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Labs 3 and 4- Yeast Genetics Introduction Baker’s yeast (Saccharomyces cereviseae) is a unicellular organism that reproduces both sexually and asexually. It is possible to distinguish all the major stages of the yeast life cycle under the microscope because the cells have a characteristic shape at each stage. A brief description and the figures on the next page will help you understand the stages observed. Haploid yeast cells can occur in two mating types (or “sexes”): mating type a and mating type a (alpha). Each of these has a spherical morphology, and the two types are indistinguishable from each other. When cells of opposite mating types come in close contact, however, they induce a change in haploid cells of the opposite mating type. This developmental change is marked by the elongation of haploid cells into egg-shaped “shmoos” as they develop into gametes. The gametes then can fuse at their narrow ends and produce zygotes, which appear as peanut-shaped cells. When cultured on a rich, nutritious growth medium, the fused peanut-shaped cells can reproduce asexually by budding (which appear as cloverleaf’s) and may grow into a visible colony that contains up to 100 million cells. In times of stress such as starvation, however, these cells cease to bud and undergo a process that produces reproductive cells called spores. Spores can survive stressful environmental conditions, and when growth conditions become favorable, the spores can germinate and reenter life stages that lead once again to reproduction by budding. n these experiments, you will use yeast of opposite mating types to observe a complete yeast life cycle and the transfer of genetic information through inheritance.
Procedures Day 1 1. Obtain from your instructor Plate 1, which contains two yeast strains: the a mating type at 12 o’clock and the a mating type at 9 o’clock. Prepare fresh cultures of both mating types on Plate 2 as follows: a. Touch the flat end of a sterile toothpick to the a mating type. b. Gently drag the toothpick across the surface of a YED medium plate to make a vertical steak about 1 cm long and 1 cm from the edge at a position of 12 o’clock. c. Discard the toothpick in the waste bag. Use a glass-marking pencil or marker to label the plate with an a near the streak. d. Use a new sterile toothpick to put a horizontal streak of a mating type at 9 o’clock on the same plate. Label this streak a. e. Label this culture Plate 2 and add your name and the date.
Be sure to use aseptic (sterile) technique when working with the yeast plates. This includes keeping the lid on the plate except when transferring yeast.
2. Prepare a mating mixture on Plate 2 as follows: a. Use a new sterile toothpick to transfer a small amount of the a mating type from the streak on Plate 1 to the middle of the agar in Plate 2. b. Use another sterile toothpick to transfer an equal amount of the a mating type to a point just next to the first spot. c. Use a third toothpick to thoroughly mix the two dots of yeast to make a mating mixture.
Be careful not to tear the surface of the agar. 3. Invert Plate 2 and incubate at 30°C for 90 minutes so that the cells can begin mating. 4. Now, return to Plate 1 and use the microscope to examine some yeast cells of each mating type. Use the following procedure. a. Put a small drop of water on a microscope slide. b. Gently touch the flat end of a sterile toothpick to the streak of either the a mating type (strain that has a red or pink phenotype) or the a mating type (strain that has a cream phenotype) on Plate 1. c. Mix the material on the toothpick with the small drop of water on the slide. d. Place a cover slip over the drop. e. Discard the toothpick in the plastic waste bag. f. Examine the cells with the high-power (dry) lens of the microscope (40X objective). g. Sketch the cells and note the phenotype of each mating type.
5. Use the procedure in Step 4 to examine the mating mixture (from Plate 2) through the microscope. a. Sketch the cells, and describe any changes as compared with the original mating types, and estimate the relative percentages of the different cell morphologies. (Hint: You should notice another cell morphology (or form), although you may have to look around the slide to find it.) b. Explain why these changes may have occurred and show (or explain) how you calculated your estimate. 6. Return Plate 2 to the incubator and incubate overnight at 30°C. (If necessary, your instructor will refrigerate the plate after this incubation.)
Day 2 7. Examine Plate 2 and answer the following questions: · What is the phenotype of the mating mixture? (If it is not obvious, refrigerate the plate for two hours.) · If you compare this phenotype with the phenotypes of the two mating types, what conclusion can you draw about the relationship between the phenotypes? 8. Use the procedure in Step 4 to examine the mating mixture through the microscope. a. Sketch the cells and describe the similarities and differences with the original mating types and with the observations you made in Step 5. What are the percentages of the different cell morphologies? b. Explain why these changes may have occurred. 9. Make a subculture of mating type a, some of mating type a, and some of the mating mixture. Use sterile toothpicks to transfer very small amounts of the yeast to an MV (minimal) medium agar plate. (The amount transferred should not be large enough to see.) Use the same pattern that you used on Plate 2. Label this subculture Plate 3 and add your name and the date. 10. Invert the plate and incubate overnight at 30°C.
Day 3 11. Examine Plate 3 and answer the following questions: · What do you observe about the subcultures on Plate 3 after incubation? · Explain how the genetic information present in the fused cells differs from the genetic information present in either one of the original mating types. 12. Make a wet mount of the freshly grown mating mixture from Plate 3, observe the cells through the microscope, and answer the following questions: · What types of cells are present? Sketch each type and determine the approximate percentage of the different types. · If any of the cell types seen in Step 8 have disappeared, explain what happened to them. 13. Make a subculture by transferring some of the mating mixture with a sterile toothpick to a fresh YED medium agar plate. a. Streak the cells in one horizontal line across the middle of the plate. b. Label this subculture Plate 4 and add your name and the date. 14. Invert the plate and incubate at 30°C overnight.
Day 4 15. On a plate of “unknown” medium, make several thick streaks of the freshly grown subculture from Plate 4. Label this culture Plate 5 and add your name and the date. 16. Invert the plate and incubate at 30°C for at least 3 days.
Day 7 17. Make a wet mount slide of the subculture from Plate 5, observe the cells through the microscope, sketch them, and answer the following questions: · What cell types are present now that were not present before? Sketch these cell types. · Compare these cell types with the cell types that you observed at other stages. · Speculate on the relative nutritional value of the “unknown” medium versus the YED and MV media. · If the cells in the sacs are most frequently found in groups of four, propose an explanation for how they were formed and why? · Explain whether the cells in the sacs are haploid or diploid. 18. Transfer some of the yeast from Plate 5 to a fresh YED medium plate, using a streaking technique that will maximize the number of single colonies that result. Label this Plate 6 and add your name and the date. 19. Invert the plate and incubate at 30°C overnight.
Day 8 20. Observe the colonies that result on Plate 6 and comment on the phenotypes. 21. Make a wet mount slide of the subculture from Plate 6, observe the cells through the microscope, sketch them, and answer the following question: · How would you explain the distribution of cells observed? · Explain how the series of experiments just completed demonstrates (a) allelic interactions and genetic variation and (b) the continuity of genetic information as seen through the life cycle of yeast. Use specific genetic terminology and relate the experiments to the concepts discussed in lecture.
Assignment Complete the work in this laboratory, including sketches, and answer all of the questions (some tasks and questions are bulleted; some are embedded in bold text in the procedural steps). You will turn in your sketches and answers. Make certain that you clearly indicate which task/question you are addressing and that you include enough of the question in your answer that the answer makes sense.
A.
Haploid yeast cells budding
Materials microscope slides cover slips dropping pipette glass-marking pencil or marker compound microscope container of sterile toothpicks bottle of water plastic waste bag MV (minimal) medium agar plate 3 YED (growth) medium agar plates “unknown” medium agar plate
References: Yeast morphology illustration from: http://www.phys.ksu.edu/gene/chapters.html |