Synergistic Effect of Quercetin with Allicin from the Ethanolic …
71
2 Materials and Methods
2.1 Microorganism
The biofilm-forming bacterial strain was collected from infected throat and was
cultivated in LB broth at 37 °C for 24 h. Characterization of the isolated organism
was done by performing 16S rRNA sequencing.
2.2 Preparation of Plant Extract
The ethanolic extract of the plant A. cepa (onion) was prepared by mixing 8 gm of
pulverized rhizome of the plant with 8 ml of 30% ethanol, followed by a treatment
with 700 µL of 80% H 2 SO 4 . After 24 h, the sample was filtered out using cotton
gauge filter and the filtrate was stored at 0–4 °C.
2.3 Biochemical Assay of Plant Extract
Biochemical assays of ethanolic extract of A. cepa were done for estimating total
flavonoid, total phenol, hydroxyl scavenging and hydrogen peroxide scavenging
activity (Debiyi and Sofowora 1978; Roopashree et al. 2008).
2.4 Minimum Inhibitory Concentration (MIC) and MBEC
Assay by Crystal Violet Method
The bactericidal and bacteriostatic effects of the bioactive compounds of A. cepa
(Onion) were detected on LB Agar spread plates for the strain of Bacillus cereus
using Azithromycin as standard antibiotic (Tang et al. 2011).
The antibiofilm effect of the ethanolic extract of A. cepa on B. cereus was checked
by microdilution method where bacterial cells (1 × 10
6 CFU/ml) were grown on the
wells (96 well-plate) leaving the control at 37 °C for 72 h to allow the formation
of biofilm (Tang et al. 2011). Diluted plant extract or antibiotic (Azithromycin)
were added with 3-[4,5-dimethyl-2-thiazolyl]-2, 5-diphenyl-2H-tetrazolium bromide
(MTT dye) to quantify the viability of the bacterial cells in the presence of the
challenge using ELISA plate reader (2018 GEN-NET). The percentage inhibition
was determined using the following equation:
[1-(A 570 of the test/A 570 of non-treated control) × 100] (Gomes et al. 2012)
71
2 Materials and Methods
2.1 Microorganism
The biofilm-forming bacterial strain was collected from infected throat and was
cultivated in LB broth at 37 °C for 24 h. Characterization of the isolated organism
was done by performing 16S rRNA sequencing.
2.2 Preparation of Plant Extract
The ethanolic extract of the plant A. cepa (onion) was prepared by mixing 8 gm of
pulverized rhizome of the plant with 8 ml of 30% ethanol, followed by a treatment
with 700 µL of 80% H 2 SO 4 . After 24 h, the sample was filtered out using cotton
gauge filter and the filtrate was stored at 0–4 °C.
2.3 Biochemical Assay of Plant Extract
Biochemical assays of ethanolic extract of A. cepa were done for estimating total
flavonoid, total phenol, hydroxyl scavenging and hydrogen peroxide scavenging
activity (Debiyi and Sofowora 1978; Roopashree et al. 2008).
2.4 Minimum Inhibitory Concentration (MIC) and MBEC
Assay by Crystal Violet Method
The bactericidal and bacteriostatic effects of the bioactive compounds of A. cepa
(Onion) were detected on LB Agar spread plates for the strain of Bacillus cereus
using Azithromycin as standard antibiotic (Tang et al. 2011).
The antibiofilm effect of the ethanolic extract of A. cepa on B. cereus was checked
by microdilution method where bacterial cells (1 × 10
6 CFU/ml) were grown on the
wells (96 well-plate) leaving the control at 37 °C for 72 h to allow the formation
of biofilm (Tang et al. 2011). Diluted plant extract or antibiotic (Azithromycin)
were added with 3-[4,5-dimethyl-2-thiazolyl]-2, 5-diphenyl-2H-tetrazolium bromide
(MTT dye) to quantify the viability of the bacterial cells in the presence of the
challenge using ELISA plate reader (2018 GEN-NET). The percentage inhibition
was determined using the following equation:
[1-(A 570 of the test/A 570 of non-treated control) × 100] (Gomes et al. 2012)