The natural compound eugenol used in the present study was
found to act by reducing the total mass of causative microorganisms
without affecting the viability of the oral bacteria,
thereby decimating the development of resistant strains or
The adherence assay was performed on a saliva-coated flatbottomed
cell culture plate. Saliva was collected [8]. Cell pellets of
S. mutants grown in BHI from mid-exponential phase were
re-suspended in TSBC (10 mmol/L Tris–HCl, 150 mmol/L NaCl,
5 mmol/L CaCl2, pH 7.6) (OD600 = 1.0). The bacterial suspension
(200 mL) was added to the saliva-coated wells with varying
concentrations of eugenol (ranging from 0.0195 mg/mL to
0.3125 mg/mL) and was incubated at 37 8C for 24 h. To remove
unattached cells, the wells were gently rinsed with 200 mL of
deionised distilled water. Adhered bacteria were fixed using 37%
formaldehyde and were stained with 200 mL of 0.1% crystal violet
(CV) for 15 min. After washing twice with 200 mL of deionised
distilled water, CV was released from the stained cells using 200 mL
of 99% ethanol. The adhered cells that were attached to the salivacoated
surface were quantified by measuring the absorbance of the
solution at 630 nm using an ELISA plate reader (iMarkTM
microplate reader; Bio-Rad). A bacterial suspension treated with
chlorhexidine was used as a positive control; a suspension without
any treatment was used as a negative control; and TSBC alone was
used as a blank control. The effect of eugenol on biofilm formation
was determined on saliva-coated plates using the protocol
described elsewhere [9].
The natural compound eugenol used in the present study wasfound to act by reducing the total mass of causative microorganismswithout affecting the viability of the oral bacteria,thereby decimating the development of resistant strains orThe adherence assay was performed on a saliva-coated flatbottomedcell culture plate. Saliva was collected [8]. Cell pellets ofS. mutants grown in BHI from mid-exponential phase werere-suspended in TSBC (10 mmol/L Tris–HCl, 150 mmol/L NaCl,5 mmol/L CaCl2, pH 7.6) (OD600 = 1.0). The bacterial suspension(200 mL) was added to the saliva-coated wells with varyingconcentrations of eugenol (ranging from 0.0195 mg/mL to0.3125 mg/mL) and was incubated at 37 8C for 24 h. To removeunattached cells, the wells were gently rinsed with 200 mL ofdeionised distilled water. Adhered bacteria were fixed using 37%formaldehyde and were stained with 200 mL of 0.1% crystal violet(CV) for 15 min. After washing twice with 200 mL of deioniseddistilled water, CV was released from the stained cells using 200 mLof 99% ethanol. The adhered cells that were attached to the salivacoatedsurface were quantified by measuring the absorbance of thesolution at 630 nm using an ELISA plate reader (iMarkTMmicroplate reader; Bio-Rad). A bacterial suspension treated withchlorhexidine was used as a positive control; a suspension withoutany treatment was used as a negative control; and TSBC alone wasused as a blank control. The effect of eugenol on biofilm formationwas determined on saliva-coated plates using the protocoldescribed elsewhere [9].
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