Nitrite Reduction Test- Principle, Procedure, Results, Limitations

The objective of the nitrite reduction test is to determine the ability of an organism to reduce nitrites to gaseous nitrogen or to other compounds containing nitrogen. This test is useful for identifying and differentiating bacteria based on their nitrate and nitrite metabolism.

Nitrite reduction test is based on the ability of some bacteria to use nitrate as an electron acceptor during anaerobic respiration. Nitrate is a highly oxidized form of nitrogen that can be reduced to various nitrogen products depending on the enzyme system of the organism and the environmental conditions.

• Brain-heart infusion broth: This is a general-purpose medium that supports the growth of a wide range of bacteria.
• Nitrite reduction medium: This is a selective and differential medium that contains peptone, beef extract, yeast extract, and dextrose as sources of carbon and nitrogen.

1. To perform the nitrite reduction test, you will need two types of media: brain-heart infusion broth and nitrite reduction medium.
2. First, prepare the media according to the manufacturer`s instructions or the recipe given above.
3. Next, inoculate the brain-heart infusion broth with a loopful of bacteria from a pure culture.
4. After 24 hours, check the broth culture for growth.
5. If there is growth in the broth culture, use a sterile loop to transfer some of the bacteria to the nitrite reduction medium.
6. Incubate the nitrite reduction medium for 48 hours.
7. After 48 hours, examine the nitrite reduction medium for gas production and nitrite presence.
8. To add the reagents, carefully remove the cap of the tube and add five drops of sulfanilic acid followed by five drops of alpha-naphthylamine.
9. Observe the color change in the medium within two minutes after adding the reagents.
10. Record your observations and interpret your results according to the provided criteria.

The results of the nitrite reduction test are based on the presence or absence of nitrite in the broth after incubation and the addition of reagents.

The nitrite reduction test has some limitations that should be considered when interpreting the results.

To ensure the accuracy and validity of the nitrite reduction test, positive and negative control organisms are used along with the test organism.

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