NNN Medium (Novy-MacNeal-Nicolle): Composition, Preparation, Uses, and Results for Leishmania Culture

A 32-year-old man from the Indian plains presents with six weeks of irregular fever, splenomegaly, and weight loss. The rK39 rapid test is positive, and the clinical picture is consistent with visceral leishmaniasis. Treatment is started with liposomal amphotericin B. Two weeks later, he is not responding as expected. The clinician wants to identify the Leishmania species definitively and check for drug resistance patterns. A splenic aspirate is sent for culture.

The aspirate is inoculated into NNN medium — a biphasic blood agar tube that recreates the temperature and nutrient environment of the sandfly gut, the natural home of Leishmania promastigotes. Within 3–7 days, free-flagellated promastigotes are visible swimming in the liquid overlay phase. Species identification and drug susceptibility testing can proceed.

The NNN (Novy-MacNeal-Nicolle) is a common culture medium for isolating the agent of the genus Leishmania, which causes leishmaniasis. The culture of this organism in suitable media gives rise to a motile, extracellular form called promastigote.

A variety of media has been used for the culture of Leishmania. These can be divided into three main categories: semisolid, biphasic, and liquid. Other in vitro culture media include RPMI 1640, Evans, and Schneider.

NNN Medium was developed by Novy, McNeal and modified by Nicolle. NNN medium consists of two phases, blood agar base (part A) and Lockes solution (part B). This medium consists of blood agar and an overlay medium. The blood agar base is a highly nutritious medium that supports the growth of fastidious organisms like Leishmania and Trypanosoma. The specimens are inoculated into the liquid phase of the biphasic medium and incubated. The amastigotes transform into promastigotes in about 24 hours.

Figure: Amastigote form of Leishmania (LD Bodies)

When to Use NNN Culture

NNN culture is not first-line diagnosis for leishmaniasis. The diagnostic hierarchy is:

Key principle: NNN culture is the reference standard for confirming Leishmania when rapid tests and microscopy are inconclusive, and the only method that provides viable organisms for downstream drug susceptibility testing or molecular typing.

Uses of Culture

• Disease diagnosis: Culture is not a common practice in the diagnostic laboratories as they perform Giemsa stain to detect the amastigote form of Leishmania or uses serological tests like rK39, DAT to make the diagnosis (in visceral leishmaniasis). But in equivocal/indeterminate cases, culture can confirm the diagnosis.
• To use in serologic studies: In vitro cultivation of Leishmania promastigotes is necessary to obtain many organisms to use in serological studies.
• Research works: To study Leishmania species’ antigenic structure, biochemical properties, and infective capabilities.

Why a Biphasic Medium?

The NNN medium's biphasic structure — a solid blood agar base (Part A) with a liquid Locke's solution overlay (Part B) — is not arbitrary. It mimics two key aspects of the Leishmania life cycle:

1. Temperature: Incubation at 21–26°C replicates the sandfly midgut temperature, which is the environment where amastigotes naturally transform into promastigotes. At 37°C (human body temperature), the organisms remain as amastigotes or die; at 22–25°C, they transform to promastigotes and multiply rapidly.
2. The interface: The moisture interface between the solid agar base and liquid overlay creates the microenvironment where Leishmania promastigotes multiply most efficiently — similar to the moist mucosal surface of the sandfly gut. The organisms colonise this interface and then swim freely in the liquid phase.

Composition of Media

Commercial suppliers supply the ready-to-use NNN medium, which you can purchase.  The constituents/ingredients of the media are:

Part A (ingredients gms/liter)

• 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

Part B (ingredients gms/liter)

• 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

Variants and Alternatives to NNN Medium

NNN is not the only medium used for Leishmania culture. Several alternatives are used depending on laboratory resources and research needs:

For routine diagnostic purposes in LMIC settings, NNN remains the standard because it uses defibrinated rabbit blood (locally available) and requires no special supplement procurement.

