Microscopic examination of the centrifuged urine using dark-field illumination is considered to be a convenient and rapid diagnostic test. Midstream urine sample is collected in a sterile bottle in a very hygienic condition. It has been reported that the rate of destruction of leptospires by urine and products of tissue autolysis is more rapid during storage at 20°C than 4°C (Miller et al., 1990). So all clinical samples including urine should be placed in an ice-container in the dark, and transported to the laboratory as soon as possible.
However, dark-field microscopic (DFM) examination is insensitive and requires a skilled observer to differentiate leptospires from artefacts. It should be considered that leptospires are very fragile and sensitive to the pH of the urine, therefore, for maximum efficiency, one-half of each urine sample should be submitted with added formalin (1 drop to 20-30 mL of urine) and the other half submitted in the fresh state. The formalin prevents bacterial overgrowth and the fresh urine sample may be used for culture.