Fletcher Leptospira HiVeg™ Medium Base (Leptospira HiVeg™ Medium Base, Fletcher)

Principle And Interpretation

The leptospires includes both free-living and parasite forms. Pathogenic species are called Leptospira interrogans. The organisms are too small to be observed in wet preparations made from fresh blood. The most reliable method for laboratory diagnosis of leptospirosis is to cultivate the organism from blood or cerebrospinal fluid during the first week of illness or from urine thereafter for several months. A few drops of heparinized or sodium chloride anticoagulated blood are inoculated into Fletchers Medium. Fletcher Leptospira Medium Base is prepared according to the formulation of Fletcher (1) and used for isolation of Leptospira from blood, urine and kidney specimens (2).

All cultures are incubated at room temperature in the dark for up to 6 weeks. The organisms grow below the surface of the medium. Material collected from a few centimeters below the surface of broth cultures should be examined weekly for the presence of growth using a direct wet preparation under dark field illumination. Leptospires will exhibit corkscrew like motility (3). The medium contains HiVeg™™ peptone and HiVeg™™ extract, which provide the necessary nutrients required for bacterial growth. Sodium chloride provides essential ions. The enrichment supplement provides carbon, vitamins and energy sources required for Leptospira growth. A low concentration of agar helps in detecting motility. Fletcher Leptospira HiVeg™™ Medium Base is prepared by using vegetable peptones in place of animal based peptones which make the media free of BSE/TSE risks. Leptospira Medium Base is enriched with addition of rabbit serum. Rabbit serum contains native haemoglobin, which along with thiamine helps in the Leptospiral growth.

Examine the tubes for growth every 5-7 days. Growth occurs as a ringed area (disk) 1-3 cm below the surface of the medium. The absences of ringed area of growth doesnt necessarily mean leptospires are not present. Remove a small amount of growth from the disk area and examine microscopically (gram stain is not satisfactory). Microcolonies can be fixed with methanol and stained with Giemsa stain to show rod forms.