Genus leshmania Culture parasitology

CULTURE TECHNIQUE FOR THE DIAGNOSES OF LESHMANIA :-

1. LESHMANIA CULTURE TECHNIQUE A J Zaahir Bsc MLT

2. Presentation Topics CULTURE TECHNIQUE FOR THE DIAGNOSES OF LESHMANIA :- It`s purpose Nam of the media Growth and requirement Result reporting and interpretation The advantages and disadvantages of media

3. Leishmania Leishmania is the genus that are responsible for the disease leishmaniasis . They are spread by sandflies of the genus Phlebotomus in the Old World, and of the genus Lutzomyia in the New World.

4. NAME OF THE MEDIUM Novy - MacNeal -Nicolle medium (NNN) is a culture medium used to grow Leishmania – needed when the amastigotes are not found by scraping. It consists of 0.6% sodium chloride ( NaCl ) added to a simple blood agar slope . NNN can also be used to grow Trypanosoma cruzi .

5. Growth and requirement Commercial suppliers supply the ready to use NNN medium which you have to purchase , The constituents/ingredients of the media are : - NEXT SLIDE

6. Part A ( ingredients gms / litre) Meat extracts 3.000 Peptic digest of animal tissue 5.000 Sodium chloride 8.000 Agar 15.000 Final pH ( at 25°C) 7.3 ± 0.2

7. Part B ( ingredients gms / litre) Sodium chloride 8.000 Potassium chloride 0.200 Calcium chloride 0.200 Monopotassium dihydrogen phosphate 0.300 Dextrose 2.500 Final pH ( at 25°C) 7.0 ± 0.2

8. Storage Keep the prepared media refrigerated until it is used ( stable for 2-4 weeks ) and bring it to room temperature right before inoculation.

9. Inoculation Inoculate the specimen  (spleen aspiration or bone marrow biopsy or lymph node aspiration) into  the liquid phase of the biphasic medium. Incubate at  21-26°C  for 4 weeks. Check for the growth of the organism and for possible fungal contamination after 4 days and continue by 4 days intervals for  next 4 weeks.

10. Leishmania donovani Promastigotes in Culture

11. Result and interpretation If positive, luxuriant growth of the organism is seen in the media. Giemsa stain the culture filtrate and observe under microscope; free, flagellated, Leishmania promastigote .

12. ADVANTAGES Effective Inexpensive Successful Effective tools to identify positive in cases where the patient shows evident clinical signs and signs compatible with the disease.

13. DISADVANTAGES Parasite culture in tubes containing Novy - MacNeal -Nicolle medium from clinical samples is difficult , requires significant technical expertise , is prone to contamination , and is time-consuming. The sensitivity of culture also tends to be low and highly variable