| Summary: | The successful culture of <i>Leptospira</i> spp. from the environment is challenging. Here, we optimized the isolation of <i>Leptospira</i> spp. from water samples spiked with different species and initial concentrations of this organism. The time periods between water sampling and the isolation process were varied (0, 2, and 4 weeks). Bacterial cultures were observed under a microscope, and cultures were graded for cell density, weekly, for 12 weeks. Most pathogenic <i>Leptospira</i> spp. were difficult to culture under all conditions. All conditions of water samples spiked with novel species of <i>Leptospira</i> subclade P1 were culture positive within 2 weeks. For <i>Leptospira</i> subclade P2, storing samples for 2 weeks prior to isolation resulted in more successful isolation compared with isolation after other storage conditions. For subclade S1, all samples with initial bacterial concentrations of more than 10<sup>3</sup> colonies/mL, under all storage conditions, were successfully cultured. These results suggest that storing contaminated water samples for 2 to 4 weeks in the dark at an ambient temperature prior to culturing can improve the isolation of <i>Leptospira</i> spp. from the samples. We implemented this protocol and collected water samples from natural sources accessed by both humans and animals. <i>Leptospira</i> spp. was identified in 32% (35/109) of water samples. The animal species using a water source influenced the likelihood of water samples being contaminated with <i>Leptospira</i> spp. Cultures of <i>Leptospira</i> spp. from environmental samples can provide useful information for understanding the complex interactions between humans, animals and the environment in the transmission of leptospirosis.
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