Biofilm plate assay
Initially, an overnight cultivation of the S. mutans R15-8
bacterial strain (a clinical isolate) was performed at 37 °C under
aerobic conditions with a 5%-10% CO2 atmosphere in BMH
(BD, Heidelberg, Germany) supplemented with 1% sucrose (MH-S).
Following this, polystyrene 96-well tissue-culture plates (Greiner
bio-one, Frickenhausen, Germany) were loaded with 100 µl of MH-S,
incorporating ten distinct concentrations (ranging from 0.019 mg/ml to
10 mg/ml) of the plant extracts under investigation. Subsequently, 5 µl
of the S. mutans overnight culture were added to each well. The
Log10 of the CFU of the S. mutans overnight
culture on CBA plates fell within the range of 108CFU/ml. These 96-well plates were then incubated for 48 hours at 37 °C
in an aerobic environment with a 5%-10% CO2atmosphere. Following the incubation period, the culture medium was
discarded, and the wells were subjected to three consecutive washes
using 300 ml of phosphate-buffered saline (PBS, Life Technologies,
Darmstadt, Germany) per plate in order to eliminate non-adherent
bacteria. Since no fixation of adherent bacterial cells within the
biofilm was deemed necessary, the plates were simply air-dried and
subsequently stained with Carbol Gentian Violet solution (Carl Roth GmbH
+ Co. KG, Karlsruhe, Germany) intended for microscopy. This staining
solution contained 0.1% - <0.25% methyl violet and was
applied for a duration of 10 minutes. After staining, excess dye was
washed away by rinsing the plates with distilled water. The plates were
then dried at 60 °C for 10 minutes. To facilitate dye resolubilization,
50 µl of absolute ethanol (99.9 vol %) was added to each well for
subsequent analysis (Merck Chemicals GmbH, Darmstadt, Germany), and the
optical density was finally measured at 595 nm using the Tecan Infinite
200 plate reader (Tecan, Crailsheim, Germany). All experimental tests
were executed in quadruplicate, and the mean values were subsequently
calculated. To validate the findings and further eliminate false
positive results, the plant extracts yielding the highest biofilm
inhibition values underwent a second screening. During the analysis, the
antibiofilm effects of each extract on S. mutans were classified
into three distinct groups, aided by two different cut-off values: no
biofilm production or C1, moderate biofilm production or C2, and high
biofilm production or C3. The low cut-off value was established by
adding three standard deviations of the blank to the negative control.
Conversely, the high cut-off value was derived after conducting the low
cut-off value measurement on three separate occasions.