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), forward and reverse primers (final concentration Navitoclax supplier of 0.4?pmol/��L for each primer), and sterile water according to the manufacturer's instructions. qPCR was performed using the MyiQ single-color real-time PCR detection system (Bio-Rad) and MyiQ System Software Version 1.0.410 (Bio Rad Laboratories Inc.). The mRNA quantity was calculated relative to the expression of two reference genes, hsp60 and tpi (Table S1). Statistical analysis For the biofilm formation assay and bacterial viability test, data were evaluated using one-way analysis of variance (ANOVA) by Prism 6.04 (La Jolla, CA, US). For qRT-PCR test, down- and upregulation were considered significant when the relative expression was decreased or increased ��4 folds. All experiments were repeated at least three times. Results Minimum inhibitory concentration (MIC) of allicin on S.?epidermidis ATCC 12228 in planktonic cultures Using the standard broth microdilution protocol, we could determine a MIC value of 12.5?��g/mL for S.?epidermidis ATCC 12228, despite the observed Bcl-2 inhibitor a slight reduction in OD values already at a concentration of 6.25?��g/mL allicin (Fig.?(Fig.1A).1A). This value was confirmed using a live/dead staining with SYTO? green and propidium iodide and a quantitative evaluation of confocal microscopy images (Fig.?(Fig.1B1B). Figure 1 Minimum inhibitory concentration (mean?��?SEM) of allicin determined in Staphylococcus epidermidis ATCC 12228 growing in planktonic cultures. (A) Results obtained with the standard CLSI broth dilution protocol; (B) Results (mean?��?SEM) ... Time- and concentration-dependent effects of allicin on biofilm formation Using the safranin staining protocol to assess biofilm formation by S.?epidermidis (ATCC 35984) after 12, 24, and 48?h, respectively, it could be shown that allicin inhibits biofilm formation in a concentration-dependent manner (Fig.?(Fig.2).2). In the tested concentration range varying from 0.78 to 25?��g/mL, significant differences in comparison to the controls were observed already during the initial 12?h of culture. Biofilm biomass was increasing during the following 12?h significantly, whereas between 24 and 48?h only a slight further increase was observed. Based on these results, 24?h biofilms were used for the forthcoming experiments on biofilm sensitivity and architecture. Figure 2 Staphylococcus epidermidis (ATCC 35984) biofilm formation within Bumetanide 48?h and after exposure to different concentrations (0.098�C25?��g/mL) of allicin. Different upper-case letters (A, B) indicate significant (P?90% when compared with OD values of controls (here at an OD value