SCHOOL OF SCIENCE AND TECHNOLOGY, SINGAPORE
INVESTIGATIVE SKILLS IN SCIENCE
Names: Chelsea Seah (01), Ee Xueqin (02), Myat Noe Pwint (05), Nehal Janakraj (06)
Group reference: H
1. Indicate the type of research that you are adopting:
[ ] Test a hypothesis: Hypothesis-driven research
e.g. Investigation of the antibacterial effect of chrysanthemum
[ ] Measure a value: Experimental research (I)
e.g. Determination of the mass of Jupiter using planetary photography
[ ] Measure a function or relationship: Experimental research (II)
e.g. Investigation of the effect of temperature on the growth of crystals
[ ] Construct a model: Theoretical sciences and applied mathematics
e.g. Modeling of the cooling curve of naphthalene
[ √ ] Observational and exploratory research
e.g. Investigation of the soil quality in School of Science and Technology, Singapore
[ ] Improve a product or process: Industrial and applied research
e.g. Development of a smart and green energy system for households
2. Write a research proposal of your interested topic in the following format:
Research Title: An investigation of the lethal DNA effects of sunlight on UV-sensitive yeast.
A. Question being addressed
When the damage caused by ultraviolet (UV) radiation to a deoxyribonucleic acid (DNA) molecule and is not repaired before the DNA replicates, the cell is likely to die before reproducing. When a cell suffers reproductive death, it cannot divide to form viable progeny. It may still be able to metabolize and grow, but is unable to divide.
In single-celled organisms such as yeast, mutations are a sublethal effect. Radiation also produces chromosomal changes that are sublethal and stimulates genetic recombination. All types of genetic changes can be studied more directly at the cellular and molecular level in yeast. Most nonlethal mutations are recessive, and are most easily studied in haploid strains, where they are expressed directly.
There are several different types of repair systems. UV-damaged cells are exposed to sunlight that has the UV wavelengths filtered out, a specific enzyme in the cell uses the energy from the visible part of the solar spectrum to reverse the reaction that produces pyrimidine dimers. If the dimers are repaired before the DNA tries to replicate them, they have no effect on the cell. However if it isn’t, the yeast cell will go through mutations due to radiation, which might result in reproductive death. Another process, called excision repair, involves a sequence of events in which the damaged portion on one of the strands of the DNA double helix is removed by enzymes, and then the gap is accurately resynthesized, with the remaining strand acting as the template. Normal yeast cells have four mechanisms for DNA repair.
Thus, in this study, we would like to observe the lethal effects of UV rays’ radiation on yeast cells, and how yeast cells are affected.
If UV-sensitive yeast is exposed to UV rays, then there would be lethal DNA effects on the yeast, causing the yeast to be unable to function properly.
C. Description in detail of method or procedures (The following are important and key items that should be included when formulating ANY AND ALL research plans.)
UV-sensitive yeast strain (mutant in several DNA repair pathways)
Yeast medium, yeast extract + dextrose (YPD)
Sterile dilution tubes
Sterile distilled water
Micro pipettes (1ml)
Permanent marker (black)
D. Procedures: Detail all procedures and experimental design to be used for data collection
Phase 1: Pouring the yeast extract peptone dextrose (YPD) Plates
1. Loosen the cap of the YPD agar bottle.
2. Heat the bottle in a microwave until the contents are completely melted.
2.1 Stop the microwave to swirl the bottle every minute to keep the contents from boiling over.
3. Pour the melted YPD agar into 10 petri dishes.
3.1 Cover them immediately after pouring so the dishes remain sterile.
3.2 Always protect your hands with heat-resistant gloves.
3.3 Pour agar and ensure the amount is just enough to cover the bottom of each plate.
3.4 Let the agar harden at room temperature overnight.
Phase 2: Streaking the Master Plate
1. To make sure your work area is clean, wipe the table with 70% alcohol.
2. Use a pair of disposable gloves to keep the inoculating loops sterile.
3. Touch the loop on an area where you can see yeast growing in the tube.
3.1 The yeast is shipped in a tube containing agar and nutrients
4. Glide the inoculating loop in a zigzag pattern across one of the YED plates that was poured previously.
5. Use a fresh loop (without yeast) to make another zigzag pattern through the first zigzag pattern. The idea is to get separate yeast cells that will grow into well-separated colonies.
