2.3 Surgical
Implantation of DMCs
1. Adult Sprague-Dawley rats (175–200 g) (see Note 2).
2. Anesthetic cocktail: Ketamine 30–50 mg/kg, xylazine
3–5 mg/kg, and acepromazine 0.75–1 mg/kg.
3. Ophthalmic ointment.
4. Electric shaver.
5. Alcohol prep pads.
6. Carprofen: 5–10 mg per kg (see Note 3).
7. 70% ethanol.
8. Surgical gloves, individually wrapped, sterile.
9. Swab stick, 2% w/v chlorhexidine gluconate in 70% isopropyl
alcohol (see Note 4).
10. Disposable serological pipets, 10 ml sterile, individually
wrapped.
11. Surgical drape, cut into 12 in. (~46 cm) squares.
12. Gauze, 4 Â 4 in., sterile (see Note 5).
13. Surgical instrument pack: one per animal, sterilized and kept
within the same sterile package, containing scalpel blade
holder, iris scissors (4 in./~10.2 cm), tissue forceps 1Â
2 tooth dissecting or Adson-Brown (5½ in./~14 cm), tissue
forceps blunt end (5½ in./~14 cm), and needle holder forceps
with built-in scissors (e.g., Olsen-Hegar, 5½ in./~14 cm) (see
Note 6).
14. 9-mm stainless steel wound closure clips and applicator.
15. Scalpel blades, No. 10.
16. Nonabsorbable suture with taper point needle attached (e.g.,
Ethicon 4-0, SH-1, 27 in. (~68.6 cm) coated VICRYL, violetbraided suture).
17. Biological safety cabinet (Biosafety Level 2; BSL-2).
18. Glass bead sterilizer, optional (see Note 6).
19. Circulating warm water blanket and pump.
20. 18-G, 1.5 in. needles attached to sterile 10 ml syringes.
3 Methods
3.1 Preparation
of Sterile Dialysis
Membrane Tubing
1. Fill three 2-L glass beakers as follows:
Beaker 1: 1–1.5-L ultrapure water.
Beaker 2: 1–1.5-L 1 mM EDTA (pH 8.0).
Beaker 3: 1–1.5-L ultrapure water.
2. Add a magnetic stir bar to each beaker, cover with heavy-duty
aluminum foil, and autoclave.
Cultivation of Leptospira interrogans within DMCs
233
Implantation of DMCs
1. Adult Sprague-Dawley rats (175–200 g) (see Note 2).
2. Anesthetic cocktail: Ketamine 30–50 mg/kg, xylazine
3–5 mg/kg, and acepromazine 0.75–1 mg/kg.
3. Ophthalmic ointment.
4. Electric shaver.
5. Alcohol prep pads.
6. Carprofen: 5–10 mg per kg (see Note 3).
7. 70% ethanol.
8. Surgical gloves, individually wrapped, sterile.
9. Swab stick, 2% w/v chlorhexidine gluconate in 70% isopropyl
alcohol (see Note 4).
10. Disposable serological pipets, 10 ml sterile, individually
wrapped.
11. Surgical drape, cut into 12 in. (~46 cm) squares.
12. Gauze, 4 Â 4 in., sterile (see Note 5).
13. Surgical instrument pack: one per animal, sterilized and kept
within the same sterile package, containing scalpel blade
holder, iris scissors (4 in./~10.2 cm), tissue forceps 1Â
2 tooth dissecting or Adson-Brown (5½ in./~14 cm), tissue
forceps blunt end (5½ in./~14 cm), and needle holder forceps
with built-in scissors (e.g., Olsen-Hegar, 5½ in./~14 cm) (see
Note 6).
14. 9-mm stainless steel wound closure clips and applicator.
15. Scalpel blades, No. 10.
16. Nonabsorbable suture with taper point needle attached (e.g.,
Ethicon 4-0, SH-1, 27 in. (~68.6 cm) coated VICRYL, violetbraided suture).
17. Biological safety cabinet (Biosafety Level 2; BSL-2).
18. Glass bead sterilizer, optional (see Note 6).
19. Circulating warm water blanket and pump.
20. 18-G, 1.5 in. needles attached to sterile 10 ml syringes.
3 Methods
3.1 Preparation
of Sterile Dialysis
Membrane Tubing
1. Fill three 2-L glass beakers as follows:
Beaker 1: 1–1.5-L ultrapure water.
Beaker 2: 1–1.5-L 1 mM EDTA (pH 8.0).
Beaker 3: 1–1.5-L ultrapure water.
2. Add a magnetic stir bar to each beaker, cover with heavy-duty
aluminum foil, and autoclave.
Cultivation of Leptospira interrogans within DMCs
233