Bacterial Immobilization for Imaging by Atomic Force Microscopy
David P. Allison, Claretta J. Sullivan, Ninell Pollas Mortensen, Scott T. Retterer, and Mitchel J. Doktycz
2011 August 10, J Vis Exp. 54: e2880; doi: 10.3791/2880.
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.
Allison DP, Sullivan CJ, Mortensen NP, Retterer ST, Doktycz M. Bacterial immobilization for imaging by atomic force microscopy. J Vis Exp. 2011 Aug 10;(54). doi:pii: 2880. 10.3791/2880. PubMed PMID: 21860374.