Detection and Inhibition of Bacterial Cell–Cell Communication - Bacterial Pathogenesis: Methods and Protocols - page 55

9. To prepare the ABt medium, add one part of Solution A to nine parts of Solution
B. Add glucose and casamino acids to a final concentration of 0.5%. Store at
room temperature away from direct sunlight ( see Note 1 ).
2.1.2. Signals and Antagonists
1. Stock solution of N -3-oxo-hexanoyl homoserine lactone (OHHL) (Sigma,
St. Louis, MO, USA), 10 μ M dissolved in dimethylsulfoxide (DMSO). Store
at -20°C.
2. High-performance liquid chromatography (HPLC) grade dichloromethane (DCM)
( see Note 2 ).
3. Stock solution of sample extract, resuspended to 5 mg/mL in 100% ethanol ( see
Note 3 ).
2.2. Inhibition of the AHL-Regulated Phenotype of Elastase
Production
1. Elastin-Congo Red: Elastin-Congo Red buffer, 0.1 M Tris-HCl (pH 7.2), 1 m M
CaCl 2 , Elastin-Congo Red (Sigma), 20 mg of powder per reaction tube containing
1 mL of Elastin-Congo buffer. The powder is insoluble in the buffer, and hence,
each reaction tube has to be measured separately.
2.3. Detection and Inhibition of AI-2 Quorum Sensing
1. Autoinducer bioassay (AB) medium (according to ref. (16) ): Solution A—0.3 M
NaCl, 0.05 M MgSO 4 , 0.2% vitamin-free casamino acids (Difco, Franklin Lakes,
NJ, USA). Make1Lofsolution A by adding the reagents above to the indicated
final concentrations in dH 2 O. Adjust pH to 7.5 with KOH, autoclave, and cool.
Add to solution A, 10 mL of 1 M potassium phosphate buffer (pH 7.0), 10 mL
of 0.1 M l-arginine, 20 mL of glycerol, 1 mL of riboflavin (10 μg/mL) and 1 mL
of thiamine (1 mg/mL).
2. Hemolysin buffer: 10 m M Tris-HCl (pH 7.2), 0.15 M NaCl, 10 m M MgCl 2 ,10m M
CaCl 2 , 0.2% (w/v) gelatin. Add the reagents above to the indicated final concen-
tration in dH 2 O. Sterilize by autoclaving.
3. Lauria Broth 20 (according to ref. (17) ):To1LofdH 2 O, add 10 g of tryptone,
5 g of yeast extract, and 20 g of NaCl. Autoclave.
3. Methods
3.1. AHL Signal, Inducer, or Antagonist Extraction and Preparation
In many cases, one may be interested in recovering either quorum-sensing
signals from particular bacteria or potential quorum-sensing inhibitors (QSIs)
from eukaryotic organisms or from microorganisms. For the isolation of QSIs
from eukaryotes, whole materials are normally extracted for active compounds,