ISOLATION of MITOCHONDRION
ISOLATION of MITOCHONDRION
AIM : Our aim is to isolate mitochondria from neat liver cells.
INTRODUCTION:
For the determination of different intracellular fractions, the cell must be lysed and each compartment separated. This phenomenon is called “cell fractionation“. For this, first the cell disintegration, then the organelle and homogenate the particles must be separated from each other.
Fractionation procedures;
-Homogenization is one of the important ways to break down soft tissues. In homogenization processes, the tissues can be chopped in the blender or in a glass container can be crushed by compressing with a teflon sleeve.
-Osmotic alterations: Organelles are more easily separated when the cells swell. When water enters the cell, the osmotic pressure increases and causes the cell to tear.
-Compression/Expansion: In this method, while the sample is under high pressure, it is suddenly switched to atmospheric pressure and thus, cell lysis is performed. Such a rapid pressure change explosion. This method is generally used for lysis of bacteria and yeast cells.
-Blender: For molecular separations, mechanical blenders are often used, varying in sophistication from household blenders to high speed blenders with chambers.
-Ultrasonication: Sonication is the act of applying sound energy to agitate particles in a sample, for various purposes such as the extraction of multiple compounds from plants, microalgae and seaweeds. Ultrasonic frequencies (>20 kHz) are usually used, leading to the process also being known as ultrasonication or ultra-sonication.
-Fractionation: is a separation process in which a certain quantity of a mixture (gas, solid, liquid, enzymes, suspension, or isotope) is divided during a phase transition, into a number of smaller quantities (fractions) in which the composition varies according to a gradient.
Mitochondria are organelles found in the cytoplasm of most cells. They are essential to healthy living as they play an important role in the way cells function in the body. Mitochondria generate energy for cells to carry out activities. This energy is in the form of adenosine triphosphate (ATP). They also take part in cell signalling and help cells sense and adapt to their environment.
MATERIALS
-Distilled water -Neat liver -Homogenization buffer -Suspension buffer -Blender -Refrigerated centrifuge -Janus green B -Hemacytometer -Microscope -96-well plate Ependorf tube -Test tube -Spatula -Micropipette -Beaker -Precision balance Weighing balance -PH replacement -NaOH solutions and HCl solutions
METHODS
Before starting our experience we calculated homogenization buffer and suspension buffer solutions. We measured HEPES, EPTA and sugar for these solutions. We measured the ph of our solutions and tehn added NaOH and HCl solutions to maket he ph 7.5. Then we mixed the liver cell in the blender and crushed it. We fractionated the liver. After mixing homogenization buffer and liver and inserting ependor tube, we waited for a while in our centrifuge machine. Then we did the washing and added the suspension solution and waited again in our centrifugal machine. After the sedimentation, we performed serial dilution and applied dyeing processes. We took some of our solution and spread it to thoma slide. We examined the thoma slide under microscope and finally mitochondria count.
RESULT
Homogenization buffer: 0.25 M sucrose in 10 mM HEPES buffer, pH 7.5
Suspansion buffer: 0.25 M sucrose in 10 mM HEPES, pH 7.5 + 1 mM EDTA
We have done the following operations for the above solutions.
For 100 ml M=n/v
n=m/MA M=m/MA×v
for sucrose 0.25M:m/342.30×0.1 m=8.5575gr
for 100ml HEPES 0.01=m/238.30×0.1 m=0.2383gr
for 100 ETDA 0.001=m/292.24×0.1 m=0.029gr 
Second area:7
Third area:6
Fourth:4
Fifth:10
Total mitochondria cell: 36
Dilution factor=solution volume/total volume
Dilution factor= volume of sample/100µl homogenate + 100µl stain + 1300µl water
Dilution factor=1/15
Live cell count=36×10000×15=5400000=5,4×106
DISCUSSION
Our experience was generally successful but we didn't' recentrifuge the supernatant at 4°C. In the last part we had difficulty counting mitochondria. we may have mistaken it because some have ink residues.
REFERENCES
https://acikders.ankara.edu.tr/pluginfile.php/4436/mod_resource/content/0/3.%20hafta.pdf
https://www.news-medical.net/life-sciences/What-are-Mitochondria.aspx
https://www.google.com/search?q=ultrasonication&spell=1&sa=X&ved=2ahUKEwj4gpK1xavmAhUJ-hQKHQ34BKcQBSgAegQIDBAp&biw=1360&bih=625
https://www.google.com/search?q=Fractionation&oq=Fractionation&aqs=chrome..69i57j0l7.1942j0j7&sourceid=chrome&ie=UTF-8
