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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

  • 1 gram of Difco Bacto-Yeast Extract
    Order #M00393A
  • 2 grams of Anhydrous dextrose (glucose)
    Order #82050231IP
  • 2 grams of Agar-agar
    Order #M00391A
  • 100mL Sterile water

  • 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:

  • A 1 day old culture of Saccharomyces cerevisiae
  • Sterile 13 x 100 mm culture tubes with caps and rack
  • 0.1 mL (100 microliter) pipettor and sterile tips
  • Sterile water
  • Sterile 1mL pipettes
  • Shortwave UV light or irradiator
  • Alcohol in 100mL beaker
  • Bunsen burner
  • Glass spreader ("rake")
  • Marking pen
  • 2 YED agar plates/group
  • PROCEDURE:

    1. 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.

    2. Obtain a quantity of yeast cells in solution and dilute until 100-200 cells can be plated.

    3. 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.

    4. 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).

    5. 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.

    6. The cover of the Petri dish is removed during irradiation, so the UV light is not filtered.

    7. Incubate both plates upside-down for two days at 30deg.C or for three days at room temperature.

    8. 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.

    9. 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

    1. 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.

    2. 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.

    3. Conduct the experiment under subdued room light (no fluorescent lights, blinds closed). Put the negative control directly into the light-proof container after plating.

    4. As soon as the positive control plate is exposed to the UV, place it in the light-proof container.

    5. 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.

    6. 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:

  • 3 YED plates/group
  • Balance
  • Six inch embroidery hoops
  • Saran wrap
  • Sunscreen of a variety of SPF's
  • PROCEDURE

    1. Tear off a piece of Saran wrap large enough to cover the embroidery hoop and attach it to the hoop.

    2. Weigh out three grams of sunscreen and evenly spread it on the Saran wrap with the glass rake.

    3. Set up a serial dilution for the number of survivors (just like experiment 1). Try a dilution that will get about 100 cells/plate.

    4. Prepare three Petri dishes for each SPF to be tested.

    5. 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).

    6. 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.

    7. Incubate the plates at 37deg.C for two days and tabulate the results.
    8. 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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