JOURNAL OF BIOCHEM

Journal Homepage: www.arzacan.edu.ph

Fractionation of Cells via Differential Centrifugation

Castro, Ethan Zachary G.*, Asprec, Aljohn Gerarld L., Lola, Ernest Nicolo G., Buenafe, Reuben James Q.**
*Student, CHM161L – A11, Mapua Institute of Technology, ezgcastro@mymail.mapua.edu.ph
**Professor, CHM161L – A11, Mapua Institute of Technology, rjbuenafe22@gmail.com
Mapua Institute of Technology, Muralla Street, Intramuros, Manila, Philippines 1002
School of Chemical Engineering, Chemistry, Biological Engineering, and Materials Science and Engineering

ABSTRACT
ARTICLE INFORMATION After the disruption of the cell membrane, the organelles of a cell may be separated by
utilizing the fact that the components have varying densities due to varying
Article History: composition. Centrifugation physically separates components according to their mass,
Volume Number: 2 density, and shape when the angular speed is varied. The experiment aims to separate
Issue Number: 1 the cellular constituents of a sample of chicken liver via differential centrifugation.
Date of Submission: July 29, 2017 The sample was homogenized using a blender, and the homogenate was subjected to
Date of Acceptance: July 29, 2017 centrifugation at a speed setting of 2900 rpm. After decantation of the sediment, the
supernatant was again centrifuged at 14700 rpm to further separate remaining
Keywords: differential centrifugation, components. The sediments and the final supernatant were tested of their components
chicken liver, nucleus, mitochondria, to determine which cellular component was isolated. It was revealed that the first
cytosol sediment most likely contained the nucleus, the second sediment the mitochondria,
and the final supernatant contains unbroken cell fragments with the cytosol.

INTRODUCTION centrifugation. In centrifugation, the lysate is rotated at a

Cell fractionation is the process of producing pure
fractions of cell components. The process involves two basic
steps: disruption of the tissue and lysis of the cells, followed
by centrifugation. The first step in cell fractionation is tissue
disruption and cell lysis. The objective is to disaggregate the
cells and break them open with minimum damage to the
cellular fraction of interest. Tissues can be broken up and cells
lysed in a number of ways. The three basic methods of
breaking up the tissues and cells are homogenization,
sonication, and osmotic lysis. The particular method one
chooses depends on the tissue, the cell type, and the particular
cell fraction of interest. Most animal and plant tissues must be
homogenized. Homogenization involves the use of a
mechanical homogenizer, like a blender or a mortar and pestle,
to break the tissue apart and lyse the cells. Sonication involves
the use of ultrasound to disrupt the cells. Sonication is often
used when prokaryotic cells are to be lysed. Osmotic lysis is
often the method of choice when dealing with cells that are
vulnerable to osmotic stresses. Red blood cells are a perfect
example of a cell that can easily be lysed through osmotic
stress. Mammalian cells are very sensitive to the tonicity of
the surrounding fluid. In vivo, mammalian cells are bathed in
isotonic fluid, in which case there is no net osmosis and the
cell neither shrinks nor swells (Campbell & Farrell, 2012).
The second step in the cell fractionation process is
centrifugation. Most of the cellular components in a cell lysate
will eventually, given time, settle to the bottom of a tube. To
accelerate this process, the lysate can be subjected to
certain speed. This rotation imposes a force on the particles
perpendicular to the axis of rotation. The force is called a
relative centrifugal force, expressed as a multiple of the force
of Earth's gravitational force. When a particle is subjected to
centrifugal force, it will migrate away from the axis of rotation
at a rate dependent on the particle's size and density (Voet,
Voet, & Pratt, 2013).
Differential centrifugation is one of two major types
of centrifugation schemes. Differential centrifugation is the
sequential centrifugation of a cell lysate at progressively
increasing centrifugation force, isolating cellular components
of decreasing size and density. The separation of the cellular
components is based solely on their sedimentation rate
through the centrifugation medium, which, in turn, is
dependent on the size and shape of the cellular components. In
differential centrifugation, each centrifugation step results in
the production of sediments, usually containing a mixture of
cellular components of the same size or density. The fluid
resting above the pellet, the supernatant, can be removed and
subjected to additional centrifugations to generate sediments
containing other cellular components of lesser size or density
(Kenkel, 2003).
After the cell fractionation process, one may opt to
perform qualitative tests to confirm the presence of the desired
organelle in the isolated sediment or supernatant. Usually, one
may detect the presence of a certain organelle by testing for its
known components. For example, the nucleus is where the
nucleic acid content of a eukaryotic cell is concentrated:

