BIOL 240W: SECTION 022
Cell Fractionation Lab
Joshua Williams
9/26/2012
Introduction:
Standard cell fractionation is a useful tool for splitting cells into their component organelles. By
utilizing destruction of the cell membrane and differential centrifugation, a cell can be broken into its
substituent parts. The theory behind this is that at slower speeds during centrifugation, heavier
organelles will fall out of the solution while at faster speeds lighter organelles will fall out of solution.
When done in a stepwise fashion the resulting pellets (solid mass that fell out of solution) should be
composed entirely of similar organelles. The question is whether this method will be effective enough to
separate chloroplasts, amyloplasts and nuclei during different centrifugations. The densities of these
three organelles are as follows; 1.6-1.63g/cm-3 for amyloplasts, 1.32g/cm-3 for Nuclei, and 1.211.24g/cm-3 (Hinchmann, 74) for chloroplasts. In other words, going off of density, amyloplasts should
be expected to fall out of solution first, nuclei second, and chloroplasts third. In this experiment, the
homogenate is centrifuged three times. Therefore in order for cell fractionation to be effective and for
the desired result of separate organelles to be observed; amyloplasts must form the pellet after the first
centrifugation, DNA must form the second, and chloroplasts must form the third. If the pellets are not
formed in this fashion then cell fractionation, as it has been set up, is not an effective solution to
organelle differentiation.
In order to differentiate organelles under a microscope cytochemical testing must be utilized.
There are specific dyes that are able to attach or alter components inside the organelles. In an
amyloplast there is generally a large amount of starch present, a molecule that gives the organelle much
of its weight. In the presence of Iodine, the amylose in starch is altered in such a way that the starch
turns a deep blue color. This allows for the easy identification of starch, and therefore amyloplasts,
while being observed under a microscope. A similar cytochemical test used is methylene blue.
Methylene blue is useful in staining DNA and RNA found within nuclei. When methylene blue is added to
a pellet the nuclei are easily recognizable as dark blue orbs. No cytochemical testing is required for
chloroplasts as these are naturally bright green due to the presence of chlorophyll. Without these
cytochemical tests, observation under the microscope would yield little information, as it would difficult
to differentiate between the organelles.
I expect the results of this experiment to indicate that cell fractionation is a viable means of separating
organelles into groups and will allow for study of these individual organelles. I expect that amyloplasts
will be removed at pellet 1, nuclei at pellet 2, and chloroplasts at pellet 3.
Methods:
We began the experiment by preparing a spinach cell homogenate. [Burpee, 2009]Once this
homogenate was prepared we moved onto differential centrifugation. In differential centrifugation the
cell homogenate was centrifuged and the resulting pellet and supernatant were separated. The
supernatant was then centrifuged again, and the pellet was re-suspended and observed under a
microscope. This procedure was repeated 3 times. The first time, the full cell homogenate was
centrifuged at 100 g for 10 minutes and 1 ml of CRB was added to the subsequent pellet (P1). The
second rotation, the supernatant was centrifuged at 800 g for 10 minutes. The resulting pellet (P2), due
to thickness, was suspended in 5 ml of CRB. The third rotation, the supernatant was centrifuged at
10000 g for 10 minutes. The resulting pellet (P3) had 5 ml of CRB added to it. The final supernatant was
not centrifuged, labeled as S3, and treated as a suspended pellet for the next step.
The next step, cytochemical microscopic analysis, involved the dyes methyl blue and iodine. Methyl blue
was used to differentiate DNA and nuclear components, and iodine was used to differentiate starch
components (amyloplasts). Because of their naturally green appearance, chloroplasts require no dye to
differentiate. Three samples of each of the three pellets and the S3 supernatant were put onto
individual microscope slides, for a total of 12 slides. One of the samples was stained with nothing, one
with methyl blue, and one with iodine. Each was observed under a compound light microscope until
individual structures could be determined. Magnifications for all samples were not the same.
The next step was determining the relative abundance of organelles in each of the samples. Instead of
counting we devised a rating system based arbitrarily on the highest concentration of . For example the
highest concentration observed was chloroplasts in P2. we assigned a nine to this sample and, relative
to that, determined the abundance of organelles for the other 11 samples.
Results:
Plant Cell Type
Abundance of Organelles Observed
Pea
Methylene Blue
Iodine
Unstained
Pellet 1
6
9
8
Pellet2
4
4
4
Pellet 3
1.5
1
1.5
Supernatant 3
0
0
0
Relative concentrations of organelles in pea differential centrifugation
Plant Cell Type
Abundance of Organelles Observed
Spinach
Methylene Blue
Iodine
Unstained
Pellet 1
6
2.5
4
Pellet 2
5
0
9
Pellet 3
1
0
1
Supernatant-3
0
0
0
Relative concentration of organelles in spinach differential centrifugation
Relative Abundance of Organelles in Spinach
10
9
8
7
6
Methylene Blue
5
Iodine
4
Unstained
3
2
1
0
Pellet 1
Pellet 2
Pellet 3
S3
Relative Abundance of Organelles in Peas
10
9
8
7
6
Methylene Blue
5
Iodine
4
Unstained
3
2
1
0
Pellet 1
Pellet 2
Pellet 3
S3
The charts indicate that amyloplasts were removed in pellet 1 for spinach. For peas, the majority of
amyloplasts were removed in pellet 1 and some in pellet 2. The results also indicate that nuclei and
chloroplasts were removed from the supernatant at similar times and at similar concentrations for both
peas and spinach. For all three organelles, there was virtually no presence in supernatant three. For
nuclei and chloroplasts, there was a very small concentration of organelles in pellet 3 for both spinach
and peas. For peas in general, there was little to no visible separation of organelles for different
centrifugations.
Discussion:
Based on the available data it has been found that cell fractionation is not a viable means of creating any
appreciable separation of these specific organelles. In peas, it was found that relative abundance was
similar across all three organelles. In spinach the similarity of DNA and chloroplast results indicate no
difference as well. By using cytochemical analysis specific structures being looked for were easily
identifiable and this increased the accuracy of the data tremendously.
I do find the results to be illogical so as to be inaccurate. I would recommend a follow up experiment to
be conducted with a few changes and improvements to keep in mind. For one, the system of counting
and determining relative abundance should be more objective and less arbitrary. Second, a standardized
addition of CRB re-suspension agent, instead of the different additions we used, should be utilized.
Finally, the speeds at which the three centrifugations occur should be checked and fixed for future
experiments so that the difference in weight can be enhanced.
I also hypothesize that because amyloplasts and chloroplasts can transform into one another Gillot et al.
(1991) that another way to create large concentrations of these specific organelles is possible. By using
the right set of conditions so that most chloroplasts are converted to amyloplasts, differential
centrifugation would give you a high enough concentration of amyloplasts in the first pellet to be of
some use. By the same token, if most are converted to chloroplasts, then there should be enough
chloroplasts in the third pallet to also be used. By cultivating these two populations, large
concentrations of these organelles can be harvested.
References:
Gillott, M.A., Erdos, G., and Buetow, D.E., 1991. Light-induced chloroplast differeentiation in soybean
cells in suspesnion culture; Untrastructural changes during the bleaching and greening cycles.
Plant Physiology, 96, 962
Hinchman, R.R. & Gordon, S. A. 1974. Amyloplast size and number in gravity- compensated oat
seedlings. Plant Physiology, 53, 398