| 产品名称： | 吲哚亚硝酸盐培养基；胰蛋白胨硝酸盐培养基 |
|---|---|
| 英文名称: | Indole Nitrite Medium；Tryptic Nitrate Medium |
| 培养基类型: | 营养培养基 |
| 级别: | for microbiology |
| 品牌: | ELITE-MEDIA |
| 产品目录号: | M1123-01、M1123-02、M1123-03 |
| 产品规格: | 27g、20支、250g |
| 产品外观: | 乳白色至浅黄色粉末。 |
| 颜色与澄清度: | 淡黄色透明溶液。 |
| 保存条件: | 密封，2-25度保存。 |
| 注意事项: | 避免呼入和皮肤接触。 |
| 相关产品: | 厌氧菌硝酸盐试剂（Anaerobe Nitrate Reagent） |
产品描述：
吲哚亚硝酸盐培养基（Indole Nitrite Medium）也称胰蛋白胨硝酸盐培养基（Tryptic Nitrate Medium），推荐用于 检测微生物的吲哚产生和硝酸盐还原能力，在微生物鉴定过程中十分重要。吲哚亚硝酸盐培养基适用于好氧菌、微好氧 菌、兼性厌氧菌、专性厌氧菌。吲哚亚硝酸盐培养基是结合了吲哚试验和硝酸盐还原试验的测试培养基，适合多种细菌。低含量的琼脂为培养基提供了不同程度的厌氧环境，满足好氧菌、兼性厌氧菌和专性厌氧菌的生长需求。吲哚亚硝酸盐培养基可以用于辅助拟杆菌、普氏菌、梭杆菌、梭状芽抱杆菌鉴别到菌种级别。吲哚亚硝酸盐培养基不推荐用于大肠杆菌和肠道菌的吲哚试验，因为 这些细菌能强烈还原硝酸盐，掩蔽吲哚试验的颜色变化，导致产生假阴性结果。
原理：
吲哚试验是一种定性操作，用于确定细菌产吲哚的能力。酪蛋白胨是色氨酸的来源。某些微生物能够产生色氨酸酶，将色氨酸转化为吲哚。吲哚与4-(二甲氨基)苯甲醛（Kovacs试剂）反应，在酸性条件下产生红色反应，表明阳性反应。硝酸盐还原试验是一种定性操作，用于确定细菌还原硝酸盐的能力。具有硝基还原酶的微生物还原硝酸盐的能力不同。 硝酸钾被还原成亚硝酸盐，后者进一步被还原成氮气或氨。硝酸盐还原的终产物取决于细菌菌种。
如果德拉姆管中没有气体存在，有必要检测检测该种微生物是否具有还原硝酸盐为亚硝酸盐的能力。亚硝酸盐的存在可 以由加入厌氧硝酸盐试剂A（对氨基苯磺酸溶液）和厌氧硝酸盐试剂B（1,6-克利弗酸溶液）后产生红色化学物来确定。 对氨基苯磺酸与亚硝酸盐反应产生一种重氮盐，继而与2-萘胺-6-磺酸结合，产生一种红色染料络合物。
加入了厌氧硝酸盐试剂A和B后，如果没有红色反应，表明两种可能性：培养基中的硝酸盐没有被微生物还原；或者微生 物已经将硝酸盐完全还原为氮气或者其它氮气衍生物，如氨，此时硝酸盐还原反应为阳性。如果一种微生物不具有硝酸盐还原酶，硝酸盐仍存在于培养基中。硝酸盐可以通过加入试剂C（锌粉）检测出来。锌能将硝酸盐转换成亚硝酸盐，形 成红色络合物，证明该种微生物不能还原硝酸盐。
另外，如果微生物已经将培养基中的硝酸盐完全还原，加入锌粉后将不会产生颜色变化。如果德拉姆管中有气体产生， 并且加入锌粉后无颜色变化，可以得出结论：硝酸盐已经被完全转变为氮气。而如果德拉姆管中无气体存在，并且加入 锌粉后无颜色变化，可以假设硝酸盐已经完全被还原为氨或其它还原型氮化合物。
测试用的分离物必须来自纯培养物，18-24h后的培养物。
硝酸盐还原颜色反应必须立即观察，因为阳性颜色反应会很快褪去（加入试剂5min后）。为了避免假阴性硝酸盐还原反 应，阴性亚硝酸盐反应必须经加入试剂C来验证。
由于氮气还原对于某些微生物来说可能需要长达48h来完成，如果要在48h之前获得结果，推荐接种多个试管中。
如果前试验结果是阴性的，最终的验证试验应该在培养48h时进行。
配方与配制方法：
| 成分 | g/L |
| 胰蛋白月示 | 20 |
| Na2HPO4 | 2 |
| D-葡萄糖 | 1 |
| KNO3 | 1 |
| 琼脂 | 1 |
| Final pH 7.2±0.2 |
配制方法：
用去离子水溶解27g培养基。
分装11ml到16×150 mm 试管中。
118°C高温蒸汽灭菌15min。
实验方法：
Method of Use:1. Allow the medium to warm to room temperature prior to inoculation. When product is being used with obligate anaerobes, it may be necessary to pre-reduce the media, by boiling for 2 minutes and allowing to cool, prior to use.
2. Select a well-isolated colony of the organism to be tested. Using a sterile loop, inoculate a tube of the prepared medium.
3. Incubate the inoculated media in aerobic atmosphere at 35ºC. for 24-48 hours. If testing for anaerobes ensure the media cap is tightened.
4. Observe for growth and the presence of gas bubbles in the Durham tube. If gas is present and the organism is known to be a non-fermenter, the test is considered positive for nitrate reduction.
5. If growth is apparent but no gas is present, or if gas is present and the organism is known to be a fermenter, proceed with the following steps:
To Test for Indole Production:
1. Aliquot approximately 1.5ml of Indole Nitrate Medium into an additional test tube. Add 4-5 drops of the Kovacs Indole Reagent and shake gently. Observe for a pink to red color, indicative of a positive result.
To Test for Nitrate Reduction:
1. Use the original tube of media to test for nitrate reduction.
2. A Durham tube is included for detecting those organisms capable of reducing nitrate to nitrogen gas. A positive result can be presumptively noted if gas is present in the Durham tube. However, it is necessary to proceed through steps 3-5 to confirm that gas production is from the complete reduction of nitrate to nitrogen gas.
3. Add five drops of Anaerobe Nitrate Reagent A and three drops of Anaerobe Nitrate Reagent B to the broth. Note: Add reagents in the order listed. Gently shake tube to mix reagents. Observe for the development of a deep red color within two minutes following application of the reagents. Color reactions with a positive test may fade rapidly (as early as five minutes).
4. If a red color does not result after step 3, add approximately 6.0mg of Reagent C to the medium. Observe for the development of a red color within 5-10 minutes following the addition of zinc dust. A red color indicates that nitrate was not reduced.
5. After the addition of zinc, the absence of a red color reaction indicates the complete reduction of nitrate. Absence of a red color upon the addition of zinc and gas in the Durham tube confirms that the gas produced is due to the reduction of nitrate to nitrogen gas. If there is no gas in the Durham tube and no color change upon the addition of zinc, it indicates that nitrate was completely reduced to ammonia.