6. Label the plate "Master Plate" with a permanent marker. Mark the date on the plate.
7. Wrap the plate in aluminum foil to protect it from light.
8. Allow the yeast to grow for two days at 27° C - 32° C.
Phase 3: Labeling the Tubes and Making a Yeast Cell Suspension
1. Label the five tubes, as follows:
- 1:10 000
2. After the two days, put on a pair of disposable gloves and use an inoculating loop to collect a mass of yeast from the master plate. The yeast should be approximately 1 mm in diameter.
3. Smear the yeast inside the tube labeled "1" toward the bottom on the side of the tube.
4. Use a 5-m∫ bulb pipette to add 5 m∫ of sterile water to the tube with the yeast.
5. Shake the tube until the yeast are suspended.
Phase 4: Making Serial Dilutions
Use a new pipette for each transfer below.
1. Use the 3.5m∫ sterile pipette to add 2.25m∫ of sterile water into each of the tubes labeled 1:10, 1:100, 1:1,000 and 1:10,000.
2. Use a clean 1m∫ pipette to transfer 0.250m∫ of yeast from the tube labeled 1 to tube labeled 1:10 and mix thoroughly.
3. Use a clean 1m∫ pipette to transfer 0.250m∫ of yeast from the tube labeled 1:10 to tube labeled 1:100.
4. Mix thoroughly.
5. Use a clean 1m∫ pipette to transfer 0.250m∫ of yeast from the tube labeled 1:100 to tube labeled 1:1,000.
6. Mix thoroughly.
7. Use a clean 1m∫ pipette to transfer 0.250 m∫ of yeast from the tube labeled 1:1,000 to tube labeled 1:10,000.
Phase 5: Spreading the Yeast onto Agar Plates
1. Label the bottoms of four agar plates with the following to prevent mixing up the agar plates when the lids are removed.
- 1:10 000/control
- 1:10 000/exposed
2. Use a 1-m∫ pipette to add 0.250m∫ of yeast suspension to the appropriately labeled plate. In other words, add yeast from the 1:1000 to two plates and 1:10 000 to two plates.
3. Spread the yeast cells evenly using a sterile L- shaped spreader .
4. Allow the plates to sit for 5 - 10 min to dry
Phase 6: Exposing the Yeast to UV Light
1. Cover all plates with black paper to prevent any UV rays from entering it.
2. Expose the plates labeled "Exposed" to the sunlight.
3. Wrap the plates labeled "Control" in aluminum foil to protect the yeast from light.
4. The time for which the yeast are exposed depends on the time of day. The plates should be positioned so that the sunlight hits the yeast from directly above. Use the guide below:
- Morning: 3 - 4 minutes
- Noon: 2 - 3 minutes
- Mid afternoon: 3 - 4 minutes
5. After exposing the yeast, cover them back with the lids.
6. Wrap the plates in aluminum foil to protect them from light and let them sit at room temperature for two days.
Phase 7: Repeating the Procedure
1. Repeat the entire procedure at least two more times to show that your results are reliable and accurate.
2. To dispose of the yeast cultures, wrap the plates and tubes in aluminum foil and dispose of them in a biohazard autoclave bag.
E. Risk and Safety
- Treat all microorganisms as potential hazards to our health. As such, always handle any laboratory equipments with gloves. (See also Microorganisms Safety Guide for more details)
- Proper disposal of yeast cultures:
- Yeast cultures, plates, and disposables that are used to manipulate the yeast should be soaked in a 10% bleach solution (10% bleach 90% water) for 1–2 hours before disposal.
- Use caution when handling the bleach.
- After soaking in bleach, the disposables may then be placed into household garbage.
- Cleaning of work space:
- Use a disinfectant or any commercial antibacterial bath/kitchen solution to thoroughly clean the surfaces of the work area after the experimenting.
- Treat disinfectant with care as they may pose as potential health hazard.
- Cover any spills or broken culture tubes with a 70% ethanol or 10% bleach solution; then cover with paper towels. After allowing the spill to sit with the disinfectant for a short time, carefully clean up and place the materials in a biohazard autoclave bag to be autoclaved.
