Name: monitoring extracellular ph in cross-kingdom biofilms using confocal microscopy
Uploaded: 2020-01-30T14:00:00
Description: Watch this Scientific Journal Video about Monitoring Extracellular pH in Cross-Kingdom Biofilms using Confocal Microscopy at JoVE.com

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 overni...

<ol>
	<li>Siqueira, J. F., Sen, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fungi+in+endodontic+infections.">Fungi in endodontic infections.</a> Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontics. 97 (5), 632-641 (2004).</li><li>Matic Petrovic, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subgingival+areas+as+potential+reservoirs+of+different+Candida+spp+in+type+2+diabetes+patients+and+healthy+subjects.">Subgingival areas as potential reservoirs of different Candida spp in type 2 diabetes patients and healthy subjects.</a> PloS One. 14 (1), 0210527 (2019).</li><li>De-La-Torre, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+Candida+colonization+in+patients+with+chronic+periodontitis.+Is+there+any+relationship.">Oral Candida colonization in patients with chronic periodontitis. Is there any relationship.</a> Revista Iberoamericana De Micologia. 35 (3), 134-139 (2018).</li><li>Xu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Streptococcal+co-infection+augments+Candida+pathogenicity+by+amplifying+the+mucosal+inflammatory+response.">Streptococcal co-infection augments Candida pathogenicity by amplifying the mucosal inflammatory response.</a> Cellular Microbiology. 16 (2), 214-231 (2014).</li><li>Xu, H., Sobue, T., Bertolini, M., Thompson, A., Dongari-Bagtzoglou, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Streptococcus+oralis+and+Candida+albicans+Synergistically+Activate+&#956;-Calpain+to+Degrade+E-cadherin+From+Oral+Epithelial+Junctions.">Streptococcus oralis and Candida albicans Synergistically Activate &#956;-Calpain to Degrade E-cadherin From Oral Epithelial Junctions.</a> The Journal of Infectious Diseases. 214 (6), 925-934 (2016).</li><li>Dongari-Bagtzoglou, A., Kashleva, H., Dwivedi, P., Diaz, P., Vasilakos, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+mucosal+Candida+albicans+biofilms.">Characterization of mucosal Candida albicans biofilms.</a> PloS One. 4 (11), 7967 (2009).</li><li>Diaz, P. 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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Symbiotic+relationship+between+Streptococcus+mutans+and+Candida+albicans+synergizes+virulence+of+plaque+biofilms+in+vivo.">Symbiotic relationship between Streptococcus mutans and Candida albicans synergizes virulence of plaque biofilms in vivo.</a> Infection and Immunity. 82 (5), 1968-1981 (2014).</li><li>Hwang, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candida+albicans+mannans+mediate+Streptococcus+mutans+exoenzyme+GtfB+binding+to+modulate+cross-kingdom+biofilm+development+in+vivo.">Candida albicans mannans mediate Streptococcus mutans exoenzyme GtfB binding to modulate cross-kingdom biofilm development in vivo.</a> PLoS Pathogens. 13 (6), 1006407 (2017).</li><li>Koo, H., Falsetta, M. L., Klein, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+exopolysaccharide+matrix:+a+virulence+determinant+of+cariogenic+biofilm.">The exopolysaccharide matrix: a virulence determinant of cariogenic biofilm.</a> Journal of Dental Research. 92 (12), 1065-1073 (2013).</li><li>Schlafer, S., Dige, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ratiometric+Imaging+of+Extracellular+pH+in+Dental+Biofilms.">Ratiometric Imaging of Extracellular pH in Dental Biofilms.</a> Journal of Visualized Experiments. (109), 53622 (2016).</li><li>Schlafer, S., Kamp, A., Garcia, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+confocal+microscopy-based+method+to+monitor+extracellular+pH+in+fungal+biofilms.">A confocal microscopy-based method to monitor extracellular pH in fungal biofilms.</a> FEMS Yeast Research. 18 (5), (2018).</li><li>Schlafer, S., B&#230;lum, V., Dige, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+pH-ratiometry+for+the+three-dimensional+mapping+of+pH+microenvironments+in+biofilms+under+flow+conditions.">Improved pH-ratiometry for the three-dimensional mapping of pH microenvironments in biofilms under flow conditions.</a> Journal of Microbiological Methods. 152, 194-200 (2018).</li><li>Hidalgo, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+tomographic+fluorescence+imaging+of+pH+microenvironments+in+microbial+biofilms+by+use+of+silica+nanoparticle+sensors.">Functional tomographic fluorescence imaging of pH microenvironments in microbial biofilms by use of silica nanoparticle sensors.</a> Applied and Environmental Microbiology. 75 (23), 7426-7435 (2009).</li><li>Vroom, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depth+Penetration+and+Detection+of+pH+Gradients+in+Biofilms+by+Two-Photon+Excitation+Microscopy.">Depth Penetration and Detection of pH Gradients in Biofilms by Two-Photon Excitation Microscopy.</a> Applied and Environmental Microbiology. 65, 3502-3511 (1999).</li><li>Lawrence, J. R., Swerhone, G. D. W., Kuhlicke, U., Neu, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+evidence+for+metabolic+and+chemical+microdomains+in+the+structured+polymer+matrix+of+bacterial+microcolonies.">In situ evidence for metabolic and chemical microdomains in the structured polymer matrix of bacterial microcolonies.</a> FEMS Microbiology Ecology. 92 (11), (2016).