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种子生活力是指种子能够萌发的潜在能力或种胚具有的生命力，其高低决定了种子品质和实用价值大小，关系到播种时的用种量，测定种子生活力常用方法为发芽实验法，即在适宜条件下让种子吸水萌发，在规定天数内统计发芽的种子占供试种子数的百分比，但是常规发芽实验法需时较长，无法用于应急需要，也无法检测休眠种子的生活力，常用比较快速的检测方法有氯化三苯基四氮唑（TTC）法、溴麝香草酚蓝（BTB）法、红墨水染色法和荧光法等。
具有生活力的种胚有呼吸作用，吸收空气中氧气放出二氧化碳，二氧化碳溶于水生成碳酸，又解离出氢离子和碳酸氢根，使得种胚周围环境的酸度增加，因此可以用溴麝香草酚蓝（BTB）来定性测定酸度的改变，BTB的变色范围为pH6.0~7.6，在酸性介质中呈黄色，在碱性介质中呈蓝色，中间色为绿色（变色点为7.1），因此可根据BTB颜色差异来判断种子的生活力；本方法有以下优点：快速、准确；颜色变化明显，易于观察。该试剂盒仅用于科研领域，不适用于临床诊断或其他用途。
自备仪器和试剂
操作步骤（仅供参考）
1、浸种：将待测种子用30℃温水浸泡2~6h，使种子充分吸胀。
2、配制BTB琼脂凝胶：取适量的BTB指示剂和琼脂粉，按BTB指示剂：琼脂粉=10ml：0.15g的比例混合，用恒温箱或微波炉加热助溶，待琼脂粉完全溶解后，趁热倒在干净的培养皿即为BTB琼脂凝胶，厚度以能浸没种子为宜，冷却后备用。
3、随机取100粒吸胀种子，种胚朝下，整齐地埋于BTB琼脂凝胶中，间距至少1cm。
4、将培养皿置于30~35℃恒温箱中培养1~4h。
5、在蓝色或绿色背景下观察，种胚附近呈现较深黄色晕圈的是活种子，否则是死种子。
6、可以用在沸水中浸泡处理的种子进行对照观察。
染色结果
| 活种子 | 种胚附近呈黄色或黄绿色 |
| 死种子 | 种胚附近无颜色变化 |
7、计算
观察统计100粒种子，计算有活力种子的百分数。其公式为：
种子生活力百分数 (%) =活种子粒数/100×100%
注意事项
1、BTB凝胶厚度取决于种子的大小，原则上需保证种胚接触培养皿底部后尚有部分露出凝胶上方，凝胶厚度应使种子稳定其中。
2、种子需完好无损，不能有破种子或霉变种子。
3、BTB指示剂应为绿色或蓝色溶液，pH在7.1~8.5较适宜，如变为黄色或黄绿色，说明pH降低，可滴加少量稀氨水或者氢氧化钠等碱性试剂调整后即可正常使用。pH值较高也不利于观察颜色的变化。
4、培养温度一般以30~35℃为宜。
5、试剂开封后请尽快使用，以防影响后续实验效果。
Seed Viability refers to the potential ability of seeds to germinate or the vitality of the seed embryo. Its level determines seed quality and practical value, and is related to the seeding rate. A common method for assessing seed viability is the germination test, where seeds are allowed to absorb water and germinate under suitable conditions, and the percentage of germinated seeds out of the total tested is counted within a specified period. However, conventional germination tests are time-consuming, cannot meet urgent needs, and cannot detect the viability of dormant seeds. Faster detection methods commonly used include the Tetrazolium Chloride (TTC) method, Bromothymol Blue (BTB) method, red ink staining method, and fluorescence method.
Viable seed embryos respire, taking up oxygen from the air and releasing carbon dioxide. The carbon dioxide dissolves in water to form carbonic acid, which dissociates into hydrogen ions and bicarbonate ions, thereby increasing the acidity around the embryo. This change in acidity can be qualitatively measured using Bromothymol Blue (BTB). The color transition range of BTB is pH 6.0–7.6: it appears yellow in an acidic medium and blue in an alkaline medium, with green as the intermediate color (transition point at pH 7.1). Therefore, seed viability can be judged based on the color difference of BTB. This method offers the following advantages: rapid, accurate; obvious color change, easy to observe. This kit is for research use only and is not suitable for clinical diagnosis or other purposes.
Reagents, consumables and Equipments not provided
Operating Steps (For Reference Only)
1. Soaking Seeds: Soak the test seeds in 30°C warm water for 2–6 hours to allow full imbibition.
2. Preparation of BTB-Agar Gel: Take appropriate amounts of BTB Indicator and agar powder. Mix at a ratio of 10 mL BTB Indicator to 0.15 g agar powder. Heat using an incubator or microwave to aid dissolution. Once the agar powder is completely dissolved, pour the hot solution into a clean culture dish to form the BTB-agar gel. The thickness should be sufficient to submerge the seeds. Let it cool for use.
3. Randomly take 100 imbibed seeds. Place them with the embryo side down, neatly embedded in the BTB-agar gel, with a spacing of at least 1 cm between seeds.
4. Place the culture dish in an incubator at 30–35°C for 1–4 hours.
5. Observe against a blue or green background. Seeds with a distinct yellow halo near the embryo are viable; otherwise, they are dead.
6. Seeds treated by boiling water immersion can be used as a control for observation.
Staining Results
| Viable Seed | Yellow or yellow-green color appears near the embryo |
| Dead Seed | No color change near the embryo |
7. Calculation:
Observe and count 100 seeds. Calculate the percentage of viable seeds using the formula:
Seed Viability Percentage (%) = (Number of viable seeds / 100) × 100%
Precautions
1. The thickness of the BTB gel depends on seed size. In principle, it should ensure that after the embryo contacts the bottom of the dish, part of the seed remains above the gel. The gel thickness should hold the seeds stably.
2. Seeds must be intact, without damage or mold.
3. The BTB Indicator should be a green or blue solution, with a pH preferably between 7.1–8.5. If it turns yellow or yellow-green, indicating a pH drop, add a small amount of dilute ammonia water or sodium hydroxide solution to adjust the pH back to normal. A pH that is too high is also not conducive to observing color changes.
4. The incubation temperature is generally 30–35°C.
5. Please use the reagent as soon as possible after opening to avoid affecting subsequent experimental results.
| S1509461 | Component | 100 mL | Storage |
| S1509461A | BTB指示剂 | 100 mL | RT. |
| S1509461B | 琼脂粉 | 1.5 g | RT. |
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0333383 |
分析证书 | S1509461 |
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