BIOCHEMISTRY (CHEM 24)

LABORATORY FORMAL REPORT

SUBCELLULAR COMPONENTS
Agustin, Ara Vanessa; Asierto, Faye Julienne; Bondoc, Jao; Bongolan, Abigail; Nicolas, Jomari;
Simsuangco, Joanna Faye; Totto, Nikki Mae
MT213 Group 5

Date Performed: January 17, 2017 Date Submitted: January 31, 2017

Institute of Arts and Sciences, Far Eastern University, Recto Ave., 395, Manila, 1008
ABSTRACT
The cell is known to be the basic unit of life or often called the building blocks of life" which is
compose of different subcellular components that have different biological molecules. All cells have DNA,
which is the hereditary material of genes, and RNA that contains the information necessary to build various
proteins such as enzymes. The objectives of this experiment are to extract cell organelles through
homogenization and to separate the different cell organelles through centrifugation specifically through
differential centrifugation. In addition to that, this experiment will give the identification of the presence of
biomolecules using different qualitative test. For the laboratory set-up, prepare a fresh chicken liver and
blend it. Transfer some on a centrifuge tube and continue with the differential centrifugation. After that,
proceed with the qualitative test; different reagents are needed to complete the experiment like NSS,
Molisch reagent, Orcinol reagent and etc. Therefore at the end of this experiment, you will learn about the
principles of separation of subcellular components. For the expected results on the qualitative test, the color
would turn into color blue for the positive result of DNA, blue to blue-green for RNA, purple/pink for proteins,
soluble red-orange for lipids and purple for carbohydrates.

1.0 INTRODUCTION specific functions inside the cell are called

Cell is the basic living unit of all
organisms. It is very small but contains all the
characteristics of life. It performs several
functions such as cell metabolism and energy
use, synthesis of molecules, communication,
reproduction and inheritance (VanPutte et al,
2016). The specialized structures that perform
organelles.
To be able to study cell and its functions,
it is important to know what components make up
the organelles of the cell. Biomolecules are
molecules that are involved in the maintenance
and metabolic processes of living organisms.

