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Method Article
Live Gram-negative and Gram-positive bacteria can be immobilized on gelatin-coated mica and imaged in liquid using Atomic Force Microscopy (AFM).
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.
1. Mica preparation:
2. Preparation of gelatin solution:
3. Mica coating:
4. Mounting bacteria on gelatin coated mica:
5. Representative Results:
Figure 2 shows example images of bacterial cells that have been mounted on gelatin-coated mica and imaged by AFM. In our laboratories, samples are typically imaged using a PicoPlus atomic force microscope (Agilent Technologies, Temple, AZ) outfitted with a 100 μm scanning head. The instrument is operated at 128-512 pixels per line scan at a scan speed of 0.5-1.0 lines/s. The cantilevers that are typically used are Veeco silicon nitride probes (MLCT-AUHW, Veeco, Santa Barbara, CA) with nominal spring constants ranging from 0.01 to 0.1 nN/nm.
Figure 1. Gelatin at a temperature of 60-70 °C is placed in a 20 ml beaker. Using a pair of tweezers freshly cleaved mica squares are totally immersed into the gelatin (a) withdrawn and placed upright on a paper towel leaning against a micro centrifuge rack (b). A droplet of bacterial sample is placed on the mica and spread with a pipet tip in both the X and Y directions (c). After allowing the sample to incubate on the mica surface for at least 10 minutes, rinse the sample in a stream of distilled water. If the immobilization worked you will be able to see an opaque spot on the mica as shown in the right panel of (d). If the immobilization did not work you will get a clear piece of mica as in the left panel. The opaque spot on the mica scales to the concentration of the bacteria in the droplet that you place on the gelatin treated mica.
Figure 2. AFM images of E. coli. In panel (a), the cells are mounted on gelatin-coated mica and imaged in 0.005 M PBS. For comparison, an image of bacterial cells mounted on gelatin-coated mica and imaged in air is shown in panel (b). Though septa are clearly visible in image collected in liquid, greater resolution, as indicated by the presence of pili (inset panel (b)), can be obtained in air.
Various factors can affect microbial cell mounting and imaging by AFM. The gelatin that is used for coating the mica is important. Commercial gelatin is isolated from a number of vertebrates including fish, cows, and pigs. Both the origin and the processing method determine the gelatin's suitability for bacterial immobilization. Numerous sources and types of gelatin were evaluated for their effectiveness in immobilizing bacteria [1]. The two most effective gelatins were found to be Sigma G-6144 and G-2625. Derived...
No conflicts of interest declared.
This research is sponsored by the Office of Biological and Environmental Research, U.S. Department of Energy and by grant funding from Virginia's Commonwealth Health Research Board. Oak Ridge National Laboratory is managed by UT-Battelle, LLC, for the U. S. Department of Energy under Contract No. DE-AC05-00OR22725.
Name | Company | Catalog Number | Comments |
Name | Company | Catalogue number | |
Gelatin | Sigma, St. Louis, MO | G6144, G2625 or G2500 | |
PicoPlus Atomic Force Microscope | Agilent Technologies, Tempe, AZ | ||
AFM cantilevers | Veeco, Santa Barbara, CA | MLCT-AUHW |
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