Cigarette Toxin Effect on Paramecia tetraurelia Cultures
By: Skyler D. Resendez
Lab Partner: Thomas Heath
Bio 305 – Genetics
11/19/11
Introduction
Paramecium tetraurelia: In this study, microorganisms known as Paramecia tetraurelia were
experimented upon. Paramecia are protozoa covered in alternating cilia and trichocysts. As with any
organism in the Paramecium genus, they are unicellular eukaryotes, approximately 100 micrometers in
length, yet they are complex enough to be capable of showing an array of information during biomedical
research (P ar a m e c i u m te t r a u r e l i a , 2 0 1 1 ) .
Movement: The movement of the Paramecia is very fluid. The organisms turn around quickly in a
motion that looks as if they’re folding over. Due to their movement pattern, Paramecia have often been
called “swimming neurons.” These microorganisms are able to sense changes in the environment.
Paramecia swim forward when they are in a state of homeostasis. However, when that state of
homeostasis is compromised, the response is a flooding of calcium ions into the organism. The calcium
channels are located at the base of the cilia. This causes the Paramecium to go backwards. This is known
as an avoidance reaction (E u k a r y o t es g e n o me s - p a r a m e c i u m t e tr a u r el i a , ( n . d . ) ,
P a r a me c i u m te t r a ur e li a , 2 0 1 1 ,) .
“Junk” DNA: Paramecium tetraurelia are “prime” organisms for research purposes. This is due to a
number of facts, including their small amount of what is often referred to as “junk” DNA (especially
when compared to primates.) Junk DNA simply refers to segments of DNA that do not code for anything.
Primates, such as humans, have a large amount of Junk DNA. This is partially due to what are known as
“jumping genes,” or transposons. Transposons, which are actually segments of DNA (not genes), have
the ability to move from place to place on the chromosomes. Transposons lengthen the amount of
“junk” DNA in primates, which partially accounts for the massive percentage of non-coding DNA in
humans. Approximately 96 percent of a human’s DNA falls under this category. This is why it is much
simpler to cause a mutation in Paramecium tetraurelia. The coding DNA of the Paramecia accounts for
about 70% of their genome. In order for a mutation to be seen, the change in the genome must occur in
a section of coding DNA. Therefore, any mutations that occur in the Paramecia have a 70% chance of
showing, compared to the measly 4% chance in humans. Note that 4% is a high estimate.
Food Supply: As it is well known that Paramecium tetraurelia are auxotrophs, they need a steady food
supply and supportive environment for them to continue to prosper. Paramecia often prey upon
different bacteria. Thus, they were constantly supplied with a sufficient amount of Klebsiella
pneumoniea. The organism Klebsiella pneumonia is a gram-negative bacterium and is also the pathogen
often responsible for urinary-tract infections. Klebsiella are rod-shaped and are facultative anaerobic
bacteria, the latter of which meaning that they will utilize oxygen when available, but can survive
without it (Klebsiella pneumoniae, (n.d.)). The Klebsiella pneumonia cultures were put into a wheat grass
solution in order for the bacteria to have a sufficient nutrient supply. With the Klebsiella feeding off the
wheat grass and the Paramecia feeding off the Klebsiella, a food chain had been produced. The food
supplies were constantly reintroduced to keep the Paramecia in top condition.
Methods and Materials
Maintaining Paramecia Cultures
In this experiment, it was necessary to have a constant source of healthy Paramecia. In order to do this,
the researchers were constantly aiding and altering the environment of the microorganisms. This is
necessary to keep nutritional sources for the Paramecia at adequate levels and to keep waste products
from filling up their limited environment. In order to first acquire two stock solutions of Paramecia, the
researchers acquired two flasks that contained approximately 10 mL of premade wheat media solution
in each (the size of the flasks isn’t of too much concern as long as there is enough room within the flask
to hold the wheat media and small amounts of Paramecia that will be added). It was then necessary for
the conductors of the experiment to inoculate the wheat media with Klebsiella pneumoniae. An agar
plate that had multiple colonies of the Klebsiella was then pulled from the refrigerator. The parafilm
keeping the lid fastened tightly to the agar plate was then removed as the researchers acquired an
inoculating loop. The inoculating loop was then heated to high temperatures via a bacti-cinerator for 10
seconds and then allowed to cool for 20. This process helps to sterilize the equipment to prevent
contamination. The inoculating loop was then used to lightly swipe a colony from the agar plate and
then was put directly into the wheat media and stirred. This process was done for both of the flasks and
then the flasks were put into an incubator over night. An alternative method to this would have been to
simply leave the flasks on the shelf for two days, rather than one day in the incubator. The following
day, the flasks were removed from the incubator and given five to ten minutes to cool off. Once the
flasks and their contents reached approximate room temperature, the researchers acquired stock flasks
of previously created Paramecia cultures. Micropipettes were used to transfer 500 microliters (uL) of
the stock Paramecia solution into each of the flasks containing Klebsiella and wheat grass solution.
