Ratiometric imaging of extracellular pH in bacterial biofilms with C-SNARF-4

• Published in: Applied and Environmental Microbiology 2015-02, Vol.81 (4), p.1267-1273
• Main Authors: Schlafer, Sebastian, Garcia, Javier E, Greve, Matilde, Raarup, Merete K, Nyvad, Bente, Dige, Irene
• Format: Article
• Language: English
• Subjects: Acidity; Bacteria; Bacteria - chemistry; Bacteria - growth & development; Benzopyrans - chemistry; Biofilms; Biofilms - growth & development; Biomass; Fluorescent Dyes - chemistry; Hydrogen-Ion Concentration; Metabolism; Methods; Microbiology; Microscopy, Fluorescence - instrumentation; Microscopy, Fluorescence - methods; Radiometry
• ISSN: 0099-2240; 1098-5336; 1098-6596
• DOI: 10.1128/AEM.02831-14

pH in the extracellular matrix of bacterial biofilms is of central importance for microbial metabolism. Biofilms possess a complex three-dimensional architecture characterized by chemically different microenvironments in close proximity. For decades, pH measurements in biofilms have been limited to monitoring bulk pH with electrodes. Although pH microelectrodes with a better spatial resolution have been developed, they do not permit the monitoring of horizontal pH gradients in biofilms in real time. Quantitative fluorescence microscopy can overcome these problems, but none of the hitherto employed methods differentiated accurately between extracellular and intracellular microbial pH and visualized extracellular pH in all areas of the biofilms. Here, we developed a method to reliably monitor extracellular biofilm pH microscopically with the ratiometric pH-sensitive dye C-SNARF-4, choosing dental biofilms as an example. Fluorescent emissions of C-SNARF-4 can be used to calculate extracellular pH irrespective of the dye concentration. We showed that at pH values of