Yaeger’s LIT Medium (for Chagas’ Disease) (33)
1. Add all the ingredients to the distilled water, and mix well with a magnetic stirrer until dissolved. Heat, if necessary, to dissolve all the ingredients.
2. Using a Whatman no. 42 filter paper in a Büchner funnel, filter with suction. Repeat this filtration one more time.
3. Adjust pH to 7.2 with 1 N NaOH or 1 N HCl.
4. Sterilize by filtration through a 0.22-µm-pore-size membrane filter.
5. Dispense 4.5 ml into each tube.
6. Label as LIT medium, with the date of preparation and an expiration date of 1 month.
Hemin Stock Solution
1. Mix triethanolamine with water, add the mixture to a tube containing hemin, shake well, and let dissolve.
2. To make the complete medium, just before inoculation, add 0.5 ml of inactivated fetal bovine serum and 0.25 ml of antibiotic solution. The final concentrations of the antibiotics are 100 U of penicillin per ml, 100 µg of streptomycin per ml, and 0.2 µg of Fungizone per ml.
USAMRU Blood Agar Medium for Leishmaniae (36)
1. Dissolve 40 g of agar in 1,000 ml of distilled water by heating.
2. Cool the solution, and add 150 ml of defibrinated rabbit blood.
3. Dispense 5-ml portions into 125-by-16-mm screw-cap tubes. Position the tubes at a slant, and allow to cool, preferably on ice, to produce moisture of condensation in the tubes.
4. Incubate the tubes at 35°C for 24 h to ensure sterility. Antibiotics (penicillin and streptomycin) can be added if necessary (see the antibiotic formula in this chapter). The final concentrations of antibiotics are 100 U of penicillin per ml and 100 µg of streptomycin per ml.
5. Inoculate the medium with aspirate material or triturated tissue from biopsy specimen.
Note This medium has been especially useful for primary isolation of the Leishmania braziliensis complex in Latin America.
Liquid Media for Cultivation of Hemoflagellates
Hendricks et al. have reported on the highly successful use of liquid culture media for the rapid cultivation of various species of Leishmania and Trypanosoma (33). Schneider’s Drosophila medium (GIBCO, Grand Island, NY), supplemented with 30% (vol/vol) fetal calf serum, has been used in making primary isolates from human and animal infections as well as for routine maintenance of a wide variety of leishmanial and trypanosomal species in the laboratory. Both Schneider’s medium and Grace’s insect tissue culture medium (GIBCO) promote better growth of organisms and are less costly than the widely used blood-agar-based media. In addition, Schneider’s medium may be freeze-dried for at least 2 years and reconstituted with distilled water in the field, and it will provide excellent culture results.
Stock Antibiotic Solution (To Be Used with All Media)
1. Mix the components thoroughly. The concentration of the stock solution is:
2. Dispense 1 ml of the antibiotic mixture into sterile screw-cap vials or sterile cryovials.
3. Label as antibiotic solution with the date of preparation and an expiration date of 1 year.
4. Store at −20°C in cryoboxes.
Axenic Culture
1. Remove tubes containing culture medium (one of NNN, modified NNN, or Tobie’s medium and one of Schneider’s for Leishmania; one of NNN or Tobie’s medium and one of LIT for T. cruzi) from 4°C, add fetal bovine serum and antibiotics if required, and incubate at 20 to 23°C for 1 to 2 h.
2. Inoculate the specimen (aspirate, scraping, or biopsy material from skin lesions from cutaneous leishmaniasis patients; bone marrow aspirates or splenic aspirates from visceral leishmaniasis patients; or normal skin biopsy specimens, lymph node aspirates, or pieces of liver and spleen from suspected or potential wild- or domestic-animal reservoirs) into the culture tubes. For Chagas’ disease, inoculate a few drops of buffy coat into the culture tubes.
3. Add 0.5 ml of overlay (either saline or other overlay, depending on the medium). The organisms will develop in the fluid condensate and overlay at the bottom of the slant (Fig. 8.5).
4. Incubate the tubes at 20 to 24°C.
5. Once every 2 to 3 days, remove a drop of medium and examine it under the low power (100×) of a microscope, preferably one equipped with phase-contrast optics.
6. If promastigotes are seen, inoculate a couple of drops of the medium into fresh culture tubes. Add a couple of drops of 0.85% saline or the overlay solution (depending on the culture medium used) to the old tube.
7. If visible contamination occurs, add antibiotics to the overlay (to contain 200 U of penicillin/ml and 200 µg of streptomycin/ml). The parasites will not proliferate if bacterial contamination is present.
8. Incubate the tubes containing the patient specimen for at least 2 weeks at 28°C or room temperature.
9. If no organisms are seen even after 2 weeks of incubation, examine several drops of fluid under the microscope for promastigotes. The culture should be observed for 1 month before being signed out as negative. Transfers to fresh medium should be made once or twice a week after the culture is established.
Quality Control for Flagellates of Blood and Tissue
The following control strains should be available when using these cultures for clinical specimens: ATCC 30883 (Leishmania mexicana) and ATCC 30160 (T. cruzi).
1. Check all reagents and media at least once a week. The media should be free of any signs of precipitation and bacterial and/or fungal contamination.
2. The microscope(s) should have been calibrated within the last 12 months, and the original optics used for the calibration should be in place on the microscope(s). The calibration factors for all objectives should be posted on the microscope for easy access.
3. Maintain stock cultures of Leishmania spp.
A. Transfer stock cultures weekly.
a. Stock organisms should always be cultured at the same time a patient specimen is inoculated into culture medium.
b. If the stock organisms multiply and remain viable during the 96 h, patient results can be reported.
B. Stain
a. Stain a slide prepared from stock culture in parallel with the patient slide.
b. Staining results are acceptable when the control organisms stain well.
Note Cultivating the organism from suspected materials provides a definitive diagnosis, but it may take as many as 3 to 7 days. Every effort, therefore, must be made to microscopically examine wet smears and/or permanent stained smears so that appropriate therapy can be instituted without delay if findings are positive (Fig. 8.6) (37,