Laboratory Investigations in Microbiology

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Chapter 13: Effect of Radiation 

Living cells are constantly exposed to a range of electromagnetic emissions, from long-wavelength radio waves to infrared, visible and UV light and gamma and X rays. Solar irradiation may be damaging (UV) but is somewhat filtered out by ozone in the atmosphere, while visible light can be converted to useful cellular energy by organisms. Radioactive isotopes on the other hand can generate high-energy particles damaging to proteins and DNA and have become useful in sterilization procedures in medicine and industry. 
Because natural background radiation and exposure to natural deposits of radioactive materials are present in the environment, survival of radiation exposure has become an adaptive trait, particularly in surface habitats. Different forms of radiation resistance include shielding (i.e. pigmentation) and enhanced DNA repair mechanisms.  The discovery of highly radiation-resistant microbes such as Deinococcus radiodurans and Thermococcus gammatolerans have shed light on another survival strategy: Manganese-based systems to protect cellular DNA repair enzymes. This allows the microbe to survive DNA damage by repairing it after exposure.
The objective of this lab is to examine the sensitivity of several bacterial strains to ultraviolet radiation.

 

Materials per lab group
Procedure 
1. Label your TSA plate with a vertical line to divide the plate in half. Label each side with the name of one strain
2. Divide the plate into 6 equal-sized sections with horizontal (dotted) lines drawn left-to-right approximately 1" apart. Label the sections 0, 30, 60, 120, 240, and 480 (seconds)
3. Using a cotton swab, inoculate each plate half by thoroughly swabbing the agar surface with each respective strain.
4. Place your TSA plate in the back of the flow hood. Remove the lid! Place a cardboard sheet on top so that it completely covers (blocks the light from hitting) the "0" seconds section.
5. After all lab groups have placed their plates, the UV lamp will be turned on for 30 seconds, exposing the rest of the plate to UV light.
6. Move the cardboard sheet 1" to cover the 0 and 30 second sections. The UV will be turned on for 30 more seconds.
7. Move the cardboard sheet 1" to cover the 60 second section as well. The UV lamp will now be turned on for 60 more seconds.
8. Move the cardboard 1" to cover the 120 second section. The UV lamp will now be turned on for 2 more minutes
9. Move the cardboard 1" to cover the 240 second section. The UV lamp will then be turned on for another 4 minutes. The CUMULATIVE exposure time of each section now corresponds to the label.
10. Incubate all plates for 46h at 30C

Data collection

1. After incubation, observe and photograph your plate.
2. Determine the number of seconds needed to kill 90% (or more) of the cells on each side of the plate. Record this number
3. Upload your data for your strains. 

 

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