Centrifugation Lab

The purpose of this experiment was to separate and identify the lower density particles in a homogenous mixture.Thus, the objective of the exercise was to realize that there is more than one structure in the cytoplasm and to isolate them using homogenization and differential centrifugation.In doing so, one can recognize and identify these particles by how the separate-which can be affected by density, shape, viscosity, the speed and/or diameter of the centrifuge.By examining the effects of the centrifugation, it allows you to see these principles for yourself, which gives you a better understanding of how the exercise works.
O Homogenization:break up pea cells in a blender to release organelles
O Filtration:pour homogenate over beaker covered with cheesecloth to filter out large fragments-set aside for observation
o Pour filtrate into 4 test tubes, evenly filled
o Place tubes in opposing holes in centrifuge
o Centrifuge for 3 minutes @ 1000 RPM
o Centrifuge remaining 3 for 10 minutes @ 3000 RPM
o Use remaining sediment for mitochondria test
o Using the sediment in the remaining two test tubes, fill 3 test tubes 1/3 with sediment layer-label A,B,C
§ Test tube A-supernatant from centrifugation 2/3 full
§ Test tube B-sucrose buffer 2/3 full
§ Test tube C (control tube)-sucrose buffer 2/3 full
o Fill each tube 1/3 full with tetrazolium
o Place test tubes in a beaker filled with water which is 37 degrees C
o Place lamp over beaker to uphold temperature

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