This article details the preparation of Legionella pneumophila samples for cryogenic electron tomography (cryo-ET) imaging. The focus is on the bacterial secretion system crucial for host infection.
Begin with a culture of Legionella pneumophila, a pathogenic bacterium.
These bacterial cells possess a secretion system on their membrane, which is essential for host infection.
Suspend the bacterial cells in sterile water to achieve the required density for cryogenic electron tomography (cryo-ET) imaging.
Add gold nanoparticles, which serve as reference points for image alignment during three-dimensional reconstruction.
Take a grid with a perforated carbon film to deposit the bacterial sample.
Place the grid inside a gas-filled chamber and apply an electric discharge to make the surface more hydrophilic.
Apply the suspension to the grid, allowing the liquid to spread uniformly on the surface and the cells to settle.
Remove excess water from the grid and plunge it into a liquid freezing medium to freeze the sample and preserve its cellular architecture.
The sample is now ready for cryo-ET imaging of the bacterial secretion system.
To prepare the electron cryotomography sample, grow a heavy patch of Legionella Pneumophila on CYE-agar-streptomycin plates for 48 hours at 37 degrees Celsius.
Resuspend the cells in water to an OD600 of about 0.7. Then add five microliters of colloidal gold particles to 20 microliters of the cell suspension. Glow discharge a holey carbon grid and pipette five microliters of the cell mixture on it.
Let it stand for one minute. Blot the grid with filter paper and freeze it in liquid ethane using a gravity-driven plunger apparatus.
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