Instructor:
Paramjeet S. Bagga
Lab Protocols
Cell & Molecular Biology
SBIO 406
Experiment 3
ISOLATION AND STUDY OF MITOCHONDRIA
One approach to the study of cellular structure and function is the histological approach,
namely the use of microscopic techniques coupled with various dyes and stains to elucidate the
structural organization of a particular cell. These studies make it readily apparent that the cell is
not a formless mass of protoplasm; on the contrary, the cell can be seen to be divided into many
different types of membrane bound compartments (nucleus, mitochondria, chloroplast, etc.)
Cell biologists still do not know all of the functions of these various subcellular component,
but a great deal of what we do know has come from studies of cell homogenates whereby
separation of the various components are achieved. Once separated, each component can be
analyzed separately and the mechanism of its biosynthetic or physiological functions determined.
In performing such studies, biologists have discovered that there is considerable biochemical
communication between compartments, especially between the nucleus and cytoplasmic
compartments.
Today we shall make our own homogenate from pea seeds in order to demonstrate one of the
most widely used techniques in cell biology; differential centrifugation.
Materials/ Reagents/ Supplies
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Ice
Overnight soaked pea seeds
Chilled Blender
Ice cold 0.25 M Sucrose made in 0.05M Potassium Phosphate buffer (pH 7.4)
Cheesecloth
250 ml beakers
50 ml plastic centrifuge tubes
Water bath set at 37 degrees Centigrade
Cold Centrifuge set at 4oC Pasteur pipette
Test tube rack
Test tubes
1% TTC (Tetrazolium Chloride) made in 0.05M Potassium Phosphate buffer (pH 7.4)
Parafilm
Microscope
Microscope slides
Cover slips
IKI solution
Rubber Bands
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Instructor:
Paramjeet S. Bagga
Lab Protocols
Cell & Molecular Biology
SBIO 406
Instructions
IMPORTANT: Unless specifically instructed, perform all the procedure at 0 - 40C.
A. Cell Fractionation
1. Five g of dry pea (Pisum sativum) seeds (soaked overnight at 370C to soften and activate
enzymes) are placed in a blender and mixed with 25 ml of ice-cold sucrose buffer. (0.25M
sucrose buffered at 7.4 pH with
0.05M potassium phosphate buffer).
2. Homogenize for 2-3 minutes in a pre-chilled blender. The cold sucrose buffer is used to
avoid excessive damage to the cell components which are liberated when the cells are
disrupted. At the end of the homogenization, you should have "pea soup".
3. Pour the homogenate through a filter (4 layers of cheesecloth or a single layer of Miracloth)
which has been stretched across the mouth of a 250 ml beaker with the help of a rubber
band. The beaker itself should be kept in a bucket of crushed ice. You are attempting to filter
out unbroken cells, fragments of seed coat, and other undesired "gunk". You will find your
filter repeatedly being filled with this gunk. When this happens, fold up the sides of the filter
material to make a sort of bag and then squeeze out the remaining fluid into the beaker (wear
gloves - it's not dangerous but messy!). Set aside the bag containing the "gunk" for later
examination. Smaller pieces of this undesired material, as well as the desired cell
components, will be found in the filtrate in the beaker. Immediately wipe up any solution
that has spilled on the work surfaces or on the floor.
4. Swirl the beaker of filtrate to suspend the solid matter and pour the filtrate into 50 ml plastic
centrifuge tube, filling it 3/4 full (no more than 35 ml per tube). Prepare a ‘balance’ tube by
adding D.W. Both the tubes must contain equal volumes so that the centrifuge will be
balanced. Place these tubes in opposing holders (i.e. across from each other) in the
centrifuge, noting the numbers of the positions your tubes are in. When all of the tubes are in
the centrifuge, close the top and turn the speed control dial on the front to a setting which
equals a speed for relative centrifugal force of 200 x g. Centrifuge at this setting for 3
minutes, and then turn the centrifuge off.
