Biology 2025 Laboratory # 10Name__________________Isolation of Plant Mitochondria & ChloroplastsIn this lab, you will work with plant tissue to learn about cell fractionation.CellFractionationis the process that isolates different components of the cell so that theirbiochemistry and structure can be studied separate from the rest of the cell. You will use ablender to disrupt corn and spinach cells and a centrifuge to isolate their mitochondria andchloroplasts respectively.Cell Fractionation part 1: HomogenizationTissues or cells grown in culture can be homogenized a number of different ways. Asshown on page 160 of your textbook, breakage can be accomplished using high frequencysound, mild detergents, mechanical shearing or by forcing the material through a tissuesieve. Plant cells are particularly difficult to disrupt due to the strength of their cell walls.Some protocols disrupt plant cells by using enzymes to degrade the cell wall then by using amild detergent to break open the plasma membrane. You will use the blades of a blender tohomogenize plant tissues and to break open the cells to release the organelles.The main problem with the blender approach, is that you never know how muchprocessing will give the highest organelle yield. If you don’t run theblender long enough, notmany of the cells will be disrupted and you won’t release enough of the organelles. If you runthe blender too long, you will not only break open the cells but you will also destroy a largenumber of the organelles, again producing a low yield. To minimize this problem, peoplegenerally will use the blender for differing amounts of time. They may first run the blender 15seconds then pour of one third of the sample. Next, they will run it for another 15 secondsthen pour off another third of the original sample. Finally, the remaining third will get another15 seconds.By subjecting the sample to three different amounts of processing, theresearcher can get a reasonable yield each time the isolation is performed.Another major concern when trying to isolate cellular organelles, is that they may notbe stable outside of the cell. When removed from the protective intracellular environment,many organelles will fall apart if steps are not taken to support them. In your isolation, thehomogenizationbufferwill help alleviate this problem. The buffer contains10 % Sucrosewhich makes the solution somewhat viscous and simulates the cytoplasm of the cell. Thebuffer also containsTriswhich buffers the pH at about 7.4, again simulating the intracellularpH of the cell. Lastly, the buffer containsEDTAwhich removes magnesium from the solutionand inactivates degradative enzymes that might disrupt the organelles. Together, thesehomogenization buffer components stabilize the organelles so they can be isolated by thenext step.
