Utility of a modified silver staining technique for detection of Leptospira

• Main Authors: C. P. Gunasekara, M. H. F. Sumaiha, M. K. S. Damayanthi, M. M. Weerasekera, S. S. N. Fernando
• Format: Article
• Language: English
• Published: Sri Lankan Society for Microbiology 2017-10-01
• Series: Sri Lankan Journal of Infectious Diseases
• Subjects: leptospira, modified fontana silver stain, urine
• Online Access: https://sljid.sljol.info/articles/8142
• ISSN: 2012-8169; 2448-9654

<p><strong>Background:</strong><em> Leptospira</em> 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 <em>Leptospira</em> in human body fluids.</p><p><strong><br /></strong></p><p><strong>Objectives:</strong> To determine the usefulness and sensitivity of a modified Fontana silver staining method for microscopic detection of <em>Leptospira</em> in clinical specimens.<strong></strong></p><p><strong><br /></strong></p><p><strong>Methodology:</strong> 6×10⁸ organisms/ml of <em>Leptospira interrogans</em> 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.</p><p><strong><br /></strong></p><p><strong>Results:</strong> <em>Leptospira</em> 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 <em>Leptospira</em> by this method.<em> Leptospires</em> could be detected by staining the spiked PBS and urine at 6x10³ – 6x10⁶ organisms/ml.</p><p><strong><br /></strong></p><p><strong>Conclusion:</strong> <em>Leptospires</em> 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.</p>