# Potential Synergistic Inhibition of _Enterococcus faecalis_ by Essential Oils and Antibiotics
## 2 Materials and Methods
### Disk Diffusion Test for EOs and Antibiotics  
The Kirby-Bauer disk diffusion test was used to determine the antimicrobial susceptibility for EOs, antibiotics, and antibiotics combined with EOs on _E. faecalis_. The baseline antimicrobial effects of EOs were tested by placing 2 mL of freshly prepared CN (CN10%, CN5%, CN2.5%, and CN1.25%) and CL (CL50%, CL25%) EO solutions in individual culture tubes and vortexed for 30 s. Then, one sterile filter disk (6 mm diameter) was dropped in each tube, vortexed for another 15 s, and placed on BEA agar plates streaked with cultured _E. faecalis_. Two to three EO-soaked filter disks were placed firmly on the agar surface per each plate (n = 6-8/condition). To determine the baseline antimicrobial effects of the antibiotics, 2 mL of the 10% (kanamycin 5 mg/mL, streptomycin 5 mg/mL, gentamicin 3 mg/mL, ampicillin 10 mg/mL) and 5% (kanamycin 2.5 mg/mL, streptomycin 2.5 mg/mL, gentamicin 1.5 mg/mL, ampicillin 5 mg/mL) solutions of kanamycin, streptomycin, gentamicin, and ampicillin were added in individual culture tubes. One sterile filter disk (6 mm diameter) was dropped in each antibiotic solution, vortexed for 15 s, and placed on BEA agar plates streaked with cultured _E. faecalis_. Two to three antibiotic-soaked filter disks were placed firmly on the agar surface per each plate (n = 4/condition).  
For the combinational antimicrobial effects of antibiotics and EOs, the solutions were prepared as follows: a 10% concentration of an antibiotic solution was combined with an equal volume of each of the four concentrations of EOs (CN10%, CN5%, CN2.5%, and CL100%). These pairings would yield a 5% concentration antibiotic solution containing a half dilution of EO solution that individually would produce a zone of inhibition that was below the CLSI Intermediate Breakpoint for _Enterococcus_ spp., but when combined would produce ZOI at or near the CLSI Intermediate Breakpoint. For ampicillin, a 1.5 mL of 10 mg/mL ampicillin (10% \(v/v\)) was placed into four culture tubes that contained one of the following EO solutions: 1.5 mL of CN10%, 1.5 mL of CN5%, 1.5 mL of CN2.5% or 1.5 mL of CL100%. This 1:1 ratio of combination yielded a combined solution with final concentration of 5 mg/mL ampicillin + CN5%, 5 mg/mL ampicillin + CN2.5%, 5 mg/mL ampicillin + CN1.25%, and 5 mg/mL ampicillin + CL50%. The above steps were repeated for kanamycin 5 mg/mL (10%), streptomycin 5 mg/mL (10%), and gentamicin 3 mg/mL (10%), where 1.5 mL of antibiotics was combined with 1.5 mL of CN10%, 1.5 mL of CN5%, 1.5 mL of CN2.5%, or 1.5 mL of CL100% (Table 1). The culture tubes were vortexed for 30 s, followed by placing a sterile filter disk in each culture tube. The culture tubes were vortexed for an additional 15 s, and the filter disks were placed on BEA agar plates streaked with cultured _E. faecalis_. Two to three antibiotic-soaked filter disks were placed firmly on the agar surface per each plate (n = 8/condition).  
All BEA agar plates with filter disks were secured with lab tape, inverted, and incubated at 37 \({}^{\circ}\)C for 24 h. The size of zone of inhibition was measured from the smallest clearings using a ruler at 1 mm scale (Figure 1). The filter disks soaked in DMSO for 15 s. were used as the control.