Assignments: Doing the assignments is helpful to understanding the concepts of this lab. The Assignments here were written to benefit a wide range of general biology classes and you should find them useful, but check with your instructor before proceedng with any Assignments.
Once you enter MitochondriaLab, you are provided with a reaction flask in which you will perform your reactions. These reaction flasks are a standard piece of glassware in a biochemistry lab because they allow biochemists to carefully control and measure reaction conditions within the flask. To begin each experiment you will start with an empty reaction flask into which you will add isolated preparations of mitochondria. In real-life applications these are typically mitochondria that have been isolated from mitochondria-rich animal tissues, such as liver or muscle, using cell fractionation techniques.
Notice that you have two racks of test tubes to choose from. One rack of tubes contains seven different substrates that you can add to your flask (ADP, ascorbate/TMPD [tetramethyl-p-phenylene diamine], fumarate, glutamate, malate, pyruvate, and succinate). The second rack of tubes contains six different metabolic inhibitors of cell respiration (antimycin, cyanide, DNP [2,4-dinitrophenol], malonate, oligomycin, and rotenone).
Notice that the chart recorder is connected to an oxygen-measuring electrode that inserts into a port on the left side of the reaction chamber. Substrates and inhibitors will be added to the flask through the port on the right side of the flask. Oxygen consumption in the flask will be determined as a measurement of the progress of reactions in the flask. The chart recorder will measure and plot oxygen concentration in the flask during the time course of your experiments.To Experiment
Click on the To Experiment button in the lower left corner of the laboratory screen to start a MitochondriaLab experiment.
To begin an experiment you must first add mitochondria to the reaction flask. Do this by clicking on the Add Mitochondria button located below the reaction flask. A running ledger of the components that you added and the time that each component was added to the flask is shown in the lower right corner of the screen. Notice that the mitochondria have been added to a 2.0 ml suspension which consists of a solution buffered to the proper pH for these reactions. This solution contains ions and other components that are required by mitochondria to undergo cell respiration.Chart Recorder/Simulation Speed
The chart recorder at the left of the screen will be used to monitor the rate of cellular respiration by the mitochondria in the flask by plotting oxygen consumption (as a percent of the starting concentration) on the x-axis against time in minutes on the y-axis. Oxygen concentration in the flask begins at 100%. Notice how the chart recording is plotting oxygen consumption in blue ink. If desired, you can change the recording speed of the simulation by using the small buttons at the upper left corner of the screen. The default value is 4x speed.Substrates Box
To add substrates, highlight a chosen substrate and click the Add button.Poisons Box
To add poisons, highlight a chosen poison and click the Add button.Added Box
This box keeps track of the components you have added to the reaction flask.View Chart
Click on the View Chart button to see an enlarged view of the plot. Use the slider bar at the right of the plot to move up or down the plot. Notice that the chart recorder labels the time when you added each component and the concentration of components added. You can export the values from this plot by clicking on the Add to Notes button in the lower left corner of the screen. A new browser window will appear with the data in your notebook. You can now print your data from this window or save your data to a disk.
Click on the Return To Experiment button to return to the experiment in progress.Clean and Reset Chamber
Reset the reaction flask by clicking on the Clean and Reset Chamber button below the flask to prepare the reaction flask for another experiment.
Click Pause to stop the experiment and evaluate your results. Click Resume to restart the experiment.
The speed of the simulation can be set to 1x, 2x, or 4x by clicking the buttons in the upper left corner of the screen. The default speed is 4x.Add to Notes
Click this button immediately after viewing the chart recorder to move the information into the Notebook. If you don't click this before going on to another experiment, the information will be discarded.Lab Bench
This returns you to the lab equipment setup if you need to review the functions of the different equipment.
As you run experiments, form hypotheses and test them, you should keep notes on your theories and findings in the MitochondriaLab notebook.