Using this process, the Paramecia cultures reached their prime approximately six to nine days
afterwards and began dying at around day fourteen. With this in mind, the researchers repeated this
entire process weekly, requiring two more flasks of wheat media. From this point on, the older flasks
containing Paramecia were checked using a microscope after micropipetting 100 uL of their contents
from each onto a microscope slide. If they contained healthy, viable cells, 500 uL were taken from each
and put into the two newer flasks. A week later, the newer flasks were checked in a similar manner, and
if they too contained healthy cells, the older flasks (now two weeks old) were discarded and the newer
flasks were used as the stock samples to repeat this whole process again.
Note: All flasks were labeled thoroughly throughout the course the experiment to ensure that they
could be found and that their contents were known.
Creating a Stock Cigarette Solution
The entire goal of this experiment was to cause visible mutations in the Paramecia cultures. These
mutations could have included changes in size (length/width), shape, movement pattern, general
structure (ex. black dots at the ends of the cells), etc. To do this, the researchers decided to use
cigarette toxins. With this concept in mind, it was necessary to find a way to expose the Paramecia to
such toxins. To do this, the researchers made a sort of “cigarette tea” and considered it their 100% stock
solution. The researchers acquired wheat media and cigarettes (the brand “West” was used) to craft
such a solution. Fifty mL of wheat media was measured out using a pipette. The media was put into a
glass container with a lid. The individuals crafting the experiment then tore apart five cigarettes and put
the contents into the container, excluding the filter and papers. This achieved the desired goal of having
a stock solution that is one cigarette per 10 mL of wheat media. This mixture was then allowed to sit
over night. The following day, the container was brought back into the lab. At this point in time, the
cigarette stock solution was poured out into a beaker (150 mL). A petri dish lid was then put over the
top of the beaker to try to keep too much of the liquid portion of the solution from evaporating out, as
the next step was to heat it to a boil. To do this, a hot plate was used. It did not take long for the media
to start boiling. This was done in hopes of further extracting the toxins into the liquid portion of the
solution. The researchers then acquired a small side-arm flask, filter paper, a Buchner funnel of
appropriate size, and parafilm. The filter paper was put inside of the Buchner funnel, and the draining
portion of the Buchner funnel was put into the side arm flask. The parafilm was used to make an air tight
seal around the funnel and side-arm flask, but an appropriately sized stopper would have probably been
more effective. The side arm flask was then attached via a hose to a vacuum apparatus used in vacuum
filtration. The vacuum was turned on and the cigarette solution was then slowly decanted into the
funnel. The pores in the funnel would get clogged easily, so it was necessary to remove the funnel of its
contents and change the filter paper often. Disposable pipettes were very helpful with this process.
Disposable rubber gloves were also used to squeeze some of the liquid out of the tobacco which was
very good at absorbing (and not releasing) the media. Through decanting, repeated filter changes, and
slow vacuum filtration, a decent yield of cigarette stock solution was obtained. After this process was
complete, it was noted that the solution contained small, red rubber chunks. The chunks must have
been from the inside of the deteriorating hose. This was a simple fix requiring a funnel, Kimwipes, and a
conical tube. The Kimwipe was put into the funnel and the funnel into the tube. The solution (now free
of all chunks besides the rubber pieces) easily filtered through the Kimwipes, leaving the rubber chunks
behind. The cigarette stock solution was then poured carefully into a beaker, covered with parafilm,
labeled, and put onto a shelf.
