DIFFERENTIAL CENTRIFUGATION OF ORGANELLES

Organelle Separation and Assays for Biochemical Activity

Lee HS, Regala PKF, Santos DJR
Date of Submission: September 4, 2014

I. Results

A sample of mouse liver was extracted and homogenized in ice-cold phosphate-buffered saline
(PBS) solution using mortar and pestle. The homogenate was transferred into centrifuge tubes containing
0.2 M sucrose solution, with a final proportion of 1:1. This mixture was differentially centrifuged at 600xg,
3000xg, and 9000xg, all for 15 minutes at 4C. Between each centrifugation, (including the last
supernatant), the pellet was resuspended, was stained with toluidine blue, heated briefly, and then
viewed under the compound microscope Nikon SE using the low power objective (40x) and the high
power objective (100x). The micrographs in Figures 1-4 were taken using a Samsung Galaxy S3 camera.
Because yellow light was used for visualization, the micrographs were digitally filtered to remove the color
yellow.


a b
Figure 1. Micrographs of mouse liver homogenate centrifuged at 600xg viewed under (a) LPO and
(b) HPO, stained with toluidine blue. Under LPO, the pointed structure appears to be individual cells,
presumably red blood cells, aggregating into violet clumps. The dots that scatter the micrograph are
isolated cells. Under HPO, the pointed structure shows the nucleus, noticeable by its dark violet filling.


c d
Figure 2. Micrographs of mouse liver homogenate centrifuged at 3000xg viewed under (a) LPO
and (b) HPO, stained with toluidine blue. Under LPO, violet clumps were again observed. However,
they appear to be smaller compared to those viewed at 600xg. Under HPO, the pointed structure again
shows the nucleus, recognized by its dark violet filling. No additional structures were observed in both
micrographs.


a b
Figure 3. Micrographs of mouse liver homogenate centrifuged at 9000xg (pellet) viewed under (a)
LPO and (b) HPO, stained with toluidine blue. Under LPO, violet clumps were again observed.
However, they appear to be much smaller and lesser in number compared to those viewed at 600xg and
3000xg. Under HPO, faint violet shapes can be seen while the dark violet nucleus previously observed is
no longer distinguishable.


a B
Figure 4. Micrographs of mouse liver homogenate centrifuged at 9000xg (supernatant) viewed
under (a) LPO and (b) HPO, stained with toluidine blue. Under LPO, numerous dark violet dots scatter
the whole micrograph along with some cell debris. Under HPO, the dark violet dots appear to be very
small fragments that assume elongated, fibrous shapes.


II. Discussion

A eukaryotic cell is composed of different organelles, all of which operate and interact with each
other within the cell. In order to study the function and structure of these organelles in more detail and
without the interference of inapplicable factors, cell biologists must be able to isolate these organelles
from one another. This can be achieved by using cell fractionation to create a cell-free system. This type
of system can be used to recreate biological processes or even to devise enzymatic pathways not found
in nature. (Alberts, et al., 2008)

In cell fractionation, viable cells are suspended in solution, lysed through physical or chemical
methods to release the organelles, and are homogenized. This experiment used grinding via mortar and
pestle for this step, but other methods such as ultrasonification and osmotic alteration can also be used
(Heidcamp, n.d.). Next, the organelles are separated through differential centrifugation. This procedure
was used since it is more effective for isolating materials from multiple density groups. Differential
centrifugation makes use of the centrifugal force to facilitate separation based on differences in the
densities of the organelles (Slish, 2013). Organelles that are heavier tend to pellet out at lower centrifuge
speeds, while those that are lighter will pellet out at higher speeds. Thus, differential centrifugation
schemes involve a stepwise increase in the centrifugation speed (Slish, 2013). Between each increase,
the pellet is collected and the supernatant is subjected to the next centrifugation process. Therefore, the
pellets collected at different points should contain different types of organelles. Table 1 shows the
corresponding organelles present at each centrifugation speed.

Table 1. Organelles effectively pelleted out by centrifugation at increasing speeds
Speed Contents of Pellet
1000 xg (10 min.)
Unbroken cells, nuclei, plasma membrane sheets, heavy mitochondria
plus smaller, trapped particles.
3000 xg (10 min.)
Heavy mitochondria, plasma membrane fragments plus smaller, trapped
particles
10000 xg (20 min.)
Mitochondria, lysosomes, peroxisome, some Golgi membranes and rough
endoplasmic reticulum.
100000 xg (60 min.)
Membrane vesicles derived from smooth and rough endoplasmic
reticulum, Golgi vesicles and plasma membrane vesicles.
Adapted from Davies, DR. (2005). Isolation and Fractionation of Subcellular Organelles. Retrieved from:
http://personal.rhul.ac.uk/uhbc/007/BS2510/Isolation%20and%20Fractionation%20of%20Subce llular%
20Organelles.doc

After the separation of the organelles, it is necessary to observe them under a microscope to
identify and characterize each component, if present. As a normal cell is usually viscous and semi -
transparent, it is difficult to distinguish one part of the cell from the other. In order to help visualize the cell,
stains or dyes are incorporated and made to bind with the cellular component, giving striking contrast
from the background and neighboring organelles. One of the more efficient ways of staining individual
organelles is by the use of metachromatic dyes, which stain organelles according to their chemical and
molecular compositions.

