Strength of NaCl on phagocytosis in Tetrahymena
Bianca Iafrate
Erin Craven, Alex Wood
L10
11 April 2014
Introduction
Tetrahymena thermophila belongs to the Ciliated Protozoa or Ciliates, a
major, successful and diversified evolutionary lineage of unicellular eukaryotes. The
cells are large: 40-50 um along the anterior-posterior axis. Like other ciliates,
Tetrahymena cells have a striking variety of highly complex and specialized cell
structures (Orias, 1997). Tetrahymena can be used to study ciliary motion, organelle
structure and function, cell morphology, cell proliferation, and cell behaviors (Leick,
et al. 1992; Wheatley et al. 1994).
An important cellular process in microbial and aquatic ecology is bacterivory
by unicellular eukaryotes or protista (Sherr and Sherr 2002). The most
comprehensive studies on the effects of NaCl on freshwater organisms have been
done with macroinvertebrates (Blasius and Merritt, 2002 and Benbow and Merritt,
2004). Bacterivorous or phagotrophic protists engulf food particles by the process
of phagocytosis and this action has important ecological consequences. When
hungry Tetrahymena encounter food, they use their cilia to sweep it into each cell’s
oral groove (Bozzone 2000). This process can be visualized by feeding stained yeast
cells India ink (Keenan 1984) or Chlorella to Tetrahymena (Bozzone 2000). Grazing
on bacteria can change the composition and activities of bacterial communities
(Simek et al. 1997). In turn, this process can potentially alter biogeochemical
pathways under the influence of bacteria (Prast et al., 2007).
Large quantities of salt cause dehydration. Many fresh-water organisms are
still able to thrive in lakes and rivers as winter comes to an end and road salts run
into the bodies of water (low concentrations). Tetrahymena thermophila is a
freshwater organism that commonly inhabits streams, lakes, and ponds. While
freshwater lacks the presence of salt, seawater has a concentration of about 3.1% to
3.8% NaCl (varies). When put in different concentrations of salt-water solution, the
fresh-water-thriving Tetrahymena will function the least in higher salt
concentrations compared to lower salt concentrations, therefore consuming less.
Materials and Methods
Knowing that the Tetrahymena were fresh-water organisms, we knew that
salt would be an interesting concentration to experiment with. We had eleven
centrifuge tubes, labeled A through K, which were filled with 50 µl of NaCl solution
that had concentrations of 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,
and 100%. With each concentration, 50 µl of Tetrahymena were added and allowed
to adapt for ten minutes. 10 µl of blue polystyrene bead suspension was then added
to each, and the Tetrahymena were allowed to feed for 10 minutes. 20 µl was then
extracted from each tube and put into another tube, all again labeled A through K.
To each, 20 µl of 0.2% glutaraldehyde was added to fix the Tetrahymena. We then
took 10 µl from each solution and pipetted it onto separate slides and viewed under
the microscope. 30 Tetrahymena were randomly chosen under a magnification of 10
and their contents of blue polystyrene beads were counted and recorded according
to the concentration of NaCl they were in. Averages and standard error were
calculated in Excel.
Results
14
Average Numer of Beads
12
10
8
6
4
2
0
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Concentrations of NaCl (%)
(Figure 1: Average number of beads consumed by Tetrahymena in each NaCl
concentration wi (+/- SE))
Tetrahymena phagocytized the most beads, 11.57(± 1.69, SE) in the 0% NaCl
solution. At a concentration of only 10%, the Tetrahymena was extremely affected.
The average intake of blue beads dropped to 0.3 (± 0.18, SE). In comparison, as NaCl
concentrations increased to 10%, the number of beads phagocytized drastically
decreased. In NaCl concentrations of 10% and 20%, Tetrahymena consumed few
than one bead (0.3± 0.18, 0.27± 0.18, SE, respectively). In all NaCl concentrations
over 20% up to 100%, no beads were phagocytized.
Discussion
Our initial observations included that the Tetrahymena moved very quickly
and rapidly, with a lot of movement in general. Their eating habits included some
eating to the point that they were full, while others did not eat at all. Going into the
experiment, there was a chance that some would not feed. However, we did not
expect for numbers to decrease so rapidly when increasing the amount of NaCl. Our
hypothesis was supported, overall. The Tetrahymena functioned best in the three
lowest concentrations (0%, 10%, 20%) of NaCl, and had no function at all in the
other NaCl concentrations (30% through 100%). At this point, the question to ask
would be: At which concentration, specifically, did the Tetrahymena lose all ability
to function?
Tetrahymena become stressed in NaCl concentrations, which makes sense
because they are freshwater organisms. With such a dramatic drop in the feeding
count, it could be possible that we did not count enough Tetrahymena in each tube.
Not being given enough time could be why they were unable to function efficiently
enough to carry out phagocytosis. If we had randomly selected 40 or 50
Tetrahymena to count, we could have found more in-take of the blue beads. In a
future experiment, more should be counted. Using multiples of 10 as our NaCl
concentrations could have also contributed to our results. Changing the numbers
used could definitely change the results significantly. In a future experiment, instead
of using 10%, 20%, 30%, etc., increments between concentrations should be 2%.
You would test at exactly what concentration between 20% and 30% the
Tetrahymena lost their ability to function, and when between 0 and 10 it begins to
decrease. Also, you could see when it decreases between 10 and 20. Perhaps they
lost function ability closer to 20% (21% or 22%) or as they got closer to a
concentration of 30% (28%, 29%).
Works Cited
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