therefore it would give a positive test in the test for
deoxyribonucleic acid.
Figure No. 1: Differential Centrifugation
If one wishes to detect the presence of carbohydrates
in general, Molisch’s test offers a sensitive chemical test
designed especially for this purpose. Molisch’s reagent is α-
naphthol dissolved in ethanol. Carbohydrates, when treated
with concentrated sulfuric acid, become dehydrated, and will
eventually yield furfural derivates. The positive test result is
the formation of a purple product at the interface of the test
solution and sulfuric acid due to the reaction between the
furfural derivative and α-naphthol (McNaught, 2006).
On the other hand, to test for the presence of a
peptide bond, the Biuret Test may be performed on the
sample. The reagent used in the Biuret Test is made of NaOH
and hydrated cupric sulfate, together with potassium sodium
tartarate, which acts as a chelating agent to stabilize the cupric
ions. In the presence of an amide linkage, such as a peptide
bond, in an alkalinic medium, cupric ions form violet-colored
coordinated complexes, indicating a positive Biuret Test.
Amino acid monomers, such as glycine, are expected to
produce a negative result since no peptide bond is present
(McNaught, 2006).
Moreover, pentose sugar may be distinguished from
hexose sugar through Bial’s test. Bial’s reagent consists of
orcinol (3,5-Dihydroxytoluene), HCl, and FeCl3. Upon
dehydration in acidic medium, pentose sugars produce
furfural, which reacts with orcinol and ferric chloride yielding
a blue-green condensation product. On the other hand, hexose
sugars form 5-hydroxymethylfurfural, which forms a green,
brown, or reddish-brown color upon reaction with Bial’s
reagent. (Shriner, 1998)
In addition, a known test to assay lipids in unknown
samples is the Sudan Red Test. Sudan Red is fat-soluble dye
used for staining lipids. The idea is to use a moderately non-
polar solvent to dissolve the dye, allowing it to partition into
the highly non- polar lipid without the solvent dissolving the
2
lipid to be stained. A red, viscous solution with the unknown
indicates a positive Sudan Red Test (Shriner, 1998).
Finally, to test for the presence of deoxyribonucleic
acid, one may either utilized the diphenylamine test, or
Schiff’s test. The first test makes use of the reaction of 2-
deoxypentose compounds with diphenylamine under acidic
conditions, as it would give a blue complex if such compounds
are present. The disadvantage of this test is that it is not
specific for DNA, and would give a positive test as long as a
2-deoxypentose compound is present. The latter requires
partial acid hydrolysis of the sample before reaction with the
reagent. Schiff’s reagent reacts with potential aldehyde
groups, which is only present in DNA after it has been
partially hydrolyzed. The reaction will yield a purple product
(Starr, Taggart, Evers, & Starr, 2009).
After knowing the components of the given sediment
or supernatant, one can predict the organelle present by
combining the data obtained and knowledge of the molecular
compositions of typical cell organelles.
EXPERIMENTAL SECTION
The procedure commenced with the washing-off of
the chicken liver sample using a few drops of the
homogenizing medium assigned for the group. In the case of
this group, water was used as the homogenizing medium.
Other groups used 0.16 M NaCl solution for that purpose. This
is to test which homogenizing medium is more effective in
disaggregating the cells comprising the tissues of the chicken
liver sample. After washing, the sample was minced and kept
in ice. 7 grams of the minced sample were placed in a beaker,
after which 35 mL of the homogenizing medium was added.
The formed mixture was then homogenized using a blender at
the lowest speed possible for 5 minutes. The homogenate was
then placed in another clean beaker and kept in ice for the
succeeding parts.
The homogenate was then centrifuged at a speed
setting of 1. The ratio used for this experiment for the
centrifuge was 1 is to 2900 rpm. This means that a speed
setting of 1 corresponds to an angular velocity of 2900 rpm.
The sample was centrifuged for 10 minutes in a refrigerated
centrifuge. After the process, the supernatant was decanted
and was labeled Supernatant I. To the sediment, 10 mL of the
homogenizing medium was added and was stirred, while kept
at a low temperature. This sediment was labeled Sediment I.
Supernatant I was then centrifuged at a speed of 14700 rpm,
corresponding to a speed setting of 5 for the centrifuge. The
operation was done for 10 minutes; again while keeping the
temperature low. The sediment from this process was labeled
Sediment II, and was treated the same way as Sediment I. The
supernatant, on the other hand, was labeled Supernatant II and
was placed in ice after decantation.
After the differential centrifugation procedure,
qualitative tests were done to check the chemical composition
of Sediment I, Sediment II, and Supernatant II. These samples
were tested for the presence of nucleic acid, carbohydrates,
proteins, and lipids.