Metachromasy is the shift in absorption to a shorter wavelength without the change in chemical
structure in certain basic aniline dyes in the presence of water and particular substances or solutions in
different concentrations. (Bergeron & Singer, 1958; Sridharan & Shankar, 2012) Special components
called chromotropes present in the tissue samples cause the metachromatic color to occur, allowing the
use of mechromatic dyes for structural assays. In non-chromotropic substances such as fibrin, gelatin,
and most cellular structures, the dye-binding sites are too far apart such that the bound dyes cannot
interact with each other (Bergeron & Singer, 1958). This causes the dyes to remain in the orthochromatic
state, as is the blue color of toluidine blue O used in the experiment.

Basic thiazine metachromatic dyes such as toluidine blue binds with high affinity with acidic and
anionic tissue components, such as tissues rich in DNA and RNA. (Sridharan & Shankar, 2012). The
color change in these regions occurs only when the dye is saturated or the pH is adjusted until the dye
molecules can bind and interact, with lower pH giving sharper contrasts (Bergeron & Singer, 1958).
Toluidine blue gains a metachromatic purple to red color when bound to carboxylated and sulphated
polysaccharides such as gelatin, cartilage and the glycoproteins found in rough endoplasmic reticulum,
and turns green when mixed with phenols. The red and purple color is caused by the formation of staked
dye ions, in which Van der Waals forces and hydrophobic effects aggregate the dye into dimers and
polymers in the presence of water (Sridharan & Shankar, 2012). Heating the slide briefly before viewing
ensures this aggregation and polymerization to occur in correct positions. These polymers show differed
absorbance property from the original compound. (Krishnamurthy, 1999). Meanwhile, the green shift in
color is likely caused by the same property except that the -OH group in the phenol-acid polysaccharide
complex lessens the binding activity, causing the wavelength shift to be less and thus appear green.
(Ramalingam & Ravindranath, 1970) Thus, the metachromatic property of toluidine blue is valuable in
aiding the determination of chemical nature of the substances in the tissue or the cell. For example,
toluidine blue can be used to highlight patients with pathological conditions that involve mast cells,
including cancer, allergic inflammations, and gastrointestinal diseases, as it can highlight rapidly dividing
DNA of cancer cells or bind to tissue components like cartilage or mucin (Sridharan & Shankar, 2012).

In the experiment, a mouse liver tissue sample was extracted and homogenized in PBS using
mortar and pestle. The homogenate was then subjected to differential centrifugation at 600xg, 3000xg,
and 9000xg. Each pellet was resuspended in PBS, stained with toluidine blue, and then viewed under
LPO and HPO.

At 600xg under LPO (Figure 1a), numerous violet clumps along with scattered isolated dots were
observed. Under HPO (Figure 1b), distinct, dark violet structures inside lighter violet boundaries were
observed. Because only cell debris, cells, and nuclei are present at 600xg, these clumps are most
likely aggregates of cells along with some cell debris, while the dots are individual cells. The presence of
these unbroken cell clumps and large cell debris was probably caused by an ineffective homogenization
process. Highly vascular tissues such as the mouse liver would require some form of perfusion to remove
blood from the vasculature prior to homogenization. Otherwise, the erythrocytes in the homogenate can
sediment at low g-forces, as it was observed in the experiment. The liver should have been injected with
PBS and rinsed several times before working. Moreover, it is possible that the mouse liver was not
homogenated completely, as mere mincing using mortar and pestle would not be sufficient to isolate
individual cells. Whole cells and cell debris should have been filtered out first through gauze to improve
resolution. (Graham, 2002) Nevertheless, the dark violet structures seen under HPO are most likely cell
nuclei due to its darker violet color. The difference in color is owed to the basic property of toluidine blue
that tends to bind to the nucleus, staining it dark blue as the dye aggregates compactly within the
nucleus, preventing polymerization and thus metachromasia (Sridharan & Shankar, 2012). The same
explanation would hold through for the generally violet color of the tissue, as the cytoplasm present in the
cell debris would contain ribonucleoproteins for which to the dye can bind as well, but to a lesser extent.
(Klaunig et al., 1981) It is also possible that the sedimented erythrocytes in the unfiltered blood hinder
with the color, causing it to appear more reddish (Graham, 2002).

