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CAP Lab 6
Comparative Animal Physiology Laboratory Experiment #6
| Question | Answer |
|---|---|
| We will be using Hemoglobin from what animal? | Chicken |
| We will be extracting hemocyanin from what animal? | Crayfish, a crustacean |
| What is the buffer? | Chick ringers (40 mL) |
| What instrument will we be using to contain the Hemoglobin? | Tonometer |
| Where in the crayfish do we extract hemocyanin from? | Blood sinus at the base of the claw |
| Why do you not want to keep the blood in the syringe for a very long time? | It will coagulate |
| How long does it take for the hemocyanin to clot? | 5-7 minutes |
| We will pool the blood from how much crayfish? | 3 to 5 |
| We break up the clot with what? | Hand homogenizer |
| We filter the hemolymph through what? | A nylon |
| How much material should you have left after filtering the hemolymph? | Over 50% of the original volume |
| Why do you hold the stem of the stopcock while you turn the stopcock? | To avoid breaking the tonometer |
| The spectrophotometer should be set to what? | 338 nm |
| How do you calibrate the spectrophotometer? | Zero it with a cuvette containing the pH-adjusted Ringers |
| How do you deoxygenate the hemoglobin? | bubble nitrogen gas (N2) over the hemoglobin solutions |
| Why is deoxygenizing the hemoglobin touchy? | Too much nitrogen will permanently alter the oxygen binding curve |
| How do you avoid the issue when deoxygenizing? | Open both valves of the tonometer and conect one of the valves to the outflow from the gas mixer - make sure the mixer is very low so pressure doesn't build up in the tonometer |
| How do you generate an oxygen binding curve? | We add back small amounts of gas with a known oxygen content using the gas mixer, and measure the optical density after allowing time for the uptake to oxygen by the hemoglobin |
| How many times should you do the oxygenation procedure? | 7-9 times, otherwise too much oxygen was introduced into the chamber |
| When you do the experiment, you use both the acidic and basic solution for what and only the middle pH for what? | hemoglobin; hemocyanin |
| How many oxygen dissociation curves at the end do you produce? | 4 |
| What do you graph? | % saturation versus partial pressure of oxygen |
| How do you calculate partial pressure? | Fraction of oxygen x [barometric pressure] |
| How do you calculate % saturation? | {[(absorption of deoxygenated hemoglobin) - (absorption after partial re-oxygenation)] / [(absorption of deoxygenated hemoglobin) - (absorption of full oxygenated hemoglobin)]}* 100 |
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