& Data Analysis
Protein gel analysis
Keeping a lab notebook
Writing research papers
Dimensions & units
Using figures (graphs)
Examples of graphs
Principles of microscopy
Solutions & dilutions
Fractionation & centrifugation
Radioisotopes and detection
Mitochondria make multicellular life possible. Period. To produce ATP in sufficient quantities to drive energy requiring reactions in most eukaryotes, including protists, mitochondria require a constant supply of oxygen. We breathe oxygen solely to support mitochondrial electron transport. Otherwise we would not want the stuff around, as oxygen itself is somewhat toxic. We have a beating heart, a circulatory system, and all sorts of regulatory processes in place for the main purpose of delivering oxygen to our tissues.
Not only are mitochondria a relevant subject for investigation, but the kinds of studies we can conduct on intact mitochondria engage students in kinds of critical thinking that are very difficult to incorporate in undergraduate laboratory courses. Here is an outline of how our studies will be organized, starting with background material and training.
The overall objective of the study itself is to evaluate the utility of isolated mitochondria for use as an experimental model. Our strategy will be to design experiments that test specific known properties of mitochondria. Based upon our understanding of the mechanisms related to electron transport, respiratory control, and oxidative phosphorylation,we will predict how a suitable model system should respond. We will then compare the actual findings with our predictions.
Begin by looking over the polarography part of pre-lab #4. The pages following this one (Next buttons) take you to information on polarography for the study of isolated mitochondria. You will probably have to start looking at mitochondria theory and mitochondria in vitro to answer the last two questions. The talk will also cover some of this material, so wait until after the talk to submit your final responses.
Part of the laboratory session will be dedicated to training session on how to calibrate and use the polarographic system. You must be prepared to recall the methods, including detailed steps that you must take to ensure success. For polarography training and, later, for data analysis and interpretation, we will count on your remembering principles of "laboratory math," pipetting, working with dimensions and units, rounding quantities appropriately, and related general laboratory skills from prior sessions.
For this part you must review the first page under mitochondria in vitro. We will prepare functional mitochondria from fresh rat liver. We will have to organize this part carefully for two reasons. First, our Institutional Animal Care and Use Committee (IACUC) has limited our use of albino rats to four animals per laboratory session. One-half liver gives a pair of students sufficient tissue for one preparation. This means that some groups will have to conduct an alternative mitochondria preparation using tissues other than liver (heart and/or brain). While we know that liver mitochondria give us good responses, we don't know that for sure about mitochondria from the other tissues. Students with liver mitochondria preparations may have to share with another group.
The other issue is that some students may not wish to participate in the dissections. Students who opt out (for any reason – we won't ask) may wait outside until the animals are put away, then re-join a group to finish the preparation.
Your chances of success will be better if you conduct the preparation quickly. Once the tissue is deprived of oxygen we have a limited amount of time in which to process it. Once the tissue is homogenized, the faster the centrifugation procedures are completed the better are the preparations. Materials in the suspensions, especially lipids, tend to ruin the preparations. When your pellets come down, separate supernatants from pellets quickly, as instructed in the laboratory.
Another pre-laboratory assignment will focus on mitochondria theory, including how mitochondria behave in vitro. Again, the talk at the beginning of the week will present key concepts and allow you the opportunity to ask questions, so wait until after the talk to submit the assignment. You will be asked to review the theory of how mitochondria conduct electron transport, generate and maintain a gradient, and exploit the gradient to make ATP. You will need to understand the concept of respiratory control. You will also need to look specifically at how mitochondria behave in vitro, and become familiar with the terms "state IV respiration," "state III respiration," "uncoupling," and "oxidative phosphorylation."
Representative polarographic traces in response to specific interventions will be provided on the website (mitotraces). Your job is to learn why oxygen consumption rates changed in the manner shown, that is, be able to explain what you see in terms of electron tranport, the chemiosmotic gradient, and function of ATP synthase. A page on the rationale behind the experiments describes questions that the experiments address. These are the same questions that you should be thinking about as you prepare for the experiments and conduct them.
The paper will be written in parts, with the materials/methods and results to be submitted first, followed by discussion, abstract, and introduction.