Part A

a. The homogenate was filtered through a cheesecloth to ensure the purity of the sample. The
cheesecloth was used to filter out bigger cellular debris which are not needed in the experiment
and can be a source of error if not removed.
b. The phosphate buffer was used to suspend subcellular components since it prevents the drastic
change in pH of a solution which means that it can provide a homeostatic environment for the
subcellular components (Stewart, 2018). The phosphate buffer was maintained cold to lower
enzyme activity of enzymes that can degrade cellular components such proteases, DNases, etc.
(Nguyen, 2017).
c. The centrifuge tubes must be both equally heavy and placed at equal interval in the rotor to
maintain the balance of the test tubes in the rotor. Running the centrifuge with unequal volume
of centrifuge tubes can cause permanent damage to centrifuge. The contents of the test tubes
can also spill in the machine which can cause loss of sample.
d. The mungbeans were germinated in the dark to decrease the photosynthetic activity of the beans
which in turn lower starch production. Starch when broken down together with glucose can
produce cellulose in plants (lynn, n.d.). Decreased cellulose in plants will lower plant rigidity which
will make it easier to homogenize the mungbeans in the experiment.
e. In the experiment, only the epicotyl and hypocotyl and mungbean were used because it contains
the genuine cell of the plant that can be viewed under the microscope.

Part B.

For the microscopic Examination of Intact Cells, thin section of celery epidermis were obtained
using sharp blade. A wet mount of the celery epidermis was prepared and viewed under the
microscope and the necessary observations were recorded. Two sections of celery epidermis
were prepared and the 1st celery epidermis was stained with acetocarmine and the 2nd was stained
with I2K2. The slides were viewed under the microscope and the results were recorded. For the
last part of the experiment, sections of coconut endosperm and celery epidermis were prepared
and both stained with Sudan Iv. The slides were viewed under the microscope and the results
were compared.
The figure 3.1 (A) shows the photomicrograph of celery epidermis that was not stained. It is
evident in the cell the presence of stomata, which are cell structure used for gas exchange.
Chloroplast was not evident in the slide which could be due to the celery used not fresh. Figure
3.1 (B) shows the celery epidermis stained with acetocarmine. Acetocarmine stains the DNA of
plants. Figure 3.1 (C) shows celery stained with I2KI. I2Ki detects presence of starch by the
change in color to blue-black. I2ki could have stained the stomata in the cell wall since guard cell
requires starch to open and close (Lawson & Morison, 2010).
The figure 3.2 (A) shows the coconut endosperm while Figure 3.2(b) shows celery epidermis
which are both stained with Sudan IV. Sudan Iv detects the presence of lipids and it can be
observed that the lipids droplets in coconut endosperm is more noticeable while the lipids in celery
epidermis is located in the periphery and not as noticeable as in coconut endosperm.
Part c.
For the Isolation and Identification of Subcellular Fractions, 50g of mungbeans were germinated
in the dark. After 3 days, 30g epicotyl and hypocotyl were obtained and placed in a pre-chilled
blender with 120 mL of 0.2 cold phosphate buffer. The sample was homogenized at high speed
and was filtered through a cheesecloth. 30 mL of crude extract (CE) was transferred into two 50
mL centrifuge tubes and the remaining crude extract was saved for cytochemical tests while the
residue was discarded. The centrifuged tubes were centrifuged for 10 min at 400xg. The
supernate (SI) was obtained and placed in 100 mL beaker and 10 mL were saved for cytochemical
tests. The test tubes were centrifuged again for 15 minutes at 2500 xg. The supernate (SII) were
obtained and pellet I(PI) and pellet II (PII) were resuspended in 5 mL of cold phosphate buffer.
After resuspension, the samples CE, SI, SII, Pi, PII were subjected to cytochemical tests such as
I2KI test, test for lipids, DPIP test, and test for purines.

The I2kI was done to detect the presence of starch in the samples. The change in color of the
sample to blue-black after addition of a drop of I2kI, which is a positive result of the test, was
caused by the “charge transfer complexes” (Goedecke, 2016). In table 3.1, it can be observed that
only SII did not test positive for I2kI test. Theoretically only Pi should test positive since starch is
a complex molecule meaning it is naturaly heavy so it should settle right away.