At 3000xg under LPO (Figure 2a), smaller violet clumps similar to that of the structure in Figure
1a were observed. Under HPO (Figure 2b), cell nuclei were again observed due to the presence of a dark
violet structure inside ligher violet boundaries. In theory, however, cell debris, cells, and nuclei should no
longer be observed at 3000xg. Therefore, some pellet residues may have been transferred during the
separation of the supernatant from the pellet. Furthermore, it may be possible that a longer centrifugation
process at 600xg was needed to completely pellet out all of the larger fragments. No other special
organelle structures were observed. Based on Table 1, expected organelles at 3000xg include heavy
mitochondria, plasma membrane fragments and smaller particles. However, because toluidine blue is a
basic dye, it does not bind strongly to mitochondria or plasma membrane (Staining and Commonly Used
Stains, n.d.). Moreover, the homogenization process might have caused mitochondria to break or remain
in the cell. Thus, these organelles cannot be visualized in this setup.

At 9000xg (pellet) under LPO (Figure 3a), even smaller and fewer violet clumps were observed.
Under HPO, the dark violet structure disappeared, while faint violet shapes were seen. The decrease in
size of the violet clumps is most likely the result of a faster centrifugation speed: faster speeds pellet out
smaller cell aggregate. However, it is expected that aggregates of cells should no longer appear in the
9000xg pellet. Again, it is possible that some pellet fragments were transferred during the separation of
the supernatant from the pellet. The faint violet shapes are most likely cell debris that broke off during
homogenization and centrifugation, as the centrifugation speed was still not fast enough for specific
organelles to pellet out.

At 9000xg (supernatant) under LPO (Figure 4a), scattered dark violet dots along with minimal cell
debris were observed. Under HPO (Figure 4b), these dark violet dots appear to be elongated, fibrous
fragments. Based on Table 1, expected organelles are vesicles derived from rough and smooth
endoplasmic reticulum, Golgi vesicles, and plasma membrane vesicles. Rough endoplasmic reticulum is
known to contain bound ribosomes that function in cotranslational protein synthesis (Alberts, et al., 2008).
Moreover, ribosomes in the RER or free, are stained by toluidine blue (Gustafson, n.d.). Thus, it would
follow that these dark violet fibrous fragments are most likely rough endoplasmic reticulum components,
or at least some of them.

Besides toluidine blue staining, there are a number of other biochemical assays currently
employed in molecular biology. These assays range from cell viability assays used to measure cell
proliferation to immunoassays that detect certain compounds in a sample. One of the common
techniques measuring cell viability is the MTT assay. MTT is a quantitative colorimetric assay based on
the cleavage of a water soluble salt, MTT, into insoluble dark blue formazan crystals. This cleavage
process only occurs in the mitochondria of living cells using the enzyme succinate dehydrogenase. The
change of color from yellow to dark blue can be quantified by measuring the absorbance (usually 500 nm
to 600 nm) using a spectrophotometer. Since this process only occurs in metabolically active
mitochondria, the change in absorbance is directly proportional to the number of whole, viable cells.
However, this method has a number of limitations influenced by 1): the physiological state of the cells and
2) variance in mitochondrial enzyme activity between living cells (Proliferation Assay, 2007). Other cell
viability assays include resazurin reduction, protease markers, and ATP detection (Benink, et al., 2013).

Immunoassays are also used to identify whether or not specific antigens or proteins are present
in a sample. For example, ELISA (enzyme-linked immunosorbent assay), quantitatively estimates a
specific antibody or antigen in a sample such as serum. First, the test serum is incubated on a solid
phase (e. g. polystyrene surface) which effectively immobilizes the antigen, if present. Then, an antibody
specific for the desired antigen or protein is added. Note that an enzyme is also covalently bound to the
antibody. Usually, these enzymes can metabolize colorless substrates into colored products
(chromagens). Thus, if the antigen is present, the antibody-enzyme complex will bind to it. Next, the
surface is washed with mild detergent to remove other proteins or unbound antigens and antibodies. The
enzyme substrate is then added, and thus, the production of colored substrates will be observed if the
antigen is present. Lastly, the optical density for the solution is measured, which is directly proportional to
how much protein was initially present in the serum (ELISA, 2002).

III. Answers to Guide Questions

1. For what other methods/techniques is a cell-free system required?
The system inside a cell is extremely complex with numerous interrelated processes and reactions.
In most experiments, the factors that are not accounted for are either isolated from the system of interest
or kept constant. For this reason, most studies regarding individual cell components require separation
from most of the other cellular debris, or introduction into a cell-free system. A common laboratory
application of cell-free systems is in the reconstruction of biological processes in the cell. It has been
used in the study of protein assemblies involved in central cellular activities such as DNA replication,
protein synthesis, and vesicle budding (Alberts, et al., 2008).

