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Death Rays: What Duration of Ultraviolet Exposure Kills Bacteria?

Difficulty
Time Required Average (6-10 days)
Prerequisites To do this project, you will need access to a laboratory with facilities for culturing bacteria. You should be familiar with sterile technique and proper handling of bacterial cultures.
Material Availability Specialty items
Cost Average ($50 - $100)
Safety Adult supervision required. Read and follow the safety note below on ultraviolet light. Follow standard precautions for handling bacterial cultures.

Abstract

Ultraviolet 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?

Objective

The purpose of this project is to observe the effects of short-term ultraviolet light exposure on bacteria.

Credits

Andrew Olson, Ph.D., Science Buddies

Sources

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Last edit date: 2013-01-10

Introduction

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, below). 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)

effects of UV light on DNA
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.

germicidal ultraviolet light
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,
  • UVA,
  • UVB,
  • UVC,
  • DNA structure,
  • pyrimidine,
  • DNA replication.

Questions

  • What are the differences between UVA, UVB, and UVC radiation?
  • How does UV light damage DNA molecules?

Bibliography

This article discusses the chemical mechanism by which ultraviolet light causes damage to DNA molecules:

These Wikipedia articles discuss ultraviolet light and its use as a germicide:

Read and follow the UV safety precautions on this website:

Materials and Equipment

To do this experiment you will need the following materials and equipment:

  • 15 nutrient agar plates:
    •  3 plates per UV light exposure duration ×
    •  5 exposure durations =
    • 15 plates total;
  • live E. coli (strain K-12) culture (commonly available in labs; E. coli can also be purchased from online suppliers),
  • 200 μL automatic pipettor with sterile tips,
  • bent glass rod for spreading bacteria on plates,
  • bunsen burner,
  • ethanol,
  • aluminum foil,
  • 37°C incubator for bacterial culture plates,
  • short wavelength UV light,
  • UV-blocking face shield,
  • lab coat,
  • gloves.

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Experimental Procedure

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.

  1. Prepare 15 plates of chosen bacteria: pipette 100 μL from the liquid bacterial suspension, and spread it on the plate. Cover the plate and wait 5 minutes for it to dry.
  2. Protect half of each plate from UV light by using aluminum foil to cover half of the lid for each plate.
  3. 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)
    1 15
    2 30
    3 60
    4 120
    5 300
  4. 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.
  5. Remove the foil coverings, then incubate the plates, inverted, overnight at 37°C (or longer if at lower temperature).
  6. Count colonies in both halves of each plate.
  7. For each group of plates, calculate the average and standard deviation of the number of colonies in each half of the plate.
  8. Make a graph showing the average number of colonies (y-axis) as a function of UV exposure time (x-axis).
  9. 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.
  10. Is the average number of control colonies consistent across the five groups of plates? Why or why not?
  11. 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?

Safe Disposal of Plates

At the conclusion of the experiment, all plates should be disinfected for safe disposal.

  1. The best way to dispose of bacterial cultures is to pressure-sterilize (autoclave) them in a heat-stable biohazard bag.
  2. If autoclaves or pressure cookers are not available, an alternative is to bleach the plates.
    1. Wear proper safety equipment (gloves, lab coat, eye protection) when working with the bleach solution; it is corrosive.
    2. Saturate the plates with a 20% household bleach solution (in other words, one part bleach and four parts water).
    3. Allow the plates to soak overnight in the bleach solution before disposing of them.
    4. Please note that the bleach solution is corrosive and needs to be thoroughly rinsed afterwards.

Bacterial Safety

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. You can also see the Microorganisms Safety Guide for more details. Additionally, many science fairs follow ISEF Rules & Guidelines, which have specific guidelines on how bacteria and other microorganisms should be handled and disposed of.

  • 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.

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Variations

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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