细胞呼吸
At the end of this lab, students should know...
生命体的能量来自何处？
绝大多数生物体的能量最终来源于太阳。光合生物将太阳能捕获并转化为生物圈可利用的形式。
细胞呼吸如何为生物体提供能量？
该过程利用生物分子（例如葡萄糖等简单糖类），将其转化为以ATP形式存在的可利用化学能。某些形式需要氧气，而另一些则不需要。
如何测量细胞呼吸作用？
可通过使用一种称为呼吸计的工具来测量。
呼吸计的工作原理是什么？
它用于测量生物体消耗氧气的量。通过使用 KOH 来吸收 CO₂2 作为O产生2 被消耗。该过程基于理想气体定律。
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结核菌素注射器1
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毛细管20
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金属垫圈20
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种子60
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玻璃珠100 gm
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试管架10
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计时器5
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标记5
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标尺5
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标签磁带5
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肥皂水200 ml
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红色食用色素2 mL
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15% KOH20 mL
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吸水棉球15
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刀/剪刀1
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热胶枪1
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水浴（最少 2 个）-1 取决于实验室大小
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温度计 （最少 2 个）-1 取决于实验室大小
- 使用微呼吸计量化呼吸作用
- 为组装实验所需的微呼吸计，首先插上热熔胶枪进行预热。
- 将结核菌素注射器的推杆完全推入，然后剪下注射器末端。
- 接下来，将毛细管插入注射器的空针端。
- 推进毛细管，直至其接触到推杆的尖端。
- 使用热熔胶枪在毛细管与结核菌素注射器之间形成气密密封。胶体干燥期间，确保毛细管保持垂直。
- 待胶体干燥后，向后拉动推杆，确认毛细管未被堵塞。
- 接下来，通过向肥皂水中加入红色食用色素来配制压差计液体。注意：洗涤剂可覆盖毛细管内壁，防止实验过程中压差计液体附着管壁。
- 将毛细管插入压差计液体中，然后拉动注射器推杆，直至整个毛细管完全充满液体。
- 随后，将压差计液体从微呼吸计中排出。
- 用一小块吸水棉塞住微呼吸计的筒体，将其插入注射器至0 mL刻度处。使用备用毛细管或一次性玻璃移液管压实棉花。
- 使用备用毛细管或一次性玻璃移液管，在棉花上加入一滴15% KOH溶液。
- 在已浸润KOH的棉花上添加一些非吸收性棉花，作为种子的隔离屏障。使用一次性移液管压实棉花。棉花填充高度应达到0 mL至1 mL刻度线之间的中点。
- 小心重新插入推杆，直至其接触棉花，然后通过挤压去除多余的KOH。
- 在每个实验台放置四个微呼吸计。
- 然后，在微呼吸计旁边放置实验标记物以及两组分别盛有种子和玻璃珠的小培养皿。
- 最后，准备两个水浴，注入约6英寸深的水。水浴中的水位应能轻松容纳两个呼吸计，且其两根毛细管完全暴露于水面之上。
- 盖上盖子，并在每个水浴中插入一支温度计以监测温度。一个水浴应调节至约25 °C，另一个调节至约40 °C。
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Q1: What materials do you need to assemble a microrespirometer?
A microrespirometer requires a tuberculin syringe, capillary tube, hot glue gun, manometer fluid (red food coloring mixed with soapy water), absorbent cotton, potassium hydroxide (KOH), nonabsorbent cotton, and glass beads. The syringe plunger is pushed in and the syringe end is cut off before inserting the capillary tube and sealing it with hot glue to create an airtight seal.
Q2: Why is detergent added to the manometer fluid?
Detergent is added to the manometer fluid because it coats the inside of the capillary tube, preventing the manometer fluid from sticking during the experiment. This coating ensures the fluid moves freely within the tube, allowing accurate measurement of gas volume changes as respiration occurs.
Q3: How should the cotton and potassium hydroxide be arranged inside the microrespirometer?
Absorbent cotton is inserted into the syringe barrel up to the 0 mL mark and packed firmly. A single drop of 15% KOH is added to this cotton, then nonabsorbent cotton is layered on top as a barrier to the seeds. The cotton should reach halfway between the 0 mL and 1 mL mark, and excess KOH is removed by pressing it out.
Q4: What is the role of potassium hydroxide in measuring cellular respiration?
Potassium hydroxide (KOH) absorbs carbon dioxide produced during cellular respiration. By removing CO2 from the system, any volume change in the capillary tube reflects only oxygen consumption, allowing direct measurement of respiration rate through the displacement of manometer fluid in the microrespirometer.
Q5: Why are two water baths set at different temperatures?
Two water baths at approximately 25°C and 40°C allow comparison of respiration rates at different temperatures. This experimental design tests how temperature affects cellular respiration in seeds, revealing the relationship between metabolic activity and environmental conditions in the microrespirometer setup.
Q6: What is the purpose of glass beads in the microrespirometer setup?
Glass beads serve as a control material in the microrespirometer. They are placed alongside seeds to account for any physical volume changes unrelated to respiration, ensuring that measured gas volume changes reflect only biological respiration rather than physical displacement or settling of materials.
Q7: How do you prepare the capillary tube before filling it with manometer fluid?
The capillary tube is inserted into the empty needle end of the syringe until it touches the plunger tip, then sealed with hot glue while standing straight. Once dry, the plunger is pulled back to verify the capillary tube is not plugged, ensuring it is ready for respiration quantification using microrespirometers procedure.