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In the experiment, mungbean seeds had to be germinated in the dark to reduce

photosynthesis and lessen starchaccumulation.The mungbean crude extract was


made by homogenizing the mungbean tissue with phosphate buffer in a blender.
Only the epicotyl and hypocotyl of the sprouts were taken because these contain
the actual cells of the mungbean.Cold phosphate buffer was used in order to stop
enzymatic reactions, prevent the cell organellles from bursting, and stop pH
changes (Huber et. al., 2003). The homogenate was then filtered through
cheesecloth to remove insoluble tissues(mrothery.co.uk ). The particles varying in
size are then separated using centrifugation at different speeds (Lehninger, et.al.,
2004) . The centrifuge was operated with two tubes of equal weight placed on
opposite sides of the rotor so that itwould be balanced while the machine is
spinning.The chemical tests are used to determine the organelles present in each
suspension. I2KI tests the presence of starch, Sudan IV tests the presence of lipids,
Janus Green tests the presence of oxidative particles (such as
mitochondria),acetocarmine tests for the presence of nucleic acids, and the biuret
test is for determining the presence of proteins (Kirby,1950) (Science and Health
Education Partnership) (Ghazi-Khansari, 2007).Prior to centrifugation, the crude
extract was subjected to the given chemical tests. It was found that it had
severalstained structures in all the tests, most of all in the I2KI, acetocarmine, and
biuret tests. This observation accords with thetheoretical result, which is that the
crude extract would contain high amounts of each type of particle. Upon the first
roundof centrifugation, separation of particles occurred, thus particles in the first
pellet would theoretically not be found in thefirst supernatant. The test results were
that there were high amounts of all particles in the SI while there was a high
amountof starch followed by nucleic acids in the PI. According to Lehninger, et. al.
(2004), PI contains whole cells, nuclei,cytoskeletons, and plasma membrane. SI on
the other hand would contain the rest of the cell components. After centrifugation of
SI, PII was separated. The results showed that PII contained mostly lipids and
nucleic acids. This is notconsistent with reference, which explains that PII contains
large particles such as lysosomes, microbodies, andmitochondria, some of which are
also oxidative . It is expected that PII then would have an abundance of stained
structuresupon application of Janus Green. This result was nonetheless reflected by
SII, containing high amounts of oxidative particles, nucleic acids, and proteins.
However, it has been noted in other sources that there is cross contamination
betweenthe PII and the consequent PIII, meaning that mitochondria show up in PIII
and lysosomes appear in PII (YCMOUElearning Drive, 2002). Thus the results are
valid, since lysosomes may contain lipids and nucleic acids (as reflective of the
relatively high amounts of Sudan IV- and acetocarmine-stained particles in PII) still
being digested, and SII contained ahigh amount of oxidative particles.Based on the
principle of differential centrifugation, the particles were isolated by sedimentation.
The heavier particles settle first, then there is a gradual separation of the lighter
particles. Thus, there are larger particles found in SI andPI than in SII and PII. This

principle is also reflected in the distribution of starch, which is a large molecule


composed of many glucose units (Berg, et. al., 2002). More starch molecules
accumulated in SI and PI than in SII and PII.Water-soluble enzymes are found in the
cytosol and are found in abundance in SII due to its small density andsolubility. DNA,
on the other hand, was sedimented in PI because of its relatively high density. Below
is a table recountingthe subcellular components and the fraction that they can be
found in abundance, as well as the reason for their accumulation.Table 1. The
evidences or bases of the occurrence of different subcellular components in the
fractions.
Subcellular components Fraction Evidence/basisPlasma membrane PI Present in
large amounts, relatively large molecular structure Nucleus PI High number of
stained structures by acetocarmine, large molecular structureRibosomes SII High
degree of hue in biuret test, small molecular structureMembranes of organelles SI
High number of stained structures byMitochondria SII High number of stained
structures by Janus GreenSoluble enzymes SII Small molecular structure, cannot be
sedimented by centrifugation due to solubilityStarch granules PI High number of
stained structures by I2KI, large molecular structureWater SII Cannot be
sedimentedSalts SII Small molecular structure
Differential centrifugation produces only a rough fractionation of cellular
components, and it is usually purified further bydensity-gradient centrifugation. A
limitation of the use of differential centrifugation is that particles with similar
weightsand densities albeit different in nature will not be isolated.