Nitrate or Nitrite Reduction Test: Introduction,Composition of Medi

Introduction of Nitrate or Nitrite Reduction Test

Table of Contents

The nitrate or nitrite reduction test uses to determine the ability of an organism to reduce nitrate to nitrite. Anaerobic metabolism requires an electron acceptor other than atmospheric oxygen. Many gram-negative bacteria use nitrate as the final electron acceptor. The nitrate reduction test is a test that determines the production of an enzyme called nitrate reductase, which results in the reduction of nitrate. This test is useful to differentiate organisms on the basis of their ability to reduce nitrate to nitrite or nitrogenous gases.

Uses of Test

The nitrate reduction test is useful to test to differentiate Enterobacteriaceae from other gram-negative bacteria since all the members of the family Enterobacteriaceae reduce nitrate.
It is helpful for differentiating Mycobacterium.
The Nitrate reduction may be coupled to anaerobic respiration in some species.
Identification of Neisseria from Moraxella and Kingella. This test is a critical test for differentiating between Neisseria gonorrhoeae and Kinglla denitrificans, particularly when strains of Kingella denitrificans appear to be the same i.e. gram-negative diplococci in gram-stained smears.
It also facilitates species identification of Corynebacterium.

Principle

Incubate nitrate broth with a heavy inoculum of test bacteria. If the bacteria are capable of producing the nitrate reductase enzyme, it reduces the nitrate, present in the broth, to nitrite. It may be further reduced to nitric oxide (NO), nitrous oxide (N2O), or nitrogen (N2). This test is based on the detection of nitrite and its ability to form a red compound when it reacts with sulfanilic acid to form a complex i.e. nitrite-sulfanilic acid which then reacts with an α-naphthylamine to give a red precipitate i.e. prontosil, that is a water-soluble azo dye.

The red color will see only when nitrate is present in the medium. If there’s no red color in the medium after the addition of sulfanilic acid and α-naphthylamine, nitrite is not present in the medium. When the nitrate may not have been reduced, the test organism is nitrate-negative. If the nitrate may have been reduced to nitrite which has then been completely reduced to nitric oxide, nitrous oxide, or nitrogen which will not react with the reagents that react with nitrite, the test bacterium is nitrate-positive.

Use of Zinc powder

It is helpful when nitrite is not detectable and therefore, it is necessary to test if the organism has reduced nitrate beyond nitrite. Add a pinch of Zinc powder to the culture. It catalyzes the reduction of nitrate to nitrite. The development of the red color with the addition of Zinc powder indicates that nitrate was not reduced by the test organism which suggests that the test organism is not capable of reducing nitrate. If there is no color change after the addition of zinc, this indicates that the organism reduced nitrate to one of the other nitrogen compounds and hence is a nitrate reducer.

Use of A Durham tube

Durham tube is placed in the nitrate broth in order to detect gas formation in the tube and to identify denitrification by organisms that produce gas by alternate pathways.

Composition of Nitrate Broth

Ingredients	Gram weight
Peptone	2.0
Potassium nitrate	0.2
Distilled water	100 ml
Final pH	7.0 ± 0.2 at 25°C

Table: Composition of Nitrate Broth

Note: Heart infusion broth uses in the case of fastidious organisms in place of peptone( 25 grams per liter)

Dispense 4-ml aliquots in 16- by 125-mm screw-cap tubes with Durham tube.
Autoclave at 121°C for 15 min.
Store at 2 to 8°C.

Preparation of Reagents

1. Reagent A ( 0.8% Sulfanilic acid )

Sulfanilic acid: 0.8 g

Distilled water: 70 ml

Glacial acetic acid: 30 ml

Mix sulfanilic acid with water; heat to dissolve.
Cool, and then add acetic acid.
Store at 2 to 8°C.
Shelf life is 3 months.

2. Reagent B (0.5% N, N-Dimethyl-alpha-naphthylamine)

Glacial acetic acid :30 ml

Distilled water:70 ml

N,N-dimethyl-α-naphthylamine :0.5 g

Combine acetic acid and water.
Add α-naphthylamine.
Store at 2 to 8°C.
Shelf life is 3 months.

Nitrate Reduction Test Reagents A and B — Fig. Nitrate Reduction Test-Reagents A and B

3. Zinc metal dust

Test Requirements

Nitrate broth
Durham tube
Reagent A
Reagent B
Zinc dust (Powder)
Test organisms
Bunsen burner
Inoculating wire
Incubator
Control strains

Escherichia coli ATCC 25922
Pseudomonas aeruginosa ATCC 27853
Acinetobacter baumannii ATCC 19606

Test procedure of Nitrate Reduction Test

Nitrate reduction to nitrite test is sometimes completed in two steps method.

First method

The reduction of nitrate to nitrite is determined by the addition of Nitrate Reagents A and B.

Second method

The reduction of nitrate beyond nitrite determination with the help of zinc powder

Inoculate the test organism in nitrate broth and incubate the tubes at the optimal temperature of 30°C or 37°C for 24 hours.
After completion of incubation look for nitrogen gas formation first before adding reagents.
Add reagent A (6-8 drops).
Similarly, add reagent B( 6-8 drops).
Observe for color development within a minute or less.
If no color develops add zinc powder.
Observe for at least 3 minutes for a red color to develop after the addition of zinc.

Result -Interpretation

Nitrate/Nitrite reduction test: Positive
Development of a cherry red color after the addition of reagents A and B
Lacking red color development on adding Zinc powder

Nitrate/Nitrite reduction test: Negative
Development of red color with the addition of Zinc powder

Control strains

Escherichia coli ATCC 25922—nitrate positive, gas negative

Pseudomonas aeruginosa ATCC 27853—nitrate positive, gas positive

Acinetobacter baumannii ATCC 19606—nitrate negative

Fig. Nitrate or Nitrite Reduction Test Positive and Negative

Limitations

Failure to recognize that the organism did not grow in the medium will result in possible false-negative test(s).
Interpretation of color reactions should be made immediately, as color reactions with a positive test may fade rapidly.
A faint pink color may be produced following the addition of nitrate reagents. This is not a positive result.
Due to the possible presence of nitrite in the culture media, a low nitrite media such as Nitrate Agar or Nitrate Broth should be used for the nitrate reduction test.
A negative zinc reduction (no color change) test, in combination with a negative nitrite reaction, is a presumptive indication that the nitrate was reduced beyond the nitrite stage. Although a very common end product of nitrate reduction is nitrogen gas, other end products may be formed. Additional testing may be required to determine the final end products of the reaction.
To avoid false-negative nitrite reduction reactions, negative nitrite reactions must be verified by the addition of zinc dust to the medium.
Excess zinc dust has been reported to cause false-positive nitrite reduction reactions due to the complete reduction of previously unreduced nitrate to ammonia.

Nitrate or Nitrite Reduction Test: Introduction, Composition of Medium, Principle, Test Requirements, Procedure, Result -Interpretation, and Limitations