What Effect Do Ultraviolet Rays Have on Yeast Colony Growth?
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Table of Contents
Abstract……………………………………………………………………………………3
Introduction………………………………………………………………………………..4
Research………………………………..………………………………………………….5
Materials and Methods…………………………………………………………………….9
Photos…………………………………………………………………………………….10
Data………………………………………………………………………………………10
Graphs……..……………………………………………………………………………..14
Discussion………………………………………………………………………………..16
Conclusion………………………………………..……………………………………...18
Acknowledgements………………………………………………………………………19
References………………………………………………………………………………..19
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Abstract
This project attempted to determine the effect of UV light on yeast. It
was hypothesized that UV light would aid the growth of yeast if exposed for a very short
amount of time, but would hinder the growth of yeast when exposed for longer periods of
time.
To perform the experiment, yeast was grown and then serial diluted to
1:100 in a yeast suspension. .25mL of this solution was pipetted onto 24 petri dishes.
These were labeled either control or exposed for 1, 3, 5, or 7 minutes, with 3 trials for
each. Taking a cardboard box and making 3 holes across the middle for the UV
flashlights created a simulation for UV light. Three petri dishes at a time were then
exposed to the light for their labeled times.
The variation in the results shows that the data is not completely reliable
because of the high standard deviation. However, overall, the 1-minute exposure showed
more growth than its control, while the 3, 5, and 7-minute exposures showed less growth
than their respective controls. This suggests that short-term exposure might be beneficial,
but long-term exposure may be damaging
If this experiment were to be repeated, more trials would be conducted
and the yeast would be exposed to UV light for more intervals of time. Also, the UV
flashlights that were used had wavelengths of 385 nanometers. If a shorter wavelength
was used, the light may have caused more damage.
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Research
Ultraviolet light is found in sunlight, and has a damaging effect on DNA when
one is exposed to it for a long period of time. In this experiment, the Saccharomyces
cerevisiae is DNA-repair deficient, so the effects of UV light are fatal.
UV light was discovered by Johann Ritter using silver chloride, which turns dark
when exposed to light. He found that the silver chloride turned even darker beyond violet
light on the spectrum, which is why it is called “ultraviolet” light. This proves that there
is light beyond visible light (Kirkland, 2007, p. 108).
Ultraviolet light consists of the range of the electromagnetic (EM) spectrum
between visible light and x-rays. EM radiation consists of photons, small bundles of
energy, which travel in waves. The bottom of the EM spectrum (radio, microwaves) has
low-energy photons, so their wavelengths are longer, while the top (x-rays, gamma rays)
has high-energy photons with short wavelengths (Ultraviolet (UV) Radiation, 2010).
There are 3 types of ultraviolet light: UV-A, UV-B, and UV-C. UV-A is closest to visible
light; it has the longest wavelength, (320-400 nanometers) and therefore penetrates the
deepest into the skin. UV-B light consists of wavelengths from 280-320 nanometers, and
UV-C light consists of wavelengths from 100-280 nanometers (Wyman and Stevenson,
n.d.).
Sunlight consists mostly of UV-A and some UV-B light, because the ozone
absorbs UV-C and some UV-B light (Ultraviolet (UV) Radiation, 2010). There is more
UV light reaching the earth’s surface today, because of the loss of ozone, a naturally
occurring chemical that block UV light from reaching the surface. Chloroflurocarbons,
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chemicals containing chlorine that are used in refrigerants and spray can propellants, are
reacting with the ozone and are depleting the protective ozone layer (MacNeal, 2007).
The UV index (UVI) rates the amount of UV rays that could damage the skin on
the Earth’s surface. It is rated from numbers 1-11; the higher the number, the more
damaging the UV rays will be. Low is 0-3, moderate is 3-6, high is 6-8, very high is 8-11
and extreme is 11 and higher (Ultraviolet (UV) Radiation, 2010).
When skin is exposed to UV light, the epidermis, the skin’s outer layer, thickens
in an attempt to block UV light. Melanocytes also produce melanin to darken the skin;
the melanin absorbs energy from UV light and helps to prevent further damage to the
skin (MacNeal, 2007). UV-A light, specifically, is what mainly causes tanning, skin
aging, and cataracts, UV-B causes sunburn, skin aging and skin cancer, and UV-C is the
strongest, and therefore most effective at killing microorganisms (Wyman and Stevenson,
n.d.). Sunburn is a short-term sign of UV damage, but skin aging and skin cancer are
long-term effects of UV damage (Ultraviolet (UV) Radiation, 2010).
