Introduction: (Initial Observation)
The sun is so hot that it gives off enormous quantities of ultraviolet (UV) photons. These can damage the DNA in your cells and cause skin cancer. The cells of all organisms have ways of repairing most of the DNA damage caused by UV photons. But occasionally the cell’s repair machinery makes a mistake which results in a mutation, an error in one of the cell’s genes. Many scientists believe that mutations in skin cells sometime cause skin cancer. While sunburn is an immediate effect of UV damage, mutations that may lead to skin cancer accumulate over a long period of time.
While studying the effects of UV on mutation, scientists often use single cell organisms such as yeast and bacteria.
Since you can easily use millions of such organisms in each of your experiments, even a very small chance of mutation is enough to successfully find mutated cells. Short life cycle and quick reproduction of such organisms make it possible to see the experiment results in relatively short times.
In this project you will study cell mutation in yeast. You may expose yeast samples to UV radiation and look for changes that may have been caused by mutation.
While studying mutation in yeast, you will need to grow yeast on nutrient agar plates. To do this you will need some basic information about growing microorganisms, in general, and yeast in specific. You may find such information online or in biology related books. The following is a list of information that you need to gather in order to be able to complete your research.
- Yeast; Bakers Yeast; Yeast life cycle and reproduction rate.
- Yeast growth media; YEPD or YPD (Yeast Extract Peptone Dextrose)
- Preparation of Nutrient Agar Plates
- Estimating the number of yeast cells in a solution
- Dilution Techniques
- Striking techniques (use of loops)
Samples of the information that you may find are shown below:
Yeast is a single-celled organism, a type of fungus. The particular species of yeast we will be using is Saccharomyces cerevisiae, the species of yeast used to bake bread. When you buy yeast for baking at the supermarket, you are really buying dried, but still living cells of Saccharomyces cerevisiae.
Yeast cells are tiny, roughly 1/100 of a millimeter in diameter and approximately round. They “grow” by budding off new cells – that is, they grow by dividing into more new yeast cells, and not by individual cell growth (like human cells do). This is shown below:
Each time the cells bud, the number of cells doubles: 1, 2, 4, 8, 16, 32, 64, 128, etc. This is called exponential growth. Budding occurs roughly every 2 hours. That means in 24 hours, one cell would give rise to roughly 4000 descendants. Between experiments, we will let the cells grow for 7 days, which would mean that one cell would give rise to a block of yeast roughly 1 mile on a side! However, the cells eventually use up the nutrients we’ve fed them and stop growing long before that.
YEPD plates – Recipe for 1 liter
- 10g yeast extract
- bring volume to 900ml in H2O
- in separate flask mix
1. 20 gm dextrose (glucose)
2. 100 ml H2O
- autoclave separately for 20 min
- combine and pour plates
You need to know
Don’t count on your memory.
While doing yeast growth experiments, label your plates with water resistant markers. Write something to identify the date, what you put on the plate, and where you put it. Be sure to write on the bottom of the plate (the part with the medium on it) since the covers can get mixed up.
Take careful notes of what you did as well as the hypotheses you were testing
Contamination by other microbes is a big problem.
The nutrient agar plates are very rich medium, so almost any other microorganism that lands there will grow. Since we grow these plates for a long time (one week), a single spore of bread mold will have grown to cover the entire plate in a week. This kind of contamination will make your results unusable. To help prevent this, or at least minimize the tragedy it causes:
Do more than one replicate of each experiment and have the replicates on different plates. Put the different plates in separate bags – one set of experiments in each bag – so that if one set gets contaminated and the contamination spreads throughout the bag, all will not be lost.
Use sterile technique to reduce the chance of contamination:
- Before starting to work, remove all extra stuff from the lab tables or work table and wipe the tabletop with alcohol using a squirt bottle and paper towels. This will kill most microorganisms on the tabletop.
- Tie back your long hair & roll up your sleeves. This will prevent dust (with the microbes in it) from falling on your plates.
- Do anything you can to keep the dust down.
- When you must open your plates, open them for only the minimum time necessary. You may want to have one person hold the lid over the plate while another takes samples, etc.
- Do not touch the nutrient medium in the plates with your fingers or anything except a sterile toothpick that you use for sampling or a sterile loop.
- Take toothpicks out of the tubes carefully. Keep the cap on until you need a toothpick. Then, take the cap off and tap the tube to shake out a toothpick. Then, be sure to hold the toothpick only by the end that will not be touching the yeast or the plates.
