Part A
1. Rationale
a. It is necessary to filter the homogenate through cheesecloth after its homogenization
in the blender in order to collect the filtrate to be used as the crude extract and to
remove the sediment since it contains unseparated and unbroken cells not necessary
for the activity.
b. After centrifugation, cold phosphate buffer was added to the pellet to suspend the
subcellular components. This was necessary since the buffer action carried out the
dissolution of the particles with higher density, larger size, and more compact shape
than of in the supernate, in order to be tested for the cytochemical test.
c. In centrifugation, the tubes must be balanced by mass and not by volume while
placed at equal intervals at the rotor in order to obtain the proper homogenization at
increasing speed. Otherwise, it could permanently damage the centrifuge and
possibilities of the sample resulting to partial homogenization.
d. The mung bean seeds were germinated in the dark simply because the light
degrades the carbonic acid gas, fixes the carbon, and expel the oxygen which results to
hardening the parts of the seed which would have provided complications in the
experiment.
e. In preparation for this exercise, the seed coat was removed and the epicotyl and
hypocotyl were obtained. This is necessary since the seed coat only contain secondary
metabolites in protection against damage and microorganisms. However, the
components needed in this experiment were within the hypocotyl and epicotyl that
contains the growing cells during the mung bean growth.
Part B: Microscopic Examination of Intact Cells Discussion
To examine the intact cells, a thin section from the celery stalk epidermis was
subjected to acetocarmine and I2KI to differentiate the nucleus and amylose coils of
starch, respectively, from other subcellular components. In Figure 3.1a, the only cell
ultrastructure distinguishable is the cell wall since it is difficult to differentiate the
components without using stains. Nevertheless, the acetocarmine in Figure 3.1b
provided a clear differentiation for each nucleus in the obtained slide as the natural red
stain embodies the circular structures that take up most of the space for each cell. On
the other hand, Figure 3.1c was stained with I2KI in order to determine the presence of
amylose coil of starch which did appeared as a blue-black color shown in the slide.
These complex were mostly observed in the locality of the stomates since the
degradeation of starch in these structure is essential for the carbon skeleton in anion
synthesis in guard cells during stomatal opening as explained by Outlaw Jr and
Manchester (1979).
For brief comparison, celery stalk epidermis and coconut endosperm were used
for the microscopic examination of intact cells as both samples were stained with Sudan
IV. In Figure 3.2a, a few dark red spots were observed in the thin section of coconut
endosperm which indicates presence of lipid accumulation. However, it is recorded in
Figure 3.2b, several stomates are localized in the celery epidermis but no lipid residue
were observed in it. Reynolds et al. (2019) identified functional genes involved for
triacylglycerol synthesis, a lipid polymer, during maturation in developing endosperms.
It cannot be observed in celery stalk epidermis since lipids such as fatty acids will
prevent the function of stomates in taking in carbon dioxide for photosynthesis.
Part C: Isolation and Identification of Subcellular Fractions
To differentiate an organelle from other subcellular components, a series of
cytochemical tests with specific reactions to different compounds were conducted on
the pellet and supernate from the centrifuged samples. Firstly, I2KI test was one of
these tests and is necessary for the presence of starch through a positive result for the
complex to have a blue-black color. Theoretically, mung bean seedlings will tend to
have the presence of starch as food storage. Among the samples, only the first
supernate was tested negative for I2KI but it appeared to be present in the second
supernate. This observation was against the theoretical result in which the starch, as a
heavy particle, would only appear in the crude extract and as a pellet after each
centrifugation. Nevertheless, this is a possible result of contamination during the
process of transferring the substance between the glasswares.
Secondly, a test for lipids was conducted through the use of Sudan IV reagent.
Every sample subjected to testing was recorded to have positive results but it can be
noticed in Table 3.1 that the second supernate tested the most amount of lipids while
the first supernate and the crude extract was the least. According to Kurz and El-Rify
(2007), lipids are essential to mung bean germination along with the essential fatty
acids, linoleic and linolenic hence, the presence of lipids in the sample.
Another test conducted was the DPIP test to determine the presence of
mitochondria by appearing blue to colorless when added to the sample. According to
Morisato, Murray, and Barnes (n.d.), it is a blue dye that act as a hydrogen and electron
acceptor. Due to these properties, it can easily detect mitochondria since it produces a
negatively charged ATP due to its electron accepting factor.
Fourthly, a test for purines involves the use of NH4OH and AgNO3 solution that
yields a gray interface with the sample. Among the samples, only pellets tested positive
since purines are larger particles that were suspended along the pellets after
centrifugation. The structure of purines are relatively larger than of pyrimidines and a
grey interface was a result of Ag precipitate after the purine bases were hydrolysed by
NH4OH.
Lastly, a test for soluble proteins was also conducted through Biuret test. The
soluble protein composition was largely present in the crude extract and relatively
greater on supernates than pellets since the larger protein particles was not soluble
enough to have a relatively more positive result for the Biuret test.
Among the samples, only the first supernate was tested negative for I2KI but it
appeared to be present in the second supernate. This observation was against the
theoretical result in which the starch, as a heavy particle, would only appear in the
crude extract and as a pellet after each centrifugation. Nevertheless, this is a possible
result of contamination during the process of transferring the substance between the
glasswares.
DNA base pairs such as purines were only found in pellets as described before
and water soluble enzymes and ribosomes as greatly abundant in the supernates.
Alongside these compounds found, water was abundant among the supernate and
lacking in the pellet while salt mostly resided in the pellet since it is a heavy particle and
suspended with the use of a cold buffer. These are present in optimum amounts that
were necessary during the germination stage of mung bean seedlings. DNA, proteins,
and ribosomes are greatly abundant during this instantaneous growth stage while water
and salt provide supplement along the germination.
The particles in the first pellet were observed to be larger than of the second
pellet. This was the result of two series of centrifugation and how the compounds
homogenized further after a longer period of centrifugation in the process.
Differential centrifugation was a significant method in this experiment. However,
it has its limitations despite its essential utility in homogenization and isolation of
subcellular components. These limitations include having poor yield of desired isolated
component since some of it can be damaged due to high agitation and most
importantly, this method is time consuming. Nonetheless, this method is an essential
step during the isolation of subcellular components.
References
Abdel-Rahman, El-Sayed & El-Fishawy, Fawzy & El-Geddawy, Mohamed & Kurz, T. & El-
Rify, Mohamed. (2007). The Changes in the Lipid Composition of Mung Bean Seeds as
Affected by Processing Methods. International Journal of Food Engineering - INT J
FOOD ENG. 3. 10.2202/1556-3758.1186.
Morisato, D., Murray, N., Barnes, A. (n.d.), Eukaryotic Cells and their Subcellular Parts:
Differential Centrifugation. Molecule to Organism Laboratory #2.
Outlaw, W. H., & Manchester, J. (1979). Guard cell starch concentration quantitatively
related to stomatal aperture. Plant physiology, 64(1), 79–82. doi:10.1104/pp.64.1.79.
Reynolds, K. B., Cullerne, D. P., El Tahchy, A., Rolland, V., Blanchard, C. L., Wood, C.
C., Petrie, J. R. (2019). Identification of Genes Involved in Lipid Biosynthesis through de
novo Transcriptome Assembly from Cocos nucifera Developing Endosperm. Plant & cell
physiology, 60(5), 945–960. doi:10.1093/pcp/pcy247.
