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RAYDAY '98

Introduction

April 21st is RAYDAY! Across the United States and Australia students and their teachers will take part in a collaborative experiment to test the amount of ultraviolet radiation UV reaching the earth's surface. Our protocol is adapted from one developed by the Gene Project at Kansas State University. It uses ultraviolet-sensitive yeast cells to assay UV-B doses at each location.

Background Information

Ultraviolet radiation is sunlight with wavelengths of from 1000 to 3800 Angstroms. Shorter than visible light, Francis these waves deliver an energy punch when they strike living cells. They damage DNA, the genetic material, and important protein molecules. Most cells have repair enzymes which routinely correct mutations in the DNA caused by UV. However, repeated damage increases the chance that a mutation will be missed by the repair mechanisms. In humans, this can cause skin cancer, wrinkles, and immune system damage. UV light is divided into longer wavelengths (UV-A) and shorter ones (UV-B). UV-A is implicated in wrinkling because it damages collagen and elastin fibers in the dermis, while UV-B is implicated in basal cell carcinoma because it damages DNA in basal cells of the epidermis. Our yeast cells lack DNA repair enzymes, and so are killed when zapped by UV light.

Much of the UV radiation in sunlight (up to 99%) is absorbed by ozone in the Earth's atmosphere before it reaches the surface. Ozone, or O3, is a kind of molecular oxygen. Formed from oxygen released into the earth's atmosphere by the first photosynthesizers, ozone accumulates in a layer in the earth's stratosphere and protects life on earth from UV damage. Ozone can be destroyed by man-made chemicals such as CFC's, leading to the thinning in the ozone layer in recent years. The amount of UV reaching the earth's surface depends on the amount of ozone the sunlight passes through, which, in turn, is affected by latitude, longitude, altitude, time of day, time of year, global pollution and weather conditions.

Our experiment today asks: What is the UV radiation at our locality today? and how does it compare to other localities in the U.S. and Australia?

RAY DAY 98 PROTOCOL

In this activity you will investigate the effects of ultraviolet light from the sun on ultraviolet sensitive yeast cells. You will compare your results with students in different geographical regions in the United States and from Australia. The yeast cells are a strain (G948-1C) of Saccharomyces cerevisiae.

Saccharomyces cerevisiae is commonly used as baker's yeast and for some types of fermentation. The strain of yeast that you will use in this experiment will die when exposed to ultraviolet light. In this experiment you will:

  1. Prepare a suspension of a known number of UV sensitive yeast cells

  2. Prepare a set of serial dilutions of the yeast cells.

  3. Spread the appropriate dilution on agar growth plates (experimental and control plates).

  4. Expose the experimental plates to sunlight.

  5. Count the surviving yeast colonies on experimental and control plates, and calculate the fraction surviving on the experimental plates.

MATERIALS:

  1. G948-1C Saccharomyces cerevisiae

  2. Yeast-extract Adenine Dextrose Medium (Yed) agar
    Order Yeast and Media from:
    Katie Noonan
    ktnoon72@ix.netcom.com
    609 Kains Ave.
    Albany, CA 94706

    $6 for yeast, media for 500 ml (about 20-25 plates), shipping and handling
    Make check payable to Katie Noonan.
    *Be sure to include the address you want the materials sent to.

    You will also need:

  3. petri plates (use plastic petri dishes, Fisher if available)

  4. Capped 13 x 100 mL culture tubes

  5. Sterile water

  6. 1-mL pipettes

  7. Glass spreader, paperclip spreader or Q-tips

  8. Autoclave or pressure cooker

PREPARATION

  1. Prepare plates for use before RAY DAY.

    1. You can obtain the medium and the yeast cells from KSU, Dept of Physics (address above) and follow directions, or make the medium by blending the following dry ingredients in a flask:

      Yeast-extract Dextrose Medium (YED)
      • 1 gram of Difco Bacto-Yeast Extract

      • 2 grams of anhydrous dextrose (glucose)

      • 2 grams of Sigma Agar (agar-agar;gum agar)

      • 8 mL of Adenine stock solution

      • 92 mL of water


      Adenine Stock Solution
      • 400 mg adenine in 400 mL water (1mg/mL)

      • Store at room temperature

    2. Cover the flask and sterilize for 10 minutes at 15 psi in an autoclave or pressure cooker

    3. Allow it to cool until you can touch it and pour each plate about half full

    4. When the agar has solidified, invert the plates and store them at room temperature in a closed plastic bag

  2. Prepare a suspension of a known number of UV-sensitive yeast cells on RAYDAY.

    1. Sterilize tubes of distilled water for use in serial dilution. Use 13 X 100mm tube with caps.

      • 2 mL of dH2O in one tube

      • 1 mL of dH2O in 6-8 tubes

      • 0.9 mL of dH2O in 10 tubes

      • sterilize pipettes

    2. Using a sterile toothpick, or a bacteria transfer loop, place a small amount of yeast cells (about the size of a head on a pin) from your source into the tube containing 2 mL of distilled water. Mix thoroughly.

