Cell Fractionation Lab Report
Amanda Popek
Ann Karras
Ara Jo
SaharYaghoobi
VolkmarGaussmann
January 14, 2011
Biology 211 Section A
I. CELL FRACTIONATION
Supernatant
containing lightest
cell particles
Solid layer of less
heavy cell
components
Solid layer of less
heavy cell
components
Solid residue
containing the most
dense cell
components and
the nucleus
Solid residue
separated from
supernatant after
first centrifugation
Solid residue
containing the most
dense cell
components and
the nucleus
Tubes 1, 2 and 3 after centrifugation.
Tube 1 is separated into three layers
• The white solid layer contains the most dense cell components including the nucleus and
heavier membranes.
• The dark green solid layer contains less heavy cell components.
• The light green liquid layer is the supernatant containing cell particles which were not
separated out during centrifugation including chloroplasts.
Tube 2 contains the remaining residue after the removal of the supernatant. This residue contains
denser organelles that were separated after the first centrifugation.
Tube 3 contains the supernatant removed from tube 2 after the second centrifugation. This tube
contains layers similar to those found in tube 1, in smaller proportions.
Slide A: Wet mount of the original cheesecloth residue after adding iodine.
Chloroplasts which are un-pigmented after staining.
Amyloplasts which have been dyed purple
Amyloplasts which
have been dyed purple
Chloroplasts which are
un-pigmented after
staining
Prior to adding the iodine several green chloroplasts could be seen as well as larger round
amyloplasts.
Slide B: White residue from Exercise I, tube 2 after the addition of Iodine
Amyloplasts dyed purple
Amyloplasts dyed
purple
Leucoplasts
Slide C: Green residue from Exercise I, tube 3 prior to the addition of iodine.
Amyloplasts
Amyloplasts
After the addition of iodine the majority of the slide turned purple indicating the presence of
amyloplasts.
Slide D: Supernatant from Exercise I, tube 3 stained with Janus green stain.
Mitochondria particles
Mitochondria
particles
Tubes 1-6 after the addition of Methylene Blue in tubes 1, 3 and 5 and Tetrazolium in tubes 2, 4
and 6.
Tubes 1-6 after sitting in a hot water bath overnight.
II. MITOCHONDRIA TEST
PURPOSE
The purpose of this experiment is to first illustrate the biological technique of isolating
and separating the organelles in cells, and second, identifying mitochondria using different
metabolic tests.
The two metabolic test indicators that the group used to identify mitochondria were
Methylene Blue and Tetrazolium Chloride. Six test tubes were labeled 1 through 6. Test tubes 1,
3 and 5 contained 4 drops of Methylene Blue indicator while test tubes 2, 4 and 6 contained 3mL
of Tetrazolium. Test tubes 1 and 2 were the control for each indicator and the three Methylene
Blue indicator test tubes were topped with 2mL of salad oil. Methylene Blue indicator was used
to determine if oxygen was being used up by the mitochondria during aerobic cell respiration. If
the test tube remains blue, then there is oxygen present in the solution, therefore
mitochondria are not present. If the indicator turns colorless (clear) after being left overnight,
then oxygen is no longer present, therefore used up by the mitochondria. The salad oil is used to
make sure oxygen from the atmosphere does not enter the solutions interfering with the testing
and adding more oxygen. The Tetrazolium Chloride indicator was used to determine whether
oxidation-reduction reactions were occurring in the respiratory enzymes of the mitochondria.
When mitochondria are present, the normally colorless oxidized form of Tetrazolium that was
added will turn a bright red/pink overnight, meaning reduction has taken place.
HYPOTHESIS
During the blending process, the cell walls and cell membranes are broken allowing the free
floating organelles that are contained in the cytoplasm to be released. Based upon the
understanding that the mitochondria contain both an inner and outer membrane and that their
relative densities are higher than most other organelles we believe we will find them in the
residue. We believe that these membranes are the primary sites for aerobic cell respiration.
Therefore, we expect to receive positive test results in test tubes 3 and 4.
PROCEDURE:
Tube 1 contained the sucrose phosphate buffer with several (4) drops of Methylene Blue.
