Nitrate Reduction Test- Principle, Procedure, Types, Results, Uses

The nitrate reduction test has two main objectives:

• To determine the ability of an organism to reduce nitrate to nitrite or other nitrogen compounds using the enzyme nitrate reductase.

• To identify the organism based on its biochemical profile and its nitrate reduction pattern.

The nitrate reduction test is useful for characterizing and differentiating bacteria that can use nitrate as an alternative electron acceptor in anaerobic respiration. Some bacteria can reduce nitrate to nitrite, some can reduce it further to ammonia or nitrogen gas, and some cannot reduce it at all. The test can help to distinguish between different groups of bacteria, such as:

• Enterobacteriaceae, which are mostly nitrate reducers and can be further differentiated by their ability to produce gas or not.

• Pseudomonas, which are also nitrate reducers but do not ferment glucose.

• Neisseria and Moraxella, which are oxidase-positive and can be differentiated by their ability to reduce nitrate or not.

• Mycobacterium, which is acid-fast and can be differentiated by its ability to reduce nitrate or not.

• Corynebacterium, which is gram-positive rods and can be differentiated by their ability to reduce nitrate or not.

The nitrate reduction test is based on the ability of some bacteria to produce an enzyme called nitrate reductase, which catalyzes the reduction of nitrate (NO₃^-) to nitrite (NO₂^-) or other nitrogenous compounds. This enzyme is usually expressed under anaerobic conditions when nitrate serves as an alternative electron acceptor for bacterial respiration.

The test involves adding two reagents, sulfanilic acid (reagent A) and α-naphthylamine (reagent B), to the nitrate broth after incubation with the test organism. If nitrate is present in the broth, it will react with sulfanilic acid to form a colorless diazonium salt, which then reacts with α-naphthylamine to form a red-colored azo dye. The formation of red color indicates a positive result for nitrate reduction to nitrite.

However, some bacteria can further reduce nitrite to other nitrogenous compounds, such as nitric oxide (NO), nitrous oxide (N₂O), ammonia (N.H. ₃), or nitrogen gas (N₂). In this case, no red color will be formed after adding reagents A and B, even though nitrate has been reduced by the bacteria. To differentiate this scenario from a negative result (no nitrate reduction at all), a small amount of zinc dust (reagent C) is added to the broth. Zinc dust can reduce any remaining nitrate to nitrite, which will then react with reagents A and B to form the red color. Therefore, if red color appears after adding zinc dust, it means that the test organism did not reduce nitrate, and the result is negative. If no color change occurs after adding zinc dust, it means that the test organism reduced nitrate beyond nitrite, and the result is positive.

A Durham tube is also used in the nitrate broth to trap any gas produced by the bacteria during nitrate reduction. The presence of gas bubbles in the Durham tube indicates that the bacteria reduced nitrate to gaseous nitrogen compounds, such as N₂, N₂O, or NO.

Nitrate reduction test is useful for differentiating and identifying various bacterial species based on their biochemical profile. For example, it can help distinguish Neisseria gonorrhoeae (nitrate positive) from Kingella denitrificans (nitrate negative) or Mycobacterium tuberculosis (nitrate positive) from Mycobacterium bovis (nitrate negative).

The nitrate reduction test requires the following materials and equipment:

• Nitrate broth: This is the culture medium that contains potassium nitrate as the sole source of nitrogen for the bacteria. It also contains peptone as a source of carbon and energy. The broth is usually prepared by dissolving 20 grams of peptone and 2 grams of potassium nitrate in 1000 mL of distilled water. The broth is then dispensed into test tubes (4 mL per tube) and sterilized by autoclaving at 121°C and 15 lbs pressure for 15 minutes. Alternatively, ready-made nitrate broth can be purchased from commercial suppliers and used according to the manufacturer`s instructions.

• Durham tubes: These are small inverted glass tubes that are inserted into the test tubes containing the nitrate broth. They serve as indicators of gas production by the bacteria. The Durham tubes must be completely submerged in the broth and free of any air bubbles before use.

