# Therapeutic Potential of Gramicidin S in the Treatment of Root Canal Infections
## 4 Materials and Methods  
### E. faecalis Strains, Antibiotics and the Determination of the Minimum Inhibitory Concentrations  
The control strain _Enterococcus faecalis_ DSM 2570 (ATCC 29212) was purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ). The WW4 isolate from cheese and the root canal isolate WW6 were a kind gift from [PERSON] (Department of Microbiology, King's College London, London, UK). The tetracycline-resistant clinical isolates TRE1-TRE5 were obtained from the medical laboratory of Staber & Kollegen, Heilbronn, Germany.  
GS was produced by fermentation, extracted from producer cells and HPLC purified as described earlier [28]. Other antibiotics (tetracycline and demeclocycline hydrochloride) were purchased form Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany).  
The bacterial strains were maintained at \(-80\)\({}^{\circ}\)C using a Cryobank(tm)System (Mast Diagnostic, Reinfeld, Germany). To refresh the bacterial cells, single beads were recovered in 10 mL BHI broth (Becton, Dickinson and Co., Sparks, MD, USA) in an overnight incubation at 37 \({}^{\circ}\)C and 200 rpm, in the 50 mL culture flasks. Single colonies were obtained by streaking these cultures on BHI agar plates, which were then stored at 4 \({}^{\circ}\)C. Subsequently, the colonies were used to inoculate 10 mL MH broth (Becton, Dickinson and Co., Sparks, MD, USA) to an optical density (OD) of 0.02 at 550 nm, and the cultures were grown overnight. The test cultures were prepared by inoculating 10 mL MH broth to an OD\({}_{550}\) of 0.2 with the overnight cultures and allowing them to grow until the bacteria reached OD\({}_{550}\) of 1-2 at 37 \({}^{\circ}\)C and 200 rpm. For inoculation, the test cultures were diluted immediately prior to the experiment in MH broth to obtain final inoculation doses of 5 \(\times\) 10\({}^{5}\) CFU/mL according to the CLSI recommendations [17].  
For the determination of the MIC, the standard broth microdilution procedure [18] was modified to obtain the uniform medium concentration after the addition of antibiotic stock solutions dissolved in sterile deionized water (tetracycline and demeclocycline) or in 50% ethanol (GS). Briefly, 50 \(\mu\)L of the double-strength MH broth was added to the upper row of the 96-well microtiter plates (Nunclon(tm), Nunc GmbH & Co., Wiesbaden, Germany). Next, 50 \(\mu\)L of the standard MH broth was added to the remaining wells, including the columns for the ethanol control and the positive (without peptides) and negative (sterility) controls. Addition of the antibiotic stock solutions (50 \(\mu\)L) to the upper wells provided a standard medium concentration and the antibiotic concentration to establish a concentration gradient. Once the two-fold dilution series of the antibiotic concentration was prepared according to [44], the plates were inoculated with 50 \(\mu\)L of bacterial suspensions from the exponentially growing culture to reach 5 \(\times\) 10\({}^{5}\) CFU/mL. The plates were incubated for 22 h at 37 \({}^{\circ}\)C and 5% CO\({}_{2}\) without agitation. To examine bacterial growth, 20 \(\mu\)L of an aqueous 80 \(\mu\)M solution of the redox indicator resazurin was added to each well. The plates were incubated for another 2 h at 37 \({}^{\circ}\)C. The respiration activity was calculated for each well as the difference in the absorbance of resorufin at 570 nm and resazurin at 600 nm using the microtiter plate reader FlashScan550 (Analytic Jena GmbH, Jena, Germany) and the WinFlash program. Positive values indicated bacterial growth and allowed the determination of the lowest peptide concentrations that inhibit bacterial growth. All results were obtained from several independent experiments, and each was performed in triplicate.