Use of Atomic Force Microscopy and Transmission Electron Microscopy for Correlative Studies of Bacterial Capsules

Abstract

Bacteria can possess an outermost assembly of polysaccharide molecules, a capsule, which is attached to their cell wall. We have used two complementary, high-resolution microscopy techniques, atomic force microscopy (AFM) and transmission electron microscopy (TEM), to study bacterial capsules of four different gram-negative bacterial strains: Escherichia coli K30, Pseudomonas aeruginosa FRD1, Shewanella oneidensis MR-4, and Geobacter sulfurreducens PCA. TEM analysis of bacterial cells using different preparative techniques (whole-cell mounts, conventional embeddings, and freeze-substitution) revealed capsules for some but not all of the strains. In contrast, the use of AFM allowed the unambiguous identification of the presence of capsules on all strains used in the present study, including those that were shown by TEM to be not encapsulated. In addition, the use of AFM phase imaging allowed the visualization of the bacterial cell within the capsule, with a depth sensitivity that decreased with increasing tapping frequency.

Are the Soft, Liquid-Like Structures Detected around Bacteria by Ambient Dynamic Atomic Force Microscopy Capsules?

“…The absolute value of the difference between the phase angle of the substratum and that of the liquid-like substances ranged from about 5°to about 31°, with no significant differences found in the three studies done with the three different bacteria. This is quite similar to the angles reported by Stukalov et al (30)…”

“…In our study, we managed to obtain good (noise-free) images using these k ϭ 3 N/m cantilevers, but this is compatible with the fact that other authors have obtained good images using softer ones, since stability during the scanning of such delicate surfaces does not rely only on the cantilever's stiffness. For example, Stukalov et al (30) used softer cantilevers. The final success or failure of the imaging experiments will rely heavily on aspects such as the wettability (its affinity for the liquid being scanned) of the tip apex, which cannot be directly and easily measured.…”

“…Indeed, AFM has proved capable of scanning the surface of liquids, and this is being currently explored in areas such as the study of wetting properties at ultrasmall scales (19). In this sense, several works have recently reported the observation of capsules using ambient AFM (25,27,30,31) in the form of tiny amounts of a liquidlike substance around bacteria. Theoretical models are being currently developed for fluid microbial capsules (3).…”

Imaging Hydrated Microbial Extracellular Polymers: Comparative Analysis by Electron Microscopy

“…Bacterial EPS has been described as "collapsing into the thick bundles" upon the exposure to traditional organic solvents during dehydration (2). Comparative reports on EPS visualization by EM (28,52,55) and studies focused on improving EPS morphology by incubation with multivalent cationic dyes, heavy metals, or gold-conjugated lectins (13,14,34) greatly improved the morphological characterization of EPS. Unfortunately, these efforts still employed complete or partial dehydration steps, which prohibited the visualization of EPS in its nearest-to-native, hydrated state.…”

Precipitation of Iron on the Surface of Leptospira interrogans Is Associated with Mutation of the Stress Response Metalloprotease HtpX

“…Highly viscous materials, such as outer membrane vesicles (OMVs), are identified as bright or illuminated features by phase-contrast AFM (51). By AFM, cells of the parental L521 strain, cultured under conditions of high or normal concentrations of iron, exhibited typical leptospiral features, such as helical morphology, periplasmic flagella, and OMVs ( Fig.…”