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Applied and Environmental Microbiology, August 1999, p. 3502-3511, Vol. 65, No. 8
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

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

Jurrien M. Vroom,1 Kees J. De Grauw,1 Hans C. Gerritsen,1 David J. Bradshaw,2,* Philip D. Marsh,2,3 G. Keith Watson,4 John J. Birmingham,4 and Clive Allison4

University of Utrecht, Utrecht, The Netherlands,1 and Centre for Applied Microbiology and Research, Salisbury SP4 0JG,2 Leeds Dental Institute, Leeds LS2 9LU,3 and Unilever Research, Port Sunlight Laboratory, Bebington, Wirral L63 3JW,4 United Kingdom

Received 9 December 1998/Accepted 15 May 1999

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.

* Corresponding author. Mailing address: Research Division, CAMR, Salisbury SP4 0JG, United Kingdom. Phone: (44) 1980 612732. Fax: (44) 1980 612731. E-mail: david.bradshaw{at}camr.org.uk.

Applied and Environmental Microbiology, August 1999, p. 3502-3511, Vol. 65, No. 8
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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