Monitoring Extracellular pH in Cross-Kingdom Biofilms using Confocal Microscopy

Summary

The protocol describes the cultivation of cross-kingdom biofilms consisting of Candida albicans and Streptococcus mutans and presents a confocal microscopy-based method for the monitoring of extracellular pH inside these biofilms.

Abstract

Cross-kingdom biofilms consisting of both fungal and bacterial cells are involved in a variety of oral diseases, such as endodontic infections, periodontitis, mucosal infections and, most notably, early childhood caries. In all of these conditions, the pH in the biofilm matrix impacts microbe-host interactions and thus the disease progression. The present protocol describes a confocal microscopy-based method to monitor pH dynamics inside cross-kingdom biofilms comprising Candida albicans and Streptococcus mutans. The pH-dependent dual-emission spectrum and the staining properties of the ratiometric probe C-SNARF-4 are exploited to determine drops in pH in extracellular areas of the biofilms. Use of pH ratiometry with the probe requires a meticulous choice of imaging parameters, a thorough calibration of the dye, and careful, threshold-based post-processing of the image data. When used correctly, the technique allows for the rapid assessment of extracellular pH in different areas of a biofilm and thus the monitoring of both horizontal and vertical pH gradients over time. While the use of confocal microscopy limits Z-profiling to thin biofilms of 75 µm or less, the use of pH ratiometry is ideally suited for the noninvasive study of an important virulence factor in cross-kingdom biofilms.

Introduction

Cross-kingdom biofilms comprising both fungal and bacterial species are involved in several pathologic conditions in the oral cavity. Candida spp. have frequently been isolated from endodontic infections¹ and from periodontal lesions^2,3. In mucosal infections, streptococcal species from the mitis group have been shown to enhance fungal biofilm formation, tissue invasion, and dissemination in both in vitro and murine models^4,5,6,7. Most interestingly, oral carriage of Candida spp. has been proven to be associated with the prevalence of caries in children⁸. As shown in rodent models, a symbiotic relationship between Streptococcus mutans and Candidas albicans increases the production of extracellular polysaccharides and leads to the formation of thicker and more cariogenic biofilms^9,10.

In all of the above-mentioned conditions, early childhood caries in particular, the biofilm pH is of importance for disease progression, and the eminent role of the biofilm matrix for the development of acidogenic microenvironments¹¹ calls for methodologies that allow studying pH changes inside cross-kingdom biofilms. Simple and accurate confocal microscopy-based approaches to monitor pH inside bacterial¹² and fungal¹³ biofilms have been developed. With the ratiometric dye C-SNARF-4 and threshold-based image post-processing, extracellular pH can be determined in real-time in all three dimensions of a biofilm¹⁴. Compared to other published techniques for microscopy-based pH-monitoring in biofilms, pH ratiometry with C-SNARF-4 is simple and cheap, because it does not require the synthesis of particles or compounds that include a reference dye¹⁵ or the use of two-photon excitation¹⁶. The use of just one dye prevents problems with probe compartmentalization, fluorescent bleed-through, and selective bleaching^16,17,18 while still allowing for a reliable differentiation between intra- and extracellular pH. Finally, incubation with the dye is performed after biofilm growth, which allows studying both laboratory and in situ-grown biofilms.

The aim of the present work is to extend the use of pH ratiometry and provide a method to study pH changes in cross-kingdom biofilms. As proof of concept, the method is used to monitor pH in dual species biofilms consisting of S. mutans and C. albicans exposed to glucose.

Protocol

The protocol for saliva collection was reviewed and approved by the Ethics Committee of Aarhus County (M-20100032).

1. Cultivation of Cross-kingdom Biofilms

1. Grow S. mutans DSM 20523 and C. albicans NCPF 3179 on blood agar plates at 37 °C under aerobic conditions.
2. Transfer single colonies of each organism to test tubes filled with 5 mL of brain heart infusion (BHI). Grow for 18 h under aerobic conditions at 37 °C.
3. Centrifuge the overnight cultures at 1,200 x g for 5 min. Discard the supernatant, resuspend the cells in physiological saline and adjust the OD_{550 nm} to 0.5 for C. albicans (~10⁷ cells/mL) and S. mutans (~10⁸ cells/mL). Dilute the S. mutans suspension 1:10 with sterile physiological saline (~10⁷ cells/mL).
4. Pipette 50 µL of sterile salivary solution, prepared according to the method of de Jong et al.¹⁹, into the wells of an optical bottom 96-well plate for microscopy. Incubate for 30 min at 37 °C. Wash the wells 3x with 100 µL of sterile physiological saline. Empty the wells.
5. Add 100 µL of C. albicans suspension to each well. Incubate at 37 °C for 90 min. Wash 3x with sterile physiological saline.
   NOTE:Do not empty the wells completely during washing. Leave a reservoir of 20 µL to avoid excessive shear forces.
6. Add 100 µL of heat-inactivated fetal bovine serum (inactivated at 56 °C for 30 min) to each well. Incubate at 37 °C for 2 h. Wash 3x with sterile physiological saline. Empty the wells, leaving a reservoir of 20 µL.
7. Add 100 µL of S. mutans suspension (prepared in step 1.3) to each well. Add 150 µL of BHI containing 5% sucrose. Incubate at 37 °C for 24 h or longer. When cultivating older biofilms, change the medium daily to fresh BHI. At the end of the cross-kingdom biofilm growth phase, wash 5x with sterile physiological saline.

