Polystyrene cuvette

The average size of live, inactivated, and inactivated split viruses was assessed in a Litesizer 500 dynamic light scattering (DLS) instrument (Anton Paar) at an angle of 175° with 10 × 12 × 45 mm polystyrene cuvettes (Sarstedt) and 1 mL of sample. Triplicate measurements were performed per sample, each with 11 runs, and processed with the Kalliope software (Anton Paar). Live and inactivated virus samples were diluted 1:1,000 in 0.22 µm filtered PBS (pH = 7.4), while inactivated split virus samples were diluted 1:100.

Measurement of P_i concentrations in the reactive spent culture filtrate was determined using the molybdenum blue assay (Murphy and Riley, 1962 ). Three reagents were initially prepared: 2.5 M sulphuric acid (H₂SO₄), 32.33 mM ammonium molybdate ((NH₄)₆Mo₇O₂₄) and 8.21 mM potassium antimony tartrate (C₈H₁₀K₂O₁₅Sb₂). With these reagents, a mixed solution was then prepared containing: 528 mg ascorbic acid (C₆H₈O₆), 50 ml 2.5 M H₂SO₄, 15 ml 32.33 (NH₄)₆Mo₇O₂₄, 5 ml C₈H₁₀K₂O₁₅Sb₂ and made up to 100ml with Milli‐Q water. P_i standards were prepared from 0‐1 mg L^‐1 P_i, using a KH₂PO₄ stock solution, and samples were diluted in Milli‐Q water prior to analysis. 0.8 ml of the mixed solution was added to 5 ml aliquots of diluted reactive spent culture filtrate or P_i standard solution and mixed for 10 min. 1ml aliquots of these solutions were then added to 3.5 ml polystyrene cuvettes (Sarstedt AG & Co, Hemer, Germany) and the absorbance at 882 nm measured using an Ultrospec 2100 pro UV/Visible Spectrophotometer (Amersham Biosciences, GE Healthcare, Little Chalfont, UK).

E. faecalis strains were inoculated in BHI medium with the indicated antibiotics and cultured overnight at 37°C. Overnight cultures were diluted in a 1:100 ratio in TSB-D with the indicated antibiotics, 10 ng/ml cCF10, and 50 ng/ml nisin and dispensed into polystyrene cuvettes (Sarstedt) in 0.9 ml triplicates. These were incubated for 24 hr at 37°C without agitation. Afterwards, the optical density of each sample was determined at 600 nm both before (OD_sup) and after (OD_mix) vigorously mixing of the bacterial culture by pipetting. The autoaggregation percentage was then calculated as follows: 100 × [1 − (OD_sup/OD_mix)] (Bhatty et al., 2015 (link); Waters and Dunny, 2001 (link)).

A thick block of mica (Ruby muscovite, Highest grade VI, Ted Pella Inc.) was cleaved using a razor blade down to a thickness of 10 μm. A drop of UV curable epoxy (Loctite 349) was placed on a No.1 thickness glass coverslip (VWR). The mica sheet was then placed over this and the epoxy was allowed to spread under its weight. It was ensured that no air bubbles were trapped in the epoxy. The epoxy was then cured under UV light for 15 min.
A cured PDMS rectangular cut-out was stuck around the mica sheet to form a chamber. The suspending oil was introduced into the chamber. Silica capillaries (OD: 100 μm, ID: 20 μm, Polymicro Technologies) were connected with suitable fittings to a syringe pump to introduce droplets of the desired size into the suspending oil phase.
Static contact angles were measured by placing the substrate in a rectangular cuvette (10 × 10 × 45 mm, polystyrene cuvettes, Sarstedt AG & Co.). The cuvette was filled with the suspending medium and a large glycerol (R ∼ 300 µm) drop was introduced using a silica capillary. The drop was allowed to settle toward the substrate and eventually spread on the substrate, after which images were taken using a camera and a ×4 objective lens. These images were then used to measure the contact angle.