Tube 2 contained the sucrose phosphate buffer with 3mL of Tetrazolium. Tube 3 had the residue
from the original centrifuge tube 1. It was centrifuged three times. The bulk of the residue had
accumulated after the first centrifugation step. To this, several (4) drops of Methylene Blue were
added. Tube 4 had the same solid material as #3, with added Tetrazolium.
The majority of tubes 3 and 4 were relatively heavy pieces that were captured by the first
centrifugation step. Tube 5 had the supernatant from the original centrifuge tube 1, after it was
centrifuged for a second time. To this, several (4) drops of Methylene Blue were added. Tube 6
had the same material as #5, with added Tetrazolium. The majority of tubes 5 and 6 were tiny
pieces. They must have been tiny because they were still floating in supernatant after being
centrifuged twice.
In summary, the process was to generate solid deposits that were centrifuged three times,
and to generate supernatant consisting of very light-weight cell pieces that were still in
suspension after two differential centrifugation processes.
RESULTS
Mitochondria Test Initial Observations
Tube
#
Contents
Observations
1
Methylene Blue/Control
Blue color
2
Tetrazolium/control
Clear
3
Methylene Blue/Residue
Dark opaque green
4
Tetrazolium/Residue
Lime green
5
Methylene Blue/Supernatant
Opaque green
6
Tetrazolium/Supernatant
Lighter lime green
Mitochondria Test Observations After Hot Water Bath
Tube
#
Contents
Observations
Presence of
Mitochondria?
1
Methylene Blue/ Control
There was an obvious separation of layers. The
bottom layer was blue colored from the methylene
blue and the top layer was made up of the salad
oil we used.
NA
2
Tetrazolium/ Control
The whole solution was completely clear.
NA
3
Methylene Blue/
Residue
Separation into two layers. The bottom layer was
a transparent lime green color with some residue
at the bottom. The top layer consisted of the salad
oil we used. The top layer was also transparent
with some blue color from the methylene blue.
Yes
4
Tetrazolium/ Residue
Orange/brown color. At the bottom of the test
tube there was residue. Upon further examination
the residue had red specks in it.
Yes
5
Methylene Blue/
Supernatant
Separation into two layers. Top layer has apparent
white residue. Between the two layers, there is a
thin layer of methylene blue, and the bottom layer
ranges in color from light yellow towards the top,
and more neon yellow towards the bottom, with
residue at the bottom.
Yes
6
Tetrazolium/Supernatant
Dark opaque crimson color.
Yes
ANALYSIS AND CONCLUSION:
It was known that the primary sites of aerobic cell respiration were located in the
mitochondria. The laboratory experiment attempted to isolate the mitochondria and identify the
correct organelles in specific spaces in the test tubes. Because the supernatant in tubes 5 and 6
changed color according to the expected reactions of the Methylene Blue and Tetrazolium
Chloride indicators, we could clearly identify that the primary sites were somewhere in these
tubes.
The group had initially predicted that the heavy inner and outer membranes of the
mitochondria had to be the place of aerobic cell respiration, therefore ending up in the bottom
residue of the test tube after 3 centrifuges. However, our hypothesis was not correct. The wet
mount of the supernatant, under the microscope, showed what looked like collections of threads.
The primary sites of aerobic cell respiration in the mitochondria were not destroyed during the
preparatory blending process (the part that was done by laboratory staff). These primary
sites must be very light-weight, which is why they were still floating in the supernatant after two
centrifugation steps. Thus, these primary sites could not be heavy membranes. They must be
light-weight cristae.
While we did receive positive results in both tubes 5 and 6, we also observed changes in
tubes 3 and 4. We concluded that this was due to many factors, but mainly human and centrifuge
error. We were using a pipet in order to extract the supernatant from the centrifuge tubes.
Unfortunately we were not able to accurately obtain it all and more than likely picked up some
residue in the process. The centrifuges themselves were old and did not have speed settings for
our use. This would affect the preciseness by which we could separate the cell components.
In conclusion, the light-weight cristae were floating in the supernatant of tubes 5 and 6,
and these cristae are the primary sites of aerobic cell respiration by the mitochondria. We know
this because of the color change when Methylene Blue and Tetrazolium Chloride indicators were
added. This lab also helped the group improve on working as a group in laboratory as well as
understand the concepts learned in lecture.