EXERCISE 3

Isolation and Identification of Subcellular Components

Name: Josephine D. Carandang Date Submitted:
February 7, 2017

Lab Section: A-3L Lab Instructor: Prof.

Adajar

I2KI Sudan IV Janus Green Acetocarmine

A B C D
Figure 3.1 Microscopic examination of cotyledon using I2KI, Sudan
IV, Janus Green and Acetocarmine cytochemical stains.

I2KI Sudan IV Janus Green Acetocarmine

A B C D
Figure 3.2 Microscopic examination of hypocotyl and epicotyl using
I2KI, Sudan IV, Janus Green and Acetocarmine cytochemical stains.

Table 3.1 Relative abundance of the subcellular component in

different fractions using I2KI, DPIP, Biuret, terpenoid and purine
cytochemical tests.
Fraction CYTOCHEMICAL TEST
I2KI DPIP Biuret Terpenoid Purine
Test Test
CE + + ++ + -
SI - - ++++ +++ -
PI ++ ++ + - +
SII - - +++++ ++ -
 P II - +++ +++ - ++
(-) - - - - -
Part A. Rationale of the following steps.

a. filtering the homogenate The insoluble substances were separated

through a cheesecloth
from the homogenate by allowing the
homogenate to pass through the
cheesecloths porous structure.
Insoluble tissues and bigger debris such
as cell wall, membranes, etc. were
removed.

(http://www.cas.miamioh.edu/mbi-
ws/ConnieSergakis/oldpages/biotech_lab_
i.htm)
cheesecloth to able to retain the cell wall
fragments, and separate it from the
nuclei and chloroplasts, as well as the
other smaller subcellular components
that will be isolated in the experiment
(Heldt, 2011).
b. a cold phosphate buffer was in order to stop enzymatic
used to suspend the subcellular reactions, prevent the
components cell organelles from bursting, and
stop pH changes
Subcellular components need to be
protected from changes in pH and
(buffer) and are kept cold to keep
degradative enzymes from
destroying components (ie.
RNases, DNAses are inactive at low
temp).
was used to suspend the subcellular
components to prevent pH changes of
the cytoplasm due to the release of
organic acids from the vacuole of the
plant cell, minimize enzymatic
changes in the homogenate which
result from bringing together
substrates and enzymes not
accessible to one another in the intact

c. Centrifuge tubes must be
equally heavy and placed at
opposite slots in the rotate
d. seeds were germinated in the
dark
cell, and minimize changes of the cell
components that are surrounded by
the semi-permeable membrane. The
resulting homogenate was filtered
through two layers of cheesecloth to
able to retain the cell wall fragments,
and separate it from the nuclei and
chloroplasts, as well as the other
smaller subcellular components that
will be isolated in the experiment
(Heldt, 2011).
So that it would be balanced while the machine is
spinning
was used to suspend the subcellular
components to prevent pH changes of the
cytoplasm due to the release of organic
acids from the vacuole of the plant cell,
minimize enzymatic changes in the
homogenate which result from bringing
together substrates and enzymes not
accessible to one another in the intact cell,
and minimize changes of the cell
components that are surrounded by the
semi-permeable membrane. The resulting
homogenate was filtered through two layers
of cheesecloth to able to retain the cell wall
fragments, and separate it from the nuclei
and chloroplasts, as well as the other smaller
subcellular components that will be isolated
in the experiment (Heldt, 2011).
The seeds were germinated in the dark for
three days to be able to reduce the
photosynthetic activities to decrease the
starch accumulation. Starch is the most
prevalent carbon storage compound in
plants. Its transient deposition in leaf
chloroplasts helps to dampen diurnal fluxes
in energy input from sunlight, whereas
longer term storage in the form of starch
granules, which accumulate in specialized
plastids termed amyloplasts, plays an
important role in nourishing germinating
embryos and supporting vegetative

e. only the hypocotyl and epicotyl
were used for the extraction
propagation. Thus, decreasing the starch
accumulation can decrease the CO2 stored
form and decrease photosynthesis since CO 2
is one of the reactants of photosynthesis and
decreasing the starch accumulation can
decrease the formation of the semi-
crystalline granules composed of amylose
and amylopectin which are glucose
polymers. Decreasing the formation of the
semi-crystalline structures can decrease the
structural integrity of the plant cell
these parts contain actual cells for
identification of the subcellular components.