In testing for the presence of nucleic acids, partial
acid hydrolysis of the sample must first be done. Five micro
test tubes were prepared, with one having a positive control of
1% DNA or 1% RNA, the three containing the
aforementioned test samples, and the fifth test tube containing
the homogenizing medium, which served as the blank. 2-mL
of the corresponding solutions was placed inside the test tubes.
Afterwards, 2-mL of 0.1 M HCl was added to each of the test
tubes. The test tubes were then covered and placed in a boiling
water bath for 30 minutes. The resulting solutions were then
tested for DNA using the Diphenylamine Test and Feulgen’s
Nucleal Reaction, and RNA using Bial’s (Orcinol) Test.
For the Diphenylamine Test, 10 drops of the partially
hydrolyzed solutions were placed in another set of five
separate test tubes. 2 drops of diphenylamine was then added,
together with 1 drop of concentrated H2SO4. The solutions
were then heated in a boiling water bath until the positive
control gave the positive test (blue-colored solution).
Meanwhile, Feulgen’s Nucleal Reaction began with
the addition of 10 drops of the partially hydrolyzed solutions
to another set of five different micro test tubes. The pH of
each solution was tested, and was neutralized with 0.1 M
NaOH. After neutralization, 2 drops of Schiff’s reagent was
added to each test solution. The test tubes were then covered
with cork and set aside for 10 minutes. The positive test is the
appearance of a purple-colored solution.
On the other hand, to test the presence of RNA using
Bial’s Reaction, 10 drops of the partially hydrolyzed solutions
were again added to 5 different micro test tubes. Addition of 5
drops of orcinol followed. The test solutions were then heated
in a boiling water bath until the positive control gave the
positive test (blue-green solution).
Furthermore, to test for the presence of
carbohydrates, the Molisch Test was utilized. In this
procedure, the positive control was 1% ribose solution. 5
drops of the positive control, Sediment I and II, Supernatant II,
and the homogenizing medium, were placed in separate micro
test tubes, after which 5 drops of Molisch reagent was added
to each. After proper shaking, 1 mL of concentrated H 2SO4
was added dropwise and slowly, allowing the formation of a
layer. The appearance of a purple junction at the interface of
the layers indicates a positive test.
Moreover, the Biuret Test was used to confirm the
presence of protein in the samples. The positive test used for
this part of the procedure is 1% albumin solution. 5 drops of
the positive control, Sediments I and II, Supernatant II, and the
blank homogenizing medium were again placed in 5 separate
micro test tubes. Addition of 5 drops of Biuret reagent with
proper shaking followed. Afterwards, 1 mL of concentrated
H2SO4 was added dropwise and slowly. The appearance of a
violet solution is the positive Biuret test.
Finally, to test for the presence of lipids, the Sudan
Red Test was used, and for this procedure, the positive test
was cooking oil. 5 drops of the positive control, Sediments I
and II, Supernatant II, and the blank homogenizing medium
were again placed in 5 separate micro test tubes. A pinch of
Sudan IV crystals were then added to the tubes. The
3
