The Nitrate/Nitrite Reduction Test

The nitrate/Nitrite reduction test determines the ability of an organism to reduce nitrate to nitrite and to Identify the different methods that nitrate can be reduced by the organism.

The Nitrate/Nitrite Reduction Test

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 nitrogen gases.Anaerobic metabolism requires an electron acceptor other than atmospheric oxygen. 

In this article, we will take you through everything you need to know about the Nitrate reduction test and how it is carried out. 

The History Of The Nitrate Reduction Test

The current test methods for nitrate and nitrite reduction would not exist if it hadn’t been for a scientist, Peter Greiss, who developed the first recognisable diazotization reaction, as he described it, in 1858. 

Peter Greiss was the son of a blacksmith and was raised on a farm in Prussia. In his 6th year at university he began to seriously study chemistry.

He obtained employment in the coal-tar distillery where the senior chemists discovered and developed the aniline dye industry. Even though the distillery was destroyed by fire, Griess had become obsessed with the chemistry of dye making.

He was recommended for a position at the Royal College of Chemistry in Great Britain on the very day of his first article on possible diazo compounds.

Griess’ first several attempts at diazotization exploded, but his commission at the Royal College was to investigate his new nitrogen intermediates, with the result that diazo benzoic acid was isolated and an entirely new class of compounds was discovered (23, 30).

Because many of these compounds were found to be stable and could be used for dying fabric without needing a mordant, Griess is heralded as the father of the modern azo dye industry.

In 1879, Griess developed a method for the detection of nitrite in solutions. The method, an acid solution of sulfanilic acid and alpha naphthylamine, undergoes a diazotization reaction with nitrites, forming a red azo dye.

Many variations of the so-called Griess test can be found in chemistry, medicine, and industry, but all are based on the production of an azo dye via the diazotization of nitrite. 

The Purpose Of The Nitrate Reduction Test

Nitrate reduction by bacteria is mediated by nitrate reductase and indicates that the organism can use NO3 – as an electron acceptor during anaerobic respiration.

Nitrite may be reduced to a variety of nitrogen products including NO, N2O, N2, and NH3, depending on the enzyme system of the organism and the atmosphere in which it is growing.

Reduction of nitrate often indicates a shift to or facilitation of anaerobic metabolism, as some organisms can use nitrate as an electron acceptor during anaerobic respiration or anaerobic chemolithotrophy.

Nitrate reduction test is used for the differentiation of members of Enterobacteriaceae on the basis of their ability to produce nitrate reductase enzyme that hydrolyzes nitrate (NO3) to nitrite (NO2).

Which may then again be degraded to various nitrogen products like nitrogen oxide, nitrous oxide and ammonia (NH3) depending on the enzyme system of the organism and the atmosphere in which it is growing.

The Objective Of The Test

The objectives of the nitrate reduction test are mainly to determine the ability of an organism to reduce nitrate to nitrite. But also to help identify the different ways that nitrate can be reduced by bacteria. 

The Principle Of The Test

The Principle Of The Test

A large quantity of the test organism is incubated into a liquid or broth that contains nitrate.

Any of the organisms that are capable of producing nitrate reductase enzymes reduce the nitrate, present in the broth, to nitrite which may then be further reduced to nitric oxide, nitrous oxide, or nitrogen.

If the organism has reduced nitrate to nitrite, the nitrites in the medium will form nitrous acid.  When sulfanilic acid is added, it will react with the nitrous acid to produce diazotized sulfanilic acid.

This reacts with the α-naphthylamine to form a red-colored compound.  Therefore, if the medium turns red after the addition of the nitrate reagents, it is considered a positive result for nitrate reduction.

The nitrate reduction 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 (nitrite-sulfanilic acid) which then reacts with a α-naphthylamine to give a red precipitate (prontosil), which is a water-soluble azo dye.

It should be noted that, only when nitrate is present in the medium, red colour will be produced. If there’s no red colour in the medium after you’ve added sulfanilic acid and α-naphthylamine means only that nitrite is not present

If the medium does not turn red after the addition of the reagents, it can mean that the organism was unable to reduce the nitrate, or the organism was able to denitrify the nitrate or nitrite to produce ammonia or molecular nitrogen.

Therefore, another step is needed in the test. Add a small amount of powdered zinc. If the tube turns red after the addition of the zinc, it means that unreduced nitrate was present*. Therefore, the red colour on the second step is a negative result

.

There are two main outcomes and observations to this particular experiment.

  1. The nitrate will not reduce; the strain therefore is nitrate-negative.
  2. The nitrate will have reduced to nitrite – nitrate has then been completely reduced to nitric oxide, nitrous oxide, or nitrogen which will not react with the reagents that react with nitrite; the strain is nitrate-positive.

Essentially when nitrite is not detected, it is necessary to test if the organism has reduced nitrate beyond nitrite. You can do this in a variety of ways, including by indirectly adding a small amount of Zinc powder to the culture.

The Zinc powder catalyses the reduction of nitrate to nitrite. The development of the red colour with the addition of Zinc indicates that nitrate was not reduced by the organism which suggests that the test organism is not capable of reducing nitrate.

If no colour change occurs after the addition of zinc, this indicates that the organism reduced nitrate to one of the other nitrogen compounds and thus is a nitrate reducer.

It is very important to note that a  Durham tube is placed in the nitrogen broth in order to detect deterioration of the broth before inoculation, as evidenced by gas formation in the tube and to identify denitrification by organisms that produce gas by alternate pathways.

The Method Used

Determination of nitrate reduction to nitrite is a two step process. First, the reduction of nitrate to nitrite is determined by the addition of Nitrate Reagents A and B, then if necessary, the reduction of nitrate beyond nitrite is determined by the addition of Nitrate Reagent C (zinc dust).

  • Inoculate nitrate broth with a heavy growth of test organism using an aseptic technique.
  • Incubate at an appropriate temperature for 24 to 48 hours
  • Add one dropper full of sulfanilic acid and one dropper full of an α-naphthylamine to each broth.
  • At this point, a colour change to RED indicates a POSITIVE nitrate reduction test. If you get a red colour, then you can stop at this point.
  • No colour change indicates the absence of nitrite. This can happen either because nitrate was not reduced or because nitrate was reduced to nitrite, then nitrite was further reduced to some other molecule. If you don’t get a red colour, then you must proceed to the next step.
  • Add a small amount of zinc to each broth. Zinc catalyses the reduction of nitrate to nitrite.
  • At this point, a colour change to RED indicates a NEGATIVE nitrate reduction test because this means that nitrate must have been present and must have been reduced to form nitrite.
  • No colour change means that no nitrate was present. Thus no colour change at this point is a POSITIVE result. 
Jennifer Dawkins

Leave a Comment

Your email address will not be published.