• Inoculating loop or needle: This is a metal wire with a loop or a needle at one end that is used to transfer a small amount of bacterial culture from a pure colony or a broth to the nitrate broth. The loop or needle must be sterilized by flaming before and after each use to avoid contamination.

• Incubator: This is a device that maintains a constant temperature and sometimes humidity for the growth of microorganisms. The incubator must be set at an appropriate temperature for the test bacteria, usually 35 ±2°C for most bacteria, or 25 to 30°C for glucose non-fermenting Gram-negative bacilli, or 35 ±2°C under anaerobic or microaerobic conditions for Campylobacter spp.

• Reagents A and B: These are chemical solutions that are used to detect the presence of nitrite in the nitrate broth after incubation. Reagent A is 0.8% sulfanilic acid in glacial acetic acid, and reagent B is 0.5% -naphthol (N, N-dimethyl- α-naphthylamine) in glacial acetic acid. These reagents can be prepared in the laboratory or purchased from commercial suppliers. They must be stored at 2 – 8°C and used within 3 months.

• Zinc dust: This is a fine powder of pure zinc metal that is used to confirm negative results of the nitrate reduction test. Zinc dust can reduce any remaining nitrate in the broth to nitrite, which can then react with reagents A and B to produce a red color. Zinc dust must be added in a small amount (not more than what adheres to the end of an applicator stick) and observed for color change within 10 minutes.

• Test organisms: These are pure cultures of bacteria that are used to perform the nitrate reduction test. The test organisms can be isolated from clinical specimens or obtained from reference collections. The test organisms must be fresh (24 hours old) and grown on appropriate media before inoculation into the nitrate broth. Some examples of test organisms are E. coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Mycobacterium tuberculosis, etc.

• Positive and negative controls: These are known cultures of bacteria that are used to check the quality of the medium and reagents, as well as the accuracy of the test procedure. Positive controls are bacteria that can reduce nitrate to nitrite or other nitrogen compounds, such as E. coli ATCC 25922 (nitrate positive, gas negative) or Pseudomonas aeruginosa ATCC 27853 (nitrate positive, gas positive). Negative controls are bacteria that cannot reduce nitrate, such as Acinetobacter baumannii ATCC 19606 (nitrate negative). The controls must be inoculated and tested in the same way as the test organisms.

These are the basic requirements for performing the nitrate reduction test. However, depending on the method (tube method, disk method, or rapid method), some additional materials and equipment may be needed, such as nitrate disks, glass slides, Petri plates, etc. These will be discussed in detail in point 7 (Procedure of Nitrate Reduction Test).

The culture media used for the nitrate reduction test is nitrate broth, which is a liquid medium that contains peptone as a source of carbon and nitrogen and potassium nitrate as a source of nitrate. The medium is clear and colorless and does not contain any indicators or inhibitors.

The composition of nitrate broth is as follows:

• Peptone: 20 grams

• Potassium nitrate: 2 grams

• Distilled water: 1000 mL

The preparation of nitrate broth involves the following steps:

• Mix the peptone and potassium nitrate in distilled water and dissolve them completely by shaking or heating.

• Transfer 4 mL of the medium into each test tube (or more medium based on the volume of the test tube or the amount that is enough to submerge the Durham tube).

• Insert an inverted Durham tube into each test tube to collect any gas produced by the bacteria.

• Tighten the screw cap or plug the tube with a cotton plug to prevent contamination.

• Autoclave the tubes at 121°C and 15 lbs pressure for 15 minutes to sterilize them.

• Let the tubes cool to room temperature before inoculating them with the test bacteria.

(Note: The composition and preparation of nitrate broth may vary slightly depending on the manufacturer or the protocol. The above-mentioned composition and preparation are taken from Leber, Amy L., editor-in-chief. (2016). Clinical microbiology procedures handbook (Fourth edition). Washington, DC: ASM Press 1752 N St., N.W., [2016]. )

Nitrate broth is used to test the bacteria`s ability to reduce nitrate to nitrite or other nitrogen compounds. The presence or absence of gas in the Durham tube and the color change after adding reagents indicate whether the bacteria are nitrate reducers or not. The details of the procedure and interpretation of results will be discussed in the following points.

