Isolation and Study of Organelles from Pea Seeds
by Differential Centrifugation and Corn to Determine Mitochondrial Activity
BIOL 230-01 Cell & Molecular Biology
October 13, 2014
Since the discovery of the cell in 1665 by Robert Hooke, man has always wondered about the complexity and potential functionality. Using pea seeds, which is readily available, we will be studying the different parts of the cell by separating it into its component parts by cell fractionation while still maintaining the properties of the individual organelles. Differences in physical characteristics of organelles, such as size, shape, and density, make it possible to separate them by spinning in a centrifuge at various speeds and for different periods of time. This process is appropriately called differential centrifugation. Using raw and boiled corn kernels we compared whether or not they contained mitochondria. In a test tube containing with mitochondria and active dehydrogenases we found a color change supporting the fact of active mitochondria. Raw kernels have active dehydrogenation because of the colorless tetrazolium turning red. We can understand that boiled kernels were absent mitochondrial activity as well as active dehydrogenases due to the absence of any pink areas coincided with our understanding. Overall, the purpose of this lab was to find mitochondrial activity and/or active dehydrogenase using differential centrifugation and cell fractionation.
Given the complexity of the cell and the limited techniques available for its exploration it has been useful for scientists to first separate the cell into parts and study each part and its function as a separate entity. This understanding can be applied to the inner workings of the cell as a whole and visualized by microscopic techniques. With this in mind, in this experiment a homogenate, a mixture of cellular materials, was made from pea seeds and the technique of differential centrifugation was used to separate the cell components of the homogenate into layers in order to visualize the structural organization and compartmentalization of this particular cell. The isolation of mitochondria from guinea pig liver in 1934 by Bensely and Hoerr marked the inception of the method of differential centrifugation. Cell fractionation is one of the most versatile techniques in cytochemistry having the capacity to isolate particulate components of the cell that other techniques simply cannot (Claude, 1946). In differential centrifugation, the homogenate is separated into layers of cell components by rounds of centrifugation. The first round of centrifugation formed a pellet of the nuclei, followed by cell materials of differing size and density, thereby separating cell components.
Aerobic respiration in eukaryotes takes place in the membrane bound organelle known as the mitochondria. We were interested in this experiment in finding the presence of mitochondria following subsequent rounds of centrifugation. Studies performed on plant seeds based on the reaction of tetrazolium and dehydrogenase will determine whether this organelle is present in the layer. The presence of active mitochondria is indicated by the activity of enzymes called dehydrogenase which can be detected experimentally using tetrazolium chloride (TTC) which in the presence of dehydrogenase in mitochondria, an environment where hydrogen ions and electrons can be gained, will become reduced. In its oxidized form, a tetrazolium test is colorless, however in its reduced form in metabolically active cells it becomes an insoluble red formazan compound (See reaction below).
In addition, nuclei were the first to pellet after centrifugation, therefore, the IKI, an iodine solution, reacted with the starch granule cellular components to form a blueblack color, indicating the presence of nuclei. After World War II new uses and availabilities of the...
Gaynor, Dr. Jack, Dr. Reginald Halaby, Dr. Sandra Adams, Dr. James Campanella, and Dr. Quinn Vega. Laboratory Manual: Cell and Molecular Biology BIOL 230 Fall 2011. New Jersey: Department of Biology and Molecular Biology Montclair State University, 2011.
Claude, A. (1946). Fractionation of mammalian liver cells by differential centrifugation: I. Problems, methods, and preparation of extract. The Journal of experimental medicine, 84(1), 51.
De Duve, C., & Beaufay, H. (1981). A short history of tissue fractionation. The Journal of cell biology, 91(3), 293s-299s.
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