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The Effects of Shortwave Ultraviolet Radiation on Yeast Growth Rate

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					The Effect of Shortwave Ultraviolet Radiation on Yeast Growth Rate
                                     LUCAS ANDERSON
                  Senior Seminar Biological Research Investigation, Crown College
                                           May 3, 2012



                                           ABSTRACT
           Intact yeast cells were treated with 15, 30, and 45 minutes of short wave
           ultraviolet radiation. The growth rates of these cultures were studied in
           conjunction with a non-radiated control sample. The cells were grown in a
           10% sugar medium, and observed for a regular period for four hours. They
           were counted at regular intervals over this period. A final count was taken
           the following day (after 15 hours) and observations were made. The 30 and
           45-minute cultures showed a considerably slower growth rate, and were
           nearly completely stagnant from 180 to 900 minutes. The 15-minute culture
           showed an initial drop in growth, but showed the strongest amount of
           growth from 240 to 900 minutes. By cell observation and analysis, a
           significant amount of cell damage was seen in the irradiated cells.
           Abnormally shaped cells, and signs of lysis were apparent in observation of
           the irradiated cells.




              INTRODUCTION                           and growth are an important topic in the
                                                     medical field today. This for this reason,
 Yeast is sold in the grocery store in jars          yeast has been thoroughly as a model
 and small packets, and is used as an                organism for human skin aging, as well as
 ingredient for our bread. One often                 the causes for cancer.
 forgets that yeast is an organism. Every
 yeast cell is a small, single celled                This experiment demonstrates the
 organism that acts independently of the             relationship of the amount of ultraviolet
 organisms around it. It is a cell that is           radiation to cell growth and reproduction.
 very useful for research as well since it is        To begin, I have hypothesized that the
 eukaryotic and very similar to human                longer the radiation exposure, the slower
 cells. It is well known that ultraviolet            the growth and reproductive rate. It is
 light is harmful to human skin cells. It can        hypothesized that a greater amount of
 cause mutations that lead to apoptosis, or          ultraviolet light will cause more DNA
 even cancer. Yeast cells are different than         damage; thus less reproduction and more
 human cells in this way: yeast cells are            cell death. In this experiment the growth
 separate organisms.         They do not             and reproductive rate of ultraviolet
 contribute to a higher organism;                    irradiated yeast cells were studied. This
 therefore, apoptosis is disadvantageous to          experiment was executed in order to
 the organism. Yet, the causes of cell death         determine the effect the ultraviolet



                                                Page 1
radiation has on yeast cells so that the            Trial II: Yeast to Solution Ratio
results can be ultimately applied to
human skin cell exposure.                           The purpose of this trial was to find the
                                                    best yeast to solution ratio that yielded
      MATERIALS AND METHODS                         consistent growth and analyzability
                                                    without running out of nutrients. Based
Trial I: Growth Medium Concentration                upon the results from trial I, a 10% sugar
This first trial was to ascertain the ideal         solution was used.       Three flasks of
growth medium concentration. The best               solution were measured, 40 mL in each
                                                    flask.   Three 50 mL beakers were
results were based upon growth
consistency and analyzability. A mostly
arbitrary (estimated) number of 0.1
grams of yeast was measured and placed
into three 50 mL beakers (0.1 g each).
Three solutions were mixed in three
different 50 mL flasks. These solutions
were composed of sugar and water. The
first solution was mixed using 2.0 g of
sugar to 40 mL of distilled water, which is
a 5% sugar solution. In the next solution,
the amount of sugar was doubled to 4.0 g
while keeping the amount of water the
same for a 10% sugar solution. For the
20% solution, the 4.0 g were doubled to
8.0 g of sugar per 40 mL of distilled water.
The room temperature solution was
added to the 40 mL beakers of yeast that                Figure 1: a 0.2 mm * 0.2 mm area is
were marked: 5%, 10% and 20%. The                       shown where the cells were counted
yeast and solution were promptly stirred
with three different glass rods for 15
                                                    obtained, and were each marked 0.4, 0.08,
seconds and placed into the incubator at
                                                    and 0.04. These markings referred to the
33˚ C as described by Ito et al (1983).
The cells were counted on a                         grams of yeast placed in each beaker. The
hemocytometer at intervals for 2 hours.             0.4 g beaker had a 1:100 yeast to solution
                                                    ratio, the 0.08 g beaker had a 1:500 ratio,
The cells were counted within the blue
                                                    and 0.04 g beaker had a 1:1000 ratio.
highlighted area as shown in figure 1,
which is a 0.2 mm*0.2 mm* 0.1 mm                    After the yeast and solution were
volume. They were counted in this area              combined, the mixture was promptly
                                                    stirred for 15 seconds with three different
because only a relative count was needed,
                                                    glass rods. The cells were then counted
and also there were too many cells to
count within the corner area of                     on a hemocytometer at intervals for 2
hemocytometer. I used the 0.2 mm * 0.2              hours. Again, the cells were counted
mm area as shown. The cells were                    within the area shown in figure 1
                                                    (0.2mm*0.2mm*0.1mm). The results and
counted, and the results and observations
                                                    observations were recorded.
were recorded.



