Name: Monitoring Extracellular pH in Cross-Kingdom Biofilms using Confocal Microscopy (Video) | JoVE Journal
Uploaded: 2020-01-30T14:00:00.000+00:00
Duration: 10 min 26 s
Description: Learn to monitor extracellular pH in cross-kingdom biofilms using confocal microscopy and pH ratiometry. This method enables real-time analysis of Candida albicans and Streptococcus mutans interactions.

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5 minutes

Anette Aakj&#230;r Thomsen and Javier E. Garcia are acknowledged for excellent technical support. The authors thank Rubens Spin-Neto for fruitful discussions on image analysis.

The protocol for saliva collection was reviewed and approved by the Ethics Committee of Aarhus County (M-20100032).
1. Cultivation of Cross-kingdom Biofilms
<ol>
	<li>Grow S. mutans DSM 20523 and C. albicans NCPF 3179 on blood agar plates at 37 &#176;C under aerobic conditions.</li>
	<li>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 &#176;C.</li>
	<li>Centrifuge the overnight cultures at 1,200 x g for 5 min. Discard the supernatant, resuspend the cells in physiological saline and adjust the OD550 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).</li>
	<li>Pipette 50 &#181;L 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 &#176;C. Wash the wells 3x with 100 &#181;L of sterile physiological saline. Empty the wells.</li>
	<li>Add 100 &#181;L of C. albicans suspension to each well. Incubate at 37 &#176;C for 90 min. Wash 3x with sterile physiological saline. 
	NOTE:Do not empty the wells completely during washing. Leave a reservoir of 20 &#181;L to avoid excessive shear forces.</li>
	<li>Add 100 &#181;L of heat-inactivated fetal bovine serum (inactivated at 56 &#176;C for 30 min) to each well. Incubate at 37 &#176;C for 2 h. Wash 3x with sterile physiological saline. Empty the wells, leaving a reservoir of 20 &#181;L.</li>
	<li>Add 100 &#181;L of S. mutans suspension (prepared in step 1.3) to each well. Add 150 &#181;L of BHI containing 5% sucrose. Incubate at 37 &#176;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.</li>
</ol>
2. Ratiometric pH Imaging
NOTE:Ratiometric pH imaging needs to be performed immediately after biofilm growth is complete.
<ol>
	<li>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 &#176;C.</li>
	<li>Set the detector to ensure the detection of green fluorescence from 576&#8722;608 nm and simultaneous detection of red fluorescence from 629&#8722;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.</li>
	<li>Set the pinhole size to 1 Airy Unit or an optical slice of ~0.8 &#181;m. 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.</li>
	<li>Prepare 100 &#181;L 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 &#181;M. 
	CAUTION: Wear nitrile gloves when handling the ratiometric dye.</li>
	<li>Empty one of the wells with a cross-kingdom biofilm, leaving a reservoir of 20 &#181;L. Add the sterile saline containing glucose and the ratiometric dye. Place the 96-well plate on the microscope stage and start imaging the biofilm.</li>
	<li>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.</li>
	<li>Repeat steps 2.4&#8211;2.6 for the analysis of each biofilm grown in a different well.</li>
</ol>
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.
<ol>
	<li>Prepare a series of 50 mM 2-morpholinoethanesulfonic acid (MES) buffer titrated to pH 4.0&#8722;7.8 in steps of 0.2 pH units at 35 &#176;C. Pipette 150 &#181;L of each buffer solution into the wells of an optical-bottom 96-well plate for microscopy.</li>
	<li>Add the dye to the buffer-filled wells of the 96-well plate to a final concentration of 30 &#181;M. Let equilibrate for 5 min.</li>
	<li>Warm the microscope stage to 35 &#176;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 &#181;m above the bottom of the well. At regular intervals, take images with the laser turned off to correct for detector offset.</li>
	<li>Perform the calibration experiment in triplicate.</li>
	<li>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.</li>
	<li>Divide the green channel images through the red channel images and calculate the average fluorescence intensities in the resulting images by clicking Analyze | Histogram.</li>
	<li>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).</li>
</ol>
4. Digital Image Analysis
NOTE:Digital image analysis can be performed at any time point after calibration of the dye and ratiometric pH imaging.
<ol>
	<li>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.</li>
	<li>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 &#39;low&#39; threshold above the fluorescence intensity of the fungal cytoplasm and the &#39;high&#39; 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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Use the &#39;Mean&#39; 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.</li>
	<li>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.</li>
</ol>

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The authors have nothing to disclose.

Different protocols for the cultivation of cross-kingdom biofilms involving C. albicans and Streptococcus spp. have been described previously9,22,23,24,25. 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...

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 lesions2,3. 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 models4,5,6,7. 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 biofilms9,10.
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 bleaching16,17,18 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.

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

monitoring extracellular ph in cross-kingdom biofilms using confocal microscopy

Monitoring Extracellular pH in Cross-Kingdom Biofilms using Confocal Microscopy

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 &#181;m or less, the use of pH ratiometry is ideally suited for the noninvasive study of an important virulence factor in cross-kingdom biofilms.

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 curve13</s...

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.

Cross-kingdom biofilms consisting of both fungal and bacterial cells are involved in a variety of oral diseases, such as endodontic infections, ...

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Learn to monitor extracellular pH in cross-kingdom biofilms using confocal microscopy and pH ratiometry. This method enables real-time analysis of Candida albicans and Streptococcus mutans interactions.

Monitoring Extracellular pH in Cross-Kingdom Biofilms using Confocal Microscopy

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