Another strategy is to inoculate EMJH agar plates, where
Leptospira colonies might be larger than contaminants’ allowing
to separate leptospires from contaminants after prolonged
incubation.
3.3 Culture from
Blood or
Cerebrospinal Fluid
Aseptically add 0.5–1 mL of whole fresh (see Note 10) blood
collected on a heparin tube in a 10-mL EMJH culture tube
named R1, and gently homogenize. Then take 1 mL to add in a
new 10-mL EMJH culture tube named R2. Similarly do two additional serial dilutions leading to R3 and R4 culture tubes (blood can
be inhibitory and dilutions are important). All four tubes must then
be incubated at 30
C aerobically (cap not tightened) in the dark for
up to 18 weeks (see Note 11). Check daily during the first 2 weeks,
then weekly for Leptospira growth by placing a drop of culture
medium on a microscope glass slide with the dark-field microscope
at 100Â (direct observation in the drop), and 200Â and/or 400Â
(with a glass coverslip) magnification. Alternatively, using fresh
serum or plasma, up to ten drops can be seeded in a 10-mL
EMJH tube. After gentle homogenization, 1 mL is transferred to
a new 10-mL EMJH tube. Both tubes are also incubated at 30
C
aerobically. Cerebrospinal fluid can be treated identically.
3.4 Culture from
Urine
Fresh urine can be used directly to inoculate EMJH culture tubes.
A volume of 500 μL of urine is usually used for this purpose (see
Note 12). If not cultured immediately, urine pH can be buffered by
adding 0.1 volume of 10Â sterile PBS. Voided urine is prone to
contamination and should be refrigerated if not cultured immediately. Cultures must be checked early and frequently. The addition
of 0.1 volume of 10Â STAFF in the culture might prove useful to
control contaminants to overgrow leptospires. If collected aseptically from the bladder, urine culture is less prone to contamination.
3.5 Culture from
Organs
In the acute stage of the infection, leptospires might be cultured
from any organ. However, most frequently the liver or kidneys will
be used as material to initiate Leptospira culture. In animals, fetus
organs, placenta, or abortion products can also be used. In kidneys,
because leptospires are located in the glomeruli and the proximal
renal tubule, a tiny amount (e.g., a thin slice cut with a sterile scalpel)
of cortical kidney tissue can be placed directly in a 10-mL EMJH
tube. Alternatively, up to 1 g of tissue can be macerated or ground
using a sterile pellet pestle and then used to inoculate serial dilutions
of EMJH. After a few days, 1 mL of the initial tube can be transferred
into a second EMJH culture tube, because tissue degradation products can be inhibitory. When organs are collected postmortem, the
organ capsule can be disinfected with 70% ethanol (for 30–60 s
before rinsing with sterile water for another 30–60 s) before a small
sample is taken. Diluted subcultures are also recommended.
Leptospira Isolation and Culture
5
Leptospira colonies might be larger than contaminants’ allowing
to separate leptospires from contaminants after prolonged
incubation.
3.3 Culture from
Blood or
Cerebrospinal Fluid
Aseptically add 0.5–1 mL of whole fresh (see Note 10) blood
collected on a heparin tube in a 10-mL EMJH culture tube
named R1, and gently homogenize. Then take 1 mL to add in a
new 10-mL EMJH culture tube named R2. Similarly do two additional serial dilutions leading to R3 and R4 culture tubes (blood can
be inhibitory and dilutions are important). All four tubes must then
be incubated at 30
C aerobically (cap not tightened) in the dark for
up to 18 weeks (see Note 11). Check daily during the first 2 weeks,
then weekly for Leptospira growth by placing a drop of culture
medium on a microscope glass slide with the dark-field microscope
at 100Â (direct observation in the drop), and 200Â and/or 400Â
(with a glass coverslip) magnification. Alternatively, using fresh
serum or plasma, up to ten drops can be seeded in a 10-mL
EMJH tube. After gentle homogenization, 1 mL is transferred to
a new 10-mL EMJH tube. Both tubes are also incubated at 30
C
aerobically. Cerebrospinal fluid can be treated identically.
3.4 Culture from
Urine
Fresh urine can be used directly to inoculate EMJH culture tubes.
A volume of 500 μL of urine is usually used for this purpose (see
Note 12). If not cultured immediately, urine pH can be buffered by
adding 0.1 volume of 10Â sterile PBS. Voided urine is prone to
contamination and should be refrigerated if not cultured immediately. Cultures must be checked early and frequently. The addition
of 0.1 volume of 10Â STAFF in the culture might prove useful to
control contaminants to overgrow leptospires. If collected aseptically from the bladder, urine culture is less prone to contamination.
3.5 Culture from
Organs
In the acute stage of the infection, leptospires might be cultured
from any organ. However, most frequently the liver or kidneys will
be used as material to initiate Leptospira culture. In animals, fetus
organs, placenta, or abortion products can also be used. In kidneys,
because leptospires are located in the glomeruli and the proximal
renal tubule, a tiny amount (e.g., a thin slice cut with a sterile scalpel)
of cortical kidney tissue can be placed directly in a 10-mL EMJH
tube. Alternatively, up to 1 g of tissue can be macerated or ground
using a sterile pellet pestle and then used to inoculate serial dilutions
of EMJH. After a few days, 1 mL of the initial tube can be transferred
into a second EMJH culture tube, because tissue degradation products can be inhibitory. When organs are collected postmortem, the
organ capsule can be disinfected with 70% ethanol (for 30–60 s
before rinsing with sterile water for another 30–60 s) before a small
sample is taken. Diluted subcultures are also recommended.
Leptospira Isolation and Culture
5