- If you even think that you’ve contaminated the toothpick (left it out too long, brushed something, etc.), do not use it & get a new one.
Ultraviolet radiation in our environment is as common as sunlight. It generates genetic diversity and kills cells. It gives us suntans and skin cancer. It appeals to our vanity and feeds our fears. In the classroom, ultraviolet radiation is a vital topic for the study of the global environment, health, genetics, evolution, chemistry, and physics (See Jagger 1985 and Part F: A Closer Look at…Ultraviolet Radiation in Our Environment).
The amount of the damage that ultraviolet radiations produce in growing cells is grossly disproportionate to the amount of energy that is absorbed. A lethal dose of radiation, for instance, will raise the temperature of a cell by only a small fraction of one degree. The reason for this incredible efficiency is that DNA is easily damaged by radiation and small injuries to the genetic material are amplified by replication and growth. In fact, the chemical DNA is even more radiation sensitive than one would guess from the sensitivity of the cell. Because of the environmental radiations that have existed on the earth for millions of years, cells have had to evolve machinery for repairing radiation damage to the DNA (Friedberg 1985).
The mechanisms for repairing radiation damage, found in diverse organisms — from yeast to humans — are remarkably similar, suggesting that these repair processes evolved very early. The universality of these mechanisms makes yeast particularly useful for studying and demonstrating how most cells respond to radiation exposure (Haynes & Kunz 1981).
Techniques for Working with Yeast
Keeping sterile things sterile
Dust is the most common carrier of contamination (bacteria and molds) to yeast cultures and media. It can come from people, from the air or from the bench or table top. To minimize contamination, do the following:
- When you are streaking or pouring plates choose a time and place where you will not be interrupted.
- If possible, choose a work area that is far from plants, animals, Drosophila (fruit fly) cultures, and other biological materials that are sources of mold.
- Choose a work area with the least amount of air turbulence possible. Do not talk, sing, whistle, cough or sneeze in the direction of sterile items or your work area.
- Before beginning to work, clean your work area with soap and water. Then wipe it down with 70% or 95% ethyl or isopropyl (rubbing) alcohol (70% is actually more effective). Wash your hands with soap and water and then wipe them with alcohol.
- Keep sterile media, plates, toothpicks, etc. covered as much as possible.
- Only touch the yeast and sterile things with other sterile things or surfaces.
Streaking yeast cells on a media plate
- Use the rounded end of a sterile toothpick to collect a very small amount of yeast.
- Gently streak the yeast in a zig-zag pattern all across the surface of the media plate. Yeast cells will be spread on the media even if you cannot see them.
Making a suspension of yeast cells
- Use the rounded end of a sterile toothpick to collect a small amount of yeast.
- Place the yeast on the side of a sterile container and add sterile water; add enough water for each media plate on which you will place the yeast. You will need 1 ml for 100 x 15 mm Petri dishes and 0.3 ml for 60 x 15 ml dishes.
- Replace the lid on the container and swirl it to mix the yeast and water. Add more yeast cells if necessary to make a visibly cloudy suspension of cells.
- You may choose to aliquot the yeast solution for each media plate into sterile, capped test tubes or dispense the yeast yourself.
Yeast cells do not survive long in water; it is best to prepare a fresh yeast suspension immediately before each use.
Yeast incubation temperature and growth rates
- The optimum growth temperature for yeast is 30º C, but it will grow satisfactorily at room temperature; the cooler the room, the slower it will grow.
- Incubate yeast in the dark as much as possible.
- For optimal growth, the plates must be aerobic. Do not tightly seal them in plastic bags while they are incubating. You may find it helpful to incubate them in open food-storage bags, which keep them from drying out too quickly and protects them from contamination.
- If the yeast has grown enough to show differences among the treatments and you need to preserve them for further observations, place the plates upside down in the refrigerator in a sealed plastic bag. This will stop the yeast from growing. If the yeast grows too much it may obscure differences among the treatment areas on the dish.
- Yeast strains can be stored on media plates in the refrigerator for up to six months or a year.
What do you want to find out? Write a statement that describes what you want to do. Use your observations and questions to write the statement.
The purpose of this project is to see if the UV light may cause mutation in yeast cells. We expect to be able to identify the mutants by possible changes in their physical or biological properties. For example mutants may have a different color; or they may demonstrate different resistance to certain conditions such as heat, cold, or radiations. Our specific question is:
How does the UV exposure time affect mutations in yeast?
