Methods for detection of Leishmania in the scraping of the affected tissues by isolation of pure culture

When sowing scraping the affected tissue of the patient to a specific nutrient medium Leishmania multiply and they can be detected by microscopy.

Reagent

Среда W.W.W. for the cultivation of Leishmania (14 g agar, 6 g sodium chloride, about 150 ml of defibrinated rabbit blood, taken directly from the heart before preparing medium, 900 ml of distilled water). In distilled water was added sodium chloride and agar. The flask containing the medium was heated until complete melting of the agar, and 20-30 min sterilized in a steam sterilizer at 147,1 kPa (15 at).

Cooled to 56 ° C agar added sterile defibrinated rabbit blood, stirred by rotating the flask and filled into sterile pipette 4 ml tubes. Flask blood agar during dispensing is a water bath at a temperature 58 ° C. The tubes with the medium tips.

After solidification of the agar, they are placed in an incubator for a day at 37 ° C for sterility control and receiving the liquid condensate. If it is produced is not enough, before sowing in sterile vials, you can add about 1 One ml of the following solutions: 0,85 % sodium chloride, 1 % peptone solution, Hanks solution, lactalbumin hydrolyzate or other nutrient media in isotonic concentrations.

Methods of detecting Leishmania in scrapings of affected tissues by isolation of pure culture

When you select a culture carefully follow the rules of sterility. The test area of ​​the skin is wiped with a cotton swab, moistened 70 % alcohol.

After evaporation of the alcohol the sharp end of a sterile ophthalmic scalpel incision tubercle length of 2-3 mm. From the bottom of the cut with sterile Pasteur pipette do scraping tissue. The resulting drop of serous-bloody fluid was transferred to a test tube with the environment. Manipulation is carried out several times, sowing material each time a new tube. Ear plugs embedded in paraffin or put them rubber caps and placed in an incubator at 22-24 ° C.

Evaluation of results

Crops are examined under a microscope on a daily basis to identify the causative agent. Explore native drugs, prepared by the method of crushed drops, the lens 40×40. The results are considered negative sowing, if in 40 days pathogen was not found. Cultures are elongated Leishmania (10-12 Microns with wiring).

Application of the culture method is most useful when tuberculoid form of leishmaniasis, diagnostics that other methods may be less effective.