The chemicals/reagents required for the nitrate reduction test are:

• 0.8% sulfanilic acid as reagent A

• 0.5% α-naphthol as reagent B

• Pure zinc metal dust

These reagents can be prepared as follows:

Preparation of 0.8% sulfanilic acid (Reagent A)

• Weigh 0.8 grams of sulfanilic acid and dissolve it in 70 mL of distilled water by heating.

• Cool the solution and add 30 mL of glacial acetic acid slowly while stirring.

• Store the reagent in a dark bottle at 2 - 8°C for up to 3 months.

Preparation of 0.5% α-naphthol (Reagent B)

• Weigh 0.5 grams of α-naphthol (N, N-dimethyl-α-naphthylamine) and dissolve it in 30 mL of glacial acetic acid by shaking.

• Add 70 mL of distilled water slowly while stirring.

• Store the reagent in a dark bottle at 2 - 8°C for up to 3 months.

Preparation of zinc dust

• Zinc dust can be purchased from a chemical supplier or prepared by grinding pure zinc metal with a mortar and pestle.

• Store the zinc dust in a dry and airtight container at room temperature.

These reagents are used to detect the presence or absence of nitrite in the culture medium after incubation. Reagent A reacts with nitrite to form a colorless diazonium salt, which then reacts with reagent B to form a red-colored azo dye. If nitrate is not present, zinc dust is added to reduce any remaining nitrate to nitrite and repeat the reaction. The formation of red color indicates a negative result for nitrate reduction, while the absence of red color indicates a positive result.

The equipment and test organisms required for the nitrate reduction test depend on the method used. There are three methods commonly used: the tube method, the disk method, and the rapid method. Each method has its own advantages and limitations.

The tube method is the most frequently used testing method. It involves inoculating a test tube containing nitrate broth and a Durham tube to collect gas. The test organisms can be any bacteria that are suspected of having nitrate reductase enzyme, which reduces nitrate to nitrite or other nitrogen compounds. Some examples of nitrate-reducing bacteria are E. coli, Pseudomonas aeruginosa, Salmonella typhimurium, Bacillus cereus, and Mycobacterium tuberculosis. The positive and negative controls for this method are E. coli ATCC 25922 as nitrate positive, gas negative; P. aeruginosa ATCC 27853 as nitrate positive, gas positive; and Acinetobacter baumannii ATCC 19606 as nitrate negative.

The disk method is used only for anaerobic organisms. It involves placing a nitrate disk on a fresh culture of the test organism and incubating it anaerobically for 24 to 48 hours. The test organisms can be any anaerobic bacteria that are capable of reducing nitrate. Some examples of anaerobic nitrate-reducing bacteria are Clostridium spp., Bacteroides spp., and Fusobacterium spp. The positive and negative controls for this method are not specified in the sources, but they should be similar to those of the tube method.

The rapid method is used for quick results if the organism is supposed to be a quick reducer of nitrate and multiply rapidly. It involves inoculating a test tube containing nitrate broth with a heavy inoculum of the test organism and incubating it at 35°C for 2 hours. The test organisms can be any bacteria that have a very short generation time and can reduce nitrate rapidly. Some examples of rapid nitrate-reducing bacteria are Enterobacter spp., Klebsiella spp., Proteus spp., and Serratia spp. The positive and negative controls for this method are not specified in the sources, but they should be similar to those of the tube method.

The equipment required for all methods is test tubes, inoculating loop, an incubator, reagents (sulfanilic acid, α-naphthylamine, zinc dust), and a glass slide or petri plate (for the disk method). The reagents are used to detect the presence of nitrite or unreduced nitrate in the medium by forming a red color.

For nitrate reduction tests, three methods, the tube method, the disk method, and the rapid method are commonly used. Among these methods, the tube method is the most frequently used testing method.

Tube Method

• Autoclave test tubes with nitrate broth and invert Durham`s tube, and let them cool to room temperature.

