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, ....

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} ....

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 curve¹³

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

List of materials used in this article

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