Quorum Sensing

5. Sterile scissor.


6. Sterile razor.


7. Sterile glass microscope slides.


8. Petri dishes.


9. Paraffin film.


10. Fresh plant extract (seeSubheading3.6).

2.5 Quantitation
of Aggregate
Formation by Crystal
Violet Assay

1. Cultures of the wild typeXhpand its QS mutants.


2. LB broth (seeSubheading2.1,item 3).


3. Minimal M9 medium [11]: 6 g/l Na 2 HPO 4 , 3 g/l KH 2 PO 4 ,
   1 g/l NH 4 Cl, 0.5 g/l NaCl, 1 mM MgSO 4 , 0.01 mM CaCl 2 ,
   200 mg/l methionine, 200 mg/l thiamine, 20 mg/l nicotinic
   acid, 2 g/l glucose, 8 g/l agar.


4. Fresh plant extract (seeSubheading3.6).


5. Spectrophotometer.


6. Incubator.


7. 24-well multidishes.


8. Crystal violet (0.1%) solution in water.


9. Ethanol 95%.

3 Methods

3.1 Bioassay
for Detection of DSF
Production

1. Scrub frozen bacterial culture from the glycerol stocks of
   Xanthomonas campestrispv.campestris8523 (biosensor strain)
   and the strains to be tested for DSF production (in this case
   Xhp305 and its QS mutants) with a sterile pipette tip or tooth-
   pick and inoculate glass tubes containing 3 ml LB broth sup-
   plemented with the appropriated antibiotics. Grow the
   bacterial cultures overnight at 28C with shaking at 150 rpm.


2. On the following day, transfer 100μl of each of the overnight
   cultures into 250 ml Erlenmeyer flasks containing 50 ml LB
   broth. Incubate at 28C with shaking at 150–180 rpm (see
   Note 4). Grow the cultures for approximately 5 h and then
   monitor the optical densities (OD) using spectrophotometer.


3. When the OD, measured at 595 nm, reaches 0.25–0.3 (early
   exponential phase of the growth), transfer 100μl of the bio-
   sensor culture and spread uniformly over surface of LB agar
   plates. Allow the plates to dry for 15 min by placing them
   uncovered in a biological hood.


4. After drying the plates, carefully lay the Whatman paper discs
   on the agar surface (Fig.3a)(seeNote 5).

246 Shulamit Manulis-Sasson and Laura Chalupowicz