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。
硝酸盐还原试剂（锌还原剂）主要成分为锌粉，可配套硝酸盐还原试验试剂使用，常用于硝酸盐还原试验（Griess试验）中的硝酸盐的还原，主要用于肠杆菌、假单胞菌等的鉴别。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。
自备试剂和耗材
- 硝酸盐培养基、硝酸盐还原试验试剂
- 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）
操作步骤（仅供参考）
1、被检细菌接种于硝酸盐培养基，35℃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：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、锌还原剂有一定危险性，需谨慎操作；不得接触水，应避光，通风处保存。
7、为了您的安全和健康，请穿实验服并戴一次性手套操作。
Microbial respiration is primarily divided into aerobic respiration and anaerobic respiration. The oxidase test, catalase test, and nitrate reduction test are key steps in aerobic or anaerobic respiration and play important roles in bacterial identification.
The nitrate reduction reaction includes two processes: First, in the synthesis process, nitrate is reduced to nitrite and ammonia, and ammonia is subsequently converted into amino acids and other intracellular nitrogen-containing compounds. Second, in the catabolic process, nitrate or nitrite substitutes oxygen as the terminal hydrogen acceptor in the respiratory enzyme system. Bacteria capable of reducing nitrate obtain oxygen from nitrate to form nitrite and other reductive products. The nitrate reduction process varies among bacteria. Some bacteria only reduce nitrate to nitrite, such as Escherichia coli; some can reduce it to nitrite and ammonium ions; some bacteria can reduce nitrate or nitrite to nitrogen, such as Pseudomonas and Serratia species; others may fully utilize the reduction products in anabolic metabolism.
Nitrate Reduction Reagent (Zinc Reducing Agent) primarily consists of zinc powder. It can be used in conjunction with nitrate reduction test reagents and is commonly employed in nitrate reduction tests (Griess test) for the reduction of nitrate. It is mainly used for the identification of Enterobacteriaceae, Pseudomonas, etc. Bacteria such as Escherichia coli only reduce nitrate to nitrite. Under acetic acid conditions, nitrite reacts with sulfanilic 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 or denitrogenation, which produces bubbles but no red color appears.
This reagent is intended for research use only and is not suitable for clinical diagnosis or other purposes.
Reagents, consumables and Equipments not provided
Nitrate medium, Nitrate reduction test reagent
Constant temperature incubator, Test tubes, Alcohol burner
Inoculation loop, Inoculation hood, Precision balance (accuracy 0.1 mg)
Procedure (For Reference Only)
Inoculate the test bacteria into nitrate medium and incubate at 35°C for 1–4 days.
Mix equal volumes of sulfanilic acid solution and α‑naphthylamine solution to prepare the nitrate reduction test reagent.
Add 0.1 mL of the nitrate reduction test reagent to the culture medium and observe the result immediately.
If no red color appears after adding the nitrate reduction test reagent, add a small amount of zinc reducer (1–3 mg) to the test tube. If red color develops, it indicates that nitrate is still present in the medium. If no red color appears, it indicates that nitrate has been reduced to ammonia or nitrogen. A small inverted tube can be placed in the medium; if bubbles are generated, it indicates nitrogen gas production, helping to rule out false negatives.
Result Interpretation
Condition
| Color Result
| Interpretation (Example Organisms)
|
Without zinc reducer
| Red
| Positive (e.g., Enterobacteriaceae, Veillonella)
|
With zinc reducer
| Red
| Negative
|
With zinc reducer
| No color change
| False negative (e.g., Pseudomonas, Serratia)
|
Note: If no color reaction occurs after adding the reagent, possible reasons are: 1. Nitrate was not reduced – true negative; 2. Nitrate was reduced to ammonia, nitrogen, or other substances, leading to a false negative. In this case, add a small amount of zinc reducer. If red color appears, the test is truly negative; if no red color appears, the test is a false negative.
Precautions
Take appropriate self‑protection measures during bacterial culture and experimental procedures.
The incubation time of the test bacteria can also affect staining. Over‑incubation, death, or lysis of bacteria often leads to negative reactions.
Some anaerobic bacteria (e.g., Veillonella) may also show positive reactions.
Results must be judged immediately after adding the reagent; otherwise, rapid fading of the color may make interpretation difficult, as with Pseudomonas aeruginosa and Stenotrophomonas maltophilia.
Sulfanilic acid solution and α‑naphthylamine solution contain acetic acid and have an irritating odor; avoid inhalation.
Zinc reducer is potentially hazardous; handle with caution. Avoid contact with water, store protected from light and in a ventilated area.
For your safety and health, please wear a lab coat and disposable gloves during operation.
ZJ26F0131445
危险品化学品经营许可证（带存储）