This cell suspension constituted the

standard starting inoculum (S) as defined by CLSI guidelines for antimicrobial susceptibility testing [68]. Double (D) and half (H) the size of the standard inoculum were used to evaluate the effect of the initial cell AZD3965 clinical trial density on the activity of biocides towards S. algae. To check the actual starting cell number, a 200 μl sample of the inoculum was serially tenfold diluted from 10−1 to 10−8. Four 10 μl drops from each dilution were spotted on agar plates and incubated. Colony formation was assessed after 24 h. Microscopy: general procedures For microscopy experiments, the bottoms of the wells of a microtiter plate were mechanically sectioned with a computer numerical control milling machine (Fagor CNC 8055 M) in order to use exactly the same substrate as in previous tests. The sectioned discs thus obtained (5.86-5.98 mm in diameter, 1.00-1.08 mm in height, data from 15 random

PLX-4720 supplier measurements) were carefully disengaged and sterilised by a brief sonication in ethanol and UV irradiation before their use in the experiments. To develop the biofilms, the discs were placed at the bottom of a 24-well microtiter plate. Two-mililiter bacterial cultures were prepared in the appropriate medium following the same procedures as described previously. After the incubation period, discs were rinsed three times with FSW and kept immersed upon their use in the microscope. Confocal Laser Scanning Microscopy Biofilms formed on polystyrene discs were fluorescently stained with acridine orange (AO), a membrane permeant nucleic acid stain that intercalates dsDNA and binds to ssDNA as well as to ssRNA through dye-base stacking to give broad spectrum fluorescence when excited Ribose-5-phosphate isomerase at 476 nm [69]. This compound stains all cells in a biofilm, live or dead, and may

also bind to nucleic acids that are present in the extracellular matrix. To stain biofilms, discs were immersed in 0.1% w/v AO (Sigma-Aldrich) in PBS for 5 min at room temperature and washed with FSW. Fluorescently labelled biofilms were placed in two drops of 0.9% FSW on the surface of a glass coverslip and were examined using an Olympus Fluoview 1000 Confocal Laser Scanning Microscope. Each biofilm was scanned at 4 positions randomly selected at the microscope stage and confocal image series were generated by optical sectioning at each of these positions. Three independent biofilm experiments were performed, and image stacks of 512×512 pixels were collected for quantification. Image combining and processing were performed with the Imaris software package, version 4.0 (Bitplane AG, Zürich, Switzerland). The biofilm structure was quantified using the software program COMSTAT [70] available as free downloadable software at http://​www.​imageanalysis.​dk. COMSTAT converts pixels from confocal image stacks into numerical values, facilitating quantitative characterization of each structural component within 3D biofilm images [71].