Each sample was tested in triplicate and results were expressed i

Each sample was tested in triplicate and results were expressed in μM. Biofilms were observed by CSLM as described below (Otto, 2008). Before imaging, the biofilm was formed in microtiter plates (24-well, Greiner Bio-One, Germany), and rinsed with

sterile 50 mM potassium phosphate buffer solution (pH 7.2; no autofluorescence detected) for 10 min before being stained with 15 μM propidium iodide (Sigma) for 5 min at room temperature to detect bacterial cells in red. After being washed in PBS, the samples were incubated with 50 mg mL−1 of fluorescein isothiocyanate-conjugated Con A (FITC–Con A) (Sigma) for 5 min at room temperature to stain the glycocalyx matrix green. The propidium iodide was excited at 520 nm, the emission was monitored RXDX-106 price at 620 nm, and FITC–Con A at 495 and 525 nm, respectively. Intact biofilms were examined nondestructively using a Fluoview FV1000 Espectral Olympus CSLM

(Olympus Latin America, Miami, FL) equipped with UPlanSApo × 100/1.40 oil UIS2 Olympus oil immersion lens. Optical sections of 0.87 μm were collected over the complete thickness of biofilms. Then, for each sample, images from three randomly selected positions were obtained and analyzed using an Olympus Fluoview FV 1000 (Zernotti et al., 2010). For image analysis, three investigators BAY 80-6946 purchase (J.A.M., I.A. and M.G.P.) evaluated the images independently in a blinded retrospective manner. All experiments were performed in triplicate and numerical data are presented as means with error bars representing SDs. The data were statistically analyzed using a one-way anova followed by the Student–Newman–Keuls test for multiple comparisons. Differences between means were assessed with a P-value <0.05 being considered statistically significant. A quantitative analysis showed that the S. aureus cells attached to 96-well plates exhibited good biofilm

formation (according to the scale described in Materials and methods) after 18 h and remained up to 24 h. However after 48 h, their attachment was significantly reduced (P<0.005) when tested under the same experimental IKBKE conditions. The ATCC was stable until 48 h (BBU=2.50), with this strain showing the best biofilm formation between 18 and 24 h. After 48 h, the biofilm of S. aureus was detached from the abiotic surface. No additional biofilm production was detected after 72 h compared with incubation at 48 h. Therefore, 18–24 h was chosen for the other assays (Fig. 1a). The production of detectable amounts of ROS and RNI (NO) by S. aureus in the biofilm was evaluated by NBT and Griess, respectively. These assays were useful in determining the relation between the intracellular and extracellular metabolite strains, determined by iROS/BBU (Fig. 1b), eROS/BBU (Fig. 1c) and NO/BBU (Fig. 1d), where the BBU were obtained from each strain of S. aureus.

Leave a Reply

Your email address will not be published. Required fields are marked *

*

You may use these HTML tags and attributes: <a href="" title=""> <abbr title=""> <acronym title=""> <b> <blockquote cite=""> <cite> <code> <del datetime=""> <em> <i> <q cite=""> <strike> <strong>