Isolation of Leptospira
Author: Miladin Kostovic

ISOLATION OF LEPTOSPIRES

The isolation of leptospires depends on the choice of the material and the stage of infection.

During the leptospiraemic phase (days 1 to 10 or so from the onset of illness) the materials are blood, vascular organs (liver, spleen and kidneys) and CSF.

BLOOD

Blood cultures should be done in the first 10 days of the illness and before antibiotics are given. Venous blood is collected by aseptic technique and inoculated at the bedside. Small inocula of one or more drops of blood are inoculated into several tubes, each containing 5 ml of suitable medium. Large inocula will inhibit the growth of leptospires.

CSF

Leptospires may be observed and isolated from cerebrospinal fluid during the end of the first week of illness. However, this procedure is not usually performed.
During the phase of leptospiruria and increasing concentrations of antibodies (after about 1 week from onset) the renal cortex and the urine are the most suitable inocula for isolation of leptospires from man but during the carrier state also to wild and domestic animals.

URINE

Fresh midstream urine is collected and inoculated immediately. One drop of undiluted urine is inoculated into the first tube containing 5 ml of medium. Some more tubes are similarly inoculated with urine in increasing 10-fold dilution. Urine should be examined within 2 hours after voiding, using culture or animal inoculation.
Media containing antibiotics are essential to reduce contamination of urine cultures.

POST MORTEM MATERIAL

In fatal cases of human and animal leptospirosis, the organisms may be seen and cultured from ground post-mortem specimens of tissues: liver, kidneys and brain are the tissues most suitable. Leptospires may also be successfully isolated from aborted animal fetuses in this manner; the tissues should not e frozen.

The isolation of leptospires is not only dependent on numbers of organisms but also on numbers of viable organisms dependent. For instance, post mortem changes can rapidly reduce the number of viable organisms. This reduction in numbers is also temperature dependent: leptospires survive well at 4C but are rapidly killed in tissues held at 20C never mind the 30 to 40C which dead fetuses are held at before being expelled by their dams.

Culturing is very time consuming and labor intensive: isolation can take up to six months to achieve and involves the use of multiple tubes of media containing 2-4 different dilutions of tissue and several different media for each dilution, all of which have to be examined by dark field microscopy at least ever two weeks with perhaps the additional problem of passages having to be made before success is achieved.

The degree of success achieved in isolating leptospires is also dependent on how the culture tubes inoculated with tissue are manipulated. It is not sufficient to merely leave them and read at regular intervals.

Culture positive with least manipulations:
- Original tube
- Original tube topped up with fresh medium
- After subculturing

The length of time for which cultures are kept also effect the degree of success achieved in isolating leptospires. Some investigators investigate their cultures for a period of 26 weeks.

Culture tubes are inoculated from 1:10, 1:100 and 1:1000 dilution. The amount of inoculum given to each tube ranges from 100 to 250 ul.

If the operator wishes to cut down on the number of tubes used, this should be done by only inoculating from 1:10 and 1:100 dilution.