Aluminium specimen stubs

In order to examine single-subspecies biofilm architecture using SEM, biofilms were grown on poly-L-lysine coated plastic (Thermanox™) coverslips in 24-well plates. Biofilms were fixed using 2.5% glutaraldehyde (Agar Scientific, Stansted, United Kingdom) in 0.1 M sodium cacodylate buffer (pH 7.4, BioWorld, Dublin, Ireland) for 10 min at room temperature. Following fixation, biofilms were dehydrated with increasing ethanol concentrations (20–100%) and incubated for 10 min at each step.
Finally, drying agent hexamethyldisilizane (Sigma-Aldrich/Merck, Darmstadt, Germany) was applied and left to evaporate overnight. Coverslips with biofilms were mounted onto aluminium specimen stubs (Agar Scientific, Stansted, United Kingdom), sputter coated with two layers of gold and visualised using a scanning electron microscope (Zeiss EVO MA10). A visible layer of biofilm was chosen after visual evaluation of each specimen under low magnification (50X) and recorded at 1000X and 5000X magnification.

Low molecular weight chitosan (>75% deacetylated, 20–300 cps (1% in 1% acetic acid)), 1,3,5-benzene tricarboxylic acid (BTC, trimesic acid, C₉H₆O_6, Mwt 210.14 g/mol), 5-fluorouracil (5-FU, ≥99% HPLC, powder, C₄H₃FN₂O_2, Mwt 130.08), dialysis tubing with an average flat width of 25 mm (1.0 inch) and molecular weight cut-off (MWCO) 3500 Da, glacial acetic acid, deuterium chloride, deuterium oxide, and ethanol were all purchased from Sigma-Aldrich, Gillingham (UK). Aluminium specimen stubs (0.5″) and carbon tabs (12 mm diameter) were purchased from Agar Scientific Ltd., Stanstead (UK). Deionised water was used for all preparations.