The strength of the UV rays is determined by geography, reflectivity, altitude,
time of year, and time of day. UV rays are the strongest near the equator because the sun
shines directly over the equator, and there is less ozone to absorb the radiation. It is also
stronger near snow, water and other reflective surfaces, and in higher altitudes due to the
lower amount of ozone. Because the sun has a more direct angle to the earth, UV rays are
strongest in the summer, and at noon because the sun is at its highest point in the sky
(Ultraviolet (UV) Radiation, 2010).
According to the Merck Manual, a small amount of UV light is healthy because it
aids in vitamin D production. Large amounts of UV light, however, are detrimental to the
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skin because they damage the skin’s DNA and could change the amounts and types of
chemicals that they produce (MacNeal, 2007).
People used to think that UV-B rays were more harmful than UV-A rays;
however, in a study published in the Journal of Investigative Dermatology, research
suggests that UV-A rays may be just as harmful, or even more harmful, than UV-B rays.
They split up UV-A rays into UVA1 rays, which have a longer wavelength, and UVA2
rays, which are closer to UV-B. Their study shows that the UVA1 rays caused the same
type of DNA damage found in skin cancer, which hadn’t really been shown before; it was
always UV-B. They took skin that had never been exposed to light and applied UVA1
and UV-B light. It was found that the damage caused by the UVA light increased as they
went deeper into the skin which could be more detrimental than the surface of the skin.
They said it may be due to “some kind of reflection process of UVA so it goes back in
and then it’s sent back” (Goodman, 2011).
Diseases caused by UV exposure include skin cancers like cutaneous malignant
melanoma, squamous cell carcinoma of the skin, and basal cell carcinoma of the skin,
photageing (a loss of skin tightness), cataracts (the eye lens becomes opaque and
eventually causes blindness), pterygium ( fleshy growth on the surface of the eye),
sqamous cell carcinoma of the cornea or conjunctiva (rare tumor on the surface of the
eye), photokeratitis (unflammation of cornea), and photoconjunctivitis (inflammation of
the conjunctiva). UV radiation also weakens the immune system by changing the cells
that trigger immune responses (Ultraviolet Radiation and Human, 2009).
In a study in Biofouling, Anat Lakretz says “UV light irradiation is being
increasingly applied as a primary process for water disinfection.”. Instead of chlorine,
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which, according to researchers from Tel Aviv University, produces carcinogenic
byproducts, UV light is being utilized to keep water bacteria-free. “The best way to
control and kill these micro-organisms was to damage their DNA,” Lakretz said . “The
damage that the UV light causes has no known negative effect on the water” (Ultraviolet
Treatment for Water, 2010).
In a 1940 study at the University of Saint Andrew, it was found that when yeast
was exposed to UV light, it caused the release of nitrogenous materials from the cell. The
material was mainly an amino-N substance, which “facilitated growth and reproduction.”
This was backed by another study in 1923 from the University of Chicago Press that
stated that UV light increases yeast production. However, prolonged UV exposure slowly
destroys the cell membrane, which would be the likely cause of the release of nitrogenous
materials, and even other materials as well. Eventually, UV light does kill yeast cells
(Highland, n.d.).
“DNA contains the instructions needed for an organism to develop, survive, and
reproduce.” It is found inside the nucleus of a cell, tightly packed in chromosomes.
During cell division, however, the DNA unwinds in order to be copied. A DNA strand is
made up of nucleotides, which consist of a phosphate group, a sugar group, and a
nitrogen base. The four types of nitrogen bases are adenine, thymine, guanine, and
cytosine. The order of the bases in a strand of DNA determines what instructions are
contained: those sequences of DNA are genes. The shape of DNA is a double helix,
which means that it is double stranded like the 2 sides of a ladder, and is twisted. The
rungs of the ladder are made up of 2 nitrogen bases linked by hydrogen bonds. Adenine
always pairs with thymine, and guanine always pairs with cytosine. This allows DNA
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replication because when the 2 strands of DNA are separated, it is easy to figure out
which sequence of bases must be paired with each half, called a template, because the
same bases always pair with each other. It is in the same way that DNA is transcribed
into RNA, which is then used to create proteins (Deoxyribonucleic Acid (DNA), 2011).
