observe their structures inside the cell by conventional electron
microscopy technique.
Here, I describe a general protocol for the in situ structural
analysis of periplasmic flagella in Leptospira spp. by electron cryotomography (ECT). ECT is an imaging technique that directly
provides 3D structures of cells and molecular complexes in their
cellular environment at nanometer resolution. EM images are 2D
projections of 3D objects. To obtain the structures of cells and
molecules, the specimen grid is tilted incrementally around an axis
perpendicular to the electron beam, e.g., from À60
to +60
with
2
increments, and an image is taken at each tilt angle. Each image
represents a different view of the 3D objects on the grid. The
images of a tilt series are aligned and are back-projected to generate
a 3D image (tomogram) of the specimen. Using the protocols
described here, the structures of periplasmic flagella are directly
visualized whereby the interaction between the periplasmic flagella
and outer membrane can be revealed [12]. In this chapter, I focus
on sample preparation, data collection, tilt series image alignment,
and tomographic reconstruction for visualization of intact periplasmic flagella in Leptospira spp.
2 Materials
Prepare all solutions using ultrapure water (resistance ¼ 18 MΩ at
25
C) and analytical grade reagents. Prepare all reagents at room
temperature. Store all reagents at 4
C. Particular attention should
be paid to the adequate disposal of materials contaminated with
Leptospira.
2.1 Bacterial Stain
and Cell Culture
1. Bacterial strain: Leptospira biflexa serovar Patoc strain Patoc I.
2. EMJH basement: Dissolve 2.3 g of Difco™ Leptospira
Medium Base EMJH (Becton Dickinson) in 800 mL of ultrapure water. Add 1 mL of glycerol (stock 10 g/100 mL glycerol) and 1 mL of sodium pyruvate (stock 10 g/100 mL
sodium pyruvate).
3. EMJH supplement: Dissolve 10 g of bovine serum albumin in
100 mL of ultrapure water. Mix 1 mL of ZnSO 4 (stock 0.4 g/
100 mL ZnSO 4 ∙7H 2 O), 1 mL of MgCl 2 (stock1.5 g/100 mL
MgCl 2 ∙6H 2 O), 1 mL of CaCl 2 (1.5 g CaCl 2 ∙2H 2 O), 12.5 mL
of Tween 80 (stock 10 g/100 mL Tween 80), 1 mL of cyanocobalamin (stock 0.02 g/100 mL cyanocobalamin), and
10 mL of FeSO 4 (0.05 g/10 mL FeSO 4 ∙7H 2 O, prepare just
before making EMJH supplement), and adjust to 198 mL with
ultrapure water (see Note 1).
4. Liquid EMJH medium: 802 mL of EMJH basement, 198 mL
of EMJH supplement, pH 7.4. Sterilize the medium by filtration with 0.22 μm pore size filter.
132
Akihiro Kawamoto
microscopy technique.
Here, I describe a general protocol for the in situ structural
analysis of periplasmic flagella in Leptospira spp. by electron cryotomography (ECT). ECT is an imaging technique that directly
provides 3D structures of cells and molecular complexes in their
cellular environment at nanometer resolution. EM images are 2D
projections of 3D objects. To obtain the structures of cells and
molecules, the specimen grid is tilted incrementally around an axis
perpendicular to the electron beam, e.g., from À60
to +60
with
2
increments, and an image is taken at each tilt angle. Each image
represents a different view of the 3D objects on the grid. The
images of a tilt series are aligned and are back-projected to generate
a 3D image (tomogram) of the specimen. Using the protocols
described here, the structures of periplasmic flagella are directly
visualized whereby the interaction between the periplasmic flagella
and outer membrane can be revealed [12]. In this chapter, I focus
on sample preparation, data collection, tilt series image alignment,
and tomographic reconstruction for visualization of intact periplasmic flagella in Leptospira spp.
2 Materials
Prepare all solutions using ultrapure water (resistance ¼ 18 MΩ at
25
C) and analytical grade reagents. Prepare all reagents at room
temperature. Store all reagents at 4
C. Particular attention should
be paid to the adequate disposal of materials contaminated with
Leptospira.
2.1 Bacterial Stain
and Cell Culture
1. Bacterial strain: Leptospira biflexa serovar Patoc strain Patoc I.
2. EMJH basement: Dissolve 2.3 g of Difco™ Leptospira
Medium Base EMJH (Becton Dickinson) in 800 mL of ultrapure water. Add 1 mL of glycerol (stock 10 g/100 mL glycerol) and 1 mL of sodium pyruvate (stock 10 g/100 mL
sodium pyruvate).
3. EMJH supplement: Dissolve 10 g of bovine serum albumin in
100 mL of ultrapure water. Mix 1 mL of ZnSO 4 (stock 0.4 g/
100 mL ZnSO 4 ∙7H 2 O), 1 mL of MgCl 2 (stock1.5 g/100 mL
MgCl 2 ∙6H 2 O), 1 mL of CaCl 2 (1.5 g CaCl 2 ∙2H 2 O), 12.5 mL
of Tween 80 (stock 10 g/100 mL Tween 80), 1 mL of cyanocobalamin (stock 0.02 g/100 mL cyanocobalamin), and
10 mL of FeSO 4 (0.05 g/10 mL FeSO 4 ∙7H 2 O, prepare just
before making EMJH supplement), and adjust to 198 mL with
ultrapure water (see Note 1).
4. Liquid EMJH medium: 802 mL of EMJH basement, 198 mL
of EMJH supplement, pH 7.4. Sterilize the medium by filtration with 0.22 μm pore size filter.
132
Akihiro Kawamoto