Making of Culture Media/Tube

Commercial suppliers mostly supply dehydrated media (in the form of free-flowing powder). You can prepare ready-to-use culture media following the procedure mentioned below.

Part A:

1. Suspend 31 grams in 1000 ml distilled water.
2. Heat to boiling to dissolve the medium completely.
3. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
4. Cool to 45°C and aseptically add sterile defibrinated rabbit or human blood.
5. Mix well and dispense in 5 ml amounts in test tubes or 25 ml amounts in flasks.
6. Allow tubed media to cool in a slanted position.

Part B :

1. Suspend 11.2 grams of Part B in 1000 ml distilled water.
2. Heat to boiling to dissolve the medium completely.
3. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
4. Cool and add approximately 2 ml in tubes or 10-15 ml in flasks over solidified Part A medium.

Storage

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

Figure: Leishmania donovani promastigotes in culture

Inoculation

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

Results and Interpretation

Positive culture: Motile promastigotes are visible in the liquid overlay phase under low-power microscopy (10× objective). Growth typically appears within 3–10 days for bone marrow or splenic aspirate specimens; skin specimens may take up to 2–4 weeks.

Promastigote morphology on Giemsa-stained culture smear:

• Elongated spindle shape, 15–25 μm in length
• Single prominent flagellum emerging from the anterior kinetoplast
• Nucleus centrally located; kinetoplast (deeply staining mitochondrial DNA) located at the anterior end
• Found free in the liquid phase (not intracellular — amastigotes are intracellular; promastigotes are extracellular)

Checking for growth: Examine the tubes every 4 days for 4 weeks. Positive growth shows turbidity or visible motile organisms in the liquid phase. Under low-power microscopy, motile flagellated forms (promastigotes) are visible as swimming organisms — their motion is distinctive (forward with a spiral rotation).

Negative culture: Absence of growth after 4 weeks with clear liquid phase. Before reporting negative, confirm microscopically that no organisms are present — early growth may be visible only under microscopy before the liquid phase becomes visibly turbid.

Contamination: Fungal contamination (fluffy growth, discolouration) or bacterial contamination (turbid, malodorous) renders the culture non-interpretable. Strict aseptic technique during inoculation is essential. Antibiotics (gentamicin or penicillin-streptomycin) are sometimes added to the overlay to reduce bacterial contamination from skin specimens.

Where Students Actually Get Confused

1. "NNN medium is used only for Leishmania." NNN medium was developed primarily for Leishmania but also supports the growth of Trypanosoma species (particularly T. cruzi and T. brucei) and has historically been used for Entamoeba histolytica culture (though Robinson's medium is preferred for amoeba). The medium's blood agar base supports any fastidious haemoflagellate.

2. "Promastigotes are found in human tissue." Promastigotes are the sandfly-stage form. In human tissues (and in culture at 22–26°C), the organisms exist as amastigotes (intracellular, non-flagellated). NNN culture works because the low incubation temperature triggers transformation from amastigote → promastigote. At 37°C (body temperature), amastigotes revert or die; promastigotes do not survive in human tissues.

3. "A negative NNN culture excludes leishmaniasis." Culture sensitivity depends heavily on specimen quality and handling. Bone marrow aspirate cultures have 53–86% sensitivity; splenic aspirate culture reaches 93–99%. Cold chain during transport is critical — Leishmania is thermolabile and does not survive extended room temperature exposure. A negative culture with a positive rK39 and consistent clinical picture should prompt repeat sampling rather than excluding the diagnosis.

4. "The incubation temperature for NNN is 37°C." No — 37°C is human body temperature, which maintains the amastigote form. NNN is incubated at 21–26°C (sandfly gut temperature) to trigger promastigote transformation. This is the most commonly examined detail about NNN medium in university papers.

5. "Part A and Part B are mixed before inoculation." Part A (blood agar base) is prepared separately, dispensed into tubes, and allowed to solidify in a slanted position. Part B (Locke's solution) is added as a liquid overlay onto the solidified Part A. They remain as separate phases — the organism inoculated into the liquid overlay grows at the interface and in the liquid phase. They are never mixed.

