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

Table of Contents

• Introduction
• Objectives
• Principle of Nitrate Reduction Test
• Requirements
• Culture Media
• Chemicals/Reagents
• Equipment
• Test organism (Sample bacteria)
• Procedure of Nitrate Reduction Test
• Tube Method
• Disk Method
• Rapid Method
• Results and Interpretation of Nitrate Reduction Test
• Quality Control
• Bacteria Showing Positive Result
• Bacteria Showing Negative Result
• Precautions 
• Applications of Nitrate Reduction Test
• Limitations of Nitrate Reduction Test

Introduction

• In anaerobic respiration, bacteria obtain oxygen from nitrate (NO3–) or use NO3– as a terminal electron acceptor.
• Nitrate is initially reduced to nitrite (NO2–) and may subsequently be reduced to molecular nitrogen gas (N2), ammonia (NH3), hydroxylamine, or other end products.
• The further reduction of nitrite depends on the bacterium's metabolism or the enzymes present.
• Bacteria capable of reducing nitrate are known as nitrate-reducing bacteria or denitrifying bacteria.
• Nitrate-reducing bacteria are crucial in soil microbiology and ecology by recycling nitrogen.
• These bacteria produce nitrate reductase enzymes that convert nitrate into nitrite.
• Many clinically significant bacteria also have the ability to reduce nitrate.
• Identifying the nitrate-reducing ability is important for distinguishing several bacterial species.
• The nitrate reduction test is a laboratory biochemical test used to determine a bacterium's ability to reduce nitrate to nitrite.
• In the laboratory, bacteria are cultured in media containing a nitrate compound.
• An acid solution of sulfanilic acid and alpha-naphthol is added to the culture to test for nitrate reduction.

Objectives

• To determine the nitrate-reducing ability (production of nitrate reductase enzyme) of the test bacteria.
• To identify the test bacteria based on its biochemical profile.

Principle of Nitrate Reduction Test

• Nitrate-reducing organisms produce the nitrate reductase enzyme, which reduces nitrate to nitrite.
• The resulting nitrite reacts with acetic acid to form nitrous acid.
• Nitrous acid is diazotized with sulfanilic acid to create a colorless diazonium salt (diazotized sulfanilic acid).
• When this colorless compound reacts with dimethyl-naphthylamine (α-naphthol), a water-soluble red-colored azo dye (p-Sulfobenzene-azo-naphthylamine) is formed.
• Some organisms can further reduce nitrite to other nitrogen compounds, preventing the formation of the red color despite nitrate reduction.
• Similarly, if nitrate is not reduced, the red color will not form.
• To differentiate these scenarios, zinc dust is added to detect unreduced nitrate.
• Zinc reduces nitrate to nitrite, leading to the formation of the red-colored azo dye.

Requirements

Culture Media

• Nitrate broth is used to test a bacterium's ability to reduce nitrate.

Composition:

• Peptone: 20 grams (If the test bacteria are fastidious, use 25 grams of heart infusion broth powder instead of peptone)
• Potassium nitrate: 2 grams
• Distilled water: 1000 mL

Preparation:

• Mix the components in the specified proportions:
• Peptone: 20 grams (if the test bacteria are fastidious, use 25 grams of heart infusion broth powder instead)
• Potassium nitrate: 2 grams
• Distilled water: 1000 mL
• If using ready-made agar, measure and mix with distilled water as directed by the manufacturer.
• Shake well to completely dissolve the mixture.
• Transfer 4 mL of the medium into a 16 × 125 mm test tube (or more medium depending on the test tube volume or enough to submerge the Durham tube).
• Tighten the screw cap (or plug the tube with a cotton plug).
• Autoclave at 121°C and 15 lbs pressure for 15 minutes.

(Note: The composition may vary based on manufacturing companies. The above-mentioned composition is from Leber, Amy L., editor-in-chief. (2016). Clinical Microbiology Procedures Handbook (Fourth edition). Washington, DC: ASM Press.)

Chemicals/Reagents

• 0.8% sulfanilic acid as reagent A
• 0.5% α-naphthol as reagent B
• Pure zinc metal dust

Preparation of 0.8% sulfanilic acid (Regent A)

• Sulfanilic acid: 0.8 grams
• Distilled water: 70 mL
• Glacial acetic acid: 30 mL

Instructions:

• Mix the sulfanilic acid with distilled water and heat until completely dissolved.
• Allow the mixture to cool.
• Slowly add the glacial acetic acid to the cooled mixture.
• Store the solution at 2–8°C for up to 3 months.

Preparation of 0.5% -naphthol (Reagent B)

• α-Naphthol (N,N-dimethyl-α-naphthylamine): 0.5 grams
• Distilled water: 70 mL
• Glacial acetic acid: 30 mL

Instructions:

• Mix the glacial acetic acid with distilled water.
• Slowly add the α-naphthol to the mixture.
• Shake well until completely mixed.
• Store the solution at 2–8°C for up to 3 months.

Equipment

• Test tubes
• Dropper
• Personal protective equipment (PPE) and other general laboratory materials
• Durham tubes
• Autoclave
• Bunsen burner
• Test tube holder
• Micropipette
• Inoculating loop
• Nitrate disk (only for the disk method of nitrate reduction test of anaerobic bacteria)

Test organism (Sample bacteria)

Positive Controls:

• E. coli ATCC 25922: Nitrate positive, gas negative
• P. aeruginosa ATCC 27853: Nitrate positive, gas positive

Negative Control:

• Acinetobacter baumannii ATCC 19606: Nitrate negative

Procedure of Nitrate Reduction Test

Nitrate reduction tests are frequently performed using three methods: the tube method, the disk method, and the fast method. The tube method is the most widely utilized testing procedure. 