Damage caused to DNA by the environment, if it is not repaired, leads to
mutation and disease. The most common example of this is that skin cancer is caused by
excessive exposure to UV light. During the cell cycle, regulators prevent mitosis until all
subsequent prerequisites are met. If the DNA is damaged, the cell will not divide, which
causes an accumulation of damage. UVA radiation specifically causes 2 types of DNA
damage: cyclobutane pyrimidine dimers and 6-4 photoproducts, both of which cause
bends in DNA structure, therefore hindering replication and transcription. These 2 types
of damage are repaired by nucleotide excision repair, in which the damage is detected,
the section of DNA that includes the damage is removed, and it is filled in with new
DNA by DNA polymerase (Clancy, 2008).
2 other ways DNA damage can occur is through deamination and depurination.
Deamination occurs when there is a loss of an amino group that changes one of the
nitrogenous bases; for example, the loss of NH2 would change cytosine to uracil.
Depurination occurs as a result of the loss of a purine (either guanine or adenine) from a
nucleotide, which leaves a “missing tooth” in the molecule of DNA (DNA Damage
From, n.d.).
Saccharomyces, a genus of heterogeneous yeast, is used for sugar fermentations,
producing vitamins, or bioengineering the yeast to carry genes from other organisms in
its DNA in order to produce large amounts of that product. Saccharomyces cells are
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usually an ellipse shape between 2.5-6.5 micrometers in width and 3.5-8.5 micrometers in
length. They reproduce asexually by mitosis, and bud from their parent cell with a full
chromosome. They are very adaptable; they have the ability to live in many different
environments and can live withstand conditions from 0 to 40 degrees Celsius, and of pH
3 or lower. Saccharomyces cerevisiae, specifically, can live in alcohols, soil, fruits, and
human skin. It is also a facultative anaerobe, which means that although it prefers
oxygen, it can function both with or without oxygen. They are used frequently as a model
organism for genetic studies because they are small, replicate quickly, their population
doubles within 2 hours, they are easily cultured, have simple nutrition requirements, go
through transformation, are good genetic models, and since they are eukaryotes they have
the basic structure of plants and animals (Maczulak, n.d.). A model organism is a
“species that has been widely studied, usually because it is easy to maintain and breed in
a laboratory setting and has particular experimental advantages” ( Twyman, 2002).
Saccharomyces cerevisiae was discovered by Antony van Leeuwenhoek, who was
the first to study beer yeast with a microscope. Louis Pasteur observed that the sugar
changed when treated with the beer yeast, and then in 1978 Albert Hinnen, James Hicks,
and Gerald Fink discovered that Saccharomyces cerevisiae was picking up outside
plasmid and incorporating it into their DNA, which they called transformation
(Maczulak, n.d.).
When using yeast, it is important to avoid contact of the nose and mouth with any
materials used with yeast cultures, and to wash hands after working with yeast. Anything
that comes in contact with the yeast should also be sterilized by being soaked in a 10%
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bleach solution for 1-2 hours. It is also important to use a disinfectant to clean any
surfaces used during experimentation (Whyte, 2009).
This experiment also involves the use of agar plates. Agar is a gelatinous
substance made up of galactose that is part of the cell wall of red algae from eastern Asia
and California. It is widely used as a culture medium for microorganisms because it
won’t be eaten by bacteria and is firmer and stronger than regular gelatin. It solidifies
from 32-40 degrees Celsius, is a gel up to 65 degrees Celsius, and melts at 85 degrees
Celsius. It is important when pouring agar plates to sterilize the neck of the agar bottle by
passing it through a flame, to cover the petri dishes immediately so that they remain
sterile, and to take care not to spill the agar or burn oneself (Liu & Usinger, n.d.).
In this experiment, the Saccharomyces cerivisiae is genetically engineered to be
DNA-repair-deficient. This means that the enzymes that normally would repair DNA
damage are knocked out so that the yeast is especially sensitive to UV light. This helps to
show what effect that gene has in yeast’s lives, and how fatal UV light would be if the
DNA damage was not repaired (Whyte, 2009).
