This experiment can be used to investigate the following concepts and phenomena:
See the Effect of Solar UV on Cells and Global Ozone video segments in Tape II. Yeast cells have four ways to repair damaged DNA. (See Biological Consequences of Ultraviolet Exposure and A Closer Look...Repair of DNA.) G948-1C is extremely sensitive because it is mutant (broken) in three of the four ways.
Numbers refer to steps in the student procedure) 2. Yeast cells usually come from the supplier growing on agar slants. To keep contamination at a minimum, it is usually best to limit the use of the original stock culture and have students work from subculture plates. For this experiment, one subculture plate will supply enough yeast to make suspensions for over one hundred plates. Store the original stock in a refrigerator to keep viable for up to nine months.
It is best to have relatively fresh yeast cultures for your experiments. You may wish to standardize the subculturing procedure for your classroom and then use the same procedure each time you subculture yeast cells.
It takes one milliliter of suspension to make one plate. Figure the number of plates you wish to make and then use the appropriate amount of water. It is possible to store the yeast suspension in the refrigerator and use it during several class periods within a day or two. A "just turbid" suspension has approximately one million cells per milliliter.
Numbers refer to steps in the student procedure
Making the UV exposure plates: 5. If the agar plates are fresh, the water will take considerably longer than ten minutes to soak into the agar. It is best if the plates are poured and then allowed to set out at room temperature for a week before using them for this experiment. You may also make a more concentrated suspension, put less than one milliliter (one tenth mL) on each plate and then use sterile paper clip spreaders to spread the suspension over the surface of fresh plates.
9. On cloudy days or in the winter, it is possible to use a 300 watt quartz-halogen security light (with the glass cover off) as a source of artificial sunlight. (See "A Closer Look... Modeling UV Effects.")
Questions and Answers: 1.How does the plate look different the day after it was exposed to the sun (3rd day)?
The lawn of yeast should be just becoming visible and some growth difference between the covered and uncovered portions should be visible. (If the plate is incubated at room temperature, the growth may not be visible until 4th day.)
2.Describe the plate on 4th day (5th day, if incubated at room temperature).
Note the amount of growth on the plate: 1) Where the paper or card shaded the cells, 2)where cells were exposed to direct sunlight through the lid, and 3)where anything was placed to test its ability to protect the cells. There should be little or no growth where the cells were fully exposed. Where they were fully shaded, there should be a heavy layer of yeast covering the agar. The experimental portion(#3) can be compared with these two extremes.
3.How can you tell if you gave the exposed half of the plate a lethal dose? Did all of the cells die?
The cells on the covered part will grow normally. If however, the exposure is great enough, all the exposed cells will be killed. If the dose is not enough to kill all of the cells, the growth may still be reduced.
4.Why did you cover a portion of the plate with a piece of opaque material?
The material will shade the cells protecting them from the sun. This is an untreated control for the experiment. It simply tells you what the cells are like when they are not being stressed by environmental conditions.
5.Why is it important to hold the plate perpendicular to the sun's rays?
The surface of the plate should be perpendicular to the rays coming from the sun to expose the cells to the maximum dose of UV.
6.How can you tell if you have the plate perpendicular to the sun's rays?
Put a card or similar object that will show a shadow behind the plate. The shadow will be smallest when the surface of the plate is perpendicular to the sun's rays and the radiation is most concentrated on the surface.
7. How does the angle of the sun affect your chance of getting asunburn?
When the angle of the sun is small, your shadow is shorter than your height and your risk of getting sunburned from prolonged exposure is greatest. If the sun's angle is great enough to make your shadow longer than your height, the risk of getting sunburned is less.
. 8. How does the angle of the sun affect the amount of UV that falls on the cells in a given amount of time?
If the sun were directly overhead (zenith angle = 0o), which only happens in the tropics, its radiation would travel the shortest possible path through the atmosphere. When the sun's zenith angle is greater than 0o, it must pass through a greater amount of atmosphere including the ozone layer. You will then need a longer exposure to see the effect on cells or to get a sunburn. (The technical term for this relative thickness of the atmosphere is the "air mass".)
9. How does the time of day and time of year affect the sun's angle and the time required for the cells to receive a lethal exposure?
Each day, the sun is highest at noon. Throughout the year, the sun is highest at the summer solstice (approximately June 22 in the northern hemisphere) and lowest at the winter solstice (approximately December 22 in the northern hemisphere). Therefore, at any particular location, assuming the sky is clear, the time to receive a lethal exposure will be shortest at noon on June 22 . 10. How does the geographic location affect the time required for the cells to receive a lethal exposure at noon on June 22, when the sky is clear?
As you travel north of the tropic of cancer (23.5o north latitude) the zenith angle of the sun increases. Therefore, you need a longer exposure time.