Creating Paramecia/Stock Cigarette Solution Mixes
When it came time to begin attempting to cause mutations, the cigarette stock solutions, the
Paramecia stock solutions (at this point being seven days old and in their prime), and a well plate were
acquired. The well plate already had its rows and columns labeled with letters and numbers. The
amount of solution in which each well could hold was determined using water and pipettes and a
volume of 500 uL was decided upon per sample. The concentrations created were 15% cigarette stock
solution and 85% Paramecia solution, 20% cigarette stock solution and 80% Paramecia solution, and
lastly, 25% cigarette stock solution and 75% Paramecia solution (from this point on the concentrations
will simply be referred to by their cigarette solution concentration.) The formula C1V1 = C2V2 was used to
figure out the volumes needed.
Ex. C1 = original cigarette solution concentration (100%); V = volume of cigarette solution needed; C2 =
desired concentration (for this example, 15%); V2 = final volume (500 uL)
C1V1 = C2V2
100% (V1) = (15%) (500uL)
100% (V1) = 7500 % uL
(V1) = 75 uL
Therefore, 75 uL of stock cigarette solution was mixed with 425 uL of Paramecia solution to get a 15%
concentration.
With this in mind, the 15% concentrations were composed of 75 uL of stock cigarette solution mixed
with 425 uL of Paramecia solution, the 20 % concentrations were composed of 100 uL of stock cigarette
solution mixed with 400 uL of Paramecia solution, and the 25% concentrations were composed of 125
uL of stock cigarette solution mixed with 375 uL of Paramecia solution. Stock wells were also created
(500 uL of stock Paramecia solution) for comparison. Wells A1-A3 were designated to the stock
solutions, B1-B3 were designated to the 15% concentrations, C1-C3 were designated to the 20%
concentrations, and D1-D3 were designated to the 25% concentrations. All of these concentrations were
created with the use of micropipettes. The wells and flasks were then put back on the shelf. The wells
were then to be left alone for three days.
Observing Results and Reintroducing a Suitable Environment
After three days, the Paramecia were checked once more with a microscope using the techniques
described previously. They were compared to the stock solutions in the wells. Abnormalities were noted
and any mutated cells (possible mutation examples are described above) were then transported to a
suitable environment using pipettes (it was made sure that only Paramecia that had been residing in the
same concentration of cigarette solution were put together in flasks). Such an environment consisted of
Klebsiella/wheat grass solutions crafted in the same way described before. The making of such
environments needed to be done ahead of time because the incubation step requires at least a day and
four days in cigarette concentrations of 15, 20, and 25% is sure to kill the Paramecia. If the
Klebsiella/wheat grass solutions had to be crafted more than a day prior, they could simply be
refrigerated after incubation to keep the Klebsiella from becoming too numerous.
Results
Throughout the course of this experiment, the researchers managed to force multiple changes on the
Paramecia. The scientists documented altered features in the 20% cigarette, 80% Paramecia cultures
over time as they were continually put into new environments for them to thrive. The changes shifted in
frequency, severity, and type over time. Each time the cultures were checked under a microscope, any
abnormal features were documented and analyzed. Changes such as abnormalities in size, shape,
motility, and other features were documented on the following dates:
17 October, 2011
On this particular date, the Paramecia cultures that were observed had been sitting in the 20% cigarette
solution for a total of three days. In this batch, the alterations to the Paramecia were plentiful. There
were cells with the black dots, usually associated with the ends of the cells, dispersed throughout the
entire organism. (The percentage was calculated by taking the area of the organism and dividing it by
the area that the black dots covered within the organism. See Table 3 and Figure 6 for data regarding
black dot coverage of the Paramecia.) Abnormal movement was also documented. Rather than the
usual “folding-over” motion of the Paramecia, they seemed to be rotating about a point. Based off of
observations taken from a 2 minute long video with multiple cells rotating in such a manner (N = 7,
where N is the number of cells observed), the researchers estimated that the Paramecia were rotating
at a rate of approximately three spins every two seconds. Some cells were also quite misshapen and
many were smaller than usual. (See Table 1 and Figures 3 and 4 for data regarding the size of the
Paramecia.)
