mL inoculum.  Do not inject air.
    1. To maintain consistency between experiments, the OD of the inoculation culture should be 0.100 (200 μL in the microtitre plate).  If the OD of the inoculum is slightly greater than 0.100, you can dilute the culture (Volume_culture at OD_current) with sterile LB (Volume_{fresh LB})according to the following relation, which will yield 10 mL of culture at an OD of 0.100:
16. Inoculate as quickly as possible, and allow the inoculated tubing to sit stagnant for 30 minutes.
17. With pump OFF, adjust the rpm for the experiment. Then unclamp the tubes and turn the pump ON. 
18. Check the flowrate after initiating the experiment and prior to each harvest.
19. Harvest biomass from tubes (see Part II) at various times during the experiment. After harvesting, place the end of the tubing from the bubble trap into a petri dish of ethanol.  This will help to keep the system clean for the remainder of the experiment.

Part II - Harvesting Tube Biofilm

1. Remove the biofilm tubing (3- or 8-in length) from the flow path. Cut off the sections of tubing where the connectors attach.  Measure the tubing length.
2. Cut the tubing into 2 or 3 pieces with a sterile blade. Wipe the outside of the tube with ethanol, and let it evaporate to dryness.
3. For 3-in biofilm tubing, place the tube pieces into a sterile microfuge tube. For 8-in biofilm tubing, place the biofilm tube pieces into a sterile 50-mL Falcon tube.  Add 800 mL of 1 x PBS.
4. Sonicate- 1 min. Ice -1 min.  Vortex on the highest setting - 1 min.  Repeat these steps a total of 4 times.
5. Remove the biofilm tube pieces, and place them in a new sterile tube.
   1. If you are using a microfuge tube, centrifuge at 9,000 rpm (7,500 x g) for 5 min to remove residual liquid or cells from the silicone tubing.
   2. If you are using a Falcon tube, you must use the Sorvall centrifuge (L382) with the SLA-1000 rotor and adaptor for the 50-mL Falcon tube.  Centrifuge at 9,500 rpm (< 7,000 x g) for 5 minutes to remove residual liquid or cells from the silicone tubing. 
   3. After centrifugation, carefully remove the biofilm tube.  Use a pipet to transfer the liquid to the other tube (containing the rest of the cells + 800 μL 1 x PBS).
6. If this is an Aearly@ timepoint (i.e., < 48 hours), multiple biofilm tubes can be combined into a single time point. It is sometimes useful to combine tubes into a single harvest so that there is enough harvested biomass for meaningful OD and RFU measurements.
7. Thoroughly vortex the tube containing the cells for 1 min. Then sonicate, ice, and vortex, 1 min each, for a total of 2 times.  Make sure that the culture looks homogeneous.  If a lot of clumps are still observed, continue the cycle (sonicate, ice, vortex) until the culture looks homogeneous.
8. Record the total liquid volume of this tube.
9. Remove 10 μL of the cell suspension for CFU enumeration.
10. Prepare a microtitre plate with 90 μL of 1 x PBS. Add the 10 μL (step 9) to the first well, and then do serial dilutions.  Prepare dilutions from 10^-1 to 10^-8.  (Fewer dilutions may be required at early time points, when the biomass coverage is low.)
11. Spot 10 μL of each dilution, in triplicate, to LB plates. Incubate upside down at 37 ^oC overnight.
12. The cell suspension still contains LB; this must be removed because it will interfere with RFU measurements.
    1. If you are using a microfuge tube, the cells must be centrifuged at 9,000 rpm (7,500 x g) for 8 min.  Remove the supernatant with a pipet. 
    2. If you are using a 50-mL Falcon tube, centrifuge at 9,500 rpm (< 7,000 x g) for 8 min.  Remove the supernatant with a pipet.
13. Resuspend the cells in 1x PBS. Add 1 mL 1x PBS or less if a low OD is expected.  (For meaningful results, 0.1<OD<0.3.  If the OD>0.3, add more PBS, and check the OD again.)
14. Record the total volume (Vol_Total). Vortex the tube containing the cells for 1 min.  Sonicate, ice, and vortex, 1 min each, for a total of 2 times.
15. Remove 10 μL of the cell suspension for CFU enumeration. As before, add 90 μL PBS to microtitre wells, and do serial dilutions.  Plate 10-μL aliquots onto LB plates, and incubate upside down at 37^oC
16. Add 200 μL of the cells resuspended in PBS to a microtitre well; blank with an equal volume of PBS. Read the OD and RFU of the microtitre samples. Optimal OD is around 0.3.  Use a sensitivity of 120 for the chromosomal strain (100 for the plasmid strain) for the RFU measurement.
17. Make a wet mount of each harvest and view it under the microscope. (epifluorescent: Hg lamp, gain=4, exposure=5s).
18. The next day, count colony forming units (CFUs) for each dilution, where possible. For the limited spot size, approximately 20 organisms is an optimum number.
19. Calculate the number of organisms per tube surface area. Average the triplicate colony counts (N) for the dilution you wish to use.  Vol_Total is the total harvest volume; this is usually $1000 μL  Vol_spot is the volume of the dilution that is plated onto the solidified LB medium (usually 10 μL).  Dilution refers to how diluted this sample is relative to the cell harvest (e.g., 10^-5).   SA refers to the surface area of the biofilm tube in mm²; this can be calculated from the tube diameter and length (recorded in the first step of this section).
20. At the very end of the experiment, run hydrogen peroxide solution through all of the tubing and bubble traps. However, if growth has occurred in the bubble traps, autoclave the entire system at the end of the experiment, and then thoroughly wash the bubble traps.