10
11
Trial #
Control
Exposed
Control
Exposed
Control
Exposed
Control
Exposed
1 min
1 min
3 min
3 min
5 min
5 min
7 min
7 min
1
34
34
18
30
34
0
34
34
2
32
30
34
0
26
33
34
0
3
0
34
34
34
34
34
34
34
Average
22
32.6667
28.6667
21.33333
31.3333
22.33333
34
22.6667
40
35
30
25
20
Series1
15
10
5
0
control 1 exposed control 3 exposed control 5 exposed control 7 exposed
min
1 min
min
3 min
min
5 min
min
7 min
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References
Cells and DNA. (2011, October 17). Retrieved from http://ghr.nlm.nih.gov/handbook/
basics?show=all#gene
Clancy, S. (2008). DNA Damage & Repair: Mechanisms for Maintaining DNA
Integrity. Retrieved from Nature Education database.
Deoxyribonucleic Acid (DNA). (2011, March 23). Retrieved from
http://www.genome.gov/25520880
DNA damage from deamination and depurination [illustration]. (n.d.). Retrieved from
Science Online database.
Genetic Engineering. (2011, May 23). Retrieved from http://www.fda.gov/
AnimalVeterinary/DevelopmentApprovalProcess/GeneticEngineering/default.htm
Goodman, B., MA. (2011, October 6). UVA Radiation May Cause DNA Damage in Skin.
Retrieved from http://www.webmd.com/healthy-beauty/news/20111006/uvaradiation-may-cause-dna-damage-in-skin
Highland, J. (n.d.). The Effects of UV Light on Yeast. Retrieved from
http://www.livestrong.com/article/252729-the-effects-of-uv-light-on-yeast/
Hockberger, P. E. (2002). History of Ultraviolet Photobiology. Retrieved from
Department of Physiology, Feinberg School of Medicine, Northwestern
University website: http://www.photobiology.info/Hockberger.html
Kirkland, K., PhD. (2007). Infrared and Ultraviolet. In Light and Optics (pp. 106-109).
New York: Facts On File, Inc.
Liu, S., & Usinger, L. (n.d.). All About Agar. Retrieved from
http://www.sciencebuddies.org/science-fair-projects/project_ideas/
MicroBio_Agar.shtml
MacNeal, R. J., MD. (2007, August). Overview of Sunlight and Skin Damage. Retrieved
from Merck website: http://www.merckmanuals.com/home/skin_disorders/
sunlight_and_skin_damage/overview_of_sunlight_and_skin_damage.html
Maczulak, A. (n.d.). Saccharomyces. In Encyclopedia of Microbiology. Retrieved from
http://www.fofweb.com/
activelink2.asp?ItemID=WE40&SID=5&iPin=EMBIO0159&SingleRecord=True
Tanner, F. W., & Ryder, E. (1923, May). Action of Ultraviolet Light on Yeast-Like
Fungi. Botanical Gazette, 75(3), 309. Retrieved from http://www.jstor.org/pss/
2470221
Twyman, R. (2002, August 28). What are ‘model organisms’? Retrieved from
http://genome.wellcome.ac.uk/doc_wtd020803.html
Ultraviolet radiation and human health. (2009, December). Retrieved from World Health
Organization database.
Ultraviolet treatment for water suggested. (2010). Biofouling. Retrieved from
http://www.fofweb.com/activelink2.asp?ItemID=WE40&SID=5&iPin=UPI-120100422-140951-bc-israel-watertreatment&SingleRecord=True
Ultraviolet (UV) Radiation. (2010, August 17). Retrieved from http://www.fda.gov/
Radiation-EmittingProducts/RadiationEmittingProductsandProcedures/Tanning/
ucm116425.htm
Whyte, D. B. (2009, December 4). Exploring DNA Damage: What Effect Do Ultraviolet
Rays Have on Yeast Colony Growth? .
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Wyman, B., & Stevenson, H. L. (n.d.). Ultraviolet radiation–A range (UV-A). In The
Facts On File Dictionary of Environmental Science, Third Edition. Retrieved
from http://www.fofweb.com/
activelink2.asp?ItemID=WE40&SID=5&iPin=DEST4675&SingleRecord=True
Wyman, B., & Stevenson, H. L. (n.d.). Ultraviolet radiation–B range (UV-B). In The
Facts On File Dictionary of Environmental Science, Third Edition. Retrieved
from http://www.fofweb.com/
activelink2.asp?ItemID=WE40&SID=5&iPin=DEST4676&SingleRecord=True
Wyman, B., & Stevenson, H. L. (n.d.). Ultraviolet radiation–C range (UV-C). In The
Facts On File Dictionary of Environmental Science, Third Edition. Retrieved
from http://www.fofweb.com/
activelink2.asp?ItemID=WE40&SID=5&iPin=DEST4677&SingleRecord=True
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