Objectives and Applications:
This section introduces the and techniques of quantitative microbiology, which involves a number of valuable concepts and skills useful for a variety of experiments, including the following: The concept of cell growth and colony formation. The ability to prepare a suspension of cells with a known cell density by visual estimation. The ability to plate a known number of cells on a petri plate. The ability to make quantitative estimates of the number of viable cells in a culture. Getting Ready: You may wish to do this step for your students. One YED subculture plate will supply enough yeast for several classes. To save lab time you may wish to make a subculture plate for each lab group. Yeast usually comes from the supplier growing on agar slants. To keep contamination at a minimum it's usually best to limit the use of the original stock culture and have students work from subculture plates.
The video segment Serial Dilution & Viable Cell Count on video tape III illustrates this procedure. You may wish to pause the tape after each step is illustrated to allow the students to complete each step before viewing the next step in the procedure.
Preparing a "just turbid" suspension:
(See Laboratory Methods: Estimating the Number of Yeast Cells in a Culture)
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Teacher tip - 2 See Laboratory Methods: Dilution Plating Procedure
You may wish to review exponential notation with your students.
10e6 = 1,000,000
10e5 = 100,000
10e4 = 10,000
10e3 = 1,000
10e2 = 100
10e1 = 10
10e0 = 1
Numbers refer to steps in the student procedure:
2. Three drops from a bulbed pipet equals one tenth milliliter. If one fills the pipet to the one milliliter mark with water and then drops out three drops there is nine tenths milliliter left in the pipet which can then be transferred to a sterile tube.
3. As you transfer one tenth milliliter to each tube be sure to return any unused suspension to the previous tube. If you want to conserve pipets it is possible to use one pipet for the entire procedure. When using one pipet thoroughly rinse the pipet in each cell suspension as the suspension is made. The pipet may also be returned to its sterile plastic sleeve when it is not in use.
4. The most useful data will come from plating the tubes with the fewest
cells. (10e6 to 10e4)
You may wish to save plates by not using the contents of the other tubes. ( 10e3 to
10e0)
Questions and answers:
1. How well do your colony counts agree with the expected dilution steps of
ten?
The best agreement will probably be between the 10e1 and 10e2 plates.
2. If the agreement isn't perfect (and we don't expect it to be, even
with a "perfect" technician) do you think the errors could be explained by
"random chance"? Explain.
See A Closer Look At... The Dilution Series and Statistics for a discussion of
possible answers.
3. Is there any pattern to the errors? Do the ratios systematically get
larger or smaller as the number of colonies increases?
Answers will vary.
4. Write down any reasons you can think of for such systematic errors
to occur in this procedure.
The ratio may get systematically smaller if you use one pipet to make the whole
dilution series and do not rinse it thoroughly in each suspension before transferring
cells to the next tube.
5. If you have a culture with 1 106 cells/mL and you wanted to dilute
it to 1 102 cells/mL, would it be better to just make a single 10,000-fold dilution or
four ten-fold dilutions? Explain the advantages and disadvantages of each method.
Single step dilution
Advantage: takes fewer steps
Disadvantage: requires large quantities of sterile water and large containers
Four step dilution
Advantage: uses small amounts of sterile water and small containers
Disadvantage: more steps, uses more sterile pipets
Objectives and Applications:
The procedure that follows assumes that individual students or lab groups will each do a serial dilution, plate suspensions from several dilution tubes and expose the plates for several exposure times. The short term goal is to determine the length of exposure for your particular geographic location and time of year that produces a surviving fraction of approximately 0.1 (a 0.1 surviving fraction is equivalent to 10% survival). This is sometimes referred to as "determining the LD10." The long term goal is to develop enough expertise so that students can efficiently monitor UV-B over a long period of time. The amount of UV-B reaching the surface of the earth at any particular location varies over the course of a day and also varies by season over the year .
Once students get skilled at these procedures two plates should be enough to make a UV reading. (one unirradiated control plate and one irradiated timed exposure plate) It's possible to envision all types of monitoring schedules. One might take one reading each week at a particular time of day and use the data to monitor the seasonal change of UV. One might take one reading each hour during a day and monitor the change of UV over a single day. Students could use this technique to quantify the sunscreen experiments they designed in the section Observing the Effects of Solar Ultraviolet Radiation on Cells.
The preparation procedures for this experiment are essentially the same as those for Serial Dilutions and Viable Cell Counts.
Procedure:
1. a. Choose several exposure times that look reasonable and will produce a surviving
fraction close to 0.1 You may want to coordinate the exposure times between the different
lab groups so that you can cover as wide a range of exposures as possible.
2. There are several video segments that illustrate this process. They use various types of pipetting equipment and various methods of plating cells. Figure 2 illustrates the pour plating method in which a separate final dilution tube is prepared for each plate and then the entire contents of the tube are poured onto the agar surface. This method has the advantage of not requiring the use of a flame. It has the disadvantage of not being as reproducible.
Note that Figure 2 shows only one final tube of each type. Your students may not choose to make all of the illustrated pouring tubes, especially the 10,000 cells per mL. If they need several plates of one type (for example 100 cell plates for controls) they need to make a separate final tube to pour onto each plate.
