stored for up to 2 months and used when needed to rapidly
prepare EMJH agar plates.
6. Because EMJH is an expensive culture medium, it can be
relevant to prepare both small volume tubes (3- or 4-mL) and
larger volumes (10-mL culture tubes or 20-mL culture flasks)
to adjust to the needs at best cost.
7. Appropriate solvents must be used for each component because
many of these molecules are not water soluble.
8. In our experience, cell culture tested fetal calf serum (FCS) also
improves growth of difficult Leptospira strains. Because FCS is
more frequently available in most laboratories, it provides an
interesting alternative to rabbit serum.
9. Only dense Leptospira cultures should be filtered through
0.22-μm pore size filters, since most leptospires will also be
retained in the filter. An alternative is to let leptospires swim
actively through a 0.22-μm pore size filter, e.g., using microfiltration columns without centrifugation.
10. Blood samples to be used for Leptospira culture must not be
refrigerated. It must be either inoculated as soon as possible or
kept at ambient temperature until used for Leptospira culture.
Leptospira isolation may be attempted from the fresh or
non-refrigerated blood of any mammal during the acute
phase of the disease, mostly during the first week of symptoms
in an acute illness. Growth is most frequently detected in tubes
R3 and/or R4. It may be obtained in R3 but not R4 if the
initial concentration was very low, but also in some cases in R4
but not R3, for example if patient was treated with antibiotics
just before blood collection.
11. Successful isolation of leptospires was also achieved from blood
seeded in BacT/Alert culture bottles for a few days [12].
12. As for blood, if the culture is not initiated promptly, the urine
sample should be kept at ambient temperature if collected
aseptically from the bladder. Leptospires can be cultured from
the urine of sick humans or animals during the second week of
symptoms, but also in chronically infected carrier animals (reservoir animals).
13. The use of 2Â EMJH will allow to initiate culture from a
significant volume of water (e.g., 2 mL of water in 2.5 mL of
2Â EMJH and addition of 500 μL of 10Â STAFF).
14. Alternatively, a larger volume of supernatant can be inoculated
in 2Â EMJH as done in a recent study [11].
15. When working from material possibly containing multiple Leptospira strains (notably water or soil samples), several colonies
might be isolated from Petri dishes [11].
8
Cyrille Goarant et al.
prepare EMJH agar plates.
6. Because EMJH is an expensive culture medium, it can be
relevant to prepare both small volume tubes (3- or 4-mL) and
larger volumes (10-mL culture tubes or 20-mL culture flasks)
to adjust to the needs at best cost.
7. Appropriate solvents must be used for each component because
many of these molecules are not water soluble.
8. In our experience, cell culture tested fetal calf serum (FCS) also
improves growth of difficult Leptospira strains. Because FCS is
more frequently available in most laboratories, it provides an
interesting alternative to rabbit serum.
9. Only dense Leptospira cultures should be filtered through
0.22-μm pore size filters, since most leptospires will also be
retained in the filter. An alternative is to let leptospires swim
actively through a 0.22-μm pore size filter, e.g., using microfiltration columns without centrifugation.
10. Blood samples to be used for Leptospira culture must not be
refrigerated. It must be either inoculated as soon as possible or
kept at ambient temperature until used for Leptospira culture.
Leptospira isolation may be attempted from the fresh or
non-refrigerated blood of any mammal during the acute
phase of the disease, mostly during the first week of symptoms
in an acute illness. Growth is most frequently detected in tubes
R3 and/or R4. It may be obtained in R3 but not R4 if the
initial concentration was very low, but also in some cases in R4
but not R3, for example if patient was treated with antibiotics
just before blood collection.
11. Successful isolation of leptospires was also achieved from blood
seeded in BacT/Alert culture bottles for a few days [12].
12. As for blood, if the culture is not initiated promptly, the urine
sample should be kept at ambient temperature if collected
aseptically from the bladder. Leptospires can be cultured from
the urine of sick humans or animals during the second week of
symptoms, but also in chronically infected carrier animals (reservoir animals).
13. The use of 2Â EMJH will allow to initiate culture from a
significant volume of water (e.g., 2 mL of water in 2.5 mL of
2Â EMJH and addition of 500 μL of 10Â STAFF).
14. Alternatively, a larger volume of supernatant can be inoculated
in 2Â EMJH as done in a recent study [11].
15. When working from material possibly containing multiple Leptospira strains (notably water or soil samples), several colonies
might be isolated from Petri dishes [11].
8
Cyrille Goarant et al.