Dorothy Rica G.

de Asis Group 3 October 4, 2016

Cell Fractulation

Introduction
In this laboratory activity, the different fractions of the cell will be observed using the
hypocotyl of the monggo plant.

Objectives
1. To be able to observe the different fractions of cells through differential centrifugation
2. To compare the particles present in different fractions

Materials
Compound microscope centrifuge centrifuge tubes scalpel glass slide
medicine dropper cover slips beaker blender alcohol lamp
0.2 M phosphate buffer Janus green I2KI Sudan IV pipette

Procedures
A. Seed Germination and sample preparation
1. Place approximately 50g of mungbean seeds on petri dishes lined with two sheets of filter
paper. Add enough water to keep the seeds moist. Cover and store in a dark place for three
days. Check the setups from time to time and add more water if necessary.
2. After three days, gather the sprouted beans. Separate the growing part (hypocotyl and
epicotyl) from the cotyledon and seed coat using a scalpel.

B. Homogenization and centrifugation

1. Place approximately 30g of the collected hypocotyl and epicotyl and 120 mL of cold 0.2 M
phosphate buffer pH 7.0 in a pre-chilled Waring blender.
2. Homogenize the mixture for 1 minute at high speed.
3. Filter the homogenate through four layers of cheesecloth and stand the filtrate for twenty
minutes. Discard the debris which will settle at the bottom of the flask.
4. Pour the resultant (crude extract) to each of 6 centrifuge tubes such that there is at least
two cm space left from the top of each tube. Weigh the tubes, making sure they are equally
heavy. Adjust by pipetting to or out of the tube, save the remainder of the crude extract for
further examination.
5. Place the tubes in the rotor at opposite positions that are of regular interval (e.g.every
other position). Centrifuge the extract at 200 x g on a refrigerated centrifuge for 10 minutes.
6. Decant the supernate into a test tube or a 50mL beaker.
7. Save the pellet (pellet I) and resuspend in 5mL of cold buffer. Perform the cytochemical
tests on the peller.
8. Save 10mL of the supernate (supernate I) for the tests. Distribute the rest into centrifuge
tubes and centrifuge at 2500 x g for 15 minutes.
9. Decant the supernate (supernate II) into a test tube or a 50mL beaker and use for the
cytochemical tests.
10. Resuspend the pellet (pellet II) in a 5mL cold buffer and do the cytochemical tests.

C. Microscopic examination of intact cells

While the extract is being centrifuged, prepare a slide of intact bean cells. Using a
scalpel or a razor blade, remove a thin layer of epidermis from the hypocotyl of the bean
sprout. Place it on a slide and add a drop of methylene blue. After two minutes, wash off
some of the stain by adding drops of water and absorbing the excess fluid with a piece of
tissue or filter paper. Examine under low and high power, sketch a representative cell (with
the nucleus visible and label the parts).

D. Microscopic examination of fractions

Prepare slides of crude extract using the following procedure. Place a drop of the
fraction to be examined on each of three spots on a glass slide. Add a drop of I 2KI to one
spot, a drop of Sudan IV to the next, and a drop of Janus green on the third. Cover each drop
with a cover slip. Examine under high power. Add a drop of the fraction on another slide. Add
one drop of acetocarmine. Apply gentle pressure using your thumb or the blunt end of an
unused pencil eraser to remove air bubbles. Pass over the flame of an alcohol lamp several
times. Make sure that the preparation will not boil. Do the same for the rest of the fractions.
Observe the reaction of each fraction to the different stains. For a given subcellular
component, draw representative structures under the HPO taking note of the relative sizes in
the different fractions. Use + marks to indicate the abundance of the observed structures in
each fraction.
E. Cytochemical test
To test for the presence of soluble proteins, add 1 mL of concentrated NaOH to 2mL
of extract. Mix and then add 2-3 drops of CuSO 4 solution. Mix again. A violet color indicates
the presence of proteins. Prepare a blank using distilled water for comparison.
Record the changes in the table using + marks to indicate the relative color reactions.
The darker the color, the more + marks.

