Quorum Sensing

7.Day Three: Examine and photograph plates. A positive result is

indicated by a loss of pigmentation in the vicinity (seeNotre 6).

An example is shown in Fig.3c.

3.2.3 Alternative Plate-
Based Assays

The aforementioned assays in Subheadings3.2.1and 3.2.2are

useful at screening various microorganisms and other tissues for

AHL- and QS-inhibiting materials. QS bioassays also lend them-

selves to the examination of other substances including plant and

food extracts [56]. Here, one can examine pigment induction in an

AHL-responsive strain (CVO26), or pigmentation inhibition

(strain 12472). Here the test material can be placed onto a small

sterile disk of filter paper (we typically use a three-hole office punch

to generate these, and then autoclave before use):

1. Prepare overnight broth cultures ofChromobacteriumstrains as
   described instep 3, Subheading3.2.2(use CVO26 for pig-
   ment induction, and 12472 for pigment inhibition).


2. Take 100μl overnight culture and spread over the surface of
   LB agar (forming a bacterial lawn).


3. Using alcohol-sterilized forceps, place filter paper disk (con-
   taining test substance) onto plate. The substance can be added
   to the filter paper disk with a pipet after the disk has been placed
   onto the agar.


4. Incubate overnight at 30C and examine for pigmentation.
   Again the effect should be most noticeable in the vicinity of the
   test material.


5. As an alternative, one can use two reporter strains simulta-
   neously [57] and investigate whether QS inhibition might be
   due to interference with AHL production (luxI-effect) or a
   transcription response (luxR-effect). In the case of the C.
   violaceumbioassay, two reporters are used: 31532 (C6-HSL
   overproducer) and CVO26 (AHL biosensor). Overnight cul-
   tures of the reporters are prepared as described above and the
   samples inoculated as shown in Fig.2b, c.


6. If larger amounts of the test substance need to be delivered,
   100 μl of an aqueous solution of the compound can be added
   to the agar plate rather than using a paper disk. Use the wide
   end of a 200μl pipette tip to create a 6 mm punch in the agar
   plate after plating the lawn. Add 100μl of the aqueous solution
   to the punch, and incubate for 24 h at the appropriate temper-
   ature (Fig.2d). Ensure that replicate plates are of uniform
   thickness by adding the same amount of agar media to each
   poured plate.

The test material is shown as a darkened circle within the Petri

plate, the bioassay strain CVO26 is shown as a solid line, and the

C6-HSL-producing strain 31532 is shown as a dashed line.

Bioassays of Quorum and Biofilm Dispersion Signals 17