Antibacterial efficacy of GIC containing propolis by plating serial dilutions of the inoculum and pernatants were decanted, treated at 60°C for 30 performing colony counts. All experiments were minutes and ﬁltered by passage through a 0.2-µm performed in duplicate while the MIC of EEP was cellulose acetate membrane ﬁlter.21 The efﬁcacy of determined. ﬁltration was assessed by culture methods. Bioﬁlm Formation: To allow the formation of Preparation of GIC Containing Propolis salivary pellicle, each disk was placed in ster- The conventional GIC (Kavitan Pro, SpofaDen- ile plastic Petri dishes containing the processed tal, Czech Republic) was used in this study. The saliva and incubated by shaking at 37°C for one test materials were: (i) GIC (PowderGIC: LiquidGIC ra- hour. The pellicle-coated disks were then rinsed tio=1:1), (ii) 25% EEP added GIC (PGIC: LGIC:LEEP twice in sterile PBS. The disks were placed in Petri ratio=1:0.75:0.25) and (iii) 50% EEP added GIC dishes containing 20 mL of the stock culture and (PGIC: LGIC:LEEP ratio=1:0.5:0.5). EEP was added af- incubated in 5% CO2 at 37°C for 42 hours. The non- ter mixing powder and liquid of GIC. After mixing adherent cells were removed by washing the disks the powder and liquid of each cement, pastes were with sterile saline solution. To collect the bioﬁlm, put into cylindrical molds (10 mm in diameter and the deposits were carefully removed with sterile 2 mm thickness), and the upper surface was ﬂat- scalpels. tened by pressing down with a glass slide. At 230C Assessment: To measure the dry bioﬁlm weight and 50% humidity, a Vickers needle (300 g, 1.12 (biomass), the collected bioﬁlm was placed on mm in diameter) was placed onto the surface of the pre-weighed glass microcoverslips. The ﬁnal the cement every 15 s, and the surface was exam- weight was recorded after incubation at 60°C for 5 ined for any imprint left by the needle. Tests were minutes. The dry weight was obtained by subtract- repeated three times for each material. ing the weight of coverslip from the ﬁnal weight. To measure the number of viable bacteria, the Agar Disk Diffusion Test collected bioﬁlm was suspended in 4 mL 0.1 N The GIC disks were placed onto a MHA sup- NaOH. The suspension was vortexed for two min- plemented with 5% deﬁbrinated sheep blood utes and soniﬁcated for one minute. The optical agar plate and inoculated with 0.1 mL of inocu- density of the bioﬁlm was determined by a spec- lum. Plates were incubated at 37°C for 48 h, and trophotometer (Pharmacia LKB-Ultrapec II, UK) at diameters of inhibition zones produced around 640nm. specimens were measured using a digital caliper (Mitsutoyo, Tokyo, Japan) at three different points. Statistical Analysis Sizes of inhibition zones were calculated by sub- Statistical analysis was performed by Kruskal tracting the diameter of the specimen (10 mm) – Wallis test, at 95% conﬁdence level using SPSS from the average of the three halo measurements. 13.0 for Windows (SPSS Inc., Chicago, IL, USA). The procedure was modiﬁed from Takahashi et al.20 Tests were repeated three times for each ma- RESULTS terial. The MIC value of the EEP was found as 25 µg/ mL. According to disk diffusion test results, the In vitro S. mutans Bioﬁlm Formation Assay experimental GICs containing EEP exhibited in- Bacterial Culture: The stock culture was pre- hibition zones (Table 1). The inhibition zone sizes pared in 60 mL Tripticase Soy Broth (Merck KGaA were not dependent upon the concentration of 64271 Darmstadt, Germany) supplemented with propolis. The pure conventional GIC did not show 5% (w/v) sucrose. S. mutans ATCC 25175 type strain any antibacterial efﬁcacy against S. mutans. concentration of the suspension was determined According to the in vitro bioﬁlm formation assay, as 1x105 cfu/mL. the experimental GICs containing EEP developed Processed Saliva: Unstimulated whole human less biomass (dry-weight) on their surface than saliva was used to coat the cement disks. The sali- the conventional GIC (P<.001). The mean biomass va was collected from the same person and stored amount of bioﬁlm formed on the 50% EEP added at -20°C before preparation. The saliva was cen- GIC was lower than the 25% EEP, but the differ- trifuged at 12.000xg for 15 minutes. The saliva su- ence was not statistically signiﬁcant (Table 2). Ac- 430 European Journal of Dentistry