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 by
Mitochondria SII High number of stained structures by Janus GreenSoluble enzymes SII Small molecular str
ucture, cannot be sedimented by centrifugation due to solubilityStarch granules PI High number of stained str
uctures 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.

References:Alkari, S. (2007)
Centrifugation
. Online Counseling Resource: YCMOU Elearning Drive. Yashwantrao ChavanMaharashtra Open
University, Nashik.Berg, J. M., L. Stryer, J. L. Tymoczko (2002).
Biochemistry
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ed. Retrieved fromhttp://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?
book=stryer=A1517#A1522 Ghazi-Khansari, M., A. Mohammadi-Bardbori, and M-J. Hosseini (2007).
Using Janus Green B to study paraquat toxicityin rat liver mitochondria. Abstract retrieved from Annals
of the New York Academy of Sciences database. doi: 10.1196Kirby, H. (1950). Materials and
methods in the study of protozoa. Retrieved fromhttp://books.google.com Lehninger, A. L., M. M.
Cox, and D. L. Nelson (2004).
Principles of Biochemistry.
W.H. Freeman & Company.Science and Health Education Partnership.
Testing of lipids, proteins, and carbohydrates.
Retrieved fromhttp://www.seplessons.org/node/362Rothery, M.
Techniques.
Retrieved fromhttp://www.mrothery.co.uk/module1/Mod%201%20techniques.htm