After you have gather background information and made preliminary observations it is time to formulate a hypothesis. A hypothesis is merely a tentative explanation proposed to account for the observed phenomena. You are speculating on how natural events will turn out, based on what you know. "Science is systematic in method because it seeks a system of prediction." That prediction is the hypothesis. Any hypothesis selected or formulated must be testable. Experiments generally test hypotheses by testing the validity of the predictions or conclusions derived from them. The primary purpose of designing scientific experiments is to test the proposed hypotheses.

It is important to know that hypotheses are never proven - they are either supported or not supported by the data from the experimental results. Borrowing from statistics, two types of hypotheses are used simultaneously: null (H0) and alternative (H1. H0 states that events will not change, not differ and H1 states that events will change, differ, from some baseline standard or control conditions. This change (dependent variable) predicted by H1 will be due to the occurrence of an experimentally controlled variable (independent variable).

Hypothesis for this Guided Research

H0: There is no measurable relationship between the length (time) of exposure to ultraviolet light of the day and the number of surviving yeast colonies.
H1: There is a measurable relationship between the length (time) of exposure to ultraviolet light of the day and the number of surviving yeast colonies.

The following network protocol is based on the Kansas State University Gene Project's Using Yeast to Measure the Intensity of Solar UV Radiation.

Protocol for Using Yeast to Measure the Intensity of Solar Ultraviolet Radiation

This is a bioassay for solar UV-B and gives a better measure of DNA-damaging UV than most physical meters because it is based directly on UV damage to yeast DNA. Most meters don't measure just those energies of UV.

In this experiment the number of yeast cells that are killed by being exposed to sunlight depends on the intensity of UV-B in the sunlight. Therefore, you can estimate the amount of UV-B by measuring how many cells are killed. You measure the killing indirectly by determining the fraction of cells in a sample that survive a particular exposure time. You will make a yeast suspension with a known number of cells per ml, and then dilute that suspension by making a set of serial dilution tubes so that the number of cells per ml in each tube is 1/10th of the number in the previous tube. You will use the diluted yeast suspension in the tubes to plate around 100 cells on each plate. The series of plates will yield between 10 and 100 survivors for a range of exposure times around an exposure that will leave a 0.1 surviving fraction.

Materials:

5 to 10 sterile culture tubes - 13 x100 mm with caps

15 polystyrene Petri dishes with YED agar

1 Alcohol wipe

1 to 10 sterile pipets

either 1-mL calibrated bulbed transfer pipets

1mL disposable serological pipets calibrated in 0.1 mL steps /w pipet pump

Yeast strain G948-1C S

Sterile water

Sterile toothpicks

Marking pens

Optional Materials: Sterile paper clip spreaders or glass spreader, alcohol in 100 mL beaker and a source of flame Micropipettor and sterile tips

Getting Ready: 48 hours before

1. Make a clean sterile work space by wiping the table or bench with an alcohol wipe. Because most contamination is airborne select a place free from drafts.

2. Open the yeast storage vial.

3. Using the broad end of a sterile toothpick, pick up a small amount of yeast from the agar slant in the vial.

4. Replace the lid. Tighten.

5. Open the YED Petri dish just enough so that you can reach into it with the toothpick full of cells.

6. Gently make several streaks of the culture on the surface of the agar. (Remember that you need not be able to see the streaks to have enough to grow into a visible culture overnight.)

7. Close the lid and incubate the culture at 30 C, or 3 days at room temperature. Most microbial cultures should be incubated with the agar side up to prevent condensation from dropping on the colonies.

Technical Tip: This preparation is important. Fresh yeast cells must be used for your exposures. You will count how many cells survive UV-exposure because when a suspension of living yeast cells is spread on agar medium, each cell will grow into a visible colony after two to three days. Cells that form colonies are called viable cells. Some of the cells exposed to UV will be killed and so will not be viable.

Dilution, Plating and Irradiation of Cells

8. Use the sample survival curve (Figure 1) to design your experiment: Choose an exposure time that looks reasonable and will produce a surviving fraction close to 0.1 Use the following formula, based on the curve in Figure 1, to find an estimated exposure time. These times are for the middle of the summer. You will need to use longer exposure times during the other seasons of the year.

9. Set up your plates in 5 rows of three plates. Each row of three plates will represent a time series for the exposure of your plates.