The test for lipids was done by adding 1 mL of sudan Iv to the sample. Sudan IV is fat soluble
which means it adheres to lipids and stains it dark red and it can detect oleosomes in the cell
(Llewellyn, 2005). In tale 3.1, only PI did not test positive for the test for lipids. Lipids have an estimated
of 1.006 g/cm3 density and they are close in density with mitochondria with 1.1 g/cm3 which
means that it will be extracted at PII (Haunerland, n.d.;Wang, n.d.)

The DPIP test was done by adding 500 microliters to the sample and letting it stand for 5 minutes
and it was used to indicate the presence of mitochondria by the change of color blue to colorless.
In table 3.1, it can be observed that only PII did not test positive for DPIP test. Theoretically
mitochondria should be only present in PII since it has an estimated density of 1.1 g/cm3 which
means it is lighter and it should not be extracted right away at PI but in PII (Wang, n.d.).

The test for purines was done by adding 3 ml of 10% NaOH and a few drops of 3% AgNO3 and
it was used to indicate the presence of nucleus by the presence of gray color interface. In table
3.1, only Pi and Pii tested positive for the test. Theoretically, nucleus should be only present in PI
since the nucleus is quite heavy with an estimated density of 1.4 g/cm3 which means it will be
precipitated easily.

The biuret test was done by adding 1 ml Naoh and 1ml of Cuso4 and it was used to detect
presence of soluble protein by change in color of the sample to violet. As seen in table 3.1 all
tested positive for biuret test but Pii is the sample that posses a strong violet color. Theoretically
soluble proteins should be present in SI and SII since organelles that contain enzyme that degrade
proteins such as lysosomes are estimated to have a density of 1.1 g/cm3 which means that they
are light and still present in SI and SII (Wang, n.d).

The errors could be due to centrifugation. The time of centrifugation could have affected the
setting of the subcellular components. Misinterpreting of the result in cytochemical test could also
cause errors in the experiment.

There was abundance of DNA in PI since as mentioned earlier nucleus is quite heavy so it would
precipitate first. Ribosomes would be more abundant in SII since ribosomes are light it and has
a diameter of only 0.02 mm which means it would not precipitate easily (wang, n.d.)Salts and
water would be more abundant in SI since they are light but ribosomes are lighter, so it would
precipitate before ribosomes.
 The pellet and supernate can be arranged in order PI>PII>SI>SII. It can be observed the heavier
subcellular components such as the nucleus will be the first to precipitate after centrifugation.
Lighter subcellular components like ribosomes will not precipitate right away and still needs longer
centrifugation. It can be concluded that when you centrifuge samples the heavier components will
precipitate first compared to lighter objects.

References
Goedecke, C. (2016). Why Does Iodine Turn Starch Blue? Retrieved September 2, 2018, from
https://www.chemistryviews.org/details/education/10128441/Why_Does_Iodine_Turn_Starch_Blue.ht
ml
Haunerland, J. (n.d.). BISC 429. Retrieved September 2, 2018, from http://www.sfu.ca/bisc/bisc-429/li

Llewellyn, B. (2005). Stainsfile - Sudan IV. Retrieved September 2, 2018, from

http://stainsfile.info/StainsFile/dyes/26105.htm

Lynn, A.. (n.d.). When Does a Plant Change Sugar to Starch? Home Guides | SF Gate. Retrieved September
1, 2018 from http://homeguides.sfgate.com/plant-change-sugar-starch-82358.html

poprotein.html

Nguyen, D. H. (2017, November 21). How Is Enzyme Activity Affected by Lower Temperatures?

Retrieved September 1, 2018, from https://education.seattlepi.com/enzyme-activity-affected-

lower-temperatures-5527.html

Stewart, D. (2018, April 29). Important Buffers in Living Systems. Retrieved September 1, 2018,

from https://sciencing.com/important-buffers-living-systems-8659835.html

Wang. (n.d.). Cell Fractionation Based on Density Gradient. Retrieved September 2, 2018, from

https://eng.umd.edu/~nsw/ench485/lab10.htm