Store at 4°C.
NIH Method for Trypanosomes and Leishmaniae
Solution 1
Solution 2 (Locke’s Solution)
1. Infuse the beef and distilled water in a water bath at 37°C for 1 h. Heat for 5 min at 80°C.
2. Filter through filter paper (Whatman no. 2V).
3. Add the rest of the above ingredients (solution 1), and adjust the pH to 7.0 to 7.4 with NaOH.
4. Autoclave at 121°C for 20 min.
5. Cool to 45°C, and aseptically add 10% defibrinated rabbit blood.
6. Dispense 5-ml quantities in sterile tubes. Slant and cool.
7. Just before inoculation, overlay with 2 ml of sterile Locke’s solution.
4 N Medium for Trypanosomes and Leishmaniae
The 4 N medium was adapted from the original formula described by Novy and McNeal (9, 34, 35).
4 N Medium for Trypanosomes and Leishmaniae
Agar base
1. Mix the agar and water, and dissolve by autoclaving or steaming.
2. Dispense liquid agar in 5-ml aliquots into 30-ml screw-cap glass bottles.
3. Autoclave again if necessary for sterility.
4. When the medium has cooled to 45°C, add aseptically to each bottle approximately 1 ml of fresh rabbit blood and allow the agar to solidify in a slant.
Overlay for 4 N medium
1. Add 1 ml of Locke’s (see NIH method for trypanosomes and leishmaniae) solution to each bottle containing 5 ml of agar.
2. Incubate the bottles at 37°C for 12 to 24 h to check for sterility.
3. Store the bottles at 4°C for 24 h or more before use.
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,
