the serum in the specimen may contain leishmanicidal or inhibitory factors that prevent the growth of organisms. Alternatively, bone marrow juice may be centrifuged for 10 min at 250 × g and the sediment may be washed in 0.85% saline by centrifugation and then inoculated into culture tubes. Buffy coat from the blood sample rather than whole blood should be inoculated. Because the leishmanias are fastidious organisms and not all isolates may grow in any one medium, it is imperative that at least two media be used; for example, use NNN or modified Tobie’s medium and Schneider’s Drosophila medium.
Specimens for culturing Trypanosoma cruzi may consist of blood or the gut contents of the triatomid bug. It is advisable to use two different media such as liver infusion-tryptose (LIT) and NNN for the initial isolation of T. cruzi. Once growth is established, use the medium in which best growth is obtained for subculture. According to James Sullivan, LIT medium, when used as an overlay on Tobie’s slants, is excellent for isolation and diagnosis (8). The major culture form is the epimastigote; occasionally, however, trypomastigotes and amastigotes are also seen.
NNN Medium (Leishmaniasis or Chagas’ Disease) (33)
1. Mix the NaCl and agar in the distilled water in a 500-ml flask.
2. Heat the mixture until the agar melts.
3. Autoclave at 121°C for 15 min.
4. Cool to about 50°C.
5. Add 10 ml of aseptically collected, defibrinated rabbit blood.
Note Although blood collected with EDTA as the anticoagulant can be used for routine stock culture subcultures, it may not be quite as effective as defibrinated blood in isolating organisms from patient specimens. Certainly, if defibrinated blood is not available, blood collected with EDTA as the anticoagulant can be used. Make sure that the rabbit blood is fresh (no older than 10 days). It should be aseptically collected and stored at 4°C until used.
6. Dispense 4 ml into 16- by 125-mm sterile screw-cap culture tubes.
7. Place the tubes at a 10° angle (shallow slant position) until the agar sets.
8. Immediately transfer the tubes into test tube stands, and let stand upright at 4°C so that the bottom portion of the slants is covered with the water of condensation. Rapid cooling increases the water of condensation (Fig. 8.5).
9. Label as NNN medium with the preparation date and an expiration date 3 weeks from the date of preparation.
10. Store at 4°C.
Figure 8.5 Illustration of a tube of NNN medium, used for the culture and recovery of Leishmania spp. (A) Fluid condensate and tissue culture medium overlay containing the developing organisms. (B) Blood agar medium. (Illustration by Sharon Belkin.) doi:10.1128/9781555819002.ch8.f5
NNN Medium, Offutt’s Modification (Leishmaniasis)
1. Heat until the agar is dissolved in the distilled water in a 500-ml flask.
2. Autoclave at 121°C for 15 min.
3. Cool to about 50°C.
4. Add 15 ml of aseptically collected, defibrinated rabbit blood.
5. Dispense 4 ml into 16- by 125-mm sterile screw-cap culture tubes.
6. Place the tubes at a 10° angle (shallow slant position) until the agar sets.
7. Immediately transfer the tubes to test tube stands, and let stand upright at 4°C so that the bottom portion of the slants is covered with the water of condensation. Rapid cooling increases the water of condensation.
8. Label as Offutt’s medium with the preparation date and an expiration date 3 weeks from the date of preparation.
9. Store at 4°C.
Overlay Solution (To Be Used with NNN or NNN Modified)
1. Autoclave at 121°C.
2. Dispense 4 ml aseptically into 16- by 125-mm sterile culture tubes.
3. Label as 0.9% saline with the preparation date and an expiration date 3 weeks from the date of preparation.
4. Store at 4°C.
Evan’s Modified Tobie’s Medium (Leishmaniasis or Chagas’ Disease) (33)
1. Mix all the ingredients in the distilled water in a large beaker with a magnetic stirrer.
2. Heat the mixture until the agar melts.
3. Dispense 5 ml into 16- by 125 mm screw-cap culture tubes.
4. Autoclave at 121°C for 15 min.
5. Cool to about 50°C.
6. Add 1.2 ml of aseptically collected, defibrinated horse blood.
7. Hold the tubes upright in the palm of your hand, and roll the tubes gently to mix the blood and the agar well.
8. Place the tubes at a 10° angle (shallow slant position) until the agar sets.
9. Immediately transfer the tubes into test tube stands, and let stand upright at 4°C so that the bottom portion of the slants will be covered with the water of condensation. Rapid cooling increases the water of condensation.
10. Label as Evan’s modified Tobie’s medium with the preparation date and an expiration date 3 weeks from the date of preparation.
11. Store at 4°C.
Overlay Solution (To Be Used with Tobie’s Medium)
1. Place 750 ml of double-distilled water in a 1-liter beaker, and add the above ingredients one at a time in the order listed until dissolved. Use a magnetic stirrer.
2. Adjust the pH to 7.2 by adding slowly, while stirring, solid Tris (white, crystalline powder, C4H11NO3 used for buffering capacity).
3. Bring up the volume to 1,000 ml with distilled water.
4. Dispense 100 ml into each of several screw-cap flasks or bottles.
5. Autoclave at 121°C for 15 min.
6. Label as overlay solution with the preparation date and an expiration date of 1 month.