BIOCHEMISTRY (CHEM 24) LABORATORY FORMAL REPORT 1

These molecules namely, nucleic acids, proteins,
lipids and carbohydrates constitute the
organelles. The isolation of pure components of
the cell is essential for these molecules to be
studied.
Subcellular fractionation is the process
of producing pure fractions of cell components.
This method is used when studying subcellular
components for it isolates different organelles
from one another in order to be subjected to
further tests and analysis.
The process involves two basic steps:
disruption of the tissue and lysis of the cells,
followed by centrifugation, (Gallik, 2011). Figure 1
at Appendix A shows the steps on subcellular
fractionation. The first ones objective is to
disaggregate the cells and break them open with
minimum damage to the cellular fraction of
interest. This can be performed in three different
methods (1.) Homogenization breaks up the
tissue apart and lysed the cells through
mechanical homogenizer, such as blender. (2.)
Sonication uses ultrasound to disrupt the cells. It
is often used when prokaryotic cells are to be
lysed. (3.) Osmotic lysis deals with the
vulnerability of cells to osmotic stresses. Red
blood cells can easily be lysed through osmotic
stress. The process of breaking tissues and cells
is shown in Figure 2 of the Appendix A.
Most of the subcellular components in a
lysed cell will eventually settle at the bottom of
BIOCHEMISTRY (CHEM 24) LABORATORY FORMAL REPORT
the tube. To accelerate this process, the lysate
can be subjected to centrifugation. In this
process, the lysate is rotated at a certain speed
expressed as rotations per minute (RPM). This
rotation imposes a force on the particles
perpendicular to the axis of rotation. The force is
called a relative centrifugal force (RCF),
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.
The specific centrifugation scheme used
in this experiment is differential centrifugation. It
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 is dependent on the size and
shape of the cellular components. Figure 3 of
Appendix A shows centrifuged cell homogenate
that are subjected to different speeds.
This experiment focuses on separating
subcellular components through the use of
subcellular fractionation. It aims to determine
what biological molecules are present on each
fraction separated and analyze how frequent is
their appearance on each fraction.
2.0 METHODOLOGY
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2.1 Materials and Reagents
Different laboratory apparatuses and
reagents were used to identify the biomolecules
present in the different fractions namely, Pyrex
test tubes, vials, 10 ml graduated cylinder,
centrifuge, blender, pipette, droppers, beaker,
iron stand, Bunsen burner, and water bath.
Reagents like Normal saline solution and distilled
water was used to prepare the chicken liver for
homogenization. Concentrated Sulfuric acid and
diphenylamine reagent was used in Dische
reaction, a general test for DNA. For the Orcinol
test, which is a test for RNA, freshly prepared
Orcinol reagent was used. Biuret test for proteins
was done by the used of 10% NaOH, 0.5%
CuSO4 solution, and 1% albumin solution for
control test. In the test for lipids, chloroform and
Sudan IV was used. Molisch test for
carbohydrates was obtained by the used of
Molisch reagent, and concentrated sulfuric acid.
Figure 1 at Appendix B shows the different
reagents used in the experiment.
2.2 Cell fractionation
Cell fractionation is used to break open
the cells and separate the various organelles. To
perform cell fractionation, the cells need to be
suspended first in solution, and then break open
the cell. Normal saline solution was used to wash
60 grams of chicken liver to irrigate tissues for
the collection of the organelles. In addition to it,
chicken liver was soaked in distilled water for
hemolysis. This will release the organelles inside.
BIOCHEMISTRY (CHEM 24) LABORATORY FORMAL REPORT
After two hours, 250 mL of NSS was added again
to the chicken liver and used blender to emulsify
it. The chicken liver is then homogenized at
liquefy speed for 10 minutes. Each group got
their samples in vials and subjected it to
differential centrifugation. By using centrifugation,
it can easily separate the various, since the
various organelles are of different mass, and
density. During centrifugation, different organelles
will form pellet at the bottom at specific speeds
based on the mass and densities of the
organelles. The sample was subjected to the first
centrifugation at 2rpm for 5 minutes. One
member of the group decants the supernatant
from the centrifugation to another vial. The group
then obtained precipitate 1 and filtrate 1. For the
second centrifugation, filtrate 1 was put in the
centrifuge and spin at 5 rpm for 15 minutes. The
supernatant was decant to another vial as the
filtrate 2, and so precipitate 2 was attained. For
the third and last centrifugation, filtrate 2 was
subjected to centrifugation at 8rpm for 20
minutes. The filtrate 3 was discarded and got the
precipitate 3. The group collected precipitate 1, 2
and 3 in different test tubes for further study. For
the cell suspension 10 ml of NSS was added to
each precipitate.
2.3 Qualitative test
2. 3. 1 Nucleic acids
2. 3. 1. 1 DNA
To 6 drops of cell suspension, 4 drops
of diphenylamine was added followed by 2 drop
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of sulfuric acid in a test tube. The mixture was 2. 4 Observation and Recording
then heated in a water bath for 2 minutes. The The positive and negative results of bio-
procedure was done 3 times with different cell molecules in each solution were observed in the
suspensions. basis of the results that was given by the
professor as shown in Table (1).
2. 3. 1. 2 RNA
Table 1. Positive results for the
To 6 drops of cell suspension, 4 drop of Qualitative tests of Subcellular Components.
freshly prepared Orcinol reagent in a test tube.
Biological Molecules Positive Result
The solution was then heated in a boiling water Blue, Crumpled DNA
DNA
bath for 2 minutes. The procedure was done 3 Fibers
Blue to Green,
times with different cell suspensions. RNA
Crumpled RNA Fibers
Protein Purple or Pink
2. 3. 2 Proteins Lipids Soluble Red-Orange
Carbohydrates Purple
In the experiment with proteins also known
as Biuret Test, 6 drops of cell suspension in a test 3.0 RESULTS AND DISCUSSIONS
tube, 6 drops of biuret reagent was added which This experiment aimed to identify the
was composed of 2 drops 10% NaOH and 0.5% principles of separation of subcellular
CuSO4 soln. 1% albumin soln. was used as a components, the principles in using differential
control test. The procedure was done 3 times configuration to isolate subcellular components
with different cell suspensions. and to the biomolecules present in different
fractions using qualitative tests.
2. 3. 3 Lipids
In the experiment with lipids, a 2 ml cell
The cell is composed of cell organelles
suspension placed in a test tube was mixed with
that have different biomolecule compositions.
4 drops of chloroform, followed by a small
Cell is the smallest unit of life that can replicate
amount of Sudan IV.
independently, and cells are often called the
2. 3. 4 Carbohydrates building blocks of life.
In the experiment with carbohydrates also
known as Molisch Test, 2ml of cell suspension in The experiment was conducted using
a test tube was mixed with 4 drops of Molisch three general procedures: Extraction, process of
reagent. 2ml of concentrated Sulfuric acid was isolating the organelles, Homogenization, a
then carefully added and the color of the ring was process to disrupt the cell and Differential
observed and noted. Centrifugation, a process to separate the cell
organelles. Figure 2 at Appendix B shows the

