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Background: Leptospira are spiral thin and highly motile pathogenic bacteria that are best
visualized by dark ground microscopy. Although these bacteria are not stained by the Gram’s
stain, the Fontana stain, which is a silver impregnation staining method, can be used
successfully for light microscopy. It is important to investigate the usefulness of Fontana
stain method for direct demonstration of Leptospirain human body fluids.
Objectives: To determine the usefulness and sensitivity of a modified Fontana silver staining
method for microscopic detection of Leptospirain clinical specimens.
Methodology: 6×108 organisms/ml of Leptospira interrogans serovar Icterohaemorrhagiae
and Canicola were spiked into PBS (Phosphate Buffered Saline), alkalinized urine and serum
in triplicate and serial dilutions were made (6×106
to 6×101 organisms/ml). Smears were
prepared using 10 µl of each dilution. In addition, centrifuged sediment of urine were also
used to prepare smears. Slides were stained by modified Fontana method as reported by
Gangadhar et al.(1998) and examined. Numbers of leptospires per field (×100) were
recorded.
Results: Leptospira spiked in PBS and urine appeared as thin slender bacteria with
characteristic hooked ends after Fontana staining under the light microscope. Serum could
not be used for the detection of Leptospiraby this method. Leptospires could be detected by
staining the spiked PBS and urine at 6x103
– 6x106
organisms/ml.
Conclusion: Leptospires could be detected by Fontana staining in spiked PBS, urine
(uncentrifuged and sediment). Serum was not suitable for detection of leptospires by Fontana
staining. The detection limit of leptospires in PBS and urine by Fontana stain was found to be
6000 organisms/ml.
