9. Measure bacteria concentration and motility in PetroffHausser chamber using 5 μl of each suspension. This step
allows to control enumeration with quantitative PCR.
10. Prepare the DNA purification kit for automated DNA extraction (see Note 3), and pipette 40 μl of the capillary suspension
for each extraction (see Note 4).
11. Prepare quantitative PCR using SYBR green mix. Prepare standard curves mix with known numbers of leptospires enumerated in Petroff-Hausser chamber.
12. Run PCR program: PCR conditions were as follows (ramp
rates of 20
C/s): initial denaturation 95
C for 600 s; followed
by 45 cycles of amplification 95
C for 10 s, 57
C for 8 s, and
72
C for 10 s; and fusion at 95
C for 360 s [23].
13. All quantitative PCR assay enumeration should be performed
in duplicate with control reaction without template. Each assay
is repeated in at least two independent experiments with three
to four capillaries in each condition tested. Use enumeration
values to quantify chemotactic response in fold change for
capillary containing chemoattractant compared to motility
buffer only containing capillary (a change of 2 or below is not
considered significant).
3.3 Videomicroscopy
Tracking Assay
Compared to previously mentioned methods, videomicroscopy
tracking assay allows a direct observation of the motile behavior.
We can track individual motile behavior, observe trajectories, pause,
and increase in motility. We can quantify speed of bacteria and
length of trajectories. It was first pioneered in 1972 in E. coli
[16]. A dark-field videomicroscope with ImageJ analysis and
MATLAB scripts can be used to track and adapt the method to
Leptospira spp. [8].
1. Grow actively motile Leptospira spp. in EMJH liquid medium
until exponential phase. Ensure that the bacteria remain motile
daily using dark-field microscopy. Harvest cell using slowspeed centrifugation (3000 Â g for 10 min).
2. Prepare motility buffer the day of the experiment. Prewarm
100 ml of motility buffer at 65
C, add 1 g of methylcellulose
(15 cP), and homogenize. Resuspend harvested bacteria in
viscous motility buffer (1% methylcellulose).
3. Prepare sample for microscopy. Add four drops (2 mm size) of
silicon grease on glass slides, and the drops should form a
square of 15 mm. Deposit 0.8 μl of bacterial suspension in
viscous motility buffer at the center of the four drops. Apply
an 18 mm-squared cover glass on top of the drops until there is
contact with the bacterial suspension drop.
128
Ambroise Lambert
allows to control enumeration with quantitative PCR.
10. Prepare the DNA purification kit for automated DNA extraction (see Note 3), and pipette 40 μl of the capillary suspension
for each extraction (see Note 4).
11. Prepare quantitative PCR using SYBR green mix. Prepare standard curves mix with known numbers of leptospires enumerated in Petroff-Hausser chamber.
12. Run PCR program: PCR conditions were as follows (ramp
rates of 20
C/s): initial denaturation 95
C for 600 s; followed
by 45 cycles of amplification 95
C for 10 s, 57
C for 8 s, and
72
C for 10 s; and fusion at 95
C for 360 s [23].
13. All quantitative PCR assay enumeration should be performed
in duplicate with control reaction without template. Each assay
is repeated in at least two independent experiments with three
to four capillaries in each condition tested. Use enumeration
values to quantify chemotactic response in fold change for
capillary containing chemoattractant compared to motility
buffer only containing capillary (a change of 2 or below is not
considered significant).
3.3 Videomicroscopy
Tracking Assay
Compared to previously mentioned methods, videomicroscopy
tracking assay allows a direct observation of the motile behavior.
We can track individual motile behavior, observe trajectories, pause,
and increase in motility. We can quantify speed of bacteria and
length of trajectories. It was first pioneered in 1972 in E. coli
[16]. A dark-field videomicroscope with ImageJ analysis and
MATLAB scripts can be used to track and adapt the method to
Leptospira spp. [8].
1. Grow actively motile Leptospira spp. in EMJH liquid medium
until exponential phase. Ensure that the bacteria remain motile
daily using dark-field microscopy. Harvest cell using slowspeed centrifugation (3000 Â g for 10 min).
2. Prepare motility buffer the day of the experiment. Prewarm
100 ml of motility buffer at 65
C, add 1 g of methylcellulose
(15 cP), and homogenize. Resuspend harvested bacteria in
viscous motility buffer (1% methylcellulose).
3. Prepare sample for microscopy. Add four drops (2 mm size) of
silicon grease on glass slides, and the drops should form a
square of 15 mm. Deposit 0.8 μl of bacterial suspension in
viscous motility buffer at the center of the four drops. Apply
an 18 mm-squared cover glass on top of the drops until there is
contact with the bacterial suspension drop.
128
Ambroise Lambert