methods used to cultivate and isolate leptospires from clinical and
environmental samples.
2 Materials
Prepare all solutions and media using ultrapure medical grade
water, notably keeping in mind that leptospires might be present
in any crude water source and able to pass through 0.2-μm pore size
filters. Thoroughly rinse all glassware after cleaning and before
autoclaving, also keeping in mind that leptospires are highly sensitive to detergents, which would kill them even in residual trace
amounts (see Note 1).
1. EMJH supplement (Table 1): Dissolve 100 g of bovine serum
albumin (BSA) in 600 mL of ultrapure sterile water in a 2-L
beaker on a magnetic stirrer (heated at 37
C). Stir gently to
avoid foaming (see Note 2). Add 10 mL of 0.4% ZnSO 4 , 10 mL
of 1.5% MgCl 2 , 10 mL of 1.5% CaCl 2 , 10 mL of 10% sodium
pyruvate, 20 mL of 20% (w/v) glycerol solution, 125 mL of
9.3% Tween 80 (11.58 mL of Tween 80 in 113.42 mL ultrapure water, see Note 3), two vials of vitamin B12 (cyanocobalamin vials at 1000 μg/2 mL), 1 mL of 0.3% CuSO 4 , and
100 mL of 0.5% FeSO 4 . Add 110-mL ultrapure sterile water
making a total volume of 1000 mL of supplement. Adjust to
pH ¼ 7.4 using 10 N NaOH or 4 N HCl. Solutions of ZnSO 4 ,
MgCl 2 , CaCl 2 , CuSO 4 , sodium pyruvate, and glycerol are dissolved in ultrapure sterile water. These solutions can be autoclaved for 20 min at 110
C and kept for 1 year at 4
C. BSA
and Tween 80 solutions must be prepared the day when the
medium will be produced. FeSO 4 solution must be prepared
not more than 30 min prior to be included in the EMJH
supplement by dissolving 0.5 g of iron sulfate in 100-mL
ultrapure sterile water in the dark. Tween must be added to
water very slowly and progressively on a magnetic stirrer. This
supplement can be aliquoted in 100-mL sterile vials, each
usable to make 1 L of EMJH. When aliquoted it can be stored
frozen at À18
C for 1 year (see Note 4).
2. EMJH base: Dissolve 2.3 g of Difco Leptospira Medium Base
EMJH (Becton Dickinson) in 900 mL of ultrapure sterile
water. Adjust pH to 7.4 if needed.
3. EMJH base (2Â): Dissolve 2.3 g of Difco Leptospira Medium
Base EMJH in 400 mL of ultrapure sterile water (see Note 5).
4. EMJH medium: 900 mL of EMJH base, 100 mL of EMJH
supplement. Using a disposable filtration device, filter at
0.22 μm before dispensing aseptically in sterile culture tubes
(see Note 6). The amount of EMJH must be adapted to the
tube, with a maximal depth of 5 cm and an air/EMJH ratio of
2
Cyrille Goarant et al.
environmental samples.
2 Materials
Prepare all solutions and media using ultrapure medical grade
water, notably keeping in mind that leptospires might be present
in any crude water source and able to pass through 0.2-μm pore size
filters. Thoroughly rinse all glassware after cleaning and before
autoclaving, also keeping in mind that leptospires are highly sensitive to detergents, which would kill them even in residual trace
amounts (see Note 1).
1. EMJH supplement (Table 1): Dissolve 100 g of bovine serum
albumin (BSA) in 600 mL of ultrapure sterile water in a 2-L
beaker on a magnetic stirrer (heated at 37
C). Stir gently to
avoid foaming (see Note 2). Add 10 mL of 0.4% ZnSO 4 , 10 mL
of 1.5% MgCl 2 , 10 mL of 1.5% CaCl 2 , 10 mL of 10% sodium
pyruvate, 20 mL of 20% (w/v) glycerol solution, 125 mL of
9.3% Tween 80 (11.58 mL of Tween 80 in 113.42 mL ultrapure water, see Note 3), two vials of vitamin B12 (cyanocobalamin vials at 1000 μg/2 mL), 1 mL of 0.3% CuSO 4 , and
100 mL of 0.5% FeSO 4 . Add 110-mL ultrapure sterile water
making a total volume of 1000 mL of supplement. Adjust to
pH ¼ 7.4 using 10 N NaOH or 4 N HCl. Solutions of ZnSO 4 ,
MgCl 2 , CaCl 2 , CuSO 4 , sodium pyruvate, and glycerol are dissolved in ultrapure sterile water. These solutions can be autoclaved for 20 min at 110
C and kept for 1 year at 4
C. BSA
and Tween 80 solutions must be prepared the day when the
medium will be produced. FeSO 4 solution must be prepared
not more than 30 min prior to be included in the EMJH
supplement by dissolving 0.5 g of iron sulfate in 100-mL
ultrapure sterile water in the dark. Tween must be added to
water very slowly and progressively on a magnetic stirrer. This
supplement can be aliquoted in 100-mL sterile vials, each
usable to make 1 L of EMJH. When aliquoted it can be stored
frozen at À18
C for 1 year (see Note 4).
2. EMJH base: Dissolve 2.3 g of Difco Leptospira Medium Base
EMJH (Becton Dickinson) in 900 mL of ultrapure sterile
water. Adjust pH to 7.4 if needed.
3. EMJH base (2Â): Dissolve 2.3 g of Difco Leptospira Medium
Base EMJH in 400 mL of ultrapure sterile water (see Note 5).
4. EMJH medium: 900 mL of EMJH base, 100 mL of EMJH
supplement. Using a disposable filtration device, filter at
0.22 μm before dispensing aseptically in sterile culture tubes
(see Note 6). The amount of EMJH must be adapted to the
tube, with a maximal depth of 5 cm and an air/EMJH ratio of
2
Cyrille Goarant et al.