                                               Page 2
Trial III: Ultraviolet Absorption                   the usual 40 mL of 10% sugar solution.
                                                    To compensate for the 20 mL of distilled
The purpose of the final trial was to test          water in the “wet” beaker, 20 mL of a
the effects of water on UV absorption in            double concentrated solution was poured
the yeast cell. Since solid substances such         into the beaker (4 g of sugar and 20 mL of
as glass and plastic block UV rays, it was          water). An initial count was done on each
predicted that water would have an effect           of the beakers in case the “wet” beaker
on UV absorbance in the yeast cells. Also,          had metabolized or reproduced during
it was important to test if UV had any              the UV exposure. To make this count
effect on dry yeast, since it is inactive in        more accurate and conclusive an
that state. Therefore, a two-part test was          extended counting procedure was
                                                    undertaken, since the effects of water
                                                    needed to be certain (rather than relative
                                                    as in the other two trials). Since the cell
                                                    concentration was still high, the 0.2 mm *
                                                    0.2 mm areas were used (0.1 mm height).
                                                    Four of these squares—one in each
                                                    corner—were used to determine the final
                                                    value as shown in figure 2. The results
                                                    were averaged as shown in this formula,
                                                    where q equals quadrant and equals the
                                                    average number of cells. Equation 1:




                                                    The averaged results were recorded as
                                                    well as any observations, especially those
 Figure 2: the counts of four 0.2 mm *              related to the condition, health, shape, etc.
 0.2 mm quadrants were averaged.                    of the cells.

executed. The first part involved a count           The second part of this trial was testing
of wet versus dry cells. Two 50 mL                  the glow emitted from a short wave
beakers were obtained and marked “wet”              fluorescent mineral. This mineral was
and “dry.” In the “wet” beaker, 0.04 g of           observed in dry and in water, under a
yeast was placed in a 50 mL beaker along            short wave ultraviolet light. The dry
with 20 mL of distilled water to prevent            mineral was placed in a dry 50 mL beaker
metabolism and reproduction. In the                 directly under the light. The wet mineral
“Dry” beaker 0.04 g of yeast were placed            was placed in 20 mL in a 50 mL beaker,
in a 50 mL beaker without any solution.             and was placed under the light. The
The 1:1000 yeast to solution mixture was            condition that yielded the most brilliant
used, since the cells were to be counted            light had more direct ultraviolet exposure.
without prior dilution. Both beakers were           The observations were recorded.
then placed under the UV lamp. Both
beakers were subjected to 30 minutes of
short-wave ultraviolet exposure.    After
the exposure, the “dry” beaker was given