When you think you know what variables may be involved, think about ways to change one at a time. If you change more than one at a time, you will not know what variable is causing your observation. Sometimes variables are linked and work together to cause something. At first, try to choose variables that you think act independently of each other.
Independent variable (also known as manipulated variable) is the exposure time in minutes. Possible values are 1 minute, 2 minutes, 3 minutes, …, 10 minutes.
Dependent variable is the rate of mutation. *
Controlled variables are temperature and other environmental conditions.
Constants are the initial culture, type and the strength of UV light, distance, and experiment method.
* Some students may want to study the effect of UV on yeast. For example they may want to study the survival rate after UV exposure. In this case the dependent variable will be death rate or survival rate.
Based on your gathered information, make an educated guess about what types of things affect the system you are working with. Identifying variables is necessary before you can make a hypothesis.
This is a sample hypothesis:
I hypothesize that a long period of exposure is required for mutation to happen. My hypothesis is based on my observation of other cells such as skin cell that can be exposed to sunlight for many hours without any indication of mutations in the form of skin cancer.
Design an experiment to test each hypothesis. Make a step-by-step list of what you will do to answer each question. This list is called an experimental procedure. For an experiment to give answers you can trust, it must have a “control.” A control is an additional experimental trial or run. It is a separate experiment, done exactly like the others. The only difference is that no experimental variables are changed. A control is a neutral “reference point” for comparison that allows you to see what changing a variable does by comparing it to not changing anything. Dependable controls are sometimes very hard to develop. They can be the hardest part of a project. Without a control you cannot be sure that changing the variable causes your observations. A series of experiments that includes a control is called a “controlled experiment.”
Introduction: In this experiment you will spread a group of genetically identical yeast cells on several nutrient agar plates. You will then expose the plates to UV light for different number of minutes. Finally, you leave all plates in a warm room until they form colonies (in about 3 days).
- Prepare 20 nutrient agar plates (petri dishes) with yeast growth media in sterile conditions.
- Dissolve 1 gram (or one cubic centimeter) of yeast in about 100 mL of warm water.
- Place one drop of the yeast solution in each of the 20 plates, and use the rounded end of a sterile toothpick to gently streak the yeast in a zig-zag pattern all across the surface of the media plate. Yeast cells will be spread on the media even if you cannot see them.
- Mark 2 of the dishes as control and do not expose them to the UV light.
- Place the remaining 18 dishes under a fluorescent 25-watt UV light. Record the time and turn on the light.
- At the end of the first minute remove two of the dishes and label them 1m.
- At the end of the fifth minute remove two other dishes and label them 5 m.
- At the end of the 10th minute remove two other dishes and label them 10 m.
- At the end of the 20th minute remove two other dishes and label them 20 m.
- At the end of the 30th minute remove two other dishes and label them 30 m.
- At the end of the 60th minute remove two other dishes and label them 60 m.
- At the end of the 120th minute remove two other dishes and label them 120 m.
- At the end of the 180th minute remove two other dishes and label them 180 m.
- At the end of the 240th minute remove two other dishes and label them 240 m.
- Keep all the dishes in a warm room for 3 days.
- At the end of the third day or later, observe all the dishes. Count the number of colonies, the shape of colonies, the size of colonies and the color of colonies. If colonies are still small, make additional observations after 5, 7 or 10 days.
You may record your results in a table like this:
|UV Exposure Time in minutes||Number of colonies||Maximum Diameter of colonies||Number of white colonies||Number of red colonies|
Materials and Equipment:
Equipment and Materials *
- Dry YEPD or MV media
- Sterile, polystyrene Petri dishes
- Deionized or distilled water
- 70% or 95% ethyl or isopropyl (rubbing) alcohol
Glass flasks with a capacity of about twice the amount of liquid media you will place in them
- Graduated cylinder
- Balance, weighing paper, and scoopulas (if not using pre-weighed media)
- Autoclave, canning pressure cooker, or microwave
- Aluminum foil (autoclave or pressure cooker) or plastic wrap (microwave)
- Sterile facial tissues (from a freshly-opened box, or several tissues down in an opened box)
- Heat-resistant gloves or potholders (for handling hot flasks)
- Waterproof, broad-tipped markers in two colors
- Rubber bands or masking tape
* Please note that your list of material and equipment may vary from the above list. For example most students do not have an autoclave and may skip sterilization process or may use boiled tap water instead of distilled water.