- Sterilize equipment and materials. All equipments such as needles, pipettes, tubes, petri dish and other items used for culturing microorganisms should be sterilized after use.
- Disinfect work areas before and after use. Use a disinfectant to clean benches and work areas both before and after working with the yeast cultures. Avoid placing such chemicals near flames as alcohol is flammable. Also,
- Wash your hands before and after experiment. Use a disinfectant soap to wash your hands before and after working with microorganisms.
- Never place your nose, eye, or mouth near any chemicals in the lab. Avoid being too near to the chemical or the microorganisms in the lab. Always wear a goggle as protection. Keep out hands out of our mouths, and wash our hand before and after lab activities. Any incidents (cuts, ingestion of chemicals or cultures from the lab) should be reported to a teacher or lab assistant immediately.
- Label everything clearly. All cultures, chemicals, disinfectants should be clearly and securely labeled with their names and dates. If they are hazardous, label them with the proper warning and hazard information.
F. Data Analysis: Describe the procedures you will use to analyze the data/results that answer research questions or hypotheses
1. Unwrap the plates and count the number of colonies on each.
1.1 Each colony is formed from a single yeast cell.
1.2 Ideally, one of the control plates should have about 100 colonies, as this number is small enough that you can count individual colonies, but large enough that you can get an accurate percentage of killed cells.
1.3 If the colonies are too close together to count, even at the 10,000-fold dilution, repeat the dilution series and add a 100,000-fold dilution.
2. Graph the number of colonies for each plate. Put the number of colonies on the y-axis and the treatment and dilution on the x-axis.
3. Calculate the percentage of cells killed by UV light. See Equation 1, below. Compare colony counts from plates with the same dilution.
3.1 Divide the number of colonies on the exposed plate by the number of colonies on the control plate.
3.2 Subtract this number from 1.
3.3 Multiply the resulting number by 100.
3.4 This yields the percentage of yeast killed by the sunlight.
3.5 Create a table based on the results you have obtained.
100 × ( 1 - colonies on exposed plate/colonies on control plate) = % killed
1. Graph the percentage of yeast killed by exposure to the sunlight
How the tables should look like:
Number of colonies for each plate
% of yeast killed by sunlight
G. Bibliography: List at least five (5) major references (e.g. science journal articles, books, internet sites) from your literature review. If you plan to use vertebrate animals, one of these references must be an animal care reference. Choose the APA format and use it consistently to reference the literature used in the research plan. List your entries in alphabetical order.
Banas, T. (2010). The Effects of Ultraviolet Radiation on Yeast. eHow. Retrieved July 9, 2014, from http://www.ehow.com/list_6375291_effects-ultraviolet-radiation-yeast.html
Exploring DNA Damage: What Effect Do Ultraviolet Rays Have on Yeast Colony Growth? (2013). Exploring DNA Damage: What Effect Do Ultraviolet Rays Have on Yeast Colony Growth?. Retrieved March 3 2014, http://www.sciencebuddies.org/science-fair-projects/project_ideas/BioChem_p036.shtml#summary
How to put bacteria you desire on a petri dish. (2014). Retrieved July 9 2014, from http://www.sciencebuddies.org/science-fair-projects/project_ideas/MicroBio_Inoculation.shtml
Microorganisms Safety Guide. (n.d.). Microorganisms Safety. Retrieved July 15 2014, from http://www.sciencebuddies.org/science-fair-projects/project_ideas/Micro_Safety.shtml
Potentially hazardous biological agents. (2014). Retrieved July 9 2014, from https://student.societyforscience.org/Potentially-Hazardous-Biological-Agents?pid=319
Science Buddies Staff. (2013). Exploring DNA Damage: What Effect Do Ultraviolet Rays Have on Yeast Colony Growth?. Retrieved July 9, 2014 from http://www.sciencebuddies.org/science-fair-projects/project_ideas/BioChem_p036.shtml
The Response of Yeast to UV Radiation. (1997). Retrieved July 17, 2014, from http://www.phys.ksu.edu/gene/a3.html
Yeast is Fussy About Temperature. (n.d.). Retrieved July 17, 2014, from https://www.exploratorium.edu/cooking/bread/yeast_temp.html