Isolation of Leptospira
Author: Miladin Kostovic

INDIRECT ISOLATION OF LEPTOSPIRES

Indirect isolation of leptospires involves the use of laboratory animals. Their efficiency varies according to their susceptibility and depends upon the leptospiral serotype and its relative pathogenicity.

Animal inoculation: The animals used for inoculation are:

- guinea pigs (150-175 grams)
- hamsters (4-6 weeks old)

The inoculation of blood and/or urine is done by intraperitoneal way (0.5-1.0 ml).

From the third day after inoculation, peritoneal fluid is examined with a dark-field microscope for the presence of leptospires. The will of a lower quadrant of the abdomen is punctured with a capillary pipette.

Peritoneal fluid rises by capillary action in the pipette and a drop is discharged on a slide, and examined under a cover slip.

This examination should be done during 6 days (= 3-10 days after inoculation).

When the inoculated animals have died, it is possible to get a sample of the liver and kidneys. These samples are used for cultures in various kinds of medium.

Therefore, a part of the liver or kidney is put in a morter, together with PBS pH 7.2 or medium, ground and a suspension is inoculated into media. Another way of tissue handling is to use sterile syringes without a needle and to push the material through the opening at the end of the syringe.