Mean biofilm thickness provides a measure of the spatial size of

Mean biofilm thickness provides a measure of the spatial size of the biofilm. Maximum thickness: the maximum thickness over a given location, ignoring pores and voids inside the biofilm. Roughness coefficient: a measure of variation in biofilm thickness across the field of view, an indicator

of biofilm heterogeneity. The percentage of adhering cells (% Coverage) was calculated using ImageJ NIH image processing software [72]. Atomic Force Microscopy Imaging and force measurements to characterise the nanomechanical properties of Shewanella algae cells were performed by AFM. In these studies every treated polystyrene disc containing the immobilised bacteria was attached to a steel sample puck by means of an adhesive tape. When measuring in liquid, 50 μL of FSW were added onto the disc prior to be placed into the AFM liquid cell. For measurements performed in air, polystyrene discs were carefully rinsed and dried in N2 atmosphere before

using. Tapping Mode: S. algae cells were imaged by AFM operating in tapping mode in air using a Multimode microscope and a Nanoscope V control unit from Bruker at a scan rate of 1.0–1.2 Hz. To this end, etched silicon tips (RTESP, 271–311 kHz, and 40–80 N/m) were used. Peak Force Tapping and force-distance analysis: Quantitative mapping were performed in FSW at room temperature using a Nanoscope V controller (Bruker). Images were

acquired in AFM contact and Peak Force Tapping Mode [73] (Peak Force-Quantitative Nanomechanics, PF-QNM). AFM probes used in these studies were silicon GDC-0449 nmr nitride probes (NP-C, Bruker) with a nominal tip radius of 20–60 nm. The spring constant of cantilevers were measured using the thermal tuning method [74], and its values ranged 0.14-0.26 N/m. Mica surfaces were selected as rigid substrates for deflection sensitivity calibration. Note that in PF-QNM measurements AFM tips were carefully calibrated before every experience as described elsewhere [74–77]. Experimental results were acquired for single bacteria or little groups of them from the PF-QNM images, excluding thus contributions due to bacteria/EPS-free substrate. Data proceeding from at least 115 units from two PD184352 (CI-1040) independent cultures were collected for each medium. Adhesion force and Young’s modulus values SAR302503 distribution has been expressed as histograms. Force-distance (FD) curves were collected using low loading forces (F < 20 nN) in order to protect both the AFM tip and the bacterial cells [59]. Data processing was carried out using the commercial Nanoscope Analysis (Bruker), WSxM (Nanotec) [78] and Gwyddeon (GNU) softwares. Statistics The effects of culture medium, incubation temperature and their interaction on the dependent variables (total cell density and biofilm formation) were assessed by a two-way ANOVA.

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