Experiments with the UV Killing Assay

Experiments with the UV Killing Assay

See Ultraviolet Killing of Bacteria and Agar Overlay Technique for techniques used in these experiments.


Ultraviolet damage can be corrected by several mechanisms in most organisms, including bacteria. One of these repair mechanisms is photoreactivation in which pyrimidine dimers are snipped apart in situ by an enzyme which uses visible light as the source of energy. This process may be demonstrated modifying the UV killing assay as follows:
1. Prepare a seeded plate by pouring 2 mL melted and cooled top agar which has been inoculated with 0.1 mL ON culture of bacteria to be tested. (see Agar Overlay Technique ) Mark with a 16 square grid.
2. Label the four crossing lanes with the exposure times to UV: 3, 1, 0.3 and 0 minutes.
3. Label the four vertical lanes with the exposure times to visible light: 27, 9, 3 and 0 minutes.
4. Expose the plate to UV according to the labeled times as directed on page 1.
5. Rotate the plate 90, expose to visible light, covering all but the 27 minute band with opaque cardboard, place under a bright visible light (glass-filtered sunlight, or 100 watt bulb at 25 cm) for 15 minutes.
6. Move the cardboard to expose the 27 and 9 minutes bands for 6 more minute.
7. Move the cardboard to expose the 27, 9 and 3 minute bands for 3 more minutes.
8. Incubate at 37C in the dark for 24 to 48 hours, note any difference in populations in the agar overlay. Propose a mechanism for any differences observed.


Paint bands of several suntan lotions of given blocking powers across the top of a plastic petri dish. Pour a top agar layer of E. coli on top of nutrient agar plate. Expose the plate as above to 0, 1, 3 and 9 minutes. Note any protection which may be observed.


Shortly before going to a tanning saloon, prepare a nutrient agar plate with a fresh lawn of E. coli in top agar (step 1 above). At the tanning bed, cover the open plate with blocking cardboard, expose for 9, 3, 1 and 0 minutes as directed on page 1. Incubate ON and read bacterial density of exposed areas. Discuss the implication for your exposed skin for these times.

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