EFFECTS OF ULTRAVIOLET LIGHT The bacterium that you will be using for this exercise is Serratia marcescens.

NOTE KEEP YOUR EYES AND YOU PROTECTED FROM THE UV LIGHT. UV light is short wavelength nonionizing radiation. Shorter wavelength is more damaging because shorter wavelength gives more energy. UV radiation at 260 nm is the optimal absorption wavelength of DNA; therefore, UV 260 is the most germicidal. UV light causes the formation of pyrimidine dimers (formation of covalent bond between neighboring thymine or cytosine molecules in the same DNA strand) that results in the distortion of the double helix DNA structure. The distortion of DNA structure can lead to mutation and death of microbes. Cells have photolyases and excision repair mechanisms that can repair the UV damages up to a certain point. Time of exposure is one of the damaging factors. The penetrating ability of UV light also plays a role. Some materials can block UV radiation from reaching the cells. Endospores are more resistant to the harmful effects of UV light than vegetative cells. Endospores are dormant; they are not replicating. Additionally, the DNA of endospores is protected by small, acid-soluble DNA-binding proteins. Serratia marcescens is gram-negative, bacillus (short rod). The colonies of Serratia marcescens appear red because it produces red pigment. The colonies may not appear red if incubated at a temperature o higher than 25 C because the production of red pigment is inhibited at higher temperature.

Procedure: Work in groups. Label the bottoms of the plates with date, name, and the UV exposure time. 2 1:10 dilution - First, mix (resuspend) the Serratia marcescens well by rolling the tube between your palms 25 times - Pipette 1 ml of Serratia marcescens to a sterile 99ml water bottle (bottle A) -2 - Mix bottle A 25 times (Bottle A is 10 ) 4 1:10 dilution - using a new sterile pipette transfer 1 ml from bottle A to a second sterile 99 ml water bottle (bottle B) -4 - Mix bottle B 25 times (Bottle B is 10 ) 6 1:10 dilution - with a new sterile pipette, transfer 1 ml from bottle B to another sterile 99 ml water bottle (bottle C) -6 - Mix bottle C 25 times (Bottle C is 10 ) Pipette 0.4 ml of Serratia marcescens suspension to NA plate. Use sterile cotton swabs to inoculate (spread) the bacteria on the surface of the nutrient agar plates as evenly as possible by swabbing in three directions plus a final sweep over the agar rim. Cover 1/2 of the plate with its lid or with an index card (while the lid removed). Place the plate under the UV lamp. Turn on the UV lamp and expose the plate for o o the intended duration. Cover the plate with its lid. Incubate the plates at 25 C or 37 C. Each group will generate 5 plates 1 (10 ), 3 (10 ), 5 (10 ), 10 (10 ), 30 (undiluted) UV Exposure Times in Second 3 5 10 30 -6 -4 -2 (10 ) (10 ) (10 ) undiluted 3 5 10 30 -6 -4 -2 (10 ) (10 ) (10 ) undiluted 3 5 10 30 -6 -4 -2 (10 ) (10 ) (10 ) undiluted 3 5 10 30 -6 -4 -2 (10 ) (10 ) (10 ) undiluted
o -6 -6 -4 -2

Group 1 Group 2 Group 3 Group 4

1 -6 (10 ) 1 -6 (10 ) 1 -6 (10 ) 1 -6 (10 )

card lid card lid

25 C 25 C 37 C 37 C
o o o

Questions: Does UV kill the bacteria? How can you tell? If UV kills bacteria, whats the % of the population killed at each UV exposure time based on your experiment? Does UV cause mutation? How can you tell? If UV radiation is mutagenic, whats the % of the population mutated at each UV exposure time based on your experiment? Does plastic (the lid of petri dish) block UV? Does paper (the index card) block UV? Is paper more protective than plastic or the other way around? Can you tell? How?