Single-Cell Culture of Bacteria Within Giant Vesicles

Abstract

Source: Morita, M., et al. Bacterial Cell Culture at the Single-cell Level Inside Giant Vesicles. J. Vis. Exp. (2019).

This video demonstrates a method for culturing and observing individual bacterial cells encapsulated inside giant vesicles anchored to a supported lipid bilayer. The system enables stable, long-term imaging of single-cell growth dynamics in a confined, physiologically relevant microenvironment.

Protocol

1. Preparation of a giant vesicle (GV) Observation System (Bacterial Cell Culture System)

1. Preparation of small vesicles (SVs) for constructing a supported bilayer membrane
   1. Pour 20 μL of the POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) solution and 4 μL of the biotin-PEG-DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethyleneglycol)-2000) solution into a glass tube.
   2. Evaporate the organic solvent by air flow to form a lipid film and place this sample in a desiccator for 1 h to completely evaporate the organic solvent.
   3. Add 200 μL of 200 mM glucose in 1x LB (Luria-Bertani broth) medium (the outer aqueous solution of GVs) to the glass vial.
   4. Wrap the opening part of the glass vial with film and sonicate it in an ultrasonic bath (120 W) for at least 1 h.
   5. Prepare SVs by the extrusion method using a mini-extruder and polycarbonate membrane with 100 nm pore size.
2. Preparation of a handmade chamber
   1. Drill a 7 mm hole with a hollow punch on a double-faced seal (10 mm x 10 mm x 1 mm).
   2. Paste the double-faced seal with the hole on a cover glass (30 mm x 40 mm, thickness 0.25–0.35 mm).
3. Preparation of a supported bilayer membrane on the cover glass in the hole of the chamber
   1. Add 30 μL of the SV solution to the hole of the chamber (prepared in section 2.2) and incubate at RT for 30 min.
   2. Gently wash the hole twice with 20 μL of 1x LB medium containing 200 mM glucose (the outer aqueous solution of GVs) by pipetting.
4. Immobilization of GVs on the supported bilayer membrane on the cover glass in the hole of the chamber
   1. Introduce 10 μL of neutravidin with the outer aqueous solution of GVs (1 mg/mL) into the hole and incubate at RT for 15 min.
   2. Gently wash the hole twice with 20 μL of 1x LB medium containing 200 mM glucose (the outer aqueous solution of GVs) by pipetting.
   3. Add all solution containing GVs into the hole of the chamber and seal with a cover glass (18 mm x 18 mm, thickness 0.13–0.17 mm) (Figure 1b).
5. Microscopic observation of bacterial cell growth inside GVs
   1. Set a microscopic heating stage system with an inverted microscope equipped with a 40x/0.6 numerical aperture (NA) objective lens with a long working distance (Figure 1b).
   2. Place the chamber on the microscopic heating stage system (Figure 1b). Incubate the GVs containing bacterial cells in the chamber under static conditions for 6 h at 37 °C.
   3. Capture and record microscope images of bacterial cell growth inside GVs every 30 min by using a scientific complementary metal oxide semiconductor (sCMOS) camera.

Results

Figure 1: The observation system of bacterial cell culture inside GVs. (a

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Bactotryptone	BD Biosciences	211705
Chloroform	Wako Pure Chemicals	032-21921
Cover glass (18 × 18 mm)	Matsunami Glass Ind.	C018181	thickness 0.13–0.17 mm
Cover glass (30 × 40 mm)	Matsunami Glass Ind.	custom-order	thickness 0.25–0.35 mm
Desktop centrifuge	Hi-Tech Co.	ATT101	swing rotor type
Double-faced seal (10 × 10 × 1 mm)	Nitoms	T4613
Glucose	Wako Pure Chemicals	049-31165
Inverted microscope	Olympus	IX-73
Methanol	Wako Pure Chemicals	133-16771
Microscopic heating stage system	TOKAI HIT	TP-110R-100
Mineral oil	Nacalai Tesque	23334-85
Neutravidin	Thermo Fisher Scientific	31000
Objective lens	Olympus	LUCPLFLN 40×/0.6 NA
Polycarbonate membranes	Avanti Polar Lipids	610005
sCMOS camera	Andor	Zyla 4.2 plus	pore size 100 nm
Sodium chloride	Wako Pure Chemicals	191-01665
Sucrose	Wako Pure Chemicals	196-00015
Ultrasonic bath	AS ONE	ASU-3D
Yeast extract	BD Biosciences	212750
0.6 mL lidded plastic tube	Watson	130-806C
1.5 mL lidded plastic tube	Sumitomo Bakelite Co.	MS4265-M
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocoline	Avanti Polar Lipids	850457P
1,2-distearoyl-snglycero-3-phosphoethanolamine-N-[biotinyl(polyethyleneglycol)-2000]	Avanti Polar Lipids	880129P	POPC
			Biotin-PEG-DSPE