Nitrate reduction test for MTB

Principle:

The nitrate reduction test, which measures enzyme activity, is a key element in differentiating M. tuberculosis from other tubercle bacilli.

Nitrate reductase enzyme converts nitrate into nitrite, which is detected by a colourimetric test. M. tuberculosis exhibits strong nitrate reduction activity, whereas M. bovis gives a negative or weak reaction; M. Africanum strains are usually negative, but approximately 20% of strains give a positive test reaction.

The test must be carried out on pure cultures otherwise it will yield false results.

Reagents and Solutions:

Note: Discard any reagent if the colour changes or a precipitate forms.

1. Sodium nitrate substrate in buffer, pH 7

Sodium nitrate (NaNO₃)                                                                          0.085 g

Potassium dihydrogen phosphate (KH₂PO₄)                                          0.117 g

Disodium hydrogen phosphate dodecahydrate (Na₂HPO₄.12H₂O)                        0.485 g

Distilled water                                                                                          100 ml

Check pH using a pH meter. Then, sterilize by autoclaving at 121°C for 15 minutes. 

2 ml aliquots of the substrate solution are aseptically dispensed into sterile screw-cap tubes when needed.

2.  Reagent 1: Hydrochloric acid solution

Concentrated hydrochloric acid (HCl, 36%)                                            10 ml

Distilled water                                                                                          10 ml

Add concentrated HCI slowly to distilled water.

3.  Reagent 2: Sulfanilamide solution, 0.2%

Sulfanilamide  (C₆H₈N₂O₂S)                                                                   0.2 g            

Distilled water                                                                                          100 ml

Dissolve sulfanilamide in distilled water and store it in an amber bottle in the dark in a refrigerator.

4.  Reagent 3: N-naphthylethylene diamine solution, 0.1%

N-naphthylethylene diamine dihydrochloride                                          0.1 g

Distilled water                                                                                          100 ml

Dissolve N-naphthylethylene diamine in distilled water.

Store in an amber bottle in the dark in a refrigerator.

Procedure:

The entire procedure must be carried out in a biological safety cabinet.

1. Add 0.2 ml of distilled water to a 16 x 100 mm screw-cap tube.

2. Take 2 loopful of colonies from positive culture and emulsify in water. 

3. Add 2.0 ml of NaNO₃ substrate to the tube and mix well.

4. Place in the water bath at 37 °C for 2 hours.

5. Remove the tube from the water bath.

6. Add 1 drop of reagent 1, 2 drops of reagent 2, and 2 drops of reagent 3.

7. Examine immediately for the development of a pink to red colour.

Result and interpretation:

Pink colour  = positive result

No colour     = negative result

Add a small amount of zinc powder to all negative tests:

• If nitrate is still present, it will be reduced by the zinc powder, and the colour will turn red (true negative). 

• If no colour develops the original reaction was positive, but nitrate was reduced beyond nitrite.

• Resource

  Standard Operating Procedures (Nitrate Reduction Test).

  Assessment