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Bacterial Immobilization for Imaging by Atomic Force Microscopy...

by David P Allison, Claretta Sullivan, Ninell P Mortensen, Scott T Retterer, Mitchel J Doktycz
Publication Type
Journal
Journal Name
Journal of Visualized Experiments : JoVE
Publication Date
Volume
N/A
Issue
54

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface. It has the ability to image cells and biomolecules, in a liquid
environment, without the need to chemically treat the sample. In order to accomplish this goal, the sample must sufficiently adhere to the
mounting surface to prevent removal by forces exerted by the scanning AFM cantilever tip. In many instances, successful imaging depends on
immobilization of the sample to the mounting surface. Optimally, immobilization should be minimally invasive to the sample such that metabolic
processes and functional attributes are not compromised. By coating freshly cleaved mica surfaces with porcine (pig) gelatin, negatively charged
bacteria can be immobilized on the surface and imaged in liquid by AFM. Immobilization of bacterial cells on gelatin-coated mica is most likely
due to electrostatic interaction between the negatively charged bacteria and the positively charged gelatin. Several factors can interfere with
bacterial immobilization, including chemical constituents of the liquid in which the bacteria are suspended, the incubation time of the bacteria on
the gelatin coated mica, surface characteristics of the bacterial strain and the medium in which the bacteria are imaged. Overall, the use of
gelatin-coated mica is found to be generally applicable for imaging microbial cells.