</li><li>Franks, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+strategy+for+three-dimensional+real-time+imaging+of+microbial+fuel+cell+communities:+monitoring+the+inhibitory+effects+of+proton+accumulation+within+the+anode+biofilm.">Novel strategy for three-dimensional real-time imaging of microbial fuel cell communities: monitoring the inhibitory effects of proton accumulation within the anode biofilm.</a> Energy Environmental Science. 2 (1), 113-119 (2009).</li><li>de Jong, M. H., van der Hoeven, J. S., van OS, J. H., Olijve, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+oral+Streptococcus+species+and+Actinomyces+viscosus+in+human+saliva.">Growth of oral Streptococcus species and Actinomyces viscosus in human saliva.</a> Applied and Environmental Microbiology. 47 (5), 901-904 (1984).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nature Methods. 9 (7), 671-675 (2012).</li><li>Daims, H., L&#252;cker, S., Wagner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Daime,+a+novel+image+analysis+program+for+microbial+ecology+and+biofilm+research.">Daime, a novel image analysis program for microbial ecology and biofilm research.</a> Environmental Microbiology. 8 (2), 200-213 (2006).</li><li>Barbosa, J. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Streptococcus+mutans+Can+Modulate+Biofilm+Formation+and+Attenuate+the+Virulence+of+Candida+albicans.">Streptococcus mutans Can Modulate Biofilm Formation and Attenuate the Virulence of Candida albicans.</a> PloS One. 11 (3), 0150457 (2016).</li><li>Thein, Z. M., Samaranayake, Y. H., Samaranayake, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+oral+bacteria+on+growth+and+survival+of+Candida+albicans+biofilms.">Effect of oral bacteria on growth and survival of Candida albicans biofilms.</a> Archives of Oral Biology. 51 (8), 672-680 (2006).</li><li>Krzy&#347;ciak, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+a+Lactobacillus+Salivarius+Probiotic+on+a+Double-Species+Streptococcus+Mutans+and+Candida+Albicans+Caries+Biofilm.">Effect of a Lactobacillus Salivarius Probiotic on a Double-Species Streptococcus Mutans and Candida Albicans Caries Biofilm.</a> Nutrients. 9 (11), 1242 (2017).</li><li>Liu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nicotine+Enhances+Interspecies+Relationship+between+Streptococcus+mutans+and+Candida+albicans.">Nicotine Enhances Interspecies Relationship between Streptococcus mutans and Candida albicans.</a> BioMed Research International. 2017, 7953920 (2017).</li><li>Schlafer, S., Meyer, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+microscopy+imaging+of+the+biofilm+matrix.">Confocal microscopy imaging of the biofilm matrix.</a> Journal of Microbiological Methods. 138, 50-59 (2017).</li><li>Schlafer, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ratiometric+imaging+of+extracellular+pH+in+bacterial+biofilms+with+C-SNARF-4.">Ratiometric imaging of extracellular pH in bacterial biofilms with C-SNARF-4.</a> Applied and Environmental Microbiology. 81 (4), 1267-1273 (2015).</li><li>Ohle, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+microsensor+measurement+of+local+metabolic+activities+in+ex+vivo+dental+biofilms+exposed+to+sucrose+and+treated+with+chlorhexidine.">Real-time microsensor measurement of local metabolic activities in ex vivo dental biofilms exposed to sucrose and treated with chlorhexidine.</a> Applied and Environmental Microbiology. 76 (7), 2326-2334 (2010).</li><li>Schlafer, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=pH+landscapes+in+a+novel+five-species+model+of+early+dental+biofilm.">pH landscapes in a novel five-species model of early dental biofilm.</a> PloS One. 6 (9), 25299 (2011).</li><li>Divaris, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Supragingival+Biofilm+in+Early+Childhood+Caries:+Clinical+and+Laboratory+Protocols+and+Bioinformatics+Pipelines+Supporting+Metagenomics,+Metatranscriptomics,+and+Metabolomics+Studies+of+the+Oral+Microbiome.">The Supragingival Biofilm in Early Childhood Caries: Clinical and Laboratory Protocols and Bioinformatics Pipelines Supporting Metagenomics, Metatranscriptomics, and Metabolomics Studies of the Oral Microbiome.</a> Methods in Molecular Biology. 1922, 525-548 (2019).</li><li>Stewart, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mini+review:+convection+around+biofilms.">Mini review: convection around biofilms.</a> Biofouling. 28 (2), 187-198 (2012).</li><li>Stoodley, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilms:+Flow+disrupts+communication.">Biofilms: Flow disrupts communication.</a> Nature Microbiology. 1, 15012 (2016).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#228;t Wien</td>
			<td>N/A</td>
			<td>Freeware; V2.1; <a href="https://dome.csb.univie.ac.at/daime" target="_blank">https://dome.csb.univie.ac.at/daime</a></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; <a href="https://imagej.nih.gov/ij" target="_blank">https://imagej.nih.gov/ij</a></td>
		</tr>
		<tr>
			<td>Java</td>
			<td>Oracle</td>
			<td>N/A</td>
			<td>Freeware necessary to run ImageJ; V8.0; <a href="https://java.com/en/download" target="_blank">https://java.com/en/download</a></td>
		</tr>
		<tr>
			<td>&#181;-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&#174;-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

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 &#181;m 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...

Watch this Scientific Journal Video about Monitoring Extracellular pH in Cross-Kingdom Biofilms using Confocal Microscopy at JoVE.com

Monitoring Extracellular pH in Cross-Kingdom Biofilms using Confocal Microscopy

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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