Part B: Discuss the following:

Paragraph 1: Complete summary of methodology.
Fifty grams of mungbean seeds were germinated in a dark place for
three days. Growing parts (hypocotyl and epicotyl) of the sprouted beans,
approximately 30 grams, were collected and placed in a pre-chilled blender
with 120 ml of 0.2 M phosphate buffer (pH 7.0). The mixture was
homogenized for a minute at high speed and filtered through 2 layers of
cheesecloth. The residue was discarded while some of filtrate (crude extract)
were poured in two 50-ml centrifuge tubes each containing 30-ml and the
remaining were saved for further examination. The tubes were then placed in
the centrifuge at opposite positons from 10 minutes at 400Xg. The supernate
I (SI) was decanted and placed in a 100-ml beaker with 10 ml of it saved for
the cytochemical tests while the rest was placed into centrifuge tubes and
spun at 3500 rpm for 25 minutes. After second centrifugation, the supernate
II (SII) was collected. Both pellet I and pellet II (P II) from the 1 st and 2nd
centrifugation, were resuspended with 2.5 ml and 5 ml cold buffer
respectively. When all the fractions were prepared-crude extract, SI, PI, SII
and PII- cytochemical test were performed. A negative control was prepared
for each test consisting of 1 ml distilled water and the corresponding amount
of the test reagents. I2KI, DPIP, Biuret, terpenoid and purine test done. For
I2KI, 1 mL of the different fractions were placed in separate test tubes. A
drop of I2KI was added and mixed. For DPIP, 1 mL of the different fractions
was placed in separate test tubes. 500 microliters of DPIP was added
(choose the concentrated solution), mixed and let stand for 5 minutes. For
Biuret test, 1 ml of concentrated NAOH in separate test tubes with different
fractions. After shaking, 1 ml of CuSO4 solution was added. For terpenoid
test,

Test for terpenoids

2.5 mL of the fraction will be mixed with 1 ml chloroform. 1.5 mL of
concentrated sulfuric acid will be added carefully to form a layer. The
presence of terpenoids is indicated by the reddish brown coloration of the
interface.
Test for purines

To 1 mL of the extract, add 1.5 mL 10 % NH4OH (the resultant solution must

be strongly basic). Add a few drops of freshly prepared 2 % AgNO3 solution.
Positive result is gray color at the interface.

Positive result in different fractions were ranked using + signs, with more +
signs indicating a more intense positive result. Use sign to indicate absence
of color reaction.

Simultaneously, the microscopic examination of intact cells was performed.

Removed thin layer of epidermis from the sprouted beans (cotyledon and
hypocotyl+epicotyl) were placed on slid with different cytochemical stains.
For I2KI, a drop of the stain. For Sudan IV ,a fresh section was added 50%
EtOH for a minute then stained (Sudan IV) for 20 minutes, then rinse in 50%
etOH for another minute. For Janus Green, a drop was added to the section
and was left stand for 5 minutes and rinsed with distilled water. For
Acetocarmine: After 3 minutes upon addition of the stain, it was run through
flame for 3-5 seconds and de-stained by adding a drop of acetic acid on the
edge of the coverslip and let it seep inside.

