Quorum Sensing

3. Grow the bacterial cultures at 37C with shaking at 200 rpm
   until an A 600 ffi2.0.


4. Record the A 600 of the bacterial cultures and recover culture
   supernatants by centrifugation at 17,000
   gfor 10 min.


5. Mix 1 mL of culture supernatants with 2 mL of Elastin-Congo
   Red suspension and incubate at 37C for 16–18 h with shaking
   at 250 rpm.


6. Centrifuge at 17,000
   gfor 10 min, transfer the supernatants
   to disposable cuvettes, and measure the absorbance at 495 nm
   wavelength (A 495 ). If the purified anti-AHL polyclonal sera are
   functional in repressing QS, A 495 values are expected to be
   lower in the samples derived from theP. aeruginosacultures
   treated with the anti-AHL sera with respect to samples derived
   from theP. aeruginosacultures treated with pre-immune and
   nonspecific antigen immunized polyclonal serum.

3.2.2 Nematode Killing
Assay Using Anti-QS
Polyclonal Sera

This assay can be performed using any wild-typeCaenorhabditis

elegansstrain. We routinely use the wild-type Bristol N2 strain and

follow the protocol described in [11].

1.C. elegansis maintained routinely on NGM agar plates seeded

with lawns ofE. coliOP50 for 3–4 weeks or longer. When

healthy mixed population of adults and larval stages are

required, small “agar chunks” with worms are transferred

onto new 90 mm NGM agar plates containing fresh lawns of

E. coliOP50. The “chunked” plates are kept upside down in a

plastic box with slightly opened lid to prevent build-up of

condensation and incubated at 15C for 5 days.

2. The worms from agar plates are resuspended in 5 mL of M9
   and centrifuged for 2 min at 600
   gin 15 mL polypropylene
   tubes.


3. The supernatant is removed carefully without disturbing the
   worm pellet and 1 mL of nematode bleaching solution is added
   to the tubes and incubated for no longer than 5 min at room
   temperature with continuous shaking.


4. The tubes are centrifuged at 600
   gfor 2 min, and the
   supernatant containing bleach solution is removed.


5. The worm pellet is washed by adding 1 mL M9 buffer. The
   suspension is centrifuged at 600
   gfor 2 min.


6. The worm pellet is finally suspended in 100μL M9 buffer and
   transferred to fresh sterile NGM agar plates and left overnight
   at 25C. Bleaching kills all the adult worms and therefore
   triggers a synchronized population of newly hatchedC. elegans
   at the L1 larval stage that the following day will be ready for the
   slow killing assay.

336 Soumya Palliyil