• In a tube, inoculate the test (sample) bacteria from an isolated colony of fresh (24 hours old) culture using an inoculating loop (or drop 2/3 drops of broth containing an overnight culture of the test organism).

• Incubate the tube at an appropriate temperature for the required time period.

• Glucose non-fermenting, Gram-negative bacilli at 25 to 30°C for 24 hours to 5 days.

• Other bacteria at 35 ±2°C for 24 hours to 5 days.

• Campylobacter spp. At 35 ±2°C for at least 3 days at anaerobic or microaerobic conditions.

• After 24 hours, look for visible growth and gas bubbles inside the Durham tube. If there is no gas and no visible growth, incubate the tube for the next 24 hours (or more based on test bacteria).

• If gas is present in the Durham tube in the culture of glucose non-fermenting bacteria, report the test as positive for nitrate reduction and gas production.

• If gas is not present in the Durham tube or if the test bacterium is a glucose fermenter, transfer 0.5 mL of well-mixed culture into another clean (need not to be sterile) test tube.

• Add 3 drops of reagent A and mix well by shaking gently.

• Add 3 drops of reagent B and mix well by shaking gently.

• Observe for the development of red color within 2 minutes.

• If no red color is developed, then add a small amount of zinc dust and observe for the development of the red color within 10 minutes.

• If there is no gas formation and no development of red color after the addition of both reagents A and B, reincubate the tubes and test accordingly after 48 hours and on the 5th day.

Disk Method

Used only for anaerobic organisms.

• On a fresh (24 hours/overnight old) culture of the test organism, place a nitrate disk in the area with heavy growth and incubate anaerobically for 24 to 48 hours.

• Place the nitrate disk on a clean glass slide or petri plate (need not be sterile).

• Add 1 drop of reagent A.

• Add 1 drop of reagent B.

• Observe for the development of red color within 2 minutes.

• If no red color is developed, then add a small amount of zinc dust and observe for the development of the red color within 5 minutes.

Rapid Method

It may not be as effective as the tube method, but it can be used for quick results if the organism is supposed to be a quick reducer of nitrate and multiply rapidly (have a very short generation time).

• Add 0.5 mL of nitrate broth in a clean test tube, autoclave it for 15 minutes at 15 lbs pressure and 121°C, and let it cool to room temperature.

• Inoculate the tube with a heavy inoculum of fresh bacterial culture.

• Incubate at 35°C for 2 hours.

• Add 2 drops of reagent A and 2 drops of reagent B and mix well.

• Observe for the development of red color within 2 minutes.

• If no red color is developed, add a small amount of zinc dust and observe for the development of the red color within 5 minutes.

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The interpretation of the nitrate reduction test is based on the color of the broth after the reagents are added. The possible outcomes are:

• Red color after adding reagents A and B: This indicates a positive result for nitrate reduction, meaning that the organism can reduce nitrate to nitrite. The nitrite reacts with the reagents to form a red azo dye. This result can be confirmed by adding reagent C (zinc dust), which should not cause any color change.

• No color change after adding reagents A and B: This indicates either a negative result or an incomplete result for nitrate reduction. There are two possibilities for this outcome:

• The organism cannot reduce nitrate at all, and the broth still contains nitrate. This can be verified by adding reagent C (zinc dust), which should cause a red color to appear, as zinc reduces nitrate to nitrite.

• The organism can reduce nitrate to nitrite and then further reduce nitrite to other nitrogen compounds, such as ammonia or nitrogen gas. In this case, there is no nitrite left in the broth to react with the reagents. This can be verified by adding reagent C (zinc dust), which should not cause any color change, as there is no nitrate left to be reduced.

• Gas production in Durham tube: This indicates a positive result for denitrification, meaning that the organism can reduce nitrate to nitrogen gas, which is trapped in the inverted tube. This result can be observed without adding any reagents, but it may not be visible until after 48 hours of incubation.

Some examples of bacteria that show different results for nitrate reduction tests are:

• Escherichia coli: Positive for nitrate reduction and gas production. The broth turns red after adding reagents A and B, and gas bubbles are present in the Durham tube.