                                               Page 3
Experimental Procedure                             mL of sugar solution each in two 1.5 mL
                                                   microtubes. The sugar solution was used
The experiment was undertaken using                so that the cells did not over-expand in a
the parameters suggested by the results            hypotonic solution. After the mixtures
of the preliminary trials. Thus, a 1:500           were stirred with different glass rods, a
yeast to solution ratio was used, with dry-        micropipette was used to extract 10 µL of
radiated yeast in a 10% sugar solution.            both yeast solution mixtures from each
To begin, 0.08 g of yeast was measured             beaker. The 10 µL sample was then
and placed into four 50 mL beakers.                pipetted into the solution in the
These beakers were marked: control, 15             microtubes. After pipetting the dilution to
                                                   mix it, a 10 µL sample of the diluted
                                                   mixtures     (control    and    15-minute
                                                   mixtures) were placed in each side of the
                                                   hemocytometer. The cultures were then
                                                   placed into the incubator at 33˚ C. An
                                                   initial count was made. This count was
                                                   made within the area shown in figure 3.
                                                   The highlighted quadrants had an area of
                                                   1 mm2 and a depth of 0.1 mm. The
                                                   average amount of cells were counted
                                                   using Equation 1, where the quadrants
                                                   are as shown in figure 3. After 30
                                                   minutes, the 30-minute beaker was
                                                   removed from under the UV lamp; and
                                                   the same procedure was applied to the
                                                   45-minute beaker at 45 minutes. The 30
                                                   and 45-minute beakers were diluted and
 Figure 3: the counts of the four 1                counted in the same method as was done
 mm*1 mm quadrants were averaged.                  with the control and 15-minute beaker.
                                                   The four beakers were alternately
minutes, 30 minutes, and 45 minutes. The           counted at intervals for four hours
15, 30, and 45-minute beakers were                 according to Table 3. One final count was
placed dry under the ultraviolet lamp.             executed the morning after (15 hours
The three samples were radiated with               from the beginning of the experiment) for
short wave rays (254 nm) rather than               a final count and observation. All results
long wave (365 nm). After 15 minutes,              and observations were recorded. The
the 15-minute beaker was removed from              averaged data was then extrapolated to
under the UV lamp. The control was set-            the rest of the 40 mL culture. This was
aside dry until the first beaker was ready         done according to Equation 2, taking into
after 15 minutes. The 10% sugar solution           account the 1:1000 dilution and where
was added to both the control and the 15-          0.1 µL is the volume of the quadrant. [1
minute beakers, 40 mL each. To get a               mm * 1 mm*0.1 mm = 0.1 mm3 = 0.1 µL]
more countable and analyzable sample,              Equation 2:
the mixtures were diluted to a 1:1000
ratio before being placed on the
hemocytometer, as advised by Dr.
Thomas. This was done by first placing 1

                                              Page 4
                RESULTS                          ratios had few clumps and were in a cell
                                                 range that I was looking for. The data for
Trial I: Growth Medium Concentration             the cell counts are shown below:
Surprisingly, the 5% solution yielded the                         Table 2
most consistent growth over the 2 hour
period. Yet, the 5% had too many clumps          Time (min)     1:1000    1:500    1:100
to allow for a good count. The 10%               Initial        59        136      Ruled
solution showed far less clumps and was          20             53        127      out, too
a much more analyzable sample. Another           40             60        140      many
surprising find was that the 20% sugar           60             62        138      cells to
solution had the least growth. The 20%           120            67        154      count
solution also had the least consistent
growth. After examining the numbers
and observing growth patterns, it was
determined that the 10% solution would
be used for the final experiment. It             Trial III: Ultraviolet Absorption
yielded the best results and seemed to           The more direct ultraviolet ray
provide enough food for reproduction.            absorption was observed in the dry yeast.
The data counts are shown in Table 1             There was difference in growth rate as
below:                                           well as cell health. Overall, there was
                 Table 1                         significantly more growth on the “wet”
                                                 beaker. Also, little abnormal growth was
Time (min) 5%            10%        20%          observed in the wet radiated cells. In the
10              254      215        56           dry cells, on the other hand, there were a
30              275      214        69           large number of abnormal cells in the dry
60              164*     196*       107          radiated cells. On top of the slow growth
120             324      253        78           rate, the dry radiated cells had a slower
*did not stir before counting.                   metabolic rate.       This was observed
                                                 because the dry radiated cells nearly had
                                                 no smell or foam on the top of the culture
                                                 when observed the next day. The wet
Trial II: Yeast to Solution Ratio                radiated culture had the yeast smell and a
In looking at the hemocytometer, the             0.5 cm layer of foam on the top. This
1:100 ratio was immediately ruled out.           shows that there was less sugar
There were far too many cells to count,          metabolism occurring in the dry radiated
                                                 than the wet radiated yeast cells.
numbering over five-hundred in the small
                                                 Unfortunately, I was required to leave the
0.2 mm*0.2 mm square. I gave up
counting the cells because it became too         laboratory in the middle of this trial.
time-consuming. The 1:500 and 1:1000             Nevertheless, the results were still for the
                                                 most part conclusive. The table showing
ratios were much more manageable. The
                                                 the average growth of the dry radiated
1:1000 plate would work for an undiluted
count, though there would still be too           versus wet radiated cells is shown:
many to count in the larger quadrants as
in figure 3. Both the 1:1000 and 1:500