BIOCHEMISTRY (CHEM 24) LABORATORY FORMAL REPORT 4

homogenized liver sample used in
experiment. Normal Saline Solution was the main
reagent used to sterilize the chicken liver. This
reagent was used so as to prevent the
dehydration of the cell. There were different
processes made to have three filtrates and three
precipitate, which were, needed for
qualitative tests that were Precipitate
Precipitate II and Precipitate III.
The three precipitates were used in
different qualitative tests which are: Nucleic acid
particularly DNA and RNA, Proteins (Biuret test),
Lipids and Carbohydrates (Molisch Test).
Dische Diphenylamine Reaction is used
to detect the presence of DNA in a substance. In
this process, the reaction between the Dische
reagent and 2-deoxypentose results in the
development of a blue color. The reaction
depends on the conversion of the pentose to w-
hydroxylaevulinic aldehyde, which then reacts
with diphenylamine to give a blue colored
complex. The intensity of the blue color is
proportional to the concentration of DNA. This
test is used to detect the presence of RNA in a
substance. This reaction will produce a blue-
green color. The students made several tests and
they yielded a cream color and it had a white
precipitate and that means this test gave a
negative result because there was no RNA
present in the solution. Dische reagent does not
react with the ribose sugar in RNA and does not
form a blue-colored complex. In precipitate I,
BIOCHEMISTRY (CHEM 24) LABORATORY FORMAL REPORT
the Dische test gave a negative result because it had
a cloudy appearance in the solution while Orcinol
test also gave a negative result since the color
produced after doing this test was light brown
solution. In precipitate II, both tests gave a
negative result because in Dische reaction it also
the had a cloudy appearance in the solution while
I, Orcinol test had a light brown solution. Lastly, in
precipitate III both were negative because the
Dische test had a cloudy appearance and the
Orcinol test had a color of cream. In addition to
the color changes here in Nucleic Acids, one of
the indication if the tests are positive is when you
have a crumpled fibers in the solution. In the
experiment, crumpled fibers were there in the
solution and that means DNA and RNA were
present.
Figure 1. The reaction with diphenylamine
caused the reaction to create a blue-colored
complex.
Figure 2. The reaction of Orcinol reagent,
which gives of the blue or blue-green
complex.
Biuret Test this test is used to detect the
presence of a dipeptide. In the presence of
5
peptides, a copper (II) ion forms violet-colored
coordination complexes in an alkaline solution.
The students made several tests using the given
solutions. The three precipitates gave positive
results in doing this test because the solution
was purple and that means there was a protein
present in the solutions.
Figure 3. The reaction of biuret reagent
causing it to violet color.
Sudan IV is classified as an oxidizing
dye, carcinogen. It is used to stain lipids and
triglycerides. Sudan IV is nonpolar, thus, it only
dissolves in nonpolar substances. Again, the
three precipitate gave a positive result because
all of them turned into reddish-orange right after
the small crystals of Sudan IV added.
Molisch test is a general test for
carbohydrates. Molisch is composed of 1%
alphanapthol in ethanol with addition of
concentrated H2SO4. The reagents reaction to
furfural or hydroxymethylfurfural formed by
adding sulphuric acid will cause the presence of
a violet ring. In precipitate II and III, this test gave
positive results because it had a purple ring at
the junction, which means there were
carbohydrates present in the solution.
BIOCHEMISTRY (CHEM 24) LABORATORY FORMAL REPORT
Unfortunately, precipitate I gave a negative result
because there was no purple color at the
junction.
Figure 4. The reactions in the Molisch test are
not specific for a certain carbohydrate but a
negative result shows good evidence of their
absence. Further test are required if a positive
(red or purple) result is seen.
The following results for precipitates I, II
and III are shown in figures 1, 2 and 3 of
Appendix C.
Students cannot avoid making mistakes
while performing an experiment. It is the
students job to study well the procedures in the
experiments for them to be able to perform the
experiment well. Accuracy is a must. The
