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Contact: Mary Jo Kirisits

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1. In a microtitre plate, add 100 mL 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 mL μL.  For example, for a concentration of lead of 1 mM in row A, add 40 mL μL of 4.88 mM lead solution to 160 mL μL of medium.   Mix the wells in row A, and transfer 100 mL μL from row A to the well in row B.  Continue until row G; transfer 100 mL μL from row G into the waste, and leave row H with 100 mL 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 mL 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 mL μL of the planktonic cultures into 900 mL 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 mL μL of cells from the eppendorf tubes and mixing it into 90 mL μL of PBS.  Plate 10 mL μL spots of each dilution in duplicate onto dry LB plates.
7. Place plates into 37ºC incubator overnight.

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