4.A. tumefaciens KYC6 (pCF218)(traM-tn5-gusA,KmR)
(3-o-C8-HSL overproducing strain used as positive control
forA. tumefaciensAHL bioassay) [51].- 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.
- ATGN medium (gives increased sensitivity forA. tumefaciens
bioassay) [52] (composition described in Subheading2.2.1). - Glass or metal spreading rod and 250–500 ml beaker filled to a
depth of 2–3 cm with 100% ethanol: Alcohol sterilize spreading
rod before use by dipping in 100% ethanol and flame (caution:
flammable—if ethanol in beaker catches fire, cover the top of
beaker with a suitable object, such as a book, in order to
quickly smother the flame). - 20 mg/ml X-gal in dimethylformide: Add 50μl and spread
over surface of agar plate prior to inoculating bacteria for
bioassay. - Other agar as appropriate for organism to be investigated (as a
cautionary note, ensure that this medium does not inhibitA.
tumefaciens). - 50C Water bath (needed for overlay assay).
- Test tubes with caps and rack for preparing soft agar (5 ml
aliquots). - Vortex mixer.
- Pipette and sterile tips (adjustable 10–200μl).
2.2.1 ATGN Medium
for Enhanced
A. tumefaciens
Bioassay [52]
- Prepare and autoclave the following stock solutions:
(a) 20AT buffer: Mix 214 g of KH 2 PO 4 in 800 ml H 2 O
(adjust pH to 7.0 with approximately 35 g solid NaOH),
and then bring the volume to 1000 ml with H 2 O.As a
safety precaution, please use suitable eye protection
when handling solid NaOH.
(b) 20ATsalts: 40 g/l (NH 4 ) 2 SO 4 , 3.2 g/l MgSO 4 ·7H 2 O,
0.2 g/l CaCl 2 ·2H 2 O, 24 mg/l iMnSO 4 ·H 2 O.
(c) 100 Iron stock (2.2 mM): Dissolve 0.611 g
FeSO 4 ·7H 2 O in 1 l of 10 mM HCl to prevent
precipitation.
(d) 50% Glucose: Dissolve 50 g of glucose in 100 ml H 2 O. - 1ATGN medium (per liter): Mix 50 ml 20AT buffer,
50 ml 20AT salts, 10 ml 50% glucose, and 10 ml 100
iron stock. If needed, add 15 g agar. Bring total volume to
1000 ml with distilled water and autoclave.
Bioassays of Quorum and Biofilm Dispersion Signals 13