2.3 Chromo-
bacterium violaceum
AHL Detection
(See Note 1)
1.C. violaceumCVO26 (biosensor strain) [11].
2.C. violaceumATCC 31532 (C6 HSL overproducer, used as
positive control) [11].- Luria-Bertani broth (LB): 10 g/l NaCl, 10 g/l tryptone, 5 g/l
yeast extract. - LB agar: LB plus 1.5% (w/v) agar.
- LB soft agar: LB plus 0.3% (w/v) agar.
- Other agar as appropriate for organism to be investigated (as a
cautionary note, ensure that this medium does not inhibit
C. violaceum). - Other materials as shown in Subheading2.2 (above).
2.4 Indirect C.
violaceum Assay
for AHLs and QS
Inhibition
1.C. violaceumATCC 12472 type strain used for pigmentation
inhibition [33].
2.P. aeruginosaPAO1 (used as positive control) [33].- Other materials as listed in Subheading2.2 (above).
2.5 Thin-Layer
Chromatography (TLC)
Detection of AHLs
- Ethyl acetate (containing 0.1% (v/v) acetic acid) (store in the
dark in volatile chemical storage). - C18 TLC plate (seeNote 2).
- C6-HSL and C8-HSL standards (seeNote 3), dissolve 5μMin
ethyl acetate. - TLC glass tank (seeNote 4).
- Paper towels: We use these to line tank during run, and the
resulting elevated humidity within the tank will prevent an
uneven migration of solvent front (“smile” or “frown”
pattern). - Methanol/water mixture (60:40)—need at least 200 ml.
- Laboratory-adjustable temperature hot plate.
- Labeling tape (approximately 2 cm wide).
- Plastic tub and cover (big enough to incubate TLC plate with
indicator bacteria). - Incubator (30C).
11.A. tumefaciensA136 (pCF218)(pCF372) bioassay strain. - X-gal solution (20 mg/ml in dimethylformamide).
2.6 Biofilm
Dispersion Screen
- Sterile microtiter plate with lid.
- Appropriate growth media for organism being tested.
- Test organism(s).
- Candidate-dispersing agents.
- Incubator with orbital shaker.
14 Starla G. Thornhill and Robert J.C. McLean