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Weeks 3-6 Yeast and UV light: read each week before lab BIOL 1107K

Yeast and UV: PART I

UV radiation is known to damage the DNA of skin cells and may ultimately lead to skin cancers. Most of this
radiation comes from sunlight, but may also come from artificial sources such as tanning beds. The amount of
UV exposure depends on the strength of the light, the time of exposure, and whether the skin is protected.
Living organisms have long been used to monitor our environment; for example, canaries were used in mines to
detect the presence of methane gas. In this lab, we will use Saccharomyces cerevisiae or budding yeast to
monitor the damage from UV exposure, since yeast cells are similar to human cells. We will subject yeast cells
to different amounts of UV light and measure the percent survival of these cells as an estimate of the amount of
UV damage.

In this lab, we will use the survival of yeast cells to test the protective nature of sunglasses, sunscreen, etc.
against UV radiation.

LEARNING GOALS
After completing the lab series, you should be able to:
1. Demonstrate an understanding of the scientific method
2. Use sterile technique to plate a culture.
3. Prepare a dilution series starting with an undiluted culture.
4. Graph and interpret a survival curve.
5. Explain the use of DNA repair enzymes.
6. Design an experiment using the survival of yeast cells to test the protective nature of sunglasses,
sunscreen, and other factors against UV radiation.

OBJECTIVE: To study the effect of Ultraviolet (UV) radiation on the survival of yeast

INTRODUCTION
1. Are yeast cells eukaryotic or prokaryotic? How do you know?

2. Why are model systems used in science?

3. Read through the instructions for Part I, below. What is your hypothesis and prediction?

Hypothesis:

Prediction:

Independent variable:

Dependent variable:

Controlled variables (at least 2):


Weeks 3-6 Yeast and UV light: read each week before lab BIOL 1107K

4. What is the control in this experiment?

Summary: Appropriate dilutions of a yeast culture will be plated on YPD plates to determine the number of
viable cells. Plates will be exposed to UV radiation for different amounts of time after plating. The following
week you will count the number of colonies that survived on the YPD plates, and thus determine the extent of
UV-induced killing.

EXPERIMENTAL PROCEDURE

A. Dilution series, plating yeast and UV exposure

1. You are given 2 microtubes containing yeast cultures


a. Wildtype 100
b. Unknown 100
Note: 100 refers to undiluted culture. This culture was grown at 30C for 24 hours.

100 ul 100ul 100ul 100ul 100ul 100ul


ulml

900ul
100 10-1 10-2 10-3 10-4 10-5 10-6 water

10ul 10ul 100ul

990ul 900ul
water water

2. To make 1ml (1000ul) of a 10-5 dilution of the wildtype strain. Follow the bottom part of the figure.

a. Label 2 sterile microtubes “W10-2” and “W10-4” and pipette 990l sterile water into each.
b. Vortex the tube containing the 100 wildtype culture and pipette 10l of the culture into the
10-2 test tube. This is the 10-2 dilution.
c. Vortex these cells well and then using a clean tip for your micropipette, pipette 10l of the10-2 dilution
into the tube labeled 10-4. This is the 10-4 dilution.
d. Label a microtube W10-5, and pipette 900l of sterile water into it. Vortex your 10-4 dilution well and
then pipette 100l of 10-4 dilution into the 10-5 tube. This is the 10-5 dilution.

IMPORTANT: Mixing cells immediately before pipetting is important, as yeast cells will rapidly settle to
the bottom of the tube.
Weeks 3-6 Yeast and UV light: read each week before lab BIOL 1107K

3. Using sterile technique, make 1ml (1000l) of a 10-3 dilution of the Unknown strain. Follow the top figure
above and dilute using sterile water. Label your tubes!

4. Gather 12 YPD plates to plate 6 of the 10-5 wildtype and 6 of the 10-3 unknown dilutions. Each plate will be
exposed for either 0, 5, 10, 15, 20 or 30 seconds duration of UV light. Label the bottom (agarose half) of each
plate either wildtype or unknown, the dilution of culture, and time exposed to UV (see below) and your group
symbol. For example: “wt, 10-5, UV 30 sec ”

5. Using sterile technique, plate 100l of the wildtype 10-5 dilution on each of 6 appropriately labeled YPD
plates. Then, plate 100l of the unknown 10-3 dilution on each of 6 YPD plates. Before plating the yeast
dilution on the YPD plate, make sure you mix the culture well by vortexing or inversion.

