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Cited by 303Bacterial biofilms and quorum sensingAdvanced Fluorescence Microscopy TechniquesCell Image Analysis Techniques

Depth Penetration and Detection of pH Gradients in Biofilms by Two-Photon Excitation Microscopy

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Vroom, Jurrien M., De Grauw, Kees J., Gerritsen, Hans C., Bradshaw, David J., Marsh, Philip D., Watson, G. Keith, Birmingham, John J., Allison, Clive·1999·Applied and Environmental Microbiology
DOI 10.1128/aem.65.8.3502-3511.1999PMC PMC91526
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Abstract

Deep microbial biofilms are a major problem in many industrial, environmental, and medical settings. Novel approaches are needed to understand the structure and metabolism of these biofilms. Two-photon excitation microscopy (TPE) and conventional confocal laser scanning microscopy (CLSM) were compared quantitatively for the ability to visualize bacteria within deep in vitro biofilms. pH gradients within these biofilms were determined by fluorescence lifetime imaging, together with TPE. A constant-depth film fermentor (CDFF) was inoculated for 8 h at 50 ml · h −1 with a defined mixed culture of 10 species of bacteria grown in continuous culture. Biofilms of fixed depths were developed in the CDFF for 10 or 11 days. The microbial compositions of the biofilms were determined by using viable counts on selective and nonselective agar media; diverse mixed-culture biofilms developed, including aerobic, facultative, and anaerobic species. TPE was able to record images four times deeper than CLSM. Importantly, in contrast to CLSM images, TPE images recorded deep within the biofilm showed no loss of contrast. The pH within the biofilms was measured directly by means of fluorescence lifetime imaging; the fluorescence decay of carboxyfluorescein was correlated with biofilm pH and was used to construct a calibration curve. pH gradients were detectable, in both the lateral and axial directions, in steady-state biofilms. When biofilms were overlaid with 14 mM sucrose for 1 h, distinct pH gradients developed. Microcolonies with pH values of below pH 3.0 were visible, in some cases adjacent to areas with a much higher pH (>5.0). TPE allowed resolution of images at significantly greater depths (as deep as 140 μm) than were possible with CLSM. Fluorescence lifetime imaging allowed the in situ, real-time imaging of pH and the detection of sharp gradients of pH within microbial biofilms.

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  • Exopolysaccharides create chemical and metabolic gradients within biofilms that affect chemical and nutrient cycling as well as diffusion, thus altering the microenvironment and pH within the biofilm consortium.

    “Furthermore, exopolysaccharides create chemical and metabolic gradients within biofilms that affect chemical and nutrient cycling as well as diffusion, thus altering the microenvironment and pH within biofilms consortium.”

    From The oral microbiome: Role of key organisms and complex networks in oral health and disease (2021) · cites this paper as [288]

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Research works printed but not linked: 4

  1. [8]Coombe R A, Tatevossian A, Wimpenny J W T. Bacterial thin films as in vitro models for dental plaque. In: Frank R M, Leach S A, editors. Surface and colloid phenomena in the oral cavity. London, United Kingdom: IRL Press; 1981. pp. 239–249. [Google Scholar]
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Authors
Vroom, J. M., De Grauw, K. J., Gerritsen, H. C., Bradshaw, D. J., Marsh, P. D., Watson, G. K., Birmingham, J. J., & Allison, C.
Affiliations
  • Utrecht University (NL)
  • <!--label omitted: 1-->University of Utrecht, Utrecht, The Netherlands,1 and
  • <!--label omitted: 2-->Centre for Applied Microbiology and Research, Salisbury SP4 0JG,2
  • Leeds Dental Hospital (GB)
  • Unilever (United Kingdom) (GB)
ORCID
Venue
Applied and Environmental Microbiology
Publisher
American Society for Microbiology
Published
1999-08
Volume
65
Issue
8
Pages
3502-3511
ISSN
  • 0099-2240 (ISSN-L)
  • 1098-5336
DOI
10.1128/aem.65.8.3502-3511.1999
PMC
PMC91526
OpenAlex
W2157806471
Cited by
303
References
37
Citations by year
2026: 7 · 2025: 8 · 2024: 7 · 2023: 9 · 2022: 11 · 2021: 15 · 2020: 10 · 2019: 9 · 2018: 22 · 2017: 13 · 2016: 12 · 2015: 11 · 2014: 14 · 2013: 12 · 2012: 7
FWCI
3.45
Type
journal article
Language
en
Open access
Licence
journals.asm.org/non-commercial-tdm-license (tdm)
Locations
  • Applied and Environmental Microbiology (publishedVersion)
  • Europe PMC (PubMed Central) (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Bacterial biofilms and quorum sensing
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Advanced Fluorescence Microscopy Techniques
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Cell Image Analysis Techniques
Keywords
biofilm, pH gradient, two-photon microscopy, fluorescence lifetime imaging, carboxyfluorescein, confocal laser scanning microscopy, penetration depth, mixed-culture biofilms, microcolonies
MeSH terms
Evaluation Studies as Topic, Humans, Hydrogen-Ion Concentration, Microscopy, Fluorescence, Sucrose, Colony Count, Microbial, Photons, Biofilms, Microscopy, Confocal

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