20 October, 2011
The Paramecia observed on this date were cells that had been transferred from the wells and put into a
flask of wheat grass/Klebsiella solution. By this point, the cells had lived in their new environment for a
total of three days. The researchers made sure to note the fact that there was still cigarette
concentration within this flask, but it was narrowed down to a very slight amount as there was very little
solution from the wells being put into a large volume of wheat grass/Klebsiella solution. When checked,
the sample was full of life, much of which was distorted anatomically once again. The most noticeable
change in the cells was that many of them had one very narrow end that looked “cone-like.” (See Table
4 for data regarding the “cone-like” Paramecia.) Another strange phenomenon that was noted was that
some of the Paramecia exhibited what seemed to be an inability to break apart right away at the end of
asexual reproduction. This was a frequent occurrence as two cells would be stuck together at a single
point, many of which were moving about, the cell in the back simply being dragged along. (See figure 1
for a photograph taken of two Paramecia stuck together in such a fashion.) It seemed as if there were
always one or two pairs in frame while looking at approximately 40 cells at a time. The researchers also
noted that the cells could primarily be found around what seemed like chunks of cigarette
toxin/content. (See Table 2 and Figure 5 for data regarding approximate percentages of Paramecia that
were located in the chunks of the wheat grass media.)
Figure 1: This is a 100x magnified (rescaled) photo taken on October 20th, 2011. The photo is of multiple
Paramecia and a few of their respective lengths (in um) and areas (in um^2). The lengths of these
Paramecia are particularly interesting due to how small they truly are compared to average Paramecia
(the average length for the control group on this day was 108.697 um). Another interesting portion of
this photo is the two cells that are stuck together toward the bottom of the picture. This is the change
noted where they had difficulty finishing asexual reproduction. The researchers coined the term
“Gemini” as a possible name for such Paramecia.
24 October, 2011
The Paramecia observed on this date were from the same environment that they were in on the 20th of
October. Despite this, their abnormalities still changed between the two dates. These observations were
made just prior to putting 500 um of the culture into a new environment for further study to be done a
few days later. The Paramecia in this flask were altered, but again, the changes were slightly different
from the previous date’s observations. It was noted that the cells still exhibited the “cone-shaped” ends
that had been seen before, but this was to a much lesser extent. The Paramecia were also smaller in size
and area than the control group, but the cells began to look relatively more average anatomically than
they appeared to be on the 20th of October.
27 October, 2011
This was the final date the researchers had set aside for observations. The Paramecia observed on this
date had been in their environment for a total of approximately three and a half days. The researchers
had transferred cells from the old flask into another new Klebsiella/wheat grass solution earlier that
week, once again lowering the cigarette toxin concentration even further (at this point, the cigarette
toxin levels were almost negligible). The changes in the Paramecia were much less frequent on this date
than they had been during previous observations. However, there were still cells that were abnormal
anatomically. Misshapen Paramecia were observed including one that had a “bubbling membrane.” (See
Figure 2 for a photograph taken of a misshapen Paramecium on this date.) The researchers also
managed to catch cells in the act of conjugation. The conjugation between the cells was to help increase
genetic diversity. The scientists observed this occurrence multiple times within the culture, indicating
that mutations had occurred and the cells were trying to rectify the situation. Lastly, abnormal
movement was once again documented, this time a strange twisting sort of motion from cells that often
died directly afterwards. Based on a minute long video, the cells seemed to twist over completely (360
degrees) once every 2.8 seconds (on average). The cells seemed to spiral until they met their demise.
Figure 2: This is a 40x magnified (rescaled) photo taken on October 27th, 2011. The picture is of two
Paramecia and their respective lengths (in um). This photo was chosen because of the comparison of
the fairly average Paramecium on the right, versus the small, abnormally shaped, altered cell on the left.
These were just a few of the changes seen throughout the course of this experiment.
Table 1: This data table shows the average sizes of Paramecia of which the researchers took pictures,
using an N number of 12 for both the control and experimental groups.