6. Expose the plates to the designated time of UV radiation. Note that the lids of plates must be removed
before placing them on the UV box. The plates marked UV= 0 do not receive UV treatment.
Immediately after exposure to UV, place the plates in a brown paper bag to prevent exposure to light. (Yeast
has a photolyase that uses the energy of visible light to “reverse” the UV-induced DNA damage; bagging
the plates prevents the photoreversal reaction. Make sure you bag the unexposed plate along with the UV
exposed plates for each dilution.)

7. Label the outside of the brown bag with your section number, lab day, date and group name. Plates will be
incubated at 30C for 2 days. Next week you will count the number of colonies that survived on each plate.
Weeks 3-6 Yeast and UV light: read each week before lab BIOL 1107K

Yeast and UV: PART II

B. Data Collection

1. Count the number of colonies on each plate and enter observations in the tables below.

2. To calculate the number of viable cells per ml that would be present if the original (100) culture had
been exposed to different amounts of UV

Cells/ ml = No. of colonies


(fraction of 1ml plated) X (dilution factor)

For example, if 100l of a 10-5 dilution gives 150 colonies (recall that each colony is derived from a single
cell), then the number of viable cells in the original culture would be

150 colonies = 1.50 X 108 cells/ ml

(10-1) X (10-5)

Table 1: Wildtype 10-5 dilution plated on YPD

Time exposed to UV (seconds) No. of colonies Viable cells /ml % survival


0 100
5
10
15
20
30

Table 2: Unknown 10-3 dilution plated on YPD

Time exposed to UV (seconds) No. of colonies Viable cells /ml % survival


0 100
5
10
15
20
30
Weeks 3-6 Yeast and UV light: read each week before lab BIOL 1107K

C. Graph and interpret a survival curve

In your notebooks, plot a single “survival curve” for both the wildtype and unknown survival data. Place %
survival on the Y-axis and time exposed to UV in seconds on the X-axis. Submit the graph and the answers to
the questions below to your instructor.

1. What is the time of UV exposure that results in a 50% survival rate for each, the wildtype and unknown?

2. What time of UV exposure results in complete killing of each, the wildtype and unknown?

3. Which is more sensitive to UV light, the wildtype or unknown?

4. Did your results support or refute your hypothesis? Why?

HOMEWORK Assignment. This is a homework assignment for each group to research. Your answer should
be maximum one page; double spaced, and should contain at least one original scientific research paper as a
reference. Submit your report on a separate sheet.

1. A culture of yeast cells grown in minimal media should contain approximately 2 x 107 cells/ ml. If you
want to plate 100l of a dilution and want to have about 20 colonies per plate, what dilution should you
plate?

2. Research Question: Determine what cellular process could be affected in the unknown yeast strain to
make it more sensitive to UV.
Weeks 3-6 Yeast and UV light: read each week before lab BIOL 1107K

Yeast and UV: Experiment II


In these labs, you will work in groups to design an experiment to test the UV protective effect of different
agents, e.g. sunscreen, sunglasses, different fabrics, or anything you wish to bring to test. Make sure you use the
scientific method with appropriate controls. See below for supplies provided.

List of supplies available to each group to design their experiment


 200 ul of a 100 culture of 922 wildtype in a microtube
 6 YPD plates
 Tube of sterile water 15ml
 Vortexer
 Sterilized microtubes
 Micropipettes p10, p100, p1000 and tips
 Spreader to plate yeast
 Ethanol to sterilize spreader
 Bunsen burner
 Sharpie
 Brown paper bags
 Saran wrap
 Sunglasses
 Sunscreens of various SPF’s 15, 30, 60.
 Various kinds of fabric- chiffon, lycra, cotton, polyester etc.
 Common area
 UV-B transilluminator box shielded with a large plexiglass
 Timer that counts seconds

HW: Write your experimental design in your notebook and have your instructor initial it before you begin.

********* NOTE**********
You will present your experiment and results to the class next week as a group oral presentation that counts for
2.5% of your total BIOL 1107K course grade.

The presentation must follow the scientific method and the layout of the primary literature article you read
earlier:
1. INTRODUCTION: question, background information, aim and hypothesis.
2. MATERIALS & METHODS: experimental design, controls, variables
3. RESULTS: graphs and explanation of trends in the data; compare experimental groups to controls
4. DISCUSSION: do the results support or refute your hypothesis? What are the implications (or bigger
meaning of the results)? What additional experiments could be done to follow up on these results or
expand this study?

REFERENCES:
Allison R. D'Costa and Irma Santoro (2008). The effect of UV radiation on the survival of yeast and its implication
to a real-life situation. Published in 2009 Proceedings of Association for Biological Laboratory Education
(ABLE).