possible sources of errors could be, the
containers used in the experiment are not that
clean or maybe there are still residues remaining
in there that is why some of the tests will give a
negative result. It might be the case that the
reagents provided by the laboratory are
6
contaminated with other solution. It also might be
because of the concentration of biomolecules for
a certain tests are not enough to show a positive
result. Most of the qualitative tests were negative
in RNA because RNA is a messy folder full of
notes that can be scattered around the cell. Also
it might be the case that the solutions given are
already expired and the laboratory technicians
did not notice it at all. Sometimes, it is all about
the students who were not able to perform the
procedures in their experiment that is why when
they perform their tasks they will get a negative
result. This only shows that it is important to read
or understand well the experiment. In the
experiment, the chicken liver might not blend
properly which might be able to be one source of
error. Also, it is suggested to heat the solution for
faster reaction like in DNA and RNA tests. Some
of the qualitative tests were negative because of
errors in the procedure.
4.0 CONCLUSIONS AND RECOMMENDATION
In this experiment, the students were
able to identify the principles used in differential
centrifugation for isolating subcellular
components and the biological molecules that
are present in the different fractions using
qualitative tests. The three general procedures in
this experiment were extraction which is the first
step toward isolating any subcellular component;
second is homogenization which involves the
breaking apart of cells; thus, releasing the
organelles and cytoplasm. This can also be done
by thawing mechanical means using mortar and
BIOCHEMISTRY (CHEM 24) LABORATORY FORMAL REPORT
pestle; third is centrifugation which is done by
putting the solution of homogenized organelles in
a variable speed centrifuge and rotating them at
a higher rate of speed. In general, nuclei are the
heaviest subcellular component. The next is
composed of the mitochondria and lastly the
supernatant is composed of the least dense
components such as Golgi apparatus,
endoplasmic reticulum, vesicles, ribosomes and
cell membrane. The nucleus gave a positive
result for the biuret test, which indicates the
presence of proteins. A positive result for Sudan
IV test indicates the presence of lipids. The
mitochondria contain the four biological
molecules, which are the carbohydrates,
proteins, lipids and nucleic acid. The supernatant
gave a positive result for the biuret test, Molisch
test and Sudan IV indicating the presence of
carbohydrates, proteins and lipids. The bigger
and/or heavier molecules are found in the
sediment, while the lighter components are on
the supernatant liquid.
In order to get positive result students
should have a proper knowledge about the
experiment to be performed. Careful execution of
the procedures could help in achieving the
positive results. Also, the reagents from the
laboratory should be prepared well in order to
prevent errors in the experiment. Another factor
is to increase the concentration of the subcellular
components to be able to attain an indication of
positive results.
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5.0 REFERENCES Cinnamon VanPutte, J. R. (2016). Seeley's
Alcantara, R. (2014, 11 30). Prezi. Retrieved 01 Essentials of Anatomy and Physiology . McGraw
28, 2017, from Hill Education .
https://prezi.com/c1dpzhcczkxz/subcellular-
component-experiment-2/ Stephen Gallik, P. D. (2011). Cell Biology OLM.
Biochemistry, T. C. (2014). Laboratory Manual Retrieved 01 28, 2017, from
and Guide Notes in Biochemistry . Brightkids http://cellbiologyolm.stevegallik.org/node
Publication . /74

6.0 APPENDIX
6.1 Appendix A (Subcellular Fractionation)

Figure 1. Subcellular Fractionation

BIOCHEMISTRY (CHEM 24) LABORATORY FORMAL REPORT 8

 Figure 2. Tissue/ Cell Homogenization

Figure 3. Differential Centrifugation

BIOCHEMISTRY (CHEM 24) LABORATORY FORMAL REPORT 9

6.2 Appendix B (Procedure of the Experiment)

Figure 1. Reagents Used in the Experiment

 Figure 2. Homogenized Liver

6.3 Appendix C (Qualitative Tests Results)

Figure 1. Results in Precipitate I (Nuclear Fraction)

Figure 2. Results in Precipitate II (Mitochondrial Fraction)

Figure 3. Results in Precipitate III (Mircosomal Fraction)