Whatman cyclopore circular membrane 0.2 m

Bacteria were incubated overnight in Mueller-Hinton broth (Oxoid, Altrincham, UK). The overnight culture was then diluted 1:100 in Mueller–Hinton broth and 200 µl applied to sterile Whatman cyclopore circular membrane 0.2 µm (Whatman plc, Maidstone, UK) placed on Mueller-Hinton agar plates (Sigma-Aldrich Company Ltd, Kent, UK) at 37 °C for 24 h to form colony biofilms. Log reduction testing was performed on these colony biofilms.
Dressings were cut into 3cm² square pieces and pre-moistened with sterile deionised water. Dressings were placed over colony biofilms on agar plates. Every 24 h a biofilm was removed, and quantification of viable bacteria within the biofilm (CFU/ml) was calculated by vortexing the biofilm with sterile 3 mm glass beads (Merck-Millipore, Watford, UK) in Dey-Engley neutralising broth (Merck-Millipore), before serial diluting (10^–1 to 10^–7). Standard plate counts were performed on Mueller-Hinton agar following 24 h incubation at 37 °C. Each treatment and time-point were repeated in triplicate and replicate plate counts for each dilution were performed.

An overnight culture of bacteria was diluted 1:100 in Mueller–Hinton broth. 200 µl of culture was added onto a sterile Whatman cyclopore circular membrane 0.2 µm (Whatman, Maidstone, UK) and placed on Mueller-Hinton agar. Biofilm plates were incubated for 72 h at 37 °C to allow the formation of a mature biofilm.
After the biofilm had matured for 3 days, 3 cm² square dressings were placed directly onto biofilms and incubated for 24 h at 37 °C. Following removal of dressings from the biofilm surface, 1 ml of PrestoBlue Cell Viability Reagent (Invitrogen, Waltham, MA) was added to the surface of each biofilm for 20 s. The surface was drained before recording the colour changes using a Nikon D2300 digital camera (Nikon UK Limited, Kingston, UK).