Determination of Spatial Distributions of Zinc and Active Biomass in Microbial Biofilms by Two-Photon Laser Scanning Microscopy

Abstract

The spatial distributions of zinc, a representative transition metal, and active biomass in bacterial biofilms were determined using two-photon laser scanning microscopy (2P-LSM). Application of 2P-LSM permits analysis of thicker biofilms than are amenable to observation with confocal laser scanning microscopy and also provides selective excitation of a smaller focal volume with greater depth localization. Thin Escherichia coli PHL628 biofilms were grown in a minimal mineral salts medium using pyruvate as the carbon and energy source under batch conditions, and thick biofilms were grown in Luria-Bertani medium using a continuousflow drip system.

Confocal Laser Microscopic Analysis of Biofilm on Newer Feldspar Ceramic

“…[22][23] CLSM presents non-invasive and non-destructive characteristics and is actually considered the tool of choice for study of in situ biofilm. [27][28] Fluorescent dyes, such as the LIVE\DEAD kit (Bac light), have been used together with CLSM to describe the architecture of biofilm, 29 quantify the viability of microorganisms 19,21,25 and evaluate the mean thickness 23,26,[29][30] and biovolume of biofilm. 26,[30][31] Quantitative data calculated using the COMSTAT program represents a way to obtain numeric information from biofilm, allowing for comparisons.…”

Chemodynamics and Bioavailability in Natural Waters

“…Simultaneously, a negative Donnan potential will increase the cation (and decrease the anion) concentrations in the gel with respect to the bulk medium (see Box 3). Although few studies have been performed that examine the result of these effects on diffusion, overall Ni and Zn transport has been shown to be substantially retarded in biofilms of Escherichia coli and Pseudomonas aeruginosa (18,19). Since the biouptake process plays the role of a continuous sink for bioaccumulating species, their concentration profiles in biofilms will be nonlinear, even under steady-state conditions.…”

Low-Load Compression Testing: a Novel Way of Measuring Biofilm Thickness

“…Good-quality CLSM images are possible only for biofilms up to about 70 m thick (7). Two-photon excitation microscopy allows imaging of biofilms that are up to 350 m thick due to improved spatial localization, deeper sectioning of the samples, and reduced fluorescent bleaching (8). However, two-photon excitation microscopy remains expensive.…”