Leptospira spp. culturing
Kidney samples from 20 euthanized brown rats were collected aseptically
for Leptospira culturing. Isolation of leptospires was performed
as previously described (Faine et al., 1999). Briefly, samples were
macerated and diluted 1:10 in phosphate-buffered saline (PBS, pH 7.4;
NaCl 0.137 M; KCl 0.0027 M; Na2HPO4 0.01 M; KH2PO4 0.0018 M/L), then 1
mL of the diluted samples was seeded into 9 mL of liquid EMJH medium
enriched with a selective supplement (Nalidixic acid, 50 mg/L;
Cycloheximide, 100 mg/L; Chloramphenicol, 5 mg/L and Neomycin, 5 mg/L).
Cultures were incubated at 30 ºC in for a period of 12 to 24 h and then
seeded in the semi-solid EMJH medium without the selective supplement.
The tubes were incubated at 28 °C for 12 weeks and examined weekly by
dark-field microscopy to evaluate the presence of spirochetes.