Death Rays: What Duration of Ultraviolet Exposure Kills Bacteria?
AbstractUltraviolet light can damage DNA molecules. If a cell's DNA repair mechanisms can't keep up with the damage, mutations are the result. As harmful mutations accumulate, the cell eventually dies. How much ultraviolet light is too much for a bacterial cell?
The purpose of this project is to observe the effects of short-term ultraviolet light exposure on bacteria.
Andrew Olson, Ph.D., Science Buddies
This project is based on:
- Beck, A.E., 2004. "What Are the Effects of Ultraviolet Light on Bacteria Mortality?" California State Science Fair Abstract [accessed September 18, 2006] http://www.usc.edu/CSSF/History/2004/Projects/J1303.pdf.
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Ultraviolet (UV) light is invisible to our eyes, and has higher energy than visible light. "When considering the effect of UV radiation on human health and the environment, the range of UV wavelengths is often subdivided into UVA (400–315 nm), also called Long Wave or 'blacklight'; UVB (315–280 nm), also called Medium Wave; and UVC (< 280 nm), also called Short Wave or 'germicidal'." (Wikipedia, 2006a) Short-wavelength UV light has enough energy to damage chemical bonds in DNA molecules, which are very stable under most conditions.
"Ultraviolet light is absorbed by a double bond in pyrimidine bases (such as thymine and cytosine in DNA), opening the bond and allowing it to react with neighboring molecules. If it is next to a second pyrimidine base, the UV-modified base forms direct covalent bonds with it. The most common reaction forms two new bonds between the neighboring bases, forming a tight four-membered ring (see Figure 1). Other times, a single bond forms between two carbon atoms on the rings, forming a '6-4 photoproduct.' These reactions are quite common: each cell in the skin might experience 50-100 reactions during every second of sunlight exposure." (Goodsell, 2001)
Figure 1. Short wavelength UV light can cause adjacent pyrimidine bases in DNA (thymine and cytosine), to bond to one another instead of the complementary DNA strand. This disrupts DNA replication.
Cells have mechanisms to repair this damage, but if the duration of exposure to UV light is sufficient, the repair mechanisms are unable to keep up with the rate of DNA modification. Short wavelength UV light can thus serve as a germicide.
Figure 2. Short wavelength UV lights are often used to prevent microbial growth on work surfaces in tissue culture hoods, like this one.
In this project, you will determine how much UV light exposure is needed to kill bacteria in culture plates.
Terms and Concepts
To do this project, you should do research that enables you to understand the following terms and concepts:
- ultraviolet (UV) light,
- DNA structure,
- DNA replication.
- What are the differences between UVA, UVB, and UVC radiation?
- How does UV light damage DNA molecules?
This article discusses the chemical mechanism by which ultraviolet light causes damage to DNA molecules:
- Goodsell, D.S., 2001. "The Molecular Perspective: Ultraviolet Light and Pyrimidine Dimers," The Oncologist 6 (No. 3, June, 2001): 298-299, [accessed September 18, 2006] http://theoncologist.alphamedpress.org/cgi/content/full/6/3/298
These Wikipedia articles discuss ultraviolet light and its use as a germicide:
- Wikipedia contributors, 2006a. "Ultraviolet," Wikipedia, The Free Encyclopedia [accessed September 18, 2006] http://en.wikipedia.org/w/index.php?title=Ultraviolet&oldid=76387342.
- Wikipedia contributors, 2006b. "Ultraviolet Germicidal Irradiation," Wikipedia, The Free Encyclopedia [accessed September 18, 2006] http://en.wikipedia.org/w/index.php?title=Ultraviolet_Germicidal_Irradiation&oldid=75437165
Read and follow the UV safety precautions on this website:
- IBC UMN, 2003. "Bio Basics Fact Sheet: UV Radiation Protection in Laboratories," Institutional Biosafety Committee, University of Minnesota [accessed April 15, 2011] http://www.dehs.umn.edu/PDFs/UVProtection.pdf
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Materials and Equipment
Note: If you are carrying out this experiment in a school laboratory, which is recommended, some of the materials and equipment listed may be more readily accessible.
- Nutrient agar plates (15)
- 3 plates per UV light exposure duration ×
- 5 exposure durations =
- 15 plates total;
- Glass spreader
- Live E. coli, strain K-12
- Safety glasses that offer UV eye protection. This is optional if you are using a face shield.
- Disposable gloves. Alternatively, these can be purchased at a local drug store or pharmacy. If you are allergic to latex, use vinyl or polyethylene gloves.
You will also need to gather these items:
- 200 μL automatic pipettor with sterile tips
- Bunsen burner
- 70% ethanol
- Aluminum foil
- Lab coat. Can be purchased at scientific supply companies, or through an online supplier like Carolina Biological Supply Company. Alternatively, old, long-sleeved protective clothing may be used instead.
- UV-blocking face shield
- Short wavelength UV light. These are often called germicidal UV-C lights.
- Timer or clock that shows seconds
- Permanent marker
- 37°C incubator for bacterial culture plates
- Lab notebook
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Death Rays: What Duration of Ultraviolet Exposure Kills Bacteria?
For health and safety reasons, science fairs regulate what kinds of biological materials can be used in science fair projects. You should check with your science fair's Scientific Review Committee before starting this experiment to make sure your science fair project complies with all local rules. Many science fairs follow Intel® International Science and Engineering Fair (ISEF) regulations. For more information, visit these Science Buddies pages: Projects Involving Potentially Hazardous Biological Agents and Scientific Review Committee. You can also visit the webpage ISEF Rules & Guidelines directly.
This science fair project involves the use of the bacteria E. coli. While E. coli is not considered a biohazardous or dangerous bacteria, it is important to always properly clean and dispose of bacteria and supplies that come in contact with it. See the Bacterial Safety guidelines for more details on how to handle bacterial cleanup and waste.
