Nunc thermanox

Synchrotron XFI was performed at the Stanford Synchrotron Radiation Lightsource (SSRL), Stanford University, Menlo Park, CA, USA on 15-μm thick sections collected on metal-free plastic coverslips (Nunc Thermanox, Thermo Scientific, USA). Whole sections were imaged on beamline 10-2, mounted at 45° to the incident beam (12.5 keV) which passed through a glass polycapillary (XOS, USA) to produce a 50 μm × 50 μm spot on the sample. The beam exposure time was 80 ms/pixel. Fluorescent energy windows were centered for iron (6.21–6.70 keV) and zinc (8.38–8.98 keV).
Regions of interest (ROI) were imaged at higher resolution using the microfocused XFI on beamline 2–3 with a 3 μm × 3 μm incident X-ray beam (7.15 keV) focused with Kirkpatrick–Baez mirrors, and a 120 ms/pixel dwell time. Fluorescence was normalized against the incident X-ray beam intensity to take into account its fluctuations. X-ray fluorescence was detected using a silicon drift detector (Hitachi) mounted 90° to the incident beam utilizing the Xpress3 signal processing system (Quantum Detectors, UK).

The multi-species biofilm consisted of the strains F. nucleatum (ATCC 10953), A. naeslundii (DSM 17233), S. gordonii (NCTC 7865) and P. gingivalis (W83). Overnight cultures of F. nucleatum were prepared in Schaedler Anaerobic broth and grown anaerobically at 37 °C. A. naeslundii, P. gingivalis and S. gordonii cultures were prepared in BHI broth. Bacteria were grown anaerobically at 37 °C, except S. gordonii, which was grown at 37 °C in 5% CO₂. The overnight cultures were diluted with PBS (0.01 M) to an optical density of 0.5 for S. gordonii and 0.2 for all other species (at 600 nm). To form the biofilms, 500 µL of A. naeslundii and S. gordonii were pipetted into a well of a 24-well plate onto a coverslip (13 mm diameter, Thermo Scientific™ Nunc™ Thermanox™), and incubated with 500 µL of artificial saliva for 24 h at 37 °C. The planktonic culture was then replaced with 500 µL of F. nucleatum and 500 µL of artificial saliva and cultured for a further 24 h. Finally, the planktonic culture was replaced with 500 µL of P. gingivalis and 1.5 mL of artificial saliva. Biofilms were incubated at 37 °C until 5 days old.

A frozen stock of PA01-N was used to grow P. aeruginosa colonies on BHI agar at 37 °C, 5% CO₂. Overnight cultures were grown by inoculating 5 mL of BHI broth with three colonies of PA01 and incubating at 37 °C, continuously shaking at 100 rpm for 18 h. The overnight culture was diluted using BHI broth to an optical density of 0.01 (at 600 nm), of which, 1 mL was placed in a well of a 24-well plate containing a coverslip (13 mm diameter, Thermo Scientific™ Nunc™ Thermanox™) and was performed in triplicate. The plate was then incubated for 3 h at 37 °C, shaking at 80 rpm to allow cells to adhere to the coverslip. The culture was removed from the wells and replaced with 1 mL of BHI broth, which was incubated for 24 h at 37 °C, shaking at 80 rpm. The fluorescent staining protocol was conducted as described above.

Scanning electron microscopy (SEM) was employed to visualize C. glabrata biofilm structures. The experiments were repeated three times per sample and at least two independent clones for each strain were examined. To allow for biofilm formation, the overnight culture was collected, washed twice with PBS, and diluted to an OD600 of 0.1 with YPD broth. Then, 2 mL of the cell suspension was added to a 24-well microtiter plate with plastic coverslips (Nunc Thermanox; Thermo Scientific). After incubation at 37°C for 24 h, the plastic coverslips were washed twice with PBS and fixed in 2.5% glutaraldehyde at 4°C overnight. The samples were washed three times with PBS and postfixed with 1% osmium tetroxide solution at 4°C for 1–2 h. Subsequently, the samples were washed three times with PBS and then dehydrated serially in increasing concentrations of ethanol: 30%, 50%, 70%, 80%, and 90% ethanol (each for 15 min); this was followed by submersion in 100% ethanol at 4°C for 20 min three times. The samples were then dried in an EM CPD300 critical point dryer (Leica, Germany), fixed to the specimen stage, and coated with a 10-nm gold layer using JEE-420 sputter coater. Finally, biofilm structures were examined and photographed under a scanning electron microscope (SU8010; Hitachi).

Single- and dual-species biofilms were formed on polystyrene coupons (NuncThermanox, Thermo Scientific, MA, USA) placed on 24-well polystyrene microtiter plates as described above, with slight modifications. The volume of bacterial suspension was adjusted to 1 mL/well instead of 200 μL/well. Expression of mCherry fluorophore and sfGFP was induced by adding 1 mM IPTG and 0.2% (v/v) of L-arabinose^{54 (link),55 (link)}, after 43 h of biofilms formation. Induction occurred during 5 h in the 48 h-old biofilms. Z-stacks were acquired on a CLSM (Olympus BX61, Model FluoView 1000, Olympus, Tokyo, Japan) equipped with 405–635 nm laser lines. Images were obtained with the FV10-Ver4.1.1.5 program (Olympus, Tokyo, Japan).