E. coli TOP10F’ cells were grown to steady‐state exponential phase. The bacteria were transferred to a 96‐well plate with a start OD660 of 0.025 in 100 µL. Next, compound VUF15259 was added to a concentration of 50, 100 and 200 µM. As controls, non‐treated cells and cells incubated with DMSO were used. For each condition, 6 wells were used. After sealing the plate, the bacteria were grown in the Synergy H1 plate reader (Biotek) at 37°C with 2 mm linear shaking for 3 h. Subsequently, the cells of each condition were collected and pelleted at 5,000 ×g for 10 min. Cells were washed in phosphate‐buffered saline (PBS) and finally resuspended in 1 ml 4% paraformaldehyde and 0.5% glutaraldehyde (EM grade) for 2 h at room temperature and post‐fixed with 1% OsO4. Subsequently, the samples were dehydrated in an alcohol series and embedded in Epon (LX‐112 resin Ladd research, Williston, VT, USA). Ultrathin (80 nm) epon sections were collected on Formvar‐coated grids and counterstained with uranyl acetate and lead citrate. In addition, intact aldehyde fixed cells were negatively stained with uranyl acetate and collected on Formvar‐coated grids. Grids were examined using a FEI Tecnai‐12 G2 Spirit Biotwin electron microscope (Fei, Eindhoven, the Netherlands).
Fei tecnai 12 g2 spirit biotwin electron microscope
Manufactured by Thermo Fisher Scientific
The FEI Tecnai-12 G2 Spirit Biotwin is a transmission electron microscope (TEM) designed for biological research applications. It operates at an acceleration voltage of 120 kV and is capable of achieving a resolution of up to 0.34 nm. The microscope features a high-brightness LaB6 electron source and can be equipped with various imaging and analytical modes, such as bright-field, dark-field, and energy-dispersive X-ray spectroscopy (EDS).
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2 protocols using fei tecnai 12 g2 spirit biotwin electron microscope
1
Electron Microscopy of E. coli Treated with VUF15259
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E. coli TOP10F’ cells were grown to steady‐state exponential phase. The bacteria were transferred to a 96‐well plate with a start OD660 of 0.025 in 100 µL. Next, compound VUF15259 was added to a concentration of 50, 100 and 200 µM. As controls, non‐treated cells and cells incubated with DMSO were used. For each condition, 6 wells were used. After sealing the plate, the bacteria were grown in the Synergy H1 plate reader (Biotek) at 37°C with 2 mm linear shaking for 3 h. Subsequently, the cells of each condition were collected and pelleted at 5,000 ×g for 10 min. Cells were washed in phosphate‐buffered saline (PBS) and finally resuspended in 1 ml 4% paraformaldehyde and 0.5% glutaraldehyde (EM grade) for 2 h at room temperature and post‐fixed with 1% OsO4. Subsequently, the samples were dehydrated in an alcohol series and embedded in Epon (LX‐112 resin Ladd research, Williston, VT, USA). Ultrathin (80 nm) epon sections were collected on Formvar‐coated grids and counterstained with uranyl acetate and lead citrate. In addition, intact aldehyde fixed cells were negatively stained with uranyl acetate and collected on Formvar‐coated grids. Grids were examined using a FEI Tecnai‐12 G2 Spirit Biotwin electron microscope (Fei, Eindhoven, the Netherlands).
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E. coli TOP10F’ cells were grown to steady‐state exponential phase. The bacteria were transferred to a 96‐well plate with a start OD660 of 0.025 in 100 µL. Next, compound VUF15259 was added to a concentration of 50, 100 and 200 µM. As controls, non‐treated cells and cells incubated with DMSO were used. For each condition, 6 wells were used. After sealing the plate, the bacteria were grown in the Synergy H1 plate reader (Biotek) at 37°C with 2 mm linear shaking for 3 h. Subsequently, the cells of each condition were collected and pelleted at 5,000 ×g for 10 min. Cells were washed in phosphate‐buffered saline (PBS) and finally resuspended in 1 ml 4% paraformaldehyde and 0.5% glutaraldehyde (EM grade) for 2 h at room temperature and post‐fixed with 1% OsO4. Subsequently, the samples were dehydrated in an alcohol series and embedded in Epon (LX‐112 resin Ladd research, Williston, VT, USA). Ultrathin (80 nm) epon sections were collected on Formvar‐coated grids and counterstained with uranyl acetate and lead citrate. In addition, intact aldehyde fixed cells were negatively stained with uranyl acetate and collected on Formvar‐coated grids. Grids were examined using a FEI Tecnai‐12 G2 Spirit Biotwin electron microscope (Fei, Eindhoven, the Netherlands).
2
Ultrastructural Analysis of Biological Samples
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After the indicated treatment, the samples were fixed in Karnovsky’s glutaraldehyde (Polysciences, Inc., Warrington, PA, USA) and post-fixed with 1% osmium tetroxide (OsO4, Electron microscopy sciences, Hatfield, PA, USA; in cacodylate buffer). Subsequently, the samples were dehydrated in an alcohol series and embedded into Epon (LX-112 resin Ladd research, Williston, VT, USA). First semi-thin (1 μm) sections were cut for overview images and stained with Richardson’s staining solution [13 (link)]. Then ultrathin (80 nm) epon sections of the desired areas were cut and collected on Formvar-coated grids, counterstained with uranyl acetate and lead citrate. Sections were examined with a FEI Tecnai-12 G2 Spirit Biotwin electron microscope (Fei, Eindhoven, The Netherlands), and images were taken with a Veleta camera using Radius software (EMSIS, Münster, Germany).
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