Single-Cell Culture of Bacteria Within Giant Vesicles

Begin with a chambered glass slide coated with a supported membrane composed of phospholipids, including biotinylated phospholipids.

Incubate with neutravidin, which binds to biotin on the bilayer. Wash to remove the excess neutravidin.

Introduce a nutrient medium containing giant vesicles or GVs. Each GV is a spherical, micron-sized lipid bilayer vesicle that encapsulates a single bacterial cell, serving as a model to study individual cell behavior in a confined environment.

Neutravidin binds to biotinylated GV lipids, securing the vesicles to the supported membrane for stable, long-term observation under static conditions.

Seal the chamber to maintain hydration and minimize contamination.

Place the slide on a microscope stage equipped with a heating platform to simulate physiological conditions and observe under a microscope.

Nutrients from the medium diffuse into the GV to support bacterial growth over time.

This setup allows real-time visualization of single-cell bacterial growth within confined vesicle microenvironments.

To immobilize giant vesicles on the supporter bi-layer membrane, introduce 10 microliters of neutravidin in outer aqueous giant vesicle solution to the hole for a 15 minute incubation at room temperature.

At the end of the incubation, gently wash the hole two times with 20 microliters of LB medium, supplemented with 200 millimolar glucose, and add the entire volume of giant vesicle solution to the hole in the chamber. Then seal the hole with an 18 by 18 millimeter cover glass. To monitor bacterial growth within the giant vesicles, select the 40X objective on an inverted microscope, and place the chamber on the microscope heating stage system.

Then incubate the bacterial cell-containing giant vesicles in the chamber under static conditions for six hours at 37 degrees Celsius, capturing and recording images of the bacterial cell growth every 30 minutes, with the scientific complementary metal oxide semiconductor camera.