Duygu KESER
1909621
Section: Wednesday Morning
04/04/2017
BIO310 Laboratory Report
EXPERIMENT 6: Preparation of Mitochondria, Lipid Determination and
Permeability Test
AIM:
Isolation of mitochondria by using differential centrifugation from liver and heart tissue, and
measurement of lipid content from mitochondria and measurement of permeability of different
molecules from mitochondrial membrane
INTRODUCTION:
Mitochondria is rod shaped organelle found in eukaryotic cells. Major function of mitochondria is
producing ATP from O2 and nutrients in aerobic conditions, second function is having an important
role of apoptosis. Mitochondria has double membrane which make it extraordinary organelle and
also it has its own genome which is coding for a few respiratory genes. and also capable of self
replication. The amount of mitochondria in a specific tissue depends on the requirement of energy so
it change tissue from tissue. Some tissues contains thousands of mitochondria, while some other
contains only a few. Mitochondria has two membrane and intermembrane space. Outer membrane
has big pores and molecules can enter however inner membrane is selective like plasma membrane
permeability depend on molecules features. Member of ETS are found in inner membrane and citric
acid cycle enzymes are in the mitochondrial matrix. Electrons are release from citric acid cycle which
occur in the inner membrane and transport to the ETS elements and generate a gradient to produce
ATP. Which is called chemiosmotic hypothesis.( Taylor R. 2005)(Detmer, 2007)
The unique structure of mitochondria is explained by endosymbiosis theory which claim that millions
years ago small prokaryotes are captured by larger eukaryotes but thay failed to digest them and
they create a symbiotic life then prokaryotes genes are translated to eukaryotic genome and
become mitochondria. Mitochondria inherited maternally in most animals so this feature is very
helpful in forensic science. (Davidson, 2015)
Cellular parts and organelles have different shape, size and density. When in order to isolate specific
organelle, this differences is used. In order to separate them firstly solid tissue must become
homogenize and cell membranes should be broken then organelles release. This breaking of cells is
called homogenization, it can be physical or chemical and isolation of organelles is called
fractionation. Fractionation is depend on centrifugation. And centrifugation is a technique to
separate molecules according to their size using rotor. The different size and mass of molecules
provide separation organelles by applying differential centrifugation. Applying several times of
centrifugation, cellular parts are separate. Big molecules precipitate easily first centrifugation pellet