For example, if your exposure time to produce a surviving fraction close to 0.1 is 4 minutes, 20 seconds, than you might set up the following series;

The first row of three plates will be your control and has no exposure.

The second row of three plates will be exposed for two minutes.

The third row of three plates will be exposed for four minutes.

The forth row of three plates will be exposed for six minutes.

The fifth row of three plates will be exposed for eight minutes.

Each of your plates will have close to 100 cells on it. This time series will give you a range of exposure, hence a range of numbers of surviving cells. Be sure to label the bottom of each plate with the exposure time.

Making a "just turbid" suspension to begin the dilution series:

Prepare your dilutions and remember it is easier to count a plate with too few colonies than one with too many! Prepare a dilution series of strain G948-1C.

1. Use a sterile pipet to place 2 mL of sterile water into one tube and 1 ml into each of six more tubes.

2. Make a turbid suspension in the first tube. Take a small amount of yeast--less than the size of a pin head--on a toothpick and select the tube that contains 2 mL of water. Without touching the mouth of the tube, place the yeast as far down the inside wall of the tube as you can.

3. Mix the suspension thoroughly by thumping it. Holding the tube loosely near its top between thumb and forefinger, and thump it near the bottom with the palm side of one or more fingers of the other hand, imparting a swirling motion. The thumping motion approximates the gesture one would make to beckon someone to come hither.

4. Add yeast until the suspension is noticeably turbid or cloudy.

5. Make a series of two-fold dilutions until you have a tube that is clear. Transfer 1 mL of the suspension from each tube to the next, beginning with the one containing the cells. Mix each tube between steps by thumping it.

6. Compare each tube with a tube that contains 1mL of clear water. The last turbid tube will contain between 1 and 2 million cells/mL (10x6). Use this tube for the rest of the experiment.

Serial dilution

For making the "just turbid" suspension the dilution steps are 1 mL into 1 mL so that each tube is twice as dilute as the previous one. Each dilution step is two-fold. To dilute the cell suspension further, for plating, dilute 0.1 mL into 0.9 mL to obtain more convenient ten-fold dilution steps Technical Tip: Three drops from the bulbed transfer pipet is equal to 0.1 mL.

7. Prepare and Plate a series of ten-fold dilutions ending with a 10x3 dilution: Start with the most dilute turbid tube (just barely turbid tube from step 6 above) Use the following procedure to make three serial ten-fold dilutions from this tube into sterile water.

8. Set up a series of dilution tubes. Pipet 0.9 ml of sterile water into two tubes and l.8 ml into a third tube. Label each tube. The first tube, of your just barely turbid cells is at 10x6 cells per ml.

3. Dilute the just turbid suspension into the first tube. Thump the tube containing the cells. Remove 0.1 ml, and transfer the 0.1 mL to one of the tubes with .9 ml of water. Then thump that tube to suspend the cells. This tube now has 10x5 cells per ml.

4. Repeat the procedure serially, using a fresh pipet for each of the remaining tubes. In the final tube, with 1.8 mls of water, place .2 ml from the tube before in the series. The third (final) dilution, which will be the third tube, should contain approximately 1000 cells/ml . Photo of the results of plating out each tube showing the serial dilution.

5. From the third and final tube, 0.1 ml should be placed on each of the YED agar plates. The 0.1 ml of cells in solution should be spread with a sterile spread over the surface of the agar. This will insure an even distribution of cells on the surface of the agar.

6. Tape the lid of each petri dish so that wind will not blow the lids off during the exposure. Each plate should be carefully labeled

Expose the plates

1. Expose all the plates to the sun except for the unirradiated control plates. Point the plates directly at the sun for the desired exposure time and then place them in a dark container such as a cardboard box or paper bag.

2. Incubate the plates until the colonies are large enough to count. At 30 C that will take about two days and at room temperature it will take three or four days.

Teacher tip Technical Tip: The concentration factor is based on the dilution series and is the ratio of the number of cells plated on the irradiated plate to the number of cells plated on the control plate. Counting Colonies and Analyzing Results

1. Count the colonies on each plate Mark the position of each colony on the bottom of the plate with a marking pen as you count it. Plates containing more than a few hundred colonies are considered "too numerous to count" (recorded as TNTC in data records). Count the colonies and tabulate your data on the table below.