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Chapter III Material and methods
Approximately 10 9 CFU/ml bacterial culture was suspended in one ml DMEM medium
(without serum and antibiotics) and added to different wells. The plate was incubated at 37°C
for 2 h in the presence of 5% CO2 in incubator. The monolayer was washed with sterile PBS
and the cells were detached by trypsinization. one ml of 0.25% Trypsin-EDTA solution
(Sigma. USA) was added to each well of six-well plate which was then incubated for 15 min
at room temperature. The cell suspension was platted on MRS agar by serial dilution for
determining the adherent bacterial cells. The plate was incubated for 24-48 h at 37°C and
colonies were counted. Bacterial cells initially added to each well of six-well plates were also
counted by serial dilution and plating on MRS agar. The results of the adhesion assay were
expressed as adhesion percentage, the ratio between adherent bacteria and added bacteria per
well. Three independent experiments (n = 3) with two replicates in each experiment with
Caco2 cells of same passage were carried out.
3.9 Screening of Lactobacillus spp. for Bacteriocin Production
Cell-free supernatants (CFS) of Lactobacillus were obtained by centrifuging the
cultures at 12000 x g for 10 min, collecting the supernatants, which were adjusted to pH 6.5
with 1 M NaOH, and filtered through a 0.22 μm filter (Millipore). Inhibitory activity from
hydrogen peroxide was avoided by the addition of catalase (5 mg/ml). Bacteriocin production
activity was determined using the well diffusion method (Motta and Brandelli, 2002) by
using Aeromonas hydrophila ATCC 7966, Yersinia enterolitica MTCC 840, Staphylococcus
aureus ATCC 9144, Enterobacter (Chronobacter) sakazakii MTCC 659, Shigella flexneri 2a
and Listeria monocytogenes ATCC 19111 as the indicator bacteria. Aliquots (20 µl) of
culture supernatants were applied to well (5 mm in diameter) on agar plates previously
inoculated with a cell suspension of indicator bacteria, which corresponded to a 0.5
McFarland turbidity standard solution.