TY - JOUR
T1 - Monitoring Extracellular pH in Cross-Kingdom Biofilms using Confocal Microscopy
AU - Schlafer, Sebastian
AU - Frost Kristensen, Mathilde
PY - 2019/1
Y1 - 2019/1
N2 - 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.
AB - 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.
KW - Bacteria
KW - Biofilm
KW - Candida albicans
KW - Confocal laser scanning microscopy
KW - Cross-kingdom
KW - Early childhood caries
KW - Fungi
KW - Immunology and infection
KW - PH ratiometry
KW - Streptococcus mutans
KW - Yeast
UR - http://www.scopus.com/inward/record.url?scp=85079516505&partnerID=8YFLogxK
U2 - 10.3791/60270
DO - 10.3791/60270
M3 - Journal article
C2 - 32065130
SN - 1940-087X
VL - 2020
JO - Journal of visualized experiments : JoVE
JF - Journal of visualized experiments : JoVE
IS - 155
M1 - e60270
ER -