    3. Perform a serial dilution using the tubes of 1 mL distilled water until you use all your tubes. (1 mL from tube1 to tube 2; 1 mL from tube 2 to tube 3, etc.) Remember to mix all tubes thoroughly between dilutions to suspend the cells evenly.
    4. Select the last suspension which is just barely turbid to the naked eye. This last suspension will contain between 1 and 2 million cells/mL. We will assume 106 cells/mL.

  3. Start a second series of dilutions to reach 10^2 cells/mL. You will need 1 mL of 10^2 cell/mL for each plate. Be sure that the cells for each experimental/control pair is from the same dilution batch :

    1. Remove 0.1 mL of the last cell suspension which was just barely turbid and place it in the tube containing 0.9 mL of distilled water. Mix thoroughly. This tube will now contain 105 cells/mL. Remove 0.1 mL from this tube and place it the next tube containing 0.9 mL of distilled water. Mix. This tube will now contain 104 cells/mL. Continue this procedure until you have 4 tubes. The fourth tube should contain 10^3 cells/mL. Mark each tube with the number of cells/mL.

    2. Add 9 mL of water to the 1 mL in the 103 cells/mL tube and mix thoroughly. You will have 10 mL of cells at 10^2 cells/ml. Aliquot 1 mL of this suspension into 6 epitubes.

      DO NOT SPREAD THE CELLS UNTIL YOU ARE JUST READY TO DO THE EXPERIMENT

    3. Pour the entire contents of each epitube onto a separate Petri dish of YED medium.

      Spread the cell suspension across the plate to distribute the cells as evenly as possible by rocking and swirling the plate, or by spreading with a sterile q-tip (use a new q-tip for each plate). (An alternate and probably more accurate method is to pipette 0.1 mL from each of the tubes and use a bacteria spreader to spread this reduced amount of material.)

    4. Allow 15 minutes to a half-hour for the yeast suspension to be absorbed by the agar. Keep plates in the dark during transportation.

PROCEDURE

  1. Expose 3 of the plates to sunlight for exactly 3 1/2 minutes. LEAVE THE LIDS ON THE PETRI DISHES! The uv light will pass through the plastic. The exposure time is set at 3 1/2 minutes in all locations except Alaska. Alaska will perform 2 experiments, one at 3 1/2 minutes and the other at 7 minutes exposure to the sun.

  2. Incubate the exposed and the control plates until the colonies are large enough to count. This should be 2 days at 30 degrees C and 3-4 days at room temperature.

  3. Count the colonies on experimental and control plates.

  4. Calculate the surviving fraction by the following method:

Surviving fraction = irradiated colonies
--------------------------------------
unirradiated colonies (control plate)

CAUTIONS

When making the dilution to 10^2 cells/mL, follow the protocol. Commonly, people doing this for the first time choose two concentrated a suspension as "barely turbid". If your next dilution of the "barely turbid" suspension shows any sign of cells, use that dilution instead as 10^6 cells/mL and start over with the series.

Pour your plates a day or too ahead of the experiment, so that the agar will have time to dry out. **Be sure not to turn the plates over after spreading the cells. Don't even move the plates until the yeast suspension has soaked into the agar (15 minutes to a half hour). DO NOT EXPOSE THE PLATES UNTIL THE YEAST HAS COMPLETELY SOAKED INTO THE AGAR. (If this step is not followed when the plates are inclined for exposure, water will run down the surface of the agar to the bottom of the plate carrying the yeast cells with it. This will concentrate most of the yeast in a pile on the bottom of the plate where the exposure will be ineffective and result in an uncountable plate.)

IT IS EXTREMELY IMPORTANT TO USE THE SAME SUSPENSION FOR YOUR EXPERIMENTAL AND CONTROL PLATES. If you are making up dilutions more than once, be sure you use cells from the same batch for each experimental/control set!!

IT IS EXTREMELY IMPORTANT TO KEEP AN EXACT RECORD OF THE TIME OF EXPOSURE (HOUR AND MINUTES), THE LENGTH OF EXPOSURE (MINUTES AND SECONDS). REMEMBER TO KEEP YOUR PLATES IN THE DARK WHILE TRANSPORTING. LIDS ON DURING EXPOSURE!

THE IDEAL TIME OF EXPOSURE IS 12 O'CLOCK NOON YOUR STANDARD TIME (NOT DAYLIGHT SAVINGS TIME!!!). ONCE DAYLIGHT SAVINGS IS IN EFFECT THEN 1 PM WILL BE THE OPTIMAL!! Be sure to indicate whether your exposure time is expressed in standard or daylight savings time. THE EXPOSURE TIME FOR ALL LOCALITIES, EXCEPT ALASKA, SHOULD BE 3 AND 1/2 MINUTES. IF YOU DEPART FROM THESE CONDITIONS, BE SURE TO NOTE IT ON YOUR DATA RETURN FORM (BELOW).

Corrections can be made by Brad for deviations, and your data will be included in the set. If you do your experiment on a different date than April 21st, he can adjust for that too.


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