USING YEAST AS AN ULTRAVIOLET
LIGHT MEASUREMENT TOOL
Kevin Conant
1993 Woodrow Wilson Biology Institute
INTRODUCTION
Living organisms are used in a variety of ways to monitor our environment. Historically, different species of organisms have been used for this purpose. Canaries were used in mines to detect methane gas. Stream quality is measured by looking at the number and type of organisms living there. Rabbits, rats, mice, and dogs are just a few of the animals that have been used in labs for years to further scientific knowledge.
Technological advances (i.e. immunoassay kits, spectrophotometers, Geiger counters, etc.) have replaced many of the older techniques with chemical tests or instrumentation. For example, ultraviolet (UV) light meters are available to measure the amount of UV light that contacts the earth's surface in that area. Because the cost of these tools is prohibitive for most science classrooms, living organisms may easily be substituted and used for these measurements. Students should realize the importance of organisms as standards of measurement and experimentation. The following labs use the yeast Saccharomyces cerviasae as the standard for comparison of the mutagenic effects of UV light. Yeast are ideal for these activities for a number of reasons. Yeast grow to observable colonies at 30 deg C overnight or at room temperature in several days (perfect for labs that fall on Fridays). Yeast cells are eukaryotic and are more similar to human cells than bacteria. Ultraviolet light is used in these activities because it is known to cause genetic damage. Because of the ozone layer controversy, damage caused skin by UV light is an important topic. Topics like sunscreens and tanning beds can also be discussed.
TEACHER PREPARATION
The three labs illustrated below may be done in succession or spread out in different areas of the biotechnology unit. The first lab is a simple survival curve that demonstrates the effects of ultraviolet light on cells. The second lab looks at repair mechanisms of the DNA molecule from visible light. The third lab uses yeast as a measurement tool to test the effectiveness of sunscreens. Additional labs can easily be done by using some of the mutated forms (red, white, and petite) that arise because of the exposure to ultraviolet light.
Depending on your class size you can have each set of students do these experiments over a range of times varying from 5 seconds to 35 seconds or you can have each set of students irradiate at different times and then the students can share the data.
TARGET AGE/ABILITY GROUP:
High School (9-12)
STUDENT/CLASS TIME REQUIRED:
Each lab requires three class periods along
with two days incubation at 30deg.C or three
days at room temperature.
TEACHER'S GUIDE FOR
PREPARATION OF MATERIALS
Streak plate some of the yeast cells at least one day before the serial dilution but not more than a week. The yeast need to be actively growing.
The serial dilution series should be prepared the day of the lab. See the activity "Serial Dilutions Made Easy." In a 50 minute class period, it is difficult to complete the serial dilutions, plate the cells and irradiate them. It is a good idea for the instructor to make a serial dilution for the whole class. Make sure that the students "thump" the tubes to distribute the yeast cells so that they don't settle to the bottom.
If irradiation is done with a UV lamp make sure that students are wearing safety glasses with side shields that block out UV light. In the reference section of this lab there is an address of a book from Tom Manney of Kansas State University that describes how to make a safe inexpensive UV box to irradiate cells.
SOURCES OF MATERIALS:
For culture 10 plates (one group of students in all three experiments)
YED agar (yeast extract dextrose medium) and Gibco BRL order number
INFORMATION REFERENCES:
Yeast strains in culture and YED agar can also be obtained in the powdered form with instructions from:
Dr. Tom Manney, Dept. of Physics, Kansas State University, Manhattan, KS, 66056. "A Handbook For Using Yeast To Teach Genetics" can also be obtained for $7.00. It includes background information, experiments, laboratory methods, instructions for making an inexpensive student-safe ultraviolet box.
Any of the adenine requiring strains of yeast will work the best. The strains of yeast are available for purchase from:
The Yeast Genetic Stock Center, Dept. of Molecular and Cellular Biology, Division of Genetics, University of California at Berkeley.
Strain: HA1, Genotype: ade 1,
Stock Center # XP300-3813
Strain: HA2,HAR, Genotype: ade 2
Stock Center #XP832-S25
Commercial UV lamps can be obtained through:
UltraViolet Products, Inc. of San Gabriel, CA.
Sargent-Welch, P.O. Box 1026, Skokie, IL 60076-1026.
UV safety goggles:
Sargent-Welch, S-44225-01($11.55).
STUDENT PAGE
EXPERIMENT 1
The Survival Curve:
A Measurement of Radiation Sensitivity
The objective of this procedure is to accurately measure the relationship between radiation dose and either survival or some genetic event such as mutation or recombination. The general strategy is to plate and irradiate cells, incubate the plates until the surviving cells have formed colonies then estimate the number of survivors.
MATERIALS:
PROCEDURE:
- Make a table of dosage, (amount of time that the
plate is irradiated) increment at 5 sec. from 5-
35 sec. and the number of cells plated.
- Obtain a quantity of yeast cells in solution and
dilute until 100-200 cells can be plated.
- Prepare dilution series, but do not plate the cells
on the medium in advance. Schedule plating so
that each plate gets irradiated within 5-10
minutes after the cells are spread. Two plates
are used at each time increment, experimental
plate and control plate. Both plates will receive
yeast cells, but only one will receive UV light.
- The cells are distributed over the surface of the
agar plate by a technique known as "Spreading a
Lawn" (your teacher will demonstrate this
technique).