Average Sizes of Paramecia by Date – Table 1
Length (um)
Area (um^2)
Control
Experimental Control
Experimental
17-Oct
112.421
93.151
3145.651
2331.854
20-Oct
108.697
97.563
3110.431
2541.187
24-Oct
118.563
99.911
3001.647
2275.108
27-Oct
111.652
103.86
3043.647
2226.562
The software program “ImageJ” was used with a standard micrometer as a scale to find both lengths (in
um) and area (in um^2). The data shown in Table 1 is a result of numerous measurements taken and
averaged from each day and shows that the experimental cells were often considerably smaller than
their control groups.
140
120
100
80
60
40
20
0
Length in um
Experimental
27-Oct
26-Oct
25-Oct
24-Oct
23-Oct
22-Oct
21-Oct
20-Oct
19-Oct
18-Oct
Length in um Control
17-Oct
Length (um)
Length of Paramecia on Various
Dates - Figure 3
Dates
Figure 3: This graph gives a visual of some of the information displayed in Table 1. It is a good visual to
see the difference between the average lengths of the control group versus the average lengths of the
experimental group on various dates. An N number of 12 was used for both the control and
experimental groups.
4000
3500
3000
2500
2000
1500
1000
500
0
27-Oct
26-Oct
25-Oct
24-Oct
23-Oct
22-Oct
21-Oct
20-Oct
19-Oct
18-Oct
Area in um^2 Control
17-Oct
Area (um^2)
Area of Paramecia on Various Dates Figure 4
Area in um^2
Experimental
Dates
Figure 4: This graph gives a visual of some of the information displayed in Table 1. It is a good visual to
see the difference between the average areas of the control group versus the average areas of the
experimental group on various dates. The difference in average area between the two groups can be
quite drastic. An N number of 12 was used for both the control and experimental groups.
Table 2: This data table shows approximate percentages of Paramecia that were located in the chunks
of the wheat grass media, using an N number of 31 for both the control and experimental groups.
Approximate Percentages of Paramecia in Solid Portions of Wheat Media –
Table 2
Approximate Percentages
Date
Control
Experimental
20-Oct
13%
90%
24-Oct
15%
85%
27-Oct
12%
40%
This solid portion of the wheat media seemed to have absorbed much of the cigarette toxin and was
often darker in appearance than in the control cultures. Due to this fact, the altered Paramecia were
often found in much higher frequencies in the solid portion than in the rest of the media. These
percentages were approximated based on numerous pictures of Paramecia located both in and out of
Approximate Percentage
these sections of the media.
Approximate Percentages of
Paramecia in Solid Portions of Wheat
Media - Figure 5
120%
100%
80%
60%
40%
20%
0%
Control
Experimental
Dates
Figure 5: This graph displays visually the information from Table 2. It shows that the Paramecia in the
experimental cultures were found gathering in and around the solid portion of the wheat media at a
much higher frequency than those of the control culture. It also shows that this trend decreased over
time as the Paramecia repaired their DNA through practices such as conjugation. An N number of 31
was used for both the control and experimental groups.
Table 3: Table 3 shows numerically the average percentage of how much each microorganism was
covered with the black dots, using an N number of 14 for both the experimental and control groups.
Black Dot Coverage Percentages – Table 3
Experimental
Control
17-Oct
76%
23%
20-Oct
64%
26%
24-Oct
48%
27%
27-Oct
44%
24%
Usually the black dots are located only at the ends of each organism and thus, they cover a much
smaller amount of the organism’s entirety. These percentages were acquired by dividing the entire area
of a Paramecium by the area taken up by the black dots within the microorganism. That number was
multiplied by 100%. This was done for multiple Paramecia from numerous pictures on each date.
Black Dot Coverage Percentages Figure 6
80%
60%
40%
Experimental
20%
Control
Dates
27-Oct
26-Oct
25-Oct
24-Oct
23-Oct
22-Oct
21-Oct
20-Oct
19-Oct
18-Oct
0%
17-Oct
Percentages
100%
Figure 6: This graph shows visually the information from Table 3. It is plain to see that the black dots
covered a greater percentage of the microorganisms in the experimental cultures than they did in the
control cultures (on average). It is also possible to see the declining slope in percentages of the
experimental group over time, once again indicating that DNA repair had occurred. An N number of 14
was used for both the control and experimental groups.
