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ARS Home » Pacific West Area » Corvallis, Oregon » Horticultural Crops Research Unit » Research » Publications at this Location » Publication #260013
Title: Subsurface examination of a foliar biofilm using scanning electron- and focused-ion-beam microscopy

Author
item WALLACE, PATRICIA - Oregon State University
item AREY, BRUCE - Pacific Northwest National Laboratory
item Mahaffee, Walter - Walt

Submitted to: Micron
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/10/2011
Publication Date: 8/1/2011
Citation: Wallace, P., Arey, B., Mahaffee, W.F. 2011. Subsurface examination of a foliar biofilm using scanning electron- and focused-ion-beam microscopy. Micron. 42:579-585.

Interpretive Summary: This research develops new approaches to understanding how microorganisms colonize plant tissues. The use of a dual beam scanning electron microscope, equipped with both a focused ion- and scanning electron- beam (FIB SEM) was shown to be useful for examining the subsurface structure of bacterial biofilms on plant surfaces by precisely removing bacterial and plant material without disturbing the subsurface structure. The suitability of chemical and cryofixation was examined for use with the FIB SEM to examine bacterial biofilms on leaf surfaces. Cryofixation was not suitable for examination of leaf biofilms because it created a frozen layer over the leaf surface that cracked when exposed to the electron beam, and the protective cap required for FIB milling could not be accurately deposited. With chemically fixed samples, it was possible to precisely FIB mill a single cross section (5 µm) or sequential cross sections from a single site without any damage to the surrounding surface. Biofilms, 7 days post-inoculation (DPI), were composed of 2 to 5 bacterial cell layers while biofilms 14 DPI ranged from 5 to greater than 30 cell layers. Empty spaces between bacteria cells in the subsurface structure were observed in biofilms 7- and 14-DPI. Sequential cross sections inferred that the empty spaces were often continuous between FP62 cells and could possibly make up a network of channels throughout the biofilm. FIB SEM was a useful tool to observe the subsurface composition of a foliar biofilm.

Technical Abstract: The dual beam scanning electron microscope, equipped with both a focused ion- and scanning electron- beam (FIB SEM) is a novel tool for the exploration of the subsurface structure of biological tissues. The FIB is capable of removing small cross sections to view the subsurface features and may be suitable to examining the subsurface structure of bacterial biofilms on the leaf surface. The suitability of chemical and cryofixation was examined for use with the FIB SEM to examine bacterial biofilms on leaf surfaces. The biological control agent, Burkholderia pyroccinia FP62, that rapidly colonizes the leaf surface and forms biofilms, was inoculated onto geranium leaves and incubated in a greenhouse for 7 or 14 days. Cryofixation was not suitable for examination of leaf biofilms because it created a frozen layer over the leaf surface that cracked when exposed to the electron beam, and the protective cap required for FIB milling could not be accurately deposited. With chemically fixed samples, it was possible to precisely FIB mill a single cross section (5 µm) or sequential cross sections from a single site without any damage to the surrounding surface. Biofilms, 7 days post-inoculation (DPI), were composed of 2 to 5 bacterial cell layers while biofilms 14 DPI ranged from 5 to greater than 30 cell layers. Empty spaces between bacteria cells in the subsurface structure were observed in biofilms 7- and 14-DPI. Sequential cross sections inferred that the empty spaces were often continuous between FP62 cells and could possibly make up a network of channels throughout the biofilm. FIB SEM was a useful tool to observe the subsurface composition of a foliar biofilm.