Ultraviolet Killing of Bacteria

See the related protocols on Agar Overlay Technique and Additional UV Experiments.

Ultraviolet light induces the formation of covalently-linked pyrimidine dimers in exposed DNA. If uncorrected, these dimers can trigger changes in base sequence during subsequent replication and thus induce genetic mutations.

In human beings, these mutations are responsible for the high incidence of skin cancers among persons who spend extended periods of time unprotected in the sunlight. In bacteria, they are responsible for the germicidal effect of UV light. This latter effect will be demonstrated in this experiment, but the importance of protecting one’s self from the genetic damage of UV light should apparent as well.

EQUIPMENT AND SUPPLIES:
UV transilluminater (or UV “Sunlamp”1 )
Nutrient agar plate (etc.)
wax pencil
Bacterial cultures2 [Handle pathogens with care]
sterile 0.l mL pipettes
stopwatch
protective eye wear to block UV rays
37°C incubator

PROTOCOL:

1. MARK THE PLATE: On the bottom of a nutrient agar plate, draw a square as large as possible. Divide vertically into 8 equal lanes, and horizontally into four equal regions. Label the eight vertical lanes with the initials each of the cultures to be streaked. Label the four horizontal regions with 135, 45, 15 and 0 to indicate the exposure time of each region in seconds.
2. SET UP THE UV LAMP: We will use a UV transilluminator. Four plates can be placed on its surface at a time. (See below for alternative UV lamps)
3. CROSS-STREAKING TECHNIQUE: Using sterile technique, streak lines of fresh O.N. bacterial cultures to be tested using a 0.1 mL sterile pipet dipped into the culture (it should pick up around 0.02 mL by capillary action and deliver 0.01 mL, or around 107 cells, per streak). As you streak, view the agar surface by reflected light to see that the agar is not scraped up and the liquid is being deposited at the streak. Do not cross-contaminate parallel streaks. Replace plate lid as soon as possible.
4. Wearing protective eye wear, turn on UV lamp, let warm up for one minute. (Do not look at the UV lamp, it is damaging to the rods and cones in your retinas).
5. Remove the cover of the plate and place the line between 90 and 30 on the edge of the transilluminator.
6. EXPOSE THE PLATE IN SUCCESSIVE STAGES to UV for 90, 30, 15 and 0 seconds as follows. (Do not expose your hand to the lamp any longer than necessary.)
a. Start a stopwatch and simultaneously push the plate over the edge of the rectangular UV light source to line between the 90 and the 30 regions, (exposing only the 90 second region). Exposed for an initial 90 second. Stop watch will read 1 minuite, 30 seconds.
b. After 90 seconds, move the plate to the, 30/10 line, expose for 30 more seconds. (the stopwatch will read 2.0 minutes
c. After 30 seconds (stopwatch says 2:00), move to the third 15/0 line, and expose for a final 15 seconds more (stopwatch will say 2 minutes 15 seconds
7. INCUBATE at 37°C for 24-48 hours, and score UV sensitivity of the various cultures tested, and illustrate the results of the exposure on bacterial growth in your notebook.

1 For UV sun tan lamps, (such as a GE 275 watt), mount the lamp 25 cm above the surface of the desk and clamp securely with a ring stand and clamp. Expose for 9, 3, 1 and 0 minutes.

2 Suggested strains
Initials       species of bacterium
Bs:             Bacillus sphaericus
EcB:           E. coli B
EcK            E. coli K12 lambda
Pa               Pseudomonas aeruginosa
Pv               Proteus vulgaris
St                Salmonella typhimurium
Sm             Serratia marcescens
Sa               Staphylococcus aureus