Quorum Sensing

3.6 Determination
of Aggregate
Formation by Confocal
Microscopy

Another approach to study the effect of QS on virulence is to

determine its effect on aggregate formation. InXanthomonasspp.

the reversible transition from biofilm to planktonic forms is

mediated by DSF. It affects the attachment to the plant cells,

migration, and colonization within the host.

1. Grow overnight cultures of GFP-labeled strains (wild type and
   its QS mutants) in LB broth until stationary phase (in case of
   Xhpafter 48 h).


2. Centrifuge the bacterial cultures for 1 min.


3. Discard the supernatant and wash twice the remained pellets
   with sterile water.


4. Prepare plant extracts as follows: cut 2 g plant stem sections (2-
   week-old pelargonium plants), place in plastic bags, and crush
   until plant tissue is completely disintegrated. Add 5 ml of
   distilled water and shake with a rotary shaker at 80 rpm for
   5 min. Filter the plant extract though a syringe-driven mem-
   brane filter.


5. Resuspend the pellet obtained instep 3by adding 3 ml of
   filtered plant extract. Vortex for approximately 1 min until the
   pellet is completely dissolved.


6. Load 200μl of the bacterial suspension on the surface of a
   sterile glass microscope slide and place it inside a Petri dish
   containing a wet filter paper.


7. Seal the Petri dish with paraffin film to avoid water evaporation
   and incubate at 28C.


8. After 3–7 days examine biofilm formation with a confocal laser
   scanning microscope.

3.7 Measurement
of Aggregate
Formation by Crystal
Violet Assay

Crystal violet assay has extensively been used to examine aggregate

formation developed by phytopathogenic bacteria [12–14]. Here

we present an example for quantitation of aggregate formation

when grown in plant extracts or minimal medium, which mimics

the apoplast (plant intercellular space) environment, in comparison

to rich media.

1. InoculateXhpand its QS mutants from frozen stocks in 3 ml of
   LB broth and incubate at 28C for 48 h.


2. Dilute culture 1:1000 in fresh LB medium and grow until
   reaching an OD 595 ¼0.5.


3. Centrifuge the cells, discard the supernatant, wash once the
   remained pellet with sterile distilled water, and then resuspend
   in 3 ml with sterile water.


4. Pipette in individual wells of multidish, 150μl of LB, minimal
   M9 media or fresh pelargonium extracts.

Quorum Sensing-Dependent Virulence of Phytopathogens 249