2. Ratiometric pH Imaging

NOTE:Ratiometric pH imaging needs to be performed immediately after biofilm growth is complete.

1. For ratiometric pH imaging, use an inverted confocal laser scanning microscope with a 63x oil or water immersion lens, a 543 nm laser line, and a spectral imaging system (i.e., META detector) to allow for the imaging of overlapping fluorescent signals. Use an incubator to warm the microscope stage to 35 °C.
2. Set the detector to ensure the detection of green fluorescence from 576−608 nm and simultaneous detection of red fluorescence from 629−661 nm. Choose an appropriate laser power and gain to avoid over- and underexposure.
   NOTE: Exposure of the images is best seen in palette images with false coloring.
3. Set the pinhole size to 1 Airy Unit or an optical slice of ~0.8 µm. Set the image size to 512 x 512 pixels and the scan speed to 2. Choose a line average of 2, using the mean option.
   NOTE:At the beginning of a series of experiments, check that the chosen microscope settings provides a clear contrast between bacterial cells, fungal cell walls, biofilm matrix, and fungal cytoplasm.
4. Prepare 100 µL of sterile physiological saline containing 0.4% (w/v) of glucose, titrated to pH 7. Prepare a stock solution of C-SNARF-4 (1 mM in dimethyl sulfoxide). Add the dye to a final concentration of 30 µM.
   CAUTION: Wear nitrile gloves when handling the ratiometric dye.
5. Empty one of the wells with a cross-kingdom biofilm, leaving a reservoir of 20 µL. Add the sterile saline containing glucose and the ratiometric dye. Place the 96-well plate on the microscope stage and start imaging the biofilm.
6. Acquire single images or Z-stacks in different locations of the biofilm. Mark the X-Y position in the microscope software to follow pH changes in particular fields of view over time. At regular intervals, take images with the laser turned off to correct for detector offset.
7. Repeat steps 2.4–2.6 for the analysis of each biofilm grown in a different well.

3. Calibration of the Ratiometric Dye

NOTE: Calibration of the dye and the fitting of a calibration curve can be performed on a different day than ratiometric pH imaging.

1. Prepare a series of 50 mM 2-morpholinoethanesulfonic acid (MES) buffer titrated to pH 4.0−7.8 in steps of 0.2 pH units at 35 °C. Pipette 150 µL of each buffer solution into the wells of an optical-bottom 96-well plate for microscopy.
2. Add the dye to the buffer-filled wells of the 96-well plate to a final concentration of 30 µM. Let equilibrate for 5 min.
3. Warm the microscope stage to 35 °C. Choose the same microscope settings as for ratiometric pH imaging. Place the 96-well plate on the microscope stage. Focus on the bottom of the wells. Acquire two images (green and red channel) for all buffer solutions, 5 µm above the bottom of the well. At regular intervals, take images with the laser turned off to correct for detector offset.
4. Perform the calibration experiment in triplicate.
5. Export all images as TIF files. Import them into dedicated image analysis software (i.e., ImageJ²⁰). Subtract the images taken with the laser turned off from the respective images of the buffer solutions by clicking Process | Image Calculator | Subtract).
   NOTE: If necessary, crop the images to eliminate artifacts at the image borders by performing rectangular selection using Image | Crop.
6. Divide the green channel images through the red channel images and calculate the average fluorescence intensities in the resulting images by clicking Analyze | Histogram.
7. From the triplicate experiments, plot the average green/red ratios against the pH. Use dedicated software to fit a function to the calibration data (i.e., MyCurveFit).

4. Digital Image Analysis

NOTE:Digital image analysis can be performed at any time point after calibration of the dye and ratiometric pH imaging.