In some experiments, cell-free systems are created to expose intracellular components to the
extracellular space like in the case of the Avery-Macleod-McCarty transformation experiment. There are
also many cases wherein cell-free systems are able to yield products from cell processes more efficiently
than when utilizing actual cells as in the case of PCR, (Alberts, et al., 2008) and a study on increasing the
hydrogen yield from the reaction of starch and water using an enzymatic pathway that does not exist in
nature. The number of such cases is growing due to the use of synthetic enzymatic pathways in which
modifications such as reduction of the amount of by-products, enabling the collaboration of enzymes from
different species, and removing regulatory components can be done (Zhang, Evans, Mielenz, Hopkins, &
Adams, 2007).

2. What other methods may be used to obtain a cell-free extract?
In order to subject the desired cell component to a cell-free system, it must first be taken out of the
cell, which is normally accomplished via homogenization. In the case of the experiment performed, this
step was completed using a mortar and a pestle. However, in situations that require more specificity
regarding the type of cell, additional methods are necessary. Most of the time, this is done using
disaggregation, which can be done by mechanically or enzymatically degrading other cells, or by
chelating the environment. This can be followed by either osmotic alteration or physical force for
homogenization to proceed. (Heidcamp, n.d.)

Physical force could be referring to the usage of a blender, French press, mortar and pestle, or
ultrasonification. The cells can also be exposed to a hypotonic solution, promoting membrane rupture and
organelle separation. Most of the time, it is also important to distinguish or isolate certain cell ular
structures from the rest. Sometimes, it is enough to let the solution stand and allow the natural separation
of certain substances due to differences in density. Most of the time, centrifugation is used to assist in the
process of separation. (Heidcamp, n.d.) Moving zone centrifugation, for example, can be used to
separate proteins and nucleic acid. It involves the centrifugation of a tube containing the sample on top
and different sucrose solutions with decreasing concentrations stacked on those with higher
concentration. Separation occurs because friction encountered by certain molecular shapes cause
differently shaped molecules to travel across the tube at different rates, forming distinct layers.
(Zwartouw, Westwood, & Appleyard, 1962) In this experiment, the type of centrifugation used is called
moving boundary centrifugation, in which sedimentation is repeated at different speeds in order to
accomplish separation. (Heidcamp, n.d.)

3. What is a metachromatic dye? What is the molecular basis of the affinity/affinities of toluidine blue with
various cellular components?
Metachromasy is the change in color by a shift in absorption to a lower wavelength without the
change in chemical structure. This property is exhibited by metachromatic dyes such as toluidine blue
and methylene blue, and is dependent on the presence of water and concentration of particular substrate.
A shift in color occurs when the dyes bind to chromotropes, causing the dyes to aggregate near the
binding sites and form polymers that absorb light differently (Bergeron & Singer, 1958; Sridharan &
Shankar, 2012). As toluidine blue is a basic aniline dye, it shows strong affinity to anionic acidic
compounds, such as the DNA and RNA found in the nucleus (Sridharan & Shankar, 2012). It will bind to
most cellular components as they contain anionic molecules such as ribonucleoproteins, but will remain
orthochromatic blue. Toluidine blue experiences metachromatic shift to red or purple when bound to
carboxylated and sulphated polysaccharides such as those found in gelatin, cartilage and rough
endoplasmic reticulum; while experiences green metachromatic shift with the exposure to phenols due to
its hydroxyl group interfering with the aggregation (Krishnamurthy, 1999; Ramalingam & Ravindranath,
1970).

4. What other assays can be used to assess biochemical activity?
In molecular biology, biochemical assays are used to determine and measure certain aspects of a
target cell, or detect the presence of certain molecules in a sample. In general, these assays involve the
addition of a certain molecule to a sample that elicits a signal if the activity/molecule is present. This
signal can then be read and measured numerically through the use of an instrument. For example, in the
MTT proliferation assay, the addition of MTT to cells induce metabolically active mitochondria to cleave
off MTT, which results to the formation of dark blue crystals. This represents the signal, which is read out
and measured using a spectrophotometer. ELISA (enzyme-linked immunosorbent assay) operates in a
similar fashion. This assay aims to detect the presence of certain molecules, such as an antigen, in a
sample. First, the sample is immobilized on a solid phase. An antibody specific for a target antigen is then
added. At the same time, this antibody is also covalently linked to an enzyme that metabolizes colorless
substrates into colored products. Thus, after washing off of unbound antigens and antibodies, colored
products should be present if the antibody is still bound to the antigen. This represents the signal, which
the spectrophotometer can measure and relate to the amount of antigen present in the sample.






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