Paragraph 2: Discussion of Figure 3.1. (Discussion of figures should

always include their
description and interpretation of what was observed).
Figure 3.1 showed the reaction of V. radiata intact cells (cotyledon)
with cytochemcial stains under HPO. In Fig. 3.1A, the I 2KI stained cotyledon
showed blue-black colored bean-like structures. They are probably amylose
coils of starch since I2KI test for presence of starch.
The stain, I2KI reacted with the starch and settled in a granule inside the cell.
The granule was the vacuole where the starch or stored products of plants are
found. The reaction between the starch and I2KI was due to iodine atoms fitting into
the helices of starch, which forms a complex and produces a blue-black color
(Campbell et al. 2000). On the other hand, Figure 3.1B shows the intact cell stained
with Sudan IV. The field of vision possesses various colorless intact cell body, but
the membrane surrounding the each cell is darkened due to the stain used. Sudan
IV is lysochrome diazo dye When Sudan red is added to a mixture of lipids and
water, the dye will move into the lipid layer, producing a scarlet red color (Rafat et
al 2008). In Figure 3.1C, the intact cells stained with Janus Green have a blue stain
dispersed inside the small granules. The blue stained granules indicated that it is a
cell component capable of redox reaction, since Janus Green is a test for the
presence of such reaction (Rothery, n.d.). And lastly, intact cells stained with
acetocarmine is shown on Figure 3.1D. The stain used was localized inside the
granulated spots and has a red color. According to Chu (1946), acetocarmine binds
to the DNA of the nucleus and this binding is indicated by the presence of red color.
It can be inferred that the granulated spots with red color is the nucleus.

Paragraph 3: Discussion of Figure 3.2.

Paragraph 4: Compare the size of particles found in PI and PII. Make a
similar comparison between SI and SII. Based on these observations, what
generalization could be made as to the separation of particles by
centrifugation?
PI
PII
SI
and
SI
According to Lodish, et. al (2003), the heavier or denser a molecule is,
the faster it will settle or form sediment. The crude extract was centrifuged
at 400xg for 10 minutes, producing PI and SI. Thus, PI is denser than
SI.Accoring

Thus, PI is consisted of the densest subcellular components from the homogenate

leaving the less dense components in the supernatant, such as mitochondria,
plastids, proteins and ribosomes. PI can contain whole cells, nuclei, cytoskeletons,
and plasma membrane (Nelson and Cox, 2008). Cytoskeletons and plasma
membrane are some of the major components of the cell and were present in higher
percentage than the other components. Compared to the other organelles
suspended at the cytosol, nucleus is relatively larger. The presence of whole cells
may be due to the less efficiency and accuracy of the filtration of the homogenate.
After the second centrifugation at 4,000 rpm for 15 minutes, PII and SII were
produced. PII contains components less dense than PI but denser than the
components of SII due to the principle that denser particles sediment faster.
Mitochondria, lysosomes and peroxisomes are the possible components of PII
leaving the microsomes, small vesicles, and large macromolecules at SII. Thus, the
particle size present in PI is greater than that of PII, and the particles size
suspended at SI is greater than SII.

Paragraph 5: Discussion of Table 3.1. Incorporate answers to GQ #3 and 4

(Revision on GQ#4:
Identify the fraction where each organelle/ultrastructure is
observed most abundantly. Exclude
CE fraction from the choices).
Do not include ribosomes, water, salts, plasma membranes, and
membranes of organelles to
the discussion.
Include terpenoids as one of the items to be discussed.
If there is discrepancy between the observed and expected result, mention
it in the discussion.
The evidence or basis can be supplemented with additional information
that you have
researched. This is especially needed if the observed result is different from
that of the
expected.
Paragraph 6: In which fraction are the following expected to be observed
most abundantly? Explain
your answer.
Ribosomes
Water
Salts
Plasma membrane
Paragraph 7: Answer GQ #5

D. While accuracy and completeness of the discussion is of prime

importance, it is also encouraged
that you observe correct grammar to enhance the clarity of your report.
Moreover, make your report
coherent by arranging your ideas logically and with continuity.
E. List all references used.