4 to maintain aerobic conditions during culture. Incubate
tubes at 37
C in the dark for 48–72 h, and then visually inspect
for possible bacterial growth to check for sterility. Tubes must
then be stored in the dark at 4
C and should be used within
less than 2 months.
5. Two times concentrated EMJH medium: 400 mL of
2Â EMJH base, 100 mL of EMJH supplement. Similarly filter
through a 0.22 μm filter.
6. Agar Noble solution: Mix 2.4 g of Agar Noble with 100 mL of
ultrapure sterile water and autoclave at 121
C for 15 min.
7. Semisolid EMJH medium: 100 mL of Agar Noble solution,
100 mL of 2Â concentrated EMJH medium. Two times concentrated EMJH medium is preheated and Agar Noble solution
is kept at 45
C. Dispense into Petri dishes; a volume of
20–25 mL is needed for each plate (to be used within
2–3 weeks). Petri dishes can be sealed with Parafilm to minimize
dehydration during storage at 4
C and incubation at 30
C.
8. STAFF (10Â): 1 mL of 4 mg/mL sulfamethoxazole, 200 μL of
10 mg/mL trimethoprim, 5 μL of 100 mg/mL amphotericin B,
1 mL of 40 mg/mL fosfomycin, and 500 μL of 20 mg/mL
solution of 5-fluorouracil (see Note 7). Add water to a final
volume of 10 mL. Filter-sterilize through a 0.22-μm pore size
filter. Aliquots can be kept frozen at À20 or À80
C for months.
Table 1
Composition of EMJH
Zinc sulfate (ZnSO 4 ) 0.4 g in 100-mL ultrapure sterile water
Supplement
Magnesium chloride
(MgCl 2 )
1.5 g in 100-mL ultrapure sterile water
Supplement
Calcium chloride
(CaCl 2 )
1.5 g in 100-mL ultrapure sterile water
Supplement
Copper sulfate
(CuSO 4 )
0.3 g in 100-mL ultrapure sterile water
Supplement
Sodium pyruvate
10 g in 100-mL ultrapure sterile water
Supplement
Glycerol
3.18 mL in 16.82-mL ultrapure sterile water or 20 g in a total
volume of 100 mL
Supplement
Tween 80
11.58-mL Tween 80 in 113.42-mL ultrapure water
Supplement
Bovine albumin
100-g bovine albumin in 600-mL ultrapure sterile water
Supplement
Iron (II) sulfate
(FeSO 4 )
0.5-g iron (II) sulfate in 100-mL ultrapure sterile water in the dark
(extemporaneous)
Supplement
Leptospira medium
Base
2.3 g per liter of 1Â EMJH (contains 100-mL supplement)
EMJH
medium
Leptospira Isolation and Culture
3