For qualitative AHL detection, the CV026 strain was used. This strain produces the purple pigment violacein in the presence of AHL compounds with N-acyl C4-C8 side chains (McClean et al., 1997). A standard disc-diffusion assay was used to detect inhibition of AHL production by phloretin, using the procedure described by Hossain et al. (2017) with slight modifications. Pb1692 and CV026 bacteria that had been grown for 16 h, 28°C were washed by centrifuging at 14,000 rpm for 5 min and the supernatant was discarded. Then, both CV026 and Pb1692 were suspended in fresh LB at a concentration of 5 × 106 CFU/mL. A small circular ring was made of Pb1692 and an outer, bigger ring was made with CV026; a paper disc was mounted in the middle of the inner ring. Thirty μL of each concentration of phloretin was gently poured onto the paper disc and dried for 30 min under a hood. Then, the plates were incubated at 28°C and the intensity of the purple pigment produced by the reporter strain was assessed.

The production of AHL was also quantified based on the ability of phloretin to inhibit the production of purple violacein pigment by CV026, as described by Hossain et al. (2017). Pb1692 at a concentration of 106 CFU/mL was cultured in fresh LB containing different concentrations of phloretin (0.1, 0.2, and 0.4 mM) and incubated for 48 h at 28°C with continuous shaking (150 rpm). The culture was then centrifuged (5223 g, 10 min, 4°C) and the supernatant was transferred into a new Eppendorf tube. CV026 that had been grown for 16 h, 28°C was washed and suspended at a concentration of 5 × 106 CFU/mL in fresh LB with an appropriate amount of antibiotic (kanamycin at 10 μg/mL). A total 500 μL of CV026 was placed with 500 μL of supernatant in a 1.7-mL Eppendorf tube and incubated for 24 h at 28°C with continuous shaking (150 rpm). The culture was then centrifuged (14,000 g, 10 min) to precipitate the insoluble violacein. The culture supernatant was discarded and the pellet was evenly re-suspended in 1 mL of dimethyl sulfoxide (DMSO). The solution was centrifuged (13,793 g, 10 min) to remove the cells and the violacein was quantified at a wavelength of 585 nm in a microplate reader (Spectra MR, Dynext Technologies, VA, United States).

The percentage of violacein inhibition was calculated using the following formula:

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.