2.2 Capillary Assay
1. Motility buffer: 7 mM Na 2 HPO 4 , 2.2 mM KH 2 PO 4 , 17.1 mM
NaCl, 4.7 mM NH 4 Cl, 14.8 μM thiamine, 0.5% bovine serum
albumin. Filter sterilize once pH adjusted to 7.4.
2. Petroff-Hausser chamber.
3. Hematocrit capillaries: 75 mm/60 μl, 0.95 mm diameter.
4. Hematocrit wax.
5. 96-Well storage plates, round bottom, 1.2 ml.
6. Automate of DNA purification.
7. DNA purification kit.
8. Real-time PCR detection system.
9. qPCR primers: use lipL32 (forward primer (45F), 5
0 -AAG
CAT TAC CGC TTG TGG TG-3
0 reverse primer (286R)
5
0 -GAA CTC CCA TTT CAG CGA TT-3
0 ) or rpoB primers
(forward primer 5
0 -ATG ATG AGA CGG ATG ACT GC-3
0 ,
reverse primer 5
0 -CGA CGA AAC GTT TGA ACC AA-3
0 ) to
enumerate L. interrogans (pathogenic strain) or L. biflexa (saprophytic strain), respectively [19, 20].
10. qPCR SYBR green kit.
11. Methylcellulose (15 cP).
12. Microcentrifuge.
2.3 Videomicroscopy
1. Fast frame rate acquisition camera.
2. Dark-field microscope.
3. MATLAB 2011 or later version.
4. Micro-Manager 1.4.
5. FIJI, ImageJ software.
3 Methods
3.1 Soft Agar Assay
Soft agar assay is often used to assess motile behavior. This test is
used as a first intention assay to evaluate chemotactic or motile
behavior of Leptospira spp. and to provide information about the
ability of Leptospira spp. to migrate once their nutrient source is
depleted. However, it does not discriminate between motility and
chemotaxis defect. Two types of soft agar assay have been developed: 0.5% agar plate and 0.3% agar plate. This strategy allows to
identify intermediate chemotactic or motile phenotypes [5].
1. To prepare soft agar EMJH medium, add 300 mg or 500 mg of
noble agar to 100 ml of freshly prepared EMJH medium for
0.3% agar plate and 0.5% agar plate, respectively. Dissolve
carefully agar using a microwave. Cool down agar and media
at 60
C for 15 m, and pour 35 ml in square Petri dish
with grid.
Leptospira spp. Toolbox for Chemotaxis Assay
125
1. Motility buffer: 7 mM Na 2 HPO 4 , 2.2 mM KH 2 PO 4 , 17.1 mM
NaCl, 4.7 mM NH 4 Cl, 14.8 μM thiamine, 0.5% bovine serum
albumin. Filter sterilize once pH adjusted to 7.4.
2. Petroff-Hausser chamber.
3. Hematocrit capillaries: 75 mm/60 μl, 0.95 mm diameter.
4. Hematocrit wax.
5. 96-Well storage plates, round bottom, 1.2 ml.
6. Automate of DNA purification.
7. DNA purification kit.
8. Real-time PCR detection system.
9. qPCR primers: use lipL32 (forward primer (45F), 5
0 -AAG
CAT TAC CGC TTG TGG TG-3
0 reverse primer (286R)
5
0 -GAA CTC CCA TTT CAG CGA TT-3
0 ) or rpoB primers
(forward primer 5
0 -ATG ATG AGA CGG ATG ACT GC-3
0 ,
reverse primer 5
0 -CGA CGA AAC GTT TGA ACC AA-3
0 ) to
enumerate L. interrogans (pathogenic strain) or L. biflexa (saprophytic strain), respectively [19, 20].
10. qPCR SYBR green kit.
11. Methylcellulose (15 cP).
12. Microcentrifuge.
2.3 Videomicroscopy
1. Fast frame rate acquisition camera.
2. Dark-field microscope.
3. MATLAB 2011 or later version.
4. Micro-Manager 1.4.
5. FIJI, ImageJ software.
3 Methods
3.1 Soft Agar Assay
Soft agar assay is often used to assess motile behavior. This test is
used as a first intention assay to evaluate chemotactic or motile
behavior of Leptospira spp. and to provide information about the
ability of Leptospira spp. to migrate once their nutrient source is
depleted. However, it does not discriminate between motility and
chemotaxis defect. Two types of soft agar assay have been developed: 0.5% agar plate and 0.3% agar plate. This strategy allows to
identify intermediate chemotactic or motile phenotypes [5].
1. To prepare soft agar EMJH medium, add 300 mg or 500 mg of
noble agar to 100 ml of freshly prepared EMJH medium for
0.3% agar plate and 0.5% agar plate, respectively. Dissolve
carefully agar using a microwave. Cool down agar and media
at 60
C for 15 m, and pour 35 ml in square Petri dish
with grid.
Leptospira spp. Toolbox for Chemotaxis Assay
125