5. Phosphate buffered saline (PBS): 137 mM NaCl, 2.7 mM KCl,
10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 .
6. 10 nm colloidal gold particle solution.
2.2 Equipment
and Electron
Microscope
1. 30
C incubator.
2. Sterile conical tubes of 15 mL capacity.
3. Microcentrifuge.
4. Tweezer.
5. Ion coater.
6. Quantifoil molybdenum 200 mesh R0.6/1.0 grid (Quantifoil
Micro Tools, Jena, Germany).
7. Vitrobot Mark IV (Thermo Fisher Scientific).
8. Titan Krios FEG transmission electron microscope (Thermo
Fisher Scientific).
9. FEI Falcon II 4k  4k CMOS direct electron detector camera
(Thermo Fisher Scientific).
2.3 Program for Data
Collection
and Structural
Analysis by ECT
1. EPU software (Thermo Fisher Scientific).
2. Xplore3D software package (Thermo Fisher Scientific).
3. UCSF Chimera [13].
4. IMOD software package [14].
3 Methods
3.1 Sample
Preparation
1. Leptospira cells are cultivated at 30
C in EMJH medium for
7–8 days.
2. The 100 μL of pre-culture is added to 5 mL EMJH medium,
and then the cells are incubated at 30
C in EMJH medium for
3–4 days until a final concentration ~2 Â 10
8 cells/mL.
3. The cells are collected by centrifugation (4600 Â g, 2 min,
25
C).
4. The pellet is suspended in 50 μL PBS.
3.2 ECT Sample
Preparation
1. Quantifoil molybdenum 200 mesh R0.6/1.0 holey carbon
grids are glow-discharged on a glass slide for 5 s.
2. A 3.0 μL of sample solution (Leptospira cells in PBS prepared in
Subheading 3.1) containing 10 nm colloidal gold particles is
applied onto the grid, and the grid is dried up at room temperature (see Note 2).
3. The pretreated grids are glow-discharged again on a glass slide
for 20 s.
Visualization of Intact Periplasmic Flagella
133
10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 .
6. 10 nm colloidal gold particle solution.
2.2 Equipment
and Electron
Microscope
1. 30
C incubator.
2. Sterile conical tubes of 15 mL capacity.
3. Microcentrifuge.
4. Tweezer.
5. Ion coater.
6. Quantifoil molybdenum 200 mesh R0.6/1.0 grid (Quantifoil
Micro Tools, Jena, Germany).
7. Vitrobot Mark IV (Thermo Fisher Scientific).
8. Titan Krios FEG transmission electron microscope (Thermo
Fisher Scientific).
9. FEI Falcon II 4k  4k CMOS direct electron detector camera
(Thermo Fisher Scientific).
2.3 Program for Data
Collection
and Structural
Analysis by ECT
1. EPU software (Thermo Fisher Scientific).
2. Xplore3D software package (Thermo Fisher Scientific).
3. UCSF Chimera [13].
4. IMOD software package [14].
3 Methods
3.1 Sample
Preparation
1. Leptospira cells are cultivated at 30
C in EMJH medium for
7–8 days.
2. The 100 μL of pre-culture is added to 5 mL EMJH medium,
and then the cells are incubated at 30
C in EMJH medium for
3–4 days until a final concentration ~2 Â 10
8 cells/mL.
3. The cells are collected by centrifugation (4600 Â g, 2 min,
25
C).
4. The pellet is suspended in 50 μL PBS.
3.2 ECT Sample
Preparation
1. Quantifoil molybdenum 200 mesh R0.6/1.0 holey carbon
grids are glow-discharged on a glass slide for 5 s.
2. A 3.0 μL of sample solution (Leptospira cells in PBS prepared in
Subheading 3.1) containing 10 nm colloidal gold particles is
applied onto the grid, and the grid is dried up at room temperature (see Note 2).
3. The pretreated grids are glow-discharged again on a glass slide
for 20 s.
Visualization of Intact Periplasmic Flagella
133