2 Materials
2.1 Prophage
Inductions
1. Incubator.
2. Ellinghausen-McCullough-Johnson-Harris (EMJH) medium:
dissolve 2.3 g of Difco Leptospira Medium Base EMJH (Becton Dickinson) into 900 mL of distilled water, sterilize by
autoclaving, and add 100 mL of Difco Leptospira Enrichment
EMJH (Becton Dickinson).
3. 400 mL flasks.
4. Fresh Leptospira cultures.
5. Spectrophotometer.
6. Spectrophotometry cuvettes.
7. Mitomycin C: 1 mg/mL solution in sterile water.
2.2 Bacteriophage
Purification
1. Polyethylene glycol 8000.
2. Sodium chloride.
3. Cesium chloride.
4. TN buffer: 10 mM Tris–HCl, pH 7.5, 100 mM NaCl.
5. Chloroform.
6. Centrifuge.
7. Ultracentrifuge.
8. Beckman Coulter rotor SW41.
9. 13 mL thin-walled polypropylene ultracentrifuge Coulter
tubes.
10. Peristaltic pump and flexible plastic tubing.
11. Ultrafiltration cassette (100 kDa).
12. 3.5 kDa cutoff dialysis cassettes.
13. 0.22 μm dead-end filters.
14. Syringes (1–5 mL) with 18-G needles.
2.3 Double Agar
Overlay Plaque
Lysis Assay
1. EMJH medium supplemented with 0.6% and 1.2% agar.
2. Water bath (43
C).
3. Petri dishes (90 Â 15 mm).
4. Polypropylene Falcon tubes (15 mL).
2.4 Leptophage
Observation
1. Electron microscope.
2. Microscopy carbon-coated copper grids.
3. Uranyl acetate: 4% solution in water.
4. Glutaraldehyde: 25% solution in water (stock solution) and 2%
solution (working solution).
68
Olivier Schiettekatte and Pascale Bourhy
2.1 Prophage
Inductions
1. Incubator.
2. Ellinghausen-McCullough-Johnson-Harris (EMJH) medium:
dissolve 2.3 g of Difco Leptospira Medium Base EMJH (Becton Dickinson) into 900 mL of distilled water, sterilize by
autoclaving, and add 100 mL of Difco Leptospira Enrichment
EMJH (Becton Dickinson).
3. 400 mL flasks.
4. Fresh Leptospira cultures.
5. Spectrophotometer.
6. Spectrophotometry cuvettes.
7. Mitomycin C: 1 mg/mL solution in sterile water.
2.2 Bacteriophage
Purification
1. Polyethylene glycol 8000.
2. Sodium chloride.
3. Cesium chloride.
4. TN buffer: 10 mM Tris–HCl, pH 7.5, 100 mM NaCl.
5. Chloroform.
6. Centrifuge.
7. Ultracentrifuge.
8. Beckman Coulter rotor SW41.
9. 13 mL thin-walled polypropylene ultracentrifuge Coulter
tubes.
10. Peristaltic pump and flexible plastic tubing.
11. Ultrafiltration cassette (100 kDa).
12. 3.5 kDa cutoff dialysis cassettes.
13. 0.22 μm dead-end filters.
14. Syringes (1–5 mL) with 18-G needles.
2.3 Double Agar
Overlay Plaque
Lysis Assay
1. EMJH medium supplemented with 0.6% and 1.2% agar.
2. Water bath (43
C).
3. Petri dishes (90 Â 15 mm).
4. Polypropylene Falcon tubes (15 mL).
2.4 Leptophage
Observation
1. Electron microscope.
2. Microscopy carbon-coated copper grids.
3. Uranyl acetate: 4% solution in water.
4. Glutaraldehyde: 25% solution in water (stock solution) and 2%
solution (working solution).
68
Olivier Schiettekatte and Pascale Bourhy