appearance of a red-orange solution indicates that the crystal
have dissolved and therefore is the positive test.
RESULTS AND DISCUSSION
A comparison between the results of homogenization
using water and 0.16 M NaCl solution as the homogenizing
agent was made, and several obvious differences may be
noticed. First, the homogenate when 0.16 M NaCl was used
was foamier than that of when water was used. After the first
centrifugation, the homogenate subjected to the saline solution
had finer precipitates at the bottom. Moreover, the second
supernatant produced after the centrifugation of the first
supernatant was cloudier for the sample where 0.16 M NaCl
was used for homogenization. The results imply that using
saline solution as homogenizing medium somehow improved
the effectiveness of the cell lysis procedure, since more foam
indicates more particulate matter dissolved, very fine
precipitate formed at the bottom of Supernatant I, and
cloudiness of Supernatant II indicates the presence of small
matter. This may have something to do with the tonicity of the
media used. Water is completely isotonic to the cells being
lysed. The movement of water into and from the cell is at
equilibrium with each other, which implies that it is not much
aid in lysing the given cell. However, in introducing a saline
solution to the surrounding of the cell, there will be a
concentration gradient (in terms of water) between the
surroundings and the inside of the cell. This becomes a driving
force for osmosis, or the movement of water into and from the
cell. Osmosis follows a simple principle: spontaneous flow of
water is from a high water concentration to a low water
concentration. This means that when a salt solution is used,
the water concentration outside of the cell is relatively low,
and water will flow out of the cell until equilibrium is reached.
Water flowing out of the cell brings about osmotic pressure,
which aids in the lysis of the cells comprising the sample.
Therefore, the salt solution is hypertonic to the given cell.
However, the tonicity of the salt solution may be considered
negligible, since only a small amount of salt is introduced (as
dictated by the concentration of the solution used). When
other solutions, however, such as detergents, were used, the
transfer of water would be more significant, compared to
when using salt, and the cell will be lysed more effectively. it
must be noted, however, that aside from more cell components
released, one factor that must be considered in cell lysis is that
only minimal damage is done to the cell. Therefore, the choice
of homogenizing medium is crucial: if the medium is too
harsh on the cell, it might completely degrade its components.
It must be noted that the procedures were done while
keeping the temperature of the system low. This is to prevent
the effects of cellular enzymes, such as protease, from
degrading cellular components significantly. If done at room
or higher temperature, the activity of these enzymes would
increase, and majority of the cellular components to be
analyzed would be destroyed. Further, higher temperatures
may cause degradation and denaturation of protein molecules
present. Denaturation of the protein components of the
organelles would affect their sedimentation, since denaturation

