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


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( ). The
particles varying in size are then separated using centrifugation at different speeds (Lehninger,,
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.

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.