Where you can buy UV lamps
A type of bulb known as a quartz-halogen lamp has a tungsten filament inside a quartz tube filled with an inert halogen gas. Quartz-halogen lamps can be heated to higher temperatures than ordinary light bulbs, and give off an intense light containing a considerable amount of UV-A and UV-B, and even a little UV-C. Such light bulbs are commonly used for automobile headlights, slide and overhead projectors, and outdoor security lights. Figure 2 shows the energy spectrum of a 300 watt quartz-halogen lamp that is available in hardware and discount stores for less than $20.
Normally, quartz-halogen lights are operated in a glass enclosure, which absorbs the damaging UV photons. If one removes their protective glass cover, however, they become a source of artificial sunlight. The spectrum of light they emit is quite similar to that emitted by the sun after it has been filtered through the atmospheric ozone. With its glass cover open, the lamp whose spectrum is illustrated in Figure 2 can be used as a substitute for sunlight. With the cover closed, it can be used as a source of visible light for photoreactivation.
Obviously when the protective glass cover is removed, a quartz-halogen lamp is hazardous, since it emits as much damaging UV energy as bright sunlight, so those working around it must protect their eyes and skin from the direct radiation.
The quartz-halogen lamp that we have used is Model 3030 Quartz Halogen Lamp Fixture manufactured by Lights of America, Walnut, CA 91789. It is distributed through K-MART stores and some hardware stores. It sells for $10 to $20 and must be wired to a power cord. Mount it on a secure stand at a distance at least 8 inches above the Petri plate, to keep the heat at an acceptable level. At this distance an exposure of 3 minutes will give a survival fraction of 0.1 with the sensitive strain G948-1C.
Fluorescent UV lamps and germicidal lamps are sold by many vendors. One source that carries all three of the lamps whose spectra are illustrated in Figure 3 is Cole Parmer Instrument Co. (800-323-4340). They sell 15 watt tubes under the following catalog numbers: G09815-55 (UV-A), G09815-63 (UV-B), and G09815-59 (UV-C). A safe enclosure for using these lamps is described in this volume.
Results of Experiment (Observation):
Experiments are often done in series. A series of experiments can be done by changing one variable a different amount each time. A series of experiments is made up of separate experimental “runs.” During each run you make a measurement of how much the variable affected the system under study. For each run, a different amount of change in the variable is used. This produces a different amount of response in the system. You measure this response, or record data, in a table for this purpose. This is considered “raw data” since it has not been processed or interpreted yet. When raw data gets processed mathematically, for example, it becomes results.
If you do any calculations, you must write your calculations in this section of your report.
Summary of Results:
Summarize what happened. This can be in the form of a table of processed numerical data, or graphs. It could also be a written statement of what occurred during experiments.
It is from calculations using recorded data that tables and graphs are made. Studying tables and graphs, we can see trends that tell us how different variables cause our observations. Based on these trends, we can draw conclusions about the system under study. These conclusions help us confirm or deny our original hypothesis. Often, mathematical equations can be made from graphs. These equations allow us to predict how a change will affect the system without the need to do additional experiments. Advanced levels of experimental science rely heavily on graphical and mathematical analysis of data. At this level, science becomes even more interesting and powerful.
Using the trends in your experimental data and your experimental observations, try to answer your original questions. Is your hypothesis correct? Now is the time to pull together what happened, and assess the experiments you did.
Related Questions & Answers:
What you have learned may allow you to answer other questions. Many questions are related. Several new questions may have occurred to you while doing experiments. You may now be able to understand or verify things that you discovered when gathering information for the project. Questions lead to more questions, which lead to additional hypothesis that need to be tested.
If you did not observe anything different than what happened with your control, the variable you changed may not affect the system you are investigating. If you did not observe a consistent, reproducible trend in your series of experimental runs there may be experimental errors affecting your results. The first thing to check is how you are making your measurements. Is the measurement method questionable or unreliable? Maybe you are reading a scale incorrectly, or maybe the measuring instrument is working erratically.
If you determine that experimental errors are influencing your results, carefully rethink the design of your experiments. Review each step of the procedure to find sources of potential errors. If possible, have a scientist review the procedure with you. Sometimes the designer of an experiment can miss the obvious.