                                            Page 5
                 Table 3                            This was most likely the cause for the
                                                    sudden jump in cell count in the control.
Time (hrs)    Dry              Wet                  Yet, the larger jump is seen between 90
              Radiated         Radiated             and 120 minutes from 88.75 to 119.0 (a
Initial       44.75            88.0                 30.25 cell jump) in the average quadrant.
0.5           51.0             87.75                By the end of the experiment, the 15-
10            53.25            98.75                minute cell count had passed the control
                                                    count. The 30 and 45 minute cultures had
On the second part of the test, the glow on         nearly entirely stagnant growth over the
the dry mineral had a significantly greater         11 hours of incubation between 240 and
glow than the one placed in 20 mL of                900 minutes. Graph 1 (in appendix)
water. It was more brightly illuminated             shows the upward curve of the 15 minute
and had a deeper purple color. The wet              culture, and the flat trend of the 30 and
mineral showed almost no illumination at            45-minute cultures. The averaged counts
all. This shows that the dry mineral was            are shown in Table 3. The counts from
much more absorbent of the ultraviolet              the four quadrants of the hemocytometer
rays than the wet one. This is because the          per time are represented. In the initial
water refracts or blocks the ultraviolet            count it is interesting to note that as the
rays from hitting the mineral. For this             amount of radiation increased, the
reason, the yeast was radiated while dry            average initial count decreased. There
rather than wet.                                    was an inverse relationship between
                                                    amount of radiation and initial cell count.
Experimental Results                                Table 4 displays the extrapolated data
The experimental results from the cell              using Equation 2 to extend the data to
count show an overall increase in control           number of cells per milliliter.
cells versus the irradiated cells. In fact,         Following the tables, graphs are shown of
the irradiated cell counts never cross with         the amount of cells per 0.1 µL versus time.
the control until the last count. All of the        This sample size is shown because it is an
cell cultures had a trend where they had a          exactly proportional, yet manageable
spike of growth around the 150 minute               sample.      Graph 1 shows the graph
mark. Subsequently, a sharp drop in cell            including the 900-minute point to show
growth was observed at the next interval            the larger trend. In Graph 2, the 900-
of 180 minutes. All of the samples                  minute points were removed from the
followed a similar trend line as shown in           graph so we could see the growth at a
Graph 2 (in appendix), yet the control              smaller scope. The first four hours of
had much more dramatic changes than                 growth are shown.          The separation
the irradiated cells in general. Table 3            between the control and the irradiated
shows the average 1 mm*1 mm quadrant                cells is more apparent in the two graphs.
count. It was observed that the 15, 30              It is interesting to note that while the 30
and 45-minute beakers more stagnant in              and 45-minute cultures stayed stagnant
growth, while the control had large jumps           in their growth overnight, the 15–minute
in growth. It should be noted that the              cell culture had a higher cell count than
averaged number 102.5 at 60 minutes in              the control by the conclusion of the
the Control beaker had a visible clump of           counting period by a small average
cells that was pipetted into the microtube.         amount of 1.5 cells.



