Quantificazione concomitante di cellulare e extracellulare componenti del biofilm

1. Preparation of Media and Reagents Make 300 ml overnight culture medium (THY; 3% Todd Hewitt Broth prepared according to manufacturer's instructions supplemented with 0.3% Yeast Extract in ultrapure water) and autoclave. Store at room temperature for up to 2 months. Make 1 L THY agar (3% Todd Hewitt Broth, 0.3% Yeast Extract, and 1.5% agar in ultrapure water), autoclave, and cool to 60 °C before pouring into Petri dishes. Store at 4 °C for up to 3 months. Make 150 ml biofilm growth medium (0.5X TY supplemented with 10 mM sucrose; 1.5% Tryptone, 0.5% Yeast Extract, and 10 mM sucrose in ultrapure water) and filter sterilize. Store at room temperature for up to 1 month. Make 1 L Phosphate Buffered Saline (PBS; 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, and 0.24 g KH2PO4 in ultrapure water) and autoclave. Store at room temperature for several months. Make 600 ml 10 mM Tris HCl buffer supplemented with 10 mM CaCl2 in ultrapure water (TC buffer; pH 7.2) and autoclave. Store at room temperature for several months. Autoclave 1 L ultrapure water. Store at room temperature for several months. 2. Specimen Fabrication Fabricate disk-shaped specimens of Point 4 (PF) and TPH3 (TP) dental resin composites in a custom-made stainless steel mold in two increments. Each increment is light-cured for 40 sec using an LED light-curing unit. The two products have slightly different compositions and filler levels, and thereby produce different surface topographies upon which the biofilms will be grown. Specimens of relevant substrates could be fabricated using established protocols from other disciplines in lieu of step 2.1. Finish and polish specimens to an acceptable final surface finish, e.g. by using a semi-automated grinder-polisher. Rinse polished specimens with ultrapure water and dry using compressed air. Sterilize specimens using ethylene oxide gas or an alternate sterilization method. 3. Biofilm Growth Inoculate a single colony of S. mutans into 4 ml overnight culture medium (step 1.1). Incubate overnight at 37 °C for 16-18 hr. The optical density (OD600) of the culture should be ≥0.9. It is best to use a colony from plate cultures that have been passaged 2x from an original stock, as this results in more consistent biofilm growth. Create a 1:100 dilution using the overnight culture (step 1.1) in 10 ml of biofilm growth medium (step 1.3). Transfer sterile resin composite specimens (e.g. PF, TP) to sterile 12-well plates. Add 2.5 ml of diluted culture medium (step 3.2) to wells for treatment (mouthwash) and control groups. Add 2.5 ml of sterile uninoculated biofilm growth medium to sterility control wells. Grow the biofilms under microaerophilic conditions. Place plates on a shaker at 100 rpm inside an incubator at 37 °C for 24 hr. After 24 hr, aseptically aspirate medium from all wells and wash twice with sterile PBS (step 1.4; 2.5 ml/well), aspirating the PBS after each wash. Replenish 2.5 ml of fresh biofilm growth medium in each well and incubate for an additional 24 hr under identical conditions as the previous step, for total biofilm growth time of 48 hr. In lieu of the above steps, biofilms of other species could be grown on substrates using established protocols. 4. Antibacterial/Mouthwash Treatments Aspirate medium after biofilm growth is complete. Add 2.5 ml Biotène PBF (BIO) or Listerine Total Care (LTO) mouthwash, or other treatment modality, in wells for the treatment groups, and add 2.5 ml sterile ultrapure water to the control group well. Immerse specimens for 60 sec, as per manufacturers' instructions, while agitating plates on an orbital shaker at 150 rpm. Immediately aspirate both the mouthwash and ultrapure water from the wells. Wash the specimens 5x for 15 sec per wash with sterile ultrapure water on the orbital shaker at 150 rpm, aspirating water after each washing step. In lieu of the above steps, other antibacterial agents could be tested using established protocols. 