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Lab 05 Effect of UV Radiation on Bacteria

Lab 05 Effect of UV Radiation on Bacteria

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Name _________________________ 
Exercise 5:Effect of Ultraviolet Radiationon Bacteria
[Adapted from: C
LAUS
,
 
G.
 
W
ILLIAM
. 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 itswavelength (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 andcarcinogenic.
1010101010101010101010
0-1-2-31234567
Wavelength (nm)
γ
UltravioletVisibleInfraredMicrowaveX
 
Figure 5-1. A portion of the electromagnetic spectrum.
Visible lightcomprises 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 interestbecause it is used in certain environments to kill microorganisms. For example, hospitalMonday, February 02, 2009 Page 39 6:33:53 PM
 
BIO 114: BIOLOGY OF NONINFECTIOUS DISEASE — LABORATORY MANUALEXERCISE 5: EFFECT OF ULTRAVIOLET RADIATION ON BACTERIAoperating rooms are often lined with UV lights, called germicidal lamps. When peopleare not using these areas, the lamps are turned on to kill microbes on the surfaces ofwalls, floors, ceilings, and bench tops, which helps to keep the work environmentrelatively sterile. Ultraviolet light is considered to be a germicide rather than a sterilantbecause it only has the potential for killing dividing cells and does not kill bacterialspores.The most germicidal region of the UV spectrum occurs from about 240 nm to 300nm. DNA absorbs light of 260 nm strongly, which is where the greatest lethal effectsoccur. Several effects of UV on DNA are known, but the most thoroughly studied is theformation of thymine dimers.Thymine is one of the four bases that are building blocks of DNA. When a UVray hits a pair of thymine molecules which are next to each other in a DNA strand, theybecome fused together into a structure known as a thymine dimer (Fig. 5-2). Thiscovalent 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 thyminepair. This results in a mutation (change) in the genetic content of the cell.
ThymineThymine DimerUV2NNNNNNOOOOOO
 
Figure 5-2. Ultraviolet radiation brings about the dimerization ofadjacent thymines in DNA.
 
Most microorganisms have enzymes which can repair damage to the DNAstrands caused by UV light. Two of these repair mechanisms are photoreactivation anddark repair. Photoreactivation is carried out by a photoreactivation enzyme (PRE) thatbinds to the dimers. In the presence of visible light, PRE breaks the bond between thepartners of the dimer and returns the damaged DNA strand to its original state. In darkrepair, enzymes called nucleases hydrolyze the damaged portion of the DNA templateso that other enzymes can resynthesize the original DNA in that region, replacing thethymine 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 onelengthens the exposure time, or increases the intensity of the UV light, an increase inthe number of unrepaired dimers and an increase in mutations probably occurs. If amutation occurs in an essential gene, the cell may die and is said to contain a lethalmutation. Massive cell death in the skin due to UV irradiation is called sunburn. If themutations occur in genes that control the rate of cell division and adherence toneighboring cells, the result is skin cancer. On the other hand, human beings areMonday, February 02, 2009 Page 40 6:33:53 PM
 
BIO 114: BIOLOGY OF NONINFECTIOUS DISEASE — LABORATORY MANUALEXERCISE 5: EFFECT OF ULTRAVIOLET RADIATION ON BACTERIAabsolutely 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 arock 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 moreharmful UV radiation) and have varying abilities at DNA repair. If you consistentlysustain sunburns outdoors, you have problems, for one reason or another. Althoughthe spread of melanocytes (pigment cells) is inducible by UV radiation, if you haven't gotany to induce, no amount of exposure is going to induce them. Similarly, if your thyminedimer repair mechanisms are poor, no amount of exposure is going to improve theirperformance. One of the case studies we have read describes children with
xeroderma pigmentosum
; patients with this disease are quite defective in thyminedimer repair and thus are very prone to skin cancer.
3
Keep all this in mind today as youdon protective clothing when working with the UV light. Also keep this in mind the nexttime you bake at the beach.Today you will be given two strains of
Escherichia coli 
, labeled W1485 andVT1306. One of these is wild-type with regard to DNA repair, and one of these isdefective for DNA repair. You will try to identify the unknowns by their relative suscepti-bility to UV light.
1
L
OOMIS
,
 
W.
 
F.
 
Rickets.
Scientific American 
 
223
(6): 77–91, December 1970.
2
This discussion has ignored the important issue of sexual selection. Do men at the beach really preferwomen with tans? Do women at the beach really prefer men with tans? Do pale-faced people havefewer descendants? These questions seem silly, but sexual selection seems to push many species inodd directions. Behold the peacock.
3
J
OHNSON
,
 
B
ONNIE
,
 
&
 
G
IOVANNA
B
REU
. Hiding from the light.
People Weekly 
 
33
(19): 50–57, 1990.
Monday, February 02, 2009 Page 41 6:33:53 PM

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