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Name _________________________

Exercise 5:
Effect of Ultraviolet Radiation
on Bacteria

[Adapted from: CLAUS, G. WILLIAM. Effect of ultraviolet radiation on DNA, cell viability,
and mutation frequency. Exercise 39 in Understanding Microbes: A Laboratory Text-
book for Microbiology. New York: W. H. Freeman and Co., 1989, pp. 333–346.]

INTRODUCTION

Radiation is the process of emitting radiant energy in the form of waves or parti-
cles. Whether radiant energy is useful or destructive to microorganisms depends on its
wavelength (Fig. 5-1). Radiation of wavelengths of 400 nm and below (ultraviolet, X,
and γ radiation) is damaging to the structure of DNA and is thus both mutagenic and
carcinogenic.

Microwave
Infrared

Visible

Ultraviolet

X
γ

-3 -2 -1 0 1 2 3 4 5 6 7
10 10 10 10 10 10 10 10 10 10 10

Wavelength (nm)

Figure 5-1. A portion of the electromagnetic spectrum. Visible light


comprises only a small range of the spectrum. Radiation of wavelength
<400 nm is mutagenic.

Ultraviolet (UV) radiation (10 to 400 nm wavelength) is of special interest


because it is used in certain environments to kill microorganisms. For example, hospital

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BIO 114: BIOLOGY OF NONINFECTIOUS DISEASE — LABORATORY MANUAL
EXERCISE 5: EFFECT OF ULTRAVIOLET RADIATION ON BACTERIA
operating rooms are often lined with UV lights, called germicidal lamps. When people
are not using these areas, the lamps are turned on to kill microbes on the surfaces of
walls, floors, ceilings, and bench tops, which helps to keep the work environment
relatively sterile. Ultraviolet light is considered to be a germicide rather than a sterilant
because it only has the potential for killing dividing cells and does not kill bacterial
spores.

The most germicidal region of the UV spectrum occurs from about 240 nm to 300
nm. DNA absorbs light of 260 nm strongly, which is where the greatest lethal effects
occur. Several effects of UV on DNA are known, but the most thoroughly studied is the
formation of thymine dimers.

Thymine is one of the four bases that are building blocks of DNA. When a UV
ray hits a pair of thymine molecules which are next to each other in a DNA strand, they
become fused together into a structure known as a thymine dimer (Fig. 5-2). This
covalent bonding distorts the shape of the DNA; when the strand is later replicated,
incorrect bases may be placed in the DNA due to the presence of the aberrant thymine
pair. This results in a mutation (change) in the genetic content of the cell.
O O O

N UV
N N
2

N O O N N O

Thymine Thymine Dimer

Figure 5-2. Ultraviolet radiation brings about the dimerization of


adjacent thymines in DNA.

Most microorganisms have enzymes which can repair damage to the DNA
strands caused by UV light. Two of these repair mechanisms are photoreactivation and
dark repair. Photoreactivation is carried out by a photoreactivation enzyme (PRE) that
binds to the dimers. In the presence of visible light, PRE breaks the bond between the
partners of the dimer and returns the damaged DNA strand to its original state. In dark
repair, enzymes called nucleases hydrolyze the damaged portion of the DNA template
so that other enzymes can resynthesize the original DNA in that region, replacing the
thymine dimers with two normal thymine molecules, thus restoring the DNA to its origi-
nal state.

Low doses of radiation may not produce any adverse affects on cells. If one
lengthens the exposure time, or increases the intensity of the UV light, an increase in
the number of unrepaired dimers and an increase in mutations probably occurs. If a
mutation occurs in an essential gene, the cell may die and is said to contain a lethal
mutation. Massive cell death in the skin due to UV irradiation is called sunburn. If the
mutations occur in genes that control the rate of cell division and adherence to
neighboring cells, the result is skin cancer. On the other hand, human beings are

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BIO 114: BIOLOGY OF NONINFECTIOUS DISEASE — LABORATORY MANUAL
EXERCISE 5: EFFECT OF ULTRAVIOLET RADIATION ON BACTERIA
absolutely dependent (without nutritional supplementation) on a minimal level of expo-
sure to UV light for the production of “vitamin” D.1 Thus people are caught between a
rock and a hard place with regard to UV exposure. Too little is bad; too much is bad.2

