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Applied and Environmental Microbiology, July 2005, p. 4014-4021, Vol. 71, No. 7
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.7.4014-4021.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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

Zhiqiang Hu,^1,2,3 Gabriela Hidalgo,¹ Paul L. Houston,² Anthony G. Hay,³ Michael L. Shuler,⁴ Héctor D. Abruña,² William C. Ghiorse,³ and Leonard W. Lion^1*

School of Civil and Environmental Engineering,¹ Department of Chemistry and Chemical Biology,² Department of Microbiology,³ School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York 14853⁴

Received 23 July 2004/ Accepted 30 January 2005

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 continuous-flow drip system. The biofilms were visualized by 2P-LSM and shown to have heterogeneous structures with dispersed dense cell clusters, rough surfaces, and void spaces. Contrary to homogeneous biofilm model predictions that active biomass would be located predominantly in the outer regions of the biofilm and inactive or dead biomass (biomass debris) in the inner regions, significant active biomass fractions were observed at all depths in biofilms (up to 350 µm) using live/dead fluorescent stains. The active fractions were dependent on biofilm thickness and are attributed to the heterogeneous characteristics of biofilm structures. A zinc-binding fluorochrome (8-hydroxy-5-dimethylsulfoamidoquinoline) was synthesized and used to visualize the spatial location of added Zn within biofilms. Zn was distributed evenly in a thin (12 µm) biofilm but was located only at the surface of thick biofilms, penetrating less than 20 µm after 1 h of exposure. The relatively slow movement of Zn into deeper biofilm layers provides direct evidence in support of the concept that thick biofilms may confer resistance to toxic metal species by binding metals at the biofilm-bulk liquid interface, thereby retarding metal diffusion into the biofilm (G. M. Teitzel and M. R. Park, Appl. Environ. Microbiol. 69:2313-2320, 2003).

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* Corresponding author. Mailing address: School of Civil and Environmental Engineering, Cornell University, Hollister Hall, Ithaca, NY 14853. Phone: (607) 255-7571. Fax: (607)-255-9004. E-mail: LWL3{at}cornell.edu.

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Applied and Environmental Microbiology, July 2005, p. 4014-4021, Vol. 71, No. 7
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.7.4014-4021.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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