The respiration of microorganisms is mainly divided into aerobic respiration and anaerobic respiration. The oxidase test, catalase test, and nitrate reduction test are important links in aerobic or anaerobic respiration, and play a vital role in bacterial identification.
Nitrate reduction reaction consists of two processes: First, in the synthetic process, nitrate is reduced to nitrite and ammonia, which are then converted into amino acids and other nitrogen-containing compounds in cells. Second, 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 process of nitrate reduction varies with different types of bacteria. Some bacteria only reduce nitrate to nitrite, such as Escherichia coli; some bacteria can reduce it to nitrite and ionic ammonium; some bacteria can reduce nitrate or nitrite to nitrogen, such as Pseudomonas and Serratia; and some bacteria can also fully utilize the reduction products in synthetic metabolism.
Nitrate Reduction Test Reagent (Griess Reagent, without zinc reductant) is also known as Griess Reagent or Nitrite Reagent. It is mainly composed of p-aminobenzenesulfonic acid solution and α-naphthylamine solution, and does not contain zinc reductant. It is mainly used for the identification of Enterobacteriaceae, Pseudomonas and other bacteria. Bacteria such as Escherichia coli can only reduce nitrate to nitrite. Under the action of acetic acid, nitrite reacts with p-aminobenzenesulfonic acid in the reagent to form diazobenzenesulfonic acid, which then combines with α-naphthylamine to produce a red product. Bacteria such as Pseudomonas and Serratia can reduce nitrate or nitrite to nitrogen or nitrogen oxides, a process known as denitrification, which produces gas bubbles but no red color. This reagent is for research use only and is not suitable for clinical diagnosis or other purposes.
Product Components and Storage Conditions:
N1510381
| Component
| 2×50mL
| Storage
|
N1510381A
| p-Aminobenzenesulfonic Acid Solution
| 50mL
| RT. Store in the dark.
|
N1510381B
| α-Naphthylamine Solution
| 50mL
| RT. Store in the dark. |
Materials to Be Prepared by the User:
Nitrate medium, constant-temperature incubator, test tubes, alcohol lamp, inoculation loop, inoculation chamber, analytical balance (accuracy: 0.1 mg), zinc reductant
Operating Procedures (For Reference Only):
1. Inoculate the test bacteria onto the nitrate medium and incubate at 35°C for 1–4 days.
2. Mix equal volumes of an appropriate amount of p-aminobenzenesulfonic 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 appears after adding the nitrate reduction test reagent, add a small amount of zinc reductant (1–3 mg) to the test tube. A red color indicates that nitrate is still present in the medium; if no red color develops, it means that nitrate has been reduced to ammonia or nitrogen. Place a small inverted tube in the medium—gas bubbles indicate nitrogen production, which can rule out false negatives.
Staining Results:
Red color without zinc reductant addition
| Positive (e.g., Enterobacteriaceae, Veillonella)
|
Red color after zinc reductant addition
| Negative
|
No color change after zinc reductant addition
| False negative (e.g., Pseudomonas, Serratia)
|
Note: If no color reaction occurs after reagent addition, the possible reasons are as follows: 1. Nitrate has not been reduced, and the test result is truly negative. 2. Nitrate has been reduced to other substances such as ammonia and nitrogen, leading to a false negative. In this case, add a small amount of zinc reductant. A red color confirms a true negative result, while no red color indicates a false negative.
Precautions:
1. Pay attention to personal protection during bacterial culture and the experiment.
2. The incubation time of the test bacteria can also affect the test results. Over-incubation, death or lysis of positive bacteria often leads to a negative reaction.
3. Certain anaerobic bacteria (e.g., Veillonella) can also yield positive results.
4. The result must be determined immediately after adding the reagent; otherwise, rapid fading of the color will make judgment difficult, such as in the case of Pseudomonas aeruginosa and Stenotrophomonas maltophilia.
5. The p-aminobenzenesulfonic acid solution and α-naphthylamine solution contain acetic acid and have an irritating odor—avoid inhaling the fumes.
6. For your safety and health, wear a lab coat and disposable gloves during operation.
7. This product is for research use only and must not be used for any other purposes.
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