Pea and Spinach Cell Fractionation and

Differential Centrifugation

Brittany Rockey
Group Memebers:
Olivia Schanz
Jin Meng
Jozef TomaszewskiI
Group #: 4
Code #: 4
9/24/09
Results

In the lab, there were 6 groups who observed plant cells. Three groups had spinach cells
and the other three had pea plant cells. Differential centrifugation was used to separate the
different parts of the cells. According to the textbook, Essential Cell Biology, “Centrifugation
separates cell components on the basis of size and density” (Alberts et al, 2004). This means
that the denser organelles are going to move to the bottom of the tube faster and sediment.
The denser sediment collected at the bottom is known as the pellet and the less dense part at
the top is called the supernatant. In the activity, the cells were centrifuged three times to
obtain 3 pellets and the supernatant from the third and final centrifugation. With each pellet
and the last supernatant, the number of chloroplasts, nuclei, and amyloplasts were observed.
It is expected that since the more dense organelles will pellet first, the nuclei should appear
more numerous in the first few pellets, but then should not appear in later pellets, and that the
less dense organelles, such as chloroplasts and amyloplasts should be present in many of the
pellets since they will pellet in the first few, but most will stay suspended until a later, more
high speed centrifugation.
To identify the nuclei and amyloplasts, cytochemistry was used. Stains, such as Lugol’s
iodine and Methylene blue, were used. Lugol’s iodine stains starch, so it would make
amyloplasts to appear as purple circles, and Methylene blue stains DNA in the nuclei, so it
makes the nuclei appear blue.
In an experiment somewhat related, a group from the University of California used
Arabidopsis thaliana to look at mRNA translation. They used differential centrifugation to
isolate different density molecules such as ribosomal subunits, ribosomes, and polysomes from
detergent-treated cell extracts. In the experiment, they found ways to isolate and quantify
polysome complexes from plant tissues (Mustroph et al 2009). This method is similar to the
way we used the centrifugation method to isolate and quantify the nuclei, chloroplasts, and
amyloplasts.
In data tables 1-4 below, the observations of each group of the organelles are
presented. Relative amounts were recorded. So, for example, very many mean thousands,
many would mean hundreds, some means around 100, and so on. The relative amount of each
observation is based on one viewing area and not the entire slide so as to make the amounts
more accurate.

Table 1: Spinach Cells/Chloroplasts

Chloroplasts Observed on Unstained Slide
Tube Code # Pellet 1 Pellet 2 Pellet 3 Supernatant 3
2 Many Many Very Few Very Few
3 Very Many Many Very Few Very Few
4 Very Many Very Many Few Very Few
Table 1 (above) shows groups that observed the spinach cells. The data shows the number of chloroplasts observed from each
pellet/supernatant
Table 2: Pea Cells/Chloroplasts
Chloroplasts Observed on Unstained Slide
Tube Code # Pellet 1 Pellet 2 Pellet 3 Supernatant 3
5 Many Very Many None None
6 Very Many Many None None
1 Many Many Very Few None
Table 2 (above) shows the groups who observed the pea cells. The data shows the number of chloroplasts (approximate) and the
pellet/supernatant they were seen in.

In Tables 1-2, the type of plant cell used (pea or spinach), was determined by the
number of chloroplasts. Since the spinach cells were darker green (more chloroplasts) it can be
expected that there will still be chloroplasts present in the final pellet and supernatant because
if there are more present, then there should be more at the end in the spinach cells than there
were in the pea cells that should have less chloroplasts. The groups with tube code numbers 5,
6, and 1 were put into the pea cell group since there were very relatively no chloroplasts
observed in Pellet 3 or Supernatant 3, and the remaining groups (2, 3, and 4) were placed into
the spinach cell category as these groups were still observing chloroplasts in the third pellet and
supernatant. Also, some of the groups have darker pellets in color. These groups were the
ones who had the spinach cells; however, the data related to color of the pellet was not
explicitly collected and recorded.

Table 3: Amyloplasts
Amyloplasts Observed on Lugol’s Iodine Stained Slide
Tube Code # Pellet 1 Pellet 2 Pellet 3 Supernatant 3
1 Many Many Few None
2 Very Many Very Many Few Very Few
3 Very Many Very Many Some Very Few
4 Many Very Many Many Some
5 Very Many Very Many Very Many Many
6 Very Many Very Many Very Many Very Many
Table 3 shows all of the groups and the relative number of amyloplasts they observed on each slide.

