bacteria are stopped [12, 13]. Additionally, Leptospira spp. are
atypical since they have only two endoflagella, each arising subterminally in the periplasm of the cell without overlapping at the
cell center. The rotation of the motors in two different directions at
each end is responsible for a forward thrust that allows the bacteria
to translate [14]. However, the regulatory mechanism for coordination of flagellar motor remains unknown to date in Leptospira
spp. Furthermore, chemotactic system regulating motility is very
diverse with more than 26 chemotactic receptors in saprophytic
strains and only 13 for most pathogenic strains [6]. On the other
hand, motility and chemotaxis has been well characterized early in
the 1960s with enterobacteria model like Escherichia coli with the
use of capillary assay [15] or even videomicroscopy tracking
[16]. We and other teams have applied and adapted those tools in
different studies to decipher chemotaxis on Leptospira spp. bacteria
model [6, 8, 17].
In this review I will describe the methods as a toolbox to
understand chemotaxis, from the first intention assay soft agar
plates that allow to characterize chemotaxis defect to chemoattractant identification assay like capillary assay, ending with quantitative
analysis of motility with videomicroscopy tracking assay.
2 Materials
2.1 Soft Agar Assay
1. EMJH supplement [18]: Dissolve 10 g of bovine serum albumin in 50 ml of prewarmed distilled water (37
C), slow agitation to prevent bubbles. Add 1 ml of ZnSO 4 (stock 0.04 g/L
ZnSO 4 heptahydrated), 1 ml of MgCl 2 (stock 0.15 g/L MgCl 2
hexahydrated), 1 ml of CaCl 2 (stock 0.15 g/L CaCl 2 dihydrated), 1 ml of sodium pyruvate (stock 1 g/L sodium pyruvate), 2 ml of glycerol, 12.5 ml of Tween 80, and 1 ml of
vitamin B12 (stock 2 mg/L vitamin B12). Add previously
prepared solution of FeSO 4 at 0.5% and adjust volume to
100 ml and pH to 7.4.
2. EMJH basal medium: Dissolve 2.3 g of Difco Leptospira
Medium Base EMJH (Becton Dickinson) in 900 ml of water.
3. EMJH medium: 100 ml of EMJH supplement, 900 ml of
EMJH basal medium. Adjust pH to 7.4 if necessary and filter
sterilize (0.22 μm filter), aliquot in flask of smaller volume,
control sterility at 37
C for 48 h, and then store at 4
C.
4. Noble agar.
5. Square Petri dish with grid.
124
Ambroise Lambert
atypical since they have only two endoflagella, each arising subterminally in the periplasm of the cell without overlapping at the
cell center. The rotation of the motors in two different directions at
each end is responsible for a forward thrust that allows the bacteria
to translate [14]. However, the regulatory mechanism for coordination of flagellar motor remains unknown to date in Leptospira
spp. Furthermore, chemotactic system regulating motility is very
diverse with more than 26 chemotactic receptors in saprophytic
strains and only 13 for most pathogenic strains [6]. On the other
hand, motility and chemotaxis has been well characterized early in
the 1960s with enterobacteria model like Escherichia coli with the
use of capillary assay [15] or even videomicroscopy tracking
[16]. We and other teams have applied and adapted those tools in
different studies to decipher chemotaxis on Leptospira spp. bacteria
model [6, 8, 17].
In this review I will describe the methods as a toolbox to
understand chemotaxis, from the first intention assay soft agar
plates that allow to characterize chemotaxis defect to chemoattractant identification assay like capillary assay, ending with quantitative
analysis of motility with videomicroscopy tracking assay.
2 Materials
2.1 Soft Agar Assay
1. EMJH supplement [18]: Dissolve 10 g of bovine serum albumin in 50 ml of prewarmed distilled water (37
C), slow agitation to prevent bubbles. Add 1 ml of ZnSO 4 (stock 0.04 g/L
ZnSO 4 heptahydrated), 1 ml of MgCl 2 (stock 0.15 g/L MgCl 2
hexahydrated), 1 ml of CaCl 2 (stock 0.15 g/L CaCl 2 dihydrated), 1 ml of sodium pyruvate (stock 1 g/L sodium pyruvate), 2 ml of glycerol, 12.5 ml of Tween 80, and 1 ml of
vitamin B12 (stock 2 mg/L vitamin B12). Add previously
prepared solution of FeSO 4 at 0.5% and adjust volume to
100 ml and pH to 7.4.
2. EMJH basal medium: Dissolve 2.3 g of Difco Leptospira
Medium Base EMJH (Becton Dickinson) in 900 ml of water.
3. EMJH medium: 100 ml of EMJH supplement, 900 ml of
EMJH basal medium. Adjust pH to 7.4 if necessary and filter
sterilize (0.22 μm filter), aliquot in flask of smaller volume,
control sterility at 37
C for 48 h, and then store at 4
C.
4. Noble agar.
5. Square Petri dish with grid.
124
Ambroise Lambert