What is Nitrate reduction test?
The nitrate reduction test is a biochemical method used to assess if a bacterium can use nitrate as an electron acceptor under anaerobic conditions, helping in the identification of bacterial species, particularly among Enterobacteriaceae and other facultative anaerobes. By detecting intermediate or end products from nitrate reduction, this test provides insight into bacteria’s respiratory abilities, aiding in more accurate microbial identification.
Table of Contents
Objective
The main purpose of the nitrate reduction test is to determine whether a bacterium can reduce nitrate (NO₃⁻) to nitrite (NO₂⁻) or to further reduced forms like nitrogen gas (N₂) or ammonia (NH₃). Bacteria with nitrate reduction capability can use nitrate in place of oxygen for respiration under anaerobic conditions, and this metabolic trait serves as a valuable tool for classifying bacterial species.
Principle
This test works by evaluating the activity of the enzyme nitrate reductase, which facilitates the transformation of nitrate into nitrite. For certain bacteria, nitrate reduction is a pathway to complete anaerobic respiration, using nitrate as the final electron acceptor in the absence of oxygen. The test detects specific end products of this reduction process, including:
Nitrate (NO₃⁻), which can be reduced to Nitrite (NO₂⁻).
Nitrite, which some bacteria can further reduce to Nitrogen gas (N₂), Nitrous oxide (N₂O), or Ammonia (NH₃), based on their enzyme systems.
Special reagents reveal the presence of nitrite or other reduction products, allowing for the detection of a bacterium’s nitrate reduction pathway.
Procedure
The nitrate reduction test involves inoculating a nitrate broth containing potassium nitrate with the test microorganism and then incubating the sample under anaerobic conditions to allow nitrate reduction. The main steps of the procedure are as follows:
Inoculation and Incubation
Inoculate the test organism in a nitrate broth medium (typically containing potassium nitrate) and incubate it at around 35–37°C for 24–48 hours. This allows the organism time to reduce nitrate if it possesses the necessary enzyme.
Adding Nitrate Reagents
After incubation, two reagents, sulfanilic acid (Reagent A) and alpha-naphthylamine (Reagent B), are added:
- If nitrite is present in the broth, it reacts with sulfanilic acid to produce a diazonium salt. This salt combines with alpha-naphthylamine, resulting in a red, water-soluble dye.
- A red color appearing immediately after the addition of these reagents indicates nitrite presence, confirming a positive result for nitrate reduction to nitrite.
Testing with Zinc
If no red color forms after the addition of the reagents, it suggests either that nitrate was not reduced, or it was reduced beyond nitrite to nitrogen gas or ammonia. In this case, zinc powder is added:
- Zinc reduces any remaining nitrate to nitrite. If nitrate remains in the medium, adding zinc will cause a red color change, indicating a negative result (showing the bacterium did not reduce nitrate on its own).
- If there’s no color change after zinc addition, this confirms that the organism has further reduced nitrate past nitrite, indicating a positive result for complete nitrate reduction.
Result Interpretation
Results from the nitrate reduction test are interpreted based on the color reactions observed after adding reagents and, if necessary, zinc:
Red color after adding nitrate reagents (sulfanilic acid and alpha-naphthylamine)
Positive result for nitrate reduction to nitrite, showing the organism reduced nitrate only to the nitrite stage.
Red color after adding zinc powder
Negative result for nitrate reduction, as zinc reduced the remaining nitrate to nitrite, indicating the bacterium did not reduce nitrate during incubation.
No color change after adding zinc
Positive for nitrate reduction beyond nitrite, indicating the organism has reduced nitrate further, likely to nitrogen gas or ammonia.
Applications of the Nitrate Reduction Test
This test is particularly valuable for identifying bacteria within the Enterobacteriaceae family and differentiating facultative anaerobes. In environmental microbiology, the nitrate reduction test is useful for studying denitrification, a key component of the nitrogen cycle. Denitrifying bacteria, which are common in soil and water, convert nitrate into gaseous forms that recycle nitrogen back into the environment, important for soil fertility and wastewater treatment.
Summary
The nitrate reduction test is a useful microbiological tool that evaluates a bacterium’s ability to use nitrate as an electron acceptor in the absence of oxygen. By observing reactions after adding specific reagents and zinc powder, microbiologists can interpret whether the organism reduced nitrate to nitrite or beyond. This straightforward test aids in bacterial identification and contributes to understanding microbial roles in nitrogen cycling and other environmental processes.
Frequently Asked Questions (FAQ)
Explain about the uses of Nitrate Acid test in short?
The nitrate reduction test is a biochemical method used to assess if a bacterium can use nitrate as an electron acceptor under anaerobic conditions, helping in the identification of bacterial species, particularly among Enterobacteriaceae and other facultative anaerobes.
How the result of Nitrate Acid test are interpreted ?
Results from the nitrate reduction test are interpreted based on the color reactions observed after adding reagents
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