Department of Molecular Biology and Genetics

Inonu Universty
CELL BIOLOGY LAB I
LAB REPORT I

Number: 03160021037 Date:22.10.2018

Name: GONCAGÜL KAYIKÇI
Title of Experiment: Mitochondria Isolation From Liver

AIM
The aim was to make insulation of mitochondrion from the liver of cattle.

INTRODUCTION

Fractionation is the process of the subsequent isolation of organelles with physical chemistry
techniques.
Homogenization is the process to make mixture of two materials by blending.
Mitochondrion is a double-membraned organelle which power house of the cell and found in eukaryotic
organisms. It has inner membrane, outer membrane, cristae and matrix. Mitochondria exist in every cell in the
body except red blood cells.
Chelation (chelating) is the process of removing coenzyme from environment to not enzymes become active by
binding them.
Janus green B dye is a basic dye. It is vital stain and used for staining mitochondria supravitally.
Desmosomes and tight junction function in intercellular communication. Links between cells are formed by
desmosomes. Unwanted materials are prevented from entering cell from another cell by tight juntions in
presence of tight water seal.
Homogenate is the product of homogenization.
Pellet is small particles that are settled bottom of the eppendorf tube.
Supernatant is the clear liquid that is above of pellet.
Cell debris is waste of cell membrane members that die by apoptosis or lysis or spalling in lab conditions.
There are homogenization techniques such as osmotic alterations, mortars and pestles, blenders,
compression/expansion and ultrasonification.
Mechanical process that is used an applied centrifugal force field to separate the components of a mixture
according to density or particle size is termed as centrifugation.

MATERIALS

Hepes Sucrose
Cattle Liver Precision Scale
Distille Water Janus Green B
Thoma Slide Microscope
Centrifuge Device Blade
Eppendorf Tubes

METHODS

Firstly, we prepared hepes buffer by adding 0.25M sucrose and 10 mM hepes in 9 ml distille water.
0.25M sucrose = m/ma.V = m/342.3*0.009L = 0.77 g sucrose.
10 mM hepes = 0.01 M hepes
0.1 M hepes = m/238.3*0.009L = 0.021 g hepes.
0.77 g sucrose and 0.021 g hepes are added in 9 ml of water and then pH is arranged 7.5.
The liver of cattle is put in hepes buffer. Liver is crumbled with homogenizator. And then homogenate is
aliquoted to the four eppendorf tubes. Then they are centrifuged for 10 minutes. Pellet is thrown away, cell
debris, and supernatant is removed and centrifuged again for 25 miutes. In second time, supernatant is thrown
away and pellet is used. And then pellet is solved in 10 ml hepes buffer. 100 µl Janus Green B dye and 100 µl
stock has been added to the thoma slide. Then we investigated mitochondria under light microscope.
RESULT
In thoma slide, there are 454 mitochondria. The stock was 2 ml.
N= AxDFx10000
A : total calculated mitochondria number in 0.1 mm3
DF : dilution factor
10000 : conversion factor
N : concentration of cell (CFU/ml)
N= 182 x 200/100 x 10000
N= 3640000 in a ml
Then in 2 ml, there are 3640000*2= 7280000 mitochondria.

HOMOGENATE

DISCUSSION

For isolation of mitochondria, we used sucrose because it does not have electrolyte and sucrose functions
retention of isotonic osmolarity. Also, 0.25-0.3 M sucrose should be used in such these experiment to prevent
flocculation.
HEPES is used for chelating, in other words, for removing coenzymes in environment such as Ca+2 in this
experiment.
Why Janus Green B dye is used for staining mitochondria has not been found yet.
We could get more clear result if we used EDTA in Hepes buffer and microscope was insufficient to count
mitochondria.
To protect mitochondria, heat is important, if solution becomes warmer, mitochondria number decrases and
buffer that used in experiment sould be recent. Homogenization must be done carefully to not spall
mitochondria.

REFENRENCES

 http://www.wikizeroo.net/index.php?
q=aHR0cHM6Ly90ci53aWtpcGVkaWEub3JnL3dpa2kvSEVQRVM
 http://www.wikizeroo.net/index.php?
q=aHR0cHM6Ly9lbi53aWtpcGVkaWEub3JnL3dpa2kvTHlzaXNfYnVmZmVy
 http://www.wikizeroo.net/index.php?
q=aHR0cHM6Ly9lbi53aWtpcGVkaWEub3JnL3dpa2kvRnJhY3Rpb25hdGlvbg
 https://www.dictionary.com/browse/homogenization
 http://www.wikizeroo.net/index.php?
q=aHR0cHM6Ly9lbi53aWtpcGVkaWEub3JnL3dpa2kvSmFudXNfR3JlZW5fQg