The respiration of microorganisms is mainly divided into aerobic respiration and anaerobic respiration. The oxidase reaction, catalase reaction, and nitrate reaction are key steps in the respiratory process and play an important role in bacterial identification. Nitrate reduction reactions are involved in two processes of microbial synthesis and decomposition: In the synthetic process, nitrate is reduced to nitrite and ammonia, which are then converted into amino acids and other nitrogen-containing intracellular compounds. In the catabolic process, nitrate or nitrite replaces oxygen as the terminal hydrogen acceptor in the respiratory enzyme system. Bacteria capable of nitrate reduction obtain oxygen from nitrate to form nitrite and other reductive products. The nitrate reduction process varies among different bacterial species. Some bacteria only reduce nitrate to nitrite, such as Escherichia coli; some can reduce nitrate to both nitrite and ionic ammonium; some bacteria can reduce nitrate or nitrite to nitrogen gas, such as species of the genera Pseudomonas and Serratia; some bacteria can also fully utilize their reductive products in synthetic metabolism.
Aladdin Nitrate Reduction Test Reagent, also known as Griess Reagent or Nitrite Reagent, mainly consists of sulfanilic acid solution, α-naphthylamine solution, and zinc reducing agent. It is primarily used for the identification of Enterobacteriaceae, Pseudomonas, and other bacterial strains. Bacteria such as Escherichia coli can only reduce nitrate to nitrite. Under the action of acetic acid, nitrite reacts with sulfanilic acid in the reagent to form diazobenzenesulfonic acid, which then binds to α-naphthylamine to produce a fuchsine to red-colored product. Bacteria such as Pseudomonas and Serratia can reduce nitrate or nitrite to nitrogen gas or nitrogen oxides; this process is called denitrification, which produces gas bubbles but no red coloration.This reagent is intended for research use only and is not suitable for clinical diagnosis or any other purposes.
Product Components and Storage Conditions:
N1510445
| Component
| 2×10mL
| 2×50mL
| Storage
|
N1510445A
| Sulfanilic Acid Solution
| 10mL
| 50mL
| RT. Store in the dark.
|
N1510445B
| α-Naphthylamine Solution
| 10mL
| 50mL
| RT. Store in the dark.
|
N1510445C
| Zinc Reducing Agent
| 0.2g | 1g | RT. Store in the dark. |
Materials Required (Self-prepared):
Nitrate medium, analytical balance (precision: 0.1 mg), constant-temperature incubator, inoculation loops, test tubes, alcohol lamps, etc.
Operating Procedures (For Reference Only):
1. Inoculate the test bacteria into the nitrate medium and incubate at 35°C for 1–4 days.
2. Mix equal volumes of an appropriate amount of sulfanilic acid solution and α-naphthylamine solution to prepare the nitrate reduction test reagent.
3. Add 0.1 mL of the nitrate reduction test reagent to the medium and observe the result immediately.
4. If no red color develops after adding the nitrate reduction test reagent, add a small amount of zinc reducing agent (1–3 mg) to the test tube, shake for 2–5 minutes to allow sufficient reaction. A red color indicates that nitrate is still present in the medium; if no red color still develops, it means the nitrate has been reduced to ammonia or nitrogen gas. Place a small inverted Durham tube inside the medium. The production of gas bubbles indicates nitrogen gas generation, which can rule out false negatives.
Staining Results:
Fuchsine to red color without adding zinc reducing agent
| Positive (Enterobacteriaceae, Veillonella)
|
Fuchsine to red color after adding zinc reducing agent
| Negative
|
No color change after adding zinc reducing agent
| False negative (Pseudomonas, Serratia, etc.) |
Note:No color reaction after reagent addition may result from two causes:
Nitrate has not been reduced, indicating a true negative result of the test.
Nitrate has been reduced to other substances such as ammonia and nitrogen, leading to a false negative result.
In this case, add a small amount of zinc reducing agent. A red color indicates a true negative result, while no red color indicates a false negative result.
Precautions:
1. Personal protection should be taken during bacterial culture and the experiment.
2. The incubation time of the test bacteria can also affect the reaction results. A positive bacterial strain may show a negative reaction if incubated for an excessively long time, or if the bacteria have died or lysed.
3. Certain anaerobic bacteria (e.g., Veillonella) can also yield a positive reaction.
4. The test results must be interpreted immediately after adding the reagent. Otherwise, rapid fading of the color will make result judgment difficult, especially for strains such as Pseudomonas aeruginosa and Stenotrophomonas maltophilia.
5. Sulfanilic acid solution and α-naphthylamine solution contain acetic acid and have a pungent odor; inhalation should be avoided.
6. Zinc reducing agent can be replaced with zinc granules. 2–8 granules or 5–20 mg of zinc granules are equivalent to 1–3 mg of zinc reducing agent.
7. For your safety, wear a lab coat and disposable gloves during the operation.
8. Please use the reagents as soon as possible after opening to avoid affecting subsequent experimental results.
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