Analysis and Interpretation

The component particles in the supernate II and pellet II were more prominent and are
equally distibuted compared to those in the supernate I and pellet I which is not seen clearly.
This is because the supernate II and pellet II have been subjected to centrifugation again
which made separated the particles from the water component.

Pellet II suspended in cold buffer Supernate II with Janus green, the

particles are large
 Supernate II with Sudan IV Supernate II with I 2KI, starch particles are

visibl

Pellet II with janus green Pellet II with Sudan IV

 Pellet II with I2KI

Supernate I in Biuret test. There were no precipirtae present

Pellet I in Biuret test. It is positive for proteins, with precipitate present

Pellet II in Biuret. Protein present. Pellet I suspended in phosphate buffer

Supernate II in Biuret, there was protein present, but only in small amount
 Supernate II

Epidermis of hypocotyl with intact cells

Conclusion
There is more protein present In the supernate II and pellet II given that they
have been positive in the Biuret test compared to supinate I and pellet I. The starch is
also more visible in the fractions in pellet II compared to supernate I and pellet I, this is
because pellet II have been centrifuged again and thus its particles have been
separated from its water components and the organelles are present there due to the
differential centrifugation.
Problems encountered during and after slide preparation
One of the problems that was encountered in this laboratory activity was the
problem with the centrifuge. The proper instruction as to the rotation of the centrifuge
was not followed because if the centrifuge was set to that certain rotation, the tubes
would be broken inside.

GUIDE QUESTIONS
1. Give the rationale for the following:
a. filtering the homogenate through cheesecloth
The homogenate must be filtered using a cheesecloth so it the impurities will be
removed, and therefore, only get pure crude extract which is the only part needed for
the experiment.
b. a cold phosphate buffer was used to suspend the subcellular components
Subcellular components need to be protected from changes in pH so it is suspended in
buffer and is kept cold to keep the degradative enzymes from destroying components
(ie. RNAses, DNAses are inactive at low temperature)
c. centrifuge tubes must be equally heavy and placed at equal intervals in the rotor.
Centrifuge tubes must be equally heavy and placed at equal intervals in the rotor
because if it is imbalanced, it will cause the centrifugal core to break. When spinning at
extremely high RPMs, the G force attributed to each tube in the centrifuge can change
drastically even with a small weight imbalance.

d. seeds were germinated in the dark

The seeds were germinated in the dark because they grow faster in the dark. The seeds
don’t have leaves yet, meaning, it doesn’t also have chlorophyll so it does not need to
grow where there is light. They germinate in the soil where it is dark and damp. It only
needs enough moisture, proper temperature and sufficient amount of oxygen.

e. only the hypocotyl and epicotyl were used for the extraction
Only the hypocotyl and epicotyl were used in the extraction because it is easier to
extract components with younger cells since plant cells that are older have thicker cell
wall.

2. Explain the reaction of I2KI with starch to produce a blue-black color.

The iodine does not turn starch into black. This is only based in the iodine clock reaction
which is the color change that occurs.

3. Compare the size of particles found in pellet I and II and between supernate I and II.
Are starch containing particles equally distributed into each of these fractions? What
generalization could be made as to the separation of particles by centrifugation?
The size of the particles in pellet II and supernate II are more visible and more
equally distributed compared to pellet I and supernate I. Because supernate II and pellet
II have been centrifuged again, the particles and subcomponents were more separated
from the water.

4. Where is DNA localized? Explain.

DNA is localized in the nucleus of the cell or specifically, in the chromosomes and
consists of genes. It carries the genetic material of an organism.

5. Which fraction contains mainly water soluble enzymes? Why?

The fraction which contains more water soluble enzymes is supernate II because there
are one of the smallest of the compounds in the cell. In differential centrifugation, the
densest organelles are gathered first in pellet I. However because of having only pellets
I and II, these water soluble enzymes couldn’t be isolated therefore remaining with the
final supernate- supernate II