5. You should see a sediment (sediment I) at the bottom of your tubes. Carefully decant (pour
off) the liquid (supernatant I) into a clean centrifuge tube without disturbing the sediment
layer. Save the sediment layer for later examination. Make sure the tube containing the D.W.
is again balanced against the tube containing supernatant I. Adjust the volumes to be equal
with a pasture pipette if necessary. Place the tubes opposite each other into the centrifuge;
again noting the position numbers in which your tubes are placed. Close the top of the
centrifuge and turn the speed set dial that equals 1,400 x g. Centrifuge for 12 minutes and
turn the centrifuge off. If during the centrifugation, the machine makes a grumbling noise
and starts walking across the floor, turn it off and check the positioning of the tubes.
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Instructor:
Paramjeet S. Bagga
Lab Protocols
Cell & Molecular Biology
SBIO 406
6. The nuclei and chloroplasts will be in sediment II in mixed buffy (nuclei) and green
(chloroplasts) layers, possibly on top of a white sediment similar to sediment I (which are
starch granules). On top of these layers will be a yellow-green translucent liquid layer
(supernatant II) which contains very small subcellular particles (e.g. mitochondria,
ribosomes, etc.). You will test this supernatant for mitochondrial activity as described below.
B. Test for Mitochondrial Activity in Supernatant II
1. Using a Pasture pipette transfer the supernatant II into a test tube without disturbing the
sediment at the bottom. Leave it on ice.
2. Add 3 ml of sucrose buufer to sediment II, mix gently to form a suspension. Leave it on ice.
3. Get a small test tube rack and place 3 small glass test tubes labeled "A", "B" and "C" in the
rack. Pipette 3 ml of sucrose-buffer solution to "A" and then add 2 ml of tetrazolium solution.
Pipette 3ml of your supernatant II liquid into test tube "B" and 2 ml of the tetrazolium
solution. To tube "C", add 3 ml of sediment II suspension and then add 2 ml of tetrazolium
test solution. Cover each tube with parafilm to form a tight closure, and mix each tube
thoroughly by inverting. Then put each tube back into the test tube rack, and place in the 37
degree water-bath incubator for 30 minutes.
A
B
C
Sucrose Buffer
1 % TTC
Supernatant II
Sediment II
3 ml
2 ml
---
-2 ml
3 ml
--
-2 ml
-3 ml
Total
5 ml
5 ml
5 ml
4. If a tube contains mitochondria with active hydrogen removing enzymes called
dehydrogenases, then both hydrogen ions and electrons will be removed from chemicals
found inside the mitochondria, Inside a cell, these dehydrogenation reactions localized
within the mitochondria are central to the process of energy production, and thus the
mitochondria has been called the "powerhouse" of the cell.
Tetrazolium chloride (TTC) is a compound used by scientists to detect the presence of
dehydrogenation activities. In its oxidized form, a tetrazolium test solution is colorless, but if
it is placed in an environment where hydrogen ions and electrons can be gained (such as
around mitochondria), the tetrazolium becomes reduced to form an insoluble red formazan
compound.
Thus:
Tetrazolium
(oxidized, colorless)
dehydrogenase
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Tetrazolium
(reduced, red formazon)
Instructor:
Paramjeet S. Bagga
Lab Protocols
Cell & Molecular Biology
SBIO 406
This test has commercial applications, for example in testing seed viability. It is assumed that if a
given seed is going to grow after being planted, it must have an active series of dehydrogenation
reactions required for energy production.
While you are waiting for the results for mitochondrial activity ,do the following with your
other cell fractions:
C. Procedure for observation of other Cell Fractions
1. Observe a small quantity of residue (gunk) diluted in a drop of water under low and high
power. What recognizable fragments do you find? Do you see cell wall fragments?
Introduce a drop of IKI under the edge of the coverslip and examine after a few minutes.
Iodine specifically reacts with starch to form a blue-black color. Can you, after staining,
identify starch granules?
2. Examine a diluted portion of sediment I under the microscope. Add a drop of IKI solution
directly to the sediment diluted in water on your slide before applying the coverslip. What
cellular components are present?
3. Examine a portion of sediment II under the microscope. What cellular components are
present?
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