Tube Method

1. Autoclave test tubes containing nitrate broth and invert Durham tubes, allowing them to cool to room temperature.
2. Inoculate the test bacteria into the tube from a fresh culture using an inoculating loop or by adding 2/3 drops of broth from an overnight culture.
3. Incubate at the appropriate temperature and duration according to the bacteria being tested.
4. Check for visible growth and gas bubbles in the Durham tube after 24 hours; if absent, continue incubation and recheck.
5. If gas is present in glucose non-fermenting bacteria, report as positive for nitrate reduction and gas production.
6. If no gas is produced or the bacterium is a glucose fermenter, transfer culture to a new tube and add reagents A and B.
7. Observe for the development of red color; if absent, add zinc dust and observe for color change.
8. If no gas or red color is observed, reincubate and retest after 48 hours and on the 5th day.

Disk Method

Only for anaerobic organisms:

1. Place a nitrate disk on heavy growth of the test organism on a fresh culture.
2. Incubate anaerobically for 24 to 48 hours.
3. Add reagents A and B and observe for red color development; if absent, add zinc dust and observe for color change.

Rapid Method

Although it might not be as efficient as the tube method, it can still be used to get results quickly if the organism is intended to grow quickly and reduce nitrate quickly (have a very fast generation time).

1. Autoclave nitrate broth in test tubes, inoculate with fresh bacterial culture, and incubate.
2. Add reagents A and B after 2 hours of incubation and observe for red color development; if absent, add zinc dust and observe for color change.

Results and Interpretation of Nitrate Reduction Test

1. Gas bubbles in the culture of glucose non-fermenting bacteria indicate nitrate reduction positive, gas positive.
2. Red color formation after the addition of reagents, along with gas in the Durham tube, indicates nitrate reduction positive, gas positive.
3. Red color formation after the addition of reagents, without gas in the Durham tube, indicates nitrate reduction positive, gas negative.
4. No red color formation after the addition of reagents A and B, and also, no red color formation after the addition of zinc dust, indicates nitrate reduction positive (nitrate is reduced to nitrite, and nitrite is further reduced to other nitrogen compounds, leaving nothing to turn red).
5. No red color formation after the addition of reagents A and B, but the subsequent formation of red color after the addition of zinc dust, indicates nitrate reduction negative (nitrate has not been reduced before adding zinc; upon adding zinc, the untouched nitrate reduces to nitrite, producing the red color).

Quality Control

To assess the quality of the medium and reagents, label test tubes as "positive control, gas positive," "positive control, gas negative," and "negative control," and inoculate them with P. aeruginosa ATCC 27853, E. coli ATCC 25922, and Acinetobacter baumannii ATCC 19606, respectively. Follow the outlined procedures and interpret the final results after the addition of reagents and zinc dust.

• "Positive control, gas positive": Inoculate with P. aeruginosa ATCC 27853. Expected results are nitrate-positive (red color formation) and gas-positive.
• "Positive control, gas negative": Inoculate with E. coli ATCC 25922. Expected results are nitrate-positive (red color formation) and gas-negative.
• "Negative control": Inoculate with Acinetobacter baumannii ATCC 19606. Expected results are nitrate negative (no red color formation).

Bacteria Showing Positive Result

E. coli, Salmonella typhimurium, Enterobacter spp., Bacillus cereus, Mycobacterium tuberculosis, Citrobacter spp., Pseudomonas spp., Klebsiella spp., Proteus spp., S. aureus, and other bacterial species.

Bacteria Showing Negative Result

Species such as Acinetobacter spp., Streptococcus spp., Mycobacterium bovis, M. africanum, and others.

Precautions 

• Ensure the medium is thoroughly sterilized before use and maintain a sterile working area. Adhere to proper personal protective equipment (PPE) guidelines and laboratory safety protocols. Exercise caution when handling α-naphthol due to its carcinogenic properties.
• Use an appropriate volume of medium in each test tube, considering the size of the tube and ensuring the Durham tube is fully submerged in the broth. Prior to testing, ensure there are no gas bubbles present in the Durham tubes.
• When adding zinc dust, avoid using excessive amounts. Only apply an amount that adheres to the end of the applicator stick, similar to a toothpick.

Applications of Nitrate Reduction Test

1. Understanding the biochemical traits of bacteria for phenotypic identification.
2. Distinguishing Moraxella catarrhalis from Neisseria spp. and Kingella spp. from Neisseria spp. (N. gonorrhoeae and K. denitrificans).
3. Confirming and identifying Enterobacterales.
4. Discriminating Mycobacterium spp.
5. Distinguishing and identifying Corynebacterium spp.

Limitations of Nitrate Reduction Test

1. Biochemical tests are essential but only constitute part of bacterial species identification, necessitating additional tests for comprehensive identification.
2. Special medium containing nitrate, like nitrate broth, is imperative, devoid of even trace amounts of nitrite.
3. Observing organism growth in nitrate broth can be challenging.
4. Verification involves adding zinc dust to avoid false negative results in case nitrite further reduces to other nitrogen compounds.
5. A higher quantity of zinc dust can lead to false positive results.
6. Determining if the organism is a glucose fermenter with gas production prior to the nitrate reduction test is crucial for accurate reporting.
7. Due to its culture-based nature, the test typically requires 24 hours to 5 days before a negative result can be reported.