2 Materials
2.1 Leptospira spp.
Culture
1. EMJH supplement [25]: Dissolve 10 g of bovine serum albumin (BSA) in 50 ml of prewarmed distilled water (37
C), with
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
dehydrated), 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 basal
EMJH (Becton Dickinson) in 900 ml of water.
3. EMJH medium: 100 ml of EMJH supplement, 890 ml of
EMJH basal medium, 10 ml normal rabbit serum, 1 g lactalbumin hydrolase, and 100 μl superoxide dismutase (stock
10 mg/ml in PBS). Adjust pH to 7.4 if necessary and filter
sterilize (0.22 μm filter), aliquot in flask of smaller volume, and
control sterility at 37
C for 48 h and then store at 4
C.
4. High-BSA EMJH: EMJH medium supplemented with an
additional 10 mg/ml BSA (serum replacement grade)—final
BSA concentration 20 mg/ml. Filter sterilize (0.22 μm filter)
(see Note 1).
5. Leptospira spp. in vitro culture in mid- to late-logarithmic
phase.
6. 15-ml polypropylene conical tubes, sterile.
7. Dark-field microscope.
8. Bacteriological incubators, at 30
C and 37
C.
9. Petroff-Hausser counting chamber.
10. BSC, biological safety cabinet (Biosafety Level 2; BSL-2).
2.2 Dialysis
Membrane Chambers’
Preparation and Filling
1. Ultrapure water.
2. EDTA: 1 mM solution in ultrapure water, pH 8.0.
3. Regenerated cellulose dialysis membrane tubing (6000 to
8000 MWCO, 32 mm width).
4. Filter units, 0.22 μm, 250 ml, sterile.
5. Three 2-L glass beakers, each containing a magnetic stir bar.
6. Hot plate with stirring option.
7. Extra-long blunt-end forceps, sterile.
8. Pipettor.
9. Scissors, iris, 4 in. (~10.2 cm).
10. Surgical gloves, individually wrapped, sterile.
11. Biological safety cabinet (Biosafety Level 2; BSL-2).
232
Andre Alex Grassmann and Melissa J. Caimano
2.1 Leptospira spp.
Culture
1. EMJH supplement [25]: Dissolve 10 g of bovine serum albumin (BSA) in 50 ml of prewarmed distilled water (37
C), with
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
dehydrated), 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 basal
EMJH (Becton Dickinson) in 900 ml of water.
3. EMJH medium: 100 ml of EMJH supplement, 890 ml of
EMJH basal medium, 10 ml normal rabbit serum, 1 g lactalbumin hydrolase, and 100 μl superoxide dismutase (stock
10 mg/ml in PBS). Adjust pH to 7.4 if necessary and filter
sterilize (0.22 μm filter), aliquot in flask of smaller volume, and
control sterility at 37
C for 48 h and then store at 4
C.
4. High-BSA EMJH: EMJH medium supplemented with an
additional 10 mg/ml BSA (serum replacement grade)—final
BSA concentration 20 mg/ml. Filter sterilize (0.22 μm filter)
(see Note 1).
5. Leptospira spp. in vitro culture in mid- to late-logarithmic
phase.
6. 15-ml polypropylene conical tubes, sterile.
7. Dark-field microscope.
8. Bacteriological incubators, at 30
C and 37
C.
9. Petroff-Hausser counting chamber.
10. BSC, biological safety cabinet (Biosafety Level 2; BSL-2).
2.2 Dialysis
Membrane Chambers’
Preparation and Filling
1. Ultrapure water.
2. EDTA: 1 mM solution in ultrapure water, pH 8.0.
3. Regenerated cellulose dialysis membrane tubing (6000 to
8000 MWCO, 32 mm width).
4. Filter units, 0.22 μm, 250 ml, sterile.
5. Three 2-L glass beakers, each containing a magnetic stir bar.
6. Hot plate with stirring option.
7. Extra-long blunt-end forceps, sterile.
8. Pipettor.
9. Scissors, iris, 4 in. (~10.2 cm).
10. Surgical gloves, individually wrapped, sterile.
11. Biological safety cabinet (Biosafety Level 2; BSL-2).
232
Andre Alex Grassmann and Melissa J. Caimano