For each assay, the XTT solution was thawed on ice and mixed with the menadione solution at 20:1 (v/v). Tokens with biofilm were gently placed BGB324 molecular weight inside another pre-sterilised flat bottomed 24-well tissue culture plate and 2 mL of the XTT solution (PBS + 200 mM glucose-XTT-menadione) were added to each well.
The plates were covered with aluminium foil and incubated in the dark under agitation at 37 °C for 3 h.22 Thereafter, the solution was centrifuged and 500 μL were transferred to spectrophotometer cuvettes. The bioactivity assay was performed using a spectrophotometer (Beckman Coulter, Indianapolis, IN, USA) and the readings were recorded at 490 nm. The bioactivity assays were performed in triplicate in three independent experiments on different days (n = 9). The tokens with biofilms were gently placed inside pre-sterilised flat bottomed 24-well tissue culture plates and stained using a Live/Dead BacLight viability kit (Invitrogen-Molecular Probes, Eugene, OR, USA). A
kit consisting of SYTO-9 and propidium iodide (PI) was used. STYO-9 is a green fluorescent nucleic acid stain, generally labelling both live and dead microorganisms. PI, in contrast, is a red-fluorescent nucleic acid stain and penetrates only the cells with damaged Protein Tyrosine Kinase inhibitor membranes, thus only the dead cells are visualised. Biofilms were incubated with SYTO-9 and PI at 30 °C for 20 min in the dark before CLSM analyses. The images of stained biofilms were captured using a CLSM system (Leica Microsystems CMS, Mannheim, Germany). A series of images were obtained for each position at 1 μm intervals in the z-axis to obtain a three dimensional view of the biofilms (from substratum to the top of the biofilms). Five representative randomly selected positions from each corner and the middle of the tokens were examined for each token, in two independent experiments on different days (n = 10). The same protocol and configurations were used for CLSM analysis (×63 objective lens without zoom) in order to obtain all images from control or experimental groups. COMSTAT analysis is a software program for quantification
of three-dimensional biofilm structures. It analyses stacks of images acquired with CLSM. Z-series images of biofilms after 48 h were collected by CLSM. The z-slices of the images were exported to COMSTAT software and analysed. The parameters analysed included bio-volume, 4-Aminobutyrate aminotransferase average thickness and black spaces of the biofilm. The bio-volume (μm3/μm2) is defined as the number of stained cell pixels in all images [(pixel size)x × (pixel size)y × (pixel size)z] divided by the substratum area of the image stack. 23 The tokens were placed inside a polypropylene tube containing 3 mL of sterilised PBS. Adherent micro-organisms were removed from the tokens by sonication at 7 W for 30 s.24 Once disaggregated, the cells were centrifuged (3000 rpm). The pellets were fixed by immersion in Karnovsky solution prepared in 0.1 M cacodylate buffer (pH 7.