2.4.2. Eradication of biofilm formation
The eradication of biofilm formation of P. betle extract was also
examined using the minimum biofilm eradication concentration
(MBEC) assay. Briefly, a 200 mL in early log phase (106 CFU/mL) of
each representative strain, S. mutans ATCC 25175 and
A. actinomycetemcomitans ATCC 33384, was inoculated into each
well of a 96-well plate and incubated for 24 h at 37 C for the
development of a multilayer biofilm. The culture was then blotted
out, and the well carefully washed 3 times with sterile PBS in order
to remove non-adherent cells.16 These biofilms were then exposed
to a 200 mL of various concentrations of P. betle extract ranged from
0.02 to 25 mg/mL, incubated for 24 h at 37 C in appropriate conditions.
At the end-point of the treatment of the biofilms with
P. betle extract, the adherent cells were washed 3 times with sterile
PBS. The numbers of surviving culture were determined using MTT
assay. Percentage eradication was calculated by using the equation
[1 (A570 of the test/A570 of non-treated control)] 100. The MBEC
value was defined as the concentrations that showed 50% and 90%
eradication of biofilm formation. The 0.1% CHX, PBS and the extract
free medium were used as the positive, non-treated and blank
controls, respectively.