Microscopy

ARTICLE IN PRESS

Ultramicroscopy 97 (2003) 209–216

AFM imaging of bacteria in liquid media immobilized on gelatin coated mica surfaces

M.J. Doktycza,b,c,*, C.J. Sullivanc, P.R. Hoytc, D.A. Pelletiera, S. Wud, D.P. Allisonc,d,e

a Oak Ridge National Laboratory, Life Sciences Division, Oak Ridge, TN 37831-6123, USA Oak Ridge National Laboratory, Condensed Matter Sciences Division, Oak Ridge, TN 37831-6123, USA c Genome Science and Technology, The University of Tennessee, Knoxville, TN 37932, USA d Molecular Imaging Inc., Tempe, AZ, USA e Department of Biochemistry & Cellular & Molecular Biology, The University of Tennessee, Knoxville, TN 37932, USA b

Received 20 October 2002; received in revised form 27 January 2003

Abstract Immobilization of particulates, especially biomolecules and cells, onto surfaces is critical for imaging with the atomic force microscope (AFM). In this paper, gelatin coated mica surfaces are shown to be suitable for immobilizing and imaging both gram positive, Staphylococcus aureus, and gram negative, Escherichia coli, bacteria in both air and liquid environments. Gelatin coated surfaces are shown to be superior to poly-l-lysine coated surfaces that are commonly used for the immobilization of cells. This cell immobilization technique is being developed primarily for live cell imaging of Rhodopseudomonas palustris. The genome of R. palustris has been sequenced and the organism is the target of intensive studies aimed at understanding genome function. Images of R. palustris grown both aerobically and anaerobically in liquid media are presented. Images in liquid media show the bacteria is rod shaped and smooth while images in air show marked irregularity and folding of the surface. Significant differences in the vertical dimension are also apparent with the height of the bacteria in liquid being substantially greater than images taken in air. In air immobilized bacterial flagella are clearly seen while in liquid this structure is not...