2. Grow Leptospira spp. to test until exponential phase in liquid
EMJH medium; first evaluate motility in dark-field microscopy
while counting using a Petroff-Hausser chamber. Then slow
speed centrifugate bacteria in EMJH medium at 3000 Â g for
10 m at room temperature. Resuspend the pellet in appropriate
volume such that 10 μl contains 10
6 bacteria.
3. Once soft agar EMJH medium has solidified, cooled, and dried
to room temperature, drill equally spaced holes in agar using
the grid of the plate as a reference (used 1 ml pipette tip to drill
the holes). Maximal capacity of square Petri dish can accommodate three rows and three columns of holes to perform
optimally soft agar assay. Inoculate each hole with 10 μl of the
previously prepared bacterial suspension to test.
4. Incubate plates for up to 15 days at 30
C for most pathogenic
Leptospira spp. and up to 7–10 days for the saprophytic model
bacterium Leptospira biflexa.
5. Measure the diameters of the zone of spread, and repeat soft
agar assays at least for three independent cultures of each
bacterial strain to test. Always add the wild-type strain if you
test mutant strain. Add for each plate assay a motility mutant
like ΔflaB [21] as a negative control.
3.2 Capillary Assay
Capillary assay was first designed for bacteria by Julius Adler in
1967, with the first chemotaxis capillary assay in the model bacterium E. coli [15]. They provide a quantitative assessment of chemotactic response to specific compounds. First capillary assay for
Leptospira spp. was designed in 1993 [22]. Additionally, another
assay was adapted in Borrelia burgdorferi [23]. This assay is optimized for Leptospira spp. with higher throughput by combining it
with quantitative PCR [6, 23], and it could be also combined with
flow cytometry.
1. Grow exponential-phase Leptospira in EMJH liquid medium
(10 ml). Suspension should be at OD 420nm ¼ 0.5, which
corresponds to approximately 5 Â 10
8 bacteria/ml. Do a
quick motility check under dark-field microscope, and most
bacteria should be actively motile (95% of cells with actively
gyrating ends). Harvest cells at room temperature at low centrifugation speed (3000 Â g for 10 m).
2. On the same day prepare the motility buffer.
3. Resuspend harvested bacteria into motility buffer to 10
8 bacteria/ml, incubate overnight (18 h) at 30
C to allow bacteria
to recover motility, and deplete nutrient carried over during
centrifugation.
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Ambroise Lambert
EMJH medium; first evaluate motility in dark-field microscopy
while counting using a Petroff-Hausser chamber. Then slow
speed centrifugate bacteria in EMJH medium at 3000 Â g for
10 m at room temperature. Resuspend the pellet in appropriate
volume such that 10 μl contains 10
6 bacteria.
3. Once soft agar EMJH medium has solidified, cooled, and dried
to room temperature, drill equally spaced holes in agar using
the grid of the plate as a reference (used 1 ml pipette tip to drill
the holes). Maximal capacity of square Petri dish can accommodate three rows and three columns of holes to perform
optimally soft agar assay. Inoculate each hole with 10 μl of the
previously prepared bacterial suspension to test.
4. Incubate plates for up to 15 days at 30
C for most pathogenic
Leptospira spp. and up to 7–10 days for the saprophytic model
bacterium Leptospira biflexa.
5. Measure the diameters of the zone of spread, and repeat soft
agar assays at least for three independent cultures of each
bacterial strain to test. Always add the wild-type strain if you
test mutant strain. Add for each plate assay a motility mutant
like ΔflaB [21] as a negative control.
3.2 Capillary Assay
Capillary assay was first designed for bacteria by Julius Adler in
1967, with the first chemotaxis capillary assay in the model bacterium E. coli [15]. They provide a quantitative assessment of chemotactic response to specific compounds. First capillary assay for
Leptospira spp. was designed in 1993 [22]. Additionally, another
assay was adapted in Borrelia burgdorferi [23]. This assay is optimized for Leptospira spp. with higher throughput by combining it
with quantitative PCR [6, 23], and it could be also combined with
flow cytometry.
1. Grow exponential-phase Leptospira in EMJH liquid medium
(10 ml). Suspension should be at OD 420nm ¼ 0.5, which
corresponds to approximately 5 Â 10
8 bacteria/ml. Do a
quick motility check under dark-field microscope, and most
bacteria should be actively motile (95% of cells with actively
gyrating ends). Harvest cells at room temperature at low centrifugation speed (3000 Â g for 10 m).
2. On the same day prepare the motility buffer.
3. Resuspend harvested bacteria into motility buffer to 10
8 bacteria/ml, incubate overnight (18 h) at 30
C to allow bacteria
to recover motility, and deplete nutrient carried over during
centrifugation.
126
Ambroise Lambert