5. Motility medium: 20 mM sodium phosphate buffer, pH 7.4.
Make 200 mM stock solution by mixing 81 mL of 200 mM
Na 2 HPO 4 and 19 mL of 200 mM NaH 2 PO 4 ; measure pH
using a pH meter; if necessary, adjust to pH 7.4 by the addition
of Na 2 HPO 4 or NaH 2 PO 4 solution and then autoclave. Dilute
the stock solution with distilled water to 20 mM.
6. Polymer solutions: Add polymers to motility medium to
increase viscosity or viscoelasticity. Ficoll (5–30 w/v%) and
methylcellulose (0.25–2 w/v%) are generally used in motility
assays for microorganisms (see Note 1).
7. MES buffer: 50 mM 2-morpholinoethanesulfonic acid,
pH 5.2.
8. 10 mM Tris–HCl buffer, pH 8.0.
2.2 Reagents
for Preparation of
Antibody-Coated
Beads
1. Polystyrene beads with a diameter of 0.2 μm.
2. Antibody against Leptospira whole cell or lipopolysaccharide
(see Note 2).
3. 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide (EDAC).
4. Centrifuge.
2.3 Microscopy
1. Dark-field microscope (see Note 3).
2. Video camera (see Note 4).
3. Computer.
4. Glass slide (0.8–1.0 mm in thickness).
5. 22 Â 24 mm coverslip (0.13–0.17 mm in thickness).
6. Double-sided tape (5–10 μm in thickness).
2.4 Software for Data
Analysis
The analyses described in this chapter are performed by ImageJ and
Microsoft Excel.
3 Methods
3.1 Sample
Preparation
1. Centrifuge 3–4 days culture at 1000 Â g for 10 min at room
temperature.
2. Suspend the pellet into motility medium by tapping a tube
without pipetting.
3. Make a flow chamber by attaching a coverslip with a glass slide
via double-sided tape (Fig. 3).
4. Infuse the cell solution into the flow chamber (see Note 5).
Motility assays for Leptospira
143
Make 200 mM stock solution by mixing 81 mL of 200 mM
Na 2 HPO 4 and 19 mL of 200 mM NaH 2 PO 4 ; measure pH
using a pH meter; if necessary, adjust to pH 7.4 by the addition
of Na 2 HPO 4 or NaH 2 PO 4 solution and then autoclave. Dilute
the stock solution with distilled water to 20 mM.
6. Polymer solutions: Add polymers to motility medium to
increase viscosity or viscoelasticity. Ficoll (5–30 w/v%) and
methylcellulose (0.25–2 w/v%) are generally used in motility
assays for microorganisms (see Note 1).
7. MES buffer: 50 mM 2-morpholinoethanesulfonic acid,
pH 5.2.
8. 10 mM Tris–HCl buffer, pH 8.0.
2.2 Reagents
for Preparation of
Antibody-Coated
Beads
1. Polystyrene beads with a diameter of 0.2 μm.
2. Antibody against Leptospira whole cell or lipopolysaccharide
(see Note 2).
3. 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide (EDAC).
4. Centrifuge.
2.3 Microscopy
1. Dark-field microscope (see Note 3).
2. Video camera (see Note 4).
3. Computer.
4. Glass slide (0.8–1.0 mm in thickness).
5. 22 Â 24 mm coverslip (0.13–0.17 mm in thickness).
6. Double-sided tape (5–10 μm in thickness).
2.4 Software for Data
Analysis
The analyses described in this chapter are performed by ImageJ and
Microsoft Excel.
3 Methods
3.1 Sample
Preparation
1. Centrifuge 3–4 days culture at 1000 Â g for 10 min at room
temperature.
2. Suspend the pellet into motility medium by tapping a tube
without pipetting.
3. Make a flow chamber by attaching a coverslip with a glass slide
via double-sided tape (Fig. 3).
4. Infuse the cell solution into the flow chamber (see Note 5).
Motility assays for Leptospira
143