An Assay for Real-Time Monitoring of Extracellular pH in Dental Biofilms

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Collection of In Situ Grown Dental Biofilm Samples

1. Select volunteers that fulfill inclusion and exclusion criteria relevant for the study. Make alginate impressions of their upper and lower dental arch. Make cast models from these impressions and manufacture an acrylic splint in the lower jaw. Design the splint with buccal acrylic flanges connected by a lingual orthodontic wire that allows the volunteer to bite into normal occlusion.
2. Drill recessions in the buccal flanges of the acrylic splint with the help of dental acrylic burs to allow insertion of glass slabs for biofilm collection. The depth of the recessions should be at least 1.5 mm, while the width and length of the recessions may vary depending on the number of glass slabs to be inserted.
3. For biofilm collection, use custom-made non-fluorescent glass slabs (4 x 4 x 1 mm³) with a surface roughness of grit 1,200 in order to mimic the colonization pattern on natural enamel.
4. Sterilize the glass slabs by autoclaving prior to mounting. Mount the glass slabs with sticky wax in the depressions in the buccal flanges of each side slightly recessed to the surface of the acrylic surface in order to protect the biofilm from shear forces exerted by movement of the cheeks.
   Note: The number of glass slabs placed in a recession may vary between 3 and 14, depending on the aim of the study.
5. Insert the appliance in the mouth of the volunteer. Instruct the volunteer to retain the appliance intra-orally throughout the experimental period. Instruct the volunteer to store the appliance in an orthodontic retainer container with a piece of wet paper tissue (to keep it humid) at room temperature during tooth brushing and intake of food and beverages other than water. Instruct the volunteer to not touch the buccal acrylic flanges with the glass slabs while placing and removing the appliance.
   Note: The experimental period may vary depending on the aim of the study (one day to several weeks).
6. Carefully remove the glass slabs from the appliance at the end of the experimental period. Remove the sticky wax around the slabs with a knife and transfer them with a pair of tweezers to a closed container, the biofilm facing upward, until microscopic analysis. Keep the container humid with wet paper tissue. Perform pH imaging within few hours after biofilm collection.

2. Biofilm pH Imaging

1. Prepare salivary solution by adding dithiothreitol to collected saliva according to the method of de Jong et al. Titrate the salivary solution to pH 7.0 and add glucose to a concentration of 0.4 % (wt/vol). Pipette 100 µl per biofilm to be analyzed into a glass-bottom 96-well plate for microscopy. Add 5 µl of the ratiometric dye per well.
2. Place the 96-well plate on the microscope stage. Turn on the microscope and the 543 nm laser line. Warm up the incubator to 37 °C. Use the same microscope settings as for the calibration of the dye. Wait for 30 min, until the 96-well plate has reached working temperature.
3. Pick up one or more glass slabs with a slim set of tweezers and place them in the saliva-filled wells, one slab per well, with the biofilms facing downward.
4. Acquire single images ("Scan Control" → "Single") or z-stacks ("Scan Control" → "Start") spanning the depth of the biofilms in different areas. To acquire z-stacks choose the number of slices to be imaged ("Scan Control" → "Z Settings" → "Num Slices") and mark the z-position for the first and the last slice in the microscope software ("Scan Control" → "Z Settings" → "Mark First"; "Mark Last").
   Note: Z-stacks with a depth of up to 75 µm can be acquired with good contrast between extracellular and intracellular areas.
5. To follow pH changes in a microscopic field of view over time, mark the x-y-position in the microscope software ("Stage and Focus Control" → "Mark Pos") and take repeated images at consecutive time points ("Scan Control" → "Single"). Regularly take images with the laser power set to zero for background subtraction.

3. Digital Image Analysis

1. To export the microscopic images as TIF files, use the file batch export of the microscope software ("Macro" → "File Batch Export"). Mark the files to be exported and save red and green channel images in separate folders as TIF-files ("Start Batch Export"). Rename the files in both folders giving them sequential numbers.
2. Import the red and green image series into software such as daime (digital image analysis in microbial ecology). Segment the green channel images with individually chosen brightness thresholds (Segment → Automatic segmentation → Custom threshold). Choose the brightness thresholds with care (typically between 20 and 80), so that all bacteria (brighter than the extracellular matrix), but not the matrix will be recognized as objects during segmentation. Verify visually that the areas recognized as objects correspond well to the bacterial biomass.
3. Transfer the object layer of the segmented green channel images to the corresponding red channel images (Segment → Transfer object layer). Use the object editor function to reject and delete all objects in the red and green channel images. Now only the extracellular matrix is left in the biofilm images. Export the processed image series as TIF files.
4. Import the image series into ImageJ (http://rsb.info.nih.gov/ij; v.1.47). Determine the average fluorescence intensity in the background images taken with the laser turned off (Analyze →Histogram). Subtract the appropriate background from the red and green images (Process →Math →Subtract).
5. Still in ImageJ, divide the green image series (G1) by itself (Process → Image calculator). Then multiply the resulting image series (G2) with the green image series (G1). This will yield an image series (G3), where NaN is assigned to all pixels belonging to areas that were recognized as objects in daime. Proceed in the same way with the red image series (R1/R1 = R2; R2 x R1 = R3).
   Note: As the bacterial biomass was removed from the images in step 3.3, the fluorescent intensity is 0 in these areas. Step 3.5 is necessary to convert the value 0 to NaN, which allows for ratio calculation in step 3.6.
6. Apply the 'Mean' filter (Process →Filters →Mean; radius: 1 pixel) to compensate for detector noise. Divide the green image series by the red image series (Process →Image calculator). This results in a green/red ratio for every remaining pixel in the extracellular space of the images. Use false coloring for graphic representation of the ratios in the images (Image →Lookup Tables). Calculate the mean ratio for each image (Analyze →Histogram).
7. Convert the green/red ratios to pH values according to the function fitted.