and clockwise. The reversal frequency of leptospires is affected by
the concentration gradient of chemotaxis-inducible chemicals
[13, 14] and the increased viscosity [15]. Even if the swimming
velocities measured as described in Subheading 1.2.2 were similar
between two strains, the reversal frequency can be different. Cells
hardly reversing can migrate longer distances, namely, the reversal
frequency affects the net migration distance. Thus, note the reversal
frequency, when assessing effects of mutations, chemicals, and
viscosity on the Leptospira motility.
1.2.4 Cell-Body Rotation
Rate (Spiral-End, PC,
Hook-End)
In externally flagellated bacteria, such as Escherichia coli and Salmonella spp., the data of flagellar rotation rate is usually combined
with those of motile fraction and swimming velocity for characterization of their motility and chemotaxis. As leptospires hide PFs
within the cell body, the cell-body rotation rate is informative
instead. Measurements of spiral-end and hook-end gyrations are
an indirect evaluation of the PF rotation [2, 16]. Measurement of
the PC rotation requires one-sided dark-field illumination [4], a
high numerical aperture (NA) objective [16], or labeling the cell
surface with probes such as microbeads and fluorescent dyes
[2]. This chapter describes the methods for measuring gyrations
of spiral-end and hook-end and the rotation of PC.
2 Materials
2.1 Bacteria
and Media
1. Leptospira biflexa strain Patoc I.
2. EMJH basement: Dissolve 2.3 g of Difco™ Leptospira
Medium Base EMJH (Becton Dickinson) in 900 mL of distilled 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
50 mL of distilled 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 98 mL with
sterilized water. Sterilize MgCl 2 , MgCl 2 , glycerol, and Tween
80 stock solutions at 121
C for 20 min and ZnSO 4 , cyanocobalamin, and sodium pyruvate stock solutions by using a filter
with 0.22 μm pore size. Stock solutions can be stored at 4
C.
4. Liquid EMJH medium: 902 mL of EMJH basement, 98 mL of
EMJH supplement, pH 7.4. Store at 4
C.
142
Shuichi Nakamura
the concentration gradient of chemotaxis-inducible chemicals
[13, 14] and the increased viscosity [15]. Even if the swimming
velocities measured as described in Subheading 1.2.2 were similar
between two strains, the reversal frequency can be different. Cells
hardly reversing can migrate longer distances, namely, the reversal
frequency affects the net migration distance. Thus, note the reversal
frequency, when assessing effects of mutations, chemicals, and
viscosity on the Leptospira motility.
1.2.4 Cell-Body Rotation
Rate (Spiral-End, PC,
Hook-End)
In externally flagellated bacteria, such as Escherichia coli and Salmonella spp., the data of flagellar rotation rate is usually combined
with those of motile fraction and swimming velocity for characterization of their motility and chemotaxis. As leptospires hide PFs
within the cell body, the cell-body rotation rate is informative
instead. Measurements of spiral-end and hook-end gyrations are
an indirect evaluation of the PF rotation [2, 16]. Measurement of
the PC rotation requires one-sided dark-field illumination [4], a
high numerical aperture (NA) objective [16], or labeling the cell
surface with probes such as microbeads and fluorescent dyes
[2]. This chapter describes the methods for measuring gyrations
of spiral-end and hook-end and the rotation of PC.
2 Materials
2.1 Bacteria
and Media
1. Leptospira biflexa strain Patoc I.
2. EMJH basement: Dissolve 2.3 g of Difco™ Leptospira
Medium Base EMJH (Becton Dickinson) in 900 mL of distilled 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
50 mL of distilled 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 98 mL with
sterilized water. Sterilize MgCl 2 , MgCl 2 , glycerol, and Tween
80 stock solutions at 121
C for 20 min and ZnSO 4 , cyanocobalamin, and sodium pyruvate stock solutions by using a filter
with 0.22 μm pore size. Stock solutions can be stored at 4
C.
4. Liquid EMJH medium: 902 mL of EMJH basement, 98 mL of
EMJH supplement, pH 7.4. Store at 4
C.
142
Shuichi Nakamura