[52]. Briefly, overnight cultures of S. epidermidis strains grown in TSB medium were diluted 1:200 and inoculated into wells of polystyrene microtiter plates (200 μl per well) and incubated at 37 °C for 24 h. After incubation, the wells were washed gently three times with 200 μl sterile PBS, air-dried and stained with 2% crystal violet for 5 min. Then, the plate was rinsed under running tap water, the crystal violet was redissolved in ethanol and the absorbance was determined at 570 nm. To determine whether lytSR affects cell viability in biofilm, bacterial cells were cultivated in cover-glass cell-culture
dish (WPI, Sarasota, FL, USA) as described previously [29]. Briefly, overnight cultures of S. epidermidis strains grown in TSB medium were diluted 1:200, then inoculated into the dish (2 ml per dish) and incubated at 37 °C. After 24 hours, the dish was washed gently three times with Batimastat cost 1 ml sterile 0.85% NaCl, HSP inhibitor then stained by SYTO 9 and PI for 15 min and examined by Leica TCS SP5 confocal microscope. Quantitative analysis of bacterial cell death inside biofilms To quantify relative viability of S. epidermidis strains, live/dead stained biofilms were scraped from the dish and dispersed
thoroughly by pipetting. The integrated intensities (1 second) of the green (SYTO 9, 535 nm) and red (PI, 625 nm) emission of suspensions excited at 485 nm were measured respectively Erastin clinical trial by Beckman Coulter DTX880 multimode detectors. The red/green fluorescence Momelotinib datasheet ratios (RatioR/G) were calculated, and a standard curve of Ratio R/G versus percentage of dead cells in the S. epidermidis suspension was plotted as described in the manuals of LIVE/DEAD® BacLight™Bacterial Viability Kit L7012 (Invitrogen, Carlsbad, USA). The percentage of dead cells inside biofilms was determined by comparison to the standard curve. Pyruvate utilization test To verify physiological changes of 1457ΔlytSR detected by GPI-vitek test system, overnight cultures of S. epidermidis
were diluted 1:200 into Pyruvate fermentation broth (Tryptone 10 g, Pyruvate 10 g, Yeast extract 5 g, Dipotassium phosphate 5 g, Sodium chloride 5 g per liter, pH 7.4) and incubated microaerobically at 37 °C [53]. The growth was detected by monitoring turbidity of the cultures at 600 nm. RNA extraction and Microarray analysis Overnight cultures of S. epidermidis 1457 and 1457ΔlytSR were diluted 1:200 into fresh TSB and grown at 37 °C to an OD600 of 3.0 (mid-exponential growth). Eight millilitres of bacterial cultures were pelleted, washed with ice-cold saline, and then homogenized using 0.1 mm Ziconia-silica beads in Mini-Beadbeater (Biospec) at a speed of 4800 rpm. The bacterial RNA was isolated using a QIAGEN RNeasy kit according to the standard QIAGEN RNeasy protocol. The custom-made S. epidermidis GeneChips (Shanghai Biochip Co.
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