Bacterial Immobilization for Imaging by Atomic Force Microscopy

Summary

Live Gram-negative and Gram-positive bacteria can be immobilized on gelatin-coated mica and imaged in liquid using Atomic Force Microscopy (AFM).

Abstract

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.

Protocol

1. Mica preparation:

1. Cut the mica (Electron Microscopy Sciences) with scissors to the size necessary to fit the AFM microscope (approx. 22 × 30 mm).
2. Cleave the mica on both sides, generally using tape to remove the outer layer, until only smooth unbroken layers remain.

2. Preparation of gelatin solution:

1. Add 100 ml of distilled water to a laboratory bottle.
2. Heat the bottle in a microwave until the water begins to boil. (If a microwave is not available, the water can be heated in a beaker on a hot plate.)
3. Weigh out 0.5 grams of the gelatin (Sigma G-6144, G-2625 or G-2500) a....

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Gelatin	Sigma-Aldrich	G6144, G2625 or G2500
PicoPlus Atomic Force Microscope	Agilent Technologies
AFM cantilevers	Veeco Instruments, Inc.	MLCT-AUHW