What Effect Do Ultraviolet Rays Have on Yeast Colony Growth?

What Effect Do Ultraviolet Rays Have on Yeast Colony Growth?

Katie Schneider

Period 1

10/21/11
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 Eearth’s surface today, because of the loss of ozone, a naturally occurring chemical that block UV light from reaching the surface. Chloroflurocarbons, 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 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), squamous cell carcinoma of the cornea or conjunctiva (rare tumor on the surface of the eye), photokeratitis (iunflammation 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, 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 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).

Two2 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 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% 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).

Methods and Procedures

First, the agar plates were poured by heating the YED agar in a microwave then pouring the agar onto 25 petri dishes, just covering the bottom of each plate. After hardening the plates overnight, one petri dish was labeled “master plate.”

Discussion

After exposing yeast to UV light for 1, 3, 5, and 7 minutes, varied results were concluded. The amount of yeast grown was calculated out of 34. The average number of yeast colonies for the 1 minute control was 22 and the 1 minute exposed was 32.67. The 3 minute control was 28.67 and the 3 minute exposed was 21.33. The 5 minute control was 31.33 and the 5 minute exposed was 22.33. The 7 minute control was 34 and the 7 minute exposed was 22.67. Out of the 3 trials for the 3, 5 and 7 minute exposed, 1 of the trials showed no0 growth, while all trials of the 1 minute exposed showed growth. Also, the 1 minute exposed showed more growth than the 1 minute control, while the 3, 5, and 7 minute exposed showed less growth than their respective controls. Based on this, the hypothesis that “a short amount of time will aid in yeast production, but a longer amount of time will kill the yeast cells” was correct because 1 minute aided in yeast production, while 3, 5, and 7 minutes hindered yeast growth. However, because of the varied results within trials, the standard deviation rates were high. These rates show that the data is not completely reliable.

To improve this experiment, it should be conducted in the summer so that sunlight can be used as UV light instead of UV flashlights. More trials should be conducted in order to obtain more accurate results. Also, in this experiment, the data was measured based on if there was growth in 34 different sections instead of just overall growth. If this experiment were to be performed again, one should use a higher serial dilution level, such as 1:1000 instead of 1:100, and an alternate method for counting the yeast growth should be used that yields more accurate results.