Approach to Imaging Bacterial Cells Encapsulated in Alginate Hydrogel Using LV-STEM

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The imaging of hydrogel-based samples presents a challenge for electron microscopy. These polymeric structures, capable of binding significant amounts of water, find applications in a wide range of industries (medicine, food science, agriculture, etc.). It is the high water content and the sensitive fine structure that make these samples difficult to observe with an electron microscope. Different preparation techniques combined with different types of scanning electron microscopy are most commonly used to describe hydrogel materials. However, if we want to study complex samples where, for example, bacterial cells are encapsulated in a hydrogel network, we are also interested in their internal structures. In our work, we focused on designing a protocol for the chemical preparation of plant growth-promoting rhizobacterium Azotobacter vinelandii encapsulated in an alginate hydrogel for imaging in a low-voltage scanning transmission electron microscope (LV-STEM) so that not only the ultrastructure of the cells but also of the hydrogel is preserved. Azotobacter vinelandii cells encapsulated in alginate hydrogel were cut to approx. 1mm3 cubes and fixed using 2.5% glutaraldehyde and 2% formaldehyde solution. To stabilize the hydrogel ultrastructure, 1% solution of CaCl2 was added to the following protocol steps. Samples were postfixed using 1% OsO4 with 1% K3[Fe(CN)]6, and then stained by a commercial uranium-free staining reagent UA-zero. Methanol was used for the dehydration of the samples since one of the most common dehydrating agents acetone and ethanol do not provide either sufficient solubility of CaCl2 or volatility for this type of porous specimen. Samples were embedded in Spurr’s resin and cured for 48h at 62°C. Ultrathin sections were briefly contrasted with Reynold’s lead citrate and imaged using a scanning electron microscope (Magellan 400/L, FEI) equipped with a STEM3+ detector. The LV-STEM images showed cells of A. vinelandii surrounded by a polymer capsule, which consists of two parts. An electron-dense exine forms a filamentous structure and an electron-transparent intine surrounding the immediate vicinity of the cell. The polymeric alginate net was also observed, not only close to the cells but also in the outer areas of the sample. The results we obtained suggest that further efforts to optimize the preparation protocol for such complex samples are relevant. The focus of the following experiments could be given to exploring the effect of different CaCl2 concentrations.

hydrogel; alginate; Azotobacter vinelandii; electron microscopy;

MRÁZOVÁ, K.; HAVLÍČKOVÁ, A.; ČERNAYOVÁ, D.; SEDLÁČEK, P.; KRZYŽÁNEK, V.

19. 5. 2025

Czechoslovak Microscopy Society

Brno

978-80-909216-1-0

Microscopy 2025. Book of abstracts.

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