Contact: Mary Jo Kirisits

Day 1

  1. Start a 5-mL culture of your strain and place in the 37ºC shaker.
  2. Set up the spinning disk system with 1400-2000 mL of medium in the medium bottle.  Place clamps after the medium bottle, before the reactor, and on the effluent tube of the reactor.  Autoclave the system.
  3. Set up the spinning disk: wash the 18 biofilm chips once with ethanol, 3 times with water, and then place the chips into the spinning disk.  Wrap the spinning disk in foil.  Autoclave, but if you autoclave the disk on the liquid cycle, place the foil-wrapped disk inside a pipet tip box to protect it from liquid. 
  4. Open the clamps between the reactor and medium bottle, and run ~250 mL of medium into the reactor.  Replace clamps afterwards.
  5. Towards the end of the day inoculate the reactor with 1 mL of the 5-mL culture.
  6. Stir the reactor at 2.5 overnight at room temperature.

Day 2

  1. Start the flow in the morning by unscrewing all the clamps to allow flow through the entire system, and start the pump at 1.6 rpm in the clockwise direction.

Day 3

  1. In the morning, stop flow, and remove the reactor from the stir plate.  Wipe down a stir bar retriever with ethanol, and remove the stir bar from the reactor. 
  2. Carefully peel back the foil packet from the autoclaved spinning disk until you are holding a mostly-exposed spinning disk, and then drop this into the tilted reactor. **Stir bar part of the disk should be on the bottom**
  3. Return the reactor to the stir plate, and resume flow and stirring.

Day 4

  1. In a microtitre plate, add 100 μL of medium to wells in rows B-H for the first four columns.  (If you used a rich medium, such as LB, in the reactor, use 1X PBS in the microtitre plate.  You do not want the cells to double during the incubation with metal/antibiotic [step 3].)  In row A, add the appropriate amount of antibiotic/metal solution to the medium for a total of 200 μL.  For example, for a concentration of lead of 1 mM in row A, add 40 μL of 4.88 mM lead solution to 160 μL of medium.   Mix the wells in row A, and transfer 100 μL from row A to the well in row B.  Continue until row G; transfer 100 μL from row G into the waste, and leave row H with 100 μL of medium only.
  2. 24 hours after the spinning disk was added to the reactor, stop the spinning disk reactor and take a 1 mL sample of the planktonic culture.  (Note:  You may need to dilute this 1-mL sample.  You want to expose the same amount of planktonic biomass and biofilm biomass to the metal/antibiotic stress in the microtitre plate.)  Remove the disk by reaching into the reactor and pulling it out without touching the chips.  Remove the chips from the disk with sterile tweezers and dip in fresh medium to remove loosely attached cells.  Place chips in columns #1 & #2 of the microtitre plate.  Transfer 10 μL of the planktonic culture into columns #3 & #4 of the microtitre plate and mix well.
  3. Incubate plate at 37ºC for 5 hours.
  4. Transfer the chips into 1 mL of PBS in eppendorf tubes.  Transfer 100 μL of the planktonic cultures into 900 μL of PBS in eppendorf tubes.
  5. Sonicate all tubes for 10 minutes and then briefly vortex.
  6. Do serial dilutions from each tube by taking 10 μL of cells from the eppendorf tubes and mixing it into 90 μL of PBS.  Plate 10 μL spots of each dilution in duplicate onto dry LB plates.
  7. Place plates into 37ºC incubator overnight.

Day 5

  1. Remove plates from incubator and count colonies.

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