The suspension must be allowed to soak into the agar before the plates are exposed to the sunlight. To speed up the process make the agar plates several days in advance and let they dry out a bit before you pour on the yeast suspension.
Video section C: Serial Dilution & Viable Cell Counts illustrates the pour method of plating.
When you want precise data, it is better to use an alternative plating method. Instead of pouring the dilutions illustrated in the second row in Figure 2 onto the plate, omit that step and accurately pipet 0.1 mL of the dilutions shown in the first row directly onto the agar. Use a sterile spreader to distribute the suspension uniformly over the surface, but avoid spreading it to the edges where it can run down between the agar and the side of the Petri dish.
Two types of spreaders:
1. No flame paper clip method:
You can easily make an inexpensive reusable spreader by straightening out two bends in
a large paper clip, leaving a hairpin-shaped end for spreading and a straight handle at
right angles to it. You can sterilize several spreaders together in covered beakers or
wrapped in foil or paper.
2. Flamed glass spreader method:
Video section D:Using Yeast to Measure Solar UV, illustrates a flamed glass spreader
method of plating.
Name of Investigators:
Date:
Time:
Location:
Yeast Strain:
Growth medium:
Incubation temperature:
Diagram of Dilution and Plating Procedure:
Results:
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Teacher tip - 3If semi-log graph paper is not available students may use linear graph paper, but it's unlikely that they will get a straight line relationship, and it will be harder for the them to compare their graphs. You may want to copy Figure 2 and have the students plot their data with the sample data. How does your survival curve compare to Figure 2? The curve in Figure 2 indicates that approximately 21 seconds of exposure produced a 0.1 surviving fraction. Does your data indicate more or less than 21 seconds are needed to produce a 0.1 surviving fraction? This will vary based on the intensity of your UV source. Compare your UV-C source to the one used to produce the data in
Figure 2. Teacher tip - 4Notice that plot of mutants/survivor in Figure 3 is made on linear graph paper. Once again you may wish to simply copy Figure 3 and have the students plot their pooled data with the sample data. How does your graph compare to Figure 3? Chapter 5 - Teacher tip - 1Materials: 5 to 10 YED agar plated in polystyrene Petri dishes: the sample data given in Table 1 uses 24 plates (eight different exposure times and three duplicate plates for each time), If everyone uses the same strain the class can pool data to make a survival curve so each group can get by with fewer than 24 plates 10 sterile pipets, either 1-mL calibrated bulbed transfer pipets or 1mL disposable serological pipets calibrated in 0.1 mL steps and pipet pump (see the discussion about pipets in the laboratory method section)$ Marking Pens: be sure to test the pens, some ink will not mark glass or plastic UV irradiation box with germicidal lamp: plans for this box are included in this handbook; you may be able to use a lab goggle sterilization cabinet as a UV-C source. DO NOT try to use germicidal lamps without some sort of safety enclosure, UV-C is very damaging to human tissue. There are several video segments that illustrate the dilution and plating processes that may be used in this experiment. They use various types of pipetting equipment and various methods of plating cells. Figure 8-1 illustrates the pour plating method in which a separate final dilution tube is prepared for each plate and then the entire contents of the tube are poured onto the agar surface. This method has the advantage of not requiring the use of a flame. It has the disadvantage of not being as reproducible. Note that Figure 1 shows only one final tube of each type. Based on their experimental design students may not need to make all of the illustrated pouring tubes. If they need several plates of one type (for example 100 cell plates for controls) they need to make extra final tubes, one for each plate. The suspension must be allowed to soak into the agar before the
plates are exposed to the UV. To speed up the process make the agar plates several days in
advance and let they dry out a bit before you pour on the yeast suspension. When you want precise data, it is better to use$ alternative plating
method. Instead of pouring the dilutions illustrated in the second row in Figure 1 onto
the plate, omit that step and accurately pipet 0.1 mL of the dilutions shown in the first
row directly onto the agar. Use a sterile spreader to distribute the suspension uniformly
over the surface, but avoid spreading it to the edges where it can run down between the
agar and the side of the Petri dish. Getting Ready:You may wish to do this step for your students. One YED subculture plate will supply enough yeast for several classes. To save lab time you may wish to make a subculture plate for each lab group. Yeast usually comes from the supplier growing on agar slants. To keep contamination of the original vial to a minimum it's usually best to limit the use of the original stock culture and have students work from subculture plates. Which strain should you use? Teacher tip - 2Procedure: Two types of spreaders: 3. Plastic and glass lids absorb most UV-C so the lids must be removed while the plates are in the UV-C irradiation box. It's important to cover the plates with a sheet of glass when you expose the plates to sunlight. The lids of most plastic Petri dishes are transparent to the UV-B in sunlight. The glass will filter out these harmful wavelengths preventing further damage to DNA. If sunlight isn't available, use a quartz-halogen light with the glass cover closed at 8 to 10 inches or several 40 watt fluorescent lights at 4 to 6 inches. Teacher tipThe table below shows an example of the results of a typical photoreactivation experiment. Example Results of a Photoreactivation Experiment
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