• Pseudomonas aeruginosa: Positive for nitrate reduction but negative for gas production. The broth turns red after adding reagents A and B, but no gas bubbles are present in the Durham tube.

• Acinetobacter baumannii: Negative for nitrate reduction and gas production. The broth does not change color after adding reagents A and B but turns red after adding reagent C (zinc dust).

• Streptococcus pyogenes: Negative for nitrate reduction and gas production. The broth does not change color after adding reagents A and B or reagent C (zinc dust).

To ensure the accuracy and reliability of the nitrate reduction test, some quality control measures should be followed:

• Use fresh and properly sterilized nitrate broth and reagents. Check the expiry date and storage conditions of the reagents before use.

• Use appropriate positive and negative control strains in each batch of nitrate reduction tests to verify the performance of the reagents and procedures. For example, you can use E. coli ATCC 25922 as a positive control for nitrate reduction without gas production, P. aeruginosa ATCC 27853 as a positive control for nitrate reduction with gas production, and Acinetobacter baumannii ATCC 19606 as a negative control for nitrate reduction.

• Inoculate the test tubes with a pure culture of the test organism from an isolated colony or a broth culture. Avoid contamination or over-inoculation that may affect the results.

• Incubate the test tubes at the optimal temperature and time for the test organism. Different bacteria may have different growth rates and nitrate reduction abilities under different conditions.

• Observe the test tubes carefully for any signs of growth and gas production before adding the reagents. If there is no growth or gas production after the recommended incubation period, extend the incubation time or repeat the test with a fresh culture.

• Add the reagents A and B in the correct order and amount, and observe the color change within 2 minutes. A red color indicates nitrite production by nitrate reduction.

• If there is no color change after adding reagents A and B, add a small amount of zinc dust to detect any unreduced nitrate in the medium. A red color after adding zinc indicates a negative result for nitrate reduction, while no color change indicates a positive result for nitrate reduction to other nitrogen compounds.

• Record and interpret the results based on the presence or absence of gas production, color change, and zinc reaction. Compare the results with the expected outcomes of the control strains and the test organism.

By following these quality control measures, you can ensure that your nitrate reduction test is valid and reliable. You can also use this test to differentiate and identify various bacteria based on their biochemical characteristics.

The nitrate reduction test is a simple and useful biochemical test to determine the nitrate-reducing ability of bacteria. However, some precautions must be taken to ensure the accuracy and reliability of the test results. Here are some of the precautions that should be followed while performing the nitrate reduction test:

• Sterilize the medium properly before use, sterilize the working area, work in a sterile zone, wear proper personal protective equipment (PPE), and follow laboratory safety rules. This will prevent contamination and cross-reactions of the medium and the reagents with unwanted microorganisms or substances.

• Be careful while using α-naphthol as it is carcinogenic. Wear gloves and avoid direct contact with skin or eyes. Dispose of the used reagents and tubes in a biohazard container according to the laboratory protocol.

• Use the appropriate amount of medium in a test tube based on the size of the available test tube and Durham tube. The Durham tube must be completely submerged in the broth. This will ensure that any gas production by the bacteria can be detected and measured accurately.

• There must not be any gas bubbles in the Durham tubes before the test. If there are any bubbles, discard the tube and use a new one. This will avoid false-positive results due to trapped air or other gases in the tube.

• Don`t add too much zinc dust. It must not exceed the amount that adheres to the end of an applicator stick (like a toothpick). Adding too much zinc dust can cause false-positive results by reducing nitrate to nitrite, even if the bacteria did not reduce it.

• Observe for the development of red color within 2 minutes after adding reagents A and B and within 5 minutes after adding zinc dust. Do not incubate or wait longer than these time intervals, as the color may fade or change due to oxidation or other reactions.

• Use fresh (24 hours old) culture of the test organism for inoculation. Do not use old or contaminated cultures, as they may have altered or lost their nitrate-reducing ability or produce other interfering substances.

• Use appropriate positive and negative control organisms to check the quality of the medium and reagents. The control organisms should give consistent and expected results as described in the results and interpretation section. If not, discard the medium and reagents and prepare new ones.

