This protocol is adapted from that detailed in the International Course on Laboratory methods for the Diagnosis of Leptospirosis, by Dr. R.A. Hartskeerl, Dr. H.L. Smits, Mr. H. Korver, Dr. M.G.A. Goris and Dr. W.J. Terpstra from the Leptospirosis Reference Center in Amsterdam, the Netherlands. It is routinely used at UMR PIMIT, St Denis de La R\u00e9union, France, for the isolation of pathogenic Leptospira from wild animals (terrestrial small mammals, bats and dogs). For Leptospira isolation from urine, go directly to step 10.