Вы находитесь на странице: 1из 3

Part A.

1. Rationale
a. Filtering the homogenate through cheesecloth- the cheesecloth has very small holes that
are very effective in filtering the homogenate into solid and liquid parts. This ensures that
the solid particles are strained and can be discarded
b. A cold phosphate buffer was used to suspend the subcellular components- since the
subcellular components are sensitive to changes in pH and temperature, a cold buffer is
used to maintain the pH of the components. The cold temperature inactivates the
degradative enzymes like proteases to prevent them from destroying the cellular
c. Centrifuge tubes must be equally heavy and placed at equal intervals in the rotor- A
balanced arrangement of centrifuge tubes is strictly placed because unequal mass will
force the centrifuge to wobble and cannot do centrifugation properly. It may also damage
the rotor
d. Only the celery head were used for the extraction-

Part B. Microscopic Examination of Intact Cells Discussion

Four thin sections of celery stalk epidermis were prepared using a razor blade. One
section was made into a wet mount and was observed for stomata, chloroplasts, and epidermal
cells. The section was drawn and labeled in Figure 3.1a. Two sections were stained each with
acetocarmine and Iodine potassium iodide (I2KI). The sections were put under the microscope.
The structures found were drawn and labeled in Figure 3.1b and 3.1c, respectively. A thin section
of coconut endosperm was obtained together with the fourth section of celery stalk epidermis.
The two samples where dyed with Sudan IV and compared under the microscope. The structures
found were drawn and labeled under the microscope in figure 3.2a and 3.2b, respectively.

Figure 3.1a contains the wet mount of the celery stalk epidermis. The mount shows
miniscule, oval-shaped, chlorophyll-containing chloroplasts that has a vital role to the plant. There
main function is to conduct photosynthesis by using the pigment chlorophyll to capture energy
from sunlight and perform basic plant processes. The mount also shows tiny pores called stomata
(which are open in the figure) that allows the intake of carbon dioxide and release of oxygen. It is
surrounded by a pair of transparent guard cells in which the chloroplasts are prominent. These
guard cells control the opening and closing of the stomata. Also seen in the figure are the
transparent, irregular, and elongated cells called epidermal cells that surround the stomata and
chloroplasts. These cells join to form the epidermis of the plant which protects its internal tissues
it from the outside environment by creating a barrier that combats water loss, injury, or infection
(Raven, Evert, & Curtis, 1981). Figure 3.1b contains a celery stalk epidermis dyed with
acetocarmine. The epidermal cells, stomata, guard cells, and chloroplasts are still seen but the
basic acetocarmine gave a red color to DNA which enabled the nucleus to be seen and observed
as labeled in the worksheet. The nucleus appears to be circular and varies in position within each
epidermal cell. The nucleus of a guard cell is also prominent in the figure. Since the mitochondria
and the chloroplast has also DNA, it can be inferred that the smaller red dots are the
aforementioned organelles. In Figure 3.1c, the celery stalk epidermis dyed with I2KI was observed.
The Iodine potassium iodide dye is used to detect the presence of starch. The dye slips itself in
the amylose coil in starch which gives the distinct blue-black color. The resulting mount became
transparent yellow, with evident epidermal cells, albeit transparent. The guard cells were stained
dark brown with black patches because they are capable of starch biosynthesis (Azoulay-Shemer,
et al., 2016)while the stomates were also stained because starch is produced during the opening
and closing of stomata. The starch is degraded to form sugar and malate, which acts as a counter-
ion for K+. Malate efflux is an additional factor for the closing of the stomata. The other colored
structures are plastids (chloroplasts and amyloplasts). Starch synthesized from the chloroplast is
stored in the amyloplasts (Wise, 2007)

The Sudan IV dye was used in Figures 3.2a and 3.2b. A coconut endosperm stained with
the dye was observed under the microscope. White polygonal stains were seen together with red
lipid droplets called oleosomes/oil bodies. Contrary to Figure 3.2a, celery epidermis stained with
Sudan IV dye in Figure 3.2b has no presence of oleosomes. This is because oleosomes serve as
storage areas of large amounts of triacylglycerols that fuels a plant seedling before it can
photosynthesize. Coconuts need oleosomes to fuel their growth because it takes about three to
six months for a shoot capable of photosynthesis to appear (germination). Celery, on the other
hand, only takes about 14-21 days to germinate, thus, it does not need any fuel source. Also,
oleosomes are only seen in seeds. The specimen from where the celery epidermis was taken is
already a mature plant capable of photosynthesizing, so the need for oleosomes is no longer
required. (Yatsu, Jacks, & Hensarling, 1971) (Cook & Doyle, 2007)

