Plants store sugars in the form of polysaccharides called starch.

There are two types of starch –

amylose and amylopectin. Natural starches, such as those of plant origin, are commonly mixtures of
amylose (10-20%) and amylopectin (80-90%). Detection of the presence of starch is usually done
using the starch test with Iodine-KI reagent. Amylose in starch is responsible for the formation of a
deep blue color when there is iodine present. If amylose is not present, then the color of the original
mixture remains. Starch amylopectin does not give off the same positive result, nor does cellulose or
disaccharides (Ophardt, 2003)

Ophardt, C.E., 2003. Virtual chembook. Elmhurst College, pp.121-125.

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.

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.