                                               Page 6
 While observing the cells under the                exclusively round shaped cells. There
 microscope, a large number of cell shape           were also signs of cell wall breakage
 abnormalities were seen in the irradiated          apparent in a few of the irradiated cells.
 cells. The most common abnormal cell               The concentration of cells in the control
 shape was an elongated rod morphology.             and 15-minute culture also had a much
 These are most apparently seen in the 30           higher concentration of cells than the 30
 and 45-minute pictures shown in Figure             and 45-minute cultures. The difference in
 4 (in appendix).       There were also             cell density can be seen in the between
 occurrences of sickle shaped cells, as well        the control and 15-minute pictures, and
 as other non-circular shapes. The control,         the 30 and 45-minute pictures shown in
 on the other hand, contained nearly                Figure 4.


                                 Table 3 – Actual Cell Count

Time (min)        Control Count       15 min. Count        30 min. Count      45 min. Count
Initial           68.25               44.25                33.0               18.75
15                70.25               48.75                59.75              37.5
30                78.5                59.5                 66.75              54.25
60                102.5               58.0                 76.75              67.0
90                88.75               79.5                 75.75              53.0
120               119.0               83.25                70.5               58.25
150               122.75              83.75                71.0               79.25
180               76.75               62.0                 65.75              52.0
240               71.0                51.5                 62.25              56.0
900 (15 hrs.)     96.25               97.75                66.0               55.0


                                   Table 4 – Cells per µL

Time (min)         Control             15 minutes           30 minutes          45 minutes
Initial            6.83x108            4.43x108             3.30x108            1.88x108
15                 7.03x108            4.88x108             5.98x108            3.75x108
30                 7.85x108            5.95x108             6.68x108            5.43x108
60                 1.03x109            5.80x108             7.68x108            6.70x108
90                 8.88x108            7.95x108             7.58x108            5.30x108
120                1.19x109            8.33x108             7.05x108            5.83x108
150                1.23x109            8.38x108             7.10x108            7.93x108
180                7.68x108            6.20x108             6.58x108            5.20x108
240                7.10x108            5.15x108             6.23x108            5.60x108
900 (15 hrs.)      9.63x108            9.78x108             6.60x108            5.50x108



                                               Page 7
               DISCUSSION

My results confirm that cells exposed to a          Ultraviolet ray DNA damage is a major,
larger amount of ultraviolet radiation will         and arguably the main cause of malignant
have a slower reproductive rate as                  melanoma, the most severe form of skin
preciously stated in the hypothesis. Cells          cancer (Young, 1998). This study is
exposed to ultraviolet rays for a longer            important for clinical applications since
period of time experienced a fairly                 the yeast genome and mechanisms are so
significant loss of growth. Furthermore,            similar to human cells.           Ultraviolet
the results show a strong correlation               radiation is also a major contributor to
between exposure time and abnormal cell             ageing. Long-term exposure to ultraviolet
growth. The cells subjected to a greater            irradiation causes premature photoageing,
amount of radiation had a higher                    pigment alteration, wrinkling and loss of
incidence of abnormal growth and                    skin tone. The photoageing is caused by
elongated morphology, and a small                   collagen damage due to exposure (Fisher
amount of cell membrane breakdown. My               et al., 1997). The clinical significance of
finding that the growth rate and cell               the effects of UV radiation is extremely
morphology is affected by ultraviolet               applicable and relevant to many of the
radiation exposure is consistent with a             cancer and ageing studies happening
study conducted by Stewart et al. In this           today. My results barely scratch the
study, yeast cells exposed to ultraviolet           surface compared to what is still to be
rays     experienced      elongated     cell        known about the effects of radiation on
morphology, and experienced a much                  cells. Much of the inconsistencies in my
slower growth rate because of reversible            results had to do with uneven stirring
s phase arrest.        The s phase is a             between cultures, which may have given
reparatory phase for DNA. If the DNA                falsely high or low counts. Another
cannot be immediately repaired, the DNA             source of error could have been unevenly
cannot be replicated (1997). S phase                distributed cells. Small errors become
arrest keeps the cell in the s phase of the         large when the data is extrapolated one-
cell cycle. This prevents any reproduction          thousand-fold. Yet, the data gave a good
in the cell. This is a possible explanation         trend.     For further study and more
for the lack of cell growth in the                  conclusive results, it would have been
irradiated cultures. The elongated cells            helpful to use Trypan blue dye, using the
are signs of aberrant cell division. When s         method described by Strober (2001).
phase arrest occurs, cell division ceases,          This dye turns the cytoplasm of non-
while growth continues (Stewart et al.,             viable cells blue, while keeping the
1991). For this reason, the cell elongates          cytoplasm of viable cells clear. This
without dividing. This is a cause of                would have been helpful in analyzing a
retarded cell growth, and also serves as a          count of live cells instead of all remaining
reason for the occurring elongation. A              cells. This would have provided a more
possible cause for this s phase arrest is           conclusive result about the UV’s affect on
pyrimidine dimers caused by molecular               viability of the cell. Another study that
lesions cut by ultraviolet rays in most             would be engaging to look into is the
often thymine nucleic acid bases. The               effect of UV exposure on telomere length,
pyrimidine residues then covalently bond            which has a direct effect on ageing using
causing cell division arrest or cell                electrophoresis. There is still much to be
mutation (Resnic and Setlow, 1971).                 learned in this fascinating area.



