3.6 Culture from
Water
Crude water samples will contain a variety of microorganisms prone
to overgrow Leptospira in culture. It is recommended to systematically use STAFF to favor selective growth of leptospires. Alternatively, water can first be filtered through a 0.45-μm or a 0.22-μm
pore size filter before being used for culture (see Note 13). Early and
frequent observation of cultures is needed, and additional filtration
steps may be needed to separate leptospires from contaminants.
3.7 Culture from Soil
Samples
Direct culture from soil relies on extracting leptospires from soil
particles in water and then using water for culture. Put ca. 2–5 g of
soil in a 15-mL conical tube. Add 5 mL of sterile 1Â PBS. Shake
vigorously. Allow soil particles to settle for 5–15 min. Filter supernatant using a 0.45-μm pore size syringe filter, and take 500 μL of
filtered supernatant to inoculate a 10-mL EMJH tube with STAFF
(see Note 14). Direct inoculation of unfiltered suernatant can also
be used. This procedure is summarized in Fig. 1.
3.8 Isolation of a
Single Colony
To obtain a pure monoclonal isolate, positive liquid cultures must
be plated on EMJH agar. Two hundred microliters of serial tenfold
dilutions are plated on EMJH agar plates. The plates are incubated
at 30
C until leptospiral colonies are visible. To subculture a clonal
isolate, select an isolated individual colony, take a small piece of the
colony using a sterile micropipette tip, and transfer it in an EMJH
culture tube (see Note 15).
4 Notes
1. Because of the very high sensitivity of leptospires to detergents,
some laboratories even use only new glass tubes for EMJH. If
tubes are recycled and washed, it is recommended that they are
rinsed three times with distilled water.
2. Not all bovine serum albumin brands and batches perform
equally for EMJH. Even in the same brand, some batches are
not as good as others. This most probably depends on the
amount of residual lipids in the albumin: the ability of lipids
to form peroxides can lead to toxicity to leptospires.
3. Tween 80 is also named Polysorbate 80. It spontaneously
undergoes autoxidation over time leading to the formation of
peroxides that are toxic to leptospires, so prefer not to stock for
prolonged periods and to store in cool conditions.
4. To freeze bottles of supplement, do not close the cap but cover
with Parafilm until frozen, and then tighten the screw cap.
5. Preparing concentrated 2Â EMJH is an interesting option
allowing to try isolation from a larger volume of water without
dilution of the culture medium. In addition, 2Â EMJH can be
6
Cyrille Goarant et al.
Water
Crude water samples will contain a variety of microorganisms prone
to overgrow Leptospira in culture. It is recommended to systematically use STAFF to favor selective growth of leptospires. Alternatively, water can first be filtered through a 0.45-μm or a 0.22-μm
pore size filter before being used for culture (see Note 13). Early and
frequent observation of cultures is needed, and additional filtration
steps may be needed to separate leptospires from contaminants.
3.7 Culture from Soil
Samples
Direct culture from soil relies on extracting leptospires from soil
particles in water and then using water for culture. Put ca. 2–5 g of
soil in a 15-mL conical tube. Add 5 mL of sterile 1Â PBS. Shake
vigorously. Allow soil particles to settle for 5–15 min. Filter supernatant using a 0.45-μm pore size syringe filter, and take 500 μL of
filtered supernatant to inoculate a 10-mL EMJH tube with STAFF
(see Note 14). Direct inoculation of unfiltered suernatant can also
be used. This procedure is summarized in Fig. 1.
3.8 Isolation of a
Single Colony
To obtain a pure monoclonal isolate, positive liquid cultures must
be plated on EMJH agar. Two hundred microliters of serial tenfold
dilutions are plated on EMJH agar plates. The plates are incubated
at 30
C until leptospiral colonies are visible. To subculture a clonal
isolate, select an isolated individual colony, take a small piece of the
colony using a sterile micropipette tip, and transfer it in an EMJH
culture tube (see Note 15).
4 Notes
1. Because of the very high sensitivity of leptospires to detergents,
some laboratories even use only new glass tubes for EMJH. If
tubes are recycled and washed, it is recommended that they are
rinsed three times with distilled water.
2. Not all bovine serum albumin brands and batches perform
equally for EMJH. Even in the same brand, some batches are
not as good as others. This most probably depends on the
amount of residual lipids in the albumin: the ability of lipids
to form peroxides can lead to toxicity to leptospires.
3. Tween 80 is also named Polysorbate 80. It spontaneously
undergoes autoxidation over time leading to the formation of
peroxides that are toxic to leptospires, so prefer not to stock for
prolonged periods and to store in cool conditions.
4. To freeze bottles of supplement, do not close the cap but cover
with Parafilm until frozen, and then tighten the screw cap.
5. Preparing concentrated 2Â EMJH is an interesting option
allowing to try isolation from a larger volume of water without
dilution of the culture medium. In addition, 2Â EMJH can be
6
Cyrille Goarant et al.