Quorum Sensing

flask. Allow the culture to grow for 18–20 h at 37Conan

orbital shaker at 180 rpm.

3. Transfer the bacterial overnight culture to two 50 ml centrifuge
   tubes and centrifuge for 10 min at 5000g. Discard the
   supernatant and re-suspended the bacterial pellet in 4.5 ml
   LB medium.


4. Mix 0.5 ml re-suspended bacterial pellet with 4.5 ml sterile
   protanal for production of beads.


5. When making the beads (seeSubheading2.2,item 4) the
   droplets are directed into a cross-linking solution of 0.1 M
   CaCl 2 in TRIS-HCL buffer (0.1 M, pH 7.0). Allow the beads
   to cure for 1 h in the calcium bath with continuous stirring at
   minimum rotation.


6. Transfer the beads to two 50 ml centrifuge tubes and centri-
   fuge for 10 min at 600g.


7. Discard the supernatant and wash the beads in 0.9% (wt/vol)
   NaCl. Centrifuge for 10 min at 600g. Do this twice.


8. Re-suspend the beads in 7.5 ml 0.9% (wt/vol) NaCl using a
   5 ml pipette. Homogenize the beads gently using a 5 ml
   pipette, then a 1 ml pipette, and last a 200μl pipette to avoid
   clumps. DO NOT USE AVORTEX FOR MIXING. Make two
   tenfold serial dilutions using two different pipette tips.


9. Plate appropriate dilutions using sterile glass spreaders on blue
   plates. Plate each dilution double. Store the beads at 4C until
   next day and incubate the plates at 37C.

3.2 Infection
Procedure

1. Count the plates and calculate the colony forming units
   (CFU)/ml.


2. Right before challenge, adjust the beads containingP. aerugi-
   nosaand the beads containing theE. colimonitor strain to the
   appropriate concentration in 0.9% (wt/vol) NaCl (seeNote 1).


3. The bead solution containing both bacteria is mixed at ratio 1:2
   (P. aeruginosa:E. coli).


4. Use epifluorescence or CSLM to verify that the beads are not
   fluorescencing green and thus insure that the monitor is not
   turned on before infection.


5. There should be three groups of mice in an experiment using a
   P. aeruginosastrain and one AHL monitor strain. One group of
   mice is infected withP. aeruginosaalone, one group of mice is
   infected with the AHL monitor strain alone, and one group of
   mice is infected with the mixture ofP. aeruginosaand the AHL
   monitor strain (seeNote 2).


6. Sedate the mice using 10 ml/kg body weight Hypnorm®/
   midazolam (seeSubheading2.4,item 1) injected subcutane-
   ously (s.c.) in the groin area (seeNote 3).

208 Louise Dahl Hultqvist et al.