Part C. Isolation and Identification of Subcellular Fractions

Sixty (60) grams of celery cut into small pieces and 120 ml of cold 0.2M phosphate buffer
with pH 7.0 was homogenized in a pre-chilled blender for 1 minute at high speed. The resulting
mixture was filtered through two layers of cheesecloth. The residue was discarded, and the filtrate
was poured equally to two (2) 50 ml centrifuge tubes and placed at opposite positions in the rotor.
It was then centrifuged at 400 x g on a refrigerated centrifuge for 10 minutes. The centrifuged
solution was decanted and labeled SI (Supernatant I) and PI (Pellet I). SI was poured into a 100 ml
beaker and subjected to cytochemical tests. The SI in the 100 ml beaker was distributed equally
into centrifuge tubes and centrifugated at 2500 x g for 15 minutes. The resulting mixture was
decanted and labeled SII (Supernatant II) and PII (Pellet II). Supernatant II was subjected to
cytochemical tests. The PI and PII were resuspended in 5 ml cold buffer and was subjected to
cytochemical tests individually. For each test except for the purine test and Biuret test, 1 ml of
different fractions were put in separate test tubes. For I2KI, a drop of the said dye was added to
the fractions. For the lipid test, 500 uL of Sudan IV was added. For DPIP test, 500 microliters of
0.01 % DPIP solution was added and left alone for 5 minutes. The test of purines involves the
addition of 3 ml 10% NH4OH to 2 ml of different fractions in separate test tubes. For biuret test, 1
ml of concentrated NaOH was placed in six test tubes and added with 2 ml of different fractions
each. After shaking the tubes, 1 ml CuSO4 was added. Observations were followed rea
In the I2KI test, only Pellet I came out positive for this test. (van Wijk, Peltier, & Giacomelli,
2007) (Morre & Mollenhauer, 1964) (Tropp, 2011) (Hajek, Honys, & Capkova, 2004) (Conlon &
Salter, 2007)

Azoulay-Shemer, T., Bagheri, A., Wang, C., Palomares, A., Stephan, A. B., Kunz, H.-H., & & Schroeder, J. I.
(2016). AzoulStarch Biosynthesis in Guard Cells But Not in Mesophyll Cells Is Involved in CO2-
Induced Stomatal Closing. Plant Physiology,. Plant Physiology, 788-798.

Conlon, H., & Salter, M. (2007). Plant Protein Extraction. Methods in Molecular Biology, 457.

Cook, O., & Doyle, C. (2007, April 1). Germinating Coconuts. Journal of Heredity, 7(4), 148-157.

Hajek, T., Honys, D., & Capkova, V. (2004). New method of plant mitochondria isolation and sub-
fractionation. Plant Science, 390.

Morre, D., & Mollenhauer, H. (1964). Isolation of the Golgi Apparatus from Plant Cells. The Journal of
Cell Biology, 299.

Raven, P. H., Evert, R. F., & Curtis, H. (1981). Biology of plants. New York: Worth Publishers.

Tropp, B. (2011). Molecular Biology: Genes to Proteins. New York: Jones & Bartlett Publishers.

van Wijk, K. J., Peltier, J.-B., & Giacomelli, L. (2007). Isolation of Chloroplast Proteins from Arabidopsis
thaliana for Proteome Analysis. Molecular Biology, 43-47. doi:10.1385/1-59745-227-0:43

Wise, R. (2007). The Diversity of Plastid Form and Function. Advances in Photosynthesis and Respiration,
23, 9. doi:10.1007/978-1-4020-4061-0_1

Yatsu, L. Y., Jacks, T. J., & Hensarling, T. P. (1971). Isolation of Spherosomes (Oleosomes) from Onion,
Cabbage, and Cottonseed Tissues. Plant Physiology, 675–682. doi:10.1104/pp.48.6.675