1. Store the green and red channel biofilm images in separate folders and rename both series of files with sequential numbers (e.g., GREEN_0001). Import the images into dedicated image analysis software (i.e., ImageJ). Click Analyze | Histogram to determine the average fluorescence intensity in the images taken with the laser off and subtract the value from the biofilm images by clicking Process | Math | Subtract.
2. Import the 2 image series into dedicated image analysis software (i.e., daime²¹). Perform a threshold-based segmentation of the red channel images (Segment | Automatic segmentation | Custom threshold). Set the 'low' threshold above the fluorescence intensity of the fungal cytoplasm and the 'high' threshold below the intensity of the fungal cell walls and the bacteria.
   NOTE:When appropriate thresholds have been chosen, only extracellular areas are recognized as objects.
3. Transfer the object layer of the segmented image series to the green channel image series. To do so, click Segment | Transfer object layer.
   NOTE: If the contrast between extracellular areas and fungal cytoplasm is too weak in the individual color channels, add the green channel image series to the red channel series prior to segmentation by clicking Edit | Image calculator | Addition. Perform the segmentation as described under 4.2 and transfer the object layer of the segmented image series to both the green and the red channel image series.
4. Employ the object editor to delete non-object pixels in the red and green channel image series (Visualizer | Object editor | In all images | Delete non-object pixels). Now the biofilm images are cleared from both bacterial and fungal cells. Export the processed image series as TIF files.
5. Import both image series to ImageJ. ImageJ assigns an intensity of 0 to all non-object pixels. Remove those pixels by dividing the red image series (R1) by itself (Process | Image calculator | Image 1: R1; Operation: Divide; Image 2: R1) and multiplying the resulting image series (R2) with the original red image series (Process | Image calculator | Image 1: R1; Operation: Multiply; Image 2: R2). A third image series (R3) is created, identical to R1, except for the fact that NaN is assigned to all pixels with an intensity of 0 in R1. Proceed in the same way with the green image series.
6. Use the 'Mean' filter (Process | Filters | Mean | Radius | 1 pixel) on the red and green channel image series to compensate for detector noise. Divide the green channel image series by the red channel image series (Process | Image calculator | Image 1: G3; Operation: Divide; Image 2: R3). The resulting image series (G3/R3) shows the green/red ratio for all object pixels.
7. Calculate the average ratio for each image (Analyze | Histogram). Apply false coloring for better visual representation of the ratios in the images (Image | Lookup Tables). Convert the green/red ratios to pH values employing the function fitted under 3.6.

Results

After 24 h and 48 h, robust cross-kingdom biofilms developed in the well plates. C. albicans showed varying degrees of filamentous growth, and S. mutans formed dense clusters of up to 35 µm in height. Single cells and chains of S. mutans grouped around fungal hyphae, and large intercellular spaces indicated the presence of a voluminous matrix (Figure S1).

Calibration of the ratiometric dye yields an asymmetrical sigmoidal curve^...

Discussion

Different protocols for the cultivation of cross-kingdom biofilms involving C. albicans and Streptococcus spp. have been described previously^{9,22,23,24,25}. However, the present setup focuses on simple growth conditions, a time schedule compatible with regular working days, a balanced species composition, and the development of a volu...

Materials

Name	Company	Catalog Number	Comments
Blood agar plates	Statens Serum Institut	677
Brain heart infusion	Oxoid	CM1135
Brain heart infusion + 5 % sucrose	BDH laboratory supplies	10274
Candida albicans	National Collection of Pathogenic Fungi	NCPF 3179
D-(+)-Glucose	Sigma-Aldrich	G8270
daime: digital image analysis in microbial ecology	Universität Wien	N/A	Freeware; V2.1; https://dome.csb.univie.ac.at/daime
Dimethyl sulfoxide	Life Technologies	D12345
Fetal bovine serum	Gibco Life technologies	10270
GS-6R refrigerated centrifuge	Beckman	N/A
ImageJ	National Institutes of Health	N/A	Freeware; V1.46r; https://imagej.nih.gov/ij
Java	Oracle	N/A	Freeware necessary to run ImageJ; V8.0; https://java.com/en/download
µ-Plate 96 Well Black	Ibidi	89626
MyCurveFit	MyAssays Ltd.	N/A
2-(N-Morpholino)ethanesulfonic acid (MES) buffer	Bioworld	700728
PHM210 pH-meter	Radiometer Analytical
Plan-Apochromat 63x oil immersion objective	Zeiss	N/A	NA=1.4
SNARF®-4F 5-(and-6)-Carboxylic Acid	Life Technologies	S23920
Sterile physiological saline	VWR	6404
Streptococcus mutans	Deutsche Sammlung von Mikroorganismen und Zellkulturen	DSM 20523
Vis-spectrophotometer V-3000PC	VWR	N/A
XL Incubator	PeCON	N/A
Zeiss LSM 510 META	Zeiss	N/A