alters chemical composition, and thus, this would alter the
specific properties of the organelle as well. This may result in
incorrect fractionation.
As for the differential centrifugation procedure, the
order of separation is expected to be by decreasing density,
size, and shape. A table showing the sizes and densities of
typical organelles is shown below.
Table No. 1: Size and Density of Typical Organelles
Organelle Diameter (μm) Specific Gravity
Nucleus 5-10 1.4
Mitochondria 3-4 1.1
Ribosomes 0.02 1.0
Lysosomes 1-2 1.1
By following the aforementioned order of separation, it is
expected that Sediment I contains the nucleus, Sediment II
contains the mitochondria (since it is bigger in shape
compared to lysosomes), and Supernatant II contains the rest
of the cellular components. To confirm this hypothesis,
qualitative tests were done to the sediments and Supernatant
II. Sediment I tested positive for DNA, RNA, carbohydrates,
lipids, and protein. This means that Sediment I indeed contains
the nucleus. The nucleus is where the nucleic acid content of
eukaryotic cells such as chicken liver is concentrated;
therefore it will give a positive test for DNA and RNA.
Further, DNA and RNA are actually made from subunits
called nucleotides. Nucleotides consist of a phosphate group, a
nitrogenous base, and a sugar. Therefore, the nucleus will give
a positive test for carbohydrates. In addition, chromosomes
found in the nucleus are DNA chains wrapped around histones
(a protein), giving a positive test for proteins. Further, cellular
nuclei contain high levels of phosphatidylcholine and
sphingomyelin, which are partially linked with cholesterol and
proteins to form lipid–protein complexes, thereby giving
positive lipid tests.
Meanwhile, the second densest organelles are the
mitochondria and the lysosomes, but since the mitochondria is
larger in diameter, it is expected that it resides under Sediment
II. True enough, Sediment II gave positive tests for all
macromolecules. The mitochondrion contains mitochondrial
DNA, which causes it to give a positive DNA test. Moreover,
it houses ribosomal RNA, giving a positive test for RNA. The
job of mitochondria is to break down carbohydrates and lipids
in order to generate energy. Therefore, carbohydrate and lipid
residues are expected to be present in the mitochondrion.
These residues are the ones reacting with the appropriate
reagents to give a positive carbohydrate and lipid test. Finally,
mitochondrial proteins are proteins that reside within the
mitochondria of cells, including within the cristae of the inner
mitochondrial membrane. Mitochondrial proteins are
generally involved in mitochondrial function, including
carrying out reactions of the electron transport chain.
Mitochondrial proteins are derived either from genes encoded
by the nuclear genome, or by DNA contained within
mitochondria. These proteins are the ones that cause Sediment
II to give a positive protein test.
4
Finally, the rest of the cellular components are
contained in Supernatant II. This includes the ribosomes, the
lysosomes, the cytosol, and others. No genetic material can be
seen in these organelles: therefore, no DNA is virtually
present. However, ribosomal RNA can be found along the
cytoplasm, therefore giving a positive RNA test. Protein
residues may also be found, as well as the unbroken transport
proteins of the cell, therefore giving positive protein test. Very
little carbohydrates and lipids are found in these organelles
such that it was not detected by the tests performed.
CONCLUSION
Fractionation of chicken liver cells was performed
successfully via differential centrifugation. This involved
homogenizing the tissues first, followed by sequential
centrifugation. The separation is expected to follow an order
of decreasing density, size, or mass. It is therefore concluded
that Sediment I contains the nucleus, Sediment II the
mitochondria, and Supernatant II the rest of the cellular
components. This assertion is further confirmed by performing
qualitative tests on the sediments and the second supernatant.
Both sediments gave positive tests for all macromolecules,
somehow supporting the aforementioned conclusion regarding
the two. Meanwhile, Supernatant II gave positive tests for
protein and RNA, which are expected to be the primary
composition of organelles such as ribosomes, lysosomes, and
the cytosol.
In addition, the effect of the homogenizing medium
used was also investigated in this experiment. The group used
water as the medium, while another group used 0.16 M NaCl.
It was found that cell lysis was more effective when the salt
solution was used due to it being a hypertonic solution. Water
will come out of the cell, and the osmotic pressure brought
about by flowing water will aid in the lysis of the cell.
REFERENCES
1. Campbell, M. K., & Farrell, S. O. (2012).
Biochemistry, 7th Edition. Belmont: Brooks/Cole,
Cengage Learning.
2. Voet, D., Voet, J. G., & Pratt, C. W. (2013).
Fundamentals of Biochemistry: Life at the Molecular
Level, 4th Edition. John Wiley and Sons, Inc.
3. Kenkel, J. (2003). Analytical Chemistry for
Technicians, 3E. New York City: Lewis Publishers.
4. McNaught, e. a. (2006). Compendium of Chemical
Terminology. IUPAC.
5. Shriner, R. L. (1998). The Systematic Identification of
Organic Compounds. New York City: John Wiley
and Sons.
 5

6. Starr, Taggart, Evers, & Starr. (2009). Biology: The

Unity and Diversity of Life . Cengage Learning.

SUPPORTING INFORMATION

Figure No. 6: Positive Diphenylamine Test

Figure No. 2: Positive Biuret Test

Figure No. 7: Schiff Color Test

Figure No. 3: Positive Bial’s Test

Figure No. 8: Centrifuge Instrumentation Scheme

Figure No. 4: Positive Molisch Test

Figure No. 5: Positive Sudan Red Test