NNN Medium: Introduction, Composition, Preparation, Culture Procedure

NNN Medium: Introduction, Composition, Preparation, Culture Procedure and Result Interpretation

Introduction of NNN Medium

NNN medium stands for Novy-MacNeal-Nicolle medium and it was developed by Novy, McNeal, and modified by Nicolle. It uses to grow parasites, Leishmania and Trypanosoma. Culture is sensitive than smear and thus used to grow– needed when the amastigotes are not found in sufficient quantities in smear.

Composition of NNN Medium

Ingredients Gms / 100 ml

Part- A

Lab Lamco powder /Meat extract: O. 3

Peptone: 0.5 .

Sodium chloride: O.8.

Agar: 1.5.

Ph : 7.3 ± 0.2

Part B

Sodium chloride: O. 8

Potassium chloride: 0.02

Calcium chloride: 0.02

Anhydrous potassium dihydrogen phosphate: 0.03

Dextrose (Glucose): 0.25

Final pH (at 25°C): 7.0 ± 0.2

Principle of NNN medium

NNN medium consists of a blood agar base and an overlay medium (Locke’s solution). The blood agar base is a highly nutritious medium that helps the growth of fastidious parasites like Leishmania and Trypanosoma. The specimens are inoculated into the liquid phase of this NNN diphasic medium and incubated. This supports the development of organisms in the insect vector. The amastigotes transform into promastigotes in about a day.

Preparation of NNN Medium

Part A Preparation

Suspend 3.1 grams in 50 ml and dissolve the medium completely by shaking.
Add remaining distilled water and mix properly.
Autoclave at 15 lbs pressure and 121°C temperature for 15 minutes.
Cool to 45-50°C and aseptically add 10% of sterile defibrinated rabbit or human blood after inactivation at 56°C for 30 minutes.
Mix well and dispense in 5 ml amounts in test tubes.
Allow tubed media to cool in a slanted position.

Part B Preparation

Suspend 1.12 grams of Part B in 100ml distilled water.
Shake to dissolve the medium completely and sterilize by autoclaving at 15 lbs pressure and 121°C temperature for 15 minutes.
Cool and add approximately 2 ml in tubes over solidified Part A medium as shown above image.

Culture Procedure ( Inoculation)

Take out NNN medium to room temperature prior to inoculation.
Inoculate the sample and that may either bone marrow biopsy or spleen aspiration or lymph node aspiration or punch biopsy into the liquid phase of the medium.
Incubate for 4 weeks at 21-26°C.
After 4 days, check for organism growth and potential other microbial contamination, and repeat every 4 days for the next 4 weeks.

Result Interpretation

Positive

Luxuriant growth on the medium
Giemsa stained smear from the culture filtrate: Presence of promastigotes
Hanging drop Preparation: Motile promastigotes

Negative

No growth on the medium
Giemsa stained smear from the culture filtrate: Lacking promastigotes
Hanging drop Preparation: absence of promastigotes and motility

Keynotes on NNN medium

Prepared media are stable for up to 4 weeks in the refrigerator (2-8°C).
It is also commercially available in dehydrated form.
Other cultural media that may support the growth of Leishmania are RPMI 1640, Evans, and Schneider.
This NNN medium is also known as a phase converter medium since it transfers amastigote (non-motile) to promastigote (motile and extracellular form).
Leishmania normally grows from the 5th to the 15th day.
Identification of promastigotes in the NNN medium is the gold standard method for laboratory diagnosis.
It is applicable for the following purposes-

To confirm diagnosis rather than other tests like Giemsa stain to detect the amastigote form of Leishmania, serological tests i.e. rK39, direct agglutination test (DAT), etc.
To obtain a large number of organisms for serological studies, in vitro cultivation of Leishmania promastigotes is needed.
To proceed research works on the antigenic structure, biochemical properties, and infective capabilities of organism, Leishmania.

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