Utility of a modified silver staining technique for detection of Leptospira

Abstract

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 Leptospira in human body fluids.Objectives: To determine the usefulness and sensitivity of a modified Fontana silver staining method for microscopic detection of Leptospira in 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 Leptospira by 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.

Keywords

• leptospira, modified fontana silver stain, urine