                                               Page 8
                                                       APPENDIX


                                         Graph 1: Cell Count vs. Time
               140


               120


               100
Cells per µL




                80
                                                                                                      Control
                                                                                                      15 min
                60
                                                                                                      30 min

                40                                                                                    45 min


                20


                 0
                     0   100    200   300      400      500     600    700   800         900   1000
                                                     Time (min)




                               Graph 2: Cell Count vs. Time (no final data point)
               140


               120


               100
Cells per µL




                80
                                                                                                      Control
                                                                                                      15 min
                60
                                                                                                      30 min
                                                                                                      45 min
                40


                20


                 0
                     0         50        100            150           200          250          300
                                                     Time (min)




                                                           Page 9
                                        Control                                         1515 minutes




                                    30 minutes                                           45 minutes




Figure 4: Pictures taken through the microscope revealing the cell shape and concentration of each of
the four yeast cultures grown. The control shows a higher density of cells, with a higher percentage of
cells with normal circular shape. The cells become decreasingly concentrated, and show a higher
incidence of abnormally shaped cells as the amount of UV radiation increased.




                                                  Page
                                                  10
                                  LITERATURE CITED

Fisher, G., Wang, Z., Datta, S., Varani, J., Kang, S., & Voorhees, J. (1997).
      Pathophysiology of premature skin aging induced by ultraviolet light. New England
      Journal of Medicine, 337(20), 1419-1429

Ito, H., Fukuda, Y., & Kimura, A. (1983). Transformation of intact yeast cells treated with
        alkali cations. JOURNAL OF BACTERIOLOGY, 153(1), 163-168.

Resnick, M., & Setlow, J. (1972). Repair of pyrimidine dimer damage induced in yeast by
      ultraviolet light.JOURNAL OF BACTERIOLOGY, 109(3), 979-986.

Stewart, E., Chapman, C., Al-Khodairy, F., Antony, M., & Enoch, T. (1997). rqh1 , a
      fission yeast gene related to the bloom's and werner's syndrome genes, is required
      for reversible s phase arrest. European Molecular Biology Organization Journal.

Strober, W. (2001). Trypan blue exclusion test of cell viability. Current Protocols in
      Immunology, 10, 1002-1010.

Young, A. (1998). Does uva cause malignant melanoma?. In A. Rougier & H. Schaefer
     (Eds.), Protection of the Skin Against Ultraviolet Radiations (pp. 25-28). Paris, France:
     John Libbey Eurotext.




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Description: Intact yeast cells were treated with 15, 30, and 45 minutes of short wave ultraviolet radiation. The growth rates of these cultures were studied in conjunction with a non-radiated control sample. The cells were grown in a 10% sugar medium, and observed for a regular period for four hours. They were counted at regular intervals over this period. A final count was taken the following day (after 15 hours) and observations were made. The 30 and 45-minute cultures showed a considerably slower growth rate, and were nearly completely stagnant from 180 to 900 minutes. The 15-minute culture showed an initial drop in growth, but showed the strongest amount of growth from 240 to 900 minutes. By cell observation and analysis, a significant amount of cell damage was seen in the irradiated cells. Abnormally shaped cells, and signs of lysis were apparent in observation of the irradiated cells.