## 2 Materials and Methods  
### Biofilm Culture on GP  
GP points 40/0.04 (VDW GmbH, Munich, Germany) were sterilized with plasma gas (low-temperature hydrogen peroxide gas plasma, Kingsport, TN, USA) and divided into two groups:  
Sterile GP points--The GP points were cut into 1 \(\times\) 1 mm GP particles using a sterile scalpel (no. 11) and an endodontic ruler under 2.5\(\times\) magnification (Orascoptic Inc., Madison, WI, USA).  
Infected GP points--The formation of biofilm on GP points was carried out according to [PERSON] et al. (2004). Three strains of bacteria, i.e., _Enterococcus faecalis_ (E.F.) ATCC 29212, _Streptococcus sanguis_ (S.S.) ATCC 10556, and _Porphyomonas gingistalis_ (P.G.) ATCC 33277, were cultured in equal amounts on GP points. The infected GP points were suspended in a growth medium containing varying percentages of human serum [14]. Each bacterial strain was harvested during the stationary phase, and 100 uL of each bacterial suspension was inoculated into 900 uL of cell culture medium, 900 uL of human serum (Biological Industries, Israel), or 900 uL of cell culture medium supplemented with 5% or 50% (vol/vol) human serum. Formalin fixation was performed at the end of the incubation process (Tissue-Tek @ O.C.T. TM Compound, Sakura, The Netherlands). The GP points were sliced at 20-40 um (Leica CM1900 Cryostat, Germany). Several segments were stained according to the [PERSON] et al. (2002) protocol [15]. Briefly, extracellular polymeric substances (EPS) were stained red with concanavalin. A conjugate to tetramethyl rhodamine isothiamate (TRITC) was used as a marker for biofilm, and DNA-binding stain SYTO 9 (green color) was used to visualize live bacterial cells. Slices were observed under a laser scanning confocal microscope (Carl Zeiss CLSM (LSM 510 META) with an Apo 60 \(\times\) 1.40 objective) to confirm the generated biofilm. Then, 3D depth images of the biofilm generated over the GP were taken (Figure 1).  
Non-infected and infected GP were incubated in primary fixative containing 2.5% GA and 2% PFA in 0.1 M sodium cacodylate buffer at pH 7.4 for 1 h at RT. Following washes in 0.1 M cacodylate buffer, the samples underwent a second fixation in 1% OsO4 for 20 min. The specimens were then dehydrated through a graded ethanol series, and the critical points were dried and coated with 10 nm chromium. A Zeiss ULTRA plus field emission scanning electron microscope was used to observe the samples (Figure 2A,B).  
After slicing the infected GP points, several segments were transferred into 2 mL sterile tubes. The vials were then shaken on a Retsch MM401 bead mill for 2.5 min at 30 Hz. Aliquots of the bead-milled suspensions were collected and then inoculated into blood culture bottles. Additional aliquots were spread on blood agar plates and PolyViteX chocolate agar plates, both of which were incubated for 5 days at 37 \({}^{\circ}\)C in 5% CO\({}_{2}\) and at 37 \({}^{\circ}\)C in an anaerobic atmosphere. Isolated bacteria were identified according to standard laboratory procedures. In all cases, viable E.F., S.S., and P.G. isolates were recovered from the infected GP points. The remaining GP points were cut using the same technique as the sterile GP points.  
Figure 1: Confocal laser scanning microscopy image of bacteria and biofilm generated over a guttapercha segment.