Safety Note: Adult supervision is required for this project. Read and follow these Ultraviolet Light Safety Precautions (IBC UMN, 2003):
- The germicidal ultraviolet (UV) light used in this project will also damage unprotected human cells. Your eyes and skin are particularly susceptible to UV damage.
- Exposure to UV light can burn the retina or irritate the cornea and the conjunctiva. This can cause a feeling of "sand in the eye" and heightened sensitivity to light. Symptoms appear from 6 to 24 hours after exposure and usually disappear within 48 hours.
- Persons who have had the lens of an eye removed (e.g. cataract surgery) can receive permanent retinal damage from UV exposure - including blindness.
- Individuals who are exposed to photosensitizing agents (e.g. some oral drugs or topically applied creams) may not be aware of heightened sensitivity to UV radiation.
- UV radiation burns skin promoting skin aging and cancer.
- If possible, the UV source for irradiating bacterial cultures should be operated remotely, so that the Investigator is not exposed to UV light.
- If this is not possible, then the UV source should be set up so as to avoid direct exposure to the Investigator (i.e., placed behind a UV-blocking barrier).
- The Investigator should also use the following personal protective equipment:
- All skin should be protected including face, neck, hands, and arms.
- Wear gloves and long sleeves covering all skin above the gloves.
- Eyes and face should be protected by a face shield designed to block the UV wavelengths used. Radiation can readily reach the eyes through the open sides of standard eye glasses, so they do not provide sufficient protection.
- Prepare 15 nutrient agar plates with the K-12 E. coli bacteria: pipette 100 μL from the liquid bacterial suspension, and use a sterile spreader to spread it on the plate. Cover the plate and wait 5 minutes for it to dry.
- Protect half of each plate from UV light by using aluminum foil to cover half of the lid for each plate.
- You will use the plates in five groups of three plates each. All plates should be at the same distance from the UV source. The following table shows the suggested UV exposure time for each group of plates. Remember to read and follow the UV light safety precautions (above) while performing this step.
|Group||UV light exposure time (seconds)|
- Immediately after the UV light exposure, use a permanent marker to indicate which half of each plate received UV light, and the duration of the exposure.
- Remove the foil coverings, then incubate the plates, inverted, overnight at 37°C (or longer if at lower temperature).
- Count colonies in both halves of each plate.
- For each group of plates, calculate the average and standard deviation of the number of colonies in each half of the plate.
- Make a graph showing the average number of colonies (y-axis) as a function of UV exposure time (x-axis).
- On the same graph, you can also use a different symbol to plot the average number of colonies on the unexposed (control) side of each plate.
- Is the average number of control colonies consistent across the five groups of plates? Why or why not?
- What duration of UV exposure results in 50% bacterial mortality? Are there any plates with 100% bacterial mortality? If so, what duration of UV exposure results in 100% bacterial mortality?
Bacteria are all around us in our daily lives and the vast majority of them are not harmful. However, for maximum safety, all bacterial cultures should always be treated as potential hazards. This means that proper handling, cleanup, and disposal are necessary. Below are a few important safety reminders.
- Keep your nose and mouth away from tubes, pipettes, or other tools that come in contact with bacterial cultures, in order to avoid ingesting or inhaling any bacteria.
- Make sure to wash your hands thoroughly after handling bacteria.
- Proper Disposal of Bacterial Cultures
- Bacterial cultures, plates, and disposables that are used to manipulate the bacteria should be soaked in a 10% bleach solution (1 part bleach to 9 parts water) for 1–2 hours.
- Use caution when handling the bleach, as it can ruin your clothes if spilled, and any disinfectant can be harmful if splashed in your eyes.
- After bleach treatment is completed, these items can be placed in your normal household garbage.
- Cleaning Your Work Area
- At the end of your experiment, use a disinfectant, such as 70% ethanol, a 10% bleach solution, or a commercial antibacterial kitchen/bath cleaning solution, to thoroughly clean any surfaces you have used.
- Be aware of the possible hazards of disinfectants and use them carefully.
If you like this project, you might enjoy exploring these related careers:
MicrobiologistMicroorganisms (bacteria, viruses, algae, and fungi) are the most common life-forms on Earth. They help us digest nutrients; make foods like yogurt, bread, and olives; and create antibiotics. Some microbes also cause diseases. Microbiologists study the growth, structure, development, and general characteristics of microorganisms to promote health, industry, and a basic understanding of cellular functions. Read more
BiochemistGrowing, aging, digesting—all of these are examples of chemical processes performed by living organisms. Biochemists study how these types of chemical actions happen in cells and tissues, and monitor what effects new substances, like food additives and medicines, have on living organisms. Read more
Biological TechnicianWhat do the sequencing of the human genome, the annual production of millions of units of life-saving vaccines, and the creation of new drought-tolerant rice varieties have in common? They were all accomplished through the hard work of biological technicians. Scientists may come up with the overarching plans, but the day-to-day labor behind biotechnology advances is often the work of skilled biological technicians. Read more
Be sure to follow the UV light safety precautions (above) for all of these experiments.
- Are some bacterial strains more susceptible to UV-induced DNA damage? Are some bacterial strains less susceptible to UV-induced DNA damage? Do background research in order to develop a hypothesis, and then design an experiment to test it.
- Is UVB light an effective bactericide? Design an experiment to compare UVB exposure to UVC exposure. Make sure that you verify the range of wavelengths produced by each light source (see manufacturer's specifications).
- How good is sunblock at blocking UVB rays? Can it protect bacteria from germicidal illumination? You can use a protocol similar to the one here, but instead of using foil, spread a uniform layer of sunblock over half of the plate lid.
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