Glacial acetic acid

Overnight cell cultures were washed and re-suspended to OD 1.0 at 540 nm, corresponding to concentrations of S. aureus (6.8 ± 2.2) x 10 8 and E. coli (7.1 ± 1.9) x 10 8 CFU/mL. Two replicate substrata were place horizontally into a Petri dish to which 30 mL of the standardised cell suspension was added and incubated for 1 h without shaking. Following incubation, the coupons were removed with sterile forceps and each washed gently with 5 mL of sterile filtered water from a bottle at a 45° angle with a 3 mm nozzle. The samples plus retained bacteria were then air-dried in a class II airflow cabinet for 1 h (Whitehead and Verran, 2007) . To allow visualisation with an epifluorescence microscope, the samples were stained for 2 min with 0.03 % acridine orange (Sigma, USA) diluted in 2 % glacial acetic acid (BDH, UK), rinsed in sterile distilled water and allowed to dry once more in a class II airflow cabinet in the dark for 30 min.
The retained cells upon the surface were visualised using epifluorescence microscopy (Nikon Eclipse E600 epifluorescence microscope, Tokyo, Japan) at 502 -526 nm wavelength. Cell-F software was used to visualise, capture and analyse the images. These images were used in the MATLAB®, Image Processing Toolbox® for the multifractal analysis (n = 20).

One hundred microlitres of fish extract was pipetted onto the stainless steel surface. Samples were air dried in a class 2 flow hood. Samples were stained with 0.03 % w/v acridine orange (Sigma, UK) in 2 % v/v glacial acetic acid (BDH, UK) for 2 min, (whilst being left in the dark to avoid bleaching of the fluorescent dye), rinsed once, gently with 5 cm 3 distilled H2O, with the distilled water bottle at a 45 o angle, with a 3 mm diameter nozzle opening. Samples were air dried for one hour in a class 2 hood and stored at 4 o C in the dark until visualised. A concentration of 0.03 % acridine orange was used since previous work in our laboratories had found that it was the optimum concentration for staining cells on stainless steel surfaces (Whitehead et al., 2007) .