Table 4: This data table attempts to quantify how extreme the “cone-shaped head” phenomenon was
on average, using an N number of 13 for both the control and experimental groups.
Average End Width Measurements – Table 4
Average Width of Head
Average Width of Tail
End (um)
End (um)
Differences (um)
Control
Experimental Control
Experimental Control
Experimental
20-Oct
9.838
6.545
11.547
12.499
1.709
5.954
24-Oct
9.906
7.488
11.803
11.384
1.897
3.896
This was done using comparisons. The researchers acquired the average width of each end of the
Paramecia in each culture on the two dates that the phenomenon had occurred (the widths were taken
approximately 5 um from the outermost point of the cell membrane.) The average differences between
the corresponding head/tail measurements (the head end being the one that was not as wide) were
then found. By looking at the average differences and noting the generally unwavering widths of the tail
end, one could clearly draw the conclusion that the experimental Paramecia had narrower heads. It is
also important to note that the phenomenon decreased in intensity between October 20th and October
24th.
Discussions
The data throughout this experiment and the statistic analysis of it consistently indicated that mutations
occurred. There was the presence of anatomic abnormalities including changes in the width at one end,
general size decreases, both in length and area, black dot dispersal, and general shape. There were also
changes in movement and abnormal behavior such as tending to stay close to where the toxin was most
concentrated. All of this was observed, analyzed, and quantified, and the numbers don’t lie. The large
amount of cells attempting to conjugate also indicates that mutations occurred. This is because the
Paramecia were attempting to increase biodiversity to help remove or rectify any negative attributes
attained due to the changes caused by the cigarette toxin. This experiment has also shown how effective
these organisms are at repairing their own DNA as the percentage and degree of changes observable
within the experimental groups decreased over time. However, it is important to always remain
somewhat skeptical. There are always alternative possibilities to what may have occurred. It was
observed that the altered cells were often found around the visible chunks of what appeared to be the
cigarette toxins. One might argue that the reason the number of altered Paramecia dropped and the
amount of average cells steadily increased is simply because the abnormal cells were not passing on the
genetic coding for such alterations. This could have occurred for a number of reasons including that they
may have been dying before reproducing, whereas the ones who avoided the toxin and remained
anatomically average did reproduce. The different mutations that were observed may have simply been
due to non-mutated offspring coming into contact with the toxins since it was impossible to fully
eliminate said chemicals from the solution. However, the concentration of cigarette toxin drastically
decreased after every transfer to a new environment. If this were the case entirely, it would mean that
second generation mutated offspring never came about. This is extremely doubtful, though it may have
contributed to some of the observed changes in the Paramecia. It is much more believable that there
was a large degree of mutated offspring being born and that DNA repairing occurred as these amazing
organisms conjugated, but it is important to keep a critical, ever-questioning mindset. Seeing changes
that were quite frequent wane almost out of existence is a good indication that the organisms
responded to a mutation by attempting to eliminate it. A good example of this is the black dots that
were spread throughout the cell. This is a potential mutation that was observed in just about all of the
experimental cultures and its presence slowly declined throughout the generations. The researchers
induced an environment that increased the amount of conjugation occurring. This is why it was so
frequent. On a final note, it is interesting to once again revisit the fact that the Paramecia could often be
found barreling through what was potentially concentrated cigarette toxin. This may have been an
indication that some of the cells were actually addicted to the chemicals, much like how people become
addicted to cigarettes.
Potential follow up experiments include ones that use different concentrations of the cigarette toxin for
varying periods of time. For example, rather than keeping the Paramecia in a twenty percent cigarette
toxin solution for approximately three days, what if they were set in a forty percent solution for only
one and a half. It would be interesting to see how the alterations to the organisms differ with such
changes. One could also try to isolate different toxins within the cigarettes and see how they individually
affect the Paramecia. Different brands of cigarettes could be tested too to see the differences between
the effects caused by each. These are just a few of the many examples of follow-up experiments that
could be performed.
Work Cited
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