5. Staining Prepare the stain dilutions for biofilm analysis. Prepare a 5 mg/ml stock solution of Concanavalin A, Alexa Fluor 647 conjugate (AF) in 0.1 M sodium bicarbonate pH 8.3. Store at -20 °C in single-use aliquots for several months because freezing and thawing is discouraged. Using this AF stock solution, prepare a 250 μg/ml dilution in TC buffer. Prepare a 10 μM dilution using the 5 mM Syto 9 stock solution (SY), as provided by the manufacturer, in TC buffer. Store the remaining SY stock solution at -20 °C for several months. Prepare a 10x dilution using the 5,000x Sypro Red stock solution (SR), as provided by the manufacturer, in sterile ultrapure water. Store the remaining SR stock solution at -20 °C for several months. Wash all biofilms 2x with TC buffer (2.5 ml/well) using a hand-swirling motion, allowing the second wash to remain in the plate for 30 min at room temperature. Aspirate. Place a 50 μl drop of AF stain on each biofilm specimen. Stain for 30 min at room temperature, protected from light. Wash 2x with TC buffer (2.5 ml/well), aspirating after each wash. Follow with a 50 μl drop of SY stain on each biofilm specimen. Stain for 30 min at room temperature, protected from light. Wash 2x with sterile ultrapure water (2.5 ml/well), aspirating after each wash Finally, place a 50 μl drop of SR stain on each biofilm specimen. Stain for 30 min at room temperature, protected from light. Wash 3x with sterile ultrapure water (2.5 ml/well), aspirating after each wash. Transfer the specimens to a 6-well plate, placing one specimen per well in 6 ml sterile ultrapure water to facilitate confocal microscopy. 6. Imaging Using Confocal Laser Scanning Microscopy Acquire images of each component (stain) within biofilms of the treatment and control groups using a confocal laser scanning microscope settings shown in Table 1. A 63X dipping lens with a 0.9 numerical aperture that has a working distance of 2.2 mm was used in this study. Table 1. Confocal Laser Scanning Microscope Settings. Set the scan size to 250 μm x 250 μm and a minimum pixel resolution of 512 x 512 to acquire CLSM images at a resolution adequate for quantitative analysis. Use the SY stain to set the top and bottom points of the biofilm image stack, then set the z-step to be adequate for quantitative analysis (0.6 μm for this study). Before collecting a scan, optimize the image and stain parameters (i.e. PMT voltage and offset) using the QLUT option. A color look-up table was used to assign pseudo-colors to each stain (green for SY, red for SR, and blue for AF) in order to make each biofilm component more distinguishable in the 3D reconstruction. Collect CLSM images at 400 Hz using sequential scanning in the "between stacks" mode to optimize capture of images of multiple stains within the same biofilm. 7. Biofilm Structural Analysis Analyze the CLSM images collected using image analysis software for biofilms. The following steps describe the use of ISA3D software12 to calculate the structural parameters of the biofilm images collected. Copy CLSM image files for each biofilm to the Images folder, as specified in the software's manual. Ensure that the naming convention for CLSM files required by the software is followed. The software permits analysis of files within subfolders in batch mode, thus facilitating analysis of treatment and control group biofilms in the same run. Enter x-, y-, and z-axis dimensions of CLSM images in the dxy and dz fields of the main dialog box. Select threshold and distance mapping settings, as described in the software's manual (Otsu and Quasi-Euclidean were used for this study, respectively). Input a suitable name for the results file, and then run the program. A results file will be produced in the ISA folder. View the results file to obtain values for twenty 3D structural parameters12 such as biovolume and mean biofilm thickness (measured in this study) that quantify the 3D distribution of each component (stain) within the biofilm. 8. Visualization of Biofilm Structure Create a reconstruction of overlaid components (stains) within each biofilm using image analysis software. The following steps describe the use of Volocity software to create 3D reconstructions. Create a library for each biofilm by copying CLSM image files into the software, as described in the manual. Use the 3D Renderer menu option to produce a reconstructed 3D image from the CLSM images of each biofilm (HR Opacity format was used for this study). Rotate the 3D image to orient all of the biofilms in a similar manner with regard to the x-, y-, and z-coordinate axes. Capture a snapshot of the correctly oriented biofilm reconstruction, and then export the snapshot in TIFF, JPEG or other suitable format. Perform visual analysis of the concurrent distribution of EPS, proteins and nucleic acid components within biofilms on the reconstructed images. Use Pearson's correlation coefficient and Manders' overlap coefficient to perform a colocalization analysis of multiple biofilm components (colocalization was not measured in this study). 9. Statistical Analysis Use separate mixed models statistical analyses to compare mean values of structural parameters between mouthwash and control groups (α=0.05). For this study, SAS software was used to perform the statistical analysis.