In Table 3, it should have appeared that amyloplasts were present in all of the samples
because they are not very dense, so some should stay suspended for the later centrifugations.
This is mainly what was observed. The opposite is true for Table 4. Here, the nuclei should be
present in large numbers in the first few pellets and not present in the last ones, because it is
the densest and therefore should have been pulled to the bottom to form the pellet at the
bottom. As seen in the table, in some of the groups, the expected results appeared, however
there was a lot of variation between the groups, and some groups even say a large number in
Supernatant 3.
Table 4: Nuclei
Nuclei Observed on Slide Stained with Methylene Blue
Tube Code # Pellet 1 Pellet 2 Pellet 3 Supernatant 3
1 Many Many Few Very Few
2 Many Very Few None None
3 Some Very Few Very Few None
4 Some Few Very Few None
5 Some Some Very Many Many
6 Many Some Many None
Table 4 shows each of the groups’ observations of the number of nuclei found in each view of a slide.

Discussion

The results that the groups obtained were fairly accurate. For the chloroplasts, the
numbers of chloroplasts observed seemed like they were a fair representation of what was
expected. Also, when observing the amyloplasts, the data there seemed to be generally what
was expected, although there was more variation between the different observances that
groups made. Lastly, looking at nuclei, there was a large amount of variation between all of the
groups. The expected results should have been that there were many/some in first 1/2 pellets
and then few/none in P3/S3. However, this was not the case.
When observing the slides and analyzing the pellets, there were some variations
between the groups in the magnification that was used and the amount of buffer solution that
was added to dissolve the pellet. To get better, more uniform results, the level of magnification
could be regulated. By applying this rule, it can be sure that all the groups are observing the
organelles present on the slide at approximately the same size, meaning that there will be
proportional numbers of organelles from group to group. So for instance, if one group was
using a 400x magnification, and one was only using a 100x, then the group with the higher
magnification will be seeing less, more detailed structured in their field of view than the groups
only using 100x magnification. Another variable in the experiment is the amount of buffer
solution used. There is really no way to regulate this, because some groups will have a more
concentrated pellet than others, but when using different amounts of buffer solution, the pellet
will become less/more concentrated and therefore a variable number of structures will be
observed. As another example, if only 1ml buffer is added to one group’s pellet versus 5ml
buffer in another, the group that added less buffer will have a more concentrated variety, and
will probably see more organelles than the other group that added more. In addition to these
two variables, to obtain yet even better results, the centrifuge speed could be slowed down and
done for a longer amount of time. In doing this, the less dense organelles would be less likely
to pellet at the bottom since the speed is slower.
This experiment captured basic concepts and ideas that are used for differential
centrifugation and should be used for future labs. Even though there are some variables in the
experiment, the number of consistencies between group observations of the slides was obvious
enough to be able to tell what should be happening in all of the pellets. In addition to
differential centrifugation, it would be interesting to do another lab using a different method of
centrifugation, such as density gradient. At the Himeji Institute of Technology in Japan,
“Spinach chloroplasts were isolated using floatation centrifugation followed by…density
gradient centrifugation after disruption of intact chloroplasts by freezing and thawing” (Koike et
al 1998). Different methods of centrifugation could allow student to explore alternative ways
to isolate cell components and organelles.
In conclusion, the spinach cells had more chloroplasts in Pellet 3 and Supernatant 3 than
the pea cells. There were (generally) more nuclei in the first 2 pellets than the last one, and
there were more amyloplasts in the last few pellets and supernatant 3 than the first few.

References

“Fractionation and Identification of Organelles from Plant Cells.” Edited by Nelson, K, and
Burpee, D(2009). Department of Biology, The Pennsylvania State University, University
Park, PA. Edited by Price, M. Siegfriend, E, and Burpee, D (2006). Adapted from original
work by Elizabeth McIntosh and Robert Moss, “Biochemistry of Cells,” was printed in
The American Biology Teacher, Vol. 57, No. 5, pp. 294-296, published by the National
Association of Biology Teachers.

Alberts, Bruce, Dennis Bray, Karen Hopkin, Julian Lewis, Martin Raff, Keith Roberts, Peter
Walter, and Alexander Johnson. Essential Cell Biology. 2nd. New York, NY: Garland
Science, 2004. Print.

Koike, H, M Yoshio, Y Kashino, K Satoh. "Polypeptide composition of envelopes of spinach

chloroplasts: two major proteins occupy 90% of outer envelope membranes.." Plant and
Cell Physiology 39. (1998): 526-32. Web. 24 Sep 2009.

Mustroph, A, P Juntawong, J Bailey-Serres. "Isolation of plant polysomal mRNA by differential

centrifugation and ribosome immunopurification methods." Methods of Molecular
Biology 553. (2009): 109-26. Web. 24 Sep 2009.