- After spreading the lawn on the two plates the
experimental plate is set under the UV light for
the specified time. Be sure that the necessary
precautions are taken when working with UV
light.
- The cover of the Petri dish is removed during
irradiation, so the UV light is not filtered.
- Incubate both plates upside-down for two days
at 30deg.C or for three days at room temperature.
- Most of the colonies should be visible by the
third day. Count the colonies from the bottom of
the Petri dish by marking each colony with a
marker. Record the number of colonies and
incubate on more day.
- On the next day count the additional colonies
that have become visible. Mark them with a
different color marking pen.
ANALYSIS OF DATA
A. Graph the percent survivors.
Percent survivors = Mean irradiated colonies
/mean control colonies x 100
B. What level of UV light results in a 50% survival
rate?
EXPERIMENT 2
REPAIR OF ULTRAVIOLET DAMAGE TO DNA
One concept that is introduced in a biology classroom is that damage done to the DNA molecule results in mutations of some sort. These mutations may be positive, negative or neutral. Something that is sometimes overlooked is that the DNA molecule has a remarkable ability to repair itself. One way this is done is through a process called photoreactivation. Most of the UV damge to DNA is in the form of pyrimidine dimers. Adjacent pyrimidine bases (thymine and cytosine) in the DNA become dimerized by covalent bonds formed between their rings. A photoreactivating enzyme, using energy from visible light, reverses this reaction, restoring the original pyrimidine bases, (Manney, T., 1991). If the UV-damaged DNA tries to replicate with these dimers present, the result is usually lethal. Therefore, the photoreactivation is only effective if the exposure to visible light occurs immediately after the UV exposure, before the cells start to replicate, (Manney, T, 1991). Although some placental mammals have shown some ability to do photoreactivation, humans have not shown this ability to do this with visible light.
MATERIALS
The materials are the same as in experiment 1 with these changes:
* 3 YED plates/group
* 20 X 20 cm glass plate
* Box for transfer and incubation of the plates in the dark
PROCEDURE
- The general procedure for irradiating the cells is
essentially the same as for the survival curve
(Experiment1). Use a dose (exposure time) that
will give approximately 10-20 percent survival
and plate enough cells to produce
approximatley100 colonies without
photoreactivation.
- Three plates are needed at each interval: the
negative control with yeast only, the positive
control with yeast and exposure to UV light, and
the experimental plate with yeast, exposure to
UV light, and exposure to sunlight.
- Conduct the experiment under subdued room
light (no fluorescent lights, blinds closed). Put
the negative control directly into the light-proof
container after plating.
- As soon as the positive control plate is exposed
to the UV, place it in the light-proof container.
- Expose the experimental plate to sunlight for ten
minutes (replace the plastic Petri dish lid with
the glass plate). At the end of ten minutes place
the lid back on the Petri dish and place it upside
down with the other plates and incubate at 37deg.C
for two days in total darkness.
- Count the colonies and tabulate data.
ANALYSIS OF DATA
a. Calculate the survival levels for the
photoreactivated samples and the controls.
b. Calculate the amount of photoreactivation.
c. Compare the results with the UV survival curve
and try to estimate the fraction of the UV
damage that was reactivated.
d. Can you tell if there was any significant
photoreactivation during the survival curve
experimental?
e. Why was the glass plate used instead the Petri
dish lid when the plate was outside?
EXPERIMENT 3
TESTING THE EFFECTIVENESS OF SUNSCREENS
The purpose of this lab is to find how well different sun protection factors (SPF's) help in preventing the effects of UV light on yeast. Sunscreen is designed to filter out the UV radiation that causes sunburn. This lab not only reflects the effects of sunscreen on yeast but also on humans. Students usually want to bring in their favorite brands of sunscreen.
MATERIALS
The materials are the same as in experiment 1 with these changes:
PROCEDURE
- Tear off a piece of Saran wrap large enough to
cover the embroidery hoop and attach it to the
hoop.
- Weigh out three grams of sunscreen and evenly
spread it on the Saran wrap with the glass rake.
- Set up a serial dilution for the number of
survivors (just like experiment 1). Try a
dilution that will get about 100 cells/plate.
- Prepare three Petri dishes for each SPF to be
tested.
- Use a dose of UV light that will result in a
survival rate of 50% (use your data from
experiments 1 and 2 to figure your dose).
- Irradiate the Petri dishes as follows:
Plate #1: Set embroidery hoop with Saran wrap
and sunscreen over the Petri dish (cover
of the Petri dish removed), and irradiate
them with UV light for the
predetermined time.
Plate #2: Set the embroidery hoop with Saran
wrap but no sunscreen and irradiate
them with UV light for the
predetermined time.
Plate #3: Do not expose to UV light.
- Incubate the plates at 37deg.C for two days and
tabulate the results.
- Make a table of your results. Class results are
also useful to increase the accuracy by
averaging results.
ANALYSIS OF DATA
A. Calculate the survival levels for each SPF.
B. Calculate the amount of protection each level
receives.
C. Determine if there was any significant difference
in sunscreen (SPF or brand).
D. Determine which plate was the negative control.
E. Determine which plate was the positive control.
E. Determine which plate was the experimental
group.
FURTHER STUDIES
A. Look for mutations in the yeast colonies that
have survived irradiation (ex. color differences
or size differences).
B. Compare the effects on yeast of commercial
tanning-beds lights and the UV lights in class.
C. Find a standard for determining the number of
yeast/mL in a culture tube using the Spec 20.
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