EXPERIMENT #1

Analysis of Subcellular Components

It is essential to obtain adequate quantities of cellular organelles to study their structure and
metabolic functions. Before this can be achieved, however, the cells must be disrupted in one of several
ways. They can be subjected to osmotic shock or exposed to high-frequency sound waves. Under certain
conditions, sucrose solution, for example, can be used to rupture the plasma membrane, leaving the
organelles intact. In this experiment, these organelles are separated by differential centrifugation. Their
composition can be determined by using common qualitative tests.

Learning Objectives:

At the end of the experiment, the student should be able to:

1. Give a concise account of the components of cells and how they may be isolated;

2. Determine the different biomolecules present in the cell; and

3. Understand and appreciate the different biochemical systems that bring about, activate and
maintain life.

Materials:

Equipment and glassware: centrifuge tube, centrifuge test tubes (20 x 100mm), test tube rack,
filter paper, water bath, pipette (5 mL), medical dropper, aspirator bulb, Bunsen burner, tripod, iron
stand, beaker (250 mL)

Reagents and chemicals: 0.025 M sucrose solution, 10% NaOH, 0.5% CuSO4, 1% albumin
solution, Sudan crystals, Molisch reagent (5% solution of alpha-naphthol in alcohol), conc. H2SO4,
diphenylamine reagent, paraffin oil, orcinol reagent.

Procedure:

1. Preparation of supernatant and sediment of the subcellular components of chicken liver

a. Thoroughly wash 7g of fresh chicken liver with enough 0.025 M sucrose solution. Cut into
small pieces and soak in distilled water for 2 hours. Blot with a piece of filter paper. Mince finely and
keep frozen until ready to be used.

b. Add 35mL of 0.025 M sucrose solution and homogenize the mixture at low speed for 5 to 10
minutes.
 c. Subject the mixture to differential centrifugation: Homogenize cells in sucrose solution,
centrifuge for 10 minutes at 600 rpm.

• Pellet (Nuclear fraction) – Supernate 1 (Centrifuge for 20 mins. at 12, 000 rpm)
• Pellet (Mitochondrial fraction) – Supernate 2 (Centrifuge for 60 mins. At 100, 000 rpm)
• Pellet (Microsomal fraction) – Supernate 3 (Microsomes, soluble proteins, enzymes and
inorganic ions)

2. Qualitative tests

a. Divide the cell suspension on each fraction into three sets. Label each test tube for each
fraction as Tube A, B and C. Perform the following tests on Tube A.

1. Proteins (Biuret test). Add 5 drops of 10% NaOH and 1 drop 0.5% CuSO4 to 5 drops of
the suspension. Observe the results. Run a control using 1% albumin solution.
2. Lipids. Add a small crystal of Sudan IV to 5 drops of cell suspension. Perform a control
test using 1% Paraffin oil.
3. Carbohydrates (Molisch test). Mix 2 mL of the cell suspension with 2 drops of Molisch
reagent (5% solution of alpha-naphthol in alcohol). Mix thoroughly. Carefully add 2 mL
of conc. H2SO4 by allowing it to flow down the side of the tube to form a layer below the
sugar solution. Observe the color that develops at the interface of the mixture of conc.
H2SO4 layer.
4. Nucleic acid (Diphenyl colorimetric reaction)
i. For every fraction labeled as Tube B, divide the cell suspensions into three
screw-cap tubes and do the following test:
▪ 1.0 mL DNA (cell suspension) + 0.35 mL 0.15M NaCl (Set on ice.)
▪ 1.0 mL DNA (cell suspension) + 0.35 mL 0.15M NaCl (Place in a boiling
bath for 5 mins. Then immediately place on ice. Unscrew the cap
enough to let the water vapor escape. Take care that the tube doesn’t
tip over.)
ii. Place 10 mL of 10% H2SO4 into each tube and heat for 15 minutes at 95˚C. Cool
to room temperature.
iii. Add 3 mL Diphenylamine reagent and place the tubes in a boiling water for 10
mins. Cool the tubes in an ice bath.
iv. Compare the relative amounts of proteins, nucleic acids, lipids and
carbohydrates in each of the pellet or cell suspension fractions. Use +++, ++ and
+ (depending on the intensity of the color of the solution) when recording and
tabulating your results.
5. RNA (Orcinol test). Add 5 drops of freshly prepared Orcinol reagent to 5 drops of cell
suspension on the remaining Tube C. Heat in a boiling water bath.
EXPERIMENT #1: POST-LABORATORY QUESTION AND RESULTS:

Name: Date:
Group: Instructor:
Section:

I. DATA AND RESULTS

A. Carbohydrates
i. Observations:

ii. Principle behind the test procedure:

B. Proteins
i. Observations:

ii. Principle behind the test procedure:

C. Lipids
i. Observations:

ii. Principle behind the test procedure:

D. Nucleic acid (DNA)

i. Observations:

ii. Principle behind the test procedure:

E. Nucleic acid (RNA)

i. Observations:

ii. Principle behind the test procedure:

II. TEST RESULT: Complete the table shown below by indicating the reaction of the sample sediments to
the test procedures. Use +++, ++ and +, depending on the intensity of the color of the solution.

Sediment Nucleic acids Proteins Lipids Carbohydrates

1
2
3
III. QUESTIONS

A. Sketch a generalized animal cell and label all the parts.

B. Make a table summarizing the different cell organelles’ compositions and functions.
C. List down the hydrolytic enzymes that are found in the lysosomes and identify their corresponding
substrates.

D. Why are mitochondria called “the powerhouse of the cell”? Give the full name and structure of the
energy-rich molecule generated primarily at the mitochondria.

E. What are membrane transport mechanisms? Explain these mechanisms.

F. Why do the different macromolecules require different centrifugation sediment rates and times in
order to be isolated?

G. Explain the differences in the amounts of proteins, lipids, carbohydrates and nucleic acids in the
hydrolyzed liver extract.