Key Exam Facts in One Table

Self-Check Questions

1. You inoculate a bone marrow aspirate from a suspected kala-azar patient into NNN medium and incubate at 37°C. Four weeks later the culture is negative. What error was made?
2. Under low-power microscopy on day 5, you see motile elongated flagellated organisms in the liquid overlay of an NNN tube. What are these, and what do you do next to confirm?
3. A student says "NNN medium mixes both parts together before inoculation." Is this correct? Explain the correct structure.
4. What does NNN stand for, and which organism is the medium primarily used to culture?
5. Why is the incubation temperature of 21–26°C critical for NNN medium, and what happens if incubation is performed at 37°C?

Answers

1. The incubation temperature was wrong. NNN medium must be incubated at 21–26°C (sandfly gut temperature) to trigger transformation of amastigotes into promastigotes. At 37°C (body temperature), amastigotes revert or die; promastigotes do not form. The culture must be repeated from fresh specimen at the correct temperature.
2. These are Leishmania promastigotes — the flagellated, extracellular form that grows in NNN culture at low temperature. To confirm: prepare a smear from the liquid overlay, air-dry, fix with methanol, and stain with Giemsa. Promastigotes appear as elongated (15–25 μm) spindle-shaped organisms with a centrally placed nucleus and an anteriorly located kinetoplast, with a single free flagellum.
3. Incorrect. Part A (blood agar base) and Part B (Locke's solution) are prepared separately. Part A is autoclaved, cooled to 45°C, blood is aseptically added, dispensed into tubes and allowed to solidify in a slanted position. Part B is then added as a liquid overlay on top of the solidified Part A. They remain as two distinct phases — inoculum is added to the liquid overlay phase.
4. NNN = Novy-MacNeal-Nicolle (the three scientists who developed and modified the medium). It is primarily used to culture Leishmania species, causing leishmaniasis. It also supports the growth of Trypanosoma species.
5. 21–26°C replicates the temperature of the sandfly midgut — the natural environment where amastigotes transform into promastigotes. This transformation is temperature-triggered: the lower temperature signals to the organism that it has entered the insect vector and initiates promastigote development. At 37°C, the organism "thinks" it is in human tissue and maintains (or attempts to maintain) the amastigote form, which does not multiply in culture medium.

References

1. Chouihi, E., Amri, F., Bouslimi, N., Siala, E., Selmi, K., Zallagua, N., Ben Abdallah, R., Bouratbine, A., & Aoun, K. (2009). Les cultures sur milieu NNN dans le diagnostic biologique des leishmanioses [Cultures on NNN medium for the diagnosis of leishmaniasis]. Pathologie-biologie, 57(3), 219–224. https://doi.org/10.1016/j.patbio.2008.03.007
2. Beltran, E., & Gutierrez Ballesteros, E. (1950). Empleo del medio N.N.N. (agar-sangre) en el cultivo de Entamoeba histolytica. Revista del Instituto de Salubridad y Enfermedades Tropicales, 11(2–3–4), 107–109.
3. Garcia, L. S. (2016). Diagnostic Medical Parasitology (6th ed.). ASM Press.
4. Sundar, S., & Rai, M. (2002). Laboratory diagnosis of visceral leishmaniasis. Clinical and Diagnostic Laboratory Immunology, 9(5), 951–958. https://doi.org/10.1128/cdli.9.5.951-958.2002
5. Bates, P. A. (1994). Complete developmental cycle of Leishmania mexicana in axenic culture. Parasitology, 108(1), 1–9. https://doi.org/10.1017/S0031182000078458
6. Maxfield, L., & Crane, J. S. (2023). Leishmaniasis. In StatPearls. StatPearls Publishing. https://www.ncbi.nlm.nih.gov/books/NBK531456/