En tercer lugar, como toda forma de participación del Estado en la economía, la

65 2.6.1 Confocal laser scanning microscopy

2.6.1.1 LIVE/DEAD analysis of microbial biofilms

The biofilm architecture of oral bacteria and CRS isolates, with a particular focus on extracellular DNA was visualized by confocal laser scanning microscopy (CLSM) using biofilms cultured on glass surfaces. Sterile 13 mm diameter glass coverslips were placed in wells of a six-well tissue culture plate containing 3 mL growth medium. Wells were inoculated with 50 µL of stock bacterial cultures and incubated statically in air at 37°C for 18-72 h. Coverslips were removed, rinsed three times with PBS, and inverted onto a rubber O-ring (10 mm diameter) that had been placed on a microscope slide and filled with LIVE/DEAD® BacLightTM stain (Molecular Probes). LIVE/DEAD BacLightTM contains two fluorescent stains, SYTO® 9 and propidium iodide (PI), that allow for the monitoring of bacterial cell viability (Boulos et al., 1999). Both of these fluorescent dyes bind to nucleic acids but SYTO® 9 has specificity for chromosomal DNA, whereas PI will stain both intracellular and extracellular DNA. Furthermore, PI displaces SYTO® 9 if it enters a compromised cell membrane. These characteristics allow the kit to be useful for visualising eDNA, as well as monitoring cell viability. Biofilms were examined using a Leica TCS SP2 confocal microscope with an argon/neon laser for visualisation of SYTO® 9 (excitation 485 nm, emission 519 nm), and PI (excitation 536 nm, emission 617 nm).

2.6.1.2 Extracellular DNA staining of voice prosthesis biofilms

Voice prosthesis biofilms were visualised using a CLSM dual stain technique developed previously (Kania et al., 2010) with the addition of 4',6-diamidino-2-phenylindole (DAPI) (Invitrogen). Used TESVs were washed three times with PBS to remove planktonic cells. Samples were then immersed in 2 mL of PBS to which 10 µL of PI (1 mg/mL) (Sigma Aldrich) were added; this was incubated in darkness, at room temperature, for 5 min on a moving plate after which excess stain was rinsed off. The valve was then incubated with 50 µg/mL Concanavalin A Alexa Fluor® 488 (Invitrogen) for 5 min at room temperature. Finally, the biofilm was stained with 2 µg/mL DAPI at room temperature, in darkness, for 15 min. Excess stain was removed and valves were mounted on vacuum grease in 6 well tissue culture microtiter plates (Greiner Bio-One). PBS was added to wells until samples were fully immersed in

66 liquid. Biofilms were examined using a Leica TCS SP2 with a diode/argon/helium-neon laser for visualisation of ConA (excitation 485 nm, emission 519 nm), PI (excitation 536 nm, emission 617 nm) and DAPI (excitation 358 nm, emission 461 nm).

2.6.1.3 Visualization of bacteria on the surface of sinus mucosa

Fluorescence in situ hybridisation (FISH) was performed using a peptide nucleic acid (PNA) probe corresponding to the well-characterised EUB338 probe (Amann et al., 1990). The probe was synthesized as a fluorescein amidite (FAM) conjugate by Panagene. Mucosal biopsy specimens were fixed in 10% formalin directly after surgery, and stored at 4°C for up to one month. For PNA-FISH analysis, specimens were transferred to 50% (v/v) ethanol and incubated for 16 h at -20°C. Biopsy material was transferred to 1 mL permeabilization buffer (10 mg/mL lysozyme in PBS) and incubated at 37°C for 30 min. Samples were immersed in 1 mL pre-warmed wash buffer (10 mM Tris-HCl pH 9.0, 1 mM EDTA) for 30 min at 55°C. Pre-warmed hybridization buffer (25 mM Tris-HCl pH 9.0, 100 mM NaCl, 0.5% SDS, 30% formamide) containing 150 pmol/mL of the PNA probe was added to samples and incubated in darkness for 90 min at 55°C. Unbound PNA probe was removed by incubating in pre- warmed wash buffer for 30 min at 55°C. Eukaryotic cells were counterstained by immersing the specimens in 1 mL PBS with DAPI in darkness at 20°C for 15 min. Samples were glue-mounted onto a plastic surface and immersed in 2 mL PBS. Visualisation of surface bacteria and eukaryotic cells was performed using a Leica TCS SP2 microscope with an argon/neon laser for imaging of FAM conjugates (excitation 495 nm, emission 520 nm), and DAPI (excitation 358 nm, emission 461 nm).

2.6.1.4 Microfluidic oral biofilm modelling

Biofilms were grown in a BioFlux (Fluxion) microfluidics device using a similar protocol used by Nance et al., (2013). Biofilms were cultured from pooled human saliva, S. mutans UA159, and S. gordonii DL1. During single-species experiments S.

mutans was grown in presence of 2% sucrose plus cell free saliva (CFS), and S. gordonii with 1% THYE plus CFS. Saliva biofilms were cultured in CFS only. After

micro-organisms were cultured for 20 h they were stained with LIVE/DEAD® BacLightTM and visualised using a Leica SPE CLSM. Afterwards images were rendered using Imaris (BitPlane) imaging software so that a 3D representation of biofilm

67 architecture was made. Image analysis was performed on a dedicated image processing computer in the Bio-imaging Unit at Newcastle University.

2.6.2 Scanning electron microscopy

Scanning electron microscopy was used to study biofilm formation on TESVs and single-species biofilms of chronic rhinosinusitis isolates grown on coverslips. Both types of samples were fixed in 2% (v/v) glutaraldehyde at 4°C for 16 h. Specimens were then rinsed twice in PBS and dehydrated through a series of ethanol washes as follows: 25% ethanol 30 min, 50% ethanol 30 min, 75% ethanol 30 min, and two washes for 1 h in 100% ethanol. Samples were dried in a critical point dryer (Bal-tec), mounted on aluminium stubs and sputter coated with gold at Electron Microscopy Research Services, Newcastle University. Biofilms were visualised using a scanning electron microscope (Cambridge Stereoscan 240).

2.6.3 Transmission electron microscopy of chronic rhinosinusitis obstructive mucin

Samples of obstructive material removed from patients during functional endoscopic sinus surgery were cut into ~1 mm3 pieces and placed into 2% glutaraldehyde, at 4°C, immediately after surgery. These samples were dehydrated through a series of ethanol washes, embedded and sectioned at Electron Microscopy Research Services, Newcastle University. Sections were analysed in a transmission electron microscope (Philips, CM100).