2-13: Experiment The Lethal Effect of Ultraviolet Light on Microbial Growth (pgs. 55-57)
Purpose: To determine the effect of Ultraviolet (UV) light on bacterial growth. Ultraviolet (UV) light: A. A type of electromagetic energy B. Travels in waves and is distinguishable by its wavelength ranging from 1nm to 400nm (or 1 to 400) C. Divided into three groups: 1. UV-A: longest wavelength from 315 to 400 nm 2. UV-B: wavelength between 280 to 315 nm 3. UV-C: wavelength from 100 to 280 nm (most detrimental to bacteria) 260 nm = wavelength that DNA and RNA concentration is determined 280 nm = wavelength that protein concentration is determined 265 nm = wavelength that is most damaging to DNA (wavelength emitted by our UV lamps) Prolonged exposure to UV light usually causes death of a bacterial organism by causing DNA damage in the form of pyrimidine dimers (thymine dimers are the most common) that cannot be repaired by the organism's DNA repair mechanisms.
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Materials: (per assigned group of students) One of the following cultures: TSB cultures of: Staphylococcus aureus Escherichia coli 0.85% Saline of: suspension Bacillus megaterium
Ex. 2-13: Ultraviolet Light: Lethal Effects
9 TSA plates (25ml) Tube of 9 sterile cotton swabs Index card and ruler
Working in your group, inoculate each of the TSA plates in the following manner with your assigned organism: Swab the entire surface of the plate in the three directions indicated and around the perimeter of the agar. You are trying to create a bacterial lawn. TAs will demonstrate the proper technique.
1. Swab plate in first direction
2. Turn plate and swab plate in second direction
3. Turn plate and swab plate in third direction and around the perimeter
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The exposure times to be used for all organisms are: 0 sec., 5 sec., 15 sec., 30 sec., 1 min., 2.5 min., 5 min., 10 min., and 10 min. with the Petri dish covered. Expose the plates keeping a distance of 5 inches between the UV lamp and the surface of the agar in the plate. Use the index card to cover half of the plate during exposure to UV light. (See fig. below) The covered half of the plate will act as a control. All UV exposed plates should be incubated at 37oC.
5 inches
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