Skin cancer is currently the most common form of cancer among humans.
Different people have different levels of skin pigmentation (higher levels absorb more
harmful UV radiation) and have varying abilities at DNA repair. If you consistently
sustain sunburns outdoors, you have problems, for one reason or another. Although
the spread of melanocytes (pigment cells) is inducible by UV radiation, if you haven't got
any to induce, no amount of exposure is going to induce them. Similarly, if your thymine
dimer repair mechanisms are poor, no amount of exposure is going to improve their
performance. One of the case studies we have read describes children with
xeroderma pigmentosum; patients with this disease are quite defective in thymine
dimer repair and thus are very prone to skin cancer.3 Keep all this in mind today as you
don protective clothing when working with the UV light. Also keep this in mind the next
time you bake at the beach.

Today you will be given two strains of Escherichia coli, labeled W1485 and
VT1306. One of these is wild-type with regard to DNA repair, and one of these is
defective for DNA repair. You will try to identify the unknowns by their relative suscepti-
bility to UV light.

1LOOMIS, W. F. Rickets. Scientific American 223(6): 77–91, December 1970.


2This discussion has ignored the important issue of sexual selection. Do men at the beach really prefer
women with tans? Do women at the beach really prefer men with tans? Do pale-faced people have
fewer descendants? These questions seem silly, but sexual selection seems to push many species in
odd directions. Behold the peacock.
3JOHNSON, BONNIE, & GIOVANNA BREU. Hiding from the light. People Weekly 33(19): 50–57, 1990.

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BIO 114: BIOLOGY OF NONINFECTIOUS DISEASE — LABORATORY MANUAL
EXERCISE 5: EFFECT OF ULTRAVIOLET RADIATION ON BACTERIA
PROCEDURE, DAY 1

1. Select two sterile nutrient agar plates, and divide each plate into four equal width
parts. Do this by drawing on the bottom with a marking pen:

2. Label each plate on the bottom with your name, date of inoculation, and the
strain of E. coli which you will put on the plate, W1485 or VT1306.

3. Inoculate each of the three plates with 0.1 mL of one of the broth cultures
provided, then aseptically spread the inoculum over the entire surface with a
glass spreading rod as demonstrated by your instructor.

4. Move to the UV work station and put on a laboratory coat, protective UV resistant
goggles, and rubber gloves.

5. Remove the cover from the Petri plate labeled W1485, and place this cover right
side up on a dust-free surface. Expose the bacteria on the agar to UV light as
follows:

a. Place a 4 x 6 index card over the top of the plate so that its edge is
aligned with the first line drawn on the plate bottom:

b. Move the plate so that it is directly under the UV light and immediately
begin timing the exposure.

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BIO 114: BIOLOGY OF NONINFECTIOUS DISEASE — LABORATORY MANUAL
EXERCISE 5: EFFECT OF ULTRAVIOLET RADIATION ON BACTERIA
c. After 15 seconds, move the card over to the second line:

d. After another 15 seconds, move the card over to the third line:

e. After another 15 seconds remove the plate from the UV light source. Note
that the exposure to UV radiation should be maximal at the left side of the
plate:

6. Repeat step 5 with plate VT1306.

7. Incubate the plates upside-down for 24 hours at 37 °C.

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BIO 114: BIOLOGY OF NONINFECTIOUS DISEASE — LABORATORY MANUAL
EXERCISE 5: EFFECT OF ULTRAVIOLET RADIATION ON BACTERIA
PROCEDURE, DAY 2

1. Using the Quebec colony counter, count the number of colonies per square
centimeter on each section of the two plates and record the results on the next
page.

0 Do not mistake a lawn of cells (too many to count) for no cells! You should
have a lawn ON BOTH PLATES at your 0-second control exposure!

2. Discard the used plates in autoclave bags.

RESULTS

Extent of Growth∗

Exposure in seconds W1485 VT1306

15

30

45

Use a roughly quantitative comparative scale, e.g. ++++, +++, ++, +, and –.

DISCUSSION

1. Which of the two strains of E. coli do you think was defective in DNA repair?

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