9. Disposable 1-L bottle-top vacuum filter with 0.22-μm pore
size membrane.
10. Sterile 90-mm diameter Petri dishes.
11. Water bath (45–50
C).
12. Disposable sterile 0.45 μm and 0.22-μm pore size syringe
filters.
13. Disposable sterile 2-mL pipettes and pipetting device.
14. 5-mL single use sterile syringe.
15. Sterile filtration microtubes.
16. Incubator set at 30
C (with optional shaker).
17. Dark field microscope with 10Â, 20Â, and 40Â objectives.
18. Sterile phosphate buffer saline (10Â PBS): 100 mM Na 2 PO 4 ,
18 mM KH 2 PO 4 , 1.37 M NaCl, 27 mM KCl, pH 7.4.
19. Rabbit serum or fetal calf serum.
20. Glass culture tubes.
3 Methods
Some Leptospira are the etiological agents of leptospirosis, a potentially severe infectious disease. In addition, crude samples (either
clinical or from the environment) may contain other infection
hazards. It is strongly recommended that all culture and isolation
procedures are conducted under a biosafety cabinet in BSL2 conditions. Leptospires are slow-growing, and cultures for isolation
must be kept and checked for up to 4 months.
3.1 Routine Culture
of Leptospira
Various Leptospira strains have different needs in culture. Routine
culture in EMJH is usually performed in aerobic static conditions at
30
C. The growth of some difficult strains may be improved by the
addition of rabbit serum (see Note 8) and/or by shaking the culture
on an orbital shaker in the incubator, because oxygen is frequently a
factor for growth. Routine subculture in liquid medium is usually
performed weekly. A volume of 500 μL to 1 mL of the old culture is
used to inoculate a new 10-mL EMJH culture tube.
3.2 Elimination
of Contaminants
Contaminants easily overgrow Leptospira in culture. Contaminants
can be eliminated chemically by treating cultures with selective
agents. Adding 0.1 volume of 10Â STAFF will prove successful in
killing most contaminants. Contaminants can also be eliminated by
filtration, taking benefit of the small size of leptospires, which will
pass through 0.22-μm pore size filters. First filter through a 0.45-μ
m pore size filter and subculture. If contaminants remain, filter
through a 0.22-μm pore size filter (see Note 9).
4
Cyrille Goarant et al.
size membrane.
10. Sterile 90-mm diameter Petri dishes.
11. Water bath (45–50
C).
12. Disposable sterile 0.45 μm and 0.22-μm pore size syringe
filters.
13. Disposable sterile 2-mL pipettes and pipetting device.
14. 5-mL single use sterile syringe.
15. Sterile filtration microtubes.
16. Incubator set at 30
C (with optional shaker).
17. Dark field microscope with 10Â, 20Â, and 40Â objectives.
18. Sterile phosphate buffer saline (10Â PBS): 100 mM Na 2 PO 4 ,
18 mM KH 2 PO 4 , 1.37 M NaCl, 27 mM KCl, pH 7.4.
19. Rabbit serum or fetal calf serum.
20. Glass culture tubes.
3 Methods
Some Leptospira are the etiological agents of leptospirosis, a potentially severe infectious disease. In addition, crude samples (either
clinical or from the environment) may contain other infection
hazards. It is strongly recommended that all culture and isolation
procedures are conducted under a biosafety cabinet in BSL2 conditions. Leptospires are slow-growing, and cultures for isolation
must be kept and checked for up to 4 months.
3.1 Routine Culture
of Leptospira
Various Leptospira strains have different needs in culture. Routine
culture in EMJH is usually performed in aerobic static conditions at
30
C. The growth of some difficult strains may be improved by the
addition of rabbit serum (see Note 8) and/or by shaking the culture
on an orbital shaker in the incubator, because oxygen is frequently a
factor for growth. Routine subculture in liquid medium is usually
performed weekly. A volume of 500 μL to 1 mL of the old culture is
used to inoculate a new 10-mL EMJH culture tube.
3.2 Elimination
of Contaminants
Contaminants easily overgrow Leptospira in culture. Contaminants
can be eliminated chemically by treating cultures with selective
agents. Adding 0.1 volume of 10Â STAFF will prove successful in
killing most contaminants. Contaminants can also be eliminated by
filtration, taking benefit of the small size of leptospires, which will
pass through 0.22-μm pore size filters. First filter through a 0.45-μ
m pore size filter and subculture. If contaminants remain, filter
through a 0.22-μm pore size filter (see Note 9).
4
Cyrille Goarant et al.