细菌的生化试验(也称生化反应)是指由于不同细菌具有各自的酶系统，对底物的分解能力不同，由此产生的代谢产物也不同，通过生物化学的方法测定这些代谢产物的过程，生化试验主要包括碳水化合物的生化试验、氨基酸和蛋白质的代谢试验、碳源和氮源的利用试验、酶类的代谢试验等，不同的细菌对蛋白质的分解能力不同，一般先由胞外酶把蛋白质分解为短肽或氨基酸，侵入细菌体内后由胞内酶把肽类分解为氨基酸苯丙氨酸脱氨酶试验试剂也称作三氯化铁试剂，主要由三氯化铁和去离子水组成，苯丙氨酸脱氨酶试验的基本原理是有些细菌能产生苯丙氨酸脱氨酶，使苯丙氨酸脱去氨基生成苯丙酮酸，苯丙酮酸能与三氯化铁作用生成绿色的化合物，常用于鉴别肠道细菌，如对大肠杆菌与变形杆菌的鉴别，变形杆菌具有苯丙氨酸脱氨酶，而大肠杆菌没有，因此可通过此性质来区分。该试剂仅用于科研领域，不宜用于临床诊断或其他用途。
自备材料：
1、苯丙氨酸琼脂斜面培养基
2、接种环、酒精灯、恒温培养箱
操作步骤(仅供参考)：
1、将待检细菌接种于苯丙氨酸琼脂斜面培养基，置于恒温培养箱35°C培养18~24h。
2、打开斜面培养基，滴加三氯化铁试剂3~4滴，自斜面上方留下。
3、立即观察并记录显色结果。
染色结果：
普通变性杆菌CMCC 49001/ATCC 13315
| 阳性，绿色
|
大肠埃希氏菌ATCC 25922
| 阴性，黄色 |
注意事项：
1、培养细菌时，注意避免污染。
2、接种菌量要足够多，否则容易出现假阴性反应。
3、滴加三氯化铁试剂后应立即观察结果，延长反应时间会引起褪色，不管阳性或阴性结果都必须在5min内做出判断。
4、为了您的安全和健康，请穿实验服并戴一次性手套操作。
5、试剂开封后请尽快使用，以防影响后续实验效果。
Biochemical tests (also called biochemical reactions) of bacteria refer to the process of determining metabolic products through biochemical methods. Different bacteria have their own enzyme systems, leading to differences in their ability to decompose substrates and thus the metabolic products produced. Biochemical tests mainly include biochemical tests for carbohydrates, metabolism tests for amino acids and proteins, utilization tests for carbon sources and nitrogen sources, and metabolism tests for enzymes. Different bacteria vary in their ability to decompose proteins. Generally, extracellular enzymes first break down proteins into short peptides or amino acids; after these substances enter bacterial cells, intracellular enzymes further decompose peptides into amino acids.
The phenylalanine deaminase test reagent, also known as ferric chloride reagent, mainly consists of ferric chloride and deionized water. The basic principle of the phenylalanine deaminase test is that some bacteria can produce phenylalanine deaminase, which removes the amino group from phenylalanine to form phenylpyruvic acid. Phenylpyruvic acid can react with ferric chloride to form a green compound. This test is often used to identify intestinal bacteria—for example, to distinguish between Escherichia coli and Proteus. Proteus produces phenylalanine deaminase, while Escherichia coli does not, so this property can be used for differentiation.
Self-Prepared Materials:
1. Phenylalanine agar slant medium
2. Inoculating loop, alcohol lamp, constant-temperature incubator
Operating Procedures (For Reference Only):
1. Inoculate the test bacteria onto the phenylalanine agar slant medium, and incubate in a constant-temperature incubator at 35°C for 18–24 hours.
2. Open the agar slant medium, add 3–4 drops of ferric chloride reagent, and let the reagent flow down from the top of the slant.
3. Immediately observe and record the color development result.
Staining Results:
Proteus vulgaris CMCC 49001/ATCC 13315
| Positive, Green
|
Escherichia coli ATCC 25922
| Negative, Yellow |
Precautions:
1. When culturing bacteria, take care to avoid contamination.
2. The amount of inoculated bacteria should be sufficient; otherwise, false-negative reactions are likely to occur.
3. After adding ferric chloride reagent, observe the results immediately. Prolonging the reaction time will cause discoloration—whether the result is positive or negative, a judgment must be made within 5 minutes.
4. For your safety and health, please wear a lab coat and disposable gloves during operation.
5. After opening the reagent, use it as soon as possible to prevent any impact on the results of subsequent experiments.
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