• Report the results based on both gas production and color development after adding reagents A and B and zinc dust. Do not report only one criterion, as it may lead to false-negative or false-positive results depending on the type of bacteria and their nitrate-reducing ability.

• Consult with a microbiologist or an expert if you are unsure about the results or have any doubts or questions regarding the nitrate reduction test. Do not make assumptions or guesses based on incomplete or ambiguous data.

By following these precautions, you can perform the nitrate reduction test with confidence and accuracy. The nitrate reduction test is a valuable tool for identifying and differentiating bacteria based on their biochemical profile. It can help in diagnosing infections, monitoring environmental conditions, studying microbial ecology, and exploring new applications of nitrate-reducing bacteria.

The nitrate reduction test is a useful method for characterizing and identifying different bacterial species based on their ability to reduce nitrate to nitrite or other nitrogen compounds. Some of the applications of nitrate reduction tests are:

• Differentiating Mycobacterium species. Some Mycobacterium species, such as M. tuberculosis, M. kansasii, and M. bovis BCG, can reduce nitrate to nitrite, while others, such as M. bovis, M. africanum, and M. avium complex, cannot.

• Identifying species of Neisseria and separating them from Moraxella and Kingella species. Most Neisseria species can reduce nitrate to nitrite, while Moraxella and Kingella species cannot. The nitrate reduction test is a critical test for differentiating between N. gonorrhoeae and K. denitrificans, particularly when strains of K. denitrificans appear to be gram-negative diplococci in stained smears.

• Facilitating species identification of Corynebacterium. Some Corynebacterium species, such as C. diphtheriae and C. pseudotuberculosis, can reduce nitrate to nitrite, while others, such as C. xerosis and C. ulcerans, cannot.

• Confirming and identifying Enterobacterales. All members of the family Enterobacterales can reduce nitrate to nitrite. Some members can further reduce nitrite to nitrogen gas or other nitrogen compounds. Nitrate reduction test can help to differentiate between genera and species within Enterobacterales based on their patterns of gas production and color change after adding reagents and zinc dust.

• Understanding the biochemical characteristics of bacteria and their role in the nitrogen cycle. Nitrate reduction tests can reveal the metabolic capabilities of bacteria and their potential impact on the environment. Nitrate-reducing bacteria can participate in anaerobic respiration, denitrification, ammonification, or nitrogen fixation, depending on the type of enzyme they produce and the end products they form. Nitrate reduction tests can help to study these processes and their ecological implications.

The nitrate reduction test is a useful method to determine the ability of bacteria to reduce nitrate to various nitrogen compounds, but it also has some limitations that should be considered. Some of the limitations are:

• The test requires a special medium containing nitrate but free of any trace of nitrite, which may interfere with the detection of nitrite production by bacteria.

• The test may not be enough for the identification of bacterial species, as it is only one of the biochemical tests, and other tests are required for complete characterization and differentiation.

• The test may give false-negative results if the bacteria do not grow well in the medium or if the nitrite produced by bacteria is further reduced to other nitrogen compounds that do not react with the reagents. To avoid this, zinc dust is added to verify the presence of unreduced nitrate in the medium, but this may also introduce errors if too much zinc dust is added or if the zinc dust is contaminated with nitrate.

• The test may give false-positive results if the medium is contaminated with nitrite or if other substances in the medium react with the reagents to produce a red color. To avoid this, negative control with an uninoculated medium should be included, and the color reactions should be observed immediately after adding the reagents, as they may fade over time.

• The test may not detect some bacteria that reduce nitrate only under anaerobic conditions or that require specific cofactors or substrates for nitrate reduction. To overcome this, different methods, such as the disk method or the rapid method, may be used for anaerobic bacteria or fastidious bacteria, respectively.

• The test may not differentiate between different types of nitrogen compounds produced by bacteria, such as nitrate, ammonia, nitric oxide, nitrous oxide, or nitrogen gas. To determine the exact end product of nitrate reduction, other tests such as gas chromatography or mass spectrometry may be required.

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