微生物的呼吸作用主要分为有氧呼吸和无氧呼吸，氧化酶试验、过氧化氢酶试验与硝酸盐还原试验为有氧呼吸或无氧呼吸的重要环节，在细菌鉴定中起着重要作用。
硝酸盐还原反应包括两个过程：一是在合成过程中，硝酸盐还原为亚硝酸盐和氨，再由氨转化氨基酸和细胞内其他含氮化合物；二是在分解代谢过程中，硝酸盐或亚硝酸盐代替氧作为呼吸酶系统中的终末受氢体，能使硝酸盐还原的细菌从硝酸盐中获得氧而形成亚硝酸盐和其他还原性产物。硝酸盐还原过程可因细菌不同而异。有的细菌仅使硝酸盐还原为亚硝酸盐，如大肠埃希氏菌等;有的细菌可使其还原为亚硝酸盐和离子态的铵；有的细菌能使硝酸盐或亚硝酸盐还原为氮，如假单胞菌和沙雷菌属等；有的细菌还可以将其还原产物在合成性代谢中完全利用。
硝酸盐还原试验试剂 (Griess试剂，不含锌还原剂) 又称作 Griess 试剂、亚硝酸盐试剂等，主要由对氨基苯磺酸溶液和α-萘胺溶液组成，不含锌还原剂，主要用于鉴别肠杆菌、假单胞菌等。大肠埃希氏菌等细菌仅能使硝酸盐还原为亚硝酸盐，在乙酸作用下亚硝酸盐与试剂中的对氨基苯磺酸作用生成重氮基苯磺酸，后者与α-萘胺结合生成红色产物；假单胞菌、沙雷菌等细菌能使硝酸盐或亚硝酸盐还原为氮或氧化氮，该作用被称为脱硝化或脱氮化作用，产生气泡，但无红色出现。该试剂仅用于科研领域，不适用于临床诊断或其他用途。
产品组分及储存条件：
N1510381
| Component
| 2×50mL
| Storage
|
N1510381A
| 对氨基苯磺酸溶液
| 50mL
| RT. Store in the dark.
|
N1510381B
| α-萘胺溶液
| 50mL
| RT. Store in the dark. |
自备材料：
硝酸盐培养基、恒温培养箱、试管、酒精灯、接种环、接种箱、精密天平 (精度值 0.1mg)、锌还原剂
操作步骤(仅供参考)：
1、被检细菌接种于硝酸盐培养基，35°C培养 1~4天。
2、取适量的对氨基苯磺酸溶液和α-萘胺溶液等量混合后即为硝酸盐还原试验试剂。
3、取 0.1ml硝酸盐还原试验试剂加入培养基中，立即观察结果。
4、若加入硝酸盐还原试验试剂不出现红色，需向试管中加入少许锌还原剂 (1~3mg)，如出现红色，则表示培养基中仍存在硝酸盐；若仍不产生红色，则表示硝酸盐已被还原为氨或氮，可在培养基内加入1支小倒管，若有气泡产生，表示有氮气生成，可排除假阴性。
染色结果：
未加锌还原剂呈红色
| 阳性 (肠杆菌科细菌、韦荣球菌）
|
加锌还原剂呈红色
| 阴性
|
加锌还原剂不变色
| 假阴性 (假单胞菌、沙雷菌等)
|
注意：加入试剂无颜色反应。可能原因：1、硝酸盐没有被还原，实验确为阴性。2、硝酸盐被还原为氨和氮等其他物质，进而导致假阴性。这时可加入少许锌还原剂，如出现红色则说明实验确为阴性，如果未出现红色则说明实验为假阴性。
注意事项：
1、取细菌培养和实验中，应注意自我防护。
2、待检细菌培养时间也会影响染色，阳性菌培养时间过长或已死亡或细菌溶解，都常呈阴性反应。
3、某些厌氧菌 (韦荣球菌) 亦呈阳性反应。
4、必须在加入试剂之后立即判定结果，否则因颜色迅速褪色而造成判定困难，如铜绿假单胞菌、嗜麦芽窄食单胞菌等。
5、对氨基苯磺酸溶液和α-萘胺溶液含有乙酸，有刺激性气味，应避免吸入。
6、为了您的安全和健康，请穿实验服并戴一次性手套操作。
7、本产品仅用